Carbohydrate Research 346 (2011) 2206–2212

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Carbohydrate Research

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New triterpenoid saponins from the roots of Gypsophila perfoliata Linn.

Qing Chen, Jian-Guang Luo, Ling-Yi Kong ⇑Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People’s Republic of China

a r t i c l e i n f o a b s t r a c t

Article history:Received 7 March 2011Received in revised form 28 July 2011Accepted 31 July 2011Available online 5 August 2011

Keywords:Gypsophila perfoliata Linn.CaryophyllaceaeTriterpenoid saponins

0008-6215/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.carres.2011.07.027

⇑ Corresponding author. Tel./fax: +86 25 8327 1405E-mail address: cpu_lykong@126.com (L.-Y. Kong)

Nine new triterpenoid saponins (1–9) have been isolated from the roots of Gypsophila perfoliata Linn.Their structures were established on the basis of extensive NMR (1H, 13C, HSQC and HMBC) and ESIMSstudies.

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1. Introduction

The genus Gypsophila, belonging to the family Caryophyllaceae,comprises about 150 species widely distributed in Asia and Europe.Of these, more than 18 species grow in mainland China, especiallyin Xinjiang province.1 Some species have applications in either tra-ditional Chinese medicines (TCMs) or folklore herbs to treat fever,consumptive disease, and infantile malnutrition syndrome.2 Triter-penoid saponins with biological activities have been reported inour previous phytochemical investigations from this genus.3–5 Gyp-sophila perfoliata Linn., a perennial herbaceous plant, that isapproximately 70 cm tall, grows in forest grasslands, wet river-sides, saline alkaline soils, steppe sands at 500–1000 m above sealevel.1 To date, there is only one report on the chemical identifica-tion of the essential oil of this plant.6 In our continuing search forbioactive triterpenoid saponin constituents, nine new triterpenoidsaponins were isolated from the roots of G. perfoliata.

2. Results and discussion

The concentrated n-BuOH-soluble fraction of the ethanol ex-tract of G. perfoliata roots was purified by precipitation with aceto-acetate. The resulting crude saponin mixture was further subjectedto multiple chromatographic steps over MCI gel and reversed-phase C18 and finally purified by prep-HPLC, yielding nine saponins(1–9, Fig. 1). Among them, saponins 4/5, 6/7 and 8/9 were obtainedas three inseparable mixtures of their (trans)- and (cis)-acylatedderivatives.

Compound 1 was obtained as an amorphous powder. Its high-resolution ESI mass spectrum (HR-ESIMS, negative-ion mode)

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exhibited a quasi-molecular ion peak at m/z 1873.7716 [M�H](calcd for C84H129O46: 1873.7760), which was consistent with themolecular formula of C84H128O46. Acid hydrolysis of 1 with 2 MHCl afforded the aglycone and monosaccharides. The monosaccha-rides were identified as L-rhamnose, D-fucose, D-galactose, D-xylose,D-quinovose, and D-glucuronic acid (1:1:1:4:1:1) by GC–MS analy-sis. The aglycone was identified as quillaic acid by co-TLC compar-ison with a standard sample, which was also confirmed on thebasis of the 1H and 13C NMR spectroscopy (Table 1) by comparingto the reported data.3 The downfield 13C NMR chemical shift at dC

83.7 and the upfield 13C NMR chemical shift at dC 175.9 suggestedthat 1 was a 3, 28-O-bidesmoside. Compound 1 showed nine ano-meric proton signals at d 6.17 (s), 5.90 (1H, d, J = 8.5 Hz), 5.50 (1H,d, J = 7.0 Hz), 5.28 (1H, d, J = 7.5 Hz), 5.11 (1H, d, J = 7.2 Hz), 5.06(1H, d, J = 8.0 Hz), 5.03 (1H, d, J = 8.5 Hz), 4.99 (1H, d, J = 7.5 Hz)and 4.85 (1H, d, J = 7.0 Hz) displayed the correlations with anomer-ic carbon signals at dC 101.4, 94.4, 104.1, 105.0, 105.4, 106.3, 105.7,106.8, and 103.7 in HSQC spectrum, respectively. The 1H and 13CNMR data (Tables 1 and 2) of the monosaccharide residues wereassigned starting from the readily identifiable anomeric protonsby means of the TOCSY, HSQC, and HMBC spectra obtained for thiscompound. The b-anomeric configurations for the D-glucuronicacid, D-fucose, D-quinovose, D-galactose and D-xylose units, thea-anomeric configurations for L-arabinose were determined bytheir large 3JH1,H2 coupling constants of 7–8 Hz, and the a-anomer-ic configuration of L-rhamnose was judged by its C-5 (dC 67.8).3 Thesequence of the sugar residues were unambiguously defined by theHMBC experiment. Cross peaks due to long-range correlationsbetween C-3 (dC 83.7) of the aglycone and H-1 of GlcA (dH 4.85),C-2 (dC 78.6) of GlcA and H-1 of Gal (dH 5.50), C-3 (dC 86.0) of GlcAand H-1 of Xyl (dH 5.28) indicated that the trisaccharide moiety of 1attached to C-3 was established to be b-D-galactopyranosyl-(1?2)-[b-D-xylopyranosyl-(1?3)]-b-D-glucuronopyranosyl. Similarly, the

