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#-Glucosidase Inhibition and Antihyperglycemic Activity of Phenolics from the Flowers of Edgeworthia gardneri Yan-Yan Ma, Deng-Gao Zhao, Ai-Yu Zhou, Yu Zhang, Zhiyun Du, and Kun Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b03081 • Publication Date (Web): 04 Sep 2015 Downloaded from http://pubs.acs.org on September 11, 2015

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Journal of Agricultural and Food Chemistry

α-Glucosidase Inhibition and Antihyperglycemic Activity of Phenolics from the Flowers of Edgeworthia gardneri Yan-Yan Ma,

†, ┴

Deng-Gao Zhao, †, ┴ Ai-Yu Zhou, † Yu Zhang, ‡ Zhiyun Du,∗, † and

Kun Zhang∗, †, ‡

†

College of Light Industry and Chemical Engineering, Guangdong University of

Technology, Guangzhou 510006, People’s Republic of China. ‡

School of chemistry and environment engineering, Wuyi University, Jiangmen

529020, People’s Republic of China.

Author Contributions ⊥

These authors contributed equally and should be considered co-first-authors.

*corresponding authors E-mail: [email protected] (Zhiyun Du), Tel/fax: +86-20-39323363; e-mail: [email protected] (Kun Zhang), Tel/fax: +86-20-39323363.

1

ACS Paragon Plus Environment


1

ABSTRACT

2

The flowers of Edgeworthia gardneri are consumed as an herbal tea in Tibet with

3

potential health benefits. To complement the current knowledge regarding the

4

chemical composition and antihyperglycemic activity of the flower of E. gardneri,

5

two new phenolics, Gardnerol A and B (1 and 2), along with nineteen known

6

phenolics were isolated from the flower of E. gardneri. All isolates were evaluated

7

for their inhibitory activity against α-glucosidase. Compound 5, identified as the

8

major constituent of the flower of E. gardneri, showed a significant α-glucosidase

9

inhibitory activity and acted as a competitive inhibitor. The oral administration of

10

compound 5 at a dose of 300 mg/kg significantly reduced the postprandial blood

11

glucose levels of normal and STZ-induced diabetic mice. Furthermore, compound 5

12

significantly decreased the fasting blood glucose levels in STZ-induced diabetic

13

mice.

14

KEYWORDS

15 16

Edgeworthia gardneri, phenolics, α-glucosidase inhibitor, antihyperglycemic activity, herbal tea

17

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18

INTRODUCTION

19

Diabetes mellitus (DM), a common and complex metabolic disease, is

20

characterized by abnormally high blood glucose levels (hyperglycemia) due to insulin

21

resistance and deficiency.1 Glycemic control is an effective therapy for diabetes,

22

minimizing the risk of long-term complications from the disease.2 α-Glucosidase, a

23

critical enzyme for the digestion of carbohydrates, catalyzes the cleavage of

24

absorbable monosaccharides, starting from disaccharides and oligosaccharides. Thus,

25

α-glucosidase inhibitors reduce postprandial hyperglycemia by slowing the digestion

26

of carbohydrates in the intestines.3 However, classic α-glucosidase inhibitors, such as

27

acarbose and miglitol, also cause gastrointestinal side effects.4, 5 The consumption of

28

natural α-glucosidase inhibitors derived from plant-based foods or supplements offers

29

an attractive strategy to control postprandial hyperglycemia due to their low cost and

30

low incidence of major undesirable side effects.6−9 Many types of fruits, vegetables

31

and drinks, including strawberries, blueberries, broccoli sprouts, green peppers, beer

32

hops, and green tea extracts were shown to display α-glucosidase inhibitory

33

activity.10−13

34

The flower of Edgeworthia gardneri Wall. Meisn., named “Lv-Luo-Hua” in

35

Chinese, has been used to prepare an herbal tea that is commonly consumed as a

36

health beverage in Tibet.14,15 E. gardneri is mainly distributed in Eastern Tibet and

37

the Northwest Yunnan province. It is claimed that drinking the herbal tea of E.

38

gardneri can alleviate the severity of many disorders, such as diabetes and

39

hyperlipidemia.14,

15

Previous studies have demonstrated that the extracts of the

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flower of E. gardneri exhibit many medicinal activities, in particular,

41

antihyperglycemic

42

α-glucosidase.15−18 Nevertheless, most constituents of E. gardneri are still evaluated

43

from complex mixtures. Furthermore, to the best of our knowledge, the

44

α-glucosidase inhibition and antihyperglycemic activity of constituents from E.

45

gardneri have yet to be investigated systematically.

activity

and

significant

inhibitory

activity

against

46

In the present work, we report the isolation and biological activity of phenolics

47

from the flower of E. gardneri. This is the first report on the α-glucosidase inhibition

48

and antihyperglycemic activity of phenolics from the flower of E. gardneri, both in

49

vitro and in vivo.

50

MATERIALS AND METHODS

51

Chemicals and Plant Materials. All organic solvents used in the study, such as

52

petroleum ether (PE), chloroform (CHCl3), ethyl acetate (EtOAc), n-butanol (BuOH),

53

methanol (CH3OH), and dimethyl sulfoxide (DMSO), were of analytical grade.

