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Pterocarpans and triterpenoids from Gueldenstaedtia verna
Pterocarpans and triterpenoids from Gueldenstaedtia verna Chengle Yin, Jinge Zhou, Yiqing Wu, Yue Cao, Tao Wu, Shoude Zhang, Honglin Li, Zhihong Cheng PII: DOI: Reference:
S0367-326X(15)30059-9 doi: 10.1016/j.fitote.2015.07.021 FITOTE 3238
To appear in:
Fitoterapia
Received date: Revised date: Accepted date:
29 June 2015 28 July 2015 31 July 2015
Please cite this article as: Chengle Yin, Jinge Zhou, Yiqing Wu, Yue Cao, Tao Wu, Shoude Zhang, Honglin Li, Zhihong Cheng, Pterocarpans and triterpenoids from Gueldenstaedtia verna, Fitoterapia (2015), doi: 10.1016/j.fitote.2015.07.021
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ACCEPTED MANUSCRIPT
Pterocarpans and triterpenoids from Gueldenstaedtia verna
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Chengle Yin a, Jinge Zhou b, Yiqing Wu a, Yue Cao a, Tao Wu b, Shoude Zhang c, Honglin Li c, Zhihong Cheng a,*
Department of Pharmacognosy, School of Pharmacy, Fudan University, Shanghai
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a
Key Laboratory of Standardization of Chinese Medicines of Ministry of Education, The
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b
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201203, China
Shanghai Key Laboratory for Compound Chinese Medicines, Institute of Chinese
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Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China
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c
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University of Science and Technology, Shanghai 200237, China
Title running-header: Pterocarpans and triterpenoids from Gueldenstaedtia verna
Contact Information of the Corresponding Author: Dr. Zhihong Cheng Tel: +86-21-51980157; Fax: +86-21-51980017; Email: [email protected] Mailing address: 826 Zhangheng Road, Department of Pharmacognosy, School of Pharmacy, Fudan University, Shanghai 201203, China
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ACCEPTED MANUSCRIPT ABSTRACT Two new pterocarpan glycosides (1–2), five new triterpenoids (3–7), and 13 known
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analogues (14–20) were isolated from the whole plants of Gueldenstaedtia verna. These
1D (1H and
13
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new compounds (1–7) were elucidated by extensive spectroscopic techniques including C) and 2D NMR experiments (COSY, HSQC, HMBC and NOESY),
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HR-ESI-MS and chemical methods. The absolute configuration of 1 was established by
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the optical rotation, the comparison of experimental and calculated electronic circular dichroism (ECD) spectra and an X-ray diffraction analysis. All the isolates were
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evaluated for their cytotoxicities against four human cancer cell lines and inhibitory
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activities on LPS-induced NO production in RAW264.7 cells.
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Keywords: Gueldenstaedtia verna; Leguminosae; Pterocarpans; Triterpenoids; Cytotoxic
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activity; Inhibition of NO production
2
ACCEPTED MANUSCRIPT 1. Introduction The genus Gueldenstaedtia Fisch. (Leguminosae) comprises approximately 20
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species that are mainly distributed in central and eastern Asia. Many Gueldenstaedtia
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species have been used in folk medicines for treatments of various inflammatory diseases [1]. G. verna (Georgi) Boriss. is a small (4-20 cm high) herbaceous perennials covered
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entirely with long, white pubescence, which produces violet flowers. The whole plants
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including roots have been used in traditional Chinese medicines (TCM) as a heat-clearing and detoxicating drug for the treatment of a wide range of discomforts, such as
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suppurative inflammation, furuncles, carbuncles, fever, gastroenteritis, and diarrhea. This herb shows significant antioxidant and antibacterial activities [2,3]. It is used as a
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substitute for Viola yedoensis in TCM. Very little is known about its phytochemical composition of G. verna with the exception of several flavonoids [2,3] and triterpenoids
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[4,5]. As part of our ongoing investigation of the chemistry and biological activity of V. yedoensis and its substitutes [6-8], we report herein the isolation and structural elucidation of the new pterocarpans (1–2) and triterpenoids (3–7) from G. verna. Compound 1 is assigned as a rare pterocarpan ribofuranoside. All the isolates were evaluated for their cytotoxic activities against four human cancer cell lines including lung (A549), ileocecal adenocarcinoma (HCT-8), breast (MCF-7), and hepatocellular carcinoma
(BEL-7402)
cell
lines.
In
addition,
their
inhibitory
effects
lipopolysaccharides-induced NO production in RAW264.7 cells were also studied. 2. Experimental 2.1. General
3
on
ACCEPTED MANUSCRIPT Optical rotations were measured at 20 ºC on a Rudolph Autopol VI polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA). Electronic circular dichroism
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(ECD) spectra were recorded on a Chirascan CD spectrometer (Applied Photophysics
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Ltd., Leatherhead, UK). IR spectra were recorded on a Nicolet Avatar 360 FT-IR spectrometer (Thermo Nicolet Corporation, Madison, WI, USA) using KBr disks. NMR
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spectra were measured on a Bruker DRX 400 spectrometer (Bruker Daltonics, Fallanden,
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Switzerland). ESI-MS spectra were measured on a Velos Pro dual-pressure linear ion trap mass spectrometer (Thermo Scientific, San Jose, CA, USA), and HR-ESI-MS spectra
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were obtained on a Bruker Daltonics APEX III 7.0 TESLA Fourier Transform mass spectrometer (Billerica, MA, USA). Semi-preparative HPLC were performed on a
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LC3000 system equipped with a P3000 pump, a UV3000 detector (Beijing Chuangxintongheng Science & Technology Co., Ltd., Beijing, China), and a Luna RP-18
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column (250 mm × 10 mm, i.d. 5 μm, Phenomenex, Torrance, CA, USA). Open column chromatography (CC) was carried out on silica gel (200-300 mesh, Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), Chromatorex ODS (30-50 μm, Fuji Silysia Chemical, Ltd., Aichi, Japan), Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden), Diaion HP-20 resin (Mitsubishi Chemical Corp., Tokyo, Japan), and D101 macroporous resin (Cangzhou Baoen Chemical Co., Ltd., Cangzhou, China). Standard D-ribose, L-ribose and L-rhamnose were purchased from Dalian Meilun Biotech Co., Ltd. (Dalian, China) with purity of over 98%.
D-rhamnose
(purity ≥ 98%) was
obtained from Carbosynth Limited (Berkshire, UK). The sugar derivatization reagents including
anhydrous
pyridine,
L-cysteine
methyl
ester
hydrochloride
and
trimethylsilylimidazole (purities ≥ 98%) were obtained from Sinopharm Chemical
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ACCEPTED MANUSCRIPT Reagent Co., Ltd. (Shanghai, China).
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2.2. Plant material The dried whole plants of Gueldenstaedtia verna were collected from Luoyang,
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Henan Provinces, China, in July 2013, and authenticated by the authors. A voucher
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specimen (CZH20130709) has been deposited in Department of Pharmacognosy, School
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of Pharmacy, Fudan University.
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2.3. Extraction and isolation
The dried whole plants of G. verna (23.5 kg) were powdered and extracted with 95%
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EtOH (120 L × 3) at 50 ºC. The plant materials were re-extracted three times with an
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additional 120 L of 70% ethanol. The 95% and 70% ethanolic extracts were combined
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and concentrated under reduced pressure to afford a dark brown residue (3.4 kg). The residue was then suspended in H2O and partitioned successively with petroleum ether (PE, 60–90 ºC), EtOAc and n-BuOH (2.0 L each × 5) to afford the PE-soluble (252.7 g), EtOAc-soluble (308.8 g) and n-BuOH-soluble (938 g) fractions, respectively. The EtOAc fraction was subjected to open column chromatography (CC) on silica gel, eluted with CH2Cl2–MeOH (100:1, 50:1, 25:1, 10:1 and 1:1) to afford five fractions (A-E). Fraction A (111.4 g) was further subjected to silica gel CC eluted with PE–EtOAc (from 20:1 to 1:1) to obtain five subfractions (A1–A5). Further separation of subfraction A3 by Sephadex LH-20 (CH2Cl2–MeOH, 1:1) and flash ODS column with a gradient mixture of MeOH–H2O (3:7 to10:0), followed by semi-preparative HPLC eluted with MeCN–H2O (3.5:6.5) afforded compounds 11 (12 mg) and 13 (100 mg). Subfraction A4 (3.5 g) was separated with silica gel CC (PE–EtOAc, 10:1 to 2:1) and flash ODS column 5
ACCEPTED MANUSCRIPT with a gradient mixture of MeOH–H2O (50% to 100%), followed by being recrystallized from methanol to yield compound 20 (8.3 mg). Compounds 9 (6 mg) was obtained from
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subfraction A5 (9.7 g) by repeated silica gel CC (PE–EtOAc, 10:1 to 1:1) and Sephadex
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LH-20 (CH2Cl2–MeOH, 1:1) purification.
Fraction B (22.3 g) was chromatographed over silica gel CC eluted with a gradient
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CH2Cl2–MeOH system (100:1, 50:1, 20:1, 10:1 and 5:1) to give five subfractions (B1–B5).
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Fraction B3 (19.6 g) was further purified by silica gel CC eluted with CH2Cl2–MeOH (50:1–0:1) to obtain five fractions (B3A–B3E). B3B (1.2 g) was recrystallized from
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methanol to give 16 (30 mg). Further separation of fractions B3C (6.2 g) and B3D (6.0 g) by silica gel CC eluted with CH2Cl2–MeOH (50:1-10:1), followed by separation on
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Sephadex LH-20 (CH2Cl2–MeOH, 1:1), afforded 15 (13.2 mg) and 19 (39.2 mg), respectively. Fraction B4 (3.7 g) was chromatographed on a Sephadex LH-20 column
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eluting with (CH2Cl2–MeOH, 1:1) to give 18 (39.4 mg). Fraction C (55.7 g) was separated by silica gel CC using a step gradient elution of CH2Cl2–MeOH (50:1, 30:1, 20:1 and 10:1) to yield 4 fractions C1–C4. Fraction C2 (5.0 g) was chromatographed on a Sephadex LH-20 column eluting with MeOH, followed by purification on a semi-preparative HPLC with MeCN–H2O (25:75) to give compound 17 (32.9 mg). Fraction C4 was chromatographed on a Diaion HP-20 column, eluted successively with 20% MeOH, 40% MeOH, 60% MeOH and 80% MeOH. The 20% and 40% MeOH elutes were combined and chromatographed on Sephadex LH-20 (MeOH), and then purified by semi-preparative HPLC separation with CH3OH–H2O (65:35) to afford compound 1 (6.2 mg). The fraction eluting with 60% MeOH (5.2 g) was re-chromatographed on a flash ODS column eluted with MeOH-H2O (8:2–3.5:6.5) to give compounds 2 (13 mg) and 14 (20 mg). The 80%
6
ACCEPTED MANUSCRIPT MeOH elute (3.8 g) was recrystallized from methanol affording 12 (80 mg), and the filtrate was then purified by flash ODS column chromatography with MeOH–H2O
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(8:2–0:10) to give three subfractions (1–3). Subfraction 1 was purified by
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semi-preparative HPLC with CH3CN–H2O (25:75) to afford 5 (5.4 mg), 6 (7.2 mg) and 7 (9.1 mg). Further separation of subfraction 2 (339.2 mg) by silica gel CC eluted with
SC
CH2Cl2–MeOH (20:1–10:1), followed by semi-preparative HPLC with MeOH–H2O
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(60:40), afforded 3 (28.2 mg). In a similar manner to that described for subfraction 2, subfraction 3 yielded 4 (90.4 mg) and 8 (106.3 mg).
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The n-BuOH fraction (938 g) was chromatographed on a D101 macroporous resin column, using a gradient of EtOH and H2O (0:100, 30:70, 50:50, 75:25 and 95:5) to give
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five subfractions (Bu-1-5). Fraction Bu-4 (332.2 g) was repeatedly subjected to silica gel
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CC eluted with CH2Cl2–MeOH–H2O (100:0:0–65:35:7), followed by a combinative
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separation of Sephadex LH-20 (MeOH) and semi-preparative HPLC (MeCN–H2O, 32:68), to give compound 10 (30 mg).
2.3.1. (-)-(6aR,11aR)-3-Hydroxy-9-methoxypterocarpan 8-O-α-D-ribofuranoside (1) Colorless needles (MeOH); [α]20D –27.0º (c 0.10, MeOH); UV(MeOH) λmax 230 (sh), 285 nm; ECD (MeOH) λmax (Δε) 211 (-17.10), 237 (-17.69), 290 (4.39) nm; 1H (400 MHz, DMSO-d6) and 13C NMR (100 MHz, DMSO-d6) data, see Table 1; ESI-MS m/z 417 [M H]-; HR-ESI-MS m/z 417.1182 [M - H]- (calcd. for C21H21O9, 417.1191). 2.3.2. Medicarpin 3-O--D-glucopyranoside 6'-acetate (2) Yellow powder; [α]20D –35.3º (c 0.12, MeOH); UV (MeOH) λmax 230 (sh), 285 nm; IR (KBr) νmax 3417, 2928, 2857, 1732, 1661, 1620, 1597, 1497, 1435, 1380, 1345, 1275, 1160, 1141, 1081, 1036, 950, 736 cm-1; ECD (MeOH) λmax (Δε) 209 (-11.53), 235 (-6.47), 7
ACCEPTED MANUSCRIPT 287 (2.98) nm; 1H (400 MHz, DMSO-d6) and 13C NMR (100 MHz, DMSO-d6) data, see Table 1; ESI-MS m/z 497 [M + Na]+; HR-ESI-MS m/z 497.1427 [M + Na]+ (calcd. for
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C24H26O10Na, 497.1418).
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2.3.3. Complogenin 22-O-α-L-rhamnopyranoside (3)
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White amorphous powder; [α]20D +55.5º (c 0.11, MeOH); IR (KBr) νmax 3370, 2926, 2854, 1649, 1452, 1375, 1036, 975 cm-1; 1H (400 MHz, CD3OD) and
13
C NMR (100
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MHz, CD3OD) data, see Table 2; ESI-MS m/z 619 [M + H]+, 641 [M + Na]+;
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HR-ESI-MS m/z 641.3996 [M + Na]+ (calcd. for C36H58O8Na, 641.4024). 2.3.4. Soyasapogenol A 21-O-α-L-rhamnopyranoside (4)
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White amorphous powder; [α]20D +28.8º (c 0.13, MeOH); IR (KBr) νmax 3463, 2970,
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2959, 1638, 1452, 1380, 1123, 1046, 992 cm-1; 1H (400 MHz, C5D5N) and 13C NMR (100
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MHz, C5D5N) data, see Table 2; ESI-MS m/z 643 [M + Na]+, 1263 [2M + Na]+; HR-ESI-MS m/z 643.4179 [M + Na]+ (calcd. for C36H60O8Na, 643.4180). 2.3.5. 3β,22β,24-Trihydroxyolean-12-en-11-one21-O-α-L-rhamnopyranoside (5) White amorphous powder; [α]20D +34.0º (c 0.10, MeOH); IR (KBr) νmax 3282, 2920, 2849, 1654, 1545, 1391, 1072, 1038 cm-1; 1H (400 MHz, C5D5N) and
13
C NMR (100
MHz, C5D5N) data, see Table 2; ESI-MS m/z 657 [M + Na]+, 1291 [2M + Na]+; HR-ESI-MS m/z 657.3960 [M + Na]+ (calcd. for C36H58O9Na, 657.3973). 2.3.6. 7β,15α-Dihydroxycomplogenin (6) White amorphous powder; [α]20D +66.0º (c 0.10, MeOH); IR (KBr) νmax 3391, 3337, 2929, 2855, 1652, 1660, 1458, 1375, 1074, 1029, 964 cm-1; 1H (400 MHz, C5D5N) and 13
C NMR (100 MHz, C5D5N) data, see Table 2; ESI-MS m/z 527 [M + Na]+, 1031 [2M + 8
ACCEPTED MANUSCRIPT Na]+; HR-ESI-MS m/z 527.3343 [M + Na]+ (calcd. for C30H48O6Na, 527.3343). 2.3.7. 7β,15α,22β,24-Tetrahydroxyolean-12-en-3,11-dione (7)
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White amorphous powder; [α]20D +64.9º (c 0.15, MeOH); IR (KBr) νmax 3375, 3258,
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2922, 2849, 1704, 1458, 1386, 1068, 964 cm-1; 1H (400 MHz, C5D5N) and 13C NMR (100
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MHz, C5D5N) data, see Table 2; ESI-MS m/z 525 [M + Na]+, 1027 [2M + Na]+;
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HR-ESI-MS m/z 525.3176 [M + Na]+ (calcd. for C30H46O6Na, 525.3187).
