New Sweet-Tasting Oleanane-Type Triterpenoid Saponins from

Subscriber access provided by Fudan University

Article

New Sweet-Tasting Oleanane-Type Triterpenoid Saponins from “Tugancao” (Derris eriocarpa How) Hongxia Zhang, Guo Sun, Jianlong Gu, and ZhiZhi Du J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00137 • Publication Date (Web): 25 Feb 2017 Downloaded from http://pubs.acs.org on February 27, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 33

Journal of Agricultural and Food Chemistry

New Sweet-Tasting Oleanane-Type Triterpenoid Saponins from “Tugancao” (Derris eriocarpa How) Hong-Xia Zhang†, Guo Sun†,‡,, Jian-Long Gu§, Zhi-Zhi Du*,†

†

Key Laboratory of Economic Plants and Biotechnology, Yunnan Key Laboratory for

Wild Plant Resources, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China ‡

University of Chinese Academy of Sciences, Beijing 100049, China

§

Yunnan Tobacco Quality Inspection & Supervision Station, Kunming 650106, China

Corresponding Author * Phone: +86-871-65223224; Fax: +86-871-65216335 E-mail address: [email protected]

1

ACS Paragon Plus Environment


1

ABSTRACT

2

Aiming to investigate the sweet-tasting compounds in Derris eriocarpa How (a

3

substitute for licorice in “Zhuang” and “Dai” ethnopharmacy in Guangxi and Yunnan

4

province of China) as well as to ascertain why the stem of D. eriocarpa can be used to

5

substitute for licorice in sweetness taste aspect, taste sensory-guided fractionation was

6

conducted to isolate sweet constituents from the extract of D. eriocarpa. Four

7

sweet-tasting triterpenoid saponins were obtained, including Millettiasaponin A (1)

8

and three new ones named as derrisaponins A-C (2-4). The sweetness potency was

9

evaluated by a human sensory panel test. The sweetness intensities of compounds 1-4

10

were determined to be approximately 150, 80, 2 and 0.5 times relative to sucrose at

11

the concentration 1%, respectively, of which 1 and 2, with a free carboxyl group at the

12

C-30 position, showed more potent sweetness intensity. In addition, 1 and 2 showed

13

no acute toxic activity in dose of 250 mg/kg bw and 400 mg/kg bw respectively

14

assessed through caudal veins injection to ICR mice. The contents of the sweetest

15

compounds in stems were analyzed quantitatively as 352.80 mg/kg for 1 and 1887.60

16

mg/kg for 2 respectively performed by UPLC–MS/MS.

17 18

KEYWORDS

19

Derris eriocarpa, sweet-tasting, triterpenoid saponins, sensory evaluation, acute

20

toxicity

2


Page 2 of 33

Page 3 of 33


21

INTRODUCTION

22

Human can detect at least five basic tastes including sweet, bitter, salty, sour and

23

umami tastes. Among them, sweetness plays a central role for it is close relationship

24

to appetitive sensations in food recognition, and is usually and naturally originated

25

from sugars. However, with the excessive intake of sugar, more and more people are

26

suffered from diseases, such as dental caries, hypertension, hyperglycemia,

27

cardiovascular diseases and obesity.1-3 Accordingly, there has been an increasing

28

demand for new highly sweet and noncaloric sucrose substitutes. The compounds, in

29

being so-called “high-potency (HP) sweeteners” and “low-calorie sweeteners” with

30

these metrics: safety, taste quality, stability, solubility, cost, and patentability,

31

especially natural sweeteners, attract many researchers’ attentions.4, 5 In addition to

32

the development of synthetic HP sweeteners, a number of highly sweet natural

33

compounds have been discovered from green plants, but only a relatively few

34

sweet-tasting plant-derived natural products have been successfully applied as sucrose

35

substitutes to date.6 Efforts to find more highly sweet plant constituents have been

36

stimulated both by a public demand for natural flavors to tackle problems of toxicity

37

and taste quality of existing synthetic HP sweeteners. By following up ethnobotanical

38

leads to assist in the selection of candidate sweet-tasting plant, particularly those used

39

medicinally by indigenous cultures, it is possible to discover new potent sweet natural

40

products.5

41

During our ethnobotanical field survey and sensory evaluation of characteristic

42

edible and medical plants in traditional medicine market on “Dragon boat festival”, an 3



43

ethnic edible and medical plant called “Tugancao” was of obvious sweet taste.

44

Traditional medicine market on “Dragon boat festival” of Jingxi County in Guangxi

45

province has been going on for more than 700 years with more than 500 species of

46

traditional medicinal herbs for sale.7 The abundant resources of traditional edible and

47

medicinal herbs provide a great material basis for our researches. By means of the

48

ethnobotanical methods (Key Informantion Interview), we got that the local people

49

immerse the “Tugancao” with other medicinal herbs into the white spirit to make

50

herbal liquor. Informants also mentioned that the herbal liquor is not only to relieve a

51

cough but also become a little licorice-like sweetness and pleasant to take.

52

The plant was taxonomically identified as Derris eriocarpa How, belonging to the

53

Leguminosae family, commonly known as “Tugancao” in “Zhuang” and “Dai”

54

ethnopharmacy in Guangxi and Yunnan provinces of China.8, 9 The “Tugancao”, the

55

stem of D. eriocarpa is used to substitute for licorice due to its similar sweet taste and

56

medicinal properties in folk medicine in Guangxi province. 10-11 In previous chemical

57

investigation, some isoflavonoids, stibenoids and coumarins were reported.12-14

58

However, to date there are no investigations on sweet-tasting components from this

59

plant.

60

Aiming to identify the sweet-tasting compounds and ascertain why the D.

61

eriocarpa can be used to substitute for licorice from the sweetness point, we carried

62

out a sensory-guided fractionation and purification of the crude extract of this plant.

63

In the present study, four triterpenoid saponins were isolated from the extract of D.

64

eriocarpa stems and they were proposed mine chemical constituents responsible for 4


Page 4 of 33

Page 5 of 33


65

the sweet taste of the plant.

66 67 68

MATERIALS AND METHODS Chemicals. The following materials were used: and

D-glucose

D-glucuronic

acid,

D-galactose,

69

L-rhamnose

(J&K Scientific Ltd. Guangzhou, China),

L-cysteine

70

methyl hydrochloride (Sigma-aldrich, Shanghai, China), N-trimethylsilylimidazole

71

(Sangon Biotech, Shanghai, China), n-hexane (Damao, Tianjin, China), HPLC

72

acetonitrile (Merck, Shanghai, China), ethyl acetate (Jige, Tianjin, China), acetic

73

anhydride (Damao, Tianjin, China), sulphuric acid (Xilong Chemical Co. Ltd,

74

Guangdong, China), hydrochloric acid (Xilong Chemical Co. Ltd), ferric chloride

75

(Damao, Tianjin, China), chloroform (Rionlon, Tianjin, China), dioxane (Sinopharm

76

chemical reagent Co. Ltd., Shanghai, China), sodium dicarbonate (Damao, Tianjin,

77

China).