2

C

CHOO

O

O

O

HOOCHOO

OO

OHO

O

OHOO

OHHOO

OHOR2O OH

O

O

FucQui

RhaXyl'

O

O

OHOHO

OH

Xyl

OOH

HOOH

OH

GlcA R2=H

R2=3

Gal

1 OHOO

OH

OHOHO

OH

Xyl''Xyl'''

OHO

OH

OH

R2=

R1

R1= H

R1= H

R1= OH

Ara

O

C

CHO

O

OHOHO

OH

O

OHOHO

OH

OHOO

R3OOC

OOOH OH

HOOH OMe

OHO

OHOHO

OH

HOH2C

OOR1

R2OO

MeO

GlcA

Xyl

Gal

Fuc

RhaXyl'

Glc

R1=

R1= R2=H

R1=H

R1=H

6

7

8

9

R2=H

R3=H

R3=H

R3=CH3

R3=CH3

R3=CH3

R3=CH3

R1=

R1= R2=H

4

5

R2=H

O

H3CO H3CO OS1= S2=

S1

S1

S1

S2

S2

S2

R2=

R2=

Figure 1. The structures of compounds 1–9.

Table 113C NMR data (d) for the aglycone moieties of compounds 1–9 (125 MHz, C5D5N)

1 2 3 4/5 6/7 8/9

1 38.2 38.1 38.1 38.1 38.1 38.12 25.2 25.3 25.2 25.2 25.2 25.23 83.7 84.7 83.7 82.4 84.6 84.64 55.2 55.0 55.1 55.1 55.0 55.15 48.4 48.7 48.6 49.6 48.9 48.86 20.6 20.7 20.7 20.6 20.7 20.67 32.0 32.5 32.6 32.6 32.5 32.68 40.3 40.1 40.2 40.2 40.2 40.29 49.2 47.8 47.8 47.8 47.8 47.8

10 36.2 36.2 36.2 36.2 36.2 36.311 23.7 23.2 23.6 22.8 23.6 23.812 122.1 122.4 122.5 122.5 122.5 122.513 144.5 144.0 144.0 144.1 144.0 144.114 41.5 42.2 42.3 42.0 42.0 41.915 36.2 28.4 28.6 28.2 28.1 28.216 74.5 23.7 23.2 23.7 23.9 23.817 47.4 47.0 47.1 46.4 46.4 46.418 42.1 41.9 42.0 42.2 42.1 42.219 46.9 46.3 46.3 47.1 47.1 47.120 30.7 30.7 30.7 30.8 30.8 30.821 35.9 33.9 33.9 33.9 33.9 33.922 32.8 32.2 32.3 32.5 32.5 32.523 209.8 210.2 210.4 210.3 210.2 210.324 10.9 11.1 11.0 11.1 11.2 11.225 15.8 15.7 15.7 15.7 15.7 15.826 17.4 17.3 17.4 17.5 17.5 17.527 27.1 25.9 25.9 26.0 26.0 26.028 175.9 176.4 176.5 176.5 176.5 176.529 33.2 33.0 33.1 33.1 33.1 33.130 24.5 23.7 23.6 23.8 23.7 23.7

Q. Chen et al. / Carbohydrate Research 346 (2011) 2206–2212 2207

hexasaccharide moiety of 1 attached to C-28 was established to beb-D-xylopyranosyl-(1?3)-b-D-xylopyranosyl-(1?3)-b-D-xylopyr-

anosyl-(1?4)-a-L-rhamnopyranosyl-(1?2)-[b-D-quinovopyrano-syl-(1?4)]-b-D-fucopyranosyl with the long-range correlationsbetween H-1 of xylose000 (dH 4.99) with C-3 of xylose00 (dC 84.3),H-1 of xylose00 (dH 5.06) with C-3 of xylose0 (dC 86.6), H-1 of xylose0

(dH 5.11) with C-4 of rhamnose (dC 83.7), H-1 of rhamnose (dH 6.17)with C-2 of fucose (dC 73.1), H-1 of quinovose (dH 5.03) with C-4 offucose (dC 83.6), and H-1 of fucose (dH 5.90) with C-28 of the agly-cone (dC 175.9) in the HMBC spectrum. Besides the above informa-tion of the sugars and aglycone, there were other signals in 1H and13C NMR spectra of 1, suggesting the presence of two acetylgroups: dH 1.94 (3H, s), dC 170.3, 20.7 and dH 2.02 (3H, s), dC

170.1, 20.5. The location of two acetyl groups at Qui-3 and Qui-4was determined by the HMBC correlations between H-3 (d 5.58)and H-4 (d 5.02) of quinovose with the carbonyl carbons of thetwo acetyl groups (d 170.3 and 170.1), respectively. On the basisof the above results, the structure of 1 was established as 3-O-b-D-galactopyranosyl-(1?2)-[b-D-xylopyranosyl-(1?3)]-b-D-glucur-onopyranosyl quillaic acid 28-O-b-D-xylopyranosyl-(1?3)-b-D-xylopyranosyl-(1?3)-b-D-xylopyranosyl-(1?4)-a-L-rhamnopyr-anosyl-(1?2)-[3,4-di-O-acetyl-b-D-quinovopyranosyl-(1?4)]-b-D-fucopyranoside.