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2.4. Computational details
The theoretical ECD calculations for compounds 1a, 1b, 2a and 2b were conducted
D
by the Gaussian 09 program package. The time-dependent density functional theory
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(TDDFT) was used to calculate the excitation energy (in nm) and rotatory strength R (velocity form Rvel and length form Rlen in 10-40 erg-esu-cm/Gauss) between different
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states, at B3LYP/6-31G (d, p) level in methanol solution. The ECD curves were then simulated by the following Gaussian functions:
Where σ is the width of the band at 1/e height and ΔEi and Ri are the excitation energies and rotatory strengths for transition i, respectively. σ = 0.2 eV and Rlen were used in this work. 2.5.
X-ray
data
of
(-)-(6aR,11aR)-3-Hydroxy-9-methoxypterocarpan
8-O-α-D-ribofuranoside (1)
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ACCEPTED MANUSCRIPT Suitable crystals for the X-ray diffraction were obtained by recrystallization from a methanol solution of 1. The crystallographic data of 1 was collected on a Bruker APEX
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DUO diffractometer equipped with a rotating Cu anode generator (λ = 1.54178 Å) and a parabolic Gӧ bel mirror. Crystal data: C21H22O9, M = 418.38, crystal size 0.21 × 0.05 ×
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0.04 mm3, monoclinic, space group P21, a = 4.8952(3) Å, b = 11.6874(8) Å, c =
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16.2106(10) Å, α = 90°, β = 90.128(4)°, γ = 90°, V = 927.44(10) Å3, T = 203(2) K, Z = 2,
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Dcalc = 1.498 mg/m3, μ(Cu Kα) = 1.000 mm-1, F(000) = 440, 4428 reflections collected (θmax = 67.996°), 2456 independent reflections (Rint = 0.0660). The final R1 [I > 2σ(I)] and
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wR2 values (F2) were 0.0813 and 0.1889, respectively. The final R1 (all data) and wR2 values (all data) were 0.0893 and 0.1959, respectively. The absolute structure parameter
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was 0.0(3). Crystallographic data for 1 have been deposited in the Cambridge Crystallographic Data Centre (CCDC) with the accession no. CCDC 1054442. These data
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can be obtained free of charge from CCDC via 12 Union Road, Cambridge, CB2 1EZ, UK (fax: +44-1223-336-033 or e-mail: [email protected]). 2.6. Analysis of the sugar moiety Each compound (2 mg) was dissolved in 2.0 mL of 2.0 N HCl and heated to 100 °C in a water bath for 4 h. The mixture was neutralized with excess Ag2CO3, and the solvent was removed under a stream of N2. The obtained residue was re-suspended in H2O (2 mL) and extracted three times with 2 mL of CHCl3. The aqueous layer was collected and evaporated to dryness using N2. The residue was re-dissolved in anhydrous pyridine (0.1 mL) and mixed with 0.06 N L-cysteine methyl ester hydrochloride (0.1 mL) in pyridine. The mixture was heated to 60 °C for 2 h, followed by addition of trimethylsilylimidazole (0.1 mL), and heating was continued at 60 °C for another 2 h. Water and organic solvent 10
ACCEPTED MANUSCRIPT were removed under reduced pressure, and the residue was concentrated to dryness and partitioned between n-hexane and H2O. The organic layer was analyzed by a Shimadzu
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GCMS-QP2010 Ultra instrument (Kyoto, Japan) using a TG-5MS column (0.32 mm × 30
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m) under the following conditions [injector temperature, 250 °C; initial temperature, 150°C (1 min), increased at 8 °C/min to 260°C, held for 10 min; carrier gas, He operated
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in the splitless mode; injection size, 1.0 μL; MS conditions: EI voltage, 70 eV;
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scanned-mass range, m/z 50-1000]. The hydrolysates of 3–5 gave peaks at 16.20, 16.21, and 16.21 min, respectively. With authentic
D-rhamnose
and L-rhamnose, peaks were
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detected at 16.38 and 16.22 min, respectively.
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2.7.1. Cytotoxic assay
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2.7. Biological assays
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The human lung cancer cell line (A549), human ileocecal adenocarcinoma cell line (HCT-8), human breast cancer cell line (MCF-7), and human hepatocellular carcinoma cell line (BEL-7402) were obtained from the American Type Culture Collection (ATCC). HCT-8 and BEL-7402 cell lines were cultured in RPMI-1640 medium. MCF-7 cell line was cultured in Dulbecco’s modified Eagle medium (DMEM), and A549 cell line was cultured in Ham’s F-12 medium. These cell lines were all supplemented with 10% fetal bovine serum (FBS). The cytotoxic activity was evaluated by the sulforhodamine B (SRB) method. Briefly, the cultured cells were harvested, counted, and diluted to 5 × 103 cells/well in aliquots of 190 L of fresh medium. After incubation for 24 h, various concentrations of the test compounds (10 L/well) were added to the 96-well plates, followed by incubation for 72 h at 37 ºC under 5% CO2 atmosphere. The cells were fixed
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ACCEPTED MANUSCRIPT with 100 μL of 10% cold trichloroacetic acid (TCA), incubated for 60 min at 4 ºC, washed three time with deionized water, and allowed to air-dry. The cells were then
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stained by 0.4% SRB (Sigma) dissolved in 1% acetic acid for 15 min and subsequently washed five times with 1% acetic acid. Plates were air-dried, and bound dye was
SC
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solubilized with 150 L of 10 mM Tris on a shaker for 5 min. The absorbance at 560 nm was measured by using a microplate reader. The concentration of test compounds
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resulting in 50% growth inhibition (IC50) was estimated. 5-FU and cisplatin were used as positive controls. All experiments were performed in three independent replicates. IC50
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values are expressed as the mean ± SD.
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2.7.2. Nitrite assay
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The inhibitory effect on NO production in RAW264.7 cells was tested by a previously reported method [9]. Briefly, RAW264.7 cells were placed in 96-well plates at
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a density of 2×105 cells/well for 24 h. Samples or the reference compound were dissolved in DMSO and diluted with serum-free DMEM into a series of concentrations. The cells were pre-treated with different concentrations of testing compounds for 30 min, and then stimulated with LPS (1 μg/mL) for 24 h. After incubation, an aliquot of the culture supernatants (100 µL) was added to a solution of Griess reagent (50 µL, 1:1 mixture of 1% sulfanilamide in 5% H3PO4 and 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride in H2O) in the 96-well plate and left for 20 min at room temperature. The optical density of the supernatant was then measured at 550 nm with a spectrophotometer. Nitrite concentrations in the supernatants were determined by comparison with a standard curve using sodium nitrite as a standard. Aminoguanidine hydrochloride was used as the positive control.
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ACCEPTED MANUSCRIPT 3. Results and discussion The dried and powdered whole plants of G. verna were successively extracted with
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95% and 70% ethanol. The combined extract was concentrated, suspended in water, and
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partitioned with petroleum ether (60-90 ºC), EtOAc and n-BuOH, successively. The
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EtOAc and n-BuOH fractions were repeatedly purified by a combination of column chromatography including silica gel, ODS, Sephadex LH-20 and semi-preparative HPLC.
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A total of 8 triterpenoids and 12 flavonoids were obtained including two new pterocarpan
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glycosides (1–2), five new triterpenoids (3–7), and 13 known compounds (8–20) (Fig. 1). The new structures were elucidated by extensive NMR (1H, 13C, COSY, HSQC, HMBC
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and NOESY) and HR-ESI-MS analysis. The sugar residues were identified by GC-MS
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analysis after acidic hydrolysis and trimethylsilylation. The known compounds were
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identified as sigmoside C (8) [10], soyasapogenol B (9) [11], soyasaponin I methyl ester (10) [12], medicarpin (11) [13], medicarpin 3-O--D-glucoside (12) [5], maackiain (13) [14], trifolirhizin (14) [15], 7,4'-dihydroxyflavone (15) [16], apigenin (16) [3], sulfuretin (17) [17], luteolin (18) [18], quercetin (19) [19], and formononetin (20) [20] by spectroscopic analysis and comparison with literature data. An isoflavone (20) and two triterpenoid glycosides (8 and 10) were isolated for the first time from the genus Gueldenstaedtia Fisch. Four flavonoids (11, 13, 14 and 17) were isolated for the first time from G. verna. Compound 1 was isolated as colorless needles from MeOH. Its molecular formula was determined to be C21H22O9 by HR-ESI-MS analysis ([M - H]- m/z 417.1182, calcd for C21H21O9, 417.1191) and NMR spectroscopic data. The UV absorption bands at 230 (sh) and 285 nm and the 1H NMR signals at δH 4.24 (m, H-6), 3.62 (m, H-6), 3.60 (m, 13
ACCEPTED MANUSCRIPT H-6a) and 5.52 (d, J = 6.4 Hz, H-11a) of 1 suggested characteristic signals for a pterocarpan derivative (Table 1) [21]. A 1,2,4-trisubstituted benzene ring was assigned
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from analysis of its 1H NMR coupling constants [δH 7.25 (d, J = 8.5 Hz, H-1), 6.48 (dd, J = 8.4, 2.4 Hz, H-2) and 6.26 (d, J = 2.3 Hz, H-4)] and on the basis of the COSY
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spectrum (Fig. 2). In addition, two distinct aromatic singlets at δH 7.11 and 6.58 in the 1H
SC
NMR spectrum suggested the presence of a 1,2,4,5-tetrasubstituted benzene ring in 1.
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The 13C NMR spectrum displayed 21 carbon signals, of which 16 signals were attributed to the pterocarpan aglycone moiety. The remaining five carbon signals (δC 102.5, 71.7,
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69.5, 86.3 and 61.6) and the corresponding proton signals (Table 1) were typical of a ribofuranosyl residue [22,23]. All these NMR information is in support of 1 being a
structure
of
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pterocarpan ribofuranoside. Further inspection of the NMR data of 1 found that the its
aglycone
moiety
was
very
similar
to
that
of
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(6aR,11aR)-3,8-dihydroxy-9-methoxypterocarpan [24], with C-8 hydroxyl group replaced by a ribofuranosyl unit. The pterocarpan skeleton assignment was verified by the HMBC correlations from H-11a to C-1a, C-4a, C-6 and C-6a, and from H-6a to C-7a, C-10a and C-6 (Fig. 2). The positions of the methoxy and ribofuranosyl groups were also established by analysis of the HMBC spectrum, where correlations of the methoxy protons at δH 3.72 with C-9 (δC 151.5), and of H-1' (δH 5.36) with C-8 (δC 139.2) were observed. Comparison of the
13
C NMR data of the furanosyl moiety of 1 with that of methyl
α-D-ribofuranoside and methyl β-D-ribofuranoside reported in the literature [23], along with the NOESY correlation between H-1' and H-3' of the ribose [25], assigned the configuration of the anomeric center (C-1') to be α. Complete 1H- and
13
C NMR
assignments of 1 were aided by the DEPT and 2D NMR experiments (COSY, HMQC and
14
ACCEPTED MANUSCRIPT HMBC). The relative configurations of C-6a and C-11a in 1 were determined by NOESY
PT
correlations and vicinal 1H-1H coupling constants. A significant NOESY correlation
RI
between H-6a and H-11a, along with a small coupling constant (3JH6a/H11a = 6.4 Hz) [26] indicated that H-6a and H-11a protons are cis-oriented. The absolute configuration at
SC
C-6a and C-11a was determined by optical rotation, and the comparison of experimental
NU
and calculated electronic circular dichroism (ECD) spectra. The attempt to obtain the aglycone through acidic hydrolysis of 1 was unsuccessful because of the unavailability of
MA
sufficient amount of 1. Therefore, the optical rotation value of 1 instead of its aglycone was measured. To address the influence of glycosidation on pterocarpan aglycones, we
D
compared the optical rotation values of medicarpin3-O-β-D-glucoside (12, c 0.37, C5H5N,
TE
[α]20D -153.2) and its aglycone medicarpin (11, c 0.15, CH3OH, [α]20D -226.0), and
AC CE P
trifolirhizin (14, c 0.15, CH3OH, [α]20D -107.6) and its aglycone maackiain (13, c 0.12, CH3OH, [α]20D -241.3). The results showed that mono-glycosidation did not alter the direction of optical rotation for pterocarpan aglycones, indicating that the contribution of the sugar part to the optical rotation might be limited. The absolute configuration at C-6a and C-11a was thus tentatively assigned to R from its negative optical rotation value [21]. Generally the absolute configuration of a chiral molecular can also be determined by its CD spectrum. A further proof of the absolute configuration of 1 was obtained from the experimental ECD spectrum. As shown in Fig. 4A, the ECD spectrum of 1 displayed negative Cotton effects at 211 and 237 nm and a positive Cotton effect at 290 nm, which is consistent with the C-6aR and C-11aR configurations of the previously reported pterocarpan derivatives [27]. The calculation of ECD spectra has been proved as a very
15
ACCEPTED MANUSCRIPT efficient tool for analysis of optically active naturally products over the past decade [28]. Therefore, the calculated ECD is also used to confirm the absolute configuration of 1.
PT
Two simplified structures (1a and 1b), in which the methyl group instead of the
RI
ribofuranosyl group in 1, were used for ECD calculation. The model compounds 1a and 1b with (C-6aR and C-11aR) and (C-6aS and C-11aS) absolute configurations were
SC
calculated by using the TD-DFT method at a B3LYP/6-31+G (d, p) level in MeOH. The
NU
calculation for C-6aR, C-11R (1a, the aglycone) configuration agreed with the experimental ECD spectrum (1, the glycoside, Fig. 4). Finally, a single crystal X-ray
MA
diffraction study of 1 was performed (Fig. 5), from which its relative and absolute configurations were confirmed. Consequently, the structure of 1 was established as 8-O-α-D-ribofuranoside.
