Isolation and Identification of Saponins from the Natural Pasturage

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Isolation and Identification of Saponins from the Natural Pasturage Asterothamnus centrali-asiaticus Employing Preparative Two-dimensional Reversed-phase Liquid Chromatography/hydrophilic Interaction Chromatography yan-ming wang, jian-qiang zhao, Junli Yang, Yan-Duo Tao, Lijuan Mei, and Yan-Ping Shi J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02096 • Publication Date (Web): 27 May 2016 Downloaded from http://pubs.acs.org on May 31, 2016

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Journal of Agricultural and Food Chemistry

Isolation and Identification of Saponins from the Natural Pasturage Asterothamnus centrali-asiaticus Employing Preparative Two-dimensional Reversed-phase Liquid Chromatography/hydrophilic Interaction Chromatography Yan-Ming Wang†,§,#, Jian-Qiang Zhao‡,§,#, Jun-Li Yang†, Yan-Duo Tao‡, Li-Juan Mei‡*, Yan-Ping Shi†* †

Key Laboratory of Chemistry of Northwestern Plant Resources of CAS and Key

Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, P. R. China ‡

Key Laboratory of Tibetan Medicine Research, Northwest Institute of Plateau Biology,

Chinese Academy of Sciences, Xining 810008, P. R. China §

University of Chinese Academy of Sciences, 100049, Beijing, P. R. China

*Corresponding author (Tel.: + 86-931-4968208; Fax: + 86-931-8277088; E-mail: [email protected])

1

ACS Paragon Plus Environment


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1

ABSTRACT

2

Asterothamnus centrali-asiaticus, a kind of a characteristic shrub abundant in grassland

3

and desert areas, has been used as forage fodder for camels and goats in Central Asia, and

4

this plant also plays a critical role in the maintenance of desert grassland ecosystems due

5

to its tolerance to poor soils and sand burial. However its chemical composition has been

6

rarely reported. In this study, phytochemical investigation of this pasturage was

7

performed and three new triterpenoid saponins, 1-3, were isolated together with nine

8

known

9

chromatography/hydrophilic interaction chromatography (2D RPLC/HILIC). Their

10

structures were elucidated via diverse spectroscopic analyses, including infrared

11

spectrometry

12

(HR-ESIMS), and 1D and 2D nuclear magnetic resonance (NMR). All isolated

13

triterpenoid saponins, 1-12, were reported from this genus for the first time and they were

14

further evaluated for their cytotoxicity against four cancer cell lines (A549, HepG2,

15

MGC-803, and MFC), which indicated that compound 11 showed potent cytotoxicity

16

against HepG2 cell line with IC50 value of 6.85 µg/mL.

ones,

4-12,

(IR),

using

preparative

high-resolution

two-dimensional

electrospray

ionization

reversed-phase

mass

liquid

spectrometry

17

Asterothamnus

18

KEYWORDS:

19

RPLC/HILIC, cytotoxicity

centrali-asiaticus,

triterpenoid

2


saponins,

2D

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20

INTRODUCTION

21

Some plants of the Compositae family have been considered as important forage fodder

22

resources in Central Asia. Asterothamnus centrali-asiaticus, belonging to the Compositae

23

family, has long been used as fodder for camels and goats. The plant is widely distributed

24

in meadows, gritty-stony foothills, stony riverbeds, open sand dunes, deserts, and

25

grasslands of Gansu, Nei Mongol, Ningxia, Qinghai, and Xinjiang provinces in

26

Northwestern China, and it is also distributed in southern Mongolia.1-2 Evaluation on its

27

nutrient content demonstrated that A. centrali-asiaticus was rich in phosphorus and

28

calcium, and the content of essential amino acids was equivalent to those in corn, wheat,

29

and barley. Furthermore, this plant also plays a critical role in the maintenance of desert

30

grassland ecosystems, due to its tolerance to poor soils and sand burial and its cold and

31

drought resistance.3 However, only a cembrane glycoside was reported from this

32

pasturage up to now,4 and the chemical constituents of this plant or even the genus

33

Asterothamnus are still not clearly understood.

