- Email: [email protected]
Cycloartane triterpenoid saponins from the herbs of Thalictrum fortunei
Accepted Manuscript Cycloartane triterpenoid saponins from the herbs of Thalictrum fortunei Si-Qi Jiang, Yu-Bo Zhang, Min Xiao, Lin Jiang, Ding Luo, Qian-Wen Niu, Yao-Lan Li, Xian-Tao Zhang, Guo-Cai Wang PII:
S0008-6215(17)30013-7
DOI:
10.1016/j.carres.2017.03.019
Reference:
CAR 7348
To appear in:
Carbohydrate Research
Received Date: 6 January 2017 Revised Date:
17 March 2017
Accepted Date: 17 March 2017
Please cite this article as: S.-Q. Jiang, Y.-B. Zhang, M. Xiao, L. Jiang, D. Luo, Q.-W. Niu, Y.-L. Li, X.-T. Zhang, G.-C. Wang, Cycloartane triterpenoid saponins from the herbs of Thalictrum fortunei, Carbohydrate Research (2017), doi: 10.1016/j.carres.2017.03.019. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
Cycloartane triterpenoid saponins from the herbs of Thalictrum fortunei Si-Qi Jiang,a,b Yu-Bo Zhang,a,b Min Xiao,c,d Lin Jiang,a,b Ding Luo,a,b Qian-Wen
Institute of Traditional Chinese Medicine & Natural Products, College of Pharmacy, Jinan
University, Guangzhou 510632, PR China b
Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs
Guangdong Institute of Traditional Chinese Medicine, Guangzhou 510520, PR China
Guangzhou University of Chinese Medicine, Guangzhou 510006, PR China
TE D
d
M AN U
Research, Jinan University, Guangzhou 510632, PR China c
SC
a
RI PT
Niu,a,b Yao-Lan Li,a,b Xian-Tao Zhang,*c,d Guo-Cai Wang*a,b
ABSTRACT: Six new cycloartane triterpenoid saponins, thalisides A-F (1-6), along
EP
with four known ones (7-10) were isolated from Thalictrum fortunei. The new
AC C
structures were elucidated by using spectroscopic data (NMR, IR, UV, and MS). Compounds 1-10 were determined for their in vitro cytotoxicity against two human cancer cell lines (HepG2, A549) and antiviral activity against influenza A virus (H1N1). However, compound 1-10 were inactive against human cancer cell lines (HepG2, A549) and influenza A virus (H1N1). (IC50>100 µM) Keywords: Thalictrum fortunei; cycloartane triterpenoid saponins; cytotoxicity; antiviral activity *
Corresponding authors: E-Mails: [email protected] (G.-C. Wang); [email protected] (X.-T. Zhang).
ACCEPTED MANUSCRIPT 1. Introduction The genus Thalictrum (Ranunculaceae), commonly called “meadow rue”, is widely distributed in south and southwest part of China.1 The genus Thalictrum
RI PT
(Ranunculaceae) have been discovered approximately 29 species which are used as traditional medicines for a long history to treat gastrosis, dysentery, and tuberculosis etc.2-7 Phytochemical investigations have revealed that triterpenoid saponins and
SC
alkaloids are the main constituents of the genus Thalictrum.8-11 The rhizomes of
M AN U
Thalictrum fortunei are frequently used as the substitutes for Coptis chinensis (an important Chinese medicinal plant) in China.12 These triterpenoid saponins displayed a wide spectrum of biological activities, including antitumor,13 antifungal,14 anti-inflammatory,15-16 and immunostimulating17 activities. In previous studies, we
TE D
have reported some new cycloartane triterpenoid saponins from the aerial part of T. fortunei.18-20 Further investigation on this plant led to the isolation of six new cycloartane triterpenoid sponins, thalisides A-F (1-6), along with four known ones
EP
(7-10). All isolates were evaluated for in vitro cytotoxicity against human cancer cells
AC C
(A549, HepG2) and antiviral activity against influenza A virus (H1N1). In this paper, we report the structure elucidation and biological activities of these compounds.
2. Results and discussion The ethanolic extract from the herbs of T. fortunei was successively partitioned into petroleum ether, EtOAc and n-butanol. The n-butanol fraction was subjected to various chromatographic methods to yield ten cycloartane triterpenoid saponins (1-10,
ACCEPTED MANUSCRIPT Fig.1). The molecular formula of 1 was determined to be C47H78O18 on the basis of its HR-ESI-MS data (m/z 929.5137 [M−H]-). Its IR spectrum indicated the absorption
RI PT
band of hydroxyl group at 3373 cm-1. The 1H NMR spectrum of 1 showed two characteristic proton signals for cyclopropane moiety [δ 0.45 (1H, d, J = 3.8 Hz), 0.28 (1H, d, J = 3.8 Hz)], five methyls at δ 1.98 (3H, s), 1.56 (3H, s), 1.20 (3H, d, J = 6.4
SC
Hz), 1.07 (3H, s), 0.89 (3H, s), and three anomeric protons at 5.26 (1H, d, J = 7.8 Hz), 13
M AN U
4.92 (1H, d, J = 7.8 Hz), 4.88 (1H, d, J = 6.3 Hz,). The
C NMR spectrum of 1
displayed 47 carbon signals including three oxygenated carbons (δC 89.6, 73.1 and 67.4) and three anomeric carbons (δC 106.3, 106.2, 102.9). The 1H and 13C NMR data of
1
(Tables
1
and
2)
were
to
those
of
(22S,24Z)-cycloart-24-en-3β,22,
TE D
3-O-β-D-glucopyranosyl-(1→4)-β-D-fucopyranosyl
similar
26,30-tetraol 26-O-β-D-glucopyranoside,18 except for the absence of the fucose moiety, and the presence of an extra xylose moiety. The types of sugar residues were
EP
confirmed by the acid hydrolysis of 1, which were detected as D-glucose and D-xylose
AC C
by GC analysis. The 3JH1-H2 coupling constants demonstrated β-glycosidic linkages of D-glucose
(J = 7.8 Hz) and D-xylose (J = 6.3 Hz). Moreover, the locations of aglycone
and glycosyl were confirmed by its HMBC correlations from δH 4.88 (H-1 of xylose) to δC 89.6 (C-3 of the aglycone), from δH 5.26 (H-1 of glucose II) to δC 79.0 (C-4 of xylose), and from δH 4.92 (H-1 of glucose I) to δC 67.4 (C-26 of the aglycone). The relative configuration of 1 was determined by the NOESY spectrum, which showed the NOE correlations between H-3 (δH 3.68) and H-29 (δH 1.56), between H-19 (δH
ACCEPTED MANUSCRIPT 0.45) and H-30 (δH 4.54)/H-18 (δH 1.07). Therefore, the structure of 1 was deduced as 3-O-β-D-glucopyranosyl-(1→4)-β-D-xylopyranosyl (22S,24Z)-cycloart-24-en-3β,22,26,30-tetraol 26-O-β-D-glucopyranoside and named
RI PT
as thaliside A. Its HR-ESI-MS data showed the molecular formula as C67H110O30 (m/z 1393.7073 [M−H]-). The IR spectrum of 2 suggested that the presence of carbonyl
resembled
those
of
M AN U
closely
SC
(1647 cm-1) and hydroxyl (3393 cm-1). The 1H and 13C NMR data of 2 (Tables 1 and 2)
3-O-α-L-arabinopyranosyl-(1→6)-β-D-glucopyranosyl-(1→4)-β-D-fucopyranosyl (22S,24Z)-cycloart-24-en-3β,22,26-triol
26-O-(6-O-acetyl)-β-D-glucopyranoside.19
The main differences were the presence of an additional glucopyranosyl and a
D-fucose
TE D
xylopyranosyl in 2. Acid hydrolysis of 2 yielded D-glucose, D-xylose, L-arabinose and in the ratio of 3:1:1:1 by GC analysis. The β anomeric configurations of
glucopyranosyl, fucopyranosyl and xylopyranosyl units were determined on the basis
EP
of their J values. Similarly, the anomeric configuration of arabinopyranosyl unit was
AC C
identified as α. The HMBC correlations from H-1 of fucose (δH 4.71) to C-3 of aglycone (δC 88.5), from H-1 of glucose II (δH 5.13) to C-4 of fucose (δC 83.0), from H-1 of arabinose (δH 4.92) to C-6 of glucose II (δC 70.2), from H-1 of xylose (δH 4.89) to C-4 of arabinose (δC 77.2), and from H-1 of glucose III (δH 4.87) to C-4 of xylose (δC 78.3) confirmed the locations of glycosyls. Thus, the structure of 2 was identified as 3-O-β-D-glucopyranosyl-(1→4)-β-D-xylopyranosyl-(1→4)-α-L-arabinopyranosyl(1→6)-β-D-glucopyranosyl-(1→4)-β-D-fucopyranosyl
(22S,24Z)-cycloart-24-en-
ACCEPTED MANUSCRIPT 3β,22,26-triol 26-O-(6-O-acetyl-β-D-glucopyranoside) and named as thaliside B. Compound 3 showed a [M+Cl]- ion at m/z 1299.6093 (C61H100O27Cl) in HR-ESI-MS. Comparison of the 1D NMR spectra of 3 with those of 2 (Tables 1 and 2)
RI PT
suggested that they were similar, except that the absence of an arabinopyranosyl in 3. The NMR data of sugar moiety indicated pentasaccharides residues, which were verified as D-glucose, D-xylose, D-fucose by GC analysis. The five anomeric proton
SC
signals at δH 4.67 (J = 7.4 Hz), 5.13 (J = 7.8 Hz), 4.83 (J = 7.8 Hz), 4.87 (J = 7.8 Hz),
M AN U
5.09 (J = 6.3 Hz) indicated the β-configurations of glycosyls. The glycosylating positions were determined by the HMBC spectrum, wherein the correlations between H-1 (δH 4.67) of Fuc and C-3 (δC 88.5) of aglycone, between H-1 (δH 5.13) of Glc II and C-4 (δC 83.0) of Fuc, between H-1 (δH 5.09) of Xyl and C-6 (δC 70.2) of Glc II,
TE D
between H-1 (δH 4.87) of Glc III and C-4 (δC 78.4 ) of Xyl were observed. Hence, the structure of 3 was established as 3-O-β-D-glucopyranosyl-(1→4)-β-D-xylopyranosyl(1→6)-β-D-glucopyranosyl-(1→4)-β-D-fucopyranosyl
(22S,24Z)-cycloart-24-en-3β,
EP
22,26-triol 26-O-(6-O-acetyl-β-D-glucopyranoside) and named as thaliside C.