Although
TE
D
(-)-(6aR,11aR)-3-hydroxy-9-methoxypterocarpan
many pterocarpans have been reported from the Leguminosae family, compound 1 is the
AC CE P
second pterocarpan with a rare glycosidic ribose from the genus Gueldenstaedtia Fisch. Compound 2 had a molecular formula of C24H26O10, as determined by HR-ESI-MS at m/z 497.1427 [M + Na]+ and the 13C NMR data. Comparison of the NMR data of 2 and 12 (medicarpin 3-O--D-glucopyranoside) revealed great similarity except for an additional acetyl group in 2 (Table 1). The acetyl group was assigned at C-6' of the glucose by the HMBC correlations between H-6' (δH 4.05, 4.26) and the carbonyl carbon (δC 170.3). The negative optical rotation value ([α]20D -35.3), and the comparison of experimental and calculated ECD spectra (Fig. 4B) suggested a (6aR,11aR) configuration. Thus, the structure of 2 was identified as medicarpin 3-O--D-glucopyranoside 6ʹ-acetate. Previously, compound 2 was proposed to be an artificial component of alfalfa (Medicago sativa) by LC-MS [29], but this compound has not been isolated as an
16
ACCEPTED MANUSCRIPT individual compound. Recently, this compound was isolated as a trace component in Sophora tonkinensis [30]. However, identification of the accurate molecular structure has
PT
not so far been performed by NMR technique. Thus, we consider that compound 2 is
RI
described in the present work for the first time.
Compound 3 was obtained as a white amorphous powder. It gave a positive
SC
Liebermann-Burchard test, indicating a triterpenoid skeleton. Its molecular formula was
NU
deduced as C36H58O8 by HR-ESI-MS at m/z 641.3996 [M + Na]+ (calcd for C36H58O8Na, 641.4024). The IR spectrum of 3 displayed a broad band centered at 3370 cm-1 and a
MA
relative sharp absorbance peak at 1649 cm-1 for hydroxyl and α,β-unsaturated carbonyl functionalities, respectively. The 1H NMR spectrum showed seven methyl singlets at δH
D
1.41, 1.21, 1.14, 1.12, 1.05, 0.97 and 0.95, a methyl doublet at δH 1.24 (d, J = 6.2 Hz), an
13
C NMR spectrum showed 36 carbon signals, which were assigned by
AC CE P
(Table 2). The
TE
olefinic broad singlet at δH 5.55, and together with an anomeric proton singlet at δH 4.79
HSQC and DEPT experiments as resonances for a ketone carbonyl carbon signal at δc 199.6 (C-11), a quaternary olefinic carbon signal at δc 169.1 (C-13), a tertiary olefinic carbon at δc 128.6 (C-12), an anomeric carbon signal at δc 98.2 (C-1'), nine methine carbons (including six oxygenated methines), nine methylene carbons (one for oxygenated methylene and eight for ordinary methylenes), and eight methyl carbons at δc 32.8, 27.6, 23.7, 23.2, 22.0, 18.6, 18.4 and 17.5 (Table 2). These above 1H- and 13C NMR spectroscopic data of 3 showed typical signals that suggested an oleane-type triterpenoid mono-glycoside framework [5]. A comparison between the
13
C NMR data of 3 and
complogenin [12] revealed that the carbon signals of the sapogenin of 3 and complogenin were almost identical, except for the C-22 and neighbor carbons. These data suggested
17
ACCEPTED MANUSCRIPT that 3 is complogenin with a glycosidic linkage at C-22, which is consistent with the observed downfield shift in C-22 by 3.9 ppm and an upfiled shift in C-21 (-6.6 ppm) with
PT
respect to the corresponding signals of complogenin [12]. In addition, the 13C NMR data
RI
together with the above anomeric proton suggested the presence of a rhamnopyranosyl moiety in 3. The linkage site of the rhamnopyranosyl moiety was further confirmed by
SC
the HMBC correlation of H-1' (δH 4.79) with C-22 at δc 78.7 (Fig. 2). The relative
NU
configurations of the protons at C-3 and C-22 were determined by a NOESY experiment. The correlations of H-3 (δH 3.35) with H-5 (δH 0.87) and H-23 (δH 1.21), and of H-22 (δH
MA
3.42) with H-29 (δH 0.95) indicated that they were cofacial and designated as -oriented. The -configuration of the rhamnopyranosyl moiety was deduced by the chemical shift
TE
D
of C-3 at δc 73.0 and C-5 at δc 70.5 [31], and by the significant HMBC correlations from H-1' to C-3' and C-5' [32] (Fig. 2). The absolute configuration of the rhamnose was
AC CE P
determined as L, by GC-MS analysis after acidic hydrolysis of 3 and trimethylsilylation. Thus, the structure of 3 was identified as complogenin 22-O--L-rhamnopyranoside. Compound 4 was obtained as a white amorphous powder. It gave a positive Liebermann-Burchard test, indicating a triterpenoid skeleton. Its molecular formula was deduced as C36H60O8 by HR-ESI-MS at m/z 643.4179 [M + Na]+ (calcd for C36H60O8Na, 643.4180). The 1H NMR spectrum of 4 showed signals for an olefinic proton at δH 5.34, an anomeric proton singlet at δH 5.51, seven oxygenated methine signals, one oxygenated methylene, and eight methyl signals at δH 1.66, 1.59, 1.39, 1.29, 1.27, 1.03, 1.01 and 0.94 (Table 2). The
13
C NMR spectrum displayed 36 carbons, including a
quaternary olefinic carbon signal at δc 144.5 (C-13), a tertiary olefinic carbon at δc 122.7 (C-12), an anomeric carbon signal at δc 104.6, seven oxygenated methine carbons at δc
18
ACCEPTED MANUSCRIPT 84.3, 80.2, 78.7, 73.9, 72.8, 72.4 and 70.5, an oxygenated methylene at δc 64.7, and eight methyl carbons at δc 31.1, 26.8, 23.7, 22.2, 22.0, 18.7, 17.0 and 16.3 (Table 2). These 12(13)
oleane-type skeleton with the same sugar moiety
PT
NMR data suggested that 4 had a
RI
as 3. Further inspection of the NMR data of 4 found that its sapogenin moiety was very
SC
close to those of soyasapogenol A [33], except for the signals assigned to C-21 exhibiting a significant glycosidation shift (+9.7 ppm) and a small upfiled shift for carbon
NU
neighboring C-22 (-0.9 ppm). These NMR data indicated that the rhamnopyranosyl unit was attached to the C-21 hydroxyl group. This linkage was confirmed by the HMBC
MA
correlation between H-1' (δH 5.51) and C-21 (δC 84.3) (Fig. 2). The relative configurations of three hydroxyl groups were determined by a NOESY experiment. The correlations
TE
D
from H-3 (δH 3.66) to H-23 (δH 1.59) and H-5 (δH 0.99), from H-21 (δH 3.84) to H-29 (δH 1.03), and from H-22 (δH 3.93) to H-21 (δH 3.84) indicated that hydroxyl groups at C-3,
AC CE P
C-21 and C-22 were all β-orientated (Fig. 3). Therefore, the structure of 4 was identified as soyasapogenol A 21-O-α-L-rhamnopyranoside. Compound 5 was obtained as white amorphous powders. The HR-ESI-MS showed an [M + Na]+ ion at m/z 657.3960, and together with the
13
C NMR data, indicating a
molecular formula of C36H58O9. The NMR data were very similar to those of 4, except for the presence of a C-11 keto signal in 5. The C-11-keto-
12(13)
group was also assigned by
comparison of the NMR data with those of 3, and further supported by the HMBC correlation of H-9 with C-11 (Fig. 2). NOESY correlations between H-3 (δH 3.70)/H-23 (δH 1.62), H-5 (δH 1.00); H-21 (δH 3.83)/H-29 (δH 1.03); and H-22 (δH 3.93)/H-21 (δH 3.83) indicated that the hydroxyl groups at C-3, C-21 and C-22 were all β-oriented. Therefore, the
structure
of
5
was
identified
as
19
3β,22β,24-trihydroxyolean-12-en-11-one
ACCEPTED MANUSCRIPT 21-O-α-L-rhamnopyranoside. Compound 6, obtained as a white amorphous powder, possessed a molecular
PT
formula of C30H48O6 as determined by the combination of the HR-ESI-MS (m/z 527.3343 [M + Na]+) and NMR data. The 1H NMR spectrum of 6 displayed signals for typical
13
C NMR signals at δc 199.0 (a ketone
SC
of a broad olefinic proton signal at δ 6.07 with
RI
triterpenoid methyl singlets at δ 1.74, 1.62, 1.57, 1.47, 1.32, 1.27 and 0.96. The presences
NU
carbonyl carbon), 170.8 (a quaternary olefinic carbon) and 129.1 (a secondary olefinic carbon), were characteristic of a C-11-keto-
12(13)
functionality of oleane-type
MA
triterpenoids [5]. The 1H NMR spectrum also showed six oxygenated protons (including four oxygenated methines) at δH 4.74 (m), 4.58 (d, J = 11.6 Hz), 4.49 (m), 3.88 (m), 3.77
TE
D
(m) and 3.73 (m) (Table 2). The 13C NMR and HSQC spectra showed the presence of 30 carbons due to seven methyls, seven methylenes, eight methines and eight quaternary
AC CE P
carbons. The HMBC correlations of H-6, H-5, Me-26 with C-7 as well as a long range COSY correlations of H-7 with H-5 and H-6 (Fig. 2), suggested that one hydroxyl group was located at C-7. The HMBC correlations from Me-27, H-16 to C-15 and the COSY correlation between H-15 and H-16 indicated the occurrence of a C-15 hydroxyl group. Similarly, the COSY correlation between H-21 and H-22 and HMBC correlations between Me-28, H-21 and C-22 indicated that another hydroxyl group was attached at C-22. Key HMBC and COSY correlations are shown in Fig. 2. NOESY correlations between H-3/H-5, H-23; H-7/H-5, H-6, H-9, Me-27; H-15/H-16, H-26, H-28; and H-22/H-16, H-21, H-29 (Fig. 3) indicated that the hydroxyl groups at C-3, C-7, C-15 and C-22 were β-, β-, α- and β-oriented, respectively. Thus, the structure of 6 was identified as 7β,15α-dihydroxycomplogenin.
20
ACCEPTED MANUSCRIPT Compound 7 exhibited a [M + Na]+ ion at m/z 525.3176 (C30H46O6Na) in the HR-ESI-MS. The
13
C NMR data of 7 were very similar to those of 6 except that a C-3
PT
keto group in 7 was observed instead of a hydroxyl group in 6 (Table 2), indicating 7 was
RI
a C-3keto derivative of 6. The HMBC correlations from H-1, H-2 and Me-23 to C-3 confirmed the assigned position (Fig. 2). Using the data shown in Figs. 2 and 3, the
SC
structure of 7 was identified as 7β,15α,22β,24-tetrahydroxyolean-12-en-3,11-dione.
NU
The cytotoxic activities of all the isolates were estimated toward A549, HCT-8, MCF-7 and BEL-7402 cell lines by the sulforhodamine B (SRB) assay, using
MA
5-fluorouracil (5-FU) and cisplatin as the positive controls. The results are expressed as IC50 values and are summarized in Table 3. Compounds 9, 17 and 18 exhibited weakly
TE
D
cytotoxic activities against all the selected cancer cell lines, while compounds 1, 6, 7, 10, 13 and 16 showed selectively inhibitory activity against only one of the cancer cell lines
AC CE P
studied.
All the isolates were further tested for their inhibitory activities against LPS-induced NO production in RAW264.7 cells using the Griess method. Among them, only compound 17 showed the slightly toxic, and displayed an over 50% inhibition of NO production at the dose of 50 μM. Compound 17 showed significant inhibition of NO production with IC50 values of 12.6 ± 1.8 M, which was comparable to the positive control aminoguanidine hydrochloride (IC50 = 8.94 ± 0.44 μM). Our finding is in agreement with the work of Shin et al. [34], who recently reported the in vitro anti-inflammatory effect of sulfuretin. Acknowledgments This work was supported by grants from the National Natural Science Foundation of 21
ACCEPTED MANUSCRIPT China (No. 81473421 and No. 81073025).
PT
References [1] Chen L, Wang JX, Su Y, Chang YH. Research advance in Gueldenstaedtia Fisch.
RI
Shaanxi J Tradit Chin Med 2001;22:184–5.
SC
[2] Huang HL, Xu B. Free radical scavenging activities and principals of
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Gueldenstaedtia multiflora Bge. J Qingdao Univ (Nat Sci Ed) 2006;19:30–5. [3] Wang JX, Zhu R. Studies on the chemical constituents of Gueldenstaedtia multiflora
MA
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[4] Han YL, Gao LM. Study on chemical constituents of Gueldenstaedtia multiflora Bge.
D
J Shanxi College Tradit Chin Med 2009;10:24–5.
TE
[5] Tsunoda Y, Okawa M, Kinjo J, Ikeda T, Nohara T. Studies on the constituents of
AC CE P
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ACCEPTED MANUSCRIPT [10] Mbafor JT, Ndom JC, Fomum ZT. Triterpenoid saponins from Erythrina sigmoidea. Phytochemistry 1997;44:1151–5.
PT
[11] Konoshima T, Kozuka M, Haruna M, Ito K, Kimura T. Studies on the constituents of
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Leguminous plants. XI. The structures of new triterpenoids from Wistaria brachybotrys Sieb. et Zucc. Chem Pharm Bull 1989;37:1550–3.
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[12] Cui B, Inoue J, Takeshita T, Kinjo J, Nohara T. Triterpene glycosides from the seeds
NU
of Astragalus sinicus L. Chem Pharm Bull 1992;40:3330–3. [13] Yoon JS, Sung SH, Park JH, Kim YC. Flavonoids from Spatholobus suberectus.
MA
Arch Pharm Res 2004;27:589–92.
[14] Abdel-Kader MS. Phenolic constituents of Ononis vaginalis roots. Planta Med
TE
D
2001;67:388–90.
[15] Zeng Y, Luo JJ, Li C. Chemical constituents from aerial part of Rumex patientia. J
AC CE P
Chin Med Mat 2013;36:57–60.
[16] Fu HZ, Han L. Chemical constituents from fruits of Gymnocladus chinensis. Chin Herbal Med 2009;1:66–70. [17] Li YL, Li J, Wang NL, Yao XS. Flavonoids and a new polyacetylene from Bidens parviflora Willd. Molecules 2008;13:1931–41. [18] Li J, Jiang H, Shi R. A new acylated quercetin glycoside from the leaves of Stevia rebaudiana Bertoni. Nat Prod Res 2009;23:1378–83. [19] Wu T, Abdulla R, Yang Y, Aisa HA. Flavonoids from Gossypium hirsutum flowers. Chem Nat Comp 2008;44:370–1. [20] Zheng Y, Liu H, Bai YJ, Ma YM, Zhao YY. Five flavonoids from Spatholobus suberectus. China J Chin Mat Med 2008;33:152–4.
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ACCEPTED MANUSCRIPT [21] Fedoreyev SA, Bulgakov VP, Grishchenko OV, Veselova MV, Krivoschekova OE, Kulesh NI, et al. Isoflavonoid composition of a callus culture of the relict tree
PT
Maackia amurensis Rupr. et Maxim. J Agric Food Chem 2008;56:7023–31.
RI
[22] Li Y, Li XF, Lee U, Kang JS, Choi HD, Son BW. A new radical scavenging anthracene glycoside, asperflavin ribofuranoside, and polyketides from a marine
SC
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[23] Sharma RK, Singh S, Tiwari R, Mandal D, Olsen CE, Parmar VS, et al. O-Aryl α,β-D-ribofuranosides: synthesis & highly efficient biocatalytic separation of
MA
anomers and evaluation of their Src kinase inhibitory activity. Bioorg Med Chem 2012;20:6821–30.
