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Chemistry and Biology of Aroma and Taste

New Sweet-tasting C21-pregnane Glycosides from the Roots of Myriopteron extensum Guo Sun, Hongxia Zhang, YongPeng Ma, MingXiang Li, and ZhiZhi Du J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02348 • Publication Date (Web): 02 Jul 2018 Downloaded from http://pubs.acs.org on July 8, 2018

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Journal of Agricultural and Food Chemistry

New Sweet-tasting C21 Pregnane Glycosides from the Roots of Myriopteron extensum

Guo Sun,† Hong-Xia Zhang,† Yong-Peng Ma,†,‡ Ming-Xiang Li,†,‡ 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, Yunnan, China ‡

University of Chinese Academy of Sciences, Beijing 100049, China

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

ACS Paragon Plus Environment


1

ABSTRACT

2

To investigate the sweet-tasting components in the roots of Myriopteron extensum,

3

the phytochemical study of its roots was conducted, which leads to the discovery of

4

twelve new C21 pregnane glycosides (extensumside M-X, 1-12) and two known ones

5

(extensumside C and extensumside E, 13-14). Their chemical structure elucidation

6

was accomplished by means of spectroscopic methods: IR, UV, ESI-MS, and NMR

7

(1H NMR, 13C NMR, HSQC, 1H-1H COSY, HMBC, HSQC-TOCSY, and ROESY), as

8

well as the chemical evidences. Sensory analysis of these compounds revealed that

9

nine of them (1, 3, 4, 5, 6, 7, 8, 13 and 14) are highly sweet-tasting compounds. Their

10

sweetness intensities are 25 to 400 times greater than that of sucrose. Analysis of the

11

structure-activity relationship (SAR) indicated that the sweet intensities of the isolated

12

compounds are closely related to the aglycone 3β,16α-dihydroxy-pregn-5-en-20-one,

13

the number and type of the monosaccharide in the sugar chain linked to C-3 and C-16

14

and the position of the mBe group.

15 16

KEYWORDS

17

Natural sweet molecules, C21 pregnane glycosides, human sensory evaluation,

18

sweet intensity, Myriopteron extensum

19


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INTRODUCTION

21

Human can percept at least five basic taste qualities, including sweet, umami, bitter,

22

salty, and sour,1 of which the sweet and umami taste affect the people’s selection of

23

foods and beverages to a large extent,1b and especially the sweet is the most favorite

24

taste for newborns and children.1b Sweeteners are the sweet-tasting substances used as

25

food additives in food and beverage industries. Sucrose, a disaccharide produced from

26

sugarcane and sugar beet, is the most widely used sweetener in the world.2

27

Additionally, other natural sugars, such as fructose, and glucose, are also used in

28

foods and beverages. However, with the widespread application and high intake of

29

sugar sweeteners in daily diet, some healthy concerns, such as dental caries,

30

hypertension, hyperglycemia, cardiovascular diseases, and obesity, have arisen in

31

recent years.3 Some researches showed that these diseases were related to the excess

32

intake of sugars to a certain extent.3-4 Due to these problems and the high calorie of

33

sugars, artificial synthetic sweeteners were widely used in food and beverage

34

industries to meet the great demand of food and beverages’ market for sweeteners.

35

The current market-available synthetic sweeteners are including acesulfame-K,

36

alitame, aspartame, cyclamate, neotame, saccharin, and sucralose, some of which are

37

regulated or banned to use as substitute for sugars in USA or European Union due to

38

their safety concerns.5 For example, cyclamate is not permitted to use for food and

39

beverages in the United States, and other countries allow its use with an ADI

40

(acceptable daily intake) of 11 mg/kg bodyweight per day.6 This impels people to turn

41

their attention to researching the low-calorie and high sweet-potency natural



42

sweeteners.

43

Although many sweet-tasting substances including low molecular compounds and

44

proteins have been found from the nature,1b, 5 only several of them are developed to be

45

market-available sweeteners, such as rebaudioside A, stevioside, steviol glycosides,

46

mogrol glycosides (Luo Han Guo sweetener), morgroside V.6 Furthermore, the

47

resource and yield of these natural high-potency and non-sugar sweeteners are very

48

limited relative to the great demands of foods and beverages industries for sweeteners.