54

Methanol-d4

55

Saccharomyces cerevisiae, p-nitrophenyl-α-glucopyranoside (PNPG), streptozotocin

56

(STZ), and acarbose were purchased from Sigma-Aldrich (St. Louis, MO, USA).

57 58

(99.8%),

dimethyl

sulfoxide-d6

(99.9%),

α-glucosidase

from

The flowers of E. gardneri were purchased from Xizang Sheng−Qi− Bao−Jian−Pin, Co., Ltd. (Tibet, China).

59

Animals. Kunming mice (4−6 weeks old) were obtained from the Experimental

60

Animal Center of Guangdong Province (Guangzhou, China). The use of mice was

61

reviewed and approved by the Ethics Committee for Animal Experimentation of the

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Guangdong University of Technology (Guangzhou, China) and was in accordance

63

with the National Institutes of Health Guide for the Care and Use of Laboratory

64

Animals.

65

To induce diabetes, the mice were treated with a single intraperitoneal injection of

66

streptozotocin (100 mg/kg) dissolved in citrate buffer (pH 4.5) under fasting

67

conditions. The blood glucose level was monitored on day 7 from the tail vein using a

68

one-touch glucometer (Lifescan, Inc., Milpitas, CA). Mice with fasting blood glucose

69

levels ≥16.0 mmol/L were classified as diabetic mice. General Experimental Procedures. Melting points were determined on an X-4

70 71

digital display micromelting point apparatus and are uncorrected. Optical rotations ([α]

72

25 D

73

Thermo Nicolet 6700 FT-IR spectrometer. UV spectra were recorded on a

74

PerkinElmer Lambda 25Shimadzu 160 UV/VIS Spectrometer. The HRESIMS spectra

75

were recorded on an Agilent 6210 series LC/MSD TOF from Agilent Technologies.

76

NMR spectra were acquired on a Bruker AVANCE HD III-400 using TMS as an

77

internal standard. The X-ray diffraction data were collected on a SuperNova, Dual,

78

Eos diffractometer; the structure was solved with the Superflip program using charge

79

flipping and refined with the ShelXL program (using graphite-monochromated Mo K

80

α radiation).Silica gel (200-300 mesh, Qingdao Marine Chemical Factory, China),

81

macroreticular resin (D-101, Sinopharm Chemical Reagent Co., Ltd., Shanghai,

82

China), Sephadex LH-20 gel (GE Healthcare, Uppsala, Sweden), and MCI gel

83

(CHP20P, 75-150 µm, Mitsubishi Chemical Industries Ltd., Tokyo, Japan) were used

) were measured on a Perkin−Elmer 341 polarimeter. IR spectra were obtained using

5



84

for column chromatography. Preparative HPLC was carried out on a Shimadzu

85

LC-6AD instrument with an SPD-20A detector, using an YMC-Pack ODS-A column

86

(250 mm×20 mm, 5µm). TLC was performed using Merck precoated plates (Si gel 60

87

F254, Germany) of 0.25 mm thickness. The spots on TLC were detected with 254 and

88

365 nm UV light and visualized by spraying with 98% H2SO4/C2H5OH (5:95, v/v)

89

followed by heating. The absorbances in the enzymatic assay were determined at 405

90

nm using a Bio-Rad Model 680 microplate reader.

91

Extraction and Isolation. The dried power of the flower of E. gardneri (12.0 kg)

92

was extracted three times with MeOH (40L) for 7 days each at room temperature. The

93

combined extracts were concentrated under reduced pressure, and the residue (862 g)

94

was partitioned into H2O and extracted with petroleum ether, EtOAc, and n-BuOH,

95

successively.

96

The EtOAc fraction (118 g) was subjected to column chromatography (CC) over

97

silica gel (petroleum ether/EtOAc, 40:1, 30:1, 20:1, 15:1, 10:1, 8:1, 5:1, 3:1, 1.5:1, 1:1,

98

and 0:1, v:v) to give nine major fractions (A−I). Fraction A (1.2 g) was

99

chromatographed over MCI (MeOH/H2O, 9:1) to yield two fractions, A1 and A2.

100

Fraction A2 (231 mg) was subjected to Sephadex LH-20 (CHCl3/MeOH, 1:1) to yield

101

compound 6 (22 mg). Fraction B (2 g) was purified by CC over MCI (MeOH/H2O,

102

9:1) to yield two major fractions, B1 and B2. Fraction B1 (506 mg) was run again by

103

CC over Sephadex LH-20 (CHCl3/MeOH, 1:1) to obtain compounds 7 (16 mg), 8 (31

104

mg), and 9 (27 mg). Fraction B2 (700 mg) was also run again by CC over Sephadex

105

LH-20 (CHCl3/MeOH, 1:1) to obtain 10 (123 mg). Fraction C (1.1 g) was purified by

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106

CC over MCI (MeOH/H2O, 9:1) to yield one fraction C1, and fraction C1 (638 mg)

107

was separated by CC over Sephadex LH-20 (CHCl3/MeOH, 1:1) to obtain compounds

108

1 (14 mg), 11 (56 mg), and 12 (21 mg). Fraction E (1.1 g) was purified by CC over