AC C
The HR-ESI-MS data of 4 suggested a molecular formula C58H96O25 (m/z 1191.6241 [M−H]-). The NMR data of 4 (Tables 1 and 2) were in good agreement with those of 3-O-β-D-glucopyranosyl-(1→4)-β-D-fucopyranosyl (22S,24Z)-cycloart24-en-3β,22,26-triol 26-O-β-D-glucopyranoside20 except for the existence of an extra xylopyranosyl and an arabinopyranosyl moieties in 4. Acid hydrolysis of 4 obtained sugar residues, which were identified as D-fucose
D-xylose, L-arabinose, D-glucose
and
by GC analysis. The HMBC correlations from H-1 of Glc II (δH 5.10) to C-4
ACCEPTED MANUSCRIPT of Fuc (δC 83.0), from H-1 of Ara (δH 4.90) to C-6 of Glc II (δC 69.9), and from H-1 of Xyl (δH 4.95) to C-4 of Ara (δC 77.2) verified the locations of the glycosyls. The values of coupling constants indicated α-anomeric configuration of arabinopyranose,
the
structure
of
4
was
assigned
as
RI PT
and β-anomeric configurations of xylopyranose, fucopyranose, glucopyranose. Thus, 3-O-β-D-xylopyranosyl-(1→4)-α-L-
arabinopyranosyl-(1→6)-β-D-glucopyranosyl-(1→4)-β-D-fucopyranosyl
(22S,24Z)-
SC
cycloart-24-en-3β,22,26-triol 26-O-β-D-glucopyranoside and named as thaliside D.
M AN U
Compound 5 had the molecular formula of C50H82O19 which exhibited a quasi-molecular ion peak at m/z 1021.5155 [M+Cl]-. The 1D NMR data of 5 (Tables 1 and 2) were almost identical to those of 3-O-β-D-glucopyranosyl-(1→4)-β-Dfucopyranosyl
(22S,24Z)-cycloart-24-en-3β,22,26,30-tetraol
26-O-β-D-
TE D
glucopyranoside.18 The main differences were that the presence of additional signals at δC 170.9 and 20.8, which indicated the existence of acetyl group. Correlations between δH 4.86 (H-1 of Fuc) and δC 89.3 (C-3 of aglycone), as well as δH 5.22 (H-1
EP
of Glc II) and δC 83.4 (C-4 of Fuc) were observed in HMBC spectrum, which
AC C
indicated that the sugar chain was 3-O-β-D-glucopyranosyl-(1→4)-β-D-fucopyranosyl. Thus, the structure of 5 was deduced to be 3-O-β-D-glucopyranosyl-(1→4)-β-Dfucopyranosyl (22S,24Z)-cycloart-24-en-3β,22,26,30-tetraol 26-O-(6-O-acetyl-β-Dglucopyranoside) and named as thaliside E. The molecular formula of 6 was established as C54H90O22 by HR-ESI-MS at m/z 1125.5639 [M+Cl]-. The 1H and
13
C NMR data of 6 (Tables 1 and 2) was similar to
those of 3-O-β-D-glucopyranosyl-(1→4)-β-D-fucopyranosyl (22S,24Z)-cycloart-24-
ACCEPTED MANUSCRIPT en-3β,22,26-triol 26-O-β-D-quinovopyranosyl-(1→6)-β-D-glucopyranoside20, except for oxygenation of C-30 at δ 63.4. Analysis of the sugar residues indicated that 6 had the
same
glycosyls
as
3-O-β-D-glucopyranosyl-(1→4)-β-D-fucopyranosyl 26-O-β-D-quinovopyranosyl-(1→6)-β-D-
RI PT
(22S,24Z)-cycloart-24-en-3β,22,26-triol
glucopyranoside. The HMBC correlations between H-1 (δH 5.04) of quinovose and C-6 (δC 69.9) of glucose II, between H-1 (δH 5.16) of glucose II and C-4 (δC 83.1) of
of
glycosyls.
Hence,
the
structure
of
6
was
elucidated
as
M AN U
linkages
SC
fucose, between H-1 (δH 4.83) of fucose and C-3 (δC 89.2) of aglycone suggested the
3-O-β-D-quinovopyranosyl-(1→6)-β-D-glucopyranosyl-(1→4)-β-D-fucopyranosyl (22S,24Z)-cycloart-24-en-3β,22,26,30-tetraol 26-O-β-D-glucopyranoside and named as thaliside F.
TE D
The four known triterpenoid saponins were identified as 3-O-β-D-glucopyranosyl (1→4)-β-D-fucopyranosyl (22S,24Z)-cycloart-24-en-3β,22,26,30-tetraol 26-O-β-Dglucopyranoside (7),18 3-O-β-D-glucopyranosyl-(1→4)-β-D-fucopyranosyl (22S,24Z)-
EP
cycloart-24-en-3β,22,26-triol
26-O-β-D-quinovopyranosyl-(1→6)-β-D-
AC C
glucopyranoside (8),20 3-O-β-D-glucopyranosyl(1→4)-β-D-fucopyranosyl (22S,24Z)cycloart-24-en-3β,22,26-triol
26-O-β-D-glucopyranoside
(9),18
3-O-β-D-
glucopyranosyl (24S)-cycloartane-3β,16β,24,25,30-pentaol 25-O-β-D-glucopyranosyl(1→6)-β-D-glucopyranoside (10)18 by comparing their spectroscopic data with the related literatures. In previous researches, the triterpenoid saponins isolated from the genus Thalictrum exhibited antitumor21 and antiviral22 activities. Therefore, all isolated
ACCEPTED MANUSCRIPT triterpenoid saponins were evaluated for the cytotoxicity against lung cancer cell (A549) and liver cancer cell (HepG2) by MTT assay15 and antiviral activities against H1N1 by cytopathic effect (CPE) reduction assay.23 However, the data showed that
concentration of 100 µM.
3. Experimental
M AN U
3.1. General experimental procedures
SC
RI PT
these compounds had no significant cytotoxic effects and antiviral activities even at a
Optical rotations were recorded on JASCO P-1020 polarimeter. UV and IR spectra were measured by JASCO V-550 UV/vis spectrophotometer and a JASCO FT/IR-480 plus infrared spectrometer with KBr pellets, respectively. The 1D and 2D
TE D
NMR spectra were performed on Bruker Avance 500 using pyridine-d5 as solvent. HR-ESI-MS data were recorded on a Micromass Q-TOF mass spectrometer (Agilent 6210). Silica gel (200-300 mesh, Qingdao Marine Chemical Plant, China), ODS silica
EP
gel (50 µm, YMC, Japan) and Sephadex LH-20 (Pharmacia Biotech, Uppsala, Sweden)
AC C
were used for chromatography column. TLC was performed on Silica gel GF254 plates (Yantai, China). Analytical HPLC was carried on a Waters system equipped with P680 pump, a photodiode array detector and a Cosmosil C18 analytical column (5 µm, 4.6 mm × 250 mm). Preparative HPLC was carried on Agilent system with a Cosmosil C18 preparative column (5 µm, 20 mm × 250 mm). GC was measured on a Shimadzu GC-2010 gas chromatograph. The spectroscopic and chromatographic grades of organic reagents were purchased from Tianjin Damao Chemical Company (Tianjin,
ACCEPTED MANUSCRIPT PR China). 3.2. Plant Material The dried herbs of Thalitrum fortunei were collected in June of 2013 from Wuhu
RI PT
City, Anhui Province of China. The species were authenticated by Prof. Guang-Xiong Zhou of Jinan University. A voucher specimen (No. 20130326) was deposited in the Institute of Traditional Chinese Medicine, Jinan University, Guangzhou, China.
SC
3.3. Extraction and isolation
M AN U
The air-dried and powdered herbs of T. fortunei (2.8 kg) were extracted with EtOH/H2O (95:5, v/v) at room temperature to afford a crude extract (0.4 kg). The extract was suspended in H2O, and then successively extracted with petroleum ether, EtOAc and n-butanol. The n-butanol part (97.0 g) was subjected to silica gel column
TE D
chromatography (CC) and eluted with CHCl3-CH3OH (100:1 to 1:1, v:v) to give 9 fractions (Fr. 1-9). Fr. 7 (45.6 g) was separated by silica gel CC (CHCl3-CH3OH, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1 and 0:1, v/v, each 15 L) to afford 6 subfractions (Fr. 7.1-7.6).
EP
The Fr. 7.3 (6.5 g) was then fractionated into five subfractions by Sephadex LH-20
AC C
CC (CHCl3-MeOH, 1:1, v/v). Subfractions 7.3.2 (200.5 mg) was purified by preparative HPLC (CH3CN-H2O, 30:70, v/v, flow rate: 7.0 mL/min) to yield 1 (20.3 mg) and 2 (12.5 mg). Fr. 4 was separated by an ODS column which was eluted with gradient mixtures (MeOH-H2O, 50:50 to 100:0, v:v) to produce Fr. 4.1-4.9. Fr. 4.5 (60.2 mg) was purified by preparative HPLC (CH3CN-H2O, 40:60, v/v, flow rate: 7.0 mL/min) to give 4 (15.4mg), 5 (8.2 mg) and 8 (16.0 mg). Fr. 4.5 (8.8 g) was subjected to Sephadex LH-20 (CHCl3-MeOH, 1:1, v/v) to afford 7 (60.4 mg). Fr. 4.2 was
ACCEPTED MANUSCRIPT chromatographied by Sephadex LH-20 (MeOH) and further purified by preparative HPLC (MeOH-H2O, 70:40, v/v, flow rate: 7.0 mL/min) to yield 3 (27.3 mg), 6 (13.7 mg), 9 (16.5 mg) and 10 (37.8 mg).
RI PT
3.3.1. Thaliside A (1) 25
White powder; [α] D +5.60 (c 0.5, MeOH); UV (MeOH) λmax 204 nm; IR (KBr) νmax 3373, 2934, 1635, 1375, 1167, 1077, 893 cm−1; HR-ESI-MS m/z 929.5137
SC
[M−H]- (calcd for C47H77O18, 929.5115); 1H and 13C NMR data see Tables 1 and 2.