49

Thus, the searching and development of new natural sweeteners remained to be a

50

significant and urgent problem that we are facing on.

51

Plants are the natural compounds library for finding new sweetener candidates or

52

leads due to the biodiversity and chemical diversity. Ethnobotanical survey combined

53

with sensory evaluation provides a good chance to find some species of plants to be

54

used for us to investigate natural sweet molecules. Thereof, Myriopteron extensum, a

55

plant of monotypic genus from the Asclepiadaceae, was selected for further study of

56

its sweet-tasting constituents because. In our previous work, we reported the

57

identification and sensory evaluation of ten new sweet-tasting C21 pregnane

58

glycosides from the pericarps of the plant through sensory-guided phytochemical

59

investigation.7 Their sweetness intensities range from 50-400 times relative to that of

60

sucrose, with all having a relatively low sweetness threshold. Furthermore, the

61

quantitation of the sweet compounds in the pericarps, stems and roots indicated that

62

all of them contain this kind of sweet components with a distinct distribution, and that

63

some different sweet constitutes may exist in the roots.7


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The objective of the present study, therefore, was to ascertain the sweet-tasting

65

components in the roots of M. extensum by means of the phytochemical fractionation

66

approach and modern spectroscopic technologies, to determine their molecular

67

structure, as well as human sensory evaluation of their sweetness intensities and

68

thresholds. Nine C21 pregnane glycosides were identified as highly sweet-tasting

69

constituents from the roots of M. extensum in which seven new sweet molecules are

70

different from the compounds found from the pericarps of the plant.

71 72

MATERIALS AND METHODS

73

Chemicals. The following materials were used: AR grade methanol (Huada,

74

Guangzhou, China), L-cysteine methyl hydrochloride (Sigma-Aldrich, Shanghai,

75

China), N-trimethylsilylimidazole (Sangon Biotech, Shanghai, China), n-hexane

76

(Damao, Tianjin, China), D-(+)-glucose (J & K Scientific, Guangzhou, China), HPLC

77

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

78

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

79

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

80

China), chloroform (Rionlon, Tianjin, China), dioxane (Sinopharm chemical reagent

81

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

82

hydroxide (Xilong Chemical Co. Ltd., Guangdong, China), vanillin (Sinopharm

83

Chemical Reagent Co., Ltd., Shanghai, China), sucrose (Dianwangyi, Yunnan, China),

84

water (Wahaha Group Co. Ltd, Hangzhou, China)

85

General Experimental Procedures. Optical rotations were measured with a Jasco



86

P-1020 polarimeter (Jasco, Tokyo, Japan). UV spectra were obtained using a

87

Shimadzu UV-2401PC spectrophotometer (Shimadzu, Tokyo, Japan). A Bruker

88

Tensor 27 spectrophotometer (Bruker, Bremen, Germany) was used for scanning IR

89

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

90

Avance III 500 and Avance III 600 spectrometer (Bruker, Bremen, Germany) at 298 K.

91

Unless otherwise specified, chemical shifts (δ) were expressed in ppm with reference

92

to the solvent signals. ESIMS were obtained on a Bruker Esquire HCT spectrometer

93

(Bruker, Bremen, Germany). HRESIMS were recorded on an Agilent G6230 TOF MS

94

spectrometer (Agilent Technologies, Santa Clara, CA). Column chromatography (CC)

95

was done using silica gel (200−300 mesh, Qingdao Marine Chemical Co. Ltd., China),

96

RP-18 silica gel (40−63 µm, Merck, Germany), macroporous adsorption resin D101

97

(Yunkai Resin Technology Co. Ltd., Tianjin, China), and Sephadex gel LH-20 (GE

98

Healthcare Biosciences AB, Sweden). TLC was performed on silica gel GF254

99

(Qingdao Marine Chemical Co. Ltd., China), and spots were visualized by heating

100

silica gel plates sprayed with 5% vanillin-H2SO4 reagent. HPLC analyses and

101

separations were performed on a Newstyle LC system (Hanbon Sci. & Tech. Co. Ltd.,

102

Jiangsu, China) equipped with two high pressure constant-current infusion pump

103

(NP7000) and a UV/Vis detector (NU3000). A lyophilizer (Virtis Benchtop K, USA)

104

was used to dry the samples and eliminate the residual solvents.