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D
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[24] Bezuidenhoudt
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[25] Horton RA, Bagnato JD, Grissom CB. Structural determination of 5'-OH α-ribofuranoside modified cobalamins via
13
C and DEPT NMR. J Org Chem 2003;
68:7108–11.
[26] van Aardt TG, van Rensburg H, Ferreira D. Synthesis of isoflavonoids. Enantiopure cis- and trans-6a-hydroxy pterocarpans and a racemic trans-pterocarpan. Tetrahedron 2001;57:7113-26. [27] Piccinelli AL, Fernandez MC, Cuesta-Rubio O, Hernandez IM, de Simone F, Rastrelli L. Isoflavonoids isolated from Cuban propolis. J Agric Food Chem 2005;53:9010–6. [28] Li XC, Ferreira D, Ding YQ. Determination of absolute configuration of natural products: theoretical calculation of electronic circular dichroism as a tool. Curr Org
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ACCEPTED MANUSCRIPT Chem 2010;14:1678–97. [29] Sumner LW, Paiva NL, Dixon RA, Geno PW. High-performance liquid
glycosides
in
Leguminous
plant
extracts.
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Spectrom
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PT
chromatography/ continuous-flow liquid secondary ion mass spectrometry of
1996;31:472–85.
SC
[30] Yoo H, Chae HS, Kim YM, Kang M, Ryu KH, Ahn HC, et al. Flavonoids and
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arylbenzofurans from the rhizomes and roots of Sophora tonkinensis with IL-6 production inhibitory activity. Bioorg Med Chem Lett 2014;24:5644–7.
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[31] Nishimura K, Miyase T, Noguchi H. Triterpenoid saponins from Ilex kudincha. J Nat Prod 1999;62:1128–33.
TE
D
[32] Zheng Q, Koike K, Han LK, Okuda H, Nikaido T. New biologically active triterpenoid saponins from Scabiosa tschiliensis. J Nat Prod 2004;67:604–13.
AC CE P
[33] Kinjo JE, Miyamoto I, Murakami K, Kida K, Tomimatsu T, Yamasaki M, et al. Oleanene-sapogenols from Puerariae Radix. Chem Pharm Bull 1985;33:1293–6. [34] Shin JS, Park YM, Choi JH, Park HJ, Shin MC, Lee YS, et al. Sulfuretin isolated from heartwood of Rhus verniciflua inhibits LPS-induced inducible nitric oxide synthase, cyclooxygenase-2, and pro-inflammatory cytokines expression via the down-regulation of NF-κB in RAW264.7 murine macrophage cells. Int Immunopharmacol 2010;10:943–50.
25
ACCEPTED MANUSCRIPT Figure legends Fig. 1. Structures of compounds 1-20.
PT
Fig. 2. Selected 1H-1H COSY and HMBC correlations of 1–7.
RI
Fig. 3. Selected NOESY correlations of 1 and 3–7.
Fig. 4. (A) Experimental ECD spectrum of 1 and the calculated ECD spectra of 1a and
SC
1b. (B) Experimental ECD spectrum of 2 and the calculated ECD spectra of 2a and 2b.
AC CE P
TE
D
MA
system is used for the structure in the text).
NU
Fig. 5. X-ray crystallographic structure of compound 1 (note: a different numbering
26
ACCEPTED MANUSCRIPT 30
29
7
10a
9
10
R1=H R1=(6'-acetyl)-Glu R1=H R1=Glu R1=H R1=Glu
R2
O
R3
3 5 6 7
5
6
O
4
HO
D-Glu
1
3
OH
HO
OH
HO
OH
HO R3 H H OH OH
TE
R2 H OH OH OH
AC CE P
15 16 18 19
R1 H H H OH
O
R2 H H OH OH
R3 H H OH OH
R1O
R4 H ORha H H
R5 Rha H H H
4 8 9 10
D-Rib
O
OH
O HO
HO
OH
OH
R1 R2 R3 H ORha H H H Glu H H H S H H
L-Rha
OH
D
O R1
R2
OH
OH R3
R2
O
HO
2
OH
HO
R1 OH OH OH =O
7
R3
OH
D-Gal
O
HO
24
OR3
15
5
23
OR5 28
MA
HO
17
9
3
R1
R2
21
13
10
R2=ORib R3=OMe R2=H R3=OMe R2=H R3=OMe R2=H R3=OMe R2+R3=-OCH2OR2+R3=-OCH2O-
D-GluA
11
1
S = 6'-methyl-D-GlcA(2→1)-D-Gal(2→1)-L-Rha
O
R4
19 8
PT
6a
H O
H 7a
RI
1a
1
6
11a
SC
3 2
0 01 2 11 12 13 14
O
4a
NU
4
R1O
OH HO
OH
HO
OH
O
O
O O 17
20
Fig. 1. Structures of compounds 1-20.
27
O
ACCEPTED MANUSCRIPT
O
1
OH
O OCH3
SC
OH O
1
O
O HO
OH OH
O
OCH3
O
AC
OH
O
OH
CH2OH
OH OH
O CH2OH 7
OH
OH HO
HO
OH OH
OH
6
CE
O
O
OH
CH2OH
PT ED
2
MA
O
NU
O O
HMBC
PT
OH
O O
H-1H COSY
RI
HO
OH
O
OH
O OH
OH
O
HO CH2OH
CH2OH
3
4
Fig. 2. Selected 1H-1H COSY and HMBC correlations of 1–7.
28
5
O
OH
O OH
OH
PT
ACCEPTED MANUSCRIPT
20
17
6 9
6a 3
11a
4
MA
1
20 21 17
14
22
AC
9
CE
18
5 4
3
3
5
8
3
4
21
21
17
22 9
6
14
9 8
14 8
5
14
4
3
PT ED
1
9
5
NU
5
2
22
SC
14
17
RI
18
21
18
15
15 16
6
7
7
4
6
Fig. 3. Selected NOESY correlations of 1 and 3–7.
29
7
16
22
22
ACCEPTED MANUSCRIPT 60
PT
20
0 250
300
350
-20 -40
200
NU
-60 250
400
RI
200
SC
Δε ( M-1 cm-1)
40
300
A
350
400
350
400
MA
wavelength (nm) 80
D
60
0 200
TE
20
250
AC CE P
Δε (M-1 cm-1)
40
-20
300
-40 -60
B
-80
200
250
300
350
400
wavelength (nm)
Fig. 4. (A) Experimental ECD spectrum of 1 and the calculated ECD spectra of 1a and 1b. (B) Experimental ECD spectrum of 2 and the calculated ECD spectra of 2a and 2b.
30
D
MA
NU
SC
RI
PT
ACCEPTED MANUSCRIPT
TE
Fig. 5. X-ray crystallographic structure of compound 1 (note: a different numbering
AC CE P
system is used for the structure in the text).
31
ACCEPTED MANUSCRIPT Table 1 NMR spectroscopic data (400 MHz, DMSO-d6) for compounds 1–2. 1
OCH3-9 COCH3-6' COCH3-6'
6.58 s 5.52 d (6.4) 5.36 d (4.3) 4.00 m 3.88 m
39.5 117.1 117.9 139.2 151.5 95.9 155.1 77.8 102.5
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4' 5' 6'
3.62 m 3.60 m 7.11 s
6.69 dd (8.5, 2.3)
4.04 m 3.45 brs
71.7 69.5 86.3 61.6
3.72 s
56.0
PT
132.0 114.2 110.4 158.2 104.0 156.2 66.0
RI
4.24 m
6.26 d (2.3)
132.0 111.4 109.7 158.7 102.8 156.3 65.7
SC
6 6 6a 7 7a 8 9 10 10a 11a 1' 2' 3'
6.48 dd (8.4, 2.4)
δC
7.39 d (8.5)
6.54 d (2.2)
4.29 dd (9.8, 3.5)
NU
7.25 d (8.5)
δH(J in Hz)
3.64 m 3.65 m 7.24 d (8.1)
MA
1 1a 2 3 4 4a
δC
D
δH(J in Hz)
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No.
2
6.44 dd (8.2, 2.0) 6.42 d (1.9) 5.60 d (6.8) 4.89 d (7.7) 3.24 t (7.9) 3.29 t (8.8) 3.15 t (9.2) 3.61 m 4.05 dd (11.8, 7.2) 4.26 dd (12.0, 1.4) 3.69 s 1.97 s
32
38.9 125.3 119.2 106.2 160.6 96.4 160.3 77.7 99.9 73.1 76.3 69.9 73.6 63.4 55.3 170.3 20.6
ACCEPTED MANUSCRIPT
PT
Table 2
a
4
3
5
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
1
1.02 m; 2.71 m
39.7
0.99 m; 1.59 m
39.0
2
1.83 m; 2.00 m
28.5
1.95 m; 2.03 m
3
3.35 m
79.9
3.66 m
56.0
0.99 m
6
1.47 m; 1.71 m
18.4
1.48 m; 1.73 m
7
1.47 m; 1.70 m
33.4
1.31 m; 1.51 m
62.2
10
37.3
11
199.6 128.6
12
5.55 s
13
169.1
14
45.4
39.7
1.58 m; 3.43 m
40.5
28.5
1.25 m; 3.27 dd (13.0, 3.0) 1.98 m; 2.24 m
39.5
28.5
1.20 m; 3.18 d (13.5) 1.97 m; 2.18 m
28.5
2.44 m; 3.01 dt (14.3, 6.0)
35.7
80.2
3.70 m
79.9
3.73 m
79.5
213.9
43.4
55.2
NU
56.0
1.24 m
52.7
1.71 m
54.3
19.2
1.48 m; 1.75 m
18.4
2.10 m; 2.36 m
28.6
1.31 m; 2.30 m
29.2
33.2
1.30 m; 1.62 m
33.2
4.49 m
72.0
4.48 dd (9.6, 4.8)
71.6
40.3
1.66 m
43.6
1.00 m
48.1
CE
43.7 2.45 s
δC
56.4
43.8
2.53 s
62.3
52.0 2.66 s
62.6
51.9 2.69 s
61.9
37.1
37.4
37.7
37.6
1.28 m; 1.90 m
24.2
199.7
199.0
198.7
5.34 m
122.7
AC
9
δH (J in Hz)
PT ED
0.87 m
8
δC
43.2
5
7
δH (J in Hz)
MA
43.6
6
δC
SC
No.
4
RI
NMR spectroscopic data (400 MHz, pyridine-d5) for compounds 3–7.
5.88 s
128.7
6.07 s
129.1
6.07 s
129.0
144.5
169.2
170.8
171.2
42.2
45.8
51.1
51.1
15
1.26 m; 1.64 m
26.5
1.20 m; 1.84 m
26.6
1.11 m; 1.97 m
26.9
4.74 m
66.6
4.73 dd (10.8, 4.5)
66.7
16
1.65 m; 1.87 m
27.4
1.07 m; 1.95 m
27.6
1.26 m; 1.31 m
30.0
1.84 m; 2.31 m
37.1
1.87 m; 2.31 m
37.1
17
37.4
39.5
36.7
37.9
37.9
18
2.34 m
45.6
2.58 m
43.6
2.67 m
44.0
2.63 m
46.6
2.67 m
46.6
19
1.09 m; 1.89 m
44.8
1.32 m; 2.06 m
47.2
1.17 m; 1.97 m
45.4
1.16 m; 1.99 m
45.1
1.18 m; 1.99 t
45.1
33
ACCEPTED MANUSCRIPT
PT
36.8
39.3
1.69 m; 1.74 m
41.9
1.69 m; 1.75 m
41.9
78.0
3.77 m
74.5
3.76 m
74.5
23.7
1.62 s
23.6
1.56 s
20.9
64.5
17.1
3.99 d (10.9); 4.38 m 1.66 s
65.2
17.4
3.88 m; 4.58 d (11.6) 1.47 s
64.5
16.3
3.77 d (8.5); 4.56 m 1.33 s
1.01 s
17.0
1.15 s
18.8
1.57 s
13.3
1.53 s
13.5
23.2
1.27 s
26.8
1.42 s
24.1
1.74 s
18.6
1.73 s
18.6
1.05 s
27.6
1.29 s
22.2
1.21 s
22.6
1.27 s
22.8
1.27 s
22.8
29
0.95 s
32.8
1.03 s
31.1
1.03 s
30.7
0.96 s
33.6
0.95 s
33.6
30
0.97 s
22.0
1.39 s
22.0
1.35 s
21.7
1.32 s
28.1
1.32 s
28.1
1'
4.79 brs
98.2
5.51 brs
104.6
5.51 brs
104.7
2'
3.78 m
72.6
4.70 m
72.4
4.70 brs
72.3
3'
3.65 m
73.0
4.61 m
72.8
4.61 m
72.7
4'
3.39 m
73.8
4.33 t (9.1)
73.9
4.34 t (9.1)
73.9
5'
3.65 m
70.5
4.54 m
70.5
4.55 m
70.5
6'
1.24 d (6.2)
18.4
1.66 d (5.0)
18.7
1.66 d (5.9)
18.6
1.44 m; 1.63 m
35.4
3.84 brs
84.3
3.83 brs
22
3.42 m
78.7
3.93 brs
78.7
3.93 brs
23
1.21 s
23.7
1.59 s
23.7
1.62 s
24
64.5 17.5
3.73 d (10.1); 4.53 m 0.94 s
64.7
25
3.41 m; 4.09 d (11.0) 1.12 s
26
1.14 s
18.6
27
1.41 s
28
83.8
AC
CE
PT ED
NU
21
RI
30.5
SC
20
a
30.9
MA
(13.5)
NMR measured in CD3OD.The assignments were based on DEPT, COSY, HSQC, and HMBC spectra.
34
31.0
16.5
ACCEPTED MANUSCRIPT Table 3 Cytotoxicity of isolates against four cancer cell lines (IC50, μM).a MCF-7
BEL-7402
> 150 > 150 > 150 97.9 ± 11.6 > 150 71.4 ± 11.0 76.9 ± 8.5 88.0 ± 19.8 99.5 ± 7.4
120.7 ± 16.4 > 150 > 150 115.3 ± 15.3 > 150 > 150 > 150 74.5 ± 10.0 108.0 ± 20.1
5-FUb
0.8 ± 0.5
3.5 ± 1.5
cisplatinb
1.8 ± 0.4
16.4 ± 1.6
6.7 ± 2.5
2.3 ± 1.2
20.3 ± 3.2
2.2 ± 0.2
Each IC50 value is expressed as the mean ± SD from three independent experiments.
D
a
RI
> 150 > 150 > 150 173.2 ± 55.1 > 150 > 150 > 150 98.2 ± 18.0 113.5 ± 26.1
SC
> 150 152.5 ± 18.6 97.9 ± 5.4 88.7 ± 16.1 108.7 ± 14.9 > 150 > 150 82.6 ± 8.4 81.6 ± 16.5
MA
1 6 7 9 10 13 16 17 18
PT
HCT-8
NU
compound A549
Positive controls.
AC CE P
b
TE
The other 11 compounds were inactive for all cell lines (IC50 > 150 μM).
