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Three triterpenoid saponins from the roots of Polygala japonica Houtt.
Fitoterapia 83 (2012) 1184–1190
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Three triterpenoid saponins from the roots of Polygala japonica Houtt. Chuangjun Li, Jing Fu, Jingzhi Yang, Dongming Zhang ⁎, Yuhe Yuan, Naihong Chen State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, PR China
a r t i c l e
i n f o
Article history: Received 18 April 2012 Accepted in revised form 4 July 2012 Available online 14 July 2012 Keywords: Polygala japonica Triterpenoid saponins Polygalasaponins Neuroprotective effect
a b s t r a c t Three new triterpenoid saponins polygalasaponins LI–LIII (1–3) with two acylation groups in oligosaccharide chain, together with three known saponins were isolated from the roots of Polygala japonica Houtt. (4–6). The neuroprotective effects of these compounds on neuron-like PC12 cells were evaluated in vitro. Compounds 5 and 6 show neuroprotective effects in Aβ25–35 model at the concentration of 10 μM. © 2012 Elsevier B.V. All rights reserved.
1. Introduction
2. Experimental part
The roots of Polygala japonica Houtt. have been used in Chinese folk herbal medicine as expectorant, anti-inflammatory, antibacterial, ataractic, and antidepressant agents [1]. This plant is a rich source of triterpenoid saponins, from which more than fifty triterpenoid saponins have been isolated [2–6,10]. Some of these compounds showed antidepressant activity [6] and neurotrophic activity on PC12 cells and cultured cortical neurons [7]. Our previous investigation on the roots of P. japonica resulted in the isolation of an oligosaccharide polyester, four triterpenoid saponins and six xanthones [8–10]. As part of our ongoing effort to study the chemical and biological diversity of this plant, we report herein the isolation and characterization of three new triterpenoid saponins polygalasaponins LI–LIII (1–3), which have two acylation groups in oligosaccharides chain, together with three known saponins (4–6). The neuroprotective effects of these compounds on neuron-like PC12 cells were evaluated in vitro, as a result, compounds 5 and 6 showed neuroprotective effects in Aβ25–35 model at the concentration of 10 μM.
2.1. General experimental procedures Optical rotations were determined on a Perkin-Elmer 241 digital polrimeter. IR spectra were recorded on an IMPACT 400 spectrometer. UV spectra were obtained on a Shimadzu UV-260 spectrophotometer. NMR spectra were run on Varian INOVA-500 spectrometer using TMS as internal standard. ESIMS and HRESIMS were measured on Agilent 1100 series LC/MSD Trap SL mass spectrometer and Autospee-Ultima ETOF, respectively. Preparative HPLC was performed on an YMC-Pack ODS-A (YMC Co., Ltd.) column. Macroporous resin D101 (26–60 mesh, Tianjin Haiguang Chemistry Company, Tianjin, China) and silica gel (100–200, 200–300 mesh, produced by Qingdao Marine Chemical company; Qingdao, China) were used for column chromatography and silica gel GF-254 (produced by Qingdao Marine Chemical company; Qingdao, China) for TLC analysis. 2.2. Plant material
⁎ Corresponding author at: Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, 1 Xian Nong Tan Street, Xicheng District, Beijing, 100050, PR China. Tel./fax: +86 10 63165227. E-mail addresses: [email protected] (C. Li), [email protected] (D. Zhang). 0367-326X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2012.07.002
The roots of P. japonica were collected in June 2003 in Jiangxi Province, China, it was identified by Professor Yongming Luo, Jiangxi College of Traditional Chinese Medicine. A voucher specimen (272400) is deposited in the Institute of Materia
C. Li et al. / Fitoterapia 83 (2012) 1184–1190 Table 1 (continued)
Table 1 1 H NMR data of compounds 1–3 in pydridine-d5 (500 MHz). position Aglycone 2 3 12 18 24 25 26 27 29 30
1 4.68 4.62 5.86 3.25 1.95 1.60 1.15 3.80 4.03 0.79 1.05
2 m d (3.0) brs brd (14.0) s s s brd (10.5) brd (10.5) s s
4.69 4.63 5.85 3.24 1.98 1.54 1.13 3.82 4.05 0.78 0.95
3 m d (3.0) brs brd (11.5) s s s brd (12.5) brd (12.5) s s
4.67 4.60 5.88 3.25 1.95 1.59 1.14 3.83 4.03 0.78 1.04
m d (3.5) brs brd s s s brd (12.5) brd (12.5) s s
position
1
2
Cinn-2 (Fuc-4) 3 5 6 7 8 OMe Benzyl-2 (Rham-4) 3 4 5 6 Acetyl
7.55 d (8.5) 7.05 d (8.5) 7.05 d (8.5) 7.55 d (8.5) 8.09 d (16.0) 6.73 d (16.0) 3.70 s (3H) 8.13 d (7.5) 7.27 t (7.5) 7.43 t (7.5) 7.27 t (7.5) 8.13 d (7.5)
7.54 7.03 7.03 7.54 8.04 6.71 3.70 8.11 7.24 7.41 7.24 8.11
a
C-3 sugar Glu-1 2 3 4 5 6
5.05 d (8.0) 4.16a 4.18a 4.14a 3.91a 4.27a 4.45 dd (11.5, 5.0)
C-28 sugar Fuc- 1 2 3 4 5 6 Rha-1(Fuc-2) 2 3 4 5 6 Rha-1(Fuc-3) 2 3 4 5 6 Xyl- 1(Fuc-4) 2 3 4 5
6.10 d (7.5) 4.65 t (9.0) 4.45a 5.97 brs 4.13a 1.35 d (6.5) 5.82 brs 4.75brs 4.48a 4.20a 4.29a 1.52 d (6.0) 5.57 brs 4.63 m 4.18a 6.00 t (9.5) 4.20a 1.68 d (6.0) 5.28 d (7.5) 3.85 t (8.0) 4.09a 4.20a 3.39 t (11.0) 4.17a
Gal- 1 (Xyl-4) 2 3 4 5 6
Ara-1 (Xyl-3) 2 3 4 5 Api-1 (Rha-3) 2 4 5
5.19 d (7.5) 4.47 m 4.05 m 4.16 m 3.62 d (11.5) 4.22a 6.10 brd 4.23a 4.08a 4.12a 3.85 d (12.0) 4.05 d (12.0)
5.04 d (7.0) 4.13a 4.17a 4.18a 3.91a 4.35 dd (11.5, 2.0) 4.56 dd (11.0)
5.06 d (7.5) 3.94a 4.15a 4.16a 3.88a 4.23 brd (11.5)
6.14 d (7.5) 4.73 t (9.0) 4.46a 5.85 brs 4.14a 1.34 d (6.5) 5.96 brs 4.69 brs 4.45a 4.42a 4.34a 1.51 d (6.0) 5.71 brs 4.66 m 4.18a 6.05 t (9.5) 4.20a 1.74 d (6.0) 5.20 d (8.0) 3.91 t (8.0) 4.13a 4.06a 3.45 t (10.0) 4.20a 4.88 d (7.0) 4.45 m 4.15 m 4.46 m 4.10 m 4.32 dd (11.5, 5.0) 4.52a
6.08 d (8.0) 4.63 t (8.0) 4.47a 5.82 d (3.0) 4.13a 1.32 d (6.0) 5.77 brs 4.67 brs 4.42a 4.45a 4.26a 1.51 d (6.0) 5.53 brs 4.66a 4.17a 5.77 t (9.5) 4.20a 1.67 d (6.0) 5.27 d (8.0) 3.91 t (8.5) 4.16a 4.06 m 3.39 t (11.0) 4.18a
4.31 dd (11.5, 4.5)
5.20 4.46 4.04 4.18 3.61 4.24 6.10 4.78 4.18 3.98 4.53 4.64
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d (7.0) m m m d (12.0) d (12.0) d (5.0) d (5.0)
d (11.0) d (11.0)
(continued on next page)
3 d (8.5) d (8.5) d (8.5) d (8.5) d (15.5) d (15.5) s (3H) d (7.5) t (7.5) t (7.5) t (7.5) d (7.5)
7.52 7.03 7.03 7.52 8.04 6.64 3.67
d (8.5) d (8.5) d (8.5) d (8.5) d (16.0) d (16.0) s (3H)
1.88 s (3H)
Overlap with other signals.
Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing.
2.3. Extraction and isolation Dried roots of P. japonica (5.0 kg) were extracted three times with 95% EtOH for 2 h each time, and gave 0.839 kg residue on removal of the solvent under reduced pressure. The residue was subjected to silica-gel column chromatography (100–200 mesh), eluted with CHCl3, EtOAc, EtOAc:acetone (1:1), EtOAc:acetone (1:3), acetone, acetone : EtOH (1:1), EtOH and MeOH, successively. The acetone: EtOH (1:1) extract (380 g) was applied to macroporous resin (D 101, 2 kg) column chromatography and washed with H2O, 30% EtOH, 60% EtOH and 95% EtOH, respectively. The 95% EtOH fraction (4 g) was separated by preparative MPLC (50%–60%–80%MeOH) and then purified by semi-preparative HPLC (CH3CN: H2O, 37.5:62.5/ 53:47/55:45+0.05% TFA, 7 ml/min) to afford 1 (15 mg), 2 (10 mg), 3 (10 mg), and 4 (48 mg). The 60% EtOH fraction was separated by silica-gel column chromatography (CHCl3:MeOH: H2O 8:2:0.2–7:3:0.5) and preparative MPLC (40%–50%–60%– 80%MeOH), then purified by semi-preparative HPLC (CH3CN: H2O, 28/72, 7 ml/min) to afford 5 (200 mg), and 6 (400 mg).
2.4. Polygalasaponin LI (1) White powder, [α]D20 −3.5 (c 0.3, MeOH); UV (MeOH) λ max: 206, 226, and 312 nm; IR ν max 3388, 2938, 1747, 1676, 1636, 1454, 1069, and 836 cm−1; For 1H and 13C NMR spectroscopic data, see Tables 1 and 2; ESIMS m/z: 1802 [M+Na]+, 1778 [M−H]−; HRESIMS m/z: 1801.7402 [M+Na]+ (cal. 1801.7455, C86H122O39Na).
2.5. Polygalasaponin LII (2) White powder, [α]D20 + 3.8 (c 0.26, MeOH); UV (MeOH) λ max: 206, 226, and 312 nm; IR ν max 3387, 2931, 1748, 1712, 1678, 1631, 1452, 1071, and 829 cm−1; For 1H and 13C NMR spectroscopic data, see Tables 1 and 2; ESIMS m/z: 1700 [M + Na] +, 1676 [M - H] −; HRESIMS m/z: 1699.7089 [M + Na] +(cal. 1699.7139, C82H116O36 Na).
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C. Li et al. / Fitoterapia 83 (2012) 1184–1190 Table 2 (continued)
Table 2 13 C NMR data of compounds 1–3 (125 MHz in pydridine-d5). Position
1
2
3
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
44.2 70.2 85.8 52.8 52.5 21.1 33.1 41.2 49.2 37.0 23.5 127.7 138.9 47.9 24.5 24.1 47.0 42.1 45.5 30.8 33.8 32.2 180.9 14.2 17.6 18.7 64.0 176.5 33.1 24.1
44.2 70.0 85.9 52.8 52.4 21.5 33.0 41.1 49.3 36.9 23.7 127.7 138.9 48.0 24.5 23.9 47.1 42.0 45.4 30.7 33.6 32.3 180.8 14.2 17.5 18.8 64.0 176.7 33.0 23.9
44.2 69.2 85.9 52.8 52.5 21.2 33.1 41.2 49.3 37.0 23.5 127.7 139.0 47.9 24.6 24.1 47.0 42.1 45.6 30.8 33.9 32.2 181.0 14.2 17.7 19.2 64.4 176.5 33.1 24.1
Cin. 1 2 3 4 5 6 7 8 9 OMe
127.4 114.9 130.6 162.1 130.6 114.9 115.7 145.8 167.4 55.4
127.4 114.9 130.6 162.1 130.6 114.9 115.7 145.8 167.4 55.4
127.4 114.9 130.5 162.1 130.5 114.9 115.7 145.8 167.3 55.4
131.1 129.9 128.7 133.1 128.7 129.9 166.4
131.0 129.9 128.7 133.1 128.7 129.9 166.4
Benzyl 1 2 3 4 5 6 7 AcO
Position
1
2
3
Rha-1(Fuc-2) 2 3 4 5 6 Rha-1(Fuc-3)
102.2 71.7 82.7 78.3 68.8 18.7 104.3 71.5 78.3 76.4 70.0 18.2 104.7 74.6 86.0 69.2 66.5 105.2 72.7 74.2 69.2 67.0
102.0 72.3 84.8 78.3 68.6 18.8 104.5 71.7 78.3 75.5 70.1 18.1 104.6 76.5 78.3 78.3 65.0
102.2 71.7 82.7 78.3 68.8 18.7 104.7 71.5 78.3 75.4 70.2 18.1 105.2 74.6 85.8 69.3 66.5 105.2 72.7 74.2 69.2 67.0
Xyl- 1(Rham-4) 2 3 4 5 Ara- 1(xyl-3) 2 3 4 5 Gal- 1(Xyl-4) 2 3 4 5 6 Api- 1(Rham-3) 2 3 4 5
104.5 78.3 75.2 69.9 77.2 62.7 111.9 77.7 79.7 74.6 64.4
111.9 77.7 80.0 74.3 64.4
2.6. Polygalasaponin LIII (3) White powder, [α] D20 + 1.1 (c 0.33, MeOH); UV (MeOH) λ max: 206, 226, and 312 nm; IR νmax 3404, 2938, 1748, 1710, 1677, 1631, 1457, 1073, and 834 cm−1; For 1H and 13C NMR spectroscopic data, see Tables 1 and 2; ESIMS m/z: 1740 [M+Na]+, 1716 [M−H]−; HRESIMS m/z:1739.7255 [M+Na]+ (cal. 1739.7181, C81H120O39). 2.7. Neuroprotection bioassays [11]
170.7 21.0
C-3 sugar Glc-1 2 3 4 5 6
105.5 74.6 78.3 71.5 78.3 62.6
106.8 74.8 78.3 71.5 78.3 62.7
105.5 74.6 78.3 71.5 78.3 62.7
C-28 sugar Fuc-1 2 3 4 5 6
94.8 73.3 80.0 69.7 72.0 16.8
94.6 73.6 81.5 69.9 71.5 16.9
94.9 73.2 80.0 69.7 72.0 16.7
