Quickly Screening for Potential Î±-Glucosidase Inhibitors from Guava

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Quickly Screening for Potential #-Glucosidase Inhibitors from Guava Leaves Tea by Bio-Affinity Ultrafiltration Coupled with HPLC-ESI-TOF/MS Method Lu Wang, Yufeng Liu, You Luo, Kuiying Huang, and Zhenqiang Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05280 • Publication Date (Web): 30 Jan 2018 Downloaded from http://pubs.acs.org on February 1, 2018

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Page 1 of 30

Journal of Agricultural and Food Chemistry

1

Quickly

Screening

for

Potential

2

Inhibitors from Guava Leaves Tea by Bio-Affinity

3

Ultrafiltration Coupled with HPLC-ESI-TOF/MS

4

Method

α-Glucosidase

5

Lu Wang†, Yufeng Liu†, You Luo†, Kuiying Huang‡, Zhenqiang Wu†*

6

† School of Biology and Biological Engineering, Guangdong Provincial Key

7

Laboratory of Fermentation and Enzyme Engineering, South China University of

8

Technology, Guangzhou 510006, P. R. China

9

‡ Guangzhou Institute of Microbiology, Guangzhou 510663, P. R. China

10 11 12 13 14 15 16 17 18 19 20 21 22

1

ACS Paragon Plus Environment


23

ABSTRACT: Guava leaves tea (GLT) has a potential anti-hyperglycemic effect.

24

Nevertheless, it is unclear which compound plays a key role in reducing blood sugar.

25

In this study, GLT extract (IC50 = 19.37 ± 0.21 µg/mL) exhibited a stronger inhibitory

26

potency against α-glucosidase than did acarbose (positive control) at IC50 = 178.52 ±

27

1.37 µg/mL. To rapidly identify the specific α-glucosidase inhibitor components from

28

GLT, an approach based on bio-affinity ultrafiltration combined with high

29

performance liquid chromatography coupled to electrospray ionization-time of

30

flight-mass spectrometry (BAUF-HPLC-ESI-TOF/MS) was developed. Under the

31

optimal bio-affinity ultrafiltration conditions, eleven corresponding potential

32

α-glucosidase inhibitors with high affinity degrees (ADs) were screened and identified

33

from the GLT extract. Quercetin (IC50 = 4.51 ± 0.71 µg/mL) and procyanidin B3 (IC50

34

= 28.67 ± 5.81 µg/mL) were determined to be primarily responsible for the

35

anti-hyperglycemic effect, which further verified the established screening method.

36

Moreover, structure-activity relationships were discussed. In conclusion, the

37

BAUF-HPLC-ESI-TOF/MS method could be applied to determine the potential

38

α-glucosidase inhibitors from complex natural products quickly.

39

Keywords: guava leaves tea, α-glucosidase inhibitors, quick screening, bio-affinity

40

ultrafiltration, HPLC-ESI-TOF/MS

41 42 43 44

2


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INTRODUCTION

46

Diabetes mellitus is a serious chronic endocrine metabolic disorder characterized by

47

high blood glucose levels. Based on the results of a World Health Organization (WHO)

48

survey, 90%-95% of over 400 million diabetes mellitus patients worldwide have type

49

2 diabetes mellitus

50

medicinal plants or food matrices have attracted increasing interest in the treatment

51

and prevention of diabetes due to its efficiency and low toxicity

52

one of the main carbohydrate hydrolysis enzymes, is responsible for the cleavage of

53

oligosaccharides and disaccharides into monosaccharides suitable for absorption in

54

the small intestine 6,7. α-Glucosidase inhibitors can diminish the absorption of glucose

55

and thus, reduce postprandial blood glucose levels

56

inhibitors have been considered a first line therapy by the International Diabetes

57

Federation (IDF) and the American Association of Clinical Endocrinologists (AACE)

58

11,12

59

natural products is an increasingly interesting and challenging research area for the

60

management of diabetes mellitus.

