Identification and Enrichment of Î±-Glucosidase-Inhibiting

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Identification and Enrichment of #-Glucosidase-Inhibiting Dihydrostilbene and Flavonoids from Glycyrrhiza uralensis Leaves Rigui Ye, Yu-Hong Fan, and Chao-Mei Ma J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 26 Dec 2016 Downloaded from http://pubs.acs.org on December 27, 2016

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Journal of Agricultural and Food Chemistry

1

Identification and Enrichment of α-Glucosidase-Inhibiting Dihydrostilbene and

2

Flavonoids from Glycyrrhiza uralensis Leaves

3 4

Rigui Ye, Yu-Hong Fan, Chao-Mei Ma*

5

School of Life Sciences, Inner Mongolia University, Huhhot, China 010021

6 7

1

ACS Paragon Plus Environment


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ABSTRACT: To exploit Glycyrrhiza uralensis resources, we examined the bioactive

9

constituents of G. uralensis leaves. Seven chemical components were isolated by

10

repeat column chromatography, and using spectroscopic methods, their structures

11

were

12

α,α'-dihydro-3,5,3',4'-tetrahydroxy-2,5'-diprenylstilbene (1); a methylated flavonoid,

13

quercetin-3-Me ether (4); and 5 prenylated flavonoids—5'-prenylquercetin (3),

14

8-[(E)-3-hydroxymethyl-2-butenyl]-eriodictyol

15

5'-prenyleriodictyol (6), and 6-prenylquercetin-3-Me ether (2). Compounds 1-7 and

16

their unprenylated counterparts, glycosides, and other related compounds (8-13) were

17

quantitatively analyzed. Using a macroporous resin column, most of these compounds

18

could be enriched in the 40% to 60% ethanol-eluted fractions. Compounds 1-7

19

showed strong radical scavenging activity toward DPPH, and most of them

20

demonstrated greater inhibitory activity against α-glucosidase than their unprenylated

21

counterparts.

determined

to

be

a

novel

prenylated

(7),

dihydrostilbene,

6-prenyleriodictyol

(5),

22 23

KEYWORDS: Glycyrrhiza, leaves of Glycyrrhiza uralensis, chemical constituents,

24

prenylated flavonoids, α,α'-dihydro-3,5,3',4'-tetrahydroxy-2,5'-diprenylstilbene

2


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INTRODUCTION

26

Glycyrrhiza uralensis Fisch (Leguminosae) and its constituents are widely used in

27

traditional prescription medicines, foods, beverages, brewing, tobacco, and

28

cosmetics.1-2 Rich and diverse chemical constituents have been reported from the

29

roots and rhizomes of Glycyrrhiza species (also known as licorice). In addition to its

30

characteristically sweet component, glycyrrhizin, licorice contains bioactive

31

flavonoids, coumarins, and other agents.2-8 The prenylated flavonoids in licorice have

32

varying metabolic properties9 that might result in disparate bioactivities compared

33

with unprenylated flavonoids.

34

G. uralensis grows in arid and semiarid desert steppes, desert edges, and loess

35

hilly regions and is one of the major plants that maintain a good ecological

36

environment in these areas. However, with the increasing demand of the roots and

37

rhizomes of G. uralensis, soil erosion, grassland desertification, and other ecological

38

environmental problems have occurred through overexploitation. Over times, the

39

exploitation and utilization of G. uralensis resources have focused primarily on its

40

underground parts; the aerial part of G. uralensis has not been used or examined

41

extensively, causing a waste of resources. An in-depth study of the chemical

42

constituents and biological activities of the aerial part of G. uralensis is important in

43

optimizing the use of G. uralensis resources.

44

Flavonoids, primarily those that are prenylated,10-18 have been isolated from the

45

leaves of Glycyrrhiza species. Flavonoids can be enriched using a macroporous

46

resin,17 which is a practical tool that has attracted much interest in the preparation of

47

bioactive compounds.19-20 Prenylated dihydrostilbene was recently identified from the

48

leaves of G. lepidota and G. glabra.18,

49

Glycyrrhiza leaves have antibacterial, antiviral and antioxidant activity.11,18,22 This

21, 22

Several chemical constituents in

3



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50

study reports the macroporous resin-based enrichment, isolation, structural

51

determination, quantification, and anti-α-glucosidase effects of a new prenylated

52

stilbene and 11 flavonoids from G. uralensis leaves.