Pheochromocytoma (PC12) cells were incubated in DMEM supplied with 5% fetal bovine serum and 5% equine serum as basic medium. PC12 cells in logarithmic phase were cultured at a density of 5000 cells per well in a 96-well microtiter plate. After 24 h incubation, the medium of model group was changed to DMEM or basic medium with 15 μM Aβ25–35 for 48 h, or basic medium with 350 μM rotenone for 24 h. Test compounds dissolved in dimethyl sulfoxide (DMSO) were added to each well for >1000 fold dilution in the model medium at the same time. Each sample was tested in triplicate. After the incubation at 37 °C in 5% CO2 for 24 h, 10 μl of MTT (5 mg/ml) was added to each well and incubated for another 4 h, then liquid in the wells was removed. DMSO (100 μl) was added to each well. The absorbance was recorded on a microplate reader (Bio-Rad model 550) at a wavelength of 570 nm. Analysis of variance (ANOVA) followed by Newman–Keuls post hoc test was performed to assess the differences between the relevant control and each experimental group. p-values of b0.05, b0.01 and
C. Li et al. / Fitoterapia 83 (2012) 1184–1190
b0.001 were regarded as statistically significant. Data were expressed as mean±SEM as indicated. 3. Results and discussion The acetone: ethanol (1:1) parts of the 95% EtOH extract of P. japonica were subjected to macroporous resin (D 101) column and washed with H2O, 30% EtOH, 60% EtOH and 95% EtOH in succession. The 95% EtOH and 60% EtOH fractions were successively subjected to silica gel column chromatography, MPLC and semi-prep HPLC to afford compounds 1–6 (Fig. 1). Compound 1 was isolated as a white amorphous powder, and its IR spectrum displayed absorptions for hydroxyl (3388 cm − 1), conjugated carbonyl (1676 cm − 1) and phenyl (1636, 1454 cm − 1) groups. The positive-ion HRESIMS of 1 showed a quasi-molecular ion peak at m/z 1801.7402 [M + Na] +, which indicated a molecular formula of C86H122O39 (cal. 1801.7455, C86H122O39Na). The 1H NMR spectrum of 1 showed signals attributable to five tertiary methyl groups at δH 0.79 (H3-29), 1.05 (H3-30), 1.15 (H3-26), 1.60 (H3-25), and 1.95 (H3-24), an isolated oxymethylene at δH 3.80 and 4.03 (1H each, d, J=10.5 Hz, H2-27), and two oxymethine protons at δ 4.62 (1H, d, J=3.0 Hz, H-1), 4.68 (1H, m,H-2), an olefinic proton at δH 5.82 (brs), combined with the information from the 13C NMR spectrum (five sp3carbons at δC 14.2, 17.6, 18.7, 24.1, 33.1 and two sp2 olefinic carbons at δC 127.9 and 138.9) indicating that the triterpenoid aglycone possessed an olean-12-ene skeleton [5]. By comparison of the 13C NMR spectral data of the aglycone part with those of polygalasaponins [2–6], the aglycone of 1 was assigned the structure of presenegenin, which exist in most of triterpenoid saponins isolated from the family Polygalaceae. The 1H NMR signals were also observed assignable to six anomeric protons of six sugar units at δH 6.10 (1H, d, J = 7.5 Hz, H-1 of Fuc) , 6.11 (1H, brs, H-1 of Api),5.82 (1H, brs, H-1 of
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Rham1), 5.57 (1H, brs, H-1 of Rham2 ), 5.28 (1H, d, J = 7.5 Hz, H-1 of Xyl), 5.19 (1H,d, J = 7.5 Hz, H-1 of Arab), 5.05 (1H, d, J = 8.0 Hz, H-1 of Glu ), and partially overlapped signals due to the oxymethylenes and oxymethines of the sugar units between δH 3.35 to 4.75. The presence of proton signals at δH 7.55 (2 H, d, J = 7.5 Hz), 7.05 (2 H, d, J = 7.5 Hz), 8.09 (1H, d, J = 16.0 Hz), 6.73 (1H, d, J = 16.0 Hz) and 3.70 (3H, s) suggested the existence of a 4-methoxycinnamic acid moiety in 1; the signals at δH 8.16 (2 H, brd, J = 7.5 Hz), 7.42 (1H, t, J = 7.5 Hz) and 7.27 (2 H, t, J = 7.5 Hz) indicated a benzoyl moiety in 1. These data suggested that the oligosaccharide part of compound 1 possessed six sugars and two acylating groups. The structure of 1 was finalized by a comprehensive analysis of its 2D NMR spectroscopic data. The proton and protonated carbon signals in the NMR spectra of 1 were assigned unequivocally (Tables 1 and 2) on the basis of HSQC spectroscopic analysis. The sequence of the oligosaccharides chain was finally achieved by an analysis of HMBC experiment (Fig. 2). For the pentasaccharide chain linked to the C-28 of the aglycone, the HMBC spectrum showed the correlations between H-1 (δH 5.19, d, J = 7.5 Hz) of terminal arabinose and C-3 (δC 86.0) of xylose, between H-1 (δH 5.28, d, J = 7.5 Hz) of xylose and C-4 of rhamnose1 (δC 78.3), between H-1 (δH 6.11, brs) of apiose and C-3 (δC 82.7) of rhamnose 1, between H-1(δH 5.82, brs) of rhamnose 1 and C-2 of fucose (δC 73.8), between H-1(δH 5.57, brs) of rhamnose 2 and C-3 of fucose (δC 80.0), between H-1 (δH 6.10, d, J = 7.5 Hz) of fucose and C-28 (δC 176.4). Another HMBC correlation was observed between H-1 (δH 5.05, d, J = 8.0 Hz) of glucose and C-3 (δC 85.8) of the aglycone, indicating the glucose was attached to C-3 of the aglycone. The attachment of 4-methoxycinnamic acid moiety was determined to be at C-4 of fucose by observing the HMBC correlation between H-4 (δH 5.87, s) of fucose and carboxyl
Fig. 1. Structures of compounds 1–6.