Compound 2 was obtained as a white, amorphous powder. Itwas assigned the molecular formula C74H114O37 from its nega-tive-ion (HR-ESIMS m/z 1593.6966 [M�H]�). Upon acid hydrolysiswith 2 M HCl 2 afforded the monosaccharides and aglycone. Themonosaccharides were identified as D-glucuronic acid, D-galactose,D-xylose, D-fucose, L-rhamnose, and D-quinovose based on GC–MSanalysis of their chiral derivatives. The aglycone was identified asgypsogenin by co-TLC comparison with standard sample, whichwas also confirmed on the basis of 1H and 13C NMR spectroscopy(Table 1) by comparison to the reported data.5 The downfield 13C

Table 21H and 13C NMR data for sugar moieties of compounds 1–3 (C5D5N)

1 2 3

3-O- 13C 1H 13C 1H 13C 1HGlcA1 103.7 4.85 (7.0) 104.3 4.82 (7.5) 105.5 5.17 (7.0)2 78.6 4.29 78.4 4.26 78.3 4.253 86.0 4.22 86.1 4.20 86.7 4.034 71.4 3.91 71.6 4.72 71.6 4.585 76.5 3.95 76.6 3.93 76.5 3.996 Nd* Nd* Nd*

Gal1 104.1 5.50 (7.0) 104.0 5.46 (7.8) 104.1 5.51 (7.2)2 73.6 4.39 73.6 4.42 73.6 4.433 75.4 4.46 75.4 4.09 75.5 3.974 70.2 3.65 70.7 4.15 70.8 4.115 76.4 4.17 76.5 4.17 76.5 3.916 61.6 4.36, 4.48 61.6 4.38/4.46 61.5 4.38, 4.49Xyl1 105.0 5.28(7.5) 105.1 5.19 (7.5) 105.1 5.21 (7.7)2 75.2 4.12 75.2 3.91 75.2 4.113 78.7 4.33 78.4 4.28 78.6 4.094 70.8 4.10 69.6 4.20 69.6 4.235 67.3 4.22, 4.23 67.7 3.71, 4.22 67.1 3.77, 4.29

28-O-Fuc1 94.4 5.90 (8.5) 94.3 5.90 (8.0) 94.3 5.94 (7.9)2 73.9 4.48 74.7 4.48 74.7 4.083 76.4 4.15 76.5 4.17 76.6 4.184 83.6 3.97 83.7 4.02 83.7 3.995 71.4 3.92 71.3 3.89 71.3 3.926 17.0 1.49 (6.0) 17.0 1.48 (6.3) 17.0 1.48 (6.3)Qui1 105.7 5.03(8.5) 105.7 5.01(7.8) 105.8 5.05 (8.1)2 73.1 3.97 73.1 3.98 73.1 4.033 76.2 5.58 76.1 5.56 76.2 5.614 74.3 5.02 74.3 5.02 74.4 5.065 70.2 3.66 70.2 3.67 70.2 3.706 17.7 1.22 (6.0) 17.6 1.22 (6.2) 17.7 1.24 (6.1)Rha1 101.4 6.17 101.5 6.20 101.5 6.232 71.7 4.67 71.3 4.38 70.1 4.563 72.3 4.57 72.4 4.56 72.4 4.564 83.7 4.29 85.3 4.24 85.4 4.265 68.3 4.35 68.3 4.37 68.1 4.366 18.5 1.53(5.5) 18.5 1.64 (6.2) 18.5 1.61 (6.1)Xyl0

1 105.4 5.11 (7.2) 107.6 4.95 (7.4) 107.1 4.96 (7.2)2 75.3 3.93 75.4 4.09 74.5 4.523 86.6 3.98 76.5 4.17 86.2 4.204 68.9 3.97 70.7 4.15 68.8 4.085 66.9 3.36, 4.12 67.3 3.47, 4.17 67.7 3.73, 4.26Xyl00

1 106.3 5.06 (8.0)2 73.0 4.463 84.3 4.004 69.6 4.225 67.2 3.66, 3.70Xyl000

1 106.8 4.99 (7.5)2 76.5 3.973 78.6 4.104 71.0 4.415 66.4 3.74, 4.54Ara1 105.0 5.282 71.4 4.734 72.9 4.425 69.3 4.316 66.9 3.45, 4.20Qui-3-OAc 170.3 170.3 170.4

20.7 1.94 20.7 1.95 20.7 1.95Qui-4-OAc 170.1 170.0 170.1

20.5 2.02 20.6 2.03 20.6 2.03

* nd = not detected.