53

MATERIAL AND METHODS

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Instruments and Reagents. High-resolution electrospray ionization mass

55

spectrometry (HR-ESI-MS) was measured on a Xevo G2 Q-TOF mass spectrometer

56

(Waters, Milford, MA., USA). Nuclear magnetic resonance (NMR) spectra were

57

recorded on a Bruker Avance III-500 MHz spectrometer (Bruker Inc., Fällanden,

58

Switzerland) with tetramethylsilane as an internal standard. UHPLC-DAD-ESI-MS

59

experiments were carried out on an Agilent 1290 infinity UHPLC-DAD system

60

(Agilent Technologies Singapore (International) Pte. Ltd., Singapore) with an Agilent

61

6340 triple quadrupole MS. The solvents used as mobile phase of UHPLC were from

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Fisher Scientific Company (Fair Lawn, NJ, USA). Solvents for extraction and

63

isolation were all of analytical grade from XiLong chemical Co. Ltd. (Guangdong,

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China). Macroporous resin D101 was from Tianjin Haiguang Chemical Co. Ltd.

65

(Tianjin, China). MCI gel CHP 20 was from Mitsubishi Chemical Co. (Tokyo, Japan).

66

Octadecylsilane (ODS, 38–63 µm) was from Wako Pure Chemical Industries, Ltd.

67

(Osaka, Japan). Sephadex LH-20 was from GE Healthcare Bio-Sciences AB (Uppsala,

68

Sweden). Silica gel (100−200 mesh) for column chromatography and precoated silica

69

gel plates for thin-layer chromatography were from Qingdao Haiyang Chemical Co.

70

(Qingdao, China). Standards luteolin (8), quercetin (9), eriodictyol (10),

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3,3',4',5,7-pentahydroxyflavanone (11), isoquercitrin (12), rutin (13), and

72

3-acetylcatechin (I. S.) were from Sigma-aldrich or our previous works.23-24

73

Plant Material. The leaves of G. uralensis were collected in Ordos, Inner

74

Mongolia, People’s Republic of China, in September, 2013 and verified by the

4


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Authors. The voucher specimen of G. uralensis (NPFFG-1) was stored in laboratory

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of natural medicines and functional foods, school of life sciences, Inner Mongolia

77

University, Huhhot, China.

78

Extraction and Isolation. The dried leaves of G. uralensis (2 kg) were extracted

79

with 95% ethanol (10 L) at room temperature for 24 hours followed by sonication for

80

30 min. The filtrate was collected and the residue was re-extracted with 95% ethanol

81

(6 L) at room temperature for 12 hours followed by sonication for 30 min.

82

Concentration in vacuum of the pooled filtrate yielded 434 g of an extract which

83

showed 50.7±4.1% of inhibition on α-glucosidase at 100 g/ml. The extract was

84

suspended in water and applied to a macroporous resin column eluted with

85

water-ethanol to obtain 7 fractions, i.e. water eluted part (E1), 20% ethanol eluted part

86

(E2), 40% ethanol eluted part (E3), 60% ethanol eluted part (E4), 80% ethanol eluted

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part (E5), 100% ethanol eluted part (E6), and acetone eluted part (E7). E1-E7

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exhibited -0.4±10.8%, -3.2±3.7%, 68.0±3.5%, 93.7±0.2%, 89.9±0.8%, 41.0±10.1%

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and 61.2±3.7% of inhibition on α-glucosidase at 100 µg/ml. E4 was most inhibitory

90

on α-glucosidase and thus was subjected to further separation.

91

E4 was applied to an ODS column eluted with water-methanol to obtain 11

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fractions (M1-11). M4, the 40% methanol eluted fraction was separated with silica gel

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column chromatography eluted with gradient petroleumether-ethylacetate, and the 7:3

94

eluted part was further separated with Sephadex-LH20 and MCI to obtain 1 (27 mg),

95

3 (34 mg), 5 (26 mg) and 6 (45 mg); the petroleumether-ethylacetate 6:4 eluted part

96

was further separated with Saphadex-LH20 (water-methanol) and MCI

97

(water-methanol) to yield 4 (15 mg) and 7 (119 mg). M5, the 50% methanol eluted

98

part was separated with silica gel column chromatography eluted with gradient

99

petroleumether-ethylacetate, and the 6:4 eluted part was purified with

5



100

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Saphadex-LH20 (55% methanol) and MCI (75% methanol) to yield compound 2. Compound 1，white powder; HR-ESIMS, m/z 381.2065 [M-H]-1 (calcd 381.2066).