25
M AN U
3.3.2. Thaliside B (2)
White powder; [α] D -5.87 (c 0.15, MeOH); UV (MeOH) λmax 204 nm; IR (KBr) νmax 3403, 2936, 2869, 1647, 1384, 1043, 890 cm−1; HR-ESI-MS m/z 1393.7073 [M−H]- (calcd for C67H109O30 , 1393.7009); 1H and 13C NMR data see Tables 1 and 2.
TE D
3.3.3. Thaliside C (3) 25
White powder; [α] D -6.40 (c 0.2, MeOH); UV (MeOH) λmax 204; IR (KBr) νmax 3390, 2937, 1652, 1378, 1071 cm−1; HR-ESI-MS m/z 1299.6093 [M+Cl]- (calcd for
EP
C61H100ClO27, 1299.6146); 1H and 13C NMR data see Tables 1 and 2.
AC C
3.3.4. Thaliside D (4)
25
White powder; [α] D +1.96 (c 0.14, MeOH); UV (MeOH) λmax 204 nm; IR (KBr)
νmax 3389, 2936, 1648, 1379, 1071, 891 cm−1; HR-ESI-MS m/z 1191.6241 [M−H](calcd for C58H95O25, 1191.6235); 1H and 13C NMR data see Tables 1 and 2. 3.3.5. Thaliside E (5) 25
White powder; [α] D +18.6 (c 0.69, MeOH); UV (MeOH) λmax 204 nm; IR (KBr) νmax 3420, 2936, 1648, 1377, 1256, 1071, 889 cm−1; HR-ESI-MS m/z 1021.5155
ACCEPTED MANUSCRIPT [M+Cl]- (calcd for C50H82ClO19, 1021.5144 ); 1H and 13C NMR data see Tables 1 and 2. 3.3.6. Thaliside F (6) 25
RI PT
White powder; [α] D +5.75 (c 0.25, MeOH); UV (MeOH) λmax 204 nm; IR (KBr) νmax 3394, 2935, 1649, 1456, 1379, 1068, 889 cm−1; HR-ESI-MS m/z 1125.5639 [M+Cl]- (calcd for C54H90ClO22, 1125.5618 ); 1H and 13C NMR data see Tables 1 and
SC
2.
M AN U
3.4. Acid hydrolysis and GC analysis of compounds 1-6
Each isolate was dissolved in 4.0 M HCl (10 mL) and refluxed at 90 °C for 6 h. After the solvent was dried, the mixture was partitioned with CHCl3-H2O (1:1, v:v). The aqueous layer was neutralized with Dowex (HCO3-) and then filtered. After
TE D
concentrated, the filtrate stirred with NaBH4 at 25 °C for 3 h. The reaction mixture was washed by 0.1% HCl (in MeOH) repeatedly and then heated at 105 °C for 30min. The dried residue was dissolved in dry pyridine (0.5 mL), followed by addition of
EP
Ac2O (0.5 mL). The mixture was reacted in a water bath at 100 °C for 1 h. The dried
AC C
product was extracted with CHCl3 (5 mL, 3 times) and then analyzed by GC-MS: column AT-SE-30 (0.32 mm i.d., 30 m, 0.5 µm film thickness), carrier gas N2(1 mL/min), column temperature 230 °C, injector temperature 250 °C and detector temperature 250 °C. The retention times of standard sugars were detected at 33.08 min (D-glucose), 34.46 min (L-glucose), 28.78 min (D-xylose), 30.70 min (L-xylose), 29.83 min (D-arabinose), 28.30 min (L-arabinose), 26.91 min (D-quinovose), 28.01 min (L-quinovose), 27.83 min (D-fucose) and 29.36 min (L-fucose).
ACCEPTED MANUSCRIPT Acknowledgments This work was supported by grants from the National Natural Science Foundation
RI PT
of China (Nos. 81273407, 81473116, 81273390, 81673319).
Supplementary data
Supplementary data associated with this article can be found, in the online version,
M AN U
SC
at http://dx.doi.org/
References
1. Meng FC, Yuan C, Huang XJ, Wang WJ, Lin LG, Zhang XT, et al. Phytochem Lett 2016;15:108-12.
2. Kuzmanov B, Dutschewska H. J Nat Prod 1982;45:766-71.
TE D
3. Mushtaq S, Rather MA, Qazi PH, Aga MA, Shah AM, Shah A, et al. J Ethnopharmacol 2016;193:221-6.
4. Li DH, Guo J, Bin W, Zhao N, Wang KB, Li JY, et al. Arch. Pharm Res.
EP
2016;39:871-7.
AC C
5. Mayeku WP, Josiah O, Hasanali A, Kiremire, B, Hertweck C. J Chem Pharm Res 2014;6:1-5.
6. Lotfipour F, Nazemiyeh H, Fathi-Azad F, Garaei N, Arami S, Talat S, et al. Iran J Basic Med Sci 2008;11:80-5.
7. Chen SB, Chen SL, Xiao PG. J Asian Nat Prod Res 2003;5:263-71. 8. Khamidullina EA, Gromova AS, Lutsky VI, Owen NL. Nat Prod Rep 2006;23:117-29. 9. Lutskii VI, Gromova AS, Khamidullina EA, Owen NL. Chem Nat Compd
ACCEPTED MANUSCRIPT 2005;41:117-40. 10. Lin LZ, Hu SF, Zaw K, Angerhofer CK, Chai H, Pezzuto JM, et al. J Nat Prod 1994;57:1430-6. 11. Sun H, Zhang XT, Wang L, Zhang XT, Wang Y, Chen SB, et al. Helv Chim Acta
RI PT
2008;91:1961-6.
12. Ma ZQ, Xin SM, Shen XC. Chin Tradit Herbal Drugs 1980;11:217-9. 13. Balandrin MF. Adv Exp Med Biol 1996;404:1-14.
SC
14. Gromova AS, Lutsky VI, Li D, Wood SG, Owen NL, Semenov AA, et al. J Nat Prod 2000;63:911-4.
M AN U
15. Zhang X, Zhao M, Chen L, Jiao H, Liu H, Wang L, et al. Molecules 2011;16:9505-19.
16. Ganenko TV, Isaev MI, Gromova AS, Abdullaev ND, Lutskii VI, Larin MF, et al. Chem Nat Compd 1986;22:288-94.
TE D
17. Varadinova TL, Shishkov SA, Ivanovska ND, Velcheva MP, Danghaaghi S, Samadanghiin Z, et al. Phytother Res 1996;10:414-7. 18. Zhang XT, Ma SW, Jiao HY, Zhang QW. J Asian Nat Prod Res 2012;14:327-32.
EP
19. Zhang XT, Wang L, Ma SW, Zhang QW, Liu X, Zhang LH, et al. J Nat Med
AC C
2013;67:375-80.
20. Zhang XT, Zhang LH, Ye WC, Zhang XL, Yin ZQ, Zhao SX, et al. Chem Pharm Bull 2006;54:107-10.
21. Devkota KP, Khan MTH, Ranjit R, Lannang AM. Nat Prod Res 2007;21:321-7. 22. Song GP, Shen XT, Li SM, Li YB, Si HZ, Fan JH, et al. Eur. J. Med. Chem. 2016;119:109-21. 23. Lin TJ, Lin CF, Chiu CH, Lee MC, Horng JT. Sci. Rep. 2016;6:1-10
ACCEPTED MANUSCRIPT 5 1.23 m, 1.53 m 2.16 m, 2.40 m 3.75 dd (11.5, 4.5) 1.44 0.97 m, 1.69 m 0.98 m, 1.04 m 1.34 1.28 m, 1.96 m 1.65 m, 1.65 m 1.29 m, 1.31 m 1.48 m, 2.17 m 2.39 m 1.08 s 0.26 d (3.7), 0.45 d (3.7) 1.66 m 1.22 d (6.3) 4.08 m 2.49 m, 2.79 m 5.85 t (7.3) 4.54 d (11.0), 4.78 d (11.0) 1.98 s 0.89 s 1.63 s 3.85 d (11.4) 4.56 d (11.4)
6 1.21 m, 1.52 m 2.16 m, 2.37 m 3.72 dd (11.6, 4.3) 1.46 0.93 m, 1.70 m 1.04 m, 0.98 m 1.34 1.96 m, 1.28 m 1.66 m, 1.67 m 1.32 m, 1.28 m 2.17 m, 1.50 m 2.39 m 1.07 s 0.48, d (3.4), 0.27, d (3.4) 1.61 m 1.20 d (6.7) 4.06 m 2.44 m, 2.77 m 5.81 t (6.4) 4.78 d (11.6), 4.54 d (11.6) 1.97 s 0.90 s 1.60 s 3.80 d (11.5) 4.56 d (11.5)
4.85 d (7.6) 4.04 t (7.6) 4.23 m 4.22 m 3.91 m 4.41 dd (11.4, 5.2), 4.52 d (11.4)
5.22 d (7.5) 4.04 t (7.5) 4.22 m 4.24 m 4.04 m 4.53 dd (11.6, 5.0), 4.38 d (11.6)
4.92 d (7.5) 4.08 t (7.5) 4.26 m 4.27 m 3.97 m 4.54 dd (11.5, 5.0), 4.46 d (11.5)
5.10 d (7.8) 3.91 t (7.8) 3.95 m 4.13 m 3.97 m 4.74 dd (11.5, 5.5), 4.24 d (11.5)
5.22 d (7.5) 3.94 t (7.5) 4.22 m 4.24 m 3.94 m 4.53 dd (11.0, 5.0), 4.38 d (11.0)
5.16 d (7.8) 3.97 t (7.8) 4.12 m 4.14 m 4.03 m 4.81 dd (11.2, 5.4), 4.36 d (11.2)
4.86 d (7.0) 4.35 dd (8.4, 7.0) 4.13 m 4.14 m 3.83 dd (13.2, 6.3) 1.65 d (6.3)