78

General Experiment Procedures. Optical rotations were measured with a Horiba

79

Sepa-300 polarimeter (Horiba, Tokyo, Japan). UV spectra were obtained using a

80

Shimadzu UV-2401A spectrophotometer (Shimadzu, Tokyo, Japan). A tensor 27

81

spectrophotometer (Bruker, Bremen, Germany) was used for scanning IR

82

spectroscopy using KBr pellets. 1D and 2D NMR spectra were measured on Bruker

83

AVANCE III 500MHz and AV Ⅲ 800 MHz spectrometers (Bruker, Bremen,

84

Germany) at 296 K. Unless otherwise specified, chemical shifts (δ) were expressed in

85

ppm with reference to the solvent signals. ESIMS data were obtained on a Bruker

86

HCT Esquire spectrometer (Bruker, Bremen, Germany). HRESIMS data were 5



87

recorded on an Agilent G6230 TOF MS spectrometer (Agilent Technologies, Santa

88

Clara, America).

89

Column chromatography (CC) was done using silica gel (200-300 mesh, Qingdao

90

Marine Chemical Co., Ltd., China), RP-18 gel (40-63µm, Merck, Germany), Diaion

91

HP-20 (Mitsubishi Chemical Corporation, Japan), resin D101 column (Shanghai

92

YuanYe Bio-Technology Co., Ltd., China) and Sephadex gel LH-20 (GE Healthcare

93

Bio-Sciences AB, Sweden). TLC was performed on silica gel GF254 (Qingdao Marine

94

Chemical Co., Ltd., China), and spots were visualized by heating silica gel plates

95

sprayed with 10% H2SO4 in ethanol. Quantitation by UPLC-MS/MS was performed

96

on a Waters Acquity UPLC system (Waters Corp., Milford, MA, USA). The

97

lyophilizer (Virtis Benchtop K, America) was used to dry the samples and eliminate

98

the residual solvents.

99

Plant Material. The fresh stems of D. eriocarpa were collected from Jingxi

100

County of Guangxi Province, China, and identified by Prof. Li-Song Wang. A voucher

101

specimen (No.11-32219) has been deposited in the Herbarium of Kunming Institute of

102

Botany (KUN), Chinese Academy of Sciences.

103

Animals. Male and female ICR mice (3-4 weeks, Institute of Cancer Research),

104

certified specific pathogen-free, were purchased from Kunming medical university

105

under the license number SCXK (DIAN) K2011-0004. Mice were kept in an IVC

106

animal room under the license No.: SYXK (DIAN) K2013-004, conditioned at a

107

temperature of 25±3℃, a relative humidity of 40-70% and 12h light/dark cycle. All

108

animals had a one-week acclimatization period before experiment started and received 6


Page 6 of 33

Page 7 of 33


109 110

basic feed and fresh water freely during the experiment period.15 Extraction, Sensory-guided Fractionation and Purification of the

111

Sweet-Tasting Compounds. The air-dried and powdered stems (6 kg) of D.

112

eriocarpa were extracted 3 times with 80% aqueous MeOH at room temperature.

113

After evaporation of MeOH under reduced pressure, the aqueous residue was

114

partitioned with CHCl3, EtOAc and n-BuOH to yield CHCl3 portion (150 g), EtOAc

115

portion (12 g), n-BuOH portion (80 g) and the aqueous layer. The n-BuOH portion

116

(80 g) and the aqueous layer showed sweet taste in sensory evaluation tests.

117

The n-BuOH portion (80 g) was subjected to a macroporous resin D101 column

118

chromatography (2 kg), eluted with H2O, 30%, 50% , 70% EtOH-Water, and EtOH

119

successively (6 L each). The elutant eluted with water was abandoned. The other

120

elutant was evaporated to remove organic solvent in reduced pressure and lyophilized

121

to give fraction B1 (about 11 g, eluted with 30% aqueous ethanol), fraction B2 (49 g,

122

eluted with 50% and 70% aqueous ethanol), fraction B3 (2 g, eluted with ethanol).

123

Fraction B2 was identified as the sweet-tasting fraction according to sensory

124

evaluation results.

125

Fraction B2 (49 g) was chromatographed on silica gel column, eluted with

126

CHCl3-MeOH-Water (C/M/W: 7/3/0, 7/3/0.5, 6/4/1) to get 4 fractions on the basis of

127

TLC analysis. According to the sensory evaluation, fraction B2-a (9.3 g) and B2-b

128

(8.5 g) showed non-sweet taste, fraction B2-c and B2-d showed interesting sweet taste.

129

Fraction B2-c (15 g) was subjected to CC over silica gel (C/M/W:7/3/0.5) to yield

130

three sub-fractions based on TLC analysis. B2-c-1 and B2-c-3 were non-sweet tasting 7



131

sub-fractions, and B2-c-2 was sweet tasting sub-fraction. B2-c-2 (3.5 g) afforded 1

132

(800 mg) after purified by CC over silica gel (C/M/W: 7/3/0.5). Fraction B2-d (16 g)

133

was subjected to CC over silica gel (C/M/W:7/3/0.5) to yield four sub-fractions based

134

on TLC analysis. B2-d-1 to B2-c-3 were non-sweet tasting sub-fractions, and B2-d-4

135

was sweet tasting sub-fraction. B2-d-4 (2.2 g) was subjected to experiments of CC

136

over silica gel (C/M/W: 7/3/0.5) to afford 2 (120 mg).

137

The aqueous layer was concentrated under vacuum to get a fraction named WP

138

(about 850 g). The fraction WP (850 g) was subjected to macroporous resin column

139

chromatography (Diaion HP-20, 2 kg) eluted with water, 30%, 50%, 70%

140

MeOH-Water, and MeOH, successively (6 L each). The elutant eluted by water was

141

abandoned. The other elutant was evaporated to remove organic solvent in reduced

142

pressure and lyophilized to give fraction WP1 (about 11.3 g, eluted with 30% aqueous

143

methanol), fraction WP2 (18.2 g, eluted with 50% aqueous methanol), fraction WP3

144

(55.5 g, eluted with 70% aqueous methanol), and fraction WP4 (25.5 g, eluted with

145

methanol). According to the sensory evaluation, only fraction WP3 showed interesting

146

sweet taste. Fraction WP3 was subjected to macroporous resins column

147

chromatography (Diaion HP-20, 2 kg) eluted with water, 50%, 70% MeOH-Water,

148

and MeOH, successively (6 L each). The solution eluted by water was abandoned.