109

MCI (MeOH/H2O, 9:1) to yield one fraction E1, and fraction E1 was recrystallized

110

from methanol to yield compound 3 (34 mg). Fraction G (1.6 g) was applied to MCI

111

(MeOH/H2O, 9:1) to give three major fractions (G1−G3). Fraction G1 (300 mg) was

112

subjected to CC over Sephadex LH-20, eluted with MeOH/H2O (1:1) and finally

113

purified by prep-HPLC (MeOH/H2O, 4:6) to yield compound 13 (11 mg). The

114

Fraction H (1.8 g) was subjected to CC over MCI (MeOH/H2O, 9:1) to yield three

115

fractions, H1−H3. Fraction H1 (1.5 g) was subjected to CC over Sephadex LH-20,

116

eluted with MeOH-H2O (1:1) and then purified by prep-HPLC (MeOH/H2O, 4:6) to

117

yield compound 14 (30 mg). Fraction I (82 g) was chromatographed over MCI

118

(MeOH/H2O, 9:1) to yield three fractions, I1−I3. Fraction I1 (61 g) was recrystallized

119

from methanol to yield compound 5 (53 g) and a mother liquor (3.6 g). The mother

120

liquor was subjected to CC over silica gel (CHCl3/MeOH, 10:1, 5:1, 3:1, 0:1) to yield

121

two subfractions, I1.1 and I1.2. Fraction I1.2 (1.4 g) was purified by CC over

122

Sephadex LH-20 (CHCl3/MeOH, 1:1) to yield compound 15 (18 mg). Fraction I2 (2.3

123

g) was subjected to CC over Sephadex LH-20 (MeOH/H2O, 1:1) to yield two

124

subfractions, I2.1 and I2.2. Fraction I2.2 (1.4 g) was purified by CC over Sephadex

125

LH-20 (CHCl3/MeOH, 1:1) to yield compound 16 (45 mg).

126

The n-BuOH fraction (118 g) was subjected to CC over D-101 macroreticular resin

127

with an H2O/EtOH gradient (1:0, 8:1, 5:1, 3:1, 1.5:1, 1:1, 0:1) to yield four major

7



128

fractions (1−4). Fraction 2 was subjected to CC over silica gel (CHCl3/MeOH, 4:1,

129

3:1, 2:1, 0: 1) to yield two fractions, 2.1 and 2.2. Fraction 2.2 (2.6 g) was purified by

130

CC over Sephadex LH-20 (CHCl3/MeOH, 1:1) to obtain compounds 17 (19 mg) and

131

18 (31 mg). Fraction 3 (42 g) was evaluated by TLC and compared with flavonoid

132

standards, including standards for compounds 5, 10, 11, 15, and 16, and the spots

133

were visualized by spraying them with a 5% H2SO4/EtOH solution followed by

134

heating. The TLC results showed that the spots displayed by fraction 3 were similar to

135

those of flavonoids (compounds 5, 15, and 16). Moreover, the content of compound 5

136

was the highest in this fraction. Fraction 3 (14 g) was subjected to CC over silica gel

137

(CHCl3/MeOH, 4:1, 3:1, 2:1, 0: 1) to yield three fractions, 3.1−3.3. Fraction 3.1 (2.6 g)

138

was purified by CC over Sephadex LH-20 (CHCl3/MeOH, 1:1) to obtain two

139

subfractions, 3.1.1 and 3.1.2. Fraction 3.1.2 (827 mg) was run again by CC over

140

Sephadex LH-20 (CHCl3/MeOH, 1:1) to obtain compounds 17 (19 mg) and 18 (31

141

mg). Fraction 3.2 (3.1 g) was applied to CC over Sephadex LH-20 (CHCl3/MeOH,

142

1:1) to yield two subfractions, 3.2.1 and 3.2.2. Fraction 3.2.1 (216 mg) was purified

143

by prep-HPLC (MeOH/H2O, 3:7) to yield compound 4 (22 mg). Fraction 3.2.2 (3.1 g)

144

was chromatographed over Sephadex LH-20 (CHCl3/MeOH, 1:1) to yield crude 19

145

(34 mg) and 20 (11 mg), and then they were separately recrystallized from MeOH.

146

Fraction 3.3 (2.9 g) was subjected to CC over Sephadex LH-20 (CHCl3/MeOH, 1:1)

147

to obtain crude 2 (41 mg) and compound 21 (8 mg), and then compound 2 was

148

recrystallized from MeOH.

149

Structural Elucidation of New Products. Gardnerol A (1): colorless crystals

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150

(MeOH); mp 191−192°C; IR (KBr) νmax 3228, 1693, 1589, 1513, 1433, 1031, 836,

151

792, 456 cm−1; UV (MeOH) λmax (log ε) 208 (4.40), 212 (4.41), 285 (3.90), 322 (4.12)

152

nm; 1H (DMSO-d6 and methanol-d4, 400 MHz) and

153

methanol-d4, 100 MHz) data, see Table 1; HRESIMS m/z 357.0970 [M + H]+ (calcd

154

for C19H17O7, 357.0969).