1,2

. Currently, carbohydrate hydrolysis enzyme inhibitors from

3-5

. α-Glucosidase,

8-10

. Natural α-glucosidase

. Thus, the rapid screening of α-glucosidase inhibitors from complex systems of

61

Conventional bio-assay guided approaches for screening bioactive components

62

from complex extracts require multiple-step extractions and separation procedures by

63

organic solvents, which are inefficient and environmental unfriendly 13,14. However,

64

decomposition, irreversible adsorption, and dilution effects of the isolated substances

65

typically leads to false positives events with correspondingly high failure risks. To

66

rapidly identify and isolate specific bioactive compounds, a combinatorial method of

3



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bio-affinity ultrafiltration and high performance liquid chromatography coupled to

68

electrospray ionization-time of flight-mass spectrometry (BAUF-HPLC-ESI-TOF/MS)

69

has been developed to identify potential novel bioactive compounds 15. In this assay,

70

the bio-active molecules (ligands) were firstly combined with α-glucosidase (receptor)

71

to form the ligand-receptor complexes, then the ultrafiltration centrifugation separates

72

the formed complexes under the optimal conditions, and the ligands released from the

73

complexes

74

HPLC-ESI-TOF/MS analysis. BAUF-HPLC-ESI-TOF/MS has been applied to screen

75

and identify a number of novel bioactive components from complex extracts systems

76

at early drug discovery stages without requiring tedious isolation and purification

77

steps. For example, Chen et al. (2016) used bio-affinity ultrafiltration technology with

78

DNA Top I (topoisomerase I) as a drug target to successfully isolate specific alkaloids

79

with potential anti-cancer activity from Lycoris radiata16. Ma et al. (2017) rapidly

80

screened out potential α-amylase inhibitors from Rhodiola rosea by affinity

81

ultrafiltration coupled with UPLC-TOF/MS based on a metabolomic method

82

Guava (Psidium guajava L.) belongs to the Myrtaceae family and is widely cultivated

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in tropical and subtropical environments. Guava leaves tea (GLT) is a commercial

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product manufactured by freshly guava leaves. The manufacturing process included

85

four stages: plucking, solar withering, indoor withering, and guava leaves tea product.

86

Pharmacology reports have confirmed that the crude extracts of guava leaves

87

possessed

88

concentrated on evaluating the total α-glucosidase inhibition activity of complex

could

strong

be

readily

identified

anti-hyperglycemic

and

effects

subsequently

18-21

.

4


While

quantified

previous

by

17

.

studies

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systems or pure chemicals isolated from guava leaves extracts, the specific bioactive

90

components responsible for the anti-hyperglycemic effects of GLT have not yet been

91

elucidated to date.

92

In the present work, α-glucosidase was selected as a drug target to establish the

93

BAUF-HPLC-ESI-TOF/MS assay method. Optimal bio-affinity ultrafiltration assay

94

conditions were investigated by metabolomic method. The developed method was

95

used to rapidly identify and recognize major α-glucosidase inhibitors from guava

96

leaves tea extracts. The present work provided useful information for rapidly

97

recognizing and identifying hypoglycemic components from complex medicinal

98

products.

99

MATERIALS AND METHODS

100

Chemicals

101

α-Glucosidase

102

p-nitrophenyl-α-D-glucopyranoside

103

compounds and acarbose (≥ 99.8%) were purchased from Sigma-Aldrich (St. Louis,

104

MO, USA). p-Hydroxycinnamic acid was used as the internal standards. Formic acid,

105

dimethylsulphoxide (DMSO) and acetonitrile (ACN) solvents were purchased from

106

Fisher Scientific (HPLC grade, 99.9%, Waltham, MA, USA). 0.5 mL centrifugal

107

filters devices in different sizes (10 kDa, 30 kDa, and 50 kDa) were purchased from

108

Millipore Co. Ltd. (Bedford, Massachusetts, USA). The deionized water was purified

109

by a Milli-Q water purification system from Millipore (Bedford, Massachusetts, USA).

from

Saccharomyces (p-NPG,

cerevisiae ≥

99.8%),

5


(≥ standard

99.8%), phenolics


110

Other analytical-grade reagents were obtained from Sigma-Aldrich (St. Louis,

111

Missouri, USA).

112

Tea Material and Extraction

113

Guava leaves tea was provided by the Jiangmen Nanyue Guava Tea farmer

114

cooperative (Jiangmen, Guangdong, China) and authenticated by a specialist, Peibiao

115

Liu (General manager of Jiangmen Nanyue Guava Tea Farmer Cooperative, Jiangmen,

116

China). One gram (dry mass, DM) of GLT powder was extracted with 10 mL of 70%

117

methanol by ultrasonic extraction (320 W, 40°C) for 30 min. The extracts was filtered

118

using a 0.45 µm Whatman No. 1 filter paper (Maidstone, UK). The filtrates were

119