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C. Li et al. / Fitoterapia 83 (2012) 1184–1190
Fig. 2. Key HMBC correlations of compound 1.
carbon (δC167.4) of methoxycinnamic acid. The HMBC correlation from the δH 6.00 (t, J=9.5 Hz) to δ 70.2 (Rham2-3) and 68.0 (Rham2-5) allowed assigning of H-4 of rhamnose 2. The HMBC correlation from δH 6.00 (Rham-4) to carboxyl carbon (δC166.4) of benzoic acid indicated that the benzoyl moiety was attached to C-4 of Rham 2. By comparing NMR spectral data of sugar moieties with previous isolated saponins [2–4,8] and biogenetic consi-derations, the configuration of the sugars moieties was determined as D-glucose, D-fucose, L-rhamnose, D-xylose, D-apiose and L-arabinose. Therefore, the structure of 1 was elucidated as 3-O-β-D-glucopyranosyl-presenegenin 28-O-α-L-arabinopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)[β-D-apiofuranosyl-(1→3)]-α-L-rhamnopyranosyl-(1→ 2)[4-O-benzoyl-α-L-rhamnopyranosyl-(1 → 3)]-[4-O-4-methoxy cinnamoyl]-β-D-fucopyranosyl ester (polygalasaponin LI). Polygalasaponin LII (2) revealed an [M+Na]+ ion m/z at 1700 in the positive ESIMS. Combined with the HRESIMS, its molecular formula was deduced to be C82H116O36. Comparison of the NMR data between compounds 2 and 1 showed that they were almost superimposable in aglycone moiety but had some differences in sugar moiety: the arabinose signals replaced by a galactose and the absence of apiose signals. The sugar chain of 2 was confirmed by HMBC spectrum. In HMBC, long-range correlations were observed between the following protons and carbons: H-1 of Glu with C-3 of aglycone, H-1 of Fuc with C-28 of aglycone, H-1 of Rham1 with C-2 of Fuc, H-1 of Xyl with C-4 of Rham1, H-1 of Gal with C-4 of Xyl, H-1 of Rham 2 with C-3 of Fuc. The HMBC spectrum of 2 also displayed correlations
from H-4 of Fuc (δH 5.85, brs) to carboxyl carbon (δC167.4) of 4-methoxycinnamic acid, H-4 of Rham2 (δH 6.00, t, J=9.5 Hz) to carboxyl carbon (δC166.4) of benzoyl acid, suggesting that 4-methoxycinnamic acid and benzoyl acid were located at C-4 of Fuc and C-4 of Rham2, respectively. For the above evidence, the structure of 2 was elucidated as 3-O-β-D-glucopyranosylpresenegenin 28-O-β-D-galactopyranosyl-(1→4)-β-D-xylopyranosyl-(1→4) -α-L-rhamnopyranosyl-(1→2)-[4-O-benzoyl-
Table 3 Neuroprotective activity of compounds 1–3 on PC12 cell models induced by rotenone.
Control Rotenone 1
2
3
4
Group (μM)
Cell survival rate (%)
0.1 1.0 10 0.1 1.0 100 1 1.0 10 0.1 1.0 10
100 67.20 ± 3.9## 54.63 ± 12.72 70.31 ± 13.81 68.74 ± 12.94 66.04 ± 12.20 74.80 ± 17.86 68.89 ± 13.46 47.88 ± 16.77 73.25 ± 15.63 66.52 ± 10.89 35.20 ± 3.39 69.25 ± 16.14 62.10 ± 12.94
*p b 0.05, **p b 0.01, ***p b 0.001 vs model. ## p b 0.05 vs control.
C. Li et al. / Fitoterapia 83 (2012) 1184–1190
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Table 4 Neuroprotective activity of compound 5 on PC12 cell models induced by Aβ25–35.
###p b 0.05 vs control, *p b 0.05, **p b 0.01, ***p b 0.001 vs model.
Table 5 Neuroprotective activity of compound 6 (PGSF) on PC12 cell models induced by Aβ25–35.
###p b 0.05 vs control, *p b 0.05, **p b 0.01, ***p b 0.001 vs model.
α-L-rhamnopyranosyl-(1→3)]-[4-O-4-methoxycinnamoyl]-βD-fucopyranosyl ester. The ESI-MS and HRESIMS of polygalasaponin LIII (3) gave the molecular formula C81H120O39 at m/z 1739.7255 [M + Na] + (cal. C81H120O39.1739.7181). The molecular weight of 3 was 42 mass units more than that of Polygalasaponin XXXII (4) [5]. Detailed comparison of the NMR data of the two compounds implied that the structure of 3 was almost identical with 4 except for an acetyl group at δH 1.88 (3H, S), which correspond to carbon signals at δC 21.0 and 170.7 in 3. The acetyl group was attached to the C-4 of Rham2 owing to the downfield chemical shift of H-4 [δH 6.00 (1H, t, J = 9.5 Hz)] of the Rham2. Thus, the structure of 3 was elucidated as 3-O-β-D-glucopyranosyl-presenegenin 28O-α-L-arabinopyranosyl-(1 → 3)-β-D-xylo-pyranosyl-(1 → 4)-[β-D-apiofuranosyl-(1→ 3)]-α-L-rhamno-pyranosyl-(1→ 2)-[4-O-acetyl-α-L-rhamnopyranosyl-(1→3)]-[4-O-4-methoxycinnamoyl]-β-D-fucopyranosyl ester. The structures of the known saponins (4–6) were identified by comparison of their NMR and MS analysis with those reported in literatures and the structures were determined as: polygalasaponins XXXII (4) [5], II (5) [2], and F (6) [6]. Compounds 1–6 were evaluated for their neuroprotective effect on neuron-like PC12 cells induced by rotenone or 11 Aβ25–35 in vitro using the MTT method. Compounds 1–4 failed
to protect cells in rotenone model at 0.1, 1.0, and 10 μM, compounds 5 and 6 showed moderate activity in Aβ25–35 model at 10 μM (Table 3, 4 and 5). Acknowledgments This research was financially supported by the National Natural Science Foundation of China (Nos. 20372087 and 81073078) and the National Science and Technology Project of China (2011ZX09307-002-01). References [1] Jiangsu New Medical College. Dictionary of traditional Chinese medicine. Shanghai Science and Technique Publishing House; 1986. p. 757. [2] Zhang DM, Miyase T, Kuroyanagi M, Umehara K, Ueno A. Studies on the constituents of Polygala japonica Houtt. I. Structures of polygalasaponins I–X. Chem Pharm Bull 1995;43:115. [3] Zhang DM, Miyase T, Kuroyanagi M, Umehara K, Ueno A. Studies on the constituents of Polygala japonica Houtt. II. Structures of polygalasaponins XX–XIX. Chem Pharm Bull 1995;43:966. [4] Zhang DM, Miyase T, Kuroyanagi M, Umehara K, Ueno A. Studies on the constituents of Polygala japonica Houtt. III. Structures of polygalasaponins XX–XXVII. Chem Pharm Bull 1996;44:173. [5] Zhang DM, Miyase T, Kuroyanagi M, Umehara K, Ueno A. Five new triterpene saponins, polygalasaponins .XXVIII–XXXII. Chem Pharm Bull 1996;44:810.
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[6] Li TZ, Zhang WD, Yang GJ, Liu WY, Chen HS, Shen YH. Saponins from Polygala japonica and their effects on a forced Swimming test in mice. J Nat Prod 2006;69:591. [7] Xua X, Zhou JF, Li TZ, Zhang ZH, Shan L, Xiang ZH, et al. Polygalasaponin G promotes neurite outgrowth of cultured neuron on myelin. Neurosci Lett 2009;460:41. [8] Xue QC, Li CJ, Li Z, Yang JZ, Zhang DM. Three new xanthones from the roots of Polygala japonica Houtt. J Asian Nat Prod Res 2009;11:465.
[9] Fu J, Zuo L, Yang JZ, et al. Oligosaccharide polyester and triterpenoid saponins from the roots of Polygala japonica. Phytochemisry 2008;69: 1617. [10] Fu J, Zhang DM, Chen RY. Three new xanthones from the roots of Polygala japonica Houtt. J Asian Nat Prod Res 2006;8:41. [11] Pytlowany M, Strosznajder JB, Jeśko H, et al. Acta Biochim Pol 2008;55: 339.