155

13

C NMR (DMSO-d6 and

Gardnerol B (2): with power (CHCl3-MeOH); [α]25D −10.5 (c 0.38, MeOH); IR (KBr)

156

νmax 3407, 3078, 2923, 1743, 1721, 1611, 839, 723 cm−1; UV (MeOH) λmax (log ε) 214

157

(4.26), 252 (3.62), 286 (3.93), 308 (3.98) nm; 1H (DMSO-d6 and methanol-d4, 400

158

MHz) and

159

HRESIMS m/z 343.0811 [M + H]+ (calcd for C18H15O7, 343.0812).

13

C NMR (DMSO-d6 and methanol-d4, 100 MHz) data, see Table 2;

160

X-ray Analysis. The measurement was collected on a SuperNova, Dual, Eos

161

diffractometer with graphite-monochromated Mo Kα radiation (λ = 0.71073 Å). The

162

structure of compound 1 was solved by direct method (SHELXS-2008).

163

Crystal Data for 1. Gardnerol A (1) was crystallized from CHCl3/ CH3OH (1:1) to

164

give colorless crystals. A single crystal of dimensions 0.35 × 0.31 × 0.24 mm3 was

165

used for X-ray measurements. Crystal data: C19H16O7, space group P 1 21/c 1, a =

166

14.7352(17) Å, b = 8.7127(9) Å, c = 14.2158(12) Å, α = 90.00°, β = 114.431(12) °, γ

167

= 90.00°, V = 1661.6(3) Å3, Z = 4, Dcalc = 1.424 g/cm3, R1 = 0.0713, wR2 = 0.2056.

168

The supplementary crystallographic data for 1 reported in this paper has been

169

deposited at the Cambridge Crystallographic Data Centre, under the reference

170

numbers CCDC 1403668. Copies of the data can be obtained, free of charge, on

171

application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK, fax:

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172

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+44 1223 336033 or e-mail: data_ [email protected].

173

α-Glucosidase Inhibition. The α-glucosidase inhibition was assessed according to

174

the slightly modified method of Jeon et al.19 The α-glucosidase (0.1 U/mL) and

175

substrate (p-NPG, 1.0 mM) were dissolved in potassium phosphate buffer (0.1 M, pH

176

6.7), and all samples were dissolved in DMSO. The inhibitor (10 µL) was

177

preincubated with α-glucosidase (40 µL) at 37 °C for 10 min, and then the substrate

178

(50 µL) was added to the reaction mixture. The enzymatic reaction was performed at

179

37 °C for 30 min. The reaction was then terminated by the addition of Na2CO3 (1 M,

180

100 µL). All samples were analyzed in triplicate with five different concentrations

181

near the IC50 values, and the absorbance at 405 nm was determined using a microplate

182

reader. The inhibition percentage (%) was calculated by the following equation:

183

Inhibition (%) = [(OD

184

OD control blank)] × 100.

185

control−

OD

control blank)

− (OD

sample−

OD

sample blank)/

(OD

control−

Type of α-Glucosidase Inhibition. The mode of inhibition of α-glucosidase was

186

investigated

with

increasing

concentrations

of

substrate

(4-nitrophenyl

187

α-D-glucopyranoside) and compound 5. Then, the inhibition type was determined by

188

a Lineweaver−Burk plot according to Michaelis−Menten kinetics. Origin (version 8.0)

189

software was used for plotting the results.

190

Oral Sucrose Tolerance Test (OSTT). The fasting normal and STZ-induced

191

diabetic mice were orally administered with compound 5 (150 and 300 mg/kg of

192

body weight), acarbose (5 mg/kg), or the control and after 30 min and were given a

193

sucrose solution (3 g/kg of body weight). Each group has six mice. Compound 5 and

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Page 11 of 32


194

acarbose were suspended in 0.5% sodium carboxymethyl cellulose (CMC-Na). The

195

control mice were administered with the same volume of 0.5% CMC-Na solution.

196

The tail vein glucose concentrations were measured with a glucometer at 0, 0.5, 1.0,

197

1.5, 2.0 and 3.0 hours after the sucrose load.

198

Hypoglycemic Activity Assay. The fasting normal and STZ-induced diabetic mice

199

(n=6 for each group) were orally administered with compound 5 (150 and 300 mg/kg

200

of body weight), glibenclamide (10 mg/kg), or the control (0.5% CMC-Na). The tail

201

vein glucose concentrations were measured with a glucometer at 0, 1.5, 3, 5, 7 and 9

202

hours after administration.

203

Statistical Analysis. The data were expressed as the mean ± SEM and were

204

analyzed using SPSS (version 19.0) statistical software (SPSS, Chicago, IL, USA).

205

The statistical significance of the differences (p < 0.05) between the mean values of

206

the treatment and control groups were obtained from a one-way analysis of variance

207

(ANOVA) followed by Tukey’s or Dunnett’s test.