34

Triterpenoid saponins have been considered as the primary chemical constituents of

35

the genus Aster5-11 which is closely related to the genus Asterothamnus. Traditional

36

chromatographic isolation and purification of triterpenoid saponins had been tedious and

37

time-consuming, especially for those with more than three sugar units. With the

38

launching of new chromatographic materials, many new high-performance liquid

39

chromatography (HPLC) methods were developed and introduced to the analysis and

40

isolation

41

chromatography (RPLC), hydrophilic interaction chromatography (HILIC), and the

42

two-dimensional (2D) HILIC/RPLC had been successfully applied to purification of

of

triterpenoid

saponins.12-14

For

instance,

3


reversed-phase

liquid


43

glycosides of terpenes and steroids in many cases.15-19 In this study, a preparative 2D

44

RPLC/HILIC orthogonal system, with XCharge C18 column as the first dimension, and

45

XAmide column as the second dimension, was used for the purification of twelve

46

triterpenoid saponins, 1-12, from A. centrali-asiaticus. Since many triterpenoid saponins

47

show cytotoxic activity against cancer cells,20 all of the isolates were evaluated for their

48

cytotoxicity against four cancer cell lines A549, HepG2, MGC-803, and MFC. The

49

isolation, structural elucidation, and cytotoxicity of these compounds are described in this

50

paper.

51 52

MATERIALS AND METHODS

53

General Experimental Procedures.

54

IR spectra were measured on a Nicolet NEXUS 670 FT-IR spectrometer Bio-Rad

55

(Nicolet, Madison, WI, USA) in dry film. HRESI-MS were run on a Bruker microTOF-Q

56

II mass spectrometer (Bruker Daltonics, Billerica, MA, USA). Optical rotations were

57

determined on a Perkin-Elmer model 341 polarimeter (PerkinElmer, Wellesley, MA,

58

USA) with a 1 dm cell. UV spectra were recorded on a T6-New Century

59

spectrophotometer (Pgeneral, Beijing, China). NMR spectra were measured in CD3OD

60

and recorded on a Bruker Avance III-400 spectrometer (Bruker, Rheinstetten, Germany)

61

at 25 °C. TMS was used as internal standard. Chemical shifts are reported in δ (ppm) and

62

coupling constants (J) are expressed in Hz. Column chromatography (CC) was preformed

63

over silica gel (100-200 mesh) (Qingdao Haiyang Chemical Co., Qingdao, China).

64

Pre-coated silica gel plates (Qingdao Haiyang Chemical Co., Qingdao, China) were used

65

for thin layer chromatography (TLC) analysis. Detection was done under UV light (254

4


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66

nm and 365 nm) and by spraying the plate with 10% sulfuric acid ethanol solution

67

followed by heating. An Agilent series 1200 (Agilent Technologies, Palo Alto, CA, USA)

68

was used for HPLC analysis. A Hanbon preparative HPLC (Hanbon Sci & Tech, Jiangsu,

69

China), XCharge C18 column (250 mm × 20 mm i.d., 10 µm, 100 Å) (Acchrom, Beijing,

70

China) and Xamide column (250 mm × 20 mm i.d., 10 µm 100 Å) (Acchrom, Beijing,

71

China) were used for preparative HPLC separations.

72

Plant Material.

73

The aerial parts of Asterothamnus centrali-asiaticus were collected in Xun-hua County of

74

Qing-hai Province, People’s Republic of China, in June 2012 and verified by Professor

75

Li-juan Mei. The voucher specimen (No.20121911) of A. centrali-asiaticus has been

76

deposited in the Key Laboratory of Tibetan Medicine Research, Norhtwest Institute of

77

Plateau Biology, Chinese Academy of Science, China.

78

Extraction and isolation.

79

The air-dried aerial parts of A. centrali-asiaticus (7 kg) powdered by a pulverizer were

80

extracted three times with 95% aqueous ethanol (3 × 50 L) at 65 °C. After filtration using

81

cotton and then concentration in vacuo, an ethanol-free residue (1 L) was obtained and

82

subjected to a liquid-liquid extraction successively with ethyl acetate (4 ×1 L) and

83

n-BuOH (4 ×1 L) against water (1 L), which yielded the dried EtOAc (238 g) and

84

n-BuOH (80 g) extract. The n-BuOH extract (80 g) was applied to silica gel CC eluting

85

with a CHCl3-MeOH-H2O gradient system (9:1:0.1, 8:2:0.2, 7:3:0.5, 6:4:1, 5:5:1, v/v/v,

86

each for 4L), to give six fractions F1-F6 based on TLC analysis. Triterpene saponins were

87

detected in F4-F6 through TLC and HPLC-DAD (diode array detection) analysis.