35
ACCEPTED MANUSCRIPT Graphical Abstract
RI
PT
Pterocarpans and triterpenoids from Gueldenstaedtia verna
SC
Chengle Yin, Jinge Zhou, Yiqing Wu, Yue Cao, Tao Wu, Shoude Zhang, Honglin Li,
AC CE P
TE
D
MA
NU
Zhihong Cheng
36
S0367-326X(15)30059-9 doi: 10.1016/j.fitote.2015.07.021 FITOTE 3238
To appear in:
Fitoterapia
Received date: Revised date: Accepted date:
29 June 2015 28 July 2015 31 July 2015
Please cite this article as: Chengle Yin, Jinge Zhou, Yiqing Wu, Yue Cao, Tao Wu, Shoude Zhang, Honglin Li, Zhihong Cheng, Pterocarpans and triterpenoids from Gueldenstaedtia verna, Fitoterapia (2015), doi: 10.1016/j.fitote.2015.07.021
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ACCEPTED MANUSCRIPT
Pterocarpans and triterpenoids from Gueldenstaedtia verna
PT
Chengle Yin a, Jinge Zhou b, Yiqing Wu a, Yue Cao a, Tao Wu b, Shoude Zhang c, Honglin Li c, Zhihong Cheng a,*
Department of Pharmacognosy, School of Pharmacy, Fudan University, Shanghai
RI
a
Key Laboratory of Standardization of Chinese Medicines of Ministry of Education, The
NU
b
SC
201203, China
Shanghai Key Laboratory for Compound Chinese Medicines, Institute of Chinese
MA
Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China
D
c
AC CE P
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University of Science and Technology, Shanghai 200237, China
Title running-header: Pterocarpans and triterpenoids from Gueldenstaedtia verna
Contact Information of the Corresponding Author: Dr. Zhihong Cheng Tel: +86-21-51980157; Fax: +86-21-51980017; Email: [email protected] Mailing address: 826 Zhangheng Road, Department of Pharmacognosy, School of Pharmacy, Fudan University, Shanghai 201203, China
1
ACCEPTED MANUSCRIPT ABSTRACT Two new pterocarpan glycosides (1–2), five new triterpenoids (3–7), and 13 known
PT
analogues (14–20) were isolated from the whole plants of Gueldenstaedtia verna. These
1D (1H and
13
RI
new compounds (1–7) were elucidated by extensive spectroscopic techniques including C) and 2D NMR experiments (COSY, HSQC, HMBC and NOESY),
SC
HR-ESI-MS and chemical methods. The absolute configuration of 1 was established by
NU
the optical rotation, the comparison of experimental and calculated electronic circular dichroism (ECD) spectra and an X-ray diffraction analysis. All the isolates were
MA
evaluated for their cytotoxicities against four human cancer cell lines and inhibitory
D
activities on LPS-induced NO production in RAW264.7 cells.
TE
Keywords: Gueldenstaedtia verna; Leguminosae; Pterocarpans; Triterpenoids; Cytotoxic
AC CE P
activity; Inhibition of NO production
2
ACCEPTED MANUSCRIPT 1. Introduction The genus Gueldenstaedtia Fisch. (Leguminosae) comprises approximately 20
PT
species that are mainly distributed in central and eastern Asia. Many Gueldenstaedtia
RI
species have been used in folk medicines for treatments of various inflammatory diseases [1]. G. verna (Georgi) Boriss. is a small (4-20 cm high) herbaceous perennials covered
SC
entirely with long, white pubescence, which produces violet flowers. The whole plants
NU
including roots have been used in traditional Chinese medicines (TCM) as a heat-clearing and detoxicating drug for the treatment of a wide range of discomforts, such as
MA
suppurative inflammation, furuncles, carbuncles, fever, gastroenteritis, and diarrhea. This herb shows significant antioxidant and antibacterial activities [2,3]. It is used as a
TE
D
substitute for Viola yedoensis in TCM. Very little is known about its phytochemical composition of G. verna with the exception of several flavonoids [2,3] and triterpenoids
AC CE P
[4,5]. As part of our ongoing investigation of the chemistry and biological activity of V. yedoensis and its substitutes [6-8], we report herein the isolation and structural elucidation of the new pterocarpans (1–2) and triterpenoids (3–7) from G. verna. Compound 1 is assigned as a rare pterocarpan ribofuranoside. All the isolates were evaluated for their cytotoxic activities against four human cancer cell lines including lung (A549), ileocecal adenocarcinoma (HCT-8), breast (MCF-7), and hepatocellular carcinoma
(BEL-7402)
cell
lines.
In
addition,
their
inhibitory
effects
lipopolysaccharides-induced NO production in RAW264.7 cells were also studied. 2. Experimental 2.1. General
3
on
ACCEPTED MANUSCRIPT Optical rotations were measured at 20 ºC on a Rudolph Autopol VI polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA). Electronic circular dichroism
PT
(ECD) spectra were recorded on a Chirascan CD spectrometer (Applied Photophysics
RI
Ltd., Leatherhead, UK). IR spectra were recorded on a Nicolet Avatar 360 FT-IR spectrometer (Thermo Nicolet Corporation, Madison, WI, USA) using KBr disks. NMR
SC
spectra were measured on a Bruker DRX 400 spectrometer (Bruker Daltonics, Fallanden,
NU
Switzerland). ESI-MS spectra were measured on a Velos Pro dual-pressure linear ion trap mass spectrometer (Thermo Scientific, San Jose, CA, USA), and HR-ESI-MS spectra
MA
were obtained on a Bruker Daltonics APEX III 7.0 TESLA Fourier Transform mass spectrometer (Billerica, MA, USA). Semi-preparative HPLC were performed on a
TE
D
LC3000 system equipped with a P3000 pump, a UV3000 detector (Beijing Chuangxintongheng Science & Technology Co., Ltd., Beijing, China), and a Luna RP-18
AC CE P
column (250 mm × 10 mm, i.d. 5 μm, Phenomenex, Torrance, CA, USA). Open column chromatography (CC) was carried out on silica gel (200-300 mesh, Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), Chromatorex ODS (30-50 μm, Fuji Silysia Chemical, Ltd., Aichi, Japan), Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden), Diaion HP-20 resin (Mitsubishi Chemical Corp., Tokyo, Japan), and D101 macroporous resin (Cangzhou Baoen Chemical Co., Ltd., Cangzhou, China). Standard D-ribose, L-ribose and L-rhamnose were purchased from Dalian Meilun Biotech Co., Ltd. (Dalian, China) with purity of over 98%.
D-rhamnose
(purity ≥ 98%) was
obtained from Carbosynth Limited (Berkshire, UK). The sugar derivatization reagents including
anhydrous
pyridine,
L-cysteine
methyl
ester
hydrochloride
and
trimethylsilylimidazole (purities ≥ 98%) were obtained from Sinopharm Chemical
4
ACCEPTED MANUSCRIPT Reagent Co., Ltd. (Shanghai, China).
PT
2.2. Plant material The dried whole plants of Gueldenstaedtia verna were collected from Luoyang,
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Henan Provinces, China, in July 2013, and authenticated by the authors. A voucher
SC
specimen (CZH20130709) has been deposited in Department of Pharmacognosy, School
NU
of Pharmacy, Fudan University.
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2.3. Extraction and isolation
The dried whole plants of G. verna (23.5 kg) were powdered and extracted with 95%
D
EtOH (120 L × 3) at 50 ºC. The plant materials were re-extracted three times with an
TE
additional 120 L of 70% ethanol. The 95% and 70% ethanolic extracts were combined
AC CE P
and concentrated under reduced pressure to afford a dark brown residue (3.4 kg). The residue was then suspended in H2O and partitioned successively with petroleum ether (PE, 60–90 ºC), EtOAc and n-BuOH (2.0 L each × 5) to afford the PE-soluble (252.7 g), EtOAc-soluble (308.8 g) and n-BuOH-soluble (938 g) fractions, respectively. The EtOAc fraction was subjected to open column chromatography (CC) on silica gel, eluted with CH2Cl2–MeOH (100:1, 50:1, 25:1, 10:1 and 1:1) to afford five fractions (A-E). Fraction A (111.4 g) was further subjected to silica gel CC eluted with PE–EtOAc (from 20:1 to 1:1) to obtain five subfractions (A1–A5). Further separation of subfraction A3 by Sephadex LH-20 (CH2Cl2–MeOH, 1:1) and flash ODS column with a gradient mixture of MeOH–H2O (3:7 to10:0), followed by semi-preparative HPLC eluted with MeCN–H2O (3.5:6.5) afforded compounds 11 (12 mg) and 13 (100 mg). Subfraction A4 (3.5 g) was separated with silica gel CC (PE–EtOAc, 10:1 to 2:1) and flash ODS column 5
ACCEPTED MANUSCRIPT with a gradient mixture of MeOH–H2O (50% to 100%), followed by being recrystallized from methanol to yield compound 20 (8.3 mg). Compounds 9 (6 mg) was obtained from
PT
subfraction A5 (9.7 g) by repeated silica gel CC (PE–EtOAc, 10:1 to 1:1) and Sephadex
RI
LH-20 (CH2Cl2–MeOH, 1:1) purification.
Fraction B (22.3 g) was chromatographed over silica gel CC eluted with a gradient
SC
CH2Cl2–MeOH system (100:1, 50:1, 20:1, 10:1 and 5:1) to give five subfractions (B1–B5).
NU
Fraction B3 (19.6 g) was further purified by silica gel CC eluted with CH2Cl2–MeOH (50:1–0:1) to obtain five fractions (B3A–B3E). B3B (1.2 g) was recrystallized from
MA
methanol to give 16 (30 mg). Further separation of fractions B3C (6.2 g) and B3D (6.0 g) by silica gel CC eluted with CH2Cl2–MeOH (50:1-10:1), followed by separation on
TE
D
Sephadex LH-20 (CH2Cl2–MeOH, 1:1), afforded 15 (13.2 mg) and 19 (39.2 mg), respectively. Fraction B4 (3.7 g) was chromatographed on a Sephadex LH-20 column
AC CE P
eluting with (CH2Cl2–MeOH, 1:1) to give 18 (39.4 mg). Fraction C (55.7 g) was separated by silica gel CC using a step gradient elution of CH2Cl2–MeOH (50:1, 30:1, 20:1 and 10:1) to yield 4 fractions C1–C4. Fraction C2 (5.0 g) was chromatographed on a Sephadex LH-20 column eluting with MeOH, followed by purification on a semi-preparative HPLC with MeCN–H2O (25:75) to give compound 17 (32.9 mg). Fraction C4 was chromatographed on a Diaion HP-20 column, eluted successively with 20% MeOH, 40% MeOH, 60% MeOH and 80% MeOH. The 20% and 40% MeOH elutes were combined and chromatographed on Sephadex LH-20 (MeOH), and then purified by semi-preparative HPLC separation with CH3OH–H2O (65:35) to afford compound 1 (6.2 mg). The fraction eluting with 60% MeOH (5.2 g) was re-chromatographed on a flash ODS column eluted with MeOH-H2O (8:2–3.5:6.5) to give compounds 2 (13 mg) and 14 (20 mg). The 80%
6
ACCEPTED MANUSCRIPT MeOH elute (3.8 g) was recrystallized from methanol affording 12 (80 mg), and the filtrate was then purified by flash ODS column chromatography with MeOH–H2O
PT
(8:2–0:10) to give three subfractions (1–3). Subfraction 1 was purified by
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semi-preparative HPLC with CH3CN–H2O (25:75) to afford 5 (5.4 mg), 6 (7.2 mg) and 7 (9.1 mg). Further separation of subfraction 2 (339.2 mg) by silica gel CC eluted with
SC
CH2Cl2–MeOH (20:1–10:1), followed by semi-preparative HPLC with MeOH–H2O
NU
(60:40), afforded 3 (28.2 mg). In a similar manner to that described for subfraction 2, subfraction 3 yielded 4 (90.4 mg) and 8 (106.3 mg).
MA
The n-BuOH fraction (938 g) was chromatographed on a D101 macroporous resin column, using a gradient of EtOH and H2O (0:100, 30:70, 50:50, 75:25 and 95:5) to give
D
five subfractions (Bu-1-5). Fraction Bu-4 (332.2 g) was repeatedly subjected to silica gel
TE
CC eluted with CH2Cl2–MeOH–H2O (100:0:0–65:35:7), followed by a combinative
AC CE P
separation of Sephadex LH-20 (MeOH) and semi-preparative HPLC (MeCN–H2O, 32:68), to give compound 10 (30 mg).
2.3.1. (-)-(6aR,11aR)-3-Hydroxy-9-methoxypterocarpan 8-O-α-D-ribofuranoside (1) Colorless needles (MeOH); [α]20D –27.0º (c 0.10, MeOH); UV(MeOH) λmax 230 (sh), 285 nm; ECD (MeOH) λmax (Δε) 211 (-17.10), 237 (-17.69), 290 (4.39) nm; 1H (400 MHz, DMSO-d6) and 13C NMR (100 MHz, DMSO-d6) data, see Table 1; ESI-MS m/z 417 [M H]-; HR-ESI-MS m/z 417.1182 [M - H]- (calcd. for C21H21O9, 417.1191). 2.3.2. Medicarpin 3-O--D-glucopyranoside 6'-acetate (2) Yellow powder; [α]20D –35.3º (c 0.12, MeOH); UV (MeOH) λmax 230 (sh), 285 nm; IR (KBr) νmax 3417, 2928, 2857, 1732, 1661, 1620, 1597, 1497, 1435, 1380, 1345, 1275, 1160, 1141, 1081, 1036, 950, 736 cm-1; ECD (MeOH) λmax (Δε) 209 (-11.53), 235 (-6.47), 7
ACCEPTED MANUSCRIPT 287 (2.98) nm; 1H (400 MHz, DMSO-d6) and 13C NMR (100 MHz, DMSO-d6) data, see Table 1; ESI-MS m/z 497 [M + Na]+; HR-ESI-MS m/z 497.1427 [M + Na]+ (calcd. for
PT
C24H26O10Na, 497.1418).
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2.3.3. Complogenin 22-O-α-L-rhamnopyranoside (3)
SC
White amorphous powder; [α]20D +55.5º (c 0.11, MeOH); IR (KBr) νmax 3370, 2926, 2854, 1649, 1452, 1375, 1036, 975 cm-1; 1H (400 MHz, CD3OD) and
13
C NMR (100
NU
MHz, CD3OD) data, see Table 2; ESI-MS m/z 619 [M + H]+, 641 [M + Na]+;
MA
HR-ESI-MS m/z 641.3996 [M + Na]+ (calcd. for C36H58O8Na, 641.4024). 2.3.4. Soyasapogenol A 21-O-α-L-rhamnopyranoside (4)
D
White amorphous powder; [α]20D +28.8º (c 0.13, MeOH); IR (KBr) νmax 3463, 2970,
TE
2959, 1638, 1452, 1380, 1123, 1046, 992 cm-1; 1H (400 MHz, C5D5N) and 13C NMR (100
AC CE P
MHz, C5D5N) data, see Table 2; ESI-MS m/z 643 [M + Na]+, 1263 [2M + Na]+; HR-ESI-MS m/z 643.4179 [M + Na]+ (calcd. for C36H60O8Na, 643.4180). 2.3.5. 3β,22β,24-Trihydroxyolean-12-en-11-one21-O-α-L-rhamnopyranoside (5) White amorphous powder; [α]20D +34.0º (c 0.10, MeOH); IR (KBr) νmax 3282, 2920, 2849, 1654, 1545, 1391, 1072, 1038 cm-1; 1H (400 MHz, C5D5N) and
13
C NMR (100
MHz, C5D5N) data, see Table 2; ESI-MS m/z 657 [M + Na]+, 1291 [2M + Na]+; HR-ESI-MS m/z 657.3960 [M + Na]+ (calcd. for C36H58O9Na, 657.3973). 2.3.6. 7β,15α-Dihydroxycomplogenin (6) White amorphous powder; [α]20D +66.0º (c 0.10, MeOH); IR (KBr) νmax 3391, 3337, 2929, 2855, 1652, 1660, 1458, 1375, 1074, 1029, 964 cm-1; 1H (400 MHz, C5D5N) and 13
C NMR (100 MHz, C5D5N) data, see Table 2; ESI-MS m/z 527 [M + Na]+, 1031 [2M + 8
ACCEPTED MANUSCRIPT Na]+; HR-ESI-MS m/z 527.3343 [M + Na]+ (calcd. for C30H48O6Na, 527.3343). 2.3.7. 7β,15α,22β,24-Tetrahydroxyolean-12-en-3,11-dione (7)
PT
White amorphous powder; [α]20D +64.9º (c 0.15, MeOH); IR (KBr) νmax 3375, 3258,
RI
2922, 2849, 1704, 1458, 1386, 1068, 964 cm-1; 1H (400 MHz, C5D5N) and 13C NMR (100
SC
MHz, C5D5N) data, see Table 2; ESI-MS m/z 525 [M + Na]+, 1027 [2M + Na]+;
NU
HR-ESI-MS m/z 525.3176 [M + Na]+ (calcd. for C30H46O6Na, 525.3187).