4.83 d (7.4) 4.31 dd (8.4, 7.2) 4.05 m 4.10 m 3.81 dd (13.5, 6.5) 1.76 d (6.5)
SC
RI PT
4 1.21 m, 1.51 m 1.90 m, 2.36 m 3.46 dd (11.4, 4.6) 1.49 0.75 m, 1.54 m 1.05 m, 1.30 m 1.32 1.09 m, 1.94 m 1.65 m, 1.65 m 1.32 m, 1.28 m 1.51 m, 2.16 m 2.34 m 1.05 s 0.48 d (3.8), 0.24 d (3.8) 1.68 m 1.18 d (6.0) 4.05 m 2.41 m, 2.74 m 5.78 t (7.4) 4.51 d (11.7), 4.72 d (11.7) 1.93 s 0.89 s 1.32 s 1.05 s
AC C
EP
TE D
M AN U
Table 1 1 H NMR data of compounds 1-6 (C5D5N, ppm, J in Hz)a,b,c,d position 1 2 3 1 1.21 m, 1.52 m 1.17 m, 1.48 m 1.17 m, 1.49 m 2 2.14 m, 2.37 m 1.88 m, 2.34 m 1.89 m, 2.34 m 3 3.68 dd (11.5, 4.5) 3.42 dd (11.2, 4.0) 3.42 dd (11.6, 4.5) 5 1.43 1.45 1.45 6 0.95 m, 1.69 m 0.71 m, 1.53 m 0.72 m, 1.51 m 7 0.97 m, 1.02 m 1.03 m, 1.25 m 1.03 m, 1.25 m 8 1.33 1.30 1.30 11 1.25 m, 1.96 m 1.03 m, 1.89 m 1.03 m, 1.89 m 12 1.69 m, 1.69 m 1.64 m, 1.64 m 1.64 m, 1.64 m 15 1.30 m, 1.31 m 1.27 m, 1.27 m 1.27 m, 1.27 m 16 1.52 m, 2.17 m 1.45 m, 2.11 m 1.45 m, 2.11 m 17 2.37 m 2.33 m 2.32 m 18 1.07 s 1.02 s 1.02 s 19 0.28 d (3.8), 0.45 d 0.44 d (3.4), 0.20 d 0.45 d (3.5), 0.21 d (3.8) (3.4) (3.5) 20 1.68 m 1.62 m 1.62 m 21 1.20 d (6.4) 1.16 d (6.2) 1.16 d (6.7) 22 4.08 m 4.02 m 4.03 m 23 2.45 m, 2.78 m 2.42 m, 2.73 m 2.42 m, 2.72 m 24 5.83 t (7.2) 5.79 t (7.0) 5.78 t (7.0) 26 4.54 d (11.7), 4.76 4.48 d (11.2), 4.73 d 4.49 d (11.5), 4.72 d d (11.7) (11.2) (11.5) 27 1.98 s 1.92 s 1.92 s 28 0.89 s 0.85 s 0.85 s 29 1.56 s 1.29 s 1.29 s 30 3.83 d (11.0) 1.00 s 1.00 s 4.54 d (11.0) Glc I 1 4.92 d (7.8) 4.90 d (7.6) 4.83 d (7.8) 2 4.08 t (7.8) 4.00 t (7.6) 4.00 t (7.8) 3 4.26 m 4.20 m 4.20 m 4 4.28 m 4.21 m 4.21 m 5 3.96 m 3.97 m 3.97 m 6 4.41 dd (11.0, 5.1), 4.93 dd (11.5, 5.2), 4.93 dd (11.0, 5.1), 4.53 d (11.0) 4.74 d (11.5) 4.74 d (11.0) Glc II 1 5.26 d (7.8) 5.13 d (7.8) 5.13 d (7.6) 2 4.08 t (7.8) 3.91 t (7.8) 3.91 t (7.6) 3 4.25 m 3.93 m 3.93 m 4 4.24 m 4.10 m 4.10 m 5 3.96 m 3.90 m 3.90 m 6 4.41 dd (11.5, 5.0), 4.75 dd (11.0, 5.0), 4.75 dd (11.2, 5.4), 4.54 d (11.5) 4.29 d (11.0) 4.29 d (11.2) Xyl l 4.88 d (6.3) 4.89 d (6.2) 5.09 d (6.0) 2 4.41 dd (6.3) 4.02 dd (6.2) 4.01 dd (6.0) 3 4.28 m 3.94 m 3.93 m 4 4.45 m 4.17 m 4.09 m 5 3.81 dd (11.0, 9.5), 4.30 dd (11.2, 9.5), 4.36 dd (10.7, 9.5), 4.46 d (11.0) 3.64 d (11.2) 4.27 d (10.7) Fuc 1 4.71 d (7.4) 4.67 d (7.2) 2 4.29 dd (8.0, 7.4) 4.29 dd (8.2, 7.2) 3 4.00 m 4.00 m 4 4.04 m 4.04 m 5 3.77 dd (13.0, 6.5) 3.77 dd (13.2, 6.5) 6 1.70 d (6.5) 1.70 d (6.4) Glc III 1 4.87 d (7.8) 4.87 d (7.4) 2 4.00 t (7.8) 4.00 t (7.4) 3 4.10 m 4.10 m 4 4.23 m 4.23 m 5 4.04 m 4.04 m 6 4.47 dd (11.0, 5.0), 4.47 dd (11.2, 5.5),
4.95 d (6.3) 4.02 dd (6.3) 3.92 m 4.14 m 4.28 dd (11.0, 9.5), 3.62 d (11.0) 4.70 d (7.3) 4.29 dd (8.0, 7.3) 4.02 m 4.05 m 3.77 dd (13.3, 6.0) 1.72 d (6.0)
ACCEPTED MANUSCRIPT 4.36 d (11.0)
a
Qui 1 2 3 4 5 6 CH3
4.36 d (11.2)
4.92 d (6.7) 4.01 dd (8.0, 6.7) 3.93 dd (8.0, 3.1) 4.10 m 4.36 d (9.9), 4.27 d (9.9)
4.90 d (6.8) 4.42 dd (8.1, 6.8) 4.14 dd (8.1, 3.4) 4.11 m 4.27 d (9.6), 3.69 d (9.6) 5.04 d (7.8) 4.12 m 3.73 m 4.05 m 3.76 m 1.60 d (5.8)
1.99 s
RI PT
Ara l 2 3 4 5
1.99 s 1
2.06 s
1
Signals were assigned on the basis of H- H COSY, HSQC, HMBC, and NOESY experiments. Spectra were measured at 500 MHz. c Signals are designated as follows: s, singlet; d, doublet; dd, double doublets; t, triplet. d Overlapped signals were reported without designating multiplicity.
AC C
EP
TE D
M AN U
SC
b
ACCEPTED MANUSCRIPT Table 2 13 C NMR data of compounds 1-6 (C5D5N, ppm)a,b 5 32.1 30.2 89.3 45.1 48.0 22.1 26.7 48.6 21.4 25.6 26.5 33.4 45.4 49.0 36.0 28.0 49.1 18.5 29.7 41.7 12.1 72.9 34.9 128.6 133.0 67.3 22.1 19.7 21.2 63.4
6 32.1 30.2 89.2 45.1 48.0 22.0 26.7 48.6 21.3 25.6 26.5 33.4 45.4 49.0 36.0 28.0 49.2 18.6 29.7 41.7 12.1 73.0 35.0 128.5 133.2 67.4 22.1 19.7 21.3 63.4
RI PT
4 32.3 30.0 88.6 41.3 48.1 21.2 26.3 47.7 20.1 26.3 26.7 33.4 45.5 49.1 35.9 28.1 49.2 18.4 29.8 41.7 12.2 73.1 34.9 128.6 133.2 67.4 22.3 19.7 25.9 15.5
SC
3 32.2 30.0 88.5 41.3 48.0 21.1 26.2 47.6 20.0 26.3 26.6 33.4 45.4 49.1 35.8 28.0 49.1 18.3 29.7 41.7 12.1 73.0 35.0 128.6 133.1 67.3 22.2 19.5 25.8 15.4
M AN U
2 32.2 30.0 88.5 41.3 48.0 21.1 26.2 47.6 20.0 26.3 26.6 33.4 45.4 49.1 35.8 28.0 49.1 18.3 29.7 41.7 12.1 73.0 35.0 128.6 133.1 67.3 22.2 19.5 25.8 15.4
TE D
102.9 75.2 78.8 71.7 78.5 62.8
102.9 75.3 78.6 71.4 77.4 64.8
102.9 75.3 78.6 71.4 77.4 64.8
102.8 75.1 78.5 71.4 78.4 62.7
102.7 75.2 78.6 71.7 76.0 64.8
102.9 75.2 78.6 71.4 78.4 62.8
106.2 75.7 78.7 71.4 78.5 62.6
106.6 75.8 78.4 71.6 78.4 70.2
106.5 75.8 78.4 71.6 78.4 70.2
106.5 75.8 78.5 71.6 77.3 69.9
106.9 74.9 78.6 71.6 78.4 62.9
106.6 75.8 78.6 71.7 77.4 69.9
106.3 73.2 75.2 79.0 65.4
106.1 74.9 75.2 78.3 67.1
105.7 74.9 75.3 78.4 67.3
105.9 74.9 78.0 71.1 67.0
106.9 73.5
106.9 73.5
106.9 73.5
106.3 73.3
106.3 73.2
AC C
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Glc I 1 2 3 4 5 6 Glc II 1 2 3 4 5 6 Xyl l 2 3 4 5 Fuc 1 2
1 32.1 30.1 89.6 45.1 48.0 22.1 26.7 48.7 21.4 25.6 26.6 33.4 45.4 49.0 36.1 28.1 49.2 18.7 29.8 41.7 12.1 73.1 35.0 128.6 133.2 67.4 22.2 19.7 21.3 63.4
EP
position
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
3 75.7 75.7 75.7 75.7 75.7 4 83.0 83.0 83.0 83.4 83.1 5 70.4 70.4 70.4 70.6 70.6 6 17.9 17.9 18.0 17.6 17.8 Glc III 1 102.7 102.7 2 75.1 75.1 3 78.6 78.6 4 71.6 71.6 5 78.4 78.4 6 62.8 62.8 Ara l 105.6 105.6 2 72.4 72.3 3 74.3 74.3 77.2 77.2 4 5 66.4 66.5 Qui 1 105.3 2 75.5 3 78.0 4 76.9 5 73.0 6 18.6 CO 170.9 170.9 170.9 CH3 20.8 20.8 20.8 a Signals were assigned on the basis of 1H-1H COSY, HSQC, HMBC, and NOESY experiments. b Spectra were measured at 125 MHz.
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
Figure 1 chemical structures of compounds 1-10
ACCEPTED MANUSCRIPT Six unknown cycloartane triterpenoid sponins, thalisides A-F, along with four known ones were isolated from Thalictrum fortunei. The new structures were elucidated by the spectroscopic analysis (IR, UV,
RI PT
HR-ESI-MS, 1D-NMR and 2D-NMR). All isolates were evaluated for in vitro cytotoxicity against human cancer cells
AC C
EP
TE D
M AN U
SC
(A549, HepG2) and antiviral activity against influenza A virus (H1N1).