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Three triterpenoid saponins from the roots of Polygala japonica Houtt. Chuangjun Li, Jing Fu, Jingzhi Yang, Dongming Zhang ⁎, Yuhe Yuan, Naihong Chen State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, PR China
a r t i c l e
i n f o
Article history: Received 18 April 2012 Accepted in revised form 4 July 2012 Available online 14 July 2012 Keywords: Polygala japonica Triterpenoid saponins Polygalasaponins Neuroprotective effect
a b s t r a c t Three new triterpenoid saponins polygalasaponins LI–LIII (1–3) with two acylation groups in oligosaccharide chain, together with three known saponins were isolated from the roots of Polygala japonica Houtt. (4–6). The neuroprotective effects of these compounds on neuron-like PC12 cells were evaluated in vitro. Compounds 5 and 6 show neuroprotective effects in Aβ25–35 model at the concentration of 10 μM. © 2012 Elsevier B.V. All rights reserved.
1. Introduction
2. Experimental part
The roots of Polygala japonica Houtt. have been used in Chinese folk herbal medicine as expectorant, anti-inflammatory, antibacterial, ataractic, and antidepressant agents [1]. This plant is a rich source of triterpenoid saponins, from which more than fifty triterpenoid saponins have been isolated [2–6,10]. Some of these compounds showed antidepressant activity [6] and neurotrophic activity on PC12 cells and cultured cortical neurons [7]. Our previous investigation on the roots of P. japonica resulted in the isolation of an oligosaccharide polyester, four triterpenoid saponins and six xanthones [8–10]. As part of our ongoing effort to study the chemical and biological diversity of this plant, we report herein the isolation and characterization of three new triterpenoid saponins polygalasaponins LI–LIII (1–3), which have two acylation groups in oligosaccharides chain, together with three known saponins (4–6). The neuroprotective effects of these compounds on neuron-like PC12 cells were evaluated in vitro, as a result, compounds 5 and 6 showed neuroprotective effects in Aβ25–35 model at the concentration of 10 μM.
2.1. General experimental procedures Optical rotations were determined on a Perkin-Elmer 241 digital polrimeter. IR spectra were recorded on an IMPACT 400 spectrometer. UV spectra were obtained on a Shimadzu UV-260 spectrophotometer. NMR spectra were run on Varian INOVA-500 spectrometer using TMS as internal standard. ESIMS and HRESIMS were measured on Agilent 1100 series LC/MSD Trap SL mass spectrometer and Autospee-Ultima ETOF, respectively. Preparative HPLC was performed on an YMC-Pack ODS-A (YMC Co., Ltd.) column. Macroporous resin D101 (26–60 mesh, Tianjin Haiguang Chemistry Company, Tianjin, China) and silica gel (100–200, 200–300 mesh, produced by Qingdao Marine Chemical company; Qingdao, China) were used for column chromatography and silica gel GF-254 (produced by Qingdao Marine Chemical company; Qingdao, China) for TLC analysis. 2.2. Plant material
⁎ Corresponding author at: Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, 1 Xian Nong Tan Street, Xicheng District, Beijing, 100050, PR China. Tel./fax: +86 10 63165227. E-mail addresses: [email protected] (C. Li), [email protected] (D. Zhang). 0367-326X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2012.07.002
The roots of P. japonica were collected in June 2003 in Jiangxi Province, China, it was identified by Professor Yongming Luo, Jiangxi College of Traditional Chinese Medicine. A voucher specimen (272400) is deposited in the Institute of Materia
C. Li et al. / Fitoterapia 83 (2012) 1184–1190 Table 1 (continued)
Table 1 1 H NMR data of compounds 1–3 in pydridine-d5 (500 MHz). position Aglycone 2 3 12 18 24 25 26 27 29 30
1 4.68 4.62 5.86 3.25 1.95 1.60 1.15 3.80 4.03 0.79 1.05
2 m d (3.0) brs brd (14.0) s s s brd (10.5) brd (10.5) s s
4.69 4.63 5.85 3.24 1.98 1.54 1.13 3.82 4.05 0.78 0.95
3 m d (3.0) brs brd (11.5) s s s brd (12.5) brd (12.5) s s
4.67 4.60 5.88 3.25 1.95 1.59 1.14 3.83 4.03 0.78 1.04
m d (3.5) brs brd s s s brd (12.5) brd (12.5) s s
position
1
2
Cinn-2 (Fuc-4) 3 5 6 7 8 OMe Benzyl-2 (Rham-4) 3 4 5 6 Acetyl
7.55 d (8.5) 7.05 d (8.5) 7.05 d (8.5) 7.55 d (8.5) 8.09 d (16.0) 6.73 d (16.0) 3.70 s (3H) 8.13 d (7.5) 7.27 t (7.5) 7.43 t (7.5) 7.27 t (7.5) 8.13 d (7.5)
7.54 7.03 7.03 7.54 8.04 6.71 3.70 8.11 7.24 7.41 7.24 8.11
a
C-3 sugar Glu-1 2 3 4 5 6
5.05 d (8.0) 4.16a 4.18a 4.14a 3.91a 4.27a 4.45 dd (11.5, 5.0)
C-28 sugar Fuc- 1 2 3 4 5 6 Rha-1(Fuc-2) 2 3 4 5 6 Rha-1(Fuc-3) 2 3 4 5 6 Xyl- 1(Fuc-4) 2 3 4 5
6.10 d (7.5) 4.65 t (9.0) 4.45a 5.97 brs 4.13a 1.35 d (6.5) 5.82 brs 4.75brs 4.48a 4.20a 4.29a 1.52 d (6.0) 5.57 brs 4.63 m 4.18a 6.00 t (9.5) 4.20a 1.68 d (6.0) 5.28 d (7.5) 3.85 t (8.0) 4.09a 4.20a 3.39 t (11.0) 4.17a
Gal- 1 (Xyl-4) 2 3 4 5 6
Ara-1 (Xyl-3) 2 3 4 5 Api-1 (Rha-3) 2 4 5
5.19 d (7.5) 4.47 m 4.05 m 4.16 m 3.62 d (11.5) 4.22a 6.10 brd 4.23a 4.08a 4.12a 3.85 d (12.0) 4.05 d (12.0)
5.04 d (7.0) 4.13a 4.17a 4.18a 3.91a 4.35 dd (11.5, 2.0) 4.56 dd (11.0)
5.06 d (7.5) 3.94a 4.15a 4.16a 3.88a 4.23 brd (11.5)
6.14 d (7.5) 4.73 t (9.0) 4.46a 5.85 brs 4.14a 1.34 d (6.5) 5.96 brs 4.69 brs 4.45a 4.42a 4.34a 1.51 d (6.0) 5.71 brs 4.66 m 4.18a 6.05 t (9.5) 4.20a 1.74 d (6.0) 5.20 d (8.0) 3.91 t (8.0) 4.13a 4.06a 3.45 t (10.0) 4.20a 4.88 d (7.0) 4.45 m 4.15 m 4.46 m 4.10 m 4.32 dd (11.5, 5.0) 4.52a
6.08 d (8.0) 4.63 t (8.0) 4.47a 5.82 d (3.0) 4.13a 1.32 d (6.0) 5.77 brs 4.67 brs 4.42a 4.45a 4.26a 1.51 d (6.0) 5.53 brs 4.66a 4.17a 5.77 t (9.5) 4.20a 1.67 d (6.0) 5.27 d (8.0) 3.91 t (8.5) 4.16a 4.06 m 3.39 t (11.0) 4.18a
4.31 dd (11.5, 4.5)
5.20 4.46 4.04 4.18 3.61 4.24 6.10 4.78 4.18 3.98 4.53 4.64
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d (7.0) m m m d (12.0) d (12.0) d (5.0) d (5.0)
d (11.0) d (11.0)
(continued on next page)
3 d (8.5) d (8.5) d (8.5) d (8.5) d (15.5) d (15.5) s (3H) d (7.5) t (7.5) t (7.5) t (7.5) d (7.5)
7.52 7.03 7.03 7.52 8.04 6.64 3.67
d (8.5) d (8.5) d (8.5) d (8.5) d (16.0) d (16.0) s (3H)
1.88 s (3H)
Overlap with other signals.
Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing.
2.3. Extraction and isolation Dried roots of P. japonica (5.0 kg) were extracted three times with 95% EtOH for 2 h each time, and gave 0.839 kg residue on removal of the solvent under reduced pressure. The residue was subjected to silica-gel column chromatography (100–200 mesh), eluted with CHCl3, EtOAc, EtOAc:acetone (1:1), EtOAc:acetone (1:3), acetone, acetone : EtOH (1:1), EtOH and MeOH, successively. The acetone: EtOH (1:1) extract (380 g) was applied to macroporous resin (D 101, 2 kg) column chromatography and washed with H2O, 30% EtOH, 60% EtOH and 95% EtOH, respectively. The 95% EtOH fraction (4 g) was separated by preparative MPLC (50%–60%–80%MeOH) and then purified by semi-preparative HPLC (CH3CN: H2O, 37.5:62.5/ 53:47/55:45+0.05% TFA, 7 ml/min) to afford 1 (15 mg), 2 (10 mg), 3 (10 mg), and 4 (48 mg). The 60% EtOH fraction was separated by silica-gel column chromatography (CHCl3:MeOH: H2O 8:2:0.2–7:3:0.5) and preparative MPLC (40%–50%–60%– 80%MeOH), then purified by semi-preparative HPLC (CH3CN: H2O, 28/72, 7 ml/min) to afford 5 (200 mg), and 6 (400 mg).
2.4. Polygalasaponin LI (1) White powder, [α]D20 −3.5 (c 0.3, MeOH); UV (MeOH) λ max: 206, 226, and 312 nm; IR ν max 3388, 2938, 1747, 1676, 1636, 1454, 1069, and 836 cm−1; For 1H and 13C NMR spectroscopic data, see Tables 1 and 2; ESIMS m/z: 1802 [M+Na]+, 1778 [M−H]−; HRESIMS m/z: 1801.7402 [M+Na]+ (cal. 1801.7455, C86H122O39Na).
2.5. Polygalasaponin LII (2) White powder, [α]D20 + 3.8 (c 0.26, MeOH); UV (MeOH) λ max: 206, 226, and 312 nm; IR ν max 3387, 2931, 1748, 1712, 1678, 1631, 1452, 1071, and 829 cm−1; For 1H and 13C NMR spectroscopic data, see Tables 1 and 2; ESIMS m/z: 1700 [M + Na] +, 1676 [M - H] −; HRESIMS m/z: 1699.7089 [M + Na] +(cal. 1699.7139, C82H116O36 Na).
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C. Li et al. / Fitoterapia 83 (2012) 1184–1190 Table 2 (continued)
Table 2 13 C NMR data of compounds 1–3 (125 MHz in pydridine-d5). Position
1
2
3
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
44.2 70.2 85.8 52.8 52.5 21.1 33.1 41.2 49.2 37.0 23.5 127.7 138.9 47.9 24.5 24.1 47.0 42.1 45.5 30.8 33.8 32.2 180.9 14.2 17.6 18.7 64.0 176.5 33.1 24.1
44.2 70.0 85.9 52.8 52.4 21.5 33.0 41.1 49.3 36.9 23.7 127.7 138.9 48.0 24.5 23.9 47.1 42.0 45.4 30.7 33.6 32.3 180.8 14.2 17.5 18.8 64.0 176.7 33.0 23.9
44.2 69.2 85.9 52.8 52.5 21.2 33.1 41.2 49.3 37.0 23.5 127.7 139.0 47.9 24.6 24.1 47.0 42.1 45.6 30.8 33.9 32.2 181.0 14.2 17.7 19.2 64.4 176.5 33.1 24.1
Cin. 1 2 3 4 5 6 7 8 9 OMe
127.4 114.9 130.6 162.1 130.6 114.9 115.7 145.8 167.4 55.4
127.4 114.9 130.6 162.1 130.6 114.9 115.7 145.8 167.4 55.4
127.4 114.9 130.5 162.1 130.5 114.9 115.7 145.8 167.3 55.4
131.1 129.9 128.7 133.1 128.7 129.9 166.4
131.0 129.9 128.7 133.1 128.7 129.9 166.4
Benzyl 1 2 3 4 5 6 7 AcO
Position
1
2
3
Rha-1(Fuc-2) 2 3 4 5 6 Rha-1(Fuc-3)
102.2 71.7 82.7 78.3 68.8 18.7 104.3 71.5 78.3 76.4 70.0 18.2 104.7 74.6 86.0 69.2 66.5 105.2 72.7 74.2 69.2 67.0
102.0 72.3 84.8 78.3 68.6 18.8 104.5 71.7 78.3 75.5 70.1 18.1 104.6 76.5 78.3 78.3 65.0
102.2 71.7 82.7 78.3 68.8 18.7 104.7 71.5 78.3 75.4 70.2 18.1 105.2 74.6 85.8 69.3 66.5 105.2 72.7 74.2 69.2 67.0
Xyl- 1(Rham-4) 2 3 4 5 Ara- 1(xyl-3) 2 3 4 5 Gal- 1(Xyl-4) 2 3 4 5 6 Api- 1(Rham-3) 2 3 4 5
104.5 78.3 75.2 69.9 77.2 62.7 111.9 77.7 79.7 74.6 64.4
111.9 77.7 80.0 74.3 64.4
2.6. Polygalasaponin LIII (3) White powder, [α] D20 + 1.1 (c 0.33, MeOH); UV (MeOH) λ max: 206, 226, and 312 nm; IR νmax 3404, 2938, 1748, 1710, 1677, 1631, 1457, 1073, and 834 cm−1; For 1H and 13C NMR spectroscopic data, see Tables 1 and 2; ESIMS m/z: 1740 [M+Na]+, 1716 [M−H]−; HRESIMS m/z:1739.7255 [M+Na]+ (cal. 1739.7181, C81H120O39). 2.7. Neuroprotection bioassays [11]
170.7 21.0
C-3 sugar Glc-1 2 3 4 5 6
105.5 74.6 78.3 71.5 78.3 62.6
106.8 74.8 78.3 71.5 78.3 62.7
105.5 74.6 78.3 71.5 78.3 62.7
C-28 sugar Fuc-1 2 3 4 5 6
94.8 73.3 80.0 69.7 72.0 16.8
94.6 73.6 81.5 69.9 71.5 16.9
94.9 73.2 80.0 69.7 72.0 16.7
Pheochromocytoma (PC12) cells were incubated in DMEM supplied with 5% fetal bovine serum and 5% equine serum as basic medium. PC12 cells in logarithmic phase were cultured at a density of 5000 cells per well in a 96-well microtiter plate. After 24 h incubation, the medium of model group was changed to DMEM or basic medium with 15 μM Aβ25–35 for 48 h, or basic medium with 350 μM rotenone for 24 h. Test compounds dissolved in dimethyl sulfoxide (DMSO) were added to each well for >1000 fold dilution in the model medium at the same time. Each sample was tested in triplicate. After the incubation at 37 °C in 5% CO2 for 24 h, 10 μl of MTT (5 mg/ml) was added to each well and incubated for another 4 h, then liquid in the wells was removed. DMSO (100 μl) was added to each well. The absorbance was recorded on a microplate reader (Bio-Rad model 550) at a wavelength of 570 nm. Analysis of variance (ANOVA) followed by Newman–Keuls post hoc test was performed to assess the differences between the relevant control and each experimental group. p-values of b0.05, b0.01 and