2208 Q. Chen et al. / Carbohydrate Research 346 (2011) 2206–2212

Q. Chen et al. / Carbohydrate Research 346 (2011) 2206–2212 2209

NMR chemical shift at dC 84.7 and the upfield 13C NMR chemicalshift at dC 176.4 suggested that 2 was a bidesmosidic saponin withglycosidic linkages at C-3 through an ether bond and at C-28through an ester bond. The anomeric proton signals at d 6.20 (s),5.90 (1H, d, J = 8.0 Hz), 5.46 (1H, d, J = 7.8 Hz), 5.19 (1H, d,J = 7.5 Hz), 5.01 (1H, d, J = 7.8 Hz), 4.95 (1H, d, J = 7.4 Hz) and 4.82(1H, d, J = 7.5 Hz) gave correlations with anomeric carbon signalsat d 101.5, 94.3, 104.0, 105.1, 105.7, 107.6 and 104.3 in HSQC spec-trum, respectively, which showed that 2 contained seven sugarunits. The 1H and 13C NMR data (Table 2) of the monosaccaride res-idues were assigned starting from the readily identifiable anomericprotons by means of the HSQC, and HMBC spectra obtained for this

Table 31H NMR data for sugar moieties of compounds 4–9 (500 MHz, C5D5N)

4 5 6

3-O-GlcA1 4.79 (7.2) 4.79 (7.2) 4.79 (7.6)2 4.21 4.21 4.233 4.16 4.16 4.194 4.27 4.27 4.215 3.96 3.96 4.376OCH3 3.71Gal1 5.46 (7.7) 5.46 (7.7) 5.44 (7.8)2 4.41 4.41 4.413 4.08 4.08 3.904 4.51 4.51 4.515 3.96 3.96 3.926 4.42, 4.51 4.42, 4.51 4.38, 4.46Xyl1 5.17 (7.8) 5.17 (7.8) 5.15 (9.2)2 3.93 3.93 3.903 4.06 4.06 4.064 4.21 4.21 4.055 3.35, 4.15 3.35, 4.15 3.35, 4.16

28-O-Fuc1 6.06 (8.0) 6.00 (8.0) 6.07 (8.0)2 4.03 4.03 4.043 4.40 4.40 4.434 5.68 5.68 5.675 4.21 4.21 4.486 1.24 1.24 1.28Rha1 6.03, s 5.96, s 6.03, s2 5.17 5.17 5.173 4.82 4.82 4.814 4.50 4.50 4.505 4.47 4.47 4.196 1.73 1.73 1.76Glc1 5.31 (7.6) 5.31 (7.6) 5.30 (7.8)2 4.40 4.40 3.903 3.96 3.96 4.374 4.04 4.04 4.035 3.86 3.86 4.096 4.15, 4.51 4.15, 4.51 4.14, 4.43Xyl1 5.38 (7.0) 5.38 (7.0) 5.39 (6.5)2 4.08 4.08 4.093 4.06 4.06 4.544 4.14 4.14 4.145 3.35, 4.15 3.35, 4.15 3.70, 4.21p-Methoxycinnamoyl100

200 ,600 7.40 (8.5) 7.94 (8.5) 7.41 (8.8)300 ,500 6.94 (8.5) 6.92(8.5) 6.92 (8.9)a 6.54 (16) 6.00 (13) 6.53 (16)b 7.87 (16) 6.85 (13) 7.87 (16)COOMe 3.62 3.61 3.62

compound. The b-anomeric configurations for the D-glucuronicacid, D-fucose, D-galactose, D-quinovose and D-xylose units weredetermined by their large 3JH1,H2 coupling constants of 7–8 Hz.And the a-anomeric configuration of L-rhamnose was judged byits C-5 resonance (dC 68.3).4 The sequence of the sugar residueswas subsequently determined by HMBC experiments. The linkageof the sugar units at C-3 of the aglycone was established fromthe following HMBC correlations: H-1 of galactose (dH 5.46) withC-2 of glucuronic acid (dC 78.4), H-1 of xylose (dH 5.19) with C-3of glucuronic acid (dC 86.1), and H-1 of glucuronic acid (dH 4.82)with C-3 of the aglycone (dC 84.7). Thus, the sugar chain attachedto C-3 was determined to be 3-O-b-D-galactopyranosyl-(1?2)-[b-

7 8 9

4.79 (7.6) 4.85 (7.3) 4.85 (7.3)4.23 4.28 4.284.19 4.19 4.194.21 4.18 4.184.37 3.99 3.99

3.71 3.73 3.73

5.44 (7.8) 5.51(7.6) 5.51 (7.6)4.41 4.45 4.453.90 4.45 4.454.51 4.55 4.553.92 3.93 3.934.38, 4.46 4.41, 4.47 4.41, 4.47

5.15 (9.2) 5.25(7.8) 5.25 (7.8)3.90 3.95 3.954.06 4.08 4.084.05 4.26 4.263.35, 4.16 3.38, 4.18 3.38, 4.18

6.02 (8.0) 6.10 (7.9) 6.05 (7.9)4.04 4.13 4.134.43 5.69 5.695.67 4.40 4.404.48 4.08 4.081.28 1.29 1.29

5.93, s 6.06, s 5.98, s5.17 5.17 5.174.81 4.82 4.824.50 4.51 4.514.19 4.51 4.511.76 1.74 1.74

5.30 (7.8) 5.31 (7.9) 5.31 (7.9)3.90 3.95 3.954.37 4.38 4.384.03 4.07 4.074.09 3.84 3.844.14, 4.43 4.18, 4.45 4.18, 4.45

5.39 (6.5) 5.39 (7.9) 5.39 (7.9)4.09 4.09 4.094.54 4.08 4.084.14 3.83 3.833.70, 4.21 3.62, 4.22 3.62, 4.22