149

According to TLC analysis, the 50% and 70% MeOH-Water portions showed the

150

same color as the sweet compounds 1 and 2 isolated from the n-BuOH portion. And

151

these two portions were combined and the combination (recorded as WP3-a, 20 g)

152

was chromatographed on silica gel column, eluted with CHCl3-MeOH-Water (8/2/0, 8


Page 8 of 33

Page 9 of 33


153

7/3/0, 7/3/0.5, 6/4/1) to get 5 fractions on the basis of TLC analysis. According to

154

sensory evaluation, fractions WP3-a-1 to WP3-a-4 were non-sweet fractions, while

155

fractions WP3-a-5 was sweet-tasting fraction and. Fractions WP3-a-5 (10.5 g) was

156

chromatographed on RP-18 silica gel reduced pressure column to get 4 fractions (30%,

157

35%, 40%, 45%, 50% MeOH-Water, 500 ml each, φ 2.50 × 6.5 cm; 20 g RP-18 silica

158

gel, Merck, Germany). Fraction WP3-a-5-b and WP3-a-5-d showed sweet taste on the

159

basis of sensory evaluation. WP3-a-5-b (2.4 g) was chromatographed on RP-18 silica

160

gel reduced pressure column (33%, 38%, 40% MeOH-Water, 300 ml each) to yield 4

161

(230 mg, eluted by 38% MeOH-Water). WP3-a-5-d (2.3 g) was chromatographed on

162

RP-18 silica gel reduced pressure column (35%, 38%, 40%, 45% MeOH-Water, 300

163

ml each) to yield 3 (150 mg, eluted by 40% MeOH-Water). All of those extractions,

164

fractions and compounds were lyophilized for two times to eliminate the residual

165

solvent prior to the sensory experiments.

166

Millettiasaponin A (1): white amorphous powder; the 1H NMR (Pyridine-d5, 500 13

167

MHz) and

168

Tables 1 and 2; the positive ESI-MS m/z 1037, [M+Na]+, 1059, [M+2Na–H]+; and the

169

negative ESI-MS m/z 1013, [M–H]–, 587, [M–2H]2–.

170

C NMR (Pyridine-d5, 125 MHz) spectroscopic data are presented in

Derrisaponin A (2): white amorphous powder; [α]22.6 –10.4 (c 0.18, H2O); UV (H2O) D

171

λmax(nm) (log ε): 190 (3.74); IR (KBr) νmax cm–1: 3423, 2972, 2934, 1713, 1615, 1265,

172

1074, 1048; the 1H NMR (Pyridine-d5, 500 MHz) and

173

MHz) spectroscopic data are presented in Tables 1 and 2; ESI-MS m/z 1175 [M–H]–;

174

HR-ESI-MS m/z 1175.5480, [M–H]– (calcd for C56H87O26–, 1175.5486).

13

C NMR (Pyridine-d5, 125

9



175

Page 10 of 33

Derrisaponin B (3): white amorphous powder; [α]22.6 –11.7 (c 0.12, H2O); UV (H2O) D

176

λmax(nm) (log ε): 190 (3.77); IR (KBr) νmax cm–1:3424 (OH), 2969, 2932, 1718, 1615,

177

1383, 1261, 1072, 1048; the 1H NMR (Pyridine-d5, 800 MHz) and

178

(Pyridine-d5, 200 MHz) spectroscopic data are presented in Tables 1 and 2; ESI-MS

179

m/z 1175 [M–H]–; HR-ESI-MS m/z 1175.5482, [M–H]– (calcd for C56H87O26–,

180

1175.5486.

13

C NMR

181

Derrisaponin C (4): white amorphous powder; [α]22.8 –4.3 (c 0.15, H2O); UV (H2O) D

182

λmax(nm) (log ε): 190 (3.71); IR (KBr) νmax cm–1:3424 (OH), 2968, 2932, 1720, 1619,

183

1383, 1263, 1073, 1049; the 1H NMR (Pyridine-d5, 800 MHz) and

184

(Pyridine-d5, 200 MHz) spectroscopic data are presented in Tables 1 and 2; ESI-MS

185

m/z 1337 [M–H]–, 1175 [M–Glc–H]–; HR-ESI-MS m/z 1337.6017, [M–H]– (calcd for

186

C62H97O31–, 1337.6014).

13

C NMR

187

Acute Toxicity Test. Three groups of mice (5 males and 5 females) fasted

188

overnight but allowed access to water. The parenteral acute toxicity tests of the

189

compounds were carried out by the method of previous investigation.18 Compounds 1

190

and 2 were dissolved in normal saline and injections were made into the caudal veins

191

one time in 24 h, at the dose of 250 mg/kg.bw and 400 mg/kg. bw for 1 and 2,

192

respectively. The animals were observed for toxic signs (convulsion, salivation,

193

diarrhoea, lethargy, sleep, coma, nervousness and so on) and deaths regularly

194

throughout the first day, then daily for at least 14 day. In these tests, animals were

195

dosed once at a time. At the end of the observation, surviving animals were sacrificed

196

cervical by taking off the cervical spine and organs (heart, lung, spleen, liver, kidney, 10


Page 11 of 33


197

uterus and right ovary) were removed, weighed and examined macroscopically.

198

Sensory Evaluation. The taste threshold and sweetness intensity relative to sucrose

199

were evaluated by a human sensory panel. Ten evaluation panelists, which were

200

sensitive to sweetness, were screened from more than 30 employees and graduate

201

students invited from Kunming Institute of Botany (KIB), CAS, according to

202

Givandan’s panelist selection procedure (taste intensity ranking test). All of them were

203

trained following ISO norms.16

204

The extracts and sub-fractions were dissolved in distilled water to prepare a water

205

solution at the concentration 0.2% (w/v). The sensory panelist consisting of seven

206

sweet sensitive taster (four women and three men, age from 24-45, Chinese only)

207

were asked to taste the sample solutions to evaluate the taste character as previously

208

described,17 and modified as needed, so that to find the sweet portions or

209

sub-fractions.

210

The compounds were dissolved in distilled water to prepare a stock solution at the

211

concentration of 0.1% (w/v). Fold dilution each stock solution to prepare a series of

212

lower concentration solutions from 0.1 to 0.0002% (w/v). The geometric mean of the

213

last and the second last concentration were calculated and taken as the individual

214

recognition threshold. Seven different panelists evaluated the threshold value in two

215

independent sessions. The sucrose solutions were prepared at concentrations 1%, 2%

216

and 4%. The panelists were asked to taste a sample solution and to estimate its

217

sweetness intensity relative to that of the sucrose solution of appropriate concentration.