88

F4 (17.5 g) was subjected to prep-HPLC with an XCharge C18 column (5%-30%

5



89

MeCN with 0.2% formic acid (FA), 60 min, flow rate 15 mL/min) to give fractions F41

90

(tR 35.5 min, 2.1 g) and F42 (tR 36.9 min, 3.2 g). F41 was further separated by

91

prep-HPLC through an XAmide column (90%-80% MeCN with 0.2% FA, 40 min, flow

92

rate 15 mL/min) to give fraction F412 (tR 38.1 min, 57 mg) and 8 (tR 33.2 min, 354 mg).

93

F412 was further chromatographed over prep-HPLC with an XCharge C18 column (23%

94

MeCN with 0.5% FA, 30 min, flow rate 15 mL/min), followed with an XCharge C18

95

column (40% MeCN with 0.2% FA, 10 min, flow rate 15 mL/min) to yield 3 (tR 8.9 min,

96

6 mg). F42 was subjected to prep-HPLC through an XAmide column (90%-80% MeCN

97

with 0.2% FA, 40 min, flow rate 15 mL/min) to give fraction F421 (tR 21.6 min, 85 mg)

98

and 2 (tR 26.0 min, 1.6 g). F421 was further chromatographed over prep-HPLC with an

99

XCharge C18 column (30% MeCN with 0.2% FA, 25 min, flow rate 15 mL/min) to give

100

4 (tR 22.8 min, 31 mg).

101

F5 (7.2 g) was separated through prep-HPLC with an XCharge C18 column

102

(5%-30% MeCN with 0.2% FA, 60 min, flow rate 15 mL/min) to give fractions F51 (tR

103

29.1 min, 228 mg) and F52 (tR 32.2 min, 1.2 g). F51 was subjected to prep-HPLC

104

through an XAmide column (90%-80% MeCN with 0.2% FA, 40 min, flow rate 15

105

mL/min) to give fraction F511 (tR 37.8 min, 85 mg), which was further purified by

106

prep-HPLC with an XCharge C18 column (24% MeCN with 0.2% FA, 40 min, flow rate

107

15 mL/min) to yield 9 (tR 38.8 min, 34 mg). F52 was subjected to prep-HPLC through an

108

XAmide column (90%-80% MeCN with 0.2% FA, 40 min, flow rate 15 mL/min) to give

109

fraction F521 (tR 39.9 min, 483 mg), which was then subjected to prep-HPLC with an

110

XCharge C18 column (24% MeCN with 0.2% FA, 45 min, flow rate 15 mL/min) to give

111

11 (tR 36.8 min, 93 mg) and 1 (tR 43.1 min, 131 mg).

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112

F6 (4.3 g) was fractionated by prep-HPLC with an XCharge C18 column (5%-30%

113

MeCN with 0.2% FA, 60 min, flow rate 15 mL/min) to obtain fractions F61 (tR 18.7 min,

114

126 mg), F62 (tR 28.1 min, 178 mg), and F63 (tR 31.8 min, 1.7 g). F61 was further

115

subjected to prep-HPLC with an XCharge C18 (20%-50% MeCN with 0.2% FA, 60 min,

116

flow rate 15 mL/min) to give 6 (tR 33.5 min, 8 mg) and 7 (tR 35.5 min, 9 mg). F62 was

117

purified with prep-HPLC through an XAmide column (92% MeCN with 0.2% FA, 30

118

min, flow rate 15 mL/min) to give 5 (tR 29.1 min, 50 mg). F63 was subjected to

119

prep-HPLC through an XAmide column (90%-80% MeCN with 0.2% FA, 60 min, flow

120

rate 15 mL/min) to give fraction F631 (tR 47.3 min, 483 mg) and 12 (tR 51.1 min, 307

121

mg). F631 was further purified by prep-HPLC on an XCharge C18 column (23% MeCN

122

with 0.2% FA, 50 min, flow rate 15 mL/min) to obtain 10 (tR 49.5 min, 39 mg).

123 124

3-O-β-D-glucopyranosyl(1→3)-β-D-glucopyranosyl-2β,3β,16α,23-tetrahydroxyolean-

125

12-en-28-oic acid

126

28-O-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosid

127

e, 1: White powder; [α]25D : 8.33 (c 0.12, CH3OH); IR (KBr) vmax (cm-1): 3400, 2943, 1736,

128

and 1648; UV (MeOH) λmax nm (log ε): 202 (3.78); 1H and 13C NMR data see Table 1;

129

HR-ESI-MS: m/z 1256.6333 [M+NH4]+ (calcd. 1256.6270).