7.94 (8.9) 7.41 (8.8) 7.95 (8.8)6.92 (9.0) 6.92 (8.9) 6.90 (8.9)6.00 (12.9) 6.54(15.9) 6.01(12.9)6.85 (12.9) 7.88(15.9) 6.86(12.9)

3.61 3.68 3.63

2210 Q. Chen et al. / Carbohydrate Research 346 (2011) 2206–2212

D-xylopyranosyl-(1?3)]-b-D-glucuronopyranosyl. Similarly, thesugar chain at C-28 was also established to be b-D-xylopyranosyl-(1?3)-a-L-rhamnopyranosyl-(1?2)-[3,4-di-O-acetyl-b-D-quinovo-pyranosyl-(1?4)]-b-D-fucopyranosyl from the following HMBCcorrelations: H-1 of xylose (dH 4.95) with C-4 of rhamnose (dC

85.3), H-1 of rhamnose (dH 6.20) with C-2 of fucose (dC 74.7), H-1of quinovose (dH 5.01) with C-4 of fucose (dC 83.7), and H-1 of fu-cose (dH 5.90) with C-28 of the aglycone (dC 176.4). There were twoadditional acetyl signals visible in the NMR spectra. Attachment ofthese groups to C-3 and C-4 of quinovose was confirmed by HMBCcorrelations between H-3 (d 5.56) and H-4 (d 5.02) of quinovose

Table 413C NMR data for sugar moieties of compounds 4–9 (125 MHz, C5D5N)

4 5 6

3-O-GlcA1 103.7 103.7 103.92 78.4 78.4 78.53 85.6 85.6 85.74 71.2 71.2 70.95 76.0 76.0 76.66 169.8 169.8 169.8OCH3 52.1Gal1 104.0 104.0 104.22 73.6 73.6 73.73 75.8 75.8 76.04 70.2 70.2 70.25 76.0 76.0 75.76 61.7 61.7 61.7Xyl1 105.0 105.0 105.12 75.3 75.3 75.33 79.4 79.4 79.44 70.8 70.8 70.75 67.1 67.1 67.1

28-O-Fuc1 94.8 94.8 94.82 74.7 74.7 74.73 72.8 72.8 72.94 74.5 74.5 74.55 69.6 69.6 69.16 16.7 16.7 16.7Rha1 102.3 102.3 102.32 71.2 71.2 71.13 82.4 82.4 82.34 78.9 78.9 78.85 69.0 69.0 69.66 19.0 19.0 19.0Glc1 105.3 105.3 105.32 73.1 73.1 75.33 76.4 76.4 76.44 71.7 71.7 71.85 78.2 78.2 78.36 62.6 62.6 62.7Xyl1 105.0 105.0 105.22 75.3 75.3 75.43 78.6 78.6 76.24 71.2 71.2 71.25 67.7 67.7 67.7p-Methoxycinnamoyl100 127.5 127.9 127.5200 ,600 130.3 133.1 130.3300 ,500 114.7 113.9 114.7400 161.9 161.0 161.9a 116.3 117.2 116.3b 145.1 143.9 145.1CO 167.6 166.6 167.6OMe 55.3 55.2 55.3

with the carbonyl carbons of the two acetyl groups (d 170.4, d170.1), respectively. On the basis of the data obtained, the struc-ture of 2 was established as 3-O-b-D-galactopyranosyl-(1?2)-[b-D-xylopyranosyl-(1?3)]-b-D-glucuronopyranosyl gypsogeninb-D-xylopyranosyl-(1?4)-a-L-rhamnopyranosyl-(1?2)-[3,4-di-O-acetyl-b-D-quinovopyranosyl-(1?4)]-b-D-fucopyranoside.

Compound 3 (Fig. 1) was obtained as a white, amorphous pow-der. It was assigned the molecular formula C79H122O41 from its neg-ative-ion (HR-ESIMS m/z 1725.7371 [M�H]�). The overall structureassignment was accomplished using the same protocol as in 1 and2. Acid hydrolysis afforded gypsogenin and L-arabinose, L-rham-

7 8 9

103.9 103.9 103.978.5 78.4 78.485.7 85.7 85.770.9 71.0 71.076.6 76.6 76.6

169.8 169.8 169.852.1 52.2 52.2

104.2 104.2 104.273.7 73.6 73.676.0 76.0 76.070.2 70.3 70.375.7 75.9 75.961.7 61.7 61.7

105.1 105.0 105.075.3 75.2 75.279.4 79.4 79.470.7 70.8 70.867.1 67.1 67.1

94.8 94.8 94.874.7 75.4 75.472.9 74.5 74.574.5 73.1 73.169.1 70.6 70.616.7 16.6 16.6

102.3 102.3 102.371.1 71.2 71.282.3 82.4 82.478.8 78.9 78.969.6 69.0 69.019.0 19.0 19.0

105.3 105.3 105.375.3 75.2 75.276.4 76.4 76.471.8 71.8 71.878.3 78.3 78.362.7 62.7 62.7

105.2 105.3 105.375.4 75.3 75.376.2 78.6 78.671.2 71.8 71.867.7 67.3 67.3

127.9 127.5 127.9133.1 130.3 133.1113.9 114.7 113.9161.0 161.9 161.0117.3 116.3 117.2143.9 145.2 143.9166.6 167.6 166.7