208

RESULTS AND DISCUSSION

209

Isolation of Compounds from the Flower of E. gardneri. The extract was

210

subjected to repeated column chromatography over silica gel, Sephadex LH-20, and

211

ODS to yield 21 compounds (Figure 1). Compounds 1 and 2 were identified as new

212

compounds. The 19 known compounds were identified as edgeworic acid (3),20

213

8-(3-(2,4-benzenediol)-propionic acid methyl ester)-coumarin-7-β-D-glucoside (4),

214

tiliroside (5), 4-hydroxybenzoic acid (6), ferulic acid (7), 4- hydroxybenzaldehyde (8),

215

trans-p-hydroxycinnamic acid (9), kaempferol (10), quercetin (11), caffeic acid (12),

11



(13),

(-)-secoisolariciresinol

(14),

isoquercetin

Page 12 of 32

216

(+)-lariciresinol

(15),

217

kaempferol-3-O-β-D-glucoside (16), rutin (17), kaempferol-3-rutinoside (18), syringin

218

(19), coniferin (20), and zingerone 4-O-β-D-glucopyranoside (21).

219

Structure Elucidation of the Two New Compounds. Gardnerol A (1) was

220

obtained as colorless crystals after crystallization from CHCl3–CH3OH. The

221

molecular formula of 1 was established as C19H16O7 from the [M + H] + ion at m/z

222

357.0907 (calcd. for C19H17O7, 357.0967) in the HRESIMS. The IR spectrum

223

exhibited strong absorption bands at 3228 (OH), 1693 (conjugated ester C=O), and

224

1619, 1582, and 1513 (aromatic, C=C) cm-1. The UV spectrum of 1 showed the

225

characteristic maxima of a coumarin structure at 231, 252, and 325 nm. The 1H NMR

226

spectrum exhibited the typical signals in the aromatic region associated with H-3 at δH

227

6.18 (d, J=9.6 Hz), H-4 at δH 7.88 (d, J=9.6 Hz), H-5 at 7.37 (d, J=8.8 Hz), and H-6 at

228

δH 6.95 (d, J=8.8 Hz, H-6) of a 7, 8-disubstituted coumarin. The HMBC correlations

229

from the hydroxy proton at δH 10.63 to C-6, C-7, and C-8 confirmed that the hydroxy

230

group was connected to C-7 (Figure 2). Additionally, the aromatic region of the 1H

231

NMR spectrum clearly showed the presence of an ABX spin system [δH 6.35 (d, J =

232

2.0 Hz, H-2′), δH 6.93 (d, J = 8.4 Hz, H-5′), and δH 6.27 (dd, J = 8.4, 2.0 Hz, H-6′)],

233

indicative of a trisubstituted aromatic ring, which was confirmed by proton-coupling

234

patterns and 1H-1H COSY correlations (Figure 2). The HMBC correlations of the

235

hydroxy proton at δH 9.37 with C-2′, C-3′, and C-4′ supported the assignment that the

236

hydroxy was attached to C-3′. In addition, one singlet at δH 3.52 (3H, s, –COOCH3)

237

and two mutually coupled triplets at δH 2.66 (2H, t, J=8.0 Hz, H-7′) and δH 2.46 (2H, t,

12


Page 13 of 32


238

J=8.0 Hz, H-8′) evidenced the presence of a methyl propionate side chain. The

239

location of this side chain at C-4′ was determined on the basis of crosspeaks from H-7′

240

to C-3′, C-4′, and C-5′ and from H-4′ to C-7′ in the HMBC spectrum. The linkage of

241

C-1′ to C-8 by one atom of oxygen was deduced from the above-mentioned reasoning

242

and the molecular weight. Thus, the structure of 1 was defined as methyl

243

3-(2-hydroxy-4-O-(7-hydroxycoumarinyl) phenyl) propanoate. An X-ray diffraction

244

analysis corroborated this proposed structure (Figure 3).

245

Gardnerol B (2) was obtained as a white powder. The HRMS data of compound 2

246

showed a molecular ion at m/z 527.1173 [M + Na]+ (calcd. 527.1160), which was 162

247

mass units (i.e., C6H10O5) more than that of compound 3 {m/z 343.0811, [M + H]+}.

248

Correspondingly, the 1H and 13C NMR data of 2 were similar to those of 3 except for

249

the presence of an additional glucopyranosyl moiety.

250

anomeric proton signal at δH 5.09 as a doublet with a coupling constant of 7.6 Hz

251

indicated the presence of the β-glucopyranosyl moiety. The HMBC correlation

252

between the anomeric proton (H-1′′′, δH 5.09) and C-7 (δC 154.7) indicated that the

253

β-glucopyranosyl moiety was attached to C-7 (Figure 2). Furthermore, to confirm the

254

structure of 2, compound 2 was hydrolyzed under acidic conditions. The sugar residue

255

of 2 was identified as D-(+)-glucose by TLC comparison with an authentic sample

256

and by its optical rotation value (see Supporting Information). On the basis of the

257

above analyses, the structure of compound 2 was identified as edgeworic

258

acid-7-O-β-D- glucoside.

259

20

The observation of the

α-Glucosidase Inhibition. The IC50 values of compounds 1− −21 and extracts from

13



260

the flower of E. gardneri are shown in Table 3. In accordance with the previous report,

261

the flower extract exhibited a significant inhibitory activity with an IC50 value of 267

262

µg/mL. Among these compounds, compound 11 was the most active, with an IC50

263

value of 5.1 µg/mL. Additionally, compound 10 showed a potent inhibitory activity

264

with an IC50 value of 56.2 µg/mL. Compound 5, the most abundant compound in the

265

extract, showed a moderate activity, with an IC50 value of 202 µg/mL. Compounds 8,

266

12–13, and 16–18 also showed moderate activities with IC50 values of 486, 957, 279,

267

179, 272, and 253 µg/mL, respectively. Unfortunately, compounds 2, 3, 7, 14, and

268

19–21 showed no activities at the maximum concentration tested (3000 µg/mL).