S0008-6215(17)30013-7
DOI:
10.1016/j.carres.2017.03.019
Reference:
CAR 7348
To appear in:
Carbohydrate Research
Received Date: 6 January 2017 Revised Date:
17 March 2017
Accepted Date: 17 March 2017
Please cite this article as: S.-Q. Jiang, Y.-B. Zhang, M. Xiao, L. Jiang, D. Luo, Q.-W. Niu, Y.-L. Li, X.-T. Zhang, G.-C. Wang, Cycloartane triterpenoid saponins from the herbs of Thalictrum fortunei, Carbohydrate Research (2017), doi: 10.1016/j.carres.2017.03.019. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
Cycloartane triterpenoid saponins from the herbs of Thalictrum fortunei Si-Qi Jiang,a,b Yu-Bo Zhang,a,b Min Xiao,c,d Lin Jiang,a,b Ding Luo,a,b Qian-Wen
Institute of Traditional Chinese Medicine & Natural Products, College of Pharmacy, Jinan
University, Guangzhou 510632, PR China b
Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs
Guangdong Institute of Traditional Chinese Medicine, Guangzhou 510520, PR China
Guangzhou University of Chinese Medicine, Guangzhou 510006, PR China
TE D
d
M AN U
Research, Jinan University, Guangzhou 510632, PR China c
SC
a
RI PT
Niu,a,b Yao-Lan Li,a,b Xian-Tao Zhang,*c,d Guo-Cai Wang*a,b
ABSTRACT: Six new cycloartane triterpenoid saponins, thalisides A-F (1-6), along
EP
with four known ones (7-10) were isolated from Thalictrum fortunei. The new
AC C
structures were elucidated by using spectroscopic data (NMR, IR, UV, and MS). Compounds 1-10 were determined for their in vitro cytotoxicity against two human cancer cell lines (HepG2, A549) and antiviral activity against influenza A virus (H1N1). However, compound 1-10 were inactive against human cancer cell lines (HepG2, A549) and influenza A virus (H1N1). (IC50>100 µM) Keywords: Thalictrum fortunei; cycloartane triterpenoid saponins; cytotoxicity; antiviral activity *
Corresponding authors: E-Mails: [email protected] (G.-C. Wang); [email protected] (X.-T. Zhang).
ACCEPTED MANUSCRIPT 1. Introduction The genus Thalictrum (Ranunculaceae), commonly called “meadow rue”, is widely distributed in south and southwest part of China.1 The genus Thalictrum
RI PT
(Ranunculaceae) have been discovered approximately 29 species which are used as traditional medicines for a long history to treat gastrosis, dysentery, and tuberculosis etc.2-7 Phytochemical investigations have revealed that triterpenoid saponins and
SC
alkaloids are the main constituents of the genus Thalictrum.8-11 The rhizomes of
M AN U
Thalictrum fortunei are frequently used as the substitutes for Coptis chinensis (an important Chinese medicinal plant) in China.12 These triterpenoid saponins displayed a wide spectrum of biological activities, including antitumor,13 antifungal,14 anti-inflammatory,15-16 and immunostimulating17 activities. In previous studies, we
TE D
have reported some new cycloartane triterpenoid saponins from the aerial part of T. fortunei.18-20 Further investigation on this plant led to the isolation of six new cycloartane triterpenoid sponins, thalisides A-F (1-6), along with four known ones
EP
(7-10). All isolates were evaluated for in vitro cytotoxicity against human cancer cells
AC C
(A549, HepG2) and antiviral activity against influenza A virus (H1N1). In this paper, we report the structure elucidation and biological activities of these compounds.
2. Results and discussion The ethanolic extract from the herbs of T. fortunei was successively partitioned into petroleum ether, EtOAc and n-butanol. The n-butanol fraction was subjected to various chromatographic methods to yield ten cycloartane triterpenoid saponins (1-10,
ACCEPTED MANUSCRIPT Fig.1). The molecular formula of 1 was determined to be C47H78O18 on the basis of its HR-ESI-MS data (m/z 929.5137 [M−H]-). Its IR spectrum indicated the absorption
RI PT
band of hydroxyl group at 3373 cm-1. The 1H NMR spectrum of 1 showed two characteristic proton signals for cyclopropane moiety [δ 0.45 (1H, d, J = 3.8 Hz), 0.28 (1H, d, J = 3.8 Hz)], five methyls at δ 1.98 (3H, s), 1.56 (3H, s), 1.20 (3H, d, J = 6.4
SC
Hz), 1.07 (3H, s), 0.89 (3H, s), and three anomeric protons at 5.26 (1H, d, J = 7.8 Hz), 13
M AN U
4.92 (1H, d, J = 7.8 Hz), 4.88 (1H, d, J = 6.3 Hz,). The
C NMR spectrum of 1
displayed 47 carbon signals including three oxygenated carbons (δC 89.6, 73.1 and 67.4) and three anomeric carbons (δC 106.3, 106.2, 102.9). The 1H and 13C NMR data of
1
(Tables
1
and
2)
were
to
those
of
(22S,24Z)-cycloart-24-en-3β,22,
TE D
3-O-β-D-glucopyranosyl-(1→4)-β-D-fucopyranosyl
similar
26,30-tetraol 26-O-β-D-glucopyranoside,18 except for the absence of the fucose moiety, and the presence of an extra xylose moiety. The types of sugar residues were
EP
confirmed by the acid hydrolysis of 1, which were detected as D-glucose and D-xylose
AC C
by GC analysis. The 3JH1-H2 coupling constants demonstrated β-glycosidic linkages of D-glucose
(J = 7.8 Hz) and D-xylose (J = 6.3 Hz). Moreover, the locations of aglycone
and glycosyl were confirmed by its HMBC correlations from δH 4.88 (H-1 of xylose) to δC 89.6 (C-3 of the aglycone), from δH 5.26 (H-1 of glucose II) to δC 79.0 (C-4 of xylose), and from δH 4.92 (H-1 of glucose I) to δC 67.4 (C-26 of the aglycone). The relative configuration of 1 was determined by the NOESY spectrum, which showed the NOE correlations between H-3 (δH 3.68) and H-29 (δH 1.56), between H-19 (δH
ACCEPTED MANUSCRIPT 0.45) and H-30 (δH 4.54)/H-18 (δH 1.07). Therefore, the structure of 1 was deduced as 3-O-β-D-glucopyranosyl-(1→4)-β-D-xylopyranosyl (22S,24Z)-cycloart-24-en-3β,22,26,30-tetraol 26-O-β-D-glucopyranoside and named
RI PT
as thaliside A. Its HR-ESI-MS data showed the molecular formula as C67H110O30 (m/z 1393.7073 [M−H]-). The IR spectrum of 2 suggested that the presence of carbonyl
resembled
those
of
M AN U
closely
SC
(1647 cm-1) and hydroxyl (3393 cm-1). The 1H and 13C NMR data of 2 (Tables 1 and 2)
3-O-α-L-arabinopyranosyl-(1→6)-β-D-glucopyranosyl-(1→4)-β-D-fucopyranosyl (22S,24Z)-cycloart-24-en-3β,22,26-triol
26-O-(6-O-acetyl)-β-D-glucopyranoside.19
The main differences were the presence of an additional glucopyranosyl and a
D-fucose
TE D
xylopyranosyl in 2. Acid hydrolysis of 2 yielded D-glucose, D-xylose, L-arabinose and in the ratio of 3:1:1:1 by GC analysis. The β anomeric configurations of
glucopyranosyl, fucopyranosyl and xylopyranosyl units were determined on the basis
EP
of their J values. Similarly, the anomeric configuration of arabinopyranosyl unit was
AC C
identified as α. The HMBC correlations from H-1 of fucose (δH 4.71) to C-3 of aglycone (δC 88.5), from H-1 of glucose II (δH 5.13) to C-4 of fucose (δC 83.0), from H-1 of arabinose (δH 4.92) to C-6 of glucose II (δC 70.2), from H-1 of xylose (δH 4.89) to C-4 of arabinose (δC 77.2), and from H-1 of glucose III (δH 4.87) to C-4 of xylose (δC 78.3) confirmed the locations of glycosyls. Thus, the structure of 2 was identified as 3-O-β-D-glucopyranosyl-(1→4)-β-D-xylopyranosyl-(1→4)-α-L-arabinopyranosyl(1→6)-β-D-glucopyranosyl-(1→4)-β-D-fucopyranosyl
(22S,24Z)-cycloart-24-en-
ACCEPTED MANUSCRIPT 3β,22,26-triol 26-O-(6-O-acetyl-β-D-glucopyranoside) and named as thaliside B. Compound 3 showed a [M+Cl]- ion at m/z 1299.6093 (C61H100O27Cl) in HR-ESI-MS. Comparison of the 1D NMR spectra of 3 with those of 2 (Tables 1 and 2)
RI PT
suggested that they were similar, except that the absence of an arabinopyranosyl in 3. The NMR data of sugar moiety indicated pentasaccharides residues, which were verified as D-glucose, D-xylose, D-fucose by GC analysis. The five anomeric proton
SC
signals at δH 4.67 (J = 7.4 Hz), 5.13 (J = 7.8 Hz), 4.83 (J = 7.8 Hz), 4.87 (J = 7.8 Hz),
M AN U
5.09 (J = 6.3 Hz) indicated the β-configurations of glycosyls. The glycosylating positions were determined by the HMBC spectrum, wherein the correlations between H-1 (δH 4.67) of Fuc and C-3 (δC 88.5) of aglycone, between H-1 (δH 5.13) of Glc II and C-4 (δC 83.0) of Fuc, between H-1 (δH 5.09) of Xyl and C-6 (δC 70.2) of Glc II,
TE D
between H-1 (δH 4.87) of Glc III and C-4 (δC 78.4 ) of Xyl were observed. Hence, the structure of 3 was established as 3-O-β-D-glucopyranosyl-(1→4)-β-D-xylopyranosyl(1→6)-β-D-glucopyranosyl-(1→4)-β-D-fucopyranosyl
(22S,24Z)-cycloart-24-en-3β,
EP
22,26-triol 26-O-(6-O-acetyl-β-D-glucopyranoside) and named as thaliside C.