101 102

1

103

1.724 (3H, s) (H-10, 10′, 11, 11′), 2.596 (2H, m, H-α′), 2.651 (2H, m, H-α), 3.201 (2H,

104

d, J=7.0 Hz, H-7), 3.245 (2H, d, J=7.0 Hz, H-7′), 5.047 (1H, t, J=7.0 Hz, H-8), 5.280

105

(1H, t, J=7.0 Hz, H-8′), 6.120 (1H, d, J=2.5 Hz, H-6), 6.146 (1H, d, J=2.5 Hz, H-4),

106

6.351 (1H, d, J=2.5 Hz, H-6′), 6.480 (1H, d, J=2.5 Hz, H-2′); 13C NMR (125 MHz,

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CD3OD): δ 17.90 and 18.21 (C-10, 10′), 25.31 (C-7), 25.98 and 26.04 (C-11, 11′),

108

29.20 (C-7′), 36.99 (C-α), 38.57 (C-α′), 101.33 (C-4), 108.63 (C-6), 113.85 (C-2′),

109

119.06 (C-2), 121.27 (C-6′), 124.35 (C-8′), 126.08 (C-8), 129.47 (C-5′), 130.51 (C-9),

110

132.58 (C-9′), 134.54 (C-1′), 142.02 (C-4′), 143.59 (C-1), 145.85 (C-3′), 156.59 (C-5),

111

157.10 (C-3).

H NMR (500 MHz, CD3OD): δ 1.656 (3H, s) and 1.698 (3H, s) and 1.718 (3H, s) and

112

Quantification of the Chemical Constituents in the Extract and Fractions. A

113

synthesized flavonoid, 3-acetylcatechin, was chosen as an internal standard (I.S.) due

114

to its structural similarity to the isolated constituents of G. uralensis leaves. Pure

115

constituents 1-7 and related compounds 8-13 were dissolved in DMSO at 1 mg/ml of

116

each analyte and 2 µg/ml of I.S., and the solution was diluted with DMSO containing

117

2 µg/ml of I.S. in a serial of 3-fold dilutions. The extract and fractions were dissolved

118

in DMSO containing 2 µg/ml of I.S. and filtered with micro-membrane before

119

applying to UHPLC-MS analysis.

120

UHPLC-QQQESIMS with a ZORBAX Eclipse XDB-C18 column (2.1×50 mm，

121

1.8 µm) at 30ºC and detection in MRM mode was used for the quantitative analysis.

122

The injection volume was 1 µL and flow rate was set at 0.4 mL/min. The mobile

123

phase comprised of 0.1% formic acid in H2O, and methanol as solvent A and B,

124

respectively, and programmed as：0–4 min, 10-39% B; 4-4.1 min, 39-41% B; 4.1-8 6


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min, 41-45% B; 8-8.1 min, 45-67% B; 8.1-12 min, 67-69% B; 12-12.1 min, 69-100%

126

B; 12.1-14 min, 100% B. MS detection was performed in negative ion mode with

127

capillary of 3.5 kV, Gas temperature of 350˚C; Gas flow of 11 L/min, and Nebulizer

128

of 45 psi. The MS/MS transitions, fragmentor voltage, and collision energy were

129

carefully optimized and the ensuring parameters are listed in Table S1 in

130

Supplementary Data.

131

Determination of Inhibition on α-Glucosidase. The assay was carried out on

132

96-well plates according to reported method.25 Briefly, 10 µl of sample (in DMSO),

133

80 µl of 4-nitrophenyl α-D-glucopyranoside (2 mM in 100 mM potassium phosphate

134

buffer, pH 7.0), and 10 µl of enzyme (0.40 U/ml, from Bacillus Stearothermophilus,

135

Sigma, Lot# 090M1360V) were mixed in each well. DMSO was added in stead of

136

samples in the control wells. After 20 min’s incubation at 37 ºC, the absorbance at 405

137

nm was measured with a DNM-9602 plate reader from Beijing Pu Long New

138

Technology Co. Ltd. (Beijing, China) and compared with that before incubation. ∆A,

139

the increased absorbance, was used for inhibition calculation:

140

Inhibition%=100×(∆Acontrol-∆Asample)/ ∆Acontrol

141

IC50 (the concentration that inhibited 50% of the enzyme activity) was calculated

142

from the inhibition%-concentration curve. Acarbose (EC50=0.1 µg/mL) was used as a

143

positive control and all samples were tested in triplicate.