55.2 55.3 55.2

Q. Chen et al. / Carbohydrate Research 346 (2011) 2206–2212 2211

nose, D-fucose, D-galactose, D-xylose, D-quinovose and D-glucuronicacid. The linkage positions were determined by the HMBC experi-ment. It was evident that 3 had the same trisaccharides linked toC-3 of the aglycone as in 1 and 2 (Table 1), as the expected sequencecorrelations were observed. The linkage of the remaining five sug-ars at C-28 was determined from the following HMBC correlationsbetween H-1 of arabinose (dH 5.28) with C-3 of xylose (dC 86.2), H-1of xylose (dH 4.96) with C-4 of rhamnose (dC 85.4), H-1 of rhamnose(dH 6.23) with C-2 of fucose (dC 73.1), H-1 of quinovose (dH 5.05)with C-4 of fucose (dC 83.7), and H-1 of fucose (dH 5.94) with C-28of the aglycone (dC 176.5). Attachment of the two acetyl groups toC-3 and C-4 of quinovose was determined from HMBC correlationsbetween H-3 (d 5.61) and H-4 (d 5.06) of quinovose with the car-bonyl carbons of the two acetyl groups (d 170.4 and 170.1), respec-tively. On the basis of all the foregoing evidence, compound 3 waselucidated as 3-O-b-D-galactopyranosyl-(1?2)-[b-D-xylopyrano-syl-(1?3)]-b-D-glucuronopyranosyl gypsogenin a-L-arabinopyran-syl-(1?3)-b-D-xylopyranosyl-(1?4)-a-L-rhamnopyranosyl-(1?2)-[3,4-di-O-acetyl-b-D-quinovopyranosyl-(1?4)]-b-D-fucopyranoside.

Each mixture 4/5, 6/7 or 8/9 (Fig. 1) was homogeneous by HPTLCbut was separated into trans and cis-isomers by HPLC, but all at-tempts to separate each saponin pair by semipreparative HPLCwere unsuccessful. This phenomenon of isomerization referred tothe effect of light on the 4-methoxycinnamoyl group in aqueousMeOH solution. Under these conditions, the geometrical isomericstructures of the p-methoxycinnamoyl groups showed tautomer-like behavior. Such a phenomenon has been previously observedand explained from Polygala senega,7 Silene jenisseensis,8 and Silenefortunei.9

Acid hydrolysis of compounds 4–9, obtained as white amor-phous powders, afforded a gypsogenin, D-glucose, D-galactose,D-xylose, L-rhamnose, D-glucuronic acid and D-fucose, which indi-cated they have same aglycone and sugar moiety.

The high-resolution ESI mass spectrometry (HRESIMS) (nega-tive-ion mode) of 4/5 exhibited a pseudomolecular ion peak atm/z 1685.7216 [M�H]� (calcd for C80H117O38, 1685.7228) consis-tent with a molecular formula of C81H120O38. The 13C NMR signalsof 4/5 (Table 4) showed six anomeric signals at d 94.8, 102.3, 103.7,104.0, 105.0, 105.3, which correlated in the HSQC spectrum with1H NMR spectrum (Table 3) at d 6.06/6.00, 6.03/5.96, 4.79, 5.46,5.17, 5.31, respectively. Similar to 1–3, the trisaccharide moietyof 4/5 attached to C-3 and C-28 was established to be b-D-galacto-pyranosyl-(1?2)-[b-D-xylopyranosyl-(1?3)]-b-D-glucuronopyr-anosyl and b-D-glucopyranosyl-(1?3)-[b-D-xylopyranosyl-(1?4)]-a-L-rhamnopyranosyl-(1?2)-b-D-fucopyranosyl, respectively,from the HMBC spectrum. In addition to those arising from thesugars and aglycone, there were other signals in 1H NMR spectrum,which suggested the presence of (E/Z)-p-methoxycinnamoyl {[(E)-MC] group: d(H) 7.87 and 6.54 [1H each, d, J = 16.0 Hz, (E)-MC–H–C(b)/C(a)]; d 7.40 and 6.94 [2H each, d, J = 8.5 Hz, (E)-MC–H–C(2),C(6) and H–C(3), C(5)]; [(Z)-MC] group: d(H) 6.85 and 6.00 [1Heach, d, J = 13.0 Hz, (Z)-MC–H–C(b)/C(a)]; d 7.94 and 6.92 [2H each,d, J = 8.5 Hz, (Z)-MC–H–C(2), C(6) and H-C(3), C(5)]}. The downfieldshifts for the Fuc H-4/Fuc C-4 resonances at dH 5.68/dC 74.5 re-vealed the secondary alcoholic functions Fuc-4-OH to be acylated.An HMBC correlation was observed between dH 5.68 (Fuc-4) and dC

167.6/166.6 (carbonyl group of the 4-methoxycinnamoyl) provingthat the 4-methoxycinnamoyl groups was attached to the Fuc unitat C-4. Study of the NMR spectra of 4/5 led to the establishment oftheir structures as 3-O-b-D-galactopyranosyl-(1?2)-[b-D-xylopyr-anosyl-(1?3)]-b-D-glucuronopyranosyl gypsogenin 28-b-D-xylo-pyranosyl-(1?4)-[b-D-glucopyranosyl-(1?3)]-a-L-rhamnopyrano-syl-(1?2)-4-O-trans-p-methoxycinnamoyl-b-D-fucopyranosyl (4)and its cis-isomer (5), as new compounds.