105

Plant Material. The roots of M. extensum were collected from Xinpin county of

106

Yunnan province, southwest of China, and identified by Professor Lisong Wang. A

107

voucher specimen (KUN 0309000) was deposited in Herbarium of Kunming Institute


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of Botany, Chinese Academy of Sciences.

109 110

Extraction and Isolation of Compounds. The air-dried roots (6.0 kg) of M.

111

extensum were ground, and extracted with 95% ethanol (18 L, industrial grade) for

112

three times (2 d each). The combined filtrate was concentrated under reduced vacuum

113

to get ethanol extract. The rest botanical residue was further extracted with 70%

114

aqueous ethanol (16 L × 2 d × 3) for three times. The combined filtrate was

115

concentrated under reduced vacuum to give 70% ethanol extract. The ethanol extract

116

was suspended in 1 L water, and then extracted with petroleum ether (1 L × 5) for 5

117

times. The combined extract solution was concentrated under reduced vacuum to

118

yield the petroleum ether portion (A, 37.77 g). The rest water layer was combined

119

with the 70% ethanol extract, and filled up with water to 2 L. Then the combined

120

water layer was further extracted with EtOAc (2 L × 5) for 5 times to yield the EtOAc

121

extract and the water extract, respectively. Both of these two extracts were

122

concentrated under reduced vacuum to obtain the EtOAc portion (B, 108.50 g) and

123

the water portion (C, 622.0 g).

124

The EtOAc portion (100.0 g) was submitted to column chromatography (φ 7.8 cm

125

× 60 cm) on silica gel, eluted with chloroform-methanol solvent system (C/M 50:1,

126

20:1, 10:1, 5:1, 2:1, 0:1, 10 L each; C: chloroform, M: methanol), to give seven

127

subfractions (Fr. B1-Fr. B7). Fr. B3 (10.4 g) was submitted to medium pressure liquid

128

chromatography (MPLC, BUCHI, φ 4.0 cm × 25 cm) on reverse phase-C18 (RP-18,

129

40-63 µm) eluted with 30%-80% aqueous methanol (3000 mL each) to give seventeen



130

subfractions (Fr. B3-1 ~ Fr. B3-17). Fr. B3-14 (553 mg) was submitted to Sephadex

131

LH-20 gel column chromatography eluted with C/M (1:1) solvent, and then to column

132

chromatography (φ 3.0 cm × 14 cm) on silica gel, eluted with C/M (40:1), to yield

133

compound 9 (425 mg). Fr. B5 (25.74 g) was chromatographed on MPLC (RP-18,

134

40-63 µm; φ 4.0 cm × 25 cm), eluted with 30%-80% aqueous methanol (3000 mL

135

each), to give twelve subfractions (Fr. B5-1 ~ Fr. B5-12). Fr. B5-8 (3.01 g) was

136

chromatographed on silica gel (φ 3.0 cm × 20 cm), eluted with C/M (8:1, 7:1, 6:1, 4:1,

137

1000 mL each), to give six subfractions (Fr. B5-8-1 ~ Fr. B5-8-6). Fr. B5-8-3 (381 mg)

138

was submitted to Sephadex LH-20 gel column chromatography eluted with C/M (1:1),

139

and then purified by semi-preparative HPLC on a YMC-Pack ODS-AQ column (250

140

mm × 10 mm I.D., flow rate 3.0 mL/min) with 85% MeOH-H2O, to yield 13 (82 mg,

141

tR = 13.90 min). Fr. B5-8-4 (448 mg) was submitted to Sephadex LH-20 gel column

142

chromatography eluted with methanol, and then purified by semi-preparative HPLC

143

on a YMC-Pack ODS-AQ column (250 mm × 10 mm I.D., flow rate 3.0 mL/min)

144

with 90% MeOH-H2O to yield 5 (26 mg, tR = 11.80 min). Fr. B5-8-6 (804 mg) was

145

chromatographed on Sephadex LH-20 gel eluted with methanol, and then purified by

146

semi-preparative HPLC on a YMC-Pack ODS-AQ column (250 mm × 10 mm I.D.,

147

flow rate 3.0 mL/min) with 80% MeOH-H2O to yield 8 (29 mg, tR = 15.80 min), 4 (25