130 131

3-O-β-D-glucopyranosyl-2β,3β,23-trihydroxyolean-12-en-28-oic acid

132

28-O-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosid

133

e, 2: White powder; [α]25D : 8.33 (c 0.24, CH3OH); IR (KBr) vmax (cm-1): 3403, 2933, 1731,

7



134

and 1672; UV (MeOH) λmax nm (log ε): 203 (3.80); 1H and 13C NMR data see Table 1;

135

HR-ESI-MS: m/z 1078.5863 [M+NH4]+ (calcd. 1078.5792).

136 137

3-O-β-D-glucopyranosyl-16α-O-formyl-2β,3β,23-trihydroxyolean-12-en-28-oic acid, 3:

138

White powder; [α]25D : 57.14 (c 0.07, CH3OH); IR (KBr) vmax (cm-1): 3415, 2927, 2721,

139

1722, 1450 and 1382; UV (MeOH) λmax nm (log ε): 203 (3.73); 1H and 13C NMR data see

140

Table 2; HR-ESI-MS: m/z 717.3833 [M+Na]+ (calcd. 717.3820).

141

Cytotoxicity assay.

142

Four cancer cell lines, human lung cancer A549 cells, human liver cancer HepG2 cells,

143

human gastric cancer MGC-803 cells, and mouse gastric cancer MFC cells were used in

144

the cytotoxicity assay. All the cells were cultured in DMEM medium (Hyclone, USA),

145

supplemented with 10% fetal bovine serum (Hyclone, USA) and 300 µL/mL levofloxacin

146

at 37 °C in a humidified atmosphere containing 95% air and 5% CO2. The cytotoxicity

147

assay was performed according to the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-

148

tetrazolium bromide) method in 96-well microplates.21-22 Test compounds were dissolved

149

in DMSO diluted with PBS to obtain the solutions with DMSO less than 1%. Taxol

150

(Sigma, USA) was used as positive control and dissolved in the same way. Cell culture

151

(180 µL) was added to each well of the 96-well cell culture plates and allowed cancer

152

cells to adhere for 24 h. Solution of test compound (20 µL) was then added to each well

153

of the 96-well cell culture plates in triplicates and cancer cells were incubated with these

154

compounds for 24 h at 37 °C. Then cancer cells were incubated with MTT (20 µL each

155

well) for another 4 h at 37 °C. DMSO (150 µL) was added to each well after the

156

supernatant liquor was removed. The optical density was measured at 490 nm on a

8


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157

microplate reader after the purple crystals totally dissolved in DMSO. IC50 values were

158

calculated by using Graphpad prism software 6.0.21

159

RESULTS AND DISCUSSION

160

Structure Elucidation of Triterpenoid Saponins

161

The air-dried aerial parts of A. centrali-asiaticus were extracted with 95% ethanol and

162

subsequently partitioned with EtOAc and n-BuOH to provide three parts. The n-BuOH

163

part was chromatographed on silica gel and 2D RPLC/HILIC orthogonal system to yield

164

twelve triterpenoid saponins, 1-12, including three new ones, 1-3 (Figure 1).

165

Compound 1 was obtained as a white amorphous powder. The molecular formula of

166

1 was established as C58H94O28 on the basis of HR-ESIMS. The IR spectrum displayed

167

absorption bands for hydroxy group (3400 cm−1), alkyl group (2934 cm−1), ester carbonyl

168

(1736 cm−1), and olefinic bond (1648 cm−1). In the 1H and 13C NMR spectra (Table 1), six

169

methyl groups, a hydroxy methylene, three oxymethines, and an olefinic methine were

170

observed as characteristic signals arising from polygalacic acid.23-24 Five anomeric

171

protons at δH 5.62 (d, J = 3.5 Hz), 5.03 (brs), 4.52, 4.50 and 4.75 (d, J = 7.8) and their

172

corresponding carbons at δC 93.9, 101.3, 106.6, 105.0, and 105.2 were observed. Thus

173

compound 1 should be a triterpenoid saponin with five sugar units. The

174

spectroscopic data of 1 showed high resemblance to those of platycoside J, 8,25 and the

175

only difference was an additional sugar unit in 1. The aglycone and each sugar unit were

176

further completed through 2D NMR spectroscopic methods, including HSQC, HMBC,

177

1

178

confirmed by 2D NMR spectra (Figure 2). The arabinose was located at C-28 of the

179

aglycone based on the HMBC correlation from the proton of Ara-1 (δH 5.62, 1H, d, J =

13

C NMR

H-1H COSY, and TOCSY spectra and the connections of sugar units were also

9



Page 10 of 30

180

7.8 Hz) to C-28 (δC 177.0). The inner glucose was directly linked to the aglycone at C-3

181

by HMBC correlation from the proton of Glc-1 (δH 4.50) to C-3 (δC 84.1). The HMBC

182

cross peak from H-1 (δH 4.57, 1H, d, J = 7.8 Hz) of the additional glucose to Glc-3 (δC

183

88.0) of the inner glucose revealed that the additional glucose was located at Glc-3.