218

The ratio of the concentration of sucrose solution and that of compounds solution is 11



219

the sweetness intensity relative to sucrose.

220

Samples were coded and randomly presented to panelists of 10 mL in each cup and

221

the total cups less than 20 at ambient temperature. The panelists were asked to rinse

222

their mouths with water in between samples and rest for some time after tasting

223

several cups of samples. The assays were performed at least in triplicate on separate

224

occasions.

225

Acid Hydrolysis of Compounds. Compounds 1-4 (each 2 mg) were dissolved in 2

226

M HCl (dioxane-H2O, 1:1, 2 mL) and heated at 95 °C for 2 h, respectively. After

227

cooling and evaporated to dryness under reduced pressure, the reaction mixture was

228

extracted with EtOAc for three times. The aqueous layer was neutralized with

229

NaHCO3 solution and evaporated under vacuum to furnish a neutral residue.19 The

230

residue was dissolved in anhydrous pyridine (1 mL), to which 2 mg of L-cysteine

231

methyl ester hydrochloride was added. The mixture was stirred at 60 °C for 2 h, and

232

after evaporation in vacuo to dryness, 0.2 mL of N-trimethylsilylimidazole was added;

233

the mixture was kept at 60°C for another 2 h.20 The reaction mixture was partitioned

234

between n-hexane and H2O (2 mL each), and the n-hexane extract was analyzed by

235

Agilent 7890Agas chromatography (Agilent Technologies, Santa Clara, America)

236

with a flame ionization detector (FID) under the following conditions: HP-5 capillary

237

column (50 m × 0.32 mm i.d., with a 0.52 µm film thickness, Agilent, Santa Clara,

238

America); FID temp., 250 °C; injector temp., 250 °C; over program, initial temp.

239

160 °C, then raised to 280 °C at 5 °C /min; carrier gas, helium; flow rate: 1.28

240

mL/min. Under these conditions, the following sugar units were confirmed by 12


Page 12 of 33

Page 13 of 33


241

comparison of the retention times of their derivatives with those of authentic sugars

242

derivatized in a similar way: tR (min) 21.6 (D-glucuronic acid), 20.5 (D-galactose),

243

14.6 (L-rhamnose), and 21.7 (D-glucose), respectively.

244

Quantitation by UPLC–MS/MS. Standard solutions were prepared in methanol at

245

concentrations of 23.300, 116.500, 233.000 and 466.000 µg/mL for 1, and 8.200,

246

49.000, 80.000, and 160.000 µg/mL for 2 to make working curves respectively. The

247

stems of D. eriocarpa were weighed 5.000 g precisely to the conical flasks (100 mL),

248

to which 60 mL methanol was added. The samples were extracted for 30 min on the

249

ultrasonic conditions and the supernatants were filtrated through a 0.45 µm filter

250

membrane to make tested solutions used.

251

Chromatographic analysis was performed on a Waters Acquity UPLC system

252

(Waters Corp., Milford, MA, USA), consisting of a binary pump solvent

253

management system, an online degasser, and an autosampler. MassLynxTM

254

software (version 4.1, Waters, Milford, MA, USA) was used to control the

255

instruments, and for data acquisition and processing. The separation was

256

performed on a reversed phase column (ZORBAX SB–C18, 1.8 µm, 2.1×50 mm,

257

Agilent, America), which was maintained at 30 ℃ . The mobile phase was

258

composed of acetonitrile/0.1% formic acid (9:1 at 0–5 min, 5:5 at 5–6 min, 9:1 at

259

6–8 min, v/v) with a flow rate set at 0.2 mL/min. The auto-sampler was

260

conditioned at 5℃ and each injection volume was 1 µL.

261

Mass spectrometry detection was performed using a Xevo Triple Quadrupole MS

262

(Waters Corp., Milford, MA, USA) equipped with an electrospray ionization source 13



263

(ESI). The ESI source was set in negative ionization mode. The parameters in the

264

source were set as follows: capillary voltage, 2.0 kV; cone Voltages, 70 V; collision

265

gas, argon; collision energy, 55 eV; desolvation gas, nitrogen; desolvation gas flow,

266

600 L/h; desolvation temperature, 250℃; cone gas flow, 150 L/h. The analytes were

267

quantitated by the multiple reactions monitoring (MRM). The mass spectra of the two

268

compounds (1 and 2) showed a relative abundant [M–H]– at m/z 1013.5 both for 1 and

269

2, while MS/MS of the ion indicated that the ions at m/z 487.41 for 1 and 645.28 for 2

270

were the most abundant product ions.

271 272 273

RESULTS AND DISCUSSION Isolation and Elucidation of Sweet Compounds. According to the sensory

274

bioassay-guided investigation, the n-BuOH portion and aqueous portion of 80%

275

aqueous MeOH extract, both of which showed licorice-like sweetness, were subjected

276

to isolation and purification on repeated column chromatography to obtain 4

277

sweet-tasting triterpenoid saponins (1–4), of which compounds 2-4 are new ones,

278

while compound 1 is identified for the first time from the genus Derris.

279

Millettiasaponin A (1) was obtained as a white amorphous powder. The molecular

280

formula was C50H78O21, with 12 degrees of unsaturation, in agreement with the

281

positive ESI-MS spectrum (m/z 1037, [M+Na]+; 1059, [M+2Na–H]+) and the negative

282

ESI-MS (m/z 1013, [M–H]–, 2027, [2M–H]–) , as well as 13C NMR spectroscopic data.

283

Obvious signals observed in the 1H NMR spectrum (Tables 1 and 2) of 1 were seven

284

tertiary methyls [δH 0.64, 0.87, 0.96, 1.30, 1.31, 1.39, 2.07 (each 3H, s)], a secondary 14


Page 14 of 33

Page 15 of 33


285

methyl [δH 1.79 (d, J=6.5)], a hydroxymethyl [δH 4.22, 3.23 (each d, J=11.5), a

286

olefinic bond [δH 5.52 (br s)], and three anomeric signals [δH 4.97 (d, J=8.0), 5.70 (d,

287

J=7.5), 6.17 (s)]. The 13C NMR spectrum (Tables 1 and 2) exhibited characteristic

288

carbon signals of an oleanane-type triterpenoid aglycone with a hydroxymethyl

289

group.21, 22 Detailed analysis of 13C NMR data with those of millettiasaponins A,21

290

which was isolated from Millettia speciosa, revealed that that it was almost the same

291

as those of millettiasaponins A. Further analysis of HSQC, HMBC, 1H-1H COSY, and

292

ROESY spectra of 1, it was determined as millettiasaponin A unambiguously. Here,

293

the 1H NMR data were assigned completely.