148

mg, tR = 19.20 min), and 14 (120 mg, tR = 21.05 min). Fr. B5-10 (1.557 g) was

149

chromatographed on silica gel (φ 3.0 cm × 17 cm), eluted with C/M (8:1, 7:1, 5:1, and

150

4:1, 800 mL each), to give five subfractions (Fr. B5-10-1~Fr. B5-10-5). Fr. B5-10-2

151

(443 mg) was purified by semi-preparative HPLC on a YMC-Pack ODS-AQ column


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(250 mm × 10 mm I.D., flow rate 3.0 mL/min) with 85% MeOH-H2O to yield 10 (135

153

mg, tR = 11.36 min). Fr. B5-10-3 (407 mg) was purified by semi-preparative HPLC on

154

a YMC-Pack ODS-AQ column (250 mm × 10 mm I.D., flow rate 3.0 mL/min) with

155

85% MeOH-H2O to yield 11 (49 mg, tR = 17.35 min). Fr. B5-10-5 (275 mg) was

156

submitted to column chromatography on Sephadex LH-20 gel eluted with methanol,

157

and then purified by semi-preparative HPLC on a YMC-Pack ODS-AQ column (250

158

mm × 10 mm I.D., flow rate 3.0 mL/min) with 80% MeOH-H2O to yield 12(35 mg, tR

159

= 22.38 min).

160

The water portion (C, 600.0 g) was chromatographed on D101 macroporous

161

absorption resin (φ 11.0 cm × 60 cm), eluted with water, 30%, 50%, 70%, and 95%

162

aqueous ethanol, respectively, to give four subfractions (Fr. C1 ~ Fr. C4). Fr. C3

163

(19.84 g) was chromatographed on silica gel (φ 6.0 cm × 21 cm), eluted with C/M

164

(10:1, 8:1, 7:1, 6:1, and 4:1, 3200 mL each), to give nine subfractions (Fr. C3-1 ~

165

Fr.C3-9). Fr. C3-2 (969 mg) was isolated by preparative HPLC on a YMC-Pack

166

ODS-A column (250 mm × 20 mm I.D., flow rate 10.0 mL/min) with MeOH-H2O

167

(80%-85%) to give seven subfractions (Fr. C3-2-1 ~ Fr. C3-2-7). Fr. C3-2-3 (129 mg)

168

was further purified by semi-preparative HPLC on a YMC-Pack ODS-AQ column

169

(250 mm × 10 mm I.D., flow rate 3.0 mL/min) with 75% MeOH-H2O to yield 2 (33

170

mg, tR = 14.41 min). Fr. C3-2-4 (133 mg) was purified by semi-preparative HPLC on

171


172

75% MeOH-H2O to yield 1 (43 mg, tR = 19.80 min). Fr. C3-4 (2.975 g) was isolated

173

by preparative HPLC on a YMC-Pack ODS-A column (250 mm × 20 mm I.D., flow



174

rate 10.0 mL/min) with MeOH-H2O (75%-85%) to give eight subfractions (Fr. C3-4-1

175

~ Fr. C3-4-8). Fr. C3-4-4 (242 mg) was further purified by semi-preparative HPLC on

176


177

76% MeOH-H2O to yield 7 (38 mg, tR = 18.06 min). Fr. C3-5 (1.219 g) was isolated

178

by preparative HPLC on a YMC-Pack ODS-A column (250 mm × 20 mm I.D., flow

179

rate 10.0 mL/min) with MeOH-H2O (75%-85%) to give seven subfractions (Fr.

180

C3-5-1 ~ Fr. C3-5-7). Fr. C3-5-3 (105 mg) was purified by semi-preparative HPLC on

181


182

75% MeOH-H2O to yield 3 (57 mg, tR = 14.22 min). Fr. C3-5-4 (90 mg) was purified

183

by semi-preparative HPLC on a YMC-Pack ODS-AQ column (250 mm × 10 mm I.D.,

184

flow rate 3.0 mL/min) with 80% MeOH-H2O to yield 6 (48 mg, tR = 8.90 min).