MA
2.4. Computational details
The theoretical ECD calculations for compounds 1a, 1b, 2a and 2b were conducted
D
by the Gaussian 09 program package. The time-dependent density functional theory
TE
(TDDFT) was used to calculate the excitation energy (in nm) and rotatory strength R (velocity form Rvel and length form Rlen in 10-40 erg-esu-cm/Gauss) between different
AC CE P
states, at B3LYP/6-31G (d, p) level in methanol solution. The ECD curves were then simulated by the following Gaussian functions:
Where σ is the width of the band at 1/e height and ΔEi and Ri are the excitation energies and rotatory strengths for transition i, respectively. σ = 0.2 eV and Rlen were used in this work. 2.5.
X-ray
data
of
(-)-(6aR,11aR)-3-Hydroxy-9-methoxypterocarpan
8-O-α-D-ribofuranoside (1)
9
ACCEPTED MANUSCRIPT Suitable crystals for the X-ray diffraction were obtained by recrystallization from a methanol solution of 1. The crystallographic data of 1 was collected on a Bruker APEX
PT
DUO diffractometer equipped with a rotating Cu anode generator (λ = 1.54178 Å) and a parabolic Gӧ bel mirror. Crystal data: C21H22O9, M = 418.38, crystal size 0.21 × 0.05 ×
RI
0.04 mm3, monoclinic, space group P21, a = 4.8952(3) Å, b = 11.6874(8) Å, c =
SC
16.2106(10) Å, α = 90°, β = 90.128(4)°, γ = 90°, V = 927.44(10) Å3, T = 203(2) K, Z = 2,
NU
Dcalc = 1.498 mg/m3, μ(Cu Kα) = 1.000 mm-1, F(000) = 440, 4428 reflections collected (θmax = 67.996°), 2456 independent reflections (Rint = 0.0660). The final R1 [I > 2σ(I)] and
MA
wR2 values (F2) were 0.0813 and 0.1889, respectively. The final R1 (all data) and wR2 values (all data) were 0.0893 and 0.1959, respectively. The absolute structure parameter
TE
D
was 0.0(3). Crystallographic data for 1 have been deposited in the Cambridge Crystallographic Data Centre (CCDC) with the accession no. CCDC 1054442. These data
AC CE P
can be obtained free of charge from CCDC via 12 Union Road, Cambridge, CB2 1EZ, UK (fax: +44-1223-336-033 or e-mail: [email protected]). 2.6. Analysis of the sugar moiety Each compound (2 mg) was dissolved in 2.0 mL of 2.0 N HCl and heated to 100 °C in a water bath for 4 h. The mixture was neutralized with excess Ag2CO3, and the solvent was removed under a stream of N2. The obtained residue was re-suspended in H2O (2 mL) and extracted three times with 2 mL of CHCl3. The aqueous layer was collected and evaporated to dryness using N2. The residue was re-dissolved in anhydrous pyridine (0.1 mL) and mixed with 0.06 N L-cysteine methyl ester hydrochloride (0.1 mL) in pyridine. The mixture was heated to 60 °C for 2 h, followed by addition of trimethylsilylimidazole (0.1 mL), and heating was continued at 60 °C for another 2 h. Water and organic solvent 10
ACCEPTED MANUSCRIPT were removed under reduced pressure, and the residue was concentrated to dryness and partitioned between n-hexane and H2O. The organic layer was analyzed by a Shimadzu
PT
GCMS-QP2010 Ultra instrument (Kyoto, Japan) using a TG-5MS column (0.32 mm × 30
RI
m) under the following conditions [injector temperature, 250 °C; initial temperature, 150°C (1 min), increased at 8 °C/min to 260°C, held for 10 min; carrier gas, He operated
SC
in the splitless mode; injection size, 1.0 μL; MS conditions: EI voltage, 70 eV;
NU
scanned-mass range, m/z 50-1000]. The hydrolysates of 3–5 gave peaks at 16.20, 16.21, and 16.21 min, respectively. With authentic
D-rhamnose
and L-rhamnose, peaks were
MA
detected at 16.38 and 16.22 min, respectively.
TE
2.7.1. Cytotoxic assay
D
2.7. Biological assays
AC CE P
The human lung cancer cell line (A549), human ileocecal adenocarcinoma cell line (HCT-8), human breast cancer cell line (MCF-7), and human hepatocellular carcinoma cell line (BEL-7402) were obtained from the American Type Culture Collection (ATCC). HCT-8 and BEL-7402 cell lines were cultured in RPMI-1640 medium. MCF-7 cell line was cultured in Dulbecco’s modified Eagle medium (DMEM), and A549 cell line was cultured in Ham’s F-12 medium. These cell lines were all supplemented with 10% fetal bovine serum (FBS). The cytotoxic activity was evaluated by the sulforhodamine B (SRB) method. Briefly, the cultured cells were harvested, counted, and diluted to 5 × 103 cells/well in aliquots of 190 L of fresh medium. After incubation for 24 h, various concentrations of the test compounds (10 L/well) were added to the 96-well plates, followed by incubation for 72 h at 37 ºC under 5% CO2 atmosphere. The cells were fixed
11
ACCEPTED MANUSCRIPT with 100 μL of 10% cold trichloroacetic acid (TCA), incubated for 60 min at 4 ºC, washed three time with deionized water, and allowed to air-dry. The cells were then
PT
stained by 0.4% SRB (Sigma) dissolved in 1% acetic acid for 15 min and subsequently washed five times with 1% acetic acid. Plates were air-dried, and bound dye was
SC
RI
solubilized with 150 L of 10 mM Tris on a shaker for 5 min. The absorbance at 560 nm was measured by using a microplate reader. The concentration of test compounds
NU
resulting in 50% growth inhibition (IC50) was estimated. 5-FU and cisplatin were used as positive controls. All experiments were performed in three independent replicates. IC50
MA
values are expressed as the mean ± SD.
D
2.7.2. Nitrite assay
TE
The inhibitory effect on NO production in RAW264.7 cells was tested by a previously reported method [9]. Briefly, RAW264.7 cells were placed in 96-well plates at
AC CE P
a density of 2×105 cells/well for 24 h. Samples or the reference compound were dissolved in DMSO and diluted with serum-free DMEM into a series of concentrations. The cells were pre-treated with different concentrations of testing compounds for 30 min, and then stimulated with LPS (1 μg/mL) for 24 h. After incubation, an aliquot of the culture supernatants (100 µL) was added to a solution of Griess reagent (50 µL, 1:1 mixture of 1% sulfanilamide in 5% H3PO4 and 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride in H2O) in the 96-well plate and left for 20 min at room temperature. The optical density of the supernatant was then measured at 550 nm with a spectrophotometer. Nitrite concentrations in the supernatants were determined by comparison with a standard curve using sodium nitrite as a standard. Aminoguanidine hydrochloride was used as the positive control.
12
ACCEPTED MANUSCRIPT 3. Results and discussion The dried and powdered whole plants of G. verna were successively extracted with
PT
95% and 70% ethanol. The combined extract was concentrated, suspended in water, and
RI
partitioned with petroleum ether (60-90 ºC), EtOAc and n-BuOH, successively. The
SC
EtOAc and n-BuOH fractions were repeatedly purified by a combination of column chromatography including silica gel, ODS, Sephadex LH-20 and semi-preparative HPLC.
NU
A total of 8 triterpenoids and 12 flavonoids were obtained including two new pterocarpan
MA
glycosides (1–2), five new triterpenoids (3–7), and 13 known compounds (8–20) (Fig. 1). The new structures were elucidated by extensive NMR (1H, 13C, COSY, HSQC, HMBC
D
and NOESY) and HR-ESI-MS analysis. The sugar residues were identified by GC-MS
TE
analysis after acidic hydrolysis and trimethylsilylation. The known compounds were
AC CE P
identified as sigmoside C (8) [10], soyasapogenol B (9) [11], soyasaponin I methyl ester (10) [12], medicarpin (11) [13], medicarpin 3-O--D-glucoside (12) [5], maackiain (13) [14], trifolirhizin (14) [15], 7,4'-dihydroxyflavone (15) [16], apigenin (16) [3], sulfuretin (17) [17], luteolin (18) [18], quercetin (19) [19], and formononetin (20) [20] by spectroscopic analysis and comparison with literature data. An isoflavone (20) and two triterpenoid glycosides (8 and 10) were isolated for the first time from the genus Gueldenstaedtia Fisch. Four flavonoids (11, 13, 14 and 17) were isolated for the first time from G. verna. Compound 1 was isolated as colorless needles from MeOH. Its molecular formula was determined to be C21H22O9 by HR-ESI-MS analysis ([M - H]- m/z 417.1182, calcd for C21H21O9, 417.1191) and NMR spectroscopic data. The UV absorption bands at 230 (sh) and 285 nm and the 1H NMR signals at δH 4.24 (m, H-6), 3.62 (m, H-6), 3.60 (m, 13
ACCEPTED MANUSCRIPT H-6a) and 5.52 (d, J = 6.4 Hz, H-11a) of 1 suggested characteristic signals for a pterocarpan derivative (Table 1) [21]. A 1,2,4-trisubstituted benzene ring was assigned
PT
from analysis of its 1H NMR coupling constants [δH 7.25 (d, J = 8.5 Hz, H-1), 6.48 (dd, J = 8.4, 2.4 Hz, H-2) and 6.26 (d, J = 2.3 Hz, H-4)] and on the basis of the COSY
RI
spectrum (Fig. 2). In addition, two distinct aromatic singlets at δH 7.11 and 6.58 in the 1H
SC
NMR spectrum suggested the presence of a 1,2,4,5-tetrasubstituted benzene ring in 1.
NU
The 13C NMR spectrum displayed 21 carbon signals, of which 16 signals were attributed to the pterocarpan aglycone moiety. The remaining five carbon signals (δC 102.5, 71.7,
MA
69.5, 86.3 and 61.6) and the corresponding proton signals (Table 1) were typical of a ribofuranosyl residue [22,23]. All these NMR information is in support of 1 being a
structure
of
TE
D
pterocarpan ribofuranoside. Further inspection of the NMR data of 1 found that the its
aglycone
moiety
was
very
similar
to
that
of
AC CE P
(6aR,11aR)-3,8-dihydroxy-9-methoxypterocarpan [24], with C-8 hydroxyl group replaced by a ribofuranosyl unit. The pterocarpan skeleton assignment was verified by the HMBC correlations from H-11a to C-1a, C-4a, C-6 and C-6a, and from H-6a to C-7a, C-10a and C-6 (Fig. 2). The positions of the methoxy and ribofuranosyl groups were also established by analysis of the HMBC spectrum, where correlations of the methoxy protons at δH 3.72 with C-9 (δC 151.5), and of H-1' (δH 5.36) with C-8 (δC 139.2) were observed. Comparison of the
13
C NMR data of the furanosyl moiety of 1 with that of methyl
α-D-ribofuranoside and methyl β-D-ribofuranoside reported in the literature [23], along with the NOESY correlation between H-1' and H-3' of the ribose [25], assigned the configuration of the anomeric center (C-1') to be α. Complete 1H- and
13
C NMR
assignments of 1 were aided by the DEPT and 2D NMR experiments (COSY, HMQC and
14
ACCEPTED MANUSCRIPT HMBC). The relative configurations of C-6a and C-11a in 1 were determined by NOESY
PT
correlations and vicinal 1H-1H coupling constants. A significant NOESY correlation
RI
between H-6a and H-11a, along with a small coupling constant (3JH6a/H11a = 6.4 Hz) [26] indicated that H-6a and H-11a protons are cis-oriented. The absolute configuration at
SC
C-6a and C-11a was determined by optical rotation, and the comparison of experimental
NU
and calculated electronic circular dichroism (ECD) spectra. The attempt to obtain the aglycone through acidic hydrolysis of 1 was unsuccessful because of the unavailability of
MA
sufficient amount of 1. Therefore, the optical rotation value of 1 instead of its aglycone was measured. To address the influence of glycosidation on pterocarpan aglycones, we
D
compared the optical rotation values of medicarpin3-O-β-D-glucoside (12, c 0.37, C5H5N,
TE
[α]20D -153.2) and its aglycone medicarpin (11, c 0.15, CH3OH, [α]20D -226.0), and
AC CE P
trifolirhizin (14, c 0.15, CH3OH, [α]20D -107.6) and its aglycone maackiain (13, c 0.12, CH3OH, [α]20D -241.3). The results showed that mono-glycosidation did not alter the direction of optical rotation for pterocarpan aglycones, indicating that the contribution of the sugar part to the optical rotation might be limited. The absolute configuration at C-6a and C-11a was thus tentatively assigned to R from its negative optical rotation value [21]. Generally the absolute configuration of a chiral molecular can also be determined by its CD spectrum. A further proof of the absolute configuration of 1 was obtained from the experimental ECD spectrum. As shown in Fig. 4A, the ECD spectrum of 1 displayed negative Cotton effects at 211 and 237 nm and a positive Cotton effect at 290 nm, which is consistent with the C-6aR and C-11aR configurations of the previously reported pterocarpan derivatives [27]. The calculation of ECD spectra has been proved as a very
15
ACCEPTED MANUSCRIPT efficient tool for analysis of optically active naturally products over the past decade [28]. Therefore, the calculated ECD is also used to confirm the absolute configuration of 1.
PT
Two simplified structures (1a and 1b), in which the methyl group instead of the
RI
ribofuranosyl group in 1, were used for ECD calculation. The model compounds 1a and 1b with (C-6aR and C-11aR) and (C-6aS and C-11aS) absolute configurations were
SC
calculated by using the TD-DFT method at a B3LYP/6-31+G (d, p) level in MeOH. The
NU
calculation for C-6aR, C-11R (1a, the aglycone) configuration agreed with the experimental ECD spectrum (1, the glycoside, Fig. 4). Finally, a single crystal X-ray
MA
diffraction study of 1 was performed (Fig. 5), from which its relative and absolute configurations were confirmed. Consequently, the structure of 1 was established as 8-O-α-D-ribofuranoside.