144

Radical Scavenging Assay. Radical scavenging activity on DPPH was

145

determined in 96-well plates using the method described in literature.24 Briefly, ten

146

microliters of a sample solution (in DMSO) was mixed with 190 µL of

147

1,1-diphenyl-2- picrylhydrazyl in ethanol (DPPH, 0.1 mM) in each well. In the color

148

control wells, 190 µL of ethanol were used instead of the DPPH solution. In the

149

control well, 10 µL of DMSO was used instead of sample solution. The absorbance

7



150

(A) was measured at 520 nm with the plate reader after 20 min at room temperature.

151

The radical scavenging activity was calculated by the following formula:

152

Effect % = 100 × [Acontrol − (Asample− Acolor)]/Acontrol

153

EC50 (the concentration that scavenged 50% of the radical) was obtained from the

154

effect%-concentration curve. Quercetin (9, EC50=8.4 µg/mL) was used as a positive

155

control and all samples were tested in triplicate.

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156 157

RESULTS AND DISCUSSION

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Structural Determination of the Chemical Constituents from G. uralensis Leaves.

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The leaves of G. uralensis were extracted with ethanol followed by fractionation on

160

macroporous resin column eluted with gradient water-ethanol. The 60% ethanol

161

eluted fraction (E4) was found to show the best activity on α-glucosidase. This

162

bio-active fraction was subsequently subjected to various column chromatography to

163

yield seven chemical constituents (1-7) (Figure 1).

164

-------------------Figure1--------------

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Compound 1 was obtained as a white powder. Its negative HR-ESIMS showed a

166

deprotonated molecular ion peak at m/z 381.2065 [M−H]−, indicating a molecular

167

formula of C24H30O4 (calcd 381.2066). Compound 1 displayed two pairs of singlet

168

methyl signals (H-10/11, 10′/11′) in its 1H NMR. In its HMBC spectrum, these methyl

169

signals showed long-range correlations with two pairs of olefinic carbon signals at δ

170

126.08 and 130.51 (C-8,9), as well as 124.35 and 132.58 (C-8′,9′). These data, in

171

addition to the HMBC correlations between δ 3.201 (H-7) and C-8,9 signals, and

172

between δ 3.245 (H-7′) and C-8′,9′ signals allowed the identification of two prenyl

173

groups. The aromatic carbon signals as well as two pairs of meta-coupling proton

174

signals at δ 6.120 (1H, d, J=2.5 Hz, H-6)/6.146 (1H, d, J=2.5 Hz, H-4), and 6.351 (1H,

8


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d, J=2.5 Hz, H-6′)/6.480 (1H, d, J=2.5 Hz, H-2′) indicated the presence of two

176

tetra-substituted phenyl groups. Two methylene signals at δ 2.596 and 2.651 (CH2-α

177

and CH2-α′) correlated with 2 phenyl carbons at δ 143.59 and 134.54 (C-1 and 1′) in

178

HMBC, indicating a dihydrostilbene skeleton of 1. The substitution positions of the

179

prenyl and hydroxyl groups were determined and the full structure was confirmed by

180

careful analysis of all the HMBC correlations as depicted in Figure 2. The structure of

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1 was thus determined as α,α'-dihydro-3,5,3',4'-tetrahydroxy-2,5'-diprenylstilbene.

182

-----------Figure 2---------

183

The structures of compounds 2-7 were determined as 6-prenylquercetin-3-Me

184

ether (2),26,27 5'-prenylquercetin (3),12 quercetin-3-Me ether (4),28 6-prenyleriodictyol

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(5),29 5'-prenyleriodictyol (6),12 8-[(E)-3-hydroxymethyl-2-butenyl]-eriodictyol (7)30

186

by comparison of their spectral data with those reported.