185 186

Acidic Hydrolysis of the Crude Glycosides. 1.0 g of crude glycosides was

187

dissolved in 20 mL of 1,4-dioxane solution with 5 mL of 0.1 M H2SO4 solution,

188

reacted at 60 °C for 4 hours. The reaction mixture was neutralized with 0.2 M NaOH

189

solution, and then extracted with EtOAc for three times. The aqueous layer was

190

concentrated under reduced vacuum to obtain the sugar residue and then submitted to

191

column chromatography on silica gel using chloroform-methanol system (C/M 100:1,

192

80:1, 60:1, 40:1, 20:1) and petroleum ether-acetone (P/A 10:1, 8:1, 6:1), respectively,

193

to give thevetose, cymarose, oleandrose, canarose, and digitoxose. Their absolute

194

configuration was determined to be D-series according to their specific optical

195

rotation: thevetose, [α]21D + 26.3 (c 0.21, H2O); cymarose [α]21 + 52.4 (c 0.21, H2O); D


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oleandrose [α]21D − 8.7 (c 0.20, H2O); canarose [α]21 + 18.9 (c 0.20, H2O); and D

197

digitoxose [α]21 + 42.6 (c 0.19, H2O).8 D

198 199

The Absolute Configuration Determination of Glucose in Glycosides. 1 mg of

200

each compound (compounds 1−12) was acid hydrolyzed by previously used method

201

to give sugar residue.7 Each sugar residue above was dissolved in anhydrous pyridine

202

(1 mL), to which 2 mg of L-cysteine methyl hydrochloride was added. The mixture

203

was stirred at 60 °C for 2 h, and after evaporation in vacuum to dryness, 0.2 mL of

204

N-trimethylsilylimidazole was added; the mixture was kept at 60 °C for another 2 h.

205

The reaction mixture was partitioned between n-hexane and H2O (2 mL each), and

206

the n-hexane extract was analyzed by Agilent 7890A gas chromatography with a

207

flame ionization detector (FID) to identify the derivative of D-(+)-glucose. A HP-5

208

capillary column (5% phenyl methyl siloxane, 50 m × 0.32 mm, 0.52 µm film

209

thickness) was used with helium as carrier gas (1.28 mL/min). Oven program: initial

210

temperature 160 °C, then raised to 280 °C at 5 °C/min. The injector and detector

211

temperature were held constant at 250 °C. The retention time of D-(+)-glucose

212

standard is 21.7 min.

213 214

Sensory Evaluation of Compounds. A total of 10 people were selected from more

215

than 30 volunteers to form a taste panel using Givaudan’s panelist selection procedure

216

(taste intensity ranking test) and trained following ISO norms.7 The sweetness

217

intensities relative to sucrose of pure compounds were evaluated by the sensory panel



218

consisting of seven sweet sensitive tasters (four women and three men, ages from 24

219

to 45, Chinese only) as previously described.7 All glycosides were dissolved in water

220

in graduated concentrations from 0.02 to 0.002% (w/v), while sucrose solutions were

221

made at concentrations of 1%, 2%, and 4%. The relative sweetness of compounds

222

compared to a 1−4% solution (w/v) of sucrose was determined by tasting its solutions

223

at different concentrations and selecting the concentration at which the taste was

224

approximately closest to that of the sucrose solution.

225

Each sample was dissolved in water to make a stock solution. Dilution solutions of

226

the samples were presented in an order of ascending concentrations (0.010, 0.015,

227

0.020, 0.025, 0.033, 0.050, 0.100, 0.200, and 0.400 mg/mL). The panelists were asked

228

to taste the sample solutions until they could not perceive a sweet taste from the

229

sample solution. The concentration of the second last tested sample solution was

230

taken as the individual recognition threshold of this compound.

231 232

RESULTS AND DISCUSSION

233

Isolation and Structure Determination of Compounds 1-14. The combined

234

extract of 95% ethanol and 70% aqueous ethanol from roots of M. extensum was

235

suspended with water, and extracted with petroleum ether, EtOAc respectively, to give

236

three portions. The EtOAc and aqueous portions were isolated through various

237

column chromatography and semi-preparative HPLC to give 14 compounds. Their

238

structures were identified as extensumsides M-X (1-12) (Figure 1), C (13), and E (14),

239

respectively, through modern spectroscopic technologies, such as IR, UV, ESI-MS,


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240

and 1D and 2D NMR data. Detailed structure elucidations of these compounds were

241

described in the supporting information.