Although
TE
D
(-)-(6aR,11aR)-3-hydroxy-9-methoxypterocarpan
many pterocarpans have been reported from the Leguminosae family, compound 1 is the
AC CE P
second pterocarpan with a rare glycosidic ribose from the genus Gueldenstaedtia Fisch. Compound 2 had a molecular formula of C24H26O10, as determined by HR-ESI-MS at m/z 497.1427 [M + Na]+ and the 13C NMR data. Comparison of the NMR data of 2 and 12 (medicarpin 3-O--D-glucopyranoside) revealed great similarity except for an additional acetyl group in 2 (Table 1). The acetyl group was assigned at C-6' of the glucose by the HMBC correlations between H-6' (δH 4.05, 4.26) and the carbonyl carbon (δC 170.3). The negative optical rotation value ([α]20D -35.3), and the comparison of experimental and calculated ECD spectra (Fig. 4B) suggested a (6aR,11aR) configuration. Thus, the structure of 2 was identified as medicarpin 3-O--D-glucopyranoside 6ʹ-acetate. Previously, compound 2 was proposed to be an artificial component of alfalfa (Medicago sativa) by LC-MS [29], but this compound has not been isolated as an
16
ACCEPTED MANUSCRIPT individual compound. Recently, this compound was isolated as a trace component in Sophora tonkinensis [30]. However, identification of the accurate molecular structure has
PT
not so far been performed by NMR technique. Thus, we consider that compound 2 is
RI
described in the present work for the first time.
Compound 3 was obtained as a white amorphous powder. It gave a positive
SC
Liebermann-Burchard test, indicating a triterpenoid skeleton. Its molecular formula was
NU
deduced as C36H58O8 by HR-ESI-MS at m/z 641.3996 [M + Na]+ (calcd for C36H58O8Na, 641.4024). The IR spectrum of 3 displayed a broad band centered at 3370 cm-1 and a
MA
relative sharp absorbance peak at 1649 cm-1 for hydroxyl and α,β-unsaturated carbonyl functionalities, respectively. The 1H NMR spectrum showed seven methyl singlets at δH
D
1.41, 1.21, 1.14, 1.12, 1.05, 0.97 and 0.95, a methyl doublet at δH 1.24 (d, J = 6.2 Hz), an
13
C NMR spectrum showed 36 carbon signals, which were assigned by
AC CE P
(Table 2). The
TE
olefinic broad singlet at δH 5.55, and together with an anomeric proton singlet at δH 4.79
HSQC and DEPT experiments as resonances for a ketone carbonyl carbon signal at δc 199.6 (C-11), a quaternary olefinic carbon signal at δc 169.1 (C-13), a tertiary olefinic carbon at δc 128.6 (C-12), an anomeric carbon signal at δc 98.2 (C-1'), nine methine carbons (including six oxygenated methines), nine methylene carbons (one for oxygenated methylene and eight for ordinary methylenes), and eight methyl carbons at δc 32.8, 27.6, 23.7, 23.2, 22.0, 18.6, 18.4 and 17.5 (Table 2). These above 1H- and 13C NMR spectroscopic data of 3 showed typical signals that suggested an oleane-type triterpenoid mono-glycoside framework [5]. A comparison between the
13
C NMR data of 3 and
complogenin [12] revealed that the carbon signals of the sapogenin of 3 and complogenin were almost identical, except for the C-22 and neighbor carbons. These data suggested
17
ACCEPTED MANUSCRIPT that 3 is complogenin with a glycosidic linkage at C-22, which is consistent with the observed downfield shift in C-22 by 3.9 ppm and an upfiled shift in C-21 (-6.6 ppm) with
PT
respect to the corresponding signals of complogenin [12]. In addition, the 13C NMR data
RI
together with the above anomeric proton suggested the presence of a rhamnopyranosyl moiety in 3. The linkage site of the rhamnopyranosyl moiety was further confirmed by
SC
the HMBC correlation of H-1' (δH 4.79) with C-22 at δc 78.7 (Fig. 2). The relative
NU
configurations of the protons at C-3 and C-22 were determined by a NOESY experiment. The correlations of H-3 (δH 3.35) with H-5 (δH 0.87) and H-23 (δH 1.21), and of H-22 (δH
MA
3.42) with H-29 (δH 0.95) indicated that they were cofacial and designated as -oriented. The -configuration of the rhamnopyranosyl moiety was deduced by the chemical shift
TE
D
of C-3 at δc 73.0 and C-5 at δc 70.5 [31], and by the significant HMBC correlations from H-1' to C-3' and C-5' [32] (Fig. 2). The absolute configuration of the rhamnose was
AC CE P
determined as L, by GC-MS analysis after acidic hydrolysis of 3 and trimethylsilylation. Thus, the structure of 3 was identified as complogenin 22-O--L-rhamnopyranoside. Compound 4 was obtained as a white amorphous powder. It gave a positive Liebermann-Burchard test, indicating a triterpenoid skeleton. Its molecular formula was deduced as C36H60O8 by HR-ESI-MS at m/z 643.4179 [M + Na]+ (calcd for C36H60O8Na, 643.4180). The 1H NMR spectrum of 4 showed signals for an olefinic proton at δH 5.34, an anomeric proton singlet at δH 5.51, seven oxygenated methine signals, one oxygenated methylene, and eight methyl signals at δH 1.66, 1.59, 1.39, 1.29, 1.27, 1.03, 1.01 and 0.94 (Table 2). The
13
C NMR spectrum displayed 36 carbons, including a
quaternary olefinic carbon signal at δc 144.5 (C-13), a tertiary olefinic carbon at δc 122.7 (C-12), an anomeric carbon signal at δc 104.6, seven oxygenated methine carbons at δc
18
ACCEPTED MANUSCRIPT 84.3, 80.2, 78.7, 73.9, 72.8, 72.4 and 70.5, an oxygenated methylene at δc 64.7, and eight methyl carbons at δc 31.1, 26.8, 23.7, 22.2, 22.0, 18.7, 17.0 and 16.3 (Table 2). These 12(13)
oleane-type skeleton with the same sugar moiety
PT
NMR data suggested that 4 had a
RI
as 3. Further inspection of the NMR data of 4 found that its sapogenin moiety was very
SC
close to those of soyasapogenol A [33], except for the signals assigned to C-21 exhibiting a significant glycosidation shift (+9.7 ppm) and a small upfiled shift for carbon
NU
neighboring C-22 (-0.9 ppm). These NMR data indicated that the rhamnopyranosyl unit was attached to the C-21 hydroxyl group. This linkage was confirmed by the HMBC
MA
correlation between H-1' (δH 5.51) and C-21 (δC 84.3) (Fig. 2). The relative configurations of three hydroxyl groups were determined by a NOESY experiment. The correlations
TE
D
from H-3 (δH 3.66) to H-23 (δH 1.59) and H-5 (δH 0.99), from H-21 (δH 3.84) to H-29 (δH 1.03), and from H-22 (δH 3.93) to H-21 (δH 3.84) indicated that hydroxyl groups at C-3,
AC CE P
C-21 and C-22 were all β-orientated (Fig. 3). Therefore, the structure of 4 was identified as soyasapogenol A 21-O-α-L-rhamnopyranoside. Compound 5 was obtained as white amorphous powders. The HR-ESI-MS showed an [M + Na]+ ion at m/z 657.3960, and together with the
13
C NMR data, indicating a
molecular formula of C36H58O9. The NMR data were very similar to those of 4, except for the presence of a C-11 keto signal in 5. The C-11-keto-
12(13)
group was also assigned by
comparison of the NMR data with those of 3, and further supported by the HMBC correlation of H-9 with C-11 (Fig. 2). NOESY correlations between H-3 (δH 3.70)/H-23 (δH 1.62), H-5 (δH 1.00); H-21 (δH 3.83)/H-29 (δH 1.03); and H-22 (δH 3.93)/H-21 (δH 3.83) indicated that the hydroxyl groups at C-3, C-21 and C-22 were all β-oriented. Therefore, the
structure
of
5
was
identified
as
19
3β,22β,24-trihydroxyolean-12-en-11-one
ACCEPTED MANUSCRIPT 21-O-α-L-rhamnopyranoside. Compound 6, obtained as a white amorphous powder, possessed a molecular
PT
formula of C30H48O6 as determined by the combination of the HR-ESI-MS (m/z 527.3343 [M + Na]+) and NMR data. The 1H NMR spectrum of 6 displayed signals for typical
13
C NMR signals at δc 199.0 (a ketone
SC
of a broad olefinic proton signal at δ 6.07 with
RI
triterpenoid methyl singlets at δ 1.74, 1.62, 1.57, 1.47, 1.32, 1.27 and 0.96. The presences
NU
carbonyl carbon), 170.8 (a quaternary olefinic carbon) and 129.1 (a secondary olefinic carbon), were characteristic of a C-11-keto-
12(13)
functionality of oleane-type
MA
triterpenoids [5]. The 1H NMR spectrum also showed six oxygenated protons (including four oxygenated methines) at δH 4.74 (m), 4.58 (d, J = 11.6 Hz), 4.49 (m), 3.88 (m), 3.77
TE
D
(m) and 3.73 (m) (Table 2). The 13C NMR and HSQC spectra showed the presence of 30 carbons due to seven methyls, seven methylenes, eight methines and eight quaternary
AC CE P
carbons. The HMBC correlations of H-6, H-5, Me-26 with C-7 as well as a long range COSY correlations of H-7 with H-5 and H-6 (Fig. 2), suggested that one hydroxyl group was located at C-7. The HMBC correlations from Me-27, H-16 to C-15 and the COSY correlation between H-15 and H-16 indicated the occurrence of a C-15 hydroxyl group. Similarly, the COSY correlation between H-21 and H-22 and HMBC correlations between Me-28, H-21 and C-22 indicated that another hydroxyl group was attached at C-22. Key HMBC and COSY correlations are shown in Fig. 2. NOESY correlations between H-3/H-5, H-23; H-7/H-5, H-6, H-9, Me-27; H-15/H-16, H-26, H-28; and H-22/H-16, H-21, H-29 (Fig. 3) indicated that the hydroxyl groups at C-3, C-7, C-15 and C-22 were β-, β-, α- and β-oriented, respectively. Thus, the structure of 6 was identified as 7β,15α-dihydroxycomplogenin.
20
ACCEPTED MANUSCRIPT Compound 7 exhibited a [M + Na]+ ion at m/z 525.3176 (C30H46O6Na) in the HR-ESI-MS. The
13
C NMR data of 7 were very similar to those of 6 except that a C-3
PT
keto group in 7 was observed instead of a hydroxyl group in 6 (Table 2), indicating 7 was
RI
a C-3keto derivative of 6. The HMBC correlations from H-1, H-2 and Me-23 to C-3 confirmed the assigned position (Fig. 2). Using the data shown in Figs. 2 and 3, the
SC
structure of 7 was identified as 7β,15α,22β,24-tetrahydroxyolean-12-en-3,11-dione.
NU
The cytotoxic activities of all the isolates were estimated toward A549, HCT-8, MCF-7 and BEL-7402 cell lines by the sulforhodamine B (SRB) assay, using
MA
5-fluorouracil (5-FU) and cisplatin as the positive controls. The results are expressed as IC50 values and are summarized in Table 3. Compounds 9, 17 and 18 exhibited weakly
TE
D
cytotoxic activities against all the selected cancer cell lines, while compounds 1, 6, 7, 10, 13 and 16 showed selectively inhibitory activity against only one of the cancer cell lines
AC CE P
studied.
All the isolates were further tested for their inhibitory activities against LPS-induced NO production in RAW264.7 cells using the Griess method. Among them, only compound 17 showed the slightly toxic, and displayed an over 50% inhibition of NO production at the dose of 50 μM. Compound 17 showed significant inhibition of NO production with IC50 values of 12.6 ± 1.8 M, which was comparable to the positive control aminoguanidine hydrochloride (IC50 = 8.94 ± 0.44 μM). Our finding is in agreement with the work of Shin et al. [34], who recently reported the in vitro anti-inflammatory effect of sulfuretin. Acknowledgments This work was supported by grants from the National Natural Science Foundation of 21
ACCEPTED MANUSCRIPT China (No. 81473421 and No. 81073025).
PT
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Shaanxi J Tradit Chin Med 2001;22:184–5.
SC
[2] Huang HL, Xu B. Free radical scavenging activities and principals of
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Gueldenstaedtia multiflora Bge. J Qingdao Univ (Nat Sci Ed) 2006;19:30–5. [3] Wang JX, Zhu R. Studies on the chemical constituents of Gueldenstaedtia multiflora
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[4] Han YL, Gao LM. Study on chemical constituents of Gueldenstaedtia multiflora Bge.
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J Shanxi College Tradit Chin Med 2009;10:24–5.
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[5] Tsunoda Y, Okawa M, Kinjo J, Ikeda T, Nohara T. Studies on the constituents of
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Gueldenstaedtia multiflora. Chem Pharm Bull 2008;56:1138–42. [6] Cao J, Yin CL, Qin Y, Cheng ZH, Chen DF. Approach to the study of flavone di-C-glycosides by high performance liquid chromatography tandem ion trap mass spectrometry and its application to characterization of flavonoid composition in Viola yedoensis. J Mass Spectrom 2014;49:1010–24. [7] Du DS, Cheng ZH, Chen DF. Anti-complement sesquiterpenes from Viola yedoensis. Fitoterapia 2015;101:73–9. [8] Qin Y, Yin CL, Cheng ZH. A new tetrahydrofuran lignan diglycoside from Viola tianshanica Maxim. Molecules 2013;18:13636–44. [9] Xiao GD, Li XK, Wu T, Cheng ZH, Tang QJ, Zhang T. Isolation of a new meroterpene and inhibitors of nitric oxide production from Psoralea corylifolia fruits guided by TLC bioautography. Fitoterapia 2012;83:1553–7. 22
ACCEPTED MANUSCRIPT [10] Mbafor JT, Ndom JC, Fomum ZT. Triterpenoid saponins from Erythrina sigmoidea. Phytochemistry 1997;44:1151–5.
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[11] Konoshima T, Kozuka M, Haruna M, Ito K, Kimura T. Studies on the constituents of
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Leguminous plants. XI. The structures of new triterpenoids from Wistaria brachybotrys Sieb. et Zucc. Chem Pharm Bull 1989;37:1550–3.
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[12] Cui B, Inoue J, Takeshita T, Kinjo J, Nohara T. Triterpene glycosides from the seeds
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of Astragalus sinicus L. Chem Pharm Bull 1992;40:3330–3. [13] Yoon JS, Sung SH, Park JH, Kim YC. Flavonoids from Spatholobus suberectus.
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Arch Pharm Res 2004;27:589–92.
[14] Abdel-Kader MS. Phenolic constituents of Ononis vaginalis roots. Planta Med
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D
2001;67:388–90.
[15] Zeng Y, Luo JJ, Li C. Chemical constituents from aerial part of Rumex patientia. J
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Chin Med Mat 2013;36:57–60.
[16] Fu HZ, Han L. Chemical constituents from fruits of Gymnocladus chinensis. Chin Herbal Med 2009;1:66–70. [17] Li YL, Li J, Wang NL, Yao XS. Flavonoids and a new polyacetylene from Bidens parviflora Willd. Molecules 2008;13:1931–41. [18] Li J, Jiang H, Shi R. A new acylated quercetin glycoside from the leaves of Stevia rebaudiana Bertoni. Nat Prod Res 2009;23:1378–83. [19] Wu T, Abdulla R, Yang Y, Aisa HA. Flavonoids from Gossypium hirsutum flowers. Chem Nat Comp 2008;44:370–1. [20] Zheng Y, Liu H, Bai YJ, Ma YM, Zhao YY. Five flavonoids from Spatholobus suberectus. China J Chin Mat Med 2008;33:152–4.