The HR-ESIMS (negative-ion mode) of compounds 6/7 exhibiteda quasimolecular ion peak at m/z 1699.7367 [M�H]� (calcd for

C81H119O38, 1699.7384), consistent with the molecular formula ofC81H120O38. The 1H and 13C NMR data of 6/7 (Tables 1, 3 and 4) as-signed from HSQC and HMBC experiments were similar to those of4/5, except for the appearance of one additional methyl group. Themethyl group was located at GlcA-C-6 by the HMBC correlation ofdH 3.71 (OCH3) and dC 169.8 (GlcA-C-6). On the basis of the aboveobservations, the structures of 6/7 were determined as 3-O-b-D-galactopyranosyl-(1?2)-[b-D-xylopyranosyl-(1?3)]-6-O-methyl-b-D-glucuronopyranosyl gypsogenin 28-b-D-xylopyranosyl-(1?4)-[b-D-glucopyranosyl-(1?3)]-a-L-rhamnopyranosyl-(1?2)-4-O-trans-p-methoxycinnamoyl-b-D-fucopyranoside (6) and itscis-isomer (7).

Compound 8/9 had the molecular formula C81H120O38, as re-vealed by HR-ESIMS (m/z 1699.7386 [M�H]�, calcd forC81H119O38, 1699.7384). The 1H and 13C NMR signals of 8/9 were al-most superimposable on those of 6/7 except for the fucose residue(Tables 1 and 2). The downfield shifts observed in the HSQC spec-trum for the Fuc-H-3/Fuc-C-3 resonances at dH 5.69/dC 74.5 indi-cated the primary alcoholic function of Fuc-3-OH to be acylated.That the 4-methoxycinnamoyl group was attached to the Fuc unitat C-3 was further proved by the HMBC correlation of dH 5.69(OCH3) and dC 167.6/166.7 (carbonyl group of the 4-methoxycinna-mate). Based on this analysis, the structure of 8/9 was establishedas 3-O-b-D-galactopyranosyl-(1?2)-[b-D-xylopyranosyl-(1?3)]-6-O-methyl-b-D-glucuronopyranosyl gypsogenin 28-b-D-xylopyrano-syl-(1?4)-[b-D-glucopyranosyl-(1?3)]-a-L-rhamnopyranosyl-(1?2)-3-O-trans-p-methoxycinnamoyl-b-D-fucopyranosyl (8) and itscis-isomer (9).

3. Experimental

3.1. General experimental procedures

Optical rotations were measured with a JASCO P-1020 polarim-eter (cell length: 1.0 dm). IR (KBr-disks) spectra were recorded byBrucker Tensor 27 spectrometer. Mass spectra were obtained ona MS Agilent 1100 Series LC/MSD Trap mass spectrometer (ESIMS)and a G1969A TOF MS (HRESIMS), respectively. 1D and 2D NMRspectra were recorded in C5D5N at 300 K on Bruker ACF-500NMR (1H: 500 MHz, 13C: 125 MHz) spectrometers, in which cou-pling constants were given in Hz. Gas chromatography was doneon a Varian CP-3800 Gas Chromatograph equipped with a Saturn2200 Mass detector. All solvents used were of analytical or chro-matographic grade (Jiangsu Hanbang Sci. & Tech. Co. Ltd). TLCwas performed on precoated silica gel 60 F254 plates (QingdaoHaiyang Chemical Co. Ltd), and detection was achieved by 10%H2SO4–EtOH for saponins. MCI gel (37–75 lm, Mitsubishi), andODS-C18 (40–63 lm, Fuji) were used for column chromatography.Preparative HPLC was carried out using Agilent 1100 Series withShim-park RP-C18 column (200 � 20 mm i.d.) and 1100 Series Mul-tiple Wavelength detector.

3.2. Plant material

The roots ofG. perfoliata Linn. were collected from Zhaosu County,Xinjiang Province, People’s Republic of China, in August 2007, andidentified by Professor Rena Kasimu (College of PharmaceuticalSciences, Xinjiang Medical University, China). Voucher specimens(No. 070806) were deposited at the Department of Natural Medici-nal Chemistry, China Pharmaceutical University, Nanjing, China.

3.3. Extraction and isolation

The air-dried plants (1.3 kg) were extracted with 70% aqueousethanol (v/v) three times (10 L, 2 h each time) at reflux. After

2212 Q. Chen et al. / Carbohydrate Research 346 (2011) 2206–2212

evaporation, the residue was suspended in H2O and partitioned,with EtOAc and n-BuOH, respectively. Then the n-BuOH fraction(36 g) was purified by chromatography over a MCI gel columneluted with 30%, 70% and 100% MeOH, respectively. The 70% MeOHportion was fractionated by RP-C18 column (MeOH–H2O, 1:9, 5:5,7:3, 8:2 and 10:0) to give five fractions (Fractions 1–5). Fraction3 was further separated by HPLC (MeCN–0.05% TFA in H2O,43:57, UV detection at 210 nm), to yield 1 (11 mg), 2 (20 mg)and 3 (13 mg), respectively. Fraction 4 was subjected to RP-C18 col-umn with MeOH–H2O (4:6?8:2) to give 3 fractions and then Frac-tion 4.1 was purified by HPLC (MeCN–0.05% TFA in H2O, 45:55, UVdetection at 210 nm) to yield 4/5 (9 mg). Fraction 4.2 was sub-jected to HPLC with 48% CH3CN in H2O to yield 6/7 (15 mg) and8/9 (12 mg), respectively.