184

Consequently,

185

3-O-β-D-glucopyranosyl(1→3)-β-D-glucopyranosyl-2β,3β,16α,23-tetrahydroxyolean-12-

186

en-28-oic

187

28-O-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside

188

(Figure 1).

was

1

characterized

as

acid

189


190

2 was established as C52H84O22 on the basis of HR-ESIMS. The IR spectrum of 2 also

191

displayed absorption bands for hydroxy group (3403 cm−1), alkyl group (2933 cm−1),

192

ester carbonyl (1731 cm−1), and double bond (1672 cm−1). Following the similar way as 1,

193

assignments of 1H and 13C NMR resonances of 2 were conducted, and all signals can be

194

identified by 1H-1H COSY, HSQC, HMBC, and TOCSY experiments (Table 1 & Figure2).

195

The NMR data for 2 was also closely resembled to those of platycoside J, 8,

196

indicated the same sugar units in 2 with those in platycoside J, 8. The difference was the

197

replacement of an oxygenated methylene at C-16 in platycoside J by a methylene (δC 23.7)

198

in

199

(2β,3β,23-trihydroxyolean-12-en-28-oic acid).5 Consequently, 2 was characterized as

200

3-O-β-D-glucopyranosyl-2β,3β,23-trihydroxyolean-12-en-28-oic

201

28-O-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside

202

(Figure 1).

2,

supporting

that

the

aglycone

of

10


2

was

a

25

which

bayogenin

acid

Page 11 of 30


203


204

3 was established as C37H58O12 on the basis of HR-ESIMS and NMR data (Table 2). IR

205

spectrum of 3 displayed absorption bands for hydroxy group (3415 cm−1), alkyl group

206

(2927 cm−1), and aldehyde group (2721 and 1722 cm−1). NMR analysis of 3 also revealed

207

the presence of six methyl groups, a hydroxy methylene, three oxymethines and an

208

olefinic methine, indicating that 3 also had a triterpene skeleton. NMR signals in 3 were

209

further assigned through 1D and 2D NMR spectroscopic methods (Figure 3). In the 1H

210

NMR spectrum of 3 only one anomeric proton signal (δH 4.43, d, J = 7.7 Hz) was

211

observed, illustrating that 3 contained one β-D-glucose unit. Cross-peaks in the HMBC

212

spectrum from H-1 of the glucose (δH 4.43) to C-3 of the aglycone (δC 84.0) and from

213

H-3 of the aglycone (δH 3.60) to C-1 of glucose (δC 105.6) confirmed that the glucose

214

was attached to the aglycone at C-3. NMR data of 3 closely resembled those of a known

215

triterpenoid, bernardioside A,26 but C-17 and C-28 of 3 could not be unambiguously

216

assigned, as was the case with bernardioside A.26 The only difference between

217

bernardioside A and 3 was that an additional aldehyde group (δC 162.9 and δH 8.21, s)

218

existed in 3, indicating that 3 could be a O-formyl derivate of bernardioside A. The

219

correlation in the HMBC spectrum from the proton of aldehyde group (δH 8.21) to C-16

220

of the aglycone (δC 78.1) confirmed that the 16-hydroxy of 3 was subjected to

221

esterification

222

3-O-β-D-glucopyranosyl-16α-O-formyl-2β,3β,23-trihydroxyolean-12-en-28-oic

223

(Figure 1). In order to improve the peak pattern, excess formic acid (0.5%, v/v) was

224

added to the mobile phase in the process of isolation.27 Compound 3 may be an artifact

225

due to hydrolysis and formic acid esterification.

with

formic

acid.