187

Quantification of the Chemical Constituents in G. uralensis Leaf Extract and

188

Fractions. UHPLC-MS in optimized condition was used to quantify the isolated

189

chemical constituents (1-7), their unprenylated counterparts (9-10) and related

190

compounds 8, 11-13. Calibration curve was plotted using the peak area ratio of a pure

191

compound to I.S as Y-axis and concentration of the pure compound as X-axis. As

192

shown in Table 1, the calibration curves of all the analytes displayed good linearity,

193

and the low limits of quantifications were all below 0.3232 µg/ml.

194

--------Table 1-----------

195

In addition to the isolated prenylated flavonoids, 5 flavonoids—the unprenylated

196

counterparts of the isolated flavonoids quercetin (9) and eriodictyol (10), and their

197

glycosides, isoquercitrin (12) and rutin (13), and luteolin (8) which has 1 fewer

198

hydroxyl group than quercetin at C-3 exist in the extract.

199

3,3',4',5,7-pentahydroxyflavanone (11), which harbors 1 more hydroxyl group at C-3

9



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than eriodictyol (10), was not detectable in G. uralensis leave extract. The ethanol

201

extract of G. uralensis leaves was especially rich in 5'-prenyleriodictyol (6),

202

6-prenylquercetin-3-Me ether (2), and

203

α,α'-dihydro-3,5,3',4'-tetrahydroxy-2,5'-diprenylstilbene (1). Prenylated and/or

204

methylated quercetin (4, 3, 2) levels were much higher than free quercetin (9), and

205

there was more prenylated eriodictyol (5-7) than free eriodictyol (10). As shown in

206

Table 2 and Figure S17, these chemical constituents were enriched in the 40% to 80%

207

ethanol-eluted fractions of a macroporous resin column chromatography (E3-E5).

208

Most prenylated flavonoids and stilbene were found in the 60% to 80% ethanol-eluted

209

fractions (E4-E5), whereas the flavonol glycosides (12-13) were detected almost

210

exclusively in the 40% fraction (E3).

211

------------Table 2----------------

212

Radical Scavenging Ability toward DPPH and Inhibition of α-Glucosidase.

213

All isolated chemical constituents of G. uralensis, 1–7, and their related compounds

214

8–13, showed strong radical scavenging activity (Table 3).

215

Except for 8-[(E)-3-hydroxymethyl-2-butenyl]-eriodictyol (7), all other prenylated

216

or methylated compounds demonstrated inhibition of α-glucosidase with

217

5'-prenylquercetin (3) being the strongest (IC50=2.3 µg/mL). Quercetin-3-Me ether (4),

218

5'-prenylquercetin (3), and 6-prenylquercetin-3-Me ether (2) were more active than

219

quercetin (9). Although eriodictyol (10) showed little inhibition (IC50˃100 µg/mL)

220

against α-glucosidase, its prenylated compounds, 6-prenyleriodictyol (5) and

221

5'-prenyleriodictyol (6), had moderate activity with IC50 values of 31.2 and 57.4

222

µg/mL, respectively (Table 4). These results suggest that the α-glucosidase inhibitory

223

activity is mediated by the lipophilic prenyl or methyl groups.

224

---------Table 3------------

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---------Table 4-----------

226

In summary, a new dihydrostilbene, and 11 flavonoids were identified from an

227

α-glucosidase inhibitory extract of G. uralensis leaves. These compounds showed

228

strong radical scavenging activity and moderate to robust inhibition of α-glucosidase.

229

The prenylated or methylated flavonoids inhibited α-glucosidase to a greater extent

230

than their unsubstituted counterpart flavonoids. Antioxidant and α-glucosidase

231

inhibitory activities are beneficial in relieving insulin-resistant status.31-34 We have

232

found that macroporous resin can enrich these bioactive compounds in the 40-80%

233

ethanol-eluted fractions. By UHPLC-MS analysis, this fraction contained large

234

amounts of flavonoids, primarily prenylated flavonoids with α-glucosidase inhibitory

235

activity. These results support the extraction of bioactive fractions or constituents

236

from G. uralensis leaves as food supplements to prevent or relieve insulin-resistant

237

status, such as type II diabetes and obesity.

238 239

ACKNOWLEDGEMENTS

240

We would like to express our sincere thank to Mr. Meng He of School of Chemistry,

241

Inner Mongolia University, for acquisition of NMR data.