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ACCEPTED MANUSCRIPT [21] Fedoreyev SA, Bulgakov VP, Grishchenko OV, Veselova MV, Krivoschekova OE, Kulesh NI, et al. Isoflavonoid composition of a callus culture of the relict tree
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Maackia amurensis Rupr. et Maxim. J Agric Food Chem 2008;56:7023–31.
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[22] Li Y, Li XF, Lee U, Kang JS, Choi HD, Son BW. A new radical scavenging anthracene glycoside, asperflavin ribofuranoside, and polyketides from a marine
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[23] Sharma RK, Singh S, Tiwari R, Mandal D, Olsen CE, Parmar VS, et al. O-Aryl α,β-D-ribofuranosides: synthesis & highly efficient biocatalytic separation of
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anomers and evaluation of their Src kinase inhibitory activity. Bioorg Med Chem 2012;20:6821–30.
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D
BCB,
EV,
Ferreira
D.
Flavonoid
analogues
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[24] Bezuidenhoudt
from Pterocarpus species. Phytochemistry 1987;26:531–5.
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[25] Horton RA, Bagnato JD, Grissom CB. Structural determination of 5'-OH α-ribofuranoside modified cobalamins via
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C and DEPT NMR. J Org Chem 2003;
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[26] van Aardt TG, van Rensburg H, Ferreira D. Synthesis of isoflavonoids. Enantiopure cis- and trans-6a-hydroxy pterocarpans and a racemic trans-pterocarpan. Tetrahedron 2001;57:7113-26. [27] Piccinelli AL, Fernandez MC, Cuesta-Rubio O, Hernandez IM, de Simone F, Rastrelli L. Isoflavonoids isolated from Cuban propolis. J Agric Food Chem 2005;53:9010–6. [28] Li XC, Ferreira D, Ding YQ. Determination of absolute configuration of natural products: theoretical calculation of electronic circular dichroism as a tool. Curr Org
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ACCEPTED MANUSCRIPT Chem 2010;14:1678–97. [29] Sumner LW, Paiva NL, Dixon RA, Geno PW. High-performance liquid
glycosides
in
Leguminous
plant
extracts.
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Mass
Spectrom
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flavonoid
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chromatography/ continuous-flow liquid secondary ion mass spectrometry of
1996;31:472–85.
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[30] Yoo H, Chae HS, Kim YM, Kang M, Ryu KH, Ahn HC, et al. Flavonoids and
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arylbenzofurans from the rhizomes and roots of Sophora tonkinensis with IL-6 production inhibitory activity. Bioorg Med Chem Lett 2014;24:5644–7.
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[32] Zheng Q, Koike K, Han LK, Okuda H, Nikaido T. New biologically active triterpenoid saponins from Scabiosa tschiliensis. J Nat Prod 2004;67:604–13.
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25
ACCEPTED MANUSCRIPT Figure legends Fig. 1. Structures of compounds 1-20.
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Fig. 2. Selected 1H-1H COSY and HMBC correlations of 1–7.
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Fig. 3. Selected NOESY correlations of 1 and 3–7.
Fig. 4. (A) Experimental ECD spectrum of 1 and the calculated ECD spectra of 1a and
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1b. (B) Experimental ECD spectrum of 2 and the calculated ECD spectra of 2a and 2b.
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D
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system is used for the structure in the text).
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Fig. 5. X-ray crystallographic structure of compound 1 (note: a different numbering
26
ACCEPTED MANUSCRIPT 30
29
7
10a
9
10
R1=H R1=(6'-acetyl)-Glu R1=H R1=Glu R1=H R1=Glu
R2
O
R3
3 5 6 7
5
6
O
4
HO
D-Glu
1
3
OH
HO
OH
HO
OH
HO R3 H H OH OH
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R2 H OH OH OH
AC CE P
15 16 18 19
R1 H H H OH
O
R2 H H OH OH
R3 H H OH OH
R1O
R4 H ORha H H
R5 Rha H H H
4 8 9 10
D-Rib
O
OH
O HO
HO
OH
OH
R1 R2 R3 H ORha H H H Glu H H H S H H
L-Rha
OH
D
O R1
R2
OH
OH R3
R2
O
HO
2
OH
HO
R1 OH OH OH =O
7
R3
OH
D-Gal
O
HO
24
OR3
15
5
23
OR5 28
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HO
17
9
3
R1
R2
21
13
10
R2=ORib R3=OMe R2=H R3=OMe R2=H R3=OMe R2=H R3=OMe R2+R3=-OCH2OR2+R3=-OCH2O-
D-GluA
11
1
S = 6'-methyl-D-GlcA(2→1)-D-Gal(2→1)-L-Rha
O
R4
19 8
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6a
H O
H 7a
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1a
1
6
11a
SC
3 2
0 01 2 11 12 13 14
O
4a
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4
R1O
OH HO
OH
HO
OH
O
O
O O 17
20
Fig. 1. Structures of compounds 1-20.
27
O
ACCEPTED MANUSCRIPT
O
1
OH
O OCH3
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OH O
1
O
O HO
OH OH
O
OCH3
O
AC
OH
O
OH
CH2OH
OH OH
O CH2OH 7
OH
OH HO
HO
OH OH
OH
6
CE
O
O
OH
CH2OH
PT ED
2
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O
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O O
HMBC
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OH
O O
H-1H COSY
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HO
OH
O
OH
O OH
OH
O
HO CH2OH
CH2OH
3
4
Fig. 2. Selected 1H-1H COSY and HMBC correlations of 1–7.
28
5
O
OH
O OH
OH
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ACCEPTED MANUSCRIPT
20
17
6 9
6a 3
11a
4
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1
20 21 17
14
22
AC
9
CE
18
5 4
3
3
5
8
3
4
21
21
17
22 9
6
14
9 8
14 8
5
14
4
3
PT ED
1
9
5
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5
2
22
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14
17
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18
21
18
15
15 16
6
7
7
4
6
Fig. 3. Selected NOESY correlations of 1 and 3–7.
29
7
16
22
22
ACCEPTED MANUSCRIPT 60
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20
0 250
300
350
-20 -40
200
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-60 250
400
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200
SC
Δε ( M-1 cm-1)
40
300
A
350
400
350
400
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wavelength (nm) 80
D
60
0 200
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20
250
AC CE P
Δε (M-1 cm-1)
40
-20
300
-40 -60
B
-80
200
250
300
350
400
wavelength (nm)
Fig. 4. (A) Experimental ECD spectrum of 1 and the calculated ECD spectra of 1a and 1b. (B) Experimental ECD spectrum of 2 and the calculated ECD spectra of 2a and 2b.
30
D
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SC
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PT
ACCEPTED MANUSCRIPT
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Fig. 5. X-ray crystallographic structure of compound 1 (note: a different numbering
AC CE P
system is used for the structure in the text).
31
ACCEPTED MANUSCRIPT Table 1 NMR spectroscopic data (400 MHz, DMSO-d6) for compounds 1–2. 1
OCH3-9 COCH3-6' COCH3-6'
6.58 s 5.52 d (6.4) 5.36 d (4.3) 4.00 m 3.88 m
39.5 117.1 117.9 139.2 151.5 95.9 155.1 77.8 102.5
AC CE P
4' 5' 6'
3.62 m 3.60 m 7.11 s
6.69 dd (8.5, 2.3)
4.04 m 3.45 brs
71.7 69.5 86.3 61.6
3.72 s
56.0
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132.0 114.2 110.4 158.2 104.0 156.2 66.0
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4.24 m
6.26 d (2.3)
132.0 111.4 109.7 158.7 102.8 156.3 65.7
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6 6 6a 7 7a 8 9 10 10a 11a 1' 2' 3'
6.48 dd (8.4, 2.4)
δC
7.39 d (8.5)
6.54 d (2.2)
4.29 dd (9.8, 3.5)
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7.25 d (8.5)
δH(J in Hz)
3.64 m 3.65 m 7.24 d (8.1)
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1 1a 2 3 4 4a
δC
D
δH(J in Hz)
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No.
2
6.44 dd (8.2, 2.0) 6.42 d (1.9) 5.60 d (6.8) 4.89 d (7.7) 3.24 t (7.9) 3.29 t (8.8) 3.15 t (9.2) 3.61 m 4.05 dd (11.8, 7.2) 4.26 dd (12.0, 1.4) 3.69 s 1.97 s
32
38.9 125.3 119.2 106.2 160.6 96.4 160.3 77.7 99.9 73.1 76.3 69.9 73.6 63.4 55.3 170.3 20.6
ACCEPTED MANUSCRIPT
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Table 2
a
4
3
5
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
1
1.02 m; 2.71 m
39.7
0.99 m; 1.59 m
39.0
2
1.83 m; 2.00 m
28.5
1.95 m; 2.03 m
3
3.35 m
79.9
3.66 m
56.0
0.99 m
6
1.47 m; 1.71 m
18.4
1.48 m; 1.73 m
7
1.47 m; 1.70 m
33.4
1.31 m; 1.51 m
62.2
10
37.3
11
199.6 128.6
12
5.55 s
13
169.1
14
45.4
39.7
1.58 m; 3.43 m
40.5
28.5
1.25 m; 3.27 dd (13.0, 3.0) 1.98 m; 2.24 m
39.5
28.5
1.20 m; 3.18 d (13.5) 1.97 m; 2.18 m
28.5
2.44 m; 3.01 dt (14.3, 6.0)
35.7
80.2
3.70 m
79.9
3.73 m
79.5
213.9
43.4
55.2
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56.0
1.24 m
52.7
1.71 m
54.3
19.2
1.48 m; 1.75 m
18.4
2.10 m; 2.36 m
28.6
1.31 m; 2.30 m
29.2
33.2
1.30 m; 1.62 m
33.2
4.49 m
72.0
4.48 dd (9.6, 4.8)
71.6
40.3
1.66 m
43.6
1.00 m
48.1
CE
43.7 2.45 s
δC
56.4
43.8
2.53 s
62.3
52.0 2.66 s
62.6
51.9 2.69 s
61.9
37.1
37.4
37.7
37.6
1.28 m; 1.90 m
24.2
199.7
199.0
198.7
5.34 m
122.7
AC
9
δH (J in Hz)
PT ED
0.87 m
8
δC
43.2
5
7
δH (J in Hz)
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43.6
6
δC
SC
No.
4
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NMR spectroscopic data (400 MHz, pyridine-d5) for compounds 3–7.
5.88 s
128.7
6.07 s
129.1
6.07 s
129.0
144.5
169.2
170.8
171.2
42.2
45.8
51.1
51.1
15
1.26 m; 1.64 m
26.5
1.20 m; 1.84 m
26.6
1.11 m; 1.97 m
26.9
4.74 m
66.6
4.73 dd (10.8, 4.5)
66.7
16
1.65 m; 1.87 m
27.4
1.07 m; 1.95 m
27.6
1.26 m; 1.31 m
30.0
1.84 m; 2.31 m
37.1
1.87 m; 2.31 m
37.1
17
37.4
39.5
36.7
37.9
37.9
18
2.34 m
45.6
2.58 m
43.6
2.67 m
44.0
2.63 m
46.6
2.67 m
46.6
19
1.09 m; 1.89 m
44.8
1.32 m; 2.06 m
47.2
1.17 m; 1.97 m
45.4
1.16 m; 1.99 m
45.1
1.18 m; 1.99 t
45.1
33
ACCEPTED MANUSCRIPT
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36.8
39.3
1.69 m; 1.74 m
41.9
1.69 m; 1.75 m
41.9
78.0
3.77 m
74.5
3.76 m
74.5
23.7
1.62 s
23.6
1.56 s
20.9
64.5
17.1
3.99 d (10.9); 4.38 m 1.66 s
65.2
17.4
3.88 m; 4.58 d (11.6) 1.47 s
64.5
16.3
3.77 d (8.5); 4.56 m 1.33 s
1.01 s
17.0
1.15 s
18.8
1.57 s
13.3
1.53 s
13.5
23.2
1.27 s
26.8
1.42 s
24.1
1.74 s
18.6
1.73 s
18.6
1.05 s
27.6
1.29 s
22.2
1.21 s
22.6
1.27 s
22.8
1.27 s
22.8
29
0.95 s
32.8
1.03 s
31.1
1.03 s
30.7
0.96 s
33.6
0.95 s
33.6
30
0.97 s
22.0
1.39 s
22.0
1.35 s
21.7
1.32 s
28.1
1.32 s
28.1
1'
4.79 brs
98.2
5.51 brs
104.6
5.51 brs
104.7
2'
3.78 m
72.6
4.70 m
72.4
4.70 brs
72.3
3'
3.65 m
73.0
4.61 m
72.8
4.61 m
72.7
4'
3.39 m
73.8
4.33 t (9.1)
73.9
4.34 t (9.1)
73.9
5'
3.65 m
70.5
4.54 m
70.5
4.55 m
70.5
6'
1.24 d (6.2)
18.4
1.66 d (5.0)
18.7
1.66 d (5.9)
18.6
1.44 m; 1.63 m
35.4
3.84 brs
84.3
3.83 brs
22
3.42 m
78.7
3.93 brs
78.7
3.93 brs
23
1.21 s
23.7
1.59 s
23.7
1.62 s
24
64.5 17.5
3.73 d (10.1); 4.53 m 0.94 s
64.7
25
3.41 m; 4.09 d (11.0) 1.12 s
26
1.14 s
18.6
27
1.41 s
28
83.8
AC
CE
PT ED
NU
21
RI
30.5
SC
20
a
30.9
MA
(13.5)
NMR measured in CD3OD.The assignments were based on DEPT, COSY, HSQC, and HMBC spectra.
34
31.0
16.5
ACCEPTED MANUSCRIPT Table 3 Cytotoxicity of isolates against four cancer cell lines (IC50, μM).a MCF-7
BEL-7402
> 150 > 150 > 150 97.9 ± 11.6 > 150 71.4 ± 11.0 76.9 ± 8.5 88.0 ± 19.8 99.5 ± 7.4
120.7 ± 16.4 > 150 > 150 115.3 ± 15.3 > 150 > 150 > 150 74.5 ± 10.0 108.0 ± 20.1
5-FUb
0.8 ± 0.5
3.5 ± 1.5
cisplatinb
1.8 ± 0.4
16.4 ± 1.6
6.7 ± 2.5
2.3 ± 1.2
20.3 ± 3.2
2.2 ± 0.2
Each IC50 value is expressed as the mean ± SD from three independent experiments.
D
a
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> 150 > 150 > 150 173.2 ± 55.1 > 150 > 150 > 150 98.2 ± 18.0 113.5 ± 26.1
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> 150 152.5 ± 18.6 97.9 ± 5.4 88.7 ± 16.1 108.7 ± 14.9 > 150 > 150 82.6 ± 8.4 81.6 ± 16.5
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1 6 7 9 10 13 16 17 18
PT
HCT-8
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compound A549
Positive controls.
AC CE P
b
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The other 11 compounds were inactive for all cell lines (IC50 > 150 μM).
35
ACCEPTED MANUSCRIPT Graphical Abstract
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PT
Pterocarpans and triterpenoids from Gueldenstaedtia verna
SC
Chengle Yin, Jinge Zhou, Yiqing Wu, Yue Cao, Tao Wu, Shoude Zhang, Honglin Li,
AC CE P
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D
MA
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Zhihong Cheng
36