Thus,

compound

11


3

was

identified

as acid


Page 12 of 30

226

The other isolates were identified as medicoside H, 4,28 bellissaponin BS6, 5,5

227

polygalacic acid-3-O-glucoside, 6,29 paradoxoside D, 7,30 platycoside J, 8,25

228

heteropappussaponin 5, 9,31 heteropappussaponin 7, 10,31 durantanin II, 11,29

229

3-O-β-D-glucopyranosyl(1→3)-β-D-glucopyranosyl-2β,3β,16α,23-tetrahydroxyolean-12-

230

en-28-oic

231

28-O-α-L-rhamnopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-[β-D-apiosyl-(1→3)]-α-L-r

232

hamnopyranosyl-(1→2)-α-L-arabinopyranoside, 12.32 Their structures were elucidated

233

through 1D and 2D NMR experiments and HRESIMS spectra as well as comparing with

234

the literature values.5, 25, 28-32

235

Purification of Triterpenoid Saponins by the Two-Dimensional Reversed-Phase Liquid

236

Chromatography/Hydrophilic Interaction Chromatography

237

In

acid

this

study,

a

preparative

two-dimensional

reversed-phase

liquid

238

chromatography/hydrophilic interaction chromatography method was employed for

239

saponins isolation. Considering the complicated purification procedure applied to

240

compound 4 which was isolated by 2D HPLC, followed by a further purification via

241

prep-HPLC with an XCharge C18 column, the purification process of saponin 4 was

242

selected to illustrate the good orthogonality of the chromatographic packing materials

243

used in the two dimensional chromatography (Figure 4). The n-BuOH fraction was

244

separated crudely through silica gel CC to afford six parts F1-6 based on TLC analysis.

245

Triterpenoid saponins were detected in F4-F6 through TLC and HPLC-DAD analysis.

246

First-dimensional purification was carried out on a preparative XCharge C18 column. F4

247

was subjected to an XCharge C18 column to give fraction F42, which provided good

248

separation of triterpenoids from other phenolic components (Figure 4A). XCharge C18

12


Page 13 of 30


249

reversed-phase column with strong retention of triterpenoid saponins made it possible for

250

triterpenoid saponins enrichment. Then an XAmide column was selected in HILIC mode.

251

F42 was then subjected to an XAmide column to give fraction F421 (Figure 4B).The

252

XAmide column with terminal amide group modified surface had strong polar and

253

hydrophilic characteristics19 and showed good orthogonality with the XCharge C18

254

column. Thus, XAmide column was appropriate for the second-dimension purification.

255

Finally, F421 was further purified by a prep-HPLC process through an XCharge C18

256

column to obtain compound 4 (Figure 4C). The results demonstrated that this approach

257

was effective and convenient for the separation of triterpenoid saponins in

258

A.centrali-asiaticus.

259

Cytotoxicity of Isolated Compounds

260

Many natural occurring triterpenoid saponins demonstrate potential anticancer

261

activities.20,

262

antiproliferative effects against four cancer cell lines, human lung cancer A549 cells,

263

human liver cancer HepG2 cells, human gastric cancer MGC-803 cells, and mouse

264

gastric cancer MFC cells via the MTT method. A primary screening showed that 1, 3, 8, 9,

265

11, and 12, exhibited cytotoxicity with the cell viability rate less than 50% (Figure 5) at

266

the concentration of 50 µg/mL. These six compounds were further assayed to obtain the

267

IC50 values (Table 3). Compounds 3 and 8 selectively inhibited proliferation of human

268

gastric cancer MGC-803 and mouse gastric cancer MFC cells, respectively. Compound

269

11 which possessed sugar chain containing five sugar units at C-28 showed cytotoxicity

270

against all these four cancer cell lines and compound 11 showed most potent cytotoxicity

271

against human liver cancer HepG2 cells. Compound 12 which also possessed a sugar

33-34

Herein, all isolated saponins were evaluated in vitro for their

13



272

chain containing five sugar units at C-28 showed cytotoxicity against HepG2, MGC-803,

273

and MFC cancer cell lines, and compound 12 showed most potent cytotoxicity against

274

mouse gastric cancer MFC cell line among all the tested compounds.

275

In summary, a preparative 2D RPLC/HILIC orthogonal system was successfully

276

applied to isolate twelve triterpenoid saponins from A.centrali-asiaticus including three

277

new ones. Moreover the antiproliferative potencies of all isolated compounds were

278

evaluated through the MTT assay, which revealed potential cytotoxicity for some

279

saponins. The structure-activity relationship discussion indicated that the sugar chain

280

possessing five sugar units at C-28 could play a critical role in the cytotoxicity of

281

saponins.

282 283

ASSOCIATED CONTENT

284

*S Supporting Information

285

Supporting Information available:

286

HR-ESIMS spectra of compounds 1-12. This material is available free of charge via the

287

Internet at http://pubs.acs.org

13

C NMR of compounds 4-12 and NMR and

288 289

AUTHOR INFORMATION

290

Corresponding Authors

291

*Key Laboratory of Chemistry of Northwestern Plant Resources of CAS and Key

292

Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical

293

Physics, Chinese Academy of Sciences, Lanzhou 730000, P. R. China; Tel.:

294

+86-931-4968208. Fax: +86-931-8277088. E-mail: [email protected].