242 243

ASSOCIATED CONTENT

244

Supporting Information

245

The Supporting Information is available free of charge on the ACS Publications

246

website.

247

1

H and 13C NMR spectra of compounds 1-7.

248 249

AUTHOR INFORMATION

11



250

Corresponding Author

251

Phone: +86-15690918829; Email: [email protected]

252

Funding

253

This work was supported by National Natural Science Foundation of China

254

(81360474).

255

Notes

256

The authors declare no competing financial interest.

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17



363 364 365

Figure captions

Figure 1.

Compounds isolated from G. uralensis and related compounds

366

Figure 2.

HMBC correlations of compound 1

Page 18 of 25

367 368

18


Page 19 of 25


Table 1.

369

Regression equation

Linear range (µg/ml)

Correlation coefficient(R2)

LOQ (µg/ml)

1

Y=29.0154X+0.047899

5.5304~0.0055

0.9996

0.0055

2

Y=28.3619X+0.199858

5.5660~0.0308

0.9993

0.0308

3

Y=1.0713X-0.0087

10.8395~0.0046

0.9996

0.0046

4

Y=14.7965X-0.012631

5.5627~0.0102

0.9999

0.0102

5

Y=39.94162X-0.082484

5.5699~0.0145

0.9998

0.0145

6

Y=27.5247X+0.094704

5.5489~0.0210

0.9998

0.0210

7

Y=17.0342X+0.054898

5.5603~0.0040

0.9998

0.0040

8

Y=14.5547X+0.021280

5.5499~0.0298

0.9998

0.0298

9

Y=3.4785X+0.017862

5.5932~0.0443

0.9987

0.0443

10

Y=12.7073X+0.017862

5.5741~0.0037

0.9997

0.0037

11

Y=2.1734X+0.436951

5.5548~0.3232

0.9938

0.3232

12

Y=17.8603X+0.229797

1.8330~0.0211

0.9956

0.0211

13

Y=14.7813X+2.163606

5.5532~0.1933

0.9978

0.1933

Analyte

370 371 372

Linear Relationships of Reference Substances

Notes: LOQ: quantification limit. Precision of the data obtained from repeated experiments (RSD%) ˂ 5.6%

19



373 374

375 376 377 378 379 380

Page 20 of 25

Quantification Results of Dihydrostilbene and Flavonoids in the Extract and Fractions of Glycyrrhiza uralensis Leaves

Table 2

Analyte

EE (µg/g)

E1 (µg/g)

E2 (µg/g)

E3 (µg/g)

E4 (µg/g)

E5 (µg/g)

E6 (µg/g)

E7 (µg/g)

1

23.40

1.63

0.04

0.30

52.81

18.84

1.30

0.14

2

12.03

0.92

0.19

0.16

21.07

17.09

1.08

0.17

3

6.09

1.27

0.61

0.50

9.53

3.77

1.26

0.12

4

3.98

0.33

0.23

24.84

5.53

0.65

0.29

0.04

5

5.02

0.59

0.37

1.20

10.03

2.57

0.50

0.22

6

13.79

1.05

0.25

0.18

24.17

22.29

1.32

0.08

7

1.76

0.17

0.50

16.69

3.18

0.40

0.12

BL

8

0.11

0.08

0.17

0.64

0.20

0.04

BL

0.01

9

0.60

1.88

BL

2.09

1.55

BL

BL

1.09

10

0.17

0.11

0.23

1.82

0.23

0.13

0.03

BL

11

BL

BL

BL

BL

BL

BL

BL

BL

12

2.28

0.02

0.13

44.19

0.66

0.09

0.09

BL

13

0.71

BL

BL

23.14

BL

BL

BL

BL

Total

69.94

8.05

2.72

115.75

128.96

65.87

5.99

1.87

Notes: BL, below the detection limit; EE, the ethanol extract of G. uralensis leaves; E1-E7 are fractions from a macroporous resin column. E1, water eluted fraction; E2, 20% ethanol eluted fraction; E3, 40% ethanol eluted fraction; E4, 60% ethanol eluted fraction; E5, 80% ethanol eluted fraction; E6, 100% ethanol eluted fraction; E7, 100% acetone eluted fraction.