294

Derrisaponin A (2) was obtained as a white amorphous powder. The molecular

295

formula of 2 was deduced to be C56H88O26, with 12 degrees of unsaturation, through

296

the HRESI-MS spectrum (m/z 1175.5480, [M–H]–), which was in agreement with the

297

results of the ESI-MS (m/z 1175, [M–H]–; 587, [M–2H]2–) and 13C NMR data analysis.

298

The IR spectrum suggested the presence of hydroxyl group (3423 cm–1), carbonyl

299

group (1713 cm–1), and olefinic group (1615 cm–1). Acid hydrolysis of 2 afforded

300

D-glucuronic

301

NMR spectra data (Tables 1 and 2) of 2 with 1, it was inferred that 3 and 1 shared the

302

same aglycone, except for the presence of the additional signals corresponding to a

303

glucosyl of C6H10O5 based on the molecular weight with162 more than that of 1. The

304

C3Gal-O-Glc was deduced based on the glycosidation shift of C-3Gal from δ 76.6 to δ

305

84.9, combined with the HMBC correlations. The assignment of all the sugar and

306

aglycone residues was determined by the HSQC, HMBC, 1H-1HCOSY and ROESY

acid, D-galactose, L-rhamnose, D-glucose. Comparing the 1H and 13C

15



307

experiments. The linkage sites of sugar units and the aglycone were determined by the

308

HMBC correlations from H-1GluA (δH 4.96) to C-3 (δC 91.8), from H-1Ga l δH (5.70) to

309

C-2GluA (δC 77.0), from H-1Rha (δH 6.10) to C-2GalδC (77.0), and from H-1Glc δH (5.04)

310

to C-3Gal (δC 84.9). From all the analysis above, the structure of 2 was established as

311

22β-acetyloxy-3β, 24-dihydroxy-olean-12-en-30-oic acid

312

3-O-β-D-glucopyranosyl-(1→3)-[α-L-rhamnopyranosyl-(1→2)]-β-D-galactopyranosyl

313

-(1→2)-β-D-glucuronopyranoside, and named derrisaponin A.

314

Derrisaponin B (3) was obtained as a white amorphous powder. The molecular

315

formula of 3 was deduced to be C56H88O26, with 13 degrees of unsaturation, on the

316

basis of HRESI-MS spectrum (m/z 1175.5482, [M–H]–), which was compatible with

317

the results of ESI-MS (m/z 1175, [M–H]–) and NMR data analysis. The IR spectrum

318

suggested the presence of hydroxyl group (3424 cm–1), carbonyl groups (1718 cm–1),

319

and olefinic group (1615 cm–1). The molecular formula (C56H88O26) suggested the

320

same molecular formula of 3 with 2. And acid hydrolysis of 3 yielded the same sugar

321

with 2. Detailed analysis of the NMR spectra data (Tables 1 and 2) for the aglycone of

322

3 and 1, indicated that 3 and 1 shared the same aglycone and C-3 sugar linkage, and

323

the significant difference between them was that the carboxyl group at C-30 was

324

glycosylated by a glucosyl on the basis of the glycosidation shift of C-30 from δ 179.9

325

to δ 177.0. The assignment of all the sugar residues and the aglycone moiety was

326

determined by the HSQC, HMBC, 1H-1H COSY and ROESY experiments. The

327

linkage of the sugar units and the aglycone were determined by the HMBC

328

correlations from H-1GluA (δH 4.96) to C-3 (δC 91.8), from the proton at δH 5.69 16


Page 16 of 33

Page 17 of 33


329

(H-1Gal) to C-2GluA (δC 76.9), from H-1Rha (δH 6.18) to the C-2Gal (δC 77.7), and from

330

H-1Glc (δH 6.10) to C-30 (δC 177.0). Therefore, the structure of 3 was determined as

331

22β-acetyloxy-3β, 24-dihydroxy-olean-12-en-30-oic acid

332

3-O-α-L-rhamnopyranosyl-(1→2)-β-D-galactopyranosyl-(1→2)-β-D-glucuronopyrano

333

syl-30-O-β-D-glucopyranoside, and named derrisaponin B.

334

Derrisaponin C (4) was obtained as a white amorphous powder. The molecular

335

formula of 4 was deduced to be C62H98O31, with 14 degrees of unsaturation, by

336

HRESI-MS spectrum (m/z 1337.6017, [M–H]–), which was compatible with the

337

results of ESI-MS (m/z 1337, [M–H]– and NMR data analysis. The IR spectrum

338

suggested the presence of hydroxyl group (3424 cm–1), carbonyl group (1720 cm–1),

339

and olefinic group (1619 cm–1). Acid hydrolysis of 4 yielded D-glucuronic acid,

340

D-galactose, L-rhamnose, D-glucose.

341

for 4 with compounds 1-3 was indicated by the analysis of 1H and 13C NMR data

342

(Tables 1 and 2). Detailed analysis of 13C NMR data of 4 with those of 2 indicated

343

that the significant difference between them was that the carboxyl group at C-30 was

344

glycosylated by a glucosyl on the basis of the glycosidation shift of C-30 from δ 180.0

345

to δ 177.1. And the C3Gal-O-glucosyl and C30-O-glucosyl can be confirmed by the

346

glycosidation shifts of C-3Gal from δ 76.6 to δ 84.8, and C-30 from δ 179.9 to δ 177.1.

347

The assignment of all the sugar residues and linkage sites of sugar units and the

348

aglycone were determined by the HSQC, HMBC, 1H-1H COSY and ROESY

349

experiments. Therefore, the structure of 4 was determined as 22β-acetyloxy-3β,

350

24-dihydroxy-olean-12-en-30-oic acid

The same aglycone of oleanane-type triterpenoid

17



351

3-O-β-D-glucopyranosyl-(1→3)-[α-L-rhamnopyranosyl-(1→2)]-β-D-galactopyranosyl

352

-(1→2)-β-D-glucuronopyranosyl-30-O-β-D-glucopyranoside, and named derrisaponin

353

C.