14


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Page 15 of 30


295

* Key Laboratory of Tibetan Medicine Research, Northwest Institute of Plateau Biology,

296

Chinese Academy of Sciences, Xining 810008, P. R. China; Tel.: +86-971-6143610. Fax:

297

+86-9716143282. E-mail: [email protected].

298

Author Contributions

299

#These authors contributed equally.

300

Author Contributions

301

Y.-D.T. and L.-J.M. executed the collection and identification of the plant material.

302

Y.-M.W., J.-Q.Z., and Y.-P.S. designed the experiments which Y.-M.W. and J.-Q.Z.

303

executed. Y.-M.W. executed the cytotoxic assay. Y.-M.W., J.-Q.Z., and J.-L.Y. elucidated

304

the structures and analyzed the experimental data. Y.-M.W., J.-Q.Z., Y.-P.S, and J.-L.Y.

305

wrote the paper.

306

ACKNOWLEDGMENTS

307

The work was financially supported by the National Nature Science Foundation of China

308

(Nos. 21375136 and 21575150), and the scientific research project of Central Asia Drug

309

Discovery and Development Centre of Chinese Academy of Sciences (No. CAM201404)

310

and the CAS Pioneer Hundred Talents Program.

311

Notes

312

The authors declare no competing financial interest.

313 314

15



315

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23. Kasai, R.; Miyakoshi, M.; Nie, R. L.; Zhou, J.; Matsumoto, K.; Morita, T.; Nishi, M.;

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24. Li, W.; Asada, Y.; Koike, K.; Nikaido, T.; Furuya, T.; Yoshikawa, T. Bellisosides A–F,

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34. Mo, S.; Xiong, H.; Shu, G.; Yang, X.; Wang, J.; Zheng, C.; Xiong, W.; Mei, Z.

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412

phaseoloides, induces apoptosis in Ec-109 esophageal cancer cells through reactive

413

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414

20


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FIGURE CAPTIONS. Figure 1. Structures of isolated compounds 1−12. Figure 2. Key 1H-1H COSY (TOCSY) and HMBC correlations of 1 and 2. Figure 3. Key 1H-1H COSY and HMBC correlations of 3. Figure 4. (A) HPLC-DAD chromatogram on XCharge C18 of n-butanol extract F4 from A.centrali-asiaticus at λ 210 nm including corresponding UV absorption spectra of certain peaks; (B) HPLC-DAD chromatogram on XAmide of n-butanol extract F42 from A.centrali-asiaticus at λ 210 nm; (C) HPLC-DAD chromatogram on XCharge C18 of n-butanol extract F421 from A.centrali-asiaticus at λ 210 nm. Figure 5. Effects of compounds 1-12 on the cell viability of four cancer cell lines at concentration of 50 µg/mL.

.

21



Page 22 of 30

Table 1. NMR Spectroscopic Data at 400 and 100 MHz Respectively of 1 and 2 in CD3OD. Position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

1 δH mult. (J in Hz) (ppm) 2.08 d (12.8) 1.17a 4.32 brs 3.62a 1.30a 1.48m 1.64a 1.35a 1.62

δC (ppm) 44.5 71.1 84.1 43.1 48.3 18.6 33.7

a

1.96a 5.37 brs 1.77a 1.40a 4.48a

40.9 48.4 37.5 24.7 123.8 144.7 42.9 36.3 74.6

3.05 dd (14.1, 3.2) 2.26 t (13.5) 1.05 m

50.3 42.1 47.6 31.3 36.4

a

1.90 1.15a 1.90a 1.77a 3.62a 3.24a

31.8 65.9

2 δH mult. (J in Hz) (ppm) 2.02 m 1.10a 4.27 brs 3.55 m 1.24a 1.46 m 1.52 m 1.28a 1.51

a

1.90a 5.26 brs 1.60a 1.08a 1.96a 1.61a 2.86 dd (13.1, 2.8) 1.65a 1.08a a

1.33 1.17a 1.69a 1.52a 3.56a 3.19a

δC (ppm) 44.3

Position

70.7 83.8 43.1 48.1

2 3 4 5

18.5 33.5

δC (ppm) 93.9

2 δH mult. (J in Hz) (ppm) 5.60 d (3.6)