242

General Fractionation Strategy and Criteria for Further Separation

243

Due to our knowledge of the UV absorption and TLC property of these C21

244

pregnane glycosides in previous research, we could choose a certain fraction for

245

further isolation and purification to obtain this type of C21 pregnane glycosides.

246

Generally, this type of C21 pregnane glycosides has two strong absorption peaks at

247

λmax 200 nm and 217 nm, and furthermore, they show a characteristic atroceruleous

248

colour on TLC plate sprayed a vanillin-sulfuric acid reagent and heated. Based on

249

these, the searching and separation of target compounds may become more

250

convenient and quicker.

251

Sweetness Potency and Threshold of Sweet Compounds by Sensory Evaluation.

252

Sensory evaluation of these compounds (Table 1) indicated that nine of them (1, 3, 4,

253

5, 6, 7, 8, 13 and 14) have sweet taste, while the rest five compounds (2, 9, 10, 11 and

254

12) do not have sweet taste, or even have bitter taste. Of the sweet compounds, 13 and

255

14, two known compounds first identified from the pericarps of M. extensum, have the

256

highest sweet intensities and the lowest recognition thresholds, followed by 5, 8 and 6,

257

which have the moderate sweet intensities and recognition threshold. Compounds

258

with the lowest sweet intensities are 1, 4, 3 and 7, and thus have the relatively high

259

recognition thresholds. In terms of the mouthfeel, four compounds, 13, 14, 5 and 8,

260

are better, and do not have off taste or bitter taste, whereas 3 and 7 have a worse

261

mouthfeel or even have some bitter taste and other off taste.



262

Structure-Activity Relationship of Sweet C21 Pregnane Glycosides. All of these

263

compounds have the same characteristics in structure, with the same aglycone,

264

3β,16α-dihydroxy-pregn-5-en-20-one, and two oligosaccharide chain linked at C-3

265

and C-16 respectively. And the significant distinctions of them are the sequence and

266

number of the sugar unit existing in the position of C-3 and C-16. Together with ten

267

similar compounds7 (two of which are the same) from the pericarps of M. extensum

268

reported by our group in 2016, now the information on the sweet intensities and the

269

thresholds of 22 compounds (Table S5) in total from this species are acquired, which

270

are helpful for us to discuss the structure-activity relationship of this type of

271

compounds, and thus can provide a theoretical basis for the rational design and

272

development of natural high-potency and non-sugar sweeteners.

273

First of all, the aglycone, 3β,16α-dihydroxy-pregn-5-en-20-one, is the basis of this

274

type of compounds to elicit sweet taste. Based on this, the sweet intensities are closely

275

related to the oligosaccharide chain at C-3 and C-16, i.e., the sequence, sort and

276

number of the sugar unit.

277

The sweet intensities of this type of compounds are related with the oligosaccharide

278

chain at C-16, and decrease dramatically with the number of glucose unit increasing

279

(1 and 3; 5, 6 and 22; 13, 14, 16 and 18).

280

Additionally, the sweet intensities are also in connection with the sequence, sort

281

and number of sugar unit at C-3, which exhibited a complicated relationship. That the

282

sugar unit (the first sugar unit) directly linked to C-3 is the thevetopyranosyl unit, is

283

necessary to elicit the sweet taste, while the sugar unit in this position is changed to


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284

another kind of sugar, the sweet taste may be disappeared. When the first sugar unit is

285

the thevetopyranosyl unit, the sweet intensities gradually decrease with the second

286

and the third sugar units changed in turn. The sort of the changed sugar affect the

287

sweet potency as well, and the trends of change for the second (1 and 5; 4, 8 and 14;

288

18 and 20; 19 and 21) and the third sugar unit (3 and 4; 5, 13 and 15; 6 and 14; 16 and

289

17; 18, 19 and 22) are somewhat different (for the second sugar, the change of

290

intensities: Cym > Dig > Ole; for the third sugar, the change of intensities: Cym >

291

Ole > Dig).