C. Li et al. / Fitoterapia 83 (2012) 1184–1190
b0.001 were regarded as statistically significant. Data were expressed as mean±SEM as indicated. 3. Results and discussion The acetone: ethanol (1:1) parts of the 95% EtOH extract of P. japonica were subjected to macroporous resin (D 101) column and washed with H2O, 30% EtOH, 60% EtOH and 95% EtOH in succession. The 95% EtOH and 60% EtOH fractions were successively subjected to silica gel column chromatography, MPLC and semi-prep HPLC to afford compounds 1–6 (Fig. 1). Compound 1 was isolated as a white amorphous powder, and its IR spectrum displayed absorptions for hydroxyl (3388 cm − 1), conjugated carbonyl (1676 cm − 1) and phenyl (1636, 1454 cm − 1) groups. The positive-ion HRESIMS of 1 showed a quasi-molecular ion peak at m/z 1801.7402 [M + Na] +, which indicated a molecular formula of C86H122O39 (cal. 1801.7455, C86H122O39Na). The 1H NMR spectrum of 1 showed signals attributable to five tertiary methyl groups at δH 0.79 (H3-29), 1.05 (H3-30), 1.15 (H3-26), 1.60 (H3-25), and 1.95 (H3-24), an isolated oxymethylene at δH 3.80 and 4.03 (1H each, d, J=10.5 Hz, H2-27), and two oxymethine protons at δ 4.62 (1H, d, J=3.0 Hz, H-1), 4.68 (1H, m,H-2), an olefinic proton at δH 5.82 (brs), combined with the information from the 13C NMR spectrum (five sp3carbons at δC 14.2, 17.6, 18.7, 24.1, 33.1 and two sp2 olefinic carbons at δC 127.9 and 138.9) indicating that the triterpenoid aglycone possessed an olean-12-ene skeleton [5]. By comparison of the 13C NMR spectral data of the aglycone part with those of polygalasaponins [2–6], the aglycone of 1 was assigned the structure of presenegenin, which exist in most of triterpenoid saponins isolated from the family Polygalaceae. The 1H NMR signals were also observed assignable to six anomeric protons of six sugar units at δH 6.10 (1H, d, J = 7.5 Hz, H-1 of Fuc) , 6.11 (1H, brs, H-1 of Api),5.82 (1H, brs, H-1 of
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Rham1), 5.57 (1H, brs, H-1 of Rham2 ), 5.28 (1H, d, J = 7.5 Hz, H-1 of Xyl), 5.19 (1H,d, J = 7.5 Hz, H-1 of Arab), 5.05 (1H, d, J = 8.0 Hz, H-1 of Glu ), and partially overlapped signals due to the oxymethylenes and oxymethines of the sugar units between δH 3.35 to 4.75. The presence of proton signals at δH 7.55 (2 H, d, J = 7.5 Hz), 7.05 (2 H, d, J = 7.5 Hz), 8.09 (1H, d, J = 16.0 Hz), 6.73 (1H, d, J = 16.0 Hz) and 3.70 (3H, s) suggested the existence of a 4-methoxycinnamic acid moiety in 1; the signals at δH 8.16 (2 H, brd, J = 7.5 Hz), 7.42 (1H, t, J = 7.5 Hz) and 7.27 (2 H, t, J = 7.5 Hz) indicated a benzoyl moiety in 1. These data suggested that the oligosaccharide part of compound 1 possessed six sugars and two acylating groups. The structure of 1 was finalized by a comprehensive analysis of its 2D NMR spectroscopic data. The proton and protonated carbon signals in the NMR spectra of 1 were assigned unequivocally (Tables 1 and 2) on the basis of HSQC spectroscopic analysis. The sequence of the oligosaccharides chain was finally achieved by an analysis of HMBC experiment (Fig. 2). For the pentasaccharide chain linked to the C-28 of the aglycone, the HMBC spectrum showed the correlations between H-1 (δH 5.19, d, J = 7.5 Hz) of terminal arabinose and C-3 (δC 86.0) of xylose, between H-1 (δH 5.28, d, J = 7.5 Hz) of xylose and C-4 of rhamnose1 (δC 78.3), between H-1 (δH 6.11, brs) of apiose and C-3 (δC 82.7) of rhamnose 1, between H-1(δH 5.82, brs) of rhamnose 1 and C-2 of fucose (δC 73.8), between H-1(δH 5.57, brs) of rhamnose 2 and C-3 of fucose (δC 80.0), between H-1 (δH 6.10, d, J = 7.5 Hz) of fucose and C-28 (δC 176.4). Another HMBC correlation was observed between H-1 (δH 5.05, d, J = 8.0 Hz) of glucose and C-3 (δC 85.8) of the aglycone, indicating the glucose was attached to C-3 of the aglycone. The attachment of 4-methoxycinnamic acid moiety was determined to be at C-4 of fucose by observing the HMBC correlation between H-4 (δH 5.87, s) of fucose and carboxyl
Fig. 1. Structures of compounds 1–6.
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C. Li et al. / Fitoterapia 83 (2012) 1184–1190
Fig. 2. Key HMBC correlations of compound 1.