AC C
The HR-ESI-MS data of 4 suggested a molecular formula C58H96O25 (m/z 1191.6241 [M−H]-). The NMR data of 4 (Tables 1 and 2) were in good agreement with those of 3-O-β-D-glucopyranosyl-(1→4)-β-D-fucopyranosyl (22S,24Z)-cycloart24-en-3β,22,26-triol 26-O-β-D-glucopyranoside20 except for the existence of an extra xylopyranosyl and an arabinopyranosyl moieties in 4. Acid hydrolysis of 4 obtained sugar residues, which were identified as D-fucose
D-xylose, L-arabinose, D-glucose
and
by GC analysis. The HMBC correlations from H-1 of Glc II (δH 5.10) to C-4
ACCEPTED MANUSCRIPT of Fuc (δC 83.0), from H-1 of Ara (δH 4.90) to C-6 of Glc II (δC 69.9), and from H-1 of Xyl (δH 4.95) to C-4 of Ara (δC 77.2) verified the locations of the glycosyls. The values of coupling constants indicated α-anomeric configuration of arabinopyranose,
the
structure
of
4
was
assigned
as
RI PT
and β-anomeric configurations of xylopyranose, fucopyranose, glucopyranose. Thus, 3-O-β-D-xylopyranosyl-(1→4)-α-L-
arabinopyranosyl-(1→6)-β-D-glucopyranosyl-(1→4)-β-D-fucopyranosyl
(22S,24Z)-
SC
cycloart-24-en-3β,22,26-triol 26-O-β-D-glucopyranoside and named as thaliside D.
M AN U
Compound 5 had the molecular formula of C50H82O19 which exhibited a quasi-molecular ion peak at m/z 1021.5155 [M+Cl]-. The 1D NMR data of 5 (Tables 1 and 2) were almost identical to those of 3-O-β-D-glucopyranosyl-(1→4)-β-Dfucopyranosyl
(22S,24Z)-cycloart-24-en-3β,22,26,30-tetraol
26-O-β-D-
TE D
glucopyranoside.18 The main differences were that the presence of additional signals at δC 170.9 and 20.8, which indicated the existence of acetyl group. Correlations between δH 4.86 (H-1 of Fuc) and δC 89.3 (C-3 of aglycone), as well as δH 5.22 (H-1
EP
of Glc II) and δC 83.4 (C-4 of Fuc) were observed in HMBC spectrum, which
AC C
indicated that the sugar chain was 3-O-β-D-glucopyranosyl-(1→4)-β-D-fucopyranosyl. Thus, the structure of 5 was deduced to be 3-O-β-D-glucopyranosyl-(1→4)-β-Dfucopyranosyl (22S,24Z)-cycloart-24-en-3β,22,26,30-tetraol 26-O-(6-O-acetyl-β-Dglucopyranoside) and named as thaliside E. The molecular formula of 6 was established as C54H90O22 by HR-ESI-MS at m/z 1125.5639 [M+Cl]-. The 1H and
13
C NMR data of 6 (Tables 1 and 2) was similar to
those of 3-O-β-D-glucopyranosyl-(1→4)-β-D-fucopyranosyl (22S,24Z)-cycloart-24-
ACCEPTED MANUSCRIPT en-3β,22,26-triol 26-O-β-D-quinovopyranosyl-(1→6)-β-D-glucopyranoside20, except for oxygenation of C-30 at δ 63.4. Analysis of the sugar residues indicated that 6 had the
same
glycosyls
as
3-O-β-D-glucopyranosyl-(1→4)-β-D-fucopyranosyl 26-O-β-D-quinovopyranosyl-(1→6)-β-D-
RI PT
(22S,24Z)-cycloart-24-en-3β,22,26-triol
glucopyranoside. The HMBC correlations between H-1 (δH 5.04) of quinovose and C-6 (δC 69.9) of glucose II, between H-1 (δH 5.16) of glucose II and C-4 (δC 83.1) of
of
glycosyls.
Hence,
the
structure
of
6
was
elucidated
as
M AN U
linkages
SC
fucose, between H-1 (δH 4.83) of fucose and C-3 (δC 89.2) of aglycone suggested the
3-O-β-D-quinovopyranosyl-(1→6)-β-D-glucopyranosyl-(1→4)-β-D-fucopyranosyl (22S,24Z)-cycloart-24-en-3β,22,26,30-tetraol 26-O-β-D-glucopyranoside and named as thaliside F.
TE D
The four known triterpenoid saponins were identified as 3-O-β-D-glucopyranosyl (1→4)-β-D-fucopyranosyl (22S,24Z)-cycloart-24-en-3β,22,26,30-tetraol 26-O-β-Dglucopyranoside (7),18 3-O-β-D-glucopyranosyl-(1→4)-β-D-fucopyranosyl (22S,24Z)-
EP
cycloart-24-en-3β,22,26-triol
26-O-β-D-quinovopyranosyl-(1→6)-β-D-
AC C
glucopyranoside (8),20 3-O-β-D-glucopyranosyl(1→4)-β-D-fucopyranosyl (22S,24Z)cycloart-24-en-3β,22,26-triol
26-O-β-D-glucopyranoside
(9),18
3-O-β-D-
glucopyranosyl (24S)-cycloartane-3β,16β,24,25,30-pentaol 25-O-β-D-glucopyranosyl(1→6)-β-D-glucopyranoside (10)18 by comparing their spectroscopic data with the related literatures. In previous researches, the triterpenoid saponins isolated from the genus Thalictrum exhibited antitumor21 and antiviral22 activities. Therefore, all isolated
ACCEPTED MANUSCRIPT triterpenoid saponins were evaluated for the cytotoxicity against lung cancer cell (A549) and liver cancer cell (HepG2) by MTT assay15 and antiviral activities against H1N1 by cytopathic effect (CPE) reduction assay.23 However, the data showed that
concentration of 100 µM.
3. Experimental
M AN U
3.1. General experimental procedures
SC
RI PT
these compounds had no significant cytotoxic effects and antiviral activities even at a
Optical rotations were recorded on JASCO P-1020 polarimeter. UV and IR spectra were measured by JASCO V-550 UV/vis spectrophotometer and a JASCO FT/IR-480 plus infrared spectrometer with KBr pellets, respectively. The 1D and 2D
TE D
NMR spectra were performed on Bruker Avance 500 using pyridine-d5 as solvent. HR-ESI-MS data were recorded on a Micromass Q-TOF mass spectrometer (Agilent 6210). Silica gel (200-300 mesh, Qingdao Marine Chemical Plant, China), ODS silica
EP
gel (50 µm, YMC, Japan) and Sephadex LH-20 (Pharmacia Biotech, Uppsala, Sweden)
AC C
were used for chromatography column. TLC was performed on Silica gel GF254 plates (Yantai, China). Analytical HPLC was carried on a Waters system equipped with P680 pump, a photodiode array detector and a Cosmosil C18 analytical column (5 µm, 4.6 mm × 250 mm). Preparative HPLC was carried on Agilent system with a Cosmosil C18 preparative column (5 µm, 20 mm × 250 mm). GC was measured on a Shimadzu GC-2010 gas chromatograph. The spectroscopic and chromatographic grades of organic reagents were purchased from Tianjin Damao Chemical Company (Tianjin,
ACCEPTED MANUSCRIPT PR China). 3.2. Plant Material The dried herbs of Thalitrum fortunei were collected in June of 2013 from Wuhu
RI PT
City, Anhui Province of China. The species were authenticated by Prof. Guang-Xiong Zhou of Jinan University. A voucher specimen (No. 20130326) was deposited in the Institute of Traditional Chinese Medicine, Jinan University, Guangzhou, China.
SC
3.3. Extraction and isolation
M AN U
The air-dried and powdered herbs of T. fortunei (2.8 kg) were extracted with EtOH/H2O (95:5, v/v) at room temperature to afford a crude extract (0.4 kg). The extract was suspended in H2O, and then successively extracted with petroleum ether, EtOAc and n-butanol. The n-butanol part (97.0 g) was subjected to silica gel column
TE D
chromatography (CC) and eluted with CHCl3-CH3OH (100:1 to 1:1, v:v) to give 9 fractions (Fr. 1-9). Fr. 7 (45.6 g) was separated by silica gel CC (CHCl3-CH3OH, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1 and 0:1, v/v, each 15 L) to afford 6 subfractions (Fr. 7.1-7.6).
EP
The Fr. 7.3 (6.5 g) was then fractionated into five subfractions by Sephadex LH-20
AC C
CC (CHCl3-MeOH, 1:1, v/v). Subfractions 7.3.2 (200.5 mg) was purified by preparative HPLC (CH3CN-H2O, 30:70, v/v, flow rate: 7.0 mL/min) to yield 1 (20.3 mg) and 2 (12.5 mg). Fr. 4 was separated by an ODS column which was eluted with gradient mixtures (MeOH-H2O, 50:50 to 100:0, v:v) to produce Fr. 4.1-4.9. Fr. 4.5 (60.2 mg) was purified by preparative HPLC (CH3CN-H2O, 40:60, v/v, flow rate: 7.0 mL/min) to give 4 (15.4mg), 5 (8.2 mg) and 8 (16.0 mg). Fr. 4.5 (8.8 g) was subjected to Sephadex LH-20 (CHCl3-MeOH, 1:1, v/v) to afford 7 (60.4 mg). Fr. 4.2 was
ACCEPTED MANUSCRIPT chromatographied by Sephadex LH-20 (MeOH) and further purified by preparative HPLC (MeOH-H2O, 70:40, v/v, flow rate: 7.0 mL/min) to yield 3 (27.3 mg), 6 (13.7 mg), 9 (16.5 mg) and 10 (37.8 mg).
RI PT
3.3.1. Thaliside A (1) 25
White powder; [α] D +5.60 (c 0.5, MeOH); UV (MeOH) λmax 204 nm; IR (KBr) νmax 3373, 2934, 1635, 1375, 1167, 1077, 893 cm−1; HR-ESI-MS m/z 929.5137
SC
[M−H]- (calcd for C47H77O18, 929.5115); 1H and 13C NMR data see Tables 1 and 2.
25
M AN U
3.3.2. Thaliside B (2)
White powder; [α] D -5.87 (c 0.15, MeOH); UV (MeOH) λmax 204 nm; IR (KBr) νmax 3403, 2936, 2869, 1647, 1384, 1043, 890 cm−1; HR-ESI-MS m/z 1393.7073 [M−H]- (calcd for C67H109O30 , 1393.7009); 1H and 13C NMR data see Tables 1 and 2.