292

Furthermore, the 3-methylbut-2-enoyl group (mBe) is also connected with the

293

sweet intensities of these compounds, and it may play a crucial role in controlling the

294

length of the oligosaccharide chain and determining the sweet intensities. When the

295

mBe group is linked to the OH of the terminal sugar unit, the synthesis of

296

oligosaccharide chain is terminated immediately, due to the great steric hindrance.

297

The mBe group is derived from 3-methylbut-2-enoyl-CoA, an isomerization product

298

of 3-methylbut-3-enoyl-CoA.9 The latter compound is directly produced from

299

3-hydroxy-3-methylglutaryl-CoA, an intermediate of the mevalonate pathway, by a

300

decarboxylation/dehydration reaction.9 When the mBe group is located at the C4-OH

301

of the terminal sugar unit, the sweet taste is maintained, whereas the sweet intensities

302

is decreased and the mouthfeel of these compounds become worse or even elicit some

303

bitter taste, when this group is transferred to the C3-OH of the terminal sugar unit (1

304

and 2; 6 and 7).

305

Based on the discussion above, it could be deduced that there may exist a new



306

compound

not

found

up

to

date,

3β,16α-dihydroxy-pregn-5-en-20-one

307

3-O-[4-O-(3-methylbut-2-enoyl)-β-D-cymaropyranosyl]-(1→4)-β-D-cymaropyranosyl

308

-(1→4)-β-D-thevetopyranosyl-16-O-β-D-glucopyranoside, with the highest sweet

309

intensities and the best mouthfeel in this type of compounds.

310 311 312

ASSOCIATED CONTENT Supporting Information

313

Structure Determination of Extensumsides M-X, C, and E (1-14);

314

Spectroscopic Data of Extensumsides M-X (1-12);

315

The 1H NMR, 13C NMR data of extensumsides M–X (1-12);

316

Sensory evaluation of new C21 pregnane glycosides and known ones;

317

Selected HMBC and COSY correlations of 1;

318

The ROESY correlations of the aglycone moiety;

319

Scheme for the isolation of compounds 1-14 from the roots of M. extensum.

320 321

The Supporting information is available free of charge on the ACS Publications website at DOI:

322 323

AUTHOR INFORMATION

324

Corresponding Author

325

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

326 327

Funding


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328

This research was supported by grants from the Natural Science Foundation of the

329

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

330

Returned Overseas Chinese Scholars from State Education Ministry and National

331

S&T Basic Work Program of China (2012FY110300).

332 333

Notes

334

The authors declare no competing financial interest.

335 336 337 338

ACKNOWLEDGMENT We thank Professor Lisong Wang for the identification of the plant. We are also very grateful to all the panelists participating in the sensory tests.



339

REFERENCES

340

1.

341

M.; Tachdjian, C.; Lia, X., Molecular mechanism of the sweet taste enhancers. Proc.

342

Natl. Acad. Sci. USA 2010, 107 (10), 4752-4757; (b) Behrens, M.; Meyerhof, W.;

343

Hellfritsch, C.; Hofmann, T., Sweet and umami taste: natural products, their

344

chemosensory targets, and beyond. Angew. Chem.-Int. Edit. 2011, 50 (10),

345

2220-2242.

346

2.

347

Natural Sources. J. Chem. Educ. 1995, 72 (8), 676-680.

348

3.

349

Anderson; Patel, M. J.; Munos, J. C.; Krebs, N. F.; Xanthakos, S. A.; Johnson, R. K.,

350

Added Sugars and Cardiovascular Disease Risk in Children: A Scientific Statement

351

From the American Heart Association. Circulation 2017, 135 (19), e1017-e1034.

352

4.

353

Public Health Nutr. 2014, 17 (10), 2148-2150; (b) Yang, Q.; Zhang, Z.; Gregg, E. W.;

354

Flanders, W. D.; Merritt, R.; Hu, F. B., Added sugar intake and cardiovascular

355

diseases mortality among US adults. JAMA Intern. Med. 2014, 174 (4), 516-524.

356

5.

357

Pharm. Res. 2002, 25 (6), 725-746.

358

6.

359

Sweetener Enhancers. Annu. Rev. Food Sci. Technol. 2012, 3 (1), 353-380.