354

Acute Toxicity Test. The acute toxic activities of 1 and 2 were assessed through

355

caudal veins injection to ICR mice. After injection, one of the animals administered

356

by compound 2 (at dose of 400 mg/kg bw) showed diaphragm spasm, but return to

357

normal 10 min later, and the other animals in this group and the other two groups

358

animals (administered by compound 1 at the dose of 250 mg.kg bw and solvent

359

respectively) did not show any unmoral signs. No treatment-related clinical signs of

360

toxicity or mortality were observed for the 14 days observation. In addition, all

361

internal organs examined at necropsy were free from any gross pathological changes

362

at the end of observation. This indicates that the maximum tolerated doses (MTD) of

363

compounds 1 and 2 are 250 mg/kg and 400 mg/kg respectively.

364

Sweetness Intensities of Compounds 1-4. The sweetness intensities of 1-4 were

365

evaluated at near sweetness detection threshold concentrations by seven

366

sweet-sensitive panelists and the results are reported. Compounds 1 and 2, with a

367

carboxyl group at C-30, showed potent sweetness (150 and 80 times, sweeter than that

368

of sucrose, and the threshold value is 0.05 mg/ml and 0.0625 mg/ml, respectively),

369

while the sweetness of 3 and 4, with a carboxyl group glycosylated by a glucose

370

group, showed only 2 and 0.5 times sweeter than that of sucrose, respectively (The

371

threshole value is 1 mg/ml and 5 mg/ml, respectively). Moreover, 1 and 2 showed the

372

most licorice-like sweetness. Accordingly, a free carboxyl group at the C-30 position 18


Page 18 of 33

Page 19 of 33


373

seems to be essential to the potent sweetness for this type of compounds.

374

Quantitation by UPLC–MS/MS. The quantitation of compounds 1 and 2 in the

375

stems of D. eriocarpa were analyzed by means of UPLC–MS/MS. The interesting

376

contents of 352.80 mg/kg for 1 and 1887.60 mg/kg for 2 respectively were calculated

377

from the tested data (Table 3). The regression equations of 1 and 2 were y = 316.72x –

378

2467.9 (r2 = 0.9991) and y = 10.882x – 40.715 (r2 = 0.9993), respectibely.

379

Based on the experimental data, it can be inferred that these four oleanane-type

380

triterpenoid saponins are major sweet-tasting components in the stem of this plant and

381

they are responsible for the licorice-like sweetness of D. eriocarpa. These studies

382

provide solid evidence that the stems of D. eriocarpa can be used for the alternatives

383

of licorice in the taste aspect. In addition, compounds 1 and 2 showed no acute

384

toxicity and the maximum tolerated doses (MTD) of them are 250 mg and 400 mg

385

respectively, suggesting that they may be potential natural sweeteners.

386 387

ASSOCIATED CONTENT

388

Supporting Information

389 390 391

The 1H NMR,

13

C NMR, and 2D-NMR (HSQC, HMBC, 1H-1H COSY, and

ROESY) and MS spectra of compounds 1-4 The Supporting information is available free of charge on the ACS Publications

392

website at DOI:

393

AUTHOR INFORMATION

394

Corresponding Author 19



395

* Phone: +86-871-65223224; E-mail address: [email protected]

396

Funding

397

This work was sponsored by grants from the Natural Science Foundation of Yunnan

398

Province (2013FB065), the 45th Scientific Research Foundation for the Returned

399

Overseas Chinese Scholars from State Education Ministry and National S&T Basic

400

Work Program of China (2012FY110300).

401

Note

402

The authors declare no competing financial interests.

403 404

ACKNOWLEDGMENTS

405

We thank Professor Lisong Wang for identification of the plant. The authors

406

appreciate the efforts of all panelists participating in the sensory tests. The authors

407

appreciate the sacrifice of all ICR mice.

20


Page 20 of 33

Page 21 of 33


408

REFERENCES

409

(1) Temussi, P. The history of sweet taste: not exactly a piece of cake. J. Mol.

410

Recognit. 2006, 19, 188-199.

411

(2) Vos, M. B.; Kaar, J. L.; Welsh, J. A. Added sugars and cardiovascular disease risk

412

in children: a scientific statement from the American Heart Association. Circulation

413

2016, 134, 1-18.

414

(3) Yang, Q. H.; Zhang, Z. F.; Gregg, E. W. et al. Added sugar intake and

415

cardiovascular diseases mortality among US adults. JAMA Intern. Med. 2014, 174,

416

516-524.

417

(4) Kim, N. C.; Kinghorn, A.D. Highly sweet compounds of plant origin. Arch. Pharm.

418

Res. 2002, 25, 725-746.

419

(5) Kinghorn, A. D.; Kennelly, E. J. Discovery of highly sweet compounds from

420

natural sources. J. Chem. Educ. 1995, 72, 676-680.

421

(6) Kinghorn A. D.; Chin Y. W.; Pan L.; Jia Z. H. Natural products as sweeteners and

422

sweetness modifiers. In Comprehensive natural products II; Chemistry and Biology;

423

Development & Modification of Bioactivity; Elsevier: Oxford, U.K, 2010; vol. 3, pp.

424

269-314.

425

(7) Huang Y. L.; Guo Z.Y.; Liu Y. J.; Wang Y. L.; Luo B. S.; Long C. L. Indigenous

426

Botanical Nomenclature Used by the Zhuang People in Jingxi County, Guangxi. Plant

427

Diversity and Resour. 2013, .35, 443-452.

428

(8) Yunnan Food and Drug Administration. The Standards of Chinese Medicinal

429

Materials in Yunnan Province, Dai Ethnophamacy ( Ⅱ ). Yunnan Science & 21



430

Technology Press: Kunming, 2005, vol. 5, pp. 7, 115.

431

(9) Guangxi Institute of Chinese Medicine & Pharmaceutical Science. Medicinal

432

Plants Directory of Guangxi. Guangxi People's Publishing House: Nanning, 1986, pp.

433

232.

434

(10) Editoria Committee of the Administration Bureau of Traditional Chinese

435

Medicine. Chinese Matrria Medica (Zhonghua Bencao). Shanghai Press of Science

436

and Technology: Shanghai, 1998, vol. 4, pp. 442.

437

(11) Yang, D. A.; Guo, L. C. Review of five Tugancao medicines with same name in

438

Guangxi. Cent. South Pharm. 2013, 11, 282-284.

439

(12) Wang, L. X.; Wu, H. G.; Zhang, H.; Lou, H. Y.; Liang, G. Y.; Jiang, W. W.; Yang,

440

Z. C.; Pan, W. D. Studies on flavonoids from Derris eriocarpa. Chin. J. Chin. Mater.

441

Med. 2015, 40, 3009-3012

442

(13) Yang, L. F.; Wang, K.; Jiang M. G.; Liu, H. C.; Wang, X.; Qin, P. Y.; Ouyang, Q.