Compound 1: White amorphous powder; ½a�25D +0.9 (c 0.09,

MeOH); IR (KBr) cm�1: 3419, 2940, 1730, 1680, 1642, 1433,1385, 1253, 1207, 1140, 1073; ESIMS m/z: 1873 [M�H]�, HR-ESIMSm/z: 1873.7716 [M�H]� (calcd for C84H129O46, 1873.7760); 1HNMR (C5D5N, 500 MHz) and 13C NMR (C5D5N, 125 MHz) data aregiven in Tables 1 and 2.

Compound 2: White amorphous powder; ½a�25D +5.1 (c 0.09,

MeOH); IR (KBr) cm�1: 3423, 2942, 1750, 1638, 1384, 1256, 1056;ESIMS m/z: 1594 [M�H]�, HR-ESIMS m/z: 1593.6961 [M�H]� (calcdfor C74H113O37: 1593.6966); 1H NMR (C5D5N, 500 MHz) and 13C NMR(C5D5N, 125 MHz) data are given in Tables 1 and 2.

Compound 3: White amorphous powder; ½a�25D �4.0 (c 0.05,

MeOH); IR (KBr) cm�1: 3423, 2934, 1750, 1728, 1633, 1385,1256, 1137, 1061; ESIMS: m/z 1725 [M�H]�, HR-ESIMS m/z:1725.7371 [M�H]� (calcd for C79H121O41: 1725.7388); 1H NMR(C5D5N, 500 MHz) and 13C NMR (C5D5N, 125 MHz) data are givenin Tables 1 and 2.

Compound 4/5: White amorphous powder; IR (KBr) cm�1: 3425,2926, 2854, 1679, 1633, 1385, 1256, 1205, 1141, 1076, 1046; ESIMS:m/z 1686 [M�H]�, HR-ESIMS m/z: 1685.7216 [M�H]� (calcd forC80H117O38: 1685.7228) 1H NMR (C5D5N, 500 MHz) and 13C NMR(C5D5N, 125 MHz) are given in Tables 1, 3 and 4.

Compound 6/7: White amorphous powder; IR (KBr) cm�1: 3423,2931, 1679, 1632, 1605, 1513, 1444, 1386, 1257, 1161, 1077, 1048,913; ESIMS: m/z 1737 [M+Cl]�, HR-ESIMS m/z: 1699.7367 [M�H]�

(calcd for C81H119O38, 1699.7384). 1H NMR (C5D5N, 500 MHz) and13C NMR (C5D5N, 125 MHz) are given in Tables 1, 3 and 4.

Compound 8/9: White amorphous powder; IR (KBr) cm�1:3442, 2938, 1633, 1513, 1385, 1256, 1161, 1076, 1047; ESIMS:m/z 1737 [M+Cl]�, HR-ESIMS m/z: 1699.7386 [M�H]� (calcd forC81H119O38, 1699.7384). 1H NMR (C5D5N, 500 MHz) and 13C NMR(C5D5N, 125 MHz) are given in Tables 1, 3 and 4.

3.4. Acid hydrolysis of compounds 1–9 and determination ofabsolute configuration of monosaccharides

Each saponin (3 mg) was heated in 2 M HCl (5 mL) at 90 �C for4 h. The reaction mixture was extracted with CHCl3 (5 mL � 3).The CHCl3 extract was purified by chromatography on SephadexLH-20 (2.0 � 100 cm). Comparing the TLC with authentic samples,the aglycone of 1 was determined as quillaic acid. Acid hydrolysisof 2–9 was operated likewise, and the aglycone of 2–9 was deter-mined to be gypsogenin. Each remaining aqueous layer was con-centrated to dryness to give a residue and dissolved in pyridine(2 mL), and then L-cysteine methyl ester hydrochloride (2 mg)was added to the solution. The mixture was heated at 60 �C for1 h, and chlorotrimethylsilane (0.5 mL) was added, followed byheating at 60 �C for 30 min. Then, the solution was concentratedto dryness and dissolved in water (1 mL � 3), followed by extrac-tion with n-hexane (1 mL � 3). The hexane extract was subjectedto GC–MS analysis. The absolute configurations of the monosac-charides were confirmed to be L-arabinose, D-quinovose, D-fucose,L-rhamnose, D-xylose, D-glucuronic acid, D-galactose, and D-glucoseby comparison of the retention times of monosaccharide deriva-tives with those of standard samples: L-arabinose (13.39 min), D-xylose (13.42 min), D-quinovose (14.00 min), L-rhamnose (14.18min), D-fucose(14.35 min), D-galactose (15.77 min), D-glucose(15.49 min), and D-glucuronic acid (17.10 min), respectively.

Acknowledgments

This research work was financially supported by the NationalNatural Science Foundation of China (30830116, 81073009), anda Project Funded by The Priority Academic Program Developmentof Jiangsu Higher Education Institutions.

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