360

7.

(a) Zhang, F.; Klebansky, B.; Fine, R. M.; Liu, H.; Xu, H.; Servant, G.; Zollera,

Kinghorn, A. D.; Kennelly, E. J., Discovery of Highly Sweet Compounds from

Vos, M. B.; Kaar, J. L.; Welsh, J. A.; Horn, L. V. V.; Feig, D. I.; Cheryl A.M.

(a) Anderson, A. S., Sugars and health - risk assessment to risk management.

Kim, N.-C.; Kinghorn, A. D., Highly sweet compounds of plant origin. Arch.

DuBois, G. E.; Prakash, I., Non-Caloric Sweeteners, Sweetness Modulators, and

Sun, G.; Dai, Q.; Zhang, H.; Li, Z.; Du, Z., New Sweet-tasting C21-pregnane


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Glycosides from Myriopteron extensum. J. Agric. Food Chem. 2016, 64 (49),

362

9381-9389.

363

8.

364

Chem. Pharm. Bull. 2000, 48 (7), 1017-1022.

365

9.

366

Müller, R., A biosynthetic pathway to isovaleryl-CoA in myxobacteria: the

367

involvement of the mevalonate pathway. Chembiochem 2005, 6 (2), 322-330.

Abe, F.; Yamauchi, T., Pregnane glycosides from the roots of Asclepias tuberosa.

Mahmud, T.; Wenzel, S. C.; Wan, E.; Wen, K. W.; Bode, H. B.; Gaitatzis, N.;

368 369 370



371

Figure captions

372

Figure 1. Structures of compounds 1-12 isolated from the roots of M. extensum

373 374


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Table 1. The sensory evaluation of compounds 1-14 NO.

R1

1

-β-D-Thev4-β-D-Ole4-β-D-Dig4-mBe

2 3

4

4

3

4

4

4

-β-D-Thev -β-D-Ole -β-D-Dig -mBe

4

Sweetness potencya

Threshold (mg/mL)b

-β-D-Glc6-β-D-Glc

50

0.10

6

-

-

25

0.20

-β-D-Glc -β-D-Glc

-β-D-Thev -β-D-Ole -β-D-Dig -mBe 4

R2

6

2

6

2

-β-D-Glc -β-D-Glc -β-D-Glc

4

4

-β-D-Thev -β-D-Ole -β-D-Cym -mBe


50

0.05

5

-β-D-Thev4-β-D-Cym4-β-D-Dig4-mBe

-β-D-Glc6-β-D-Glc

100

0.0667

6

-β-D-Thev4-β-D-Cym4-β-D-Dig4-mBe

-β-D-Glc6-β-D-Glc2-β-D-Glc

7

4

4

3

-β-D-Thev -β-D-Cym -β-D-Dig -mBe 4

4

4

75

0.08

6

2

25

0.10

6

2


8

-β-D-Thev -β-D-Dig -β-D-Cym -mBe


100

0.0667

9

-β-D-Dig4-β-D-Cym4-β-D-AcCan4-β-D-Dig4-mBe

-β-D-Glc

-

-

6

-

-

10

4

4

4

4

-β-D-Glc -β-D-Glc

4

4

4

3

6

-β-D-Dig -β-D-Cym -β-D-AcCan -β-D-Dig -mBe

11

-β-D-Dig -β-D-Cym -β-D-AcCan -β-D-Dig -mBe

-β-D-Glc -β-D-Glc

-

-

12

-β-D-Dig4-β-D-Cym4-β-D-AcCan4-β-D-Dig4-mBe

-β-D-Glc6-β-D-Glc2-β-D-Glc

-

-

13

-β-D-Thev4-β-D-Cym4-β-D-Cym4-mBe

-β-D-Glc6-β-D-Glc

400

0.02

200

0.025

14

4

4

4

-β-D-Thev -β-D-Cym -β-D-Cym -mBe

6

2


a

sweet intensity relative to the sucrose solution (1%, w/w);

b

minimum concentration that human can percept;



Figure 1


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TOC graphic Phytochemical investigation

why ? Sweet

Human sensory analysis


New Sweet-tasting C21 Pregnane Glycosides from ... - ACS Publications

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