TE D
3.3.3. Thaliside C (3) 25
White powder; [α] D -6.40 (c 0.2, MeOH); UV (MeOH) λmax 204; IR (KBr) νmax 3390, 2937, 1652, 1378, 1071 cm−1; HR-ESI-MS m/z 1299.6093 [M+Cl]- (calcd for
EP
C61H100ClO27, 1299.6146); 1H and 13C NMR data see Tables 1 and 2.
AC C
3.3.4. Thaliside D (4)
25
White powder; [α] D +1.96 (c 0.14, MeOH); UV (MeOH) λmax 204 nm; IR (KBr)
νmax 3389, 2936, 1648, 1379, 1071, 891 cm−1; HR-ESI-MS m/z 1191.6241 [M−H](calcd for C58H95O25, 1191.6235); 1H and 13C NMR data see Tables 1 and 2. 3.3.5. Thaliside E (5) 25
White powder; [α] D +18.6 (c 0.69, MeOH); UV (MeOH) λmax 204 nm; IR (KBr) νmax 3420, 2936, 1648, 1377, 1256, 1071, 889 cm−1; HR-ESI-MS m/z 1021.5155
ACCEPTED MANUSCRIPT [M+Cl]- (calcd for C50H82ClO19, 1021.5144 ); 1H and 13C NMR data see Tables 1 and 2. 3.3.6. Thaliside F (6) 25
RI PT
White powder; [α] D +5.75 (c 0.25, MeOH); UV (MeOH) λmax 204 nm; IR (KBr) νmax 3394, 2935, 1649, 1456, 1379, 1068, 889 cm−1; HR-ESI-MS m/z 1125.5639 [M+Cl]- (calcd for C54H90ClO22, 1125.5618 ); 1H and 13C NMR data see Tables 1 and
SC
2.
M AN U
3.4. Acid hydrolysis and GC analysis of compounds 1-6
Each isolate was dissolved in 4.0 M HCl (10 mL) and refluxed at 90 °C for 6 h. After the solvent was dried, the mixture was partitioned with CHCl3-H2O (1:1, v:v). The aqueous layer was neutralized with Dowex (HCO3-) and then filtered. After
TE D
concentrated, the filtrate stirred with NaBH4 at 25 °C for 3 h. The reaction mixture was washed by 0.1% HCl (in MeOH) repeatedly and then heated at 105 °C for 30min. The dried residue was dissolved in dry pyridine (0.5 mL), followed by addition of
EP
Ac2O (0.5 mL). The mixture was reacted in a water bath at 100 °C for 1 h. The dried
AC C
product was extracted with CHCl3 (5 mL, 3 times) and then analyzed by GC-MS: column AT-SE-30 (0.32 mm i.d., 30 m, 0.5 µm film thickness), carrier gas N2(1 mL/min), column temperature 230 °C, injector temperature 250 °C and detector temperature 250 °C. The retention times of standard sugars were detected at 33.08 min (D-glucose), 34.46 min (L-glucose), 28.78 min (D-xylose), 30.70 min (L-xylose), 29.83 min (D-arabinose), 28.30 min (L-arabinose), 26.91 min (D-quinovose), 28.01 min (L-quinovose), 27.83 min (D-fucose) and 29.36 min (L-fucose).
ACCEPTED MANUSCRIPT Acknowledgments This work was supported by grants from the National Natural Science Foundation
RI PT
of China (Nos. 81273407, 81473116, 81273390, 81673319).
Supplementary data
Supplementary data associated with this article can be found, in the online version,
M AN U
SC
at http://dx.doi.org/
References
1. Meng FC, Yuan C, Huang XJ, Wang WJ, Lin LG, Zhang XT, et al. Phytochem Lett 2016;15:108-12.
2. Kuzmanov B, Dutschewska H. J Nat Prod 1982;45:766-71.
TE D
3. Mushtaq S, Rather MA, Qazi PH, Aga MA, Shah AM, Shah A, et al. J Ethnopharmacol 2016;193:221-6.
4. Li DH, Guo J, Bin W, Zhao N, Wang KB, Li JY, et al. Arch. Pharm Res.
EP
2016;39:871-7.
AC C
5. Mayeku WP, Josiah O, Hasanali A, Kiremire, B, Hertweck C. J Chem Pharm Res 2014;6:1-5.
6. Lotfipour F, Nazemiyeh H, Fathi-Azad F, Garaei N, Arami S, Talat S, et al. Iran J Basic Med Sci 2008;11:80-5.
7. Chen SB, Chen SL, Xiao PG. J Asian Nat Prod Res 2003;5:263-71. 8. Khamidullina EA, Gromova AS, Lutsky VI, Owen NL. Nat Prod Rep 2006;23:117-29. 9. Lutskii VI, Gromova AS, Khamidullina EA, Owen NL. Chem Nat Compd
ACCEPTED MANUSCRIPT 2005;41:117-40. 10. Lin LZ, Hu SF, Zaw K, Angerhofer CK, Chai H, Pezzuto JM, et al. J Nat Prod 1994;57:1430-6. 11. Sun H, Zhang XT, Wang L, Zhang XT, Wang Y, Chen SB, et al. Helv Chim Acta
RI PT
2008;91:1961-6.
12. Ma ZQ, Xin SM, Shen XC. Chin Tradit Herbal Drugs 1980;11:217-9. 13. Balandrin MF. Adv Exp Med Biol 1996;404:1-14.
SC
14. Gromova AS, Lutsky VI, Li D, Wood SG, Owen NL, Semenov AA, et al. J Nat Prod 2000;63:911-4.
M AN U
15. Zhang X, Zhao M, Chen L, Jiao H, Liu H, Wang L, et al. Molecules 2011;16:9505-19.
16. Ganenko TV, Isaev MI, Gromova AS, Abdullaev ND, Lutskii VI, Larin MF, et al. Chem Nat Compd 1986;22:288-94.
TE D
17. Varadinova TL, Shishkov SA, Ivanovska ND, Velcheva MP, Danghaaghi S, Samadanghiin Z, et al. Phytother Res 1996;10:414-7. 18. Zhang XT, Ma SW, Jiao HY, Zhang QW. J Asian Nat Prod Res 2012;14:327-32.
EP
19. Zhang XT, Wang L, Ma SW, Zhang QW, Liu X, Zhang LH, et al. J Nat Med
AC C
2013;67:375-80.
20. Zhang XT, Zhang LH, Ye WC, Zhang XL, Yin ZQ, Zhao SX, et al. Chem Pharm Bull 2006;54:107-10.
21. Devkota KP, Khan MTH, Ranjit R, Lannang AM. Nat Prod Res 2007;21:321-7. 22. Song GP, Shen XT, Li SM, Li YB, Si HZ, Fan JH, et al. Eur. J. Med. Chem. 2016;119:109-21. 23. Lin TJ, Lin CF, Chiu CH, Lee MC, Horng JT. Sci. Rep. 2016;6:1-10
ACCEPTED MANUSCRIPT 5 1.23 m, 1.53 m 2.16 m, 2.40 m 3.75 dd (11.5, 4.5) 1.44 0.97 m, 1.69 m 0.98 m, 1.04 m 1.34 1.28 m, 1.96 m 1.65 m, 1.65 m 1.29 m, 1.31 m 1.48 m, 2.17 m 2.39 m 1.08 s 0.26 d (3.7), 0.45 d (3.7) 1.66 m 1.22 d (6.3) 4.08 m 2.49 m, 2.79 m 5.85 t (7.3) 4.54 d (11.0), 4.78 d (11.0) 1.98 s 0.89 s 1.63 s 3.85 d (11.4) 4.56 d (11.4)
6 1.21 m, 1.52 m 2.16 m, 2.37 m 3.72 dd (11.6, 4.3) 1.46 0.93 m, 1.70 m 1.04 m, 0.98 m 1.34 1.96 m, 1.28 m 1.66 m, 1.67 m 1.32 m, 1.28 m 2.17 m, 1.50 m 2.39 m 1.07 s 0.48, d (3.4), 0.27, d (3.4) 1.61 m 1.20 d (6.7) 4.06 m 2.44 m, 2.77 m 5.81 t (6.4) 4.78 d (11.6), 4.54 d (11.6) 1.97 s 0.90 s 1.60 s 3.80 d (11.5) 4.56 d (11.5)
4.85 d (7.6) 4.04 t (7.6) 4.23 m 4.22 m 3.91 m 4.41 dd (11.4, 5.2), 4.52 d (11.4)
5.22 d (7.5) 4.04 t (7.5) 4.22 m 4.24 m 4.04 m 4.53 dd (11.6, 5.0), 4.38 d (11.6)
4.92 d (7.5) 4.08 t (7.5) 4.26 m 4.27 m 3.97 m 4.54 dd (11.5, 5.0), 4.46 d (11.5)
5.10 d (7.8) 3.91 t (7.8) 3.95 m 4.13 m 3.97 m 4.74 dd (11.5, 5.5), 4.24 d (11.5)
5.22 d (7.5) 3.94 t (7.5) 4.22 m 4.24 m 3.94 m 4.53 dd (11.0, 5.0), 4.38 d (11.0)
5.16 d (7.8) 3.97 t (7.8) 4.12 m 4.14 m 4.03 m 4.81 dd (11.2, 5.4), 4.36 d (11.2)
4.86 d (7.0) 4.35 dd (8.4, 7.0) 4.13 m 4.14 m 3.83 dd (13.2, 6.3) 1.65 d (6.3)
4.83 d (7.4) 4.31 dd (8.4, 7.2) 4.05 m 4.10 m 3.81 dd (13.5, 6.5) 1.76 d (6.5)
SC
RI PT