269

Previous literature reported that flavonoids with 7, 3, 3′, and 4′ hydroxy groups

270

showed significant α-glucosidase inhibitory activity.21 Our results revealed that the 3′

271

hydroxy groups are responsible for increased activity because compounds 10 and 11,

272

with a 3′ hydroxy group, were more active than compounds 5 and 15–18, without a 3′

273

hydroxy group. Compounds 2 and 3 showed significantly less activity than

274

compounds 1 and 4. This result suggests that the methyl ester group has an impact on

275

the inhibitory activity. When compound 4 was compared to compound 1, the

276

inhibitory activity was reduced because of the absence the hydroxy groups at the C-5

277

position.

278

Types of α-Glucosidase Inhibition. To clarify the α-glucosidase inhibition mode

279

of compound 5, which was the most abundant compound in the extract,

280

Lineweaver-Burk plots were generated (Figure 4).22 As shown in Figure 4, the value

281

of vertical axis intercept (1/Vmax) remained unchanged with the increase of the

14


Page 14 of 32

Page 15 of 32


282

concentrations of compound 5, indicating that compound 5 was a competitive

283

inhibitor. According to Michaelis−Menten kinetics, the value of inhibition constant

284

(Ki) is 0.473 mM.

285

Antihyperglycemic Effects of Compound 5 on Oral Sucrose Tolerance in

286

Normal and STZ -induced Diabetic Mice. The oral sucrose tolerance test (OSTT) is

287

usually performed to evaluate the efficacy of a drug in inhibiting intestinal

288

α-glucosidase in vivo.23 Compound 5 was a major constituent of the extract of E.

289

gardneri, and 53 g of compound 5 were obtained from the extract. In addition,

290

compound 5 showed significant α-glucosidase inhibitory activity in vitro. Thus,

291

compound 5 was evaluated for its antihyperglycemic effects using an OSTT in both

292

normal and STZ-induced diabetic mice. In comparison with the vehicle, the oral

293

administration of compound 5 at a dose of 300 mg/kg significantly (p < 0.05) reduced

294

the postprandial blood glucose level of normal mice (Figure 5A). The

295

antihyperglycemic effect was observed at 30, 60, and 90 min after sucrose loading and

296

was compared with that of acarbose (5 mg/kg). In contrast, the 150 mg/kg dose did

297

not show a significant decrease in glycemia throughout the experiment. The OSTT

298

was repeated in STZ-induced diabetic mice. These results were similar to those for

299

normal mice (Figure 5B). According to the results of the OSTT, compound 5 inhibited

300

the activity of intestinal α-glucosidase.

301

The Hypoglycemic Effect of Compound 5 on Normal and STZ-induced 24, 25

302

Diabetic Mice. According to previous methods,

the hypoglycemic activity of

303

compound 5 was evaluated in both normal and STZ-induced diabetic mice. As shown

15



Page 16 of 32

304

in Figure 6, compound 5, which had decreased fasting glucose levels in STZ-induced

305

diabetic mice, did not lower the fasting glucose levels in normal mice. In diabetic

306

mice, the oral administration of compound 5 at 150 and 300 mg/kg at both doses

307

significantly decreased the fasting glucose levels when compared with the

308

vehicle-treated groups (p < 0.05). The dose at 300 mg/kg resulted in a significant

309

decrease in glucose level at 1.5 h (−9.7%), 3 h (−10.9%), 5 h (−18.5%), 7 h (−31.5%),

310

and 9 h (−37.8%). After an administration of 150 mg/kg of compound 5, the glucose

311

level decreased by 4.7% at 1.5 h, 10.5% at 3 h, 20.2% at 5 h, 24.4% at 7 h, and 36.4%

312

at 9 h. Because compound 5 displayed a hypoglycemic effect in diabetic but not in

313

normal mice, in contrast with glibenclamide, these results indicate that compound 5

314

may not act directly via insulin liberation. Previous studies suggested that kaempferol,

315

the

316

hyperglycemia-impaired pancreatic β-cell viability and insulin-secretory function.26

317

Therefore, the hypoglycemic action of compound 5 probably involves the protection

318

of pancreatic β-cell survival and function. Further studies are necessary to

319

demonstrate the mechanisms by which compound 5 decreased the fasting glucose

320

levels in STZ-induced diabetic mice.

hydrolysis

products

of

compound

5,

improved

the

chronic

321

In summary, two new phenolics, along with 19 known phenolics, were isolated

322

from the flower of E. gardneri. Their inhibitory activity against α-glucosidase from

323

Saccharomyces cerevisiae was evaluated, and the data showed that the flower of E.