δC (ppm) 93.8

75.6 71.0 66.9 63.6

Rha-1 2

3.77a 3.88a 3.85a 3.89a 3.50a 5.03 brs 3.86a

101.3 72.1

3.76a 3.84a 3.80a 3.85a 3.46a 5.03 brs 3.81a

40.7 49.2 37.4 24.6 123.8 144.8 43.1 28.9

3 4 5 6 Xyl-1 2 3 4

3.85a 3.56a 3.70a 1.28a 4.52a 3.22a 3.31a 3.47a

72.3 83.3 68.9 18.8 106.6 75.9 78.1 71.1

3.81a 3.49a 3.67a 1.24a 4.44 d (7.6) 3.17a 3.29a 3.42a

72.3 83.6 68.8 18.1 106.7 75.9 78.1 71.0

23.7

5

67.2

Glc-1 2 3

105.0 74.7 88.0

3.80a 3.14a 4.39 d (7.7) 3.22a 3.32a

67.1

48.2 42.6 47.1

3.84a 3.19a 4.50a 3.74a 3.56a

31.5 34.9

4 5

3.28a 3.32a

71.5 77.3

3.35a 3.24a

71.0 77.6

33.3

6

62.0

3.75a 3.65a

62.2

65.2

Glc-1 (termina l) 2 3 4 5 6

3.88a 3.63a 4.57 d (7.8)

24 25 26 27 28

0.94 s 1.29 s 0.78 s 1.36 s

14.7 17.6 18.0 27.4 177.0

0.89 s 1.23 s 0.74 s 1.12 s

14.7 17.5 17.9 26.4 177.8

29 30

0.87 s 0.96 s

33.4 25.1

0.86 s 0.89 s

33.5 24.0

a

Ara-1

1 δH mult. (J in Hz) (ppm) 5.62 d (3.5)

3.29a 3.39a 3.48a 3.33a 3.80a 3.72a

Overlapped signals

22


105.2

75.5 78.1 69.6 77.8 62.2

75.5 71.2 67.1 63.6 101.2 72.0

105.3 75.3 78.1

Page 23 of 30


Table 2. NMR Spectroscopic Data at 400 and 100 MHz of 3 in CD3OD. Position

δH mult. (J in Hz) (ppm) 2.07 m 1.16a 4.32 m

δC (ppm) 44.4

No.

71.2

21

3 4

3.60a

84.0 42.6

22 23

5 6 7

1.30a 1.48a 1.27a 1.60a

48.2 18.6 33.6

24 25 26

40.8 48.4 37.6 24.6 124.9 143.8 43.1 33.6

27 28 29 30 CHO Glc-1 2 3

78.1

4 5 6

1 2

8 9 10 11 12 13 14 15 16 17 18 19 a

1.62a 1.98a 5.39 brs 2.02a 1.44a 5.77 brs

b

3.13 dd (14.4, 3.7)

41.7

2.15 m 1.13a

47.4

δH mult. (J in Hz) (ppm)

δC (ppm) 31.5

1.58a 1.18a 1.79 m 3.60 m 3.21 m 0.94 s 1.28 s 0.82 s

36.2

14.7 17.5 17.8

1.30 s

27.1

0.91 s 0.99 s 8.21 s 4.43 d (7.7) 3.25a 3.36a

33.4 24.7 162.9 105.6 75.6 78.3

3.35a 3.27a 3.81 dd (11.8, 2.1) 3.70 dd (11.9, 4.6)

71.2 77.8 62.6

20

Overlapped signals; bUnambiguous assignment not possible.

23


32.5 65.6

b


Table 3. Cytotoxicity of triterpenoid saponins against four cancer cell lines. IC50a (µg/mL)

Compound A549 b

HepG2

MFC

22.59

37.33

1

>50

3

>50b

>50b

43.05

>50b

8

>50b

>50b

>50b

35.77

9

47.32

>50b

>50b

30.04

11

30.2

6.86

13.85

19.77

12

>50b

36.76

25.22

17.90

Taxol

31.31

7.17

0.097

>50b

a

>50

MGC-803

b

Values are the mean values; bThe IC50 value of the sample was higher than 50 µg/mL.

24


Page 24 of 30

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Figure 1

25



Figure 2

26


Page 26 of 30

Page 27 of 30


Figure 3

27



Figure 4

28


Page 28 of 30

Page 29 of 30


Figure 5

29



TABLE OF CONTENTS GRAPHICS

30


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Isolation and Identification of Saponins from the Natural Pasturage

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