20


Page 21 of 25


381 382

Table 3. Radical Scavenging Activities of the Chemical Constituents in G. uralensis Leaves Effect % at five different concentrations (mean ± SD)

EC50

compound

383 384

50µg/mL

25 µg/mL

12.5µg/mL

6.25µg/mL

3.125µg/mL

（µg/mL）

1

n.t.

78.5 ± 2.4

79.3 ± 3.3

65.5 ± 1.9

34.7 ± 1.0

4.5

2

77.1 ± 0.8

71.7 ± 0.2

51.1 ± 4.7

24.9 ± 0.2

n.t.

13.9

3

n.t.

75.0 ± 0.3

78.7 ± 0.7

63.1 ±0.3

47.9 ± 0.2

3.5

4

n.t.

76.6 ± 0.5

72.6 ± 1.5

49.9 ± 7.1

32.6 ± 4.6

5.7

5

n.t.

82.5 ± 1.5

82.6 ± 0.4

53.2 ± 2.8

37.6 ± 1.9

5.0

6

n.t.

81.4 ± 1.4

83.4 ± 0.3

51.1 ± 2.6

31.3 ± 1.6

5.5

7

n.t.

82.9 ± 1.0

83.2 ± 1.0

78.2 ± 2.4

59.9 ± 1.8

2.3

8

80.2 ± 1.1

68.4 ± 1.8

51.2 ± 4.1

35.8 ± 0.7

21.4± 1.9

11.6

9

87.9 ±0.1

77.1 ±3.0

57.5 ±5.4

46.2 ±0.4

26.0 ±0.7

8.4

10

63.5 ±3.7

59.6 ±0.4

45.8 ±0.1

28.6±1.8

0.1±0.6

19.8

11

63.8 ±0.7

54.5 ±1.5

40.6 ±1.4

21.7 ±0.5

11.7 ±0.8

22.4

12

62.9 ±2.6

59.4 ±0.4

23.8 ±0.5

12.2 ±0.2

20.2

13

50.3 ±1.6

30.7 ±0.6

8.9 ±1.1

4.0 ±0.1

37.1

40.6 ±0.7 16.3 ±0.2

Notes: n.t., not tested. Bioassays were carried out in triplicate for the tested samples.

21



385 386

Table 4.

α-Glucosidase Inhibitory Activity of the Chemical Constituents from G. uralensis Leaves

compound

387

Page 22 of 25

Inhibition% (mean ± SD)

IC50 （µg/mL）

1

100 µg/mL 64.6 ± 8.9

25 µg/mL 71.8 ± 2.8

6.25 µg/mL 36.8 ± 0.9

1.56 µg/mL 15.2 ± 1.1

2

75.6 ± 2.2

54.2 ± 5.7

29.6 ± 4.3

3.3 ± 1.0

21.3

3

109.5 ± 6.3

93.9 ± 2.3

68.9 ± 0.4

38.6 ± 8.0

2.3

4

64.5 ± 3.3

41.1 ±8.2

45.0 ±6.7

29.9 ± 9.3

24.5

5

73.1 ± 8.0

43.3 ± 0.5

21.4 ± 2.3

28.9 ± 4.4

31.2

6

60.1 ± 7.2

33.8 ± 2.5

22.3 ± 1.0

26.4 ± 6.9

57.4

7

40.3 ± 0.6

33.5 ± 1.0

25.5 ± 6.1

16.5 ± 6.5

>100.0

8

19.4± 4.4

34.7± 5.7

-5.3± 3.1

-9.7± 6.6

>100.0

9

78.9±0.1

40.7±5.8

27.0±1.8

2.6±9.4

25.8

10

-16.9±8.8

16.3±4.4

16.9±2.7

1.9±7.1

>100.0

11

64.1±0.4

19.1±2.1

0.5±3.2

-23.1±0.2

84.7

12

-22.1± 4.6

-1.8± 7.5

22.1± 5.4

5.3± 7.5

>100.0

13

5.9 ± 3.9

1.0 ± 1.3

12.4 ± 0.9

10.5 ± 4.8

>100.0

15.4

Note: All samples were tested in triplicate.

22


Page 23 of 25


388

23



Page 24 of 25

389 390 391

24


Page 25 of 25


392 393

Graphic for table of contents

394 395

25


Identification and Enrichment of Î±-Glucosidase-Inhibiting

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