324

gardneri is a rich source of natural α-glucosidase inhibitors. Compound 5, the most

325

abundant compound in the extract, showed a significant α-glucosidase inhibitory

16


Page 17 of 32


326

activity and acted as a competitive inhibitor of α-glucosidase. Furthermore, compound

327

5 is effective in vivo for reducing the fasting and postprandial blood glucose levels in

328

STZ-induced diabetic mice. In conclusion, the present study complements the current

329

knowledge about the chemical composition and antihyperglycemic activity of the

330

flower of E. gardneri, and it provides scientific evidence to substantiate the use of this

331

flower for traditional therapeutic and dietary uses.

332 333

334

Acknowledgment. This work was supported by the National Natural Science Foundation of China (No.21402030, 21402031 and 21272043). Supporting Information Available: Acid hydrolysis procedure, the HRESIMS,

335

IR, 1H,

13

336

available free of charge via the Internet at http://pubs.acs.org.

C, and 2D NMR spectrum of compounds 1 and 2. This information is

17



337

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338

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12. Gonçalves, R.; Mateus, N.; de Freitas, V. Inhibition of α-amylase activity by

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13. Liu, M.; Yin, H.; Liu, G.; Dong, J.J.; Qian, Zh.H.; Miao, J.L. Xanthohumol, a

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effect of water extracts from Edgeworthia gardneri (Wall.) Meissn on type 2 diabetic

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mice. Nat. Prod. Res. Dev. 2014, 26, 1385−1388.

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16. Geng, Y.; Yang, H.-M.; Xu, H.-Y.; Shi, J.-S. α-Glucosidase inhibitory activity of

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the alabastrum of Edgeworthia gardneri (Wall.) Meissn. Journal of Food Science

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17. Gao, D.; Zhang, Y.-L.; Xu, P.; Lin, Y.-X.; Yang, F.-Q.; Liu, J.-H.; Zhu, H.-W.;

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Xia, Z.-N. In vitro evaluation of dual agonists for PPARγ/β from the flower of

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Edgeworthia gardneri (wall.)Meisn. J. Ethnopharmacol. 2015, 162, 14−19.

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18. Li, S.-S.; Gao, Z.; Feng, X.; Hecht, S. M. Biscoumarin derivatives from

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Edgeworthia gardneri that inhibit the lyase activity of DNA polymerase β. J. Nat.

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Antiglycation Activity of Laccase-Catalyzed Catechin Polymers. J. Agric. Food Chem.

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2013, 61, 4577−4584.

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Edgeworthia chrysantha. Molecules 2014, 19: 2042−2048.

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Mata, R. α-Glucosidase Inhibitors from Brickellia cavanillesii. J. Nat. Prod. 2012,

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22. Yan, J.; Zhang, G.; Pan, J.; Wang, Y. α-Glucosidase inhibition by luteolin:

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23. Brindis, F.; Rodríguez, R.; Bye, R.; Gonzalez-Andrade, M.; Mata, R.

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24. Nuñez-López, A. M.; Paredes-López, O.; Reynoso-Camacho, R. Functional and

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Hypoglycemic Properties of Nopal Cladodes (O. ficusindica) at Different Maturity

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Stages Using in Vitro and in Vivo Tests. J. Agric. Food Chem. 2013, 61,

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10981−10986.

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25. Narváez-Mastache, M. J.; Garduño-Ramírez, L. M.; Alvarez, L.; Delgado, G.

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Antihyperglycemic Activity and Chemical Constituents of Eysenhardtia platycarpa. J.

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Nat. Prod. 2006, 69, 1687−1691.

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26.

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hyperglycemia-impaired pancreatic beta-cell viability and insulin secretory function.

412

Eur. J. Pharmacol. 2011, 670, 325–332.

Zhang,

Y.;

Liu,

D.

Flavonol

kaempferol

21


improves

chronic


413

Figure captions

414

Figure 1. Structures of compounds 1−21.

415

Figure 2. Key 1H-1H COSY and HMBC correlations for compounds 1 and 2.

416

Figure 3. X-ray structure of compound 1.

417

Figure 4. (A) Lineweaver–Burk plots of the reaction of α-glucosidase at different

418

concentrations of substrate and compound 5. (B) A partially enlarged view of Figure

419

4A.

420

Figure 5. Effects of compound 5 on blood glucose levels in normal (A) and STZ

421

-induced diabetic mice (B) using the OSTT. Data are the means ±SEM for 6 mice in

422

each group. * P < 0.05 by one-way ANOVA with post-hoc test compared with

423

control.

424

Figure 6. Hypoglycemic effect of compound 5 on normal (A) and STZ-induced

425

diabetic mice (B). Data are the means ±SEM for 6 mice in each group. * P < 0.05 by

426

one-way ANOVA with post-hoc test compared with control.