443

L. Isolation and characterization of a new bioactive isoflavone from Derris eriocarpa.

444

J. Asian. Nat. Prod. Res. 2015, 17, 1002-1009.

445

(14) Zhang, H. X.; Lunga, P. K.; Li, Z. J.; Dai, Q.; Du, Z. Z. Flavonoids and

446

stilbenoids from Derris eriocarpa. Fitoterapia 2014, 95, 147-153.

447

(15) Ren W. K.; Chen S.; Yin J.; Duan J. L.; Li T. J.; Liu G.; Feng Z. M.; Tan B.; Yin

448

Y. L.;, Wu G. Y. Dietary arginine supplementation of mice alters the microbial

449

population and activates intestinal innate immunity. J. Nutr. 2014, 144, 988-995.

450

(16) ISO 8586: 2012. Sensory Analysis—General guidelines for the selection, training

451

and monitoring of selected assessors and expert sensory assessors, 2012. 22


Page 22 of 33

Page 23 of 33


452

(17) Jia, Z. H.; Yang, X. G. A minor, sweet cucurbitane glycoside from Siraitia

453

grosvenorii. Nat. Prod. Commun. 2009, 4, 769-772.

454

(18) Fairhurst, S.; Marrs, T. C.; Parker, H. C.; Scawin J. W.; Swanston D. W. Acute

455

toxicity of T2 toxin in rats, mice, guinea pigs, and pigeons. Toxicology 1987, 43,

456

31-49.

457

(19) Wang J. S.; Yang X. W.; Di Y. T.; Wang Y. H.; Shen Y. M,; Hao, X. J. Isoflavone

458

Diglycosides from Glycosmis pentaphylla. J. Nat. Prod. 2006, 69, 778-782.

459

(20) Liang, D.; Hao, Z. Y.; Zhang, G. J.; Zhang, Q. J.; Chen, R. Y.; Yu, D. Q.

460

Cytotoxic triterpenoid saponins from Lysimachia clethroides. J. Nat. Prod. 2011, 74,

461

2128-2136.

462

(21) Uchiyama, T.; Furukawa, M.; Isobe, S.; Makino, M.; Akiyama, T.; Koyama, T.;

463

Fujimoto, Y. New oleanane-type triterpene saponins from Millettia speciosa.

464

Heterocycles 2003, 60, 655-661.

465

(22) Mahato S. B.; Kundu A. P. 13C NMR spectrumof pentacylclic triterpenoids — a

466

compilation and some salient features. Phytochemistry 1994, 37, 1517-1575.

23



467

Figure captions

468

Figure 1. The structure of compounds 1-4

469

Figure 2. The selected HMBC (H

470

Figure 3. ROESY correlations of the aglycone moiety

C) and 1H-1H COSY (

24


) correlations of 4

Page 24 of 33

Page 25 of 33


Table 1. 1H and 13C NMR spectroscopic data of the aglycone moieties of compounds 1-4 in pyridine-d5 (δ in ppm, J in Hz) 1

2

3

4

NO. 1

δC

δH

δC

δH

δC

δH

δC

δH

38.9, t

1.29 (m), 0.76 (m)

39.0, t

1.33 (m), 0.79 (m)

38.8, t

1.34 (m), 0.84 (m)

38.9, t

1.37 (m), 0.86 (m)

2

27.0, t

2.09 (m), 1.74 (m)

27.1, t

2.10 (m), 1.95 (m)

26.9, t

2.13 (m), 1.84 (m)

27.0, t

2.14 (m), 1.84 (m)

3

91.5, d

3.35 (dd, 11.5, 3.5)

91.8, d

3.35 (dd, 11.5, 4.0)

91.4, d

3.35 (dd, 12.0, 4.0)

91.7, d

3.36 (dd, 11.2, 4.0)

4

44.2, s

/

44.3, s

/

44.2, s

/

44.2, s

/

5

56.3, d

0.80 (m)

56.5, d

0.84 (m)

56.2, d

0.81 (m)

56.3, d

0.84 (m)

6

18.8, t

1.51 (m), 1.20 (m)

19.0, t

1.57 (m), 1.29 (m)

18.8, t

1.52 (m), 1.22 (m)

19.0, t

1.56 (m), 1.27 (m)

7

33.1, t

1.45 (m), 1.24 (m)

33.3, t

1.50 (m), 1.33 (m)

33.1, t

1.43 (m), 1.21 (m)

33.1, t

1.45 (m), 1.26 (m)

8

40.5, s

/

40.6, s

/

40.3, s

/

40.4, s

/

9

48.0, d

1.57 (m)

48.1, d

1.59 (m)

47.9, d

1.54 (m)

48.0, d

1.55 (m)

10

36.8, s

/

36.9, s

/

36.7, s

/

36.9, s

/

11

24.4, t

1.76 (2H)

24.5, t

1.79 (2H)

24.2

1.70 (2H)

24.4, t

1.72 (2H)

12

123.6, d

5.52 (brs)

123.7, d

5.52 (brs)

123.7, d

5.40 (brs)

123.8, d

5.42 (brs)

13

144.5, s

/

144.6, s

/

144.0, s

/

144.2, s

/

14

42.3, s

/

42.4, s

/

42.1, s

/

42.1, s

/

15

26.6, t

1.74 (m), 0.98 (m)

26.8, t

1.77 (m), 1.01 (m)

26.5, t

1.68 (m), 0.94 (m)

26.5, t

1.70 (m), 0.96 (m)

16

26.6, t

1.93 (m), 0.98 (m)

26.7, t

1.95 (m), 1.01 (m)

26.4, t

1.83 (m), 0.94 (m)

26.4, t

1.83 (m), 0.96 (m)

17

36.6, s

/

36.7, s

/

36.6, s

/

36.7, s

/

18

44.4, d

2.93 (dd, 12.5, 3.5)

44.6, d

2.93 (dd, 12.5, 4.0)

44.1, d

2.84 (dd, 12.0, 4.0)

44.1, d

2.84 (dd, 12.0, 4.0)

19

42.0, t

20

41.2, s

2.30 (d, 11.5) 1.82 (m) /

42.2, t 41.3, s

2.30 (d, 10.5) 1.82 (m) /

41.3, t 41.4, s

25


2.17 (d, 12.0) 1.75 (m) /

41.4, t 41.5, s

2.19 (dd, 11.2, 5.6) 1.75 (m) /


2.81 (d, 14.0),

2.82 (d, 13.0)

22

78.6, d

4.83 (brs)

78.5, d

4.84 (brs)

77.4, d

4.88 (brs)

77.6, d

4.90 (brs)

23

23.3, q

1.39 (s)

23.4, q

1.38 (s)