4 1.21 m, 1.51 m 1.90 m, 2.36 m 3.46 dd (11.4, 4.6) 1.49 0.75 m, 1.54 m 1.05 m, 1.30 m 1.32 1.09 m, 1.94 m 1.65 m, 1.65 m 1.32 m, 1.28 m 1.51 m, 2.16 m 2.34 m 1.05 s 0.48 d (3.8), 0.24 d (3.8) 1.68 m 1.18 d (6.0) 4.05 m 2.41 m, 2.74 m 5.78 t (7.4) 4.51 d (11.7), 4.72 d (11.7) 1.93 s 0.89 s 1.32 s 1.05 s
AC C
EP
TE D
M AN U
Table 1 1 H NMR data of compounds 1-6 (C5D5N, ppm, J in Hz)a,b,c,d position 1 2 3 1 1.21 m, 1.52 m 1.17 m, 1.48 m 1.17 m, 1.49 m 2 2.14 m, 2.37 m 1.88 m, 2.34 m 1.89 m, 2.34 m 3 3.68 dd (11.5, 4.5) 3.42 dd (11.2, 4.0) 3.42 dd (11.6, 4.5) 5 1.43 1.45 1.45 6 0.95 m, 1.69 m 0.71 m, 1.53 m 0.72 m, 1.51 m 7 0.97 m, 1.02 m 1.03 m, 1.25 m 1.03 m, 1.25 m 8 1.33 1.30 1.30 11 1.25 m, 1.96 m 1.03 m, 1.89 m 1.03 m, 1.89 m 12 1.69 m, 1.69 m 1.64 m, 1.64 m 1.64 m, 1.64 m 15 1.30 m, 1.31 m 1.27 m, 1.27 m 1.27 m, 1.27 m 16 1.52 m, 2.17 m 1.45 m, 2.11 m 1.45 m, 2.11 m 17 2.37 m 2.33 m 2.32 m 18 1.07 s 1.02 s 1.02 s 19 0.28 d (3.8), 0.45 d 0.44 d (3.4), 0.20 d 0.45 d (3.5), 0.21 d (3.8) (3.4) (3.5) 20 1.68 m 1.62 m 1.62 m 21 1.20 d (6.4) 1.16 d (6.2) 1.16 d (6.7) 22 4.08 m 4.02 m 4.03 m 23 2.45 m, 2.78 m 2.42 m, 2.73 m 2.42 m, 2.72 m 24 5.83 t (7.2) 5.79 t (7.0) 5.78 t (7.0) 26 4.54 d (11.7), 4.76 4.48 d (11.2), 4.73 d 4.49 d (11.5), 4.72 d d (11.7) (11.2) (11.5) 27 1.98 s 1.92 s 1.92 s 28 0.89 s 0.85 s 0.85 s 29 1.56 s 1.29 s 1.29 s 30 3.83 d (11.0) 1.00 s 1.00 s 4.54 d (11.0) Glc I 1 4.92 d (7.8) 4.90 d (7.6) 4.83 d (7.8) 2 4.08 t (7.8) 4.00 t (7.6) 4.00 t (7.8) 3 4.26 m 4.20 m 4.20 m 4 4.28 m 4.21 m 4.21 m 5 3.96 m 3.97 m 3.97 m 6 4.41 dd (11.0, 5.1), 4.93 dd (11.5, 5.2), 4.93 dd (11.0, 5.1), 4.53 d (11.0) 4.74 d (11.5) 4.74 d (11.0) Glc II 1 5.26 d (7.8) 5.13 d (7.8) 5.13 d (7.6) 2 4.08 t (7.8) 3.91 t (7.8) 3.91 t (7.6) 3 4.25 m 3.93 m 3.93 m 4 4.24 m 4.10 m 4.10 m 5 3.96 m 3.90 m 3.90 m 6 4.41 dd (11.5, 5.0), 4.75 dd (11.0, 5.0), 4.75 dd (11.2, 5.4), 4.54 d (11.5) 4.29 d (11.0) 4.29 d (11.2) Xyl l 4.88 d (6.3) 4.89 d (6.2) 5.09 d (6.0) 2 4.41 dd (6.3) 4.02 dd (6.2) 4.01 dd (6.0) 3 4.28 m 3.94 m 3.93 m 4 4.45 m 4.17 m 4.09 m 5 3.81 dd (11.0, 9.5), 4.30 dd (11.2, 9.5), 4.36 dd (10.7, 9.5), 4.46 d (11.0) 3.64 d (11.2) 4.27 d (10.7) Fuc 1 4.71 d (7.4) 4.67 d (7.2) 2 4.29 dd (8.0, 7.4) 4.29 dd (8.2, 7.2) 3 4.00 m 4.00 m 4 4.04 m 4.04 m 5 3.77 dd (13.0, 6.5) 3.77 dd (13.2, 6.5) 6 1.70 d (6.5) 1.70 d (6.4) Glc III 1 4.87 d (7.8) 4.87 d (7.4) 2 4.00 t (7.8) 4.00 t (7.4) 3 4.10 m 4.10 m 4 4.23 m 4.23 m 5 4.04 m 4.04 m 6 4.47 dd (11.0, 5.0), 4.47 dd (11.2, 5.5),
4.95 d (6.3) 4.02 dd (6.3) 3.92 m 4.14 m 4.28 dd (11.0, 9.5), 3.62 d (11.0) 4.70 d (7.3) 4.29 dd (8.0, 7.3) 4.02 m 4.05 m 3.77 dd (13.3, 6.0) 1.72 d (6.0)
ACCEPTED MANUSCRIPT 4.36 d (11.0)
a
Qui 1 2 3 4 5 6 CH3
4.36 d (11.2)
4.92 d (6.7) 4.01 dd (8.0, 6.7) 3.93 dd (8.0, 3.1) 4.10 m 4.36 d (9.9), 4.27 d (9.9)
4.90 d (6.8) 4.42 dd (8.1, 6.8) 4.14 dd (8.1, 3.4) 4.11 m 4.27 d (9.6), 3.69 d (9.6) 5.04 d (7.8) 4.12 m 3.73 m 4.05 m 3.76 m 1.60 d (5.8)
1.99 s
RI PT
Ara l 2 3 4 5
1.99 s 1
2.06 s
1
Signals were assigned on the basis of H- H COSY, HSQC, HMBC, and NOESY experiments. Spectra were measured at 500 MHz. c Signals are designated as follows: s, singlet; d, doublet; dd, double doublets; t, triplet. d Overlapped signals were reported without designating multiplicity.
AC C
EP
TE D
M AN U
SC
b
ACCEPTED MANUSCRIPT Table 2 13 C NMR data of compounds 1-6 (C5D5N, ppm)a,b 5 32.1 30.2 89.3 45.1 48.0 22.1 26.7 48.6 21.4 25.6 26.5 33.4 45.4 49.0 36.0 28.0 49.1 18.5 29.7 41.7 12.1 72.9 34.9 128.6 133.0 67.3 22.1 19.7 21.2 63.4
6 32.1 30.2 89.2 45.1 48.0 22.0 26.7 48.6 21.3 25.6 26.5 33.4 45.4 49.0 36.0 28.0 49.2 18.6 29.7 41.7 12.1 73.0 35.0 128.5 133.2 67.4 22.1 19.7 21.3 63.4
RI PT
4 32.3 30.0 88.6 41.3 48.1 21.2 26.3 47.7 20.1 26.3 26.7 33.4 45.5 49.1 35.9 28.1 49.2 18.4 29.8 41.7 12.2 73.1 34.9 128.6 133.2 67.4 22.3 19.7 25.9 15.5
SC
3 32.2 30.0 88.5 41.3 48.0 21.1 26.2 47.6 20.0 26.3 26.6 33.4 45.4 49.1 35.8 28.0 49.1 18.3 29.7 41.7 12.1 73.0 35.0 128.6 133.1 67.3 22.2 19.5 25.8 15.4
M AN U
2 32.2 30.0 88.5 41.3 48.0 21.1 26.2 47.6 20.0 26.3 26.6 33.4 45.4 49.1 35.8 28.0 49.1 18.3 29.7 41.7 12.1 73.0 35.0 128.6 133.1 67.3 22.2 19.5 25.8 15.4
TE D
102.9 75.2 78.8 71.7 78.5 62.8
102.9 75.3 78.6 71.4 77.4 64.8
102.9 75.3 78.6 71.4 77.4 64.8
102.8 75.1 78.5 71.4 78.4 62.7
102.7 75.2 78.6 71.7 76.0 64.8
102.9 75.2 78.6 71.4 78.4 62.8
106.2 75.7 78.7 71.4 78.5 62.6
106.6 75.8 78.4 71.6 78.4 70.2
106.5 75.8 78.4 71.6 78.4 70.2
106.5 75.8 78.5 71.6 77.3 69.9
106.9 74.9 78.6 71.6 78.4 62.9
106.6 75.8 78.6 71.7 77.4 69.9
106.3 73.2 75.2 79.0 65.4
106.1 74.9 75.2 78.3 67.1
105.7 74.9 75.3 78.4 67.3
105.9 74.9 78.0 71.1 67.0
106.9 73.5
106.9 73.5
106.9 73.5
106.3 73.3
106.3 73.2
AC C
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Glc I 1 2 3 4 5 6 Glc II 1 2 3 4 5 6 Xyl l 2 3 4 5 Fuc 1 2
1 32.1 30.1 89.6 45.1 48.0 22.1 26.7 48.7 21.4 25.6 26.6 33.4 45.4 49.0 36.1 28.1 49.2 18.7 29.8 41.7 12.1 73.1 35.0 128.6 133.2 67.4 22.2 19.7 21.3 63.4
EP
position
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
3 75.7 75.7 75.7 75.7 75.7 4 83.0 83.0 83.0 83.4 83.1 5 70.4 70.4 70.4 70.6 70.6 6 17.9 17.9 18.0 17.6 17.8 Glc III 1 102.7 102.7 2 75.1 75.1 3 78.6 78.6 4 71.6 71.6 5 78.4 78.4 6 62.8 62.8 Ara l 105.6 105.6 2 72.4 72.3 3 74.3 74.3 77.2 77.2 4 5 66.4 66.5 Qui 1 105.3 2 75.5 3 78.0 4 76.9 5 73.0 6 18.6 CO 170.9 170.9 170.9 CH3 20.8 20.8 20.8 a Signals were assigned on the basis of 1H-1H COSY, HSQC, HMBC, and NOESY experiments. b Spectra were measured at 125 MHz.
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
Figure 1 chemical structures of compounds 1-10
ACCEPTED MANUSCRIPT Six unknown cycloartane triterpenoid sponins, thalisides A-F, along with four known ones were isolated from Thalictrum fortunei. The new structures were elucidated by the spectroscopic analysis (IR, UV,
RI PT
HR-ESI-MS, 1D-NMR and 2D-NMR). All isolates were evaluated for in vitro cytotoxicity against human cancer cells
AC C
EP
TE D
M AN U
SC
(A549, HepG2) and antiviral activity against influenza A virus (H1N1).