427

22


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Page 23 of 32


Table 1. 1H and 13C NMR Data for Compound 1 in Methanol-d4 and DMSO-d6 Methanol-d4 position

δC

2 3 4 4a 5 6 7 8 8a 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 9′C-OMe 7-OH 3′-OH

162.7, C 112.3, CH 146.1, CH 114.1, C 126.2, CH 115.0, CH 156.0, C 130.4, C 149.9, C 158.8, C 103.2, CH 157.3, C 122.1, C 131.3, CH 106.8, CH 26.5, CH2 35.1, CH2 175.8, C 51.9, CH3

DMSO-d6

δH mult (J in Hz) 6.19, d (9.6) 7.88, d (9.6) 7.37, d (8.8) 6.95, d (8.8)

6.35, d (2.4)

6.93, d (8.4) 6.27, dd (8.4, 2.4) a (2.80, m), b (2.80, m) a (2.56, m), b (2.56, m) 3.62, s

δC 159.7, C 111.7, CH 144.7, CH 112.2, C 125.3, CH 113.6, CH 154.2, C 128.2, C 148.3, C 156.8, C 101.5, CH 155.9, C 120.2, C 130.1, CH 105.3, CH 24.8, CH2 33.5, CH2 172.9, C 51.1, CH3

23



6.24, d (2.4)

6.89, d (8.4) 6.19, dd (8.4, 2.4) a (2.66, m), b (2.66, m) a (2.46, m), b (2.46, m) 3.52, s 10.63, s 9.37, s


Page 24 of 32

Table 2. 1H and 13C NMR Data for Compound 2 in Methanol-d4 and DMSO-d6 Methanol-d4 position

δC

2 3 4 4a 5 6 7 8 8a 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 1′′ 2′′ 3′′ 4′′ 5′′ 6′′

162.2, C 114.7, CH 145.6, CH 116.3, C 126.0, CH 114.1, CH 154.7, C 132.4, C 149.3, C 158.7, C 103.8, CH 157.1, C 122.9, C 131.4, CH 107.3, CH 26.5, CH2 35.8, CH2 178.7, C 101.8, CH 74.6, CH 77.8, CH 70.9, CH 78.2, CH 62.2, CH2

DMSO-d6


6.39, d (2.4)

6.96, d (8.4) 6.31, dd (8.4, 2.4) a (2.79, m), b (2.79, m) a (2.53, m), b (2.53, m) 5.09, d (7.6) 3.37, overlap 3.45, overlap 3.37, overlap 3.45, overlap a 3.86, m b 3.69, dd (12.0, 5.2)

δC 159.5, C 113.5, CH 144.4, CH 114.3, C 125.1, CH 112.5, CH 153.4, C 130.2, C 148.3, C 156.9, C 102.4, CH 155.8, C 121.0, C 130.0, CH 105.7, CH 25.1, CH2 34.6, CH2 175.1, C 100.2, CH 73.0, CH 76.7, CH 69.4, CH 77.2, CH 60.5, CH2

24



6.33, d (2.4)

6.94, d (8.4) 6.27, dd (8.4, 2.4) a (2.65, m), b (2.65, m) a (2.40, m), b (2.40, m) 5.07, d (7.6) 3.16, overlap 3.25, m 3.13, overlap 3.36, m a 3.67, m b 3.45, dd (12.0, 5.2)

Page 25 of 32


Table 3. α-Glucosidase Inhibition by Extract and Compounds 1−21a Extract 1 2 3 4 5 6 7 8 9 10 11 a

IC50（µg/mL） 267±19 517±24 > 2000b > 2000 897±59 202±12 1200±134 > 2000 486±45 541±32 56.2±4.1 5.1±0.3

12 13 14 15 16 17 18 19 20 21 Acarbose

IC50（µg/mL） 957±36 279±14 > 2000 1233±87 179±9.2 272±15 253±19 > 2000 > 2000 > 2000 465±37

Values are the means ± SEM from at least three independent experiments. b Exceeds

maximum concentration tested.

25



5

6

4

4a

Page 26 of 32

3

Glc O

2

7

R1

8a O 1

8

O 1'

2'

O

R1 OH

O

O

O OH

6'

R2

R2

5'

HO 3'

4'

HO

7'

R2

8' 9'

O

R2=OCH3 R2=OH R2=OH R2=OCH3

HO

1'

20 R1=H 7 R1=OCH3 R2=OH 9 R1=H R2=OH 21 R =H 1 12 R1=OH R2=OH

R2=

OH

R2= O

HO

O

O OH 4'

OH H

O H

OH

HO OH

7 5 4 OH O

OH

19 R1=OCH3 R2=

R1

H 8

R2 1 O

O

6

O

1 R1=OH 2 R1=O-Glc 3 R1=OH 4 R1=O-Glc

OH

R1

HO

HO O

O

13

14 OH

5 R1=O-S1 10 R1=OH 11 R1=OH 15 R1=O-Glc 16 R1=O-Glc 17 R1=O-S2 18 R1=O-S2

R2=H R2=H R2=OH R2=OH R2=H R2=OH R2=H

O O

O

OH HO

HO

O

OH

OH

HO

OH

OH Glc

O

O

O

S1

Figure 1

26


OH HO OH S2

OH OH

Page 27 of 32


OH HO

O

O

O

O

O

O

O

O HO

OH OH

HO

O

HO

O

O 1

H-1H COSY

OH

HMBC

Figure 2

27



Figure 3

28


Page 28 of 32

Page 29 of 32


Figure 4

29



Figure 5

30


Page 30 of 32

Page 31 of 32


Figure 6

31



Table of Contents Graphic

32


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Î±-Glucosidase Inhibition and Antihyperglycemic ... - ACS Publications

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