carbon (δC167.4) of methoxycinnamic acid. The HMBC correlation from the δH 6.00 (t, J=9.5 Hz) to δ 70.2 (Rham2-3) and 68.0 (Rham2-5) allowed assigning of H-4 of rhamnose 2. The HMBC correlation from δH 6.00 (Rham-4) to carboxyl carbon (δC166.4) of benzoic acid indicated that the benzoyl moiety was attached to C-4 of Rham 2. By comparing NMR spectral data of sugar moieties with previous isolated saponins [2–4,8] and biogenetic consi-derations, the configuration of the sugars moieties was determined as D-glucose, D-fucose, L-rhamnose, D-xylose, D-apiose and L-arabinose. Therefore, the structure of 1 was elucidated as 3-O-β-D-glucopyranosyl-presenegenin 28-O-α-L-arabinopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)[β-D-apiofuranosyl-(1→3)]-α-L-rhamnopyranosyl-(1→ 2)[4-O-benzoyl-α-L-rhamnopyranosyl-(1 → 3)]-[4-O-4-methoxy cinnamoyl]-β-D-fucopyranosyl ester (polygalasaponin LI). Polygalasaponin LII (2) revealed an [M+Na]+ ion m/z at 1700 in the positive ESIMS. Combined with the HRESIMS, its molecular formula was deduced to be C82H116O36. Comparison of the NMR data between compounds 2 and 1 showed that they were almost superimposable in aglycone moiety but had some differences in sugar moiety: the arabinose signals replaced by a galactose and the absence of apiose signals. The sugar chain of 2 was confirmed by HMBC spectrum. In HMBC, long-range correlations were observed between the following protons and carbons: H-1 of Glu with C-3 of aglycone, H-1 of Fuc with C-28 of aglycone, H-1 of Rham1 with C-2 of Fuc, H-1 of Xyl with C-4 of Rham1, H-1 of Gal with C-4 of Xyl, H-1 of Rham 2 with C-3 of Fuc. The HMBC spectrum of 2 also displayed correlations
from H-4 of Fuc (δH 5.85, brs) to carboxyl carbon (δC167.4) of 4-methoxycinnamic acid, H-4 of Rham2 (δH 6.00, t, J=9.5 Hz) to carboxyl carbon (δC166.4) of benzoyl acid, suggesting that 4-methoxycinnamic acid and benzoyl acid were located at C-4 of Fuc and C-4 of Rham2, respectively. For the above evidence, the structure of 2 was elucidated as 3-O-β-D-glucopyranosylpresenegenin 28-O-β-D-galactopyranosyl-(1→4)-β-D-xylopyranosyl-(1→4) -α-L-rhamnopyranosyl-(1→2)-[4-O-benzoyl-
Table 3 Neuroprotective activity of compounds 1–3 on PC12 cell models induced by rotenone.
Control Rotenone 1
2
3
4
Group (μM)
Cell survival rate (%)
0.1 1.0 10 0.1 1.0 100 1 1.0 10 0.1 1.0 10
100 67.20 ± 3.9## 54.63 ± 12.72 70.31 ± 13.81 68.74 ± 12.94 66.04 ± 12.20 74.80 ± 17.86 68.89 ± 13.46 47.88 ± 16.77 73.25 ± 15.63 66.52 ± 10.89 35.20 ± 3.39 69.25 ± 16.14 62.10 ± 12.94
*p b 0.05, **p b 0.01, ***p b 0.001 vs model. ## p b 0.05 vs control.
C. Li et al. / Fitoterapia 83 (2012) 1184–1190
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Table 4 Neuroprotective activity of compound 5 on PC12 cell models induced by Aβ25–35.
###p b 0.05 vs control, *p b 0.05, **p b 0.01, ***p b 0.001 vs model.
Table 5 Neuroprotective activity of compound 6 (PGSF) on PC12 cell models induced by Aβ25–35.
###p b 0.05 vs control, *p b 0.05, **p b 0.01, ***p b 0.001 vs model.
α-L-rhamnopyranosyl-(1→3)]-[4-O-4-methoxycinnamoyl]-βD-fucopyranosyl ester. The ESI-MS and HRESIMS of polygalasaponin LIII (3) gave the molecular formula C81H120O39 at m/z 1739.7255 [M + Na] + (cal. C81H120O39.1739.7181). The molecular weight of 3 was 42 mass units more than that of Polygalasaponin XXXII (4) [5]. Detailed comparison of the NMR data of the two compounds implied that the structure of 3 was almost identical with 4 except for an acetyl group at δH 1.88 (3H, S), which correspond to carbon signals at δC 21.0 and 170.7 in 3. The acetyl group was attached to the C-4 of Rham2 owing to the downfield chemical shift of H-4 [δH 6.00 (1H, t, J = 9.5 Hz)] of the Rham2. Thus, the structure of 3 was elucidated as 3-O-β-D-glucopyranosyl-presenegenin 28O-α-L-arabinopyranosyl-(1 → 3)-β-D-xylo-pyranosyl-(1 → 4)-[β-D-apiofuranosyl-(1→ 3)]-α-L-rhamno-pyranosyl-(1→ 2)-[4-O-acetyl-α-L-rhamnopyranosyl-(1→3)]-[4-O-4-methoxycinnamoyl]-β-D-fucopyranosyl ester. The structures of the known saponins (4–6) were identified by comparison of their NMR and MS analysis with those reported in literatures and the structures were determined as: polygalasaponins XXXII (4) [5], II (5) [2], and F (6) [6]. Compounds 1–6 were evaluated for their neuroprotective effect on neuron-like PC12 cells induced by rotenone or 11 Aβ25–35 in vitro using the MTT method. Compounds 1–4 failed
to protect cells in rotenone model at 0.1, 1.0, and 10 μM, compounds 5 and 6 showed moderate activity in Aβ25–35 model at 10 μM (Table 3, 4 and 5). Acknowledgments This research was financially supported by the National Natural Science Foundation of China (Nos. 20372087 and 81073078) and the National Science and Technology Project of China (2011ZX09307-002-01). References [1] Jiangsu New Medical College. Dictionary of traditional Chinese medicine. Shanghai Science and Technique Publishing House; 1986. p. 757. [2] Zhang DM, Miyase T, Kuroyanagi M, Umehara K, Ueno A. Studies on the constituents of Polygala japonica Houtt. I. Structures of polygalasaponins I–X. Chem Pharm Bull 1995;43:115. [3] Zhang DM, Miyase T, Kuroyanagi M, Umehara K, Ueno A. Studies on the constituents of Polygala japonica Houtt. II. Structures of polygalasaponins XX–XIX. Chem Pharm Bull 1995;43:966. [4] Zhang DM, Miyase T, Kuroyanagi M, Umehara K, Ueno A. Studies on the constituents of Polygala japonica Houtt. III. Structures of polygalasaponins XX–XXVII. Chem Pharm Bull 1996;44:173. [5] Zhang DM, Miyase T, Kuroyanagi M, Umehara K, Ueno A. Five new triterpene saponins, polygalasaponins .XXVIII–XXXII. Chem Pharm Bull 1996;44:810.
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[6] Li TZ, Zhang WD, Yang GJ, Liu WY, Chen HS, Shen YH. Saponins from Polygala japonica and their effects on a forced Swimming test in mice. J Nat Prod 2006;69:591. [7] Xua X, Zhou JF, Li TZ, Zhang ZH, Shan L, Xiang ZH, et al. Polygalasaponin G promotes neurite outgrowth of cultured neuron on myelin. Neurosci Lett 2009;460:41. [8] Xue QC, Li CJ, Li Z, Yang JZ, Zhang DM. Three new xanthones from the roots of Polygala japonica Houtt. J Asian Nat Prod Res 2009;11:465.
[9] Fu J, Zuo L, Yang JZ, et al. Oligosaccharide polyester and triterpenoid saponins from the roots of Polygala japonica. Phytochemisry 2008;69: 1617. [10] Fu J, Zhang DM, Chen RY. Three new xanthones from the roots of Polygala japonica Houtt. J Asian Nat Prod Res 2006;8:41. [11] Pytlowany M, Strosznajder JB, Jeśko H, et al. Acta Biochim Pol 2008;55: 339.