23.2, q

1.40 (s)

23.2, q

1.39 (s)

24

63.8, t

25

16.1, q

0.64 (s)

16.1, q

0.67 (s)

16.0, q

0.67 (s)

26

17.1, q

0.87 (s)

17.3, q

0.90 (s)

17.0, q

0.85 (s)

17.1, q

0.88 (s)

27

27.1, q

1.30 (s)

27.0, q

1.33 (s)

26.8, q

1.23 (s)

26.9, q

1.25 (s)

28

21.8, q

0.96 (s)

21.9, q

0.98 (s)

21.6, q

0.86 (s)

21.7, q

0.88 (s)

29

30.2, q

1.31 (s)

30.3, q

1.33 (s)

30.0, q

1.27 (s)

30.0, q

1.29 (s)

30

179.9, s

/

180.0, s

/

177.0, s

/

177.1, s

/

171.1, s

/

171.0, s

/

171.4, s

/

171.6, s

/

21.6, q

2.07 (s)

21.5, q

2.07 (s)

22.0, q

2.38 (s)

22.1, q

2.39 (s)

4.22 (d, 11.5); 3.23 (d, 11.5)

64.0, t

4.23 (m) 3.29 (d, 11.5)

63.6, t

26


1.71 (m)

4.22 (m) 3.23 (d, 11.2)

35.7, t

2.66 (d, 13.6)

35.6, t

1.78 (m)

35.7, t

2.64 (d, 13.6)

21

1.77 (m)

35.7, t

Page 26 of 33

63.7, t 16.1, q

1.73 (m)

4.24 (m) 3.30 (d, 11.2) 0.70 (s)

Page 27 of 33


Table 2. 1H and 13C NMR spectroscopic data of the sugar moieties of compounds 1-4 in pyridine-d5 (δ in ppm, J in Hz) NO.

1

2

3

4

δC

δH

δC

δH

δC

δH

δC

δH

1

105.8

4.97 (d, 8.0)

105.8

4.96 (m)

105.7

4.96 (d, 7.2)

105.8

4.95 (d, 8.0)

2

77.0

4.52 (m)

77.0

4.52 (m)

76.9

4.52 (t, 8.8)

76.8

4.54 (t, 8.8)

3

78.3

4.67 (m)

78.6

4.57 (m)

78.2

4.66 (m)

78.3

4.65 (m)

4

74.2

4.44 (m)

74.2

4.42 (m)

74.1

4.44 (m)

74.1

4.45 (m)

5

78.0

4.67 (m)

78.0

4.63 (m)

77.9

4.66 (m)

78.0

4.65 (m)

6

172.8

/

172.9

/

172.6

/

172.8

/

1

102.1

5.70 (d, 7.5)

102.1

5.70 (d, 7.5)

102.0

5.69 (d, 7.2)

102.0

5.69 (d, 7.2)

2

77.8

4.49 (m)

77.0

4.48 (m)

77.7

4.49 (dd, 9.6, 7.2)

76.7

4.48 (m)

3

76.6

4.10 (dd, 9.5, 3.0)

84.9

4.08 (dd, 9.5, 2.0)

76.5

4.08 (dd, 9.6, 3.2)

84.8

4.05 (m)

4

71.3

4.39 (m)

71.3

4.72 (m)

71.2

4.40 (d, 3.2)

70.9

4.79 (m)

5

76.6

3.97 (m)

76.6

3.96 (m)

76.4

3.95 (t, 6.4)

76.5

4.03 (t, 6.4)

6

61.7

62.2

4.25 (2H, m)

61.7

3-O-GluA

Gal

4.37(dd, 9.5, 3.0) 4.30 (dd, 11.0, 6.0)

4.42 (2H, m), 4.34 (m)

61.7

4.30 (2H, m) 4.27 (m)

Rha 1

102.6

6.17 (s)

102.7

6.10 (s)

102.4

6.18 (s)

102.6

6.13 (s)

2

72.7

4.81 (m)

72.7

4.87 (brs)

72.6

4.83 (dd, 3.2, 1.6)

72.5

4.92 (d, 3.2)

3

72.8

4.80 (dd, 9.5, 3.0)

73.0

4.69 (m)

72.7

4.79 (dd, 9.6, 3.2)

72.7

4.79 (m)

4

74.7

4.36 (m)

74.7

4.30 (m)

74.5

4.36 (m)

74.6

4.34 (m)

5

69.8

5.01 (m)

69.8

4.93 (m)

69.6

5.0 (dq, 12.0, 6.0)

69.6

4.97 (m)

6

19.3

1.79 (d, 6.5)

19.3

1.72 (d, 6.0)

19.2

1.76 (d, 6.4)

19.3

1.73 (d, 5.6)

27



Page 28 of 33

Glc I 1

106.3

5.04 (d, 8.0)

106.3

5.06 (d, 7.2)

2

75.4

3.95 (m)

75.2

3.98 (t, 8.8)

3

78.7

4.21 (m)

78.5

4.32 (m)

4

71.8

4.12 (t, 9.5)

71.7

4.12 (t, 9.6)

5

78.7

3.86 (m)

78.7

3.92 (m)

6

62.7

4.40 (m), 4.29 (m)

62.3

4.34 (m), 4.27 (m)

30-O-Glc II 1

96.6

6.10 (d, 8.0)

96.7

6.11 (d, 8.0)

2

74.0

4.20 (m)

74.2

4.22 (m)

3

78.7

4.31 (m)

78.2

4.32 (m)

4

71.4

4.33 (m)

71.4

4.33 (m)

5

79.5

4.05 (m)

79.6

4.06 (m)

6

62.3

4.37 (m), 4.29 (m)

62.5

4.42 (2H, m)

28


Page 29 of 33


Table 3. Content of 1 and 2 in the stems of D. eriocarpa Trace (m/z) parent

daughter

RT (min)

1

1013.5

645.28

3.29

7288.672

29.4

352.8

2

1175.9

645.3

2.70

1659.176

157.3

1887.6

Compounds

Area

Content in sample solution (µg/ml)

Content in stem (mg/kg)

29



Figure 1.

30


Page 30 of 33

Page 31 of 33


Figure 2.

31



Figure 3.

32


Page 32 of 33

Page 33 of 33


TOC graphic O 30

29

？

Licorice-like sweetness Substitute for licorice（The stems of Derris eriocarpa）

OR2 19 13

11 25

26

1

23

17

22

OAc 28

14 10

9 15

3

R1O

21 18

27 5

7

CH2OH

24

Sweet-Tasting Compounds 1-4

33


New Sweet-Tasting Oleanane-Type Triterpenoid Saponins from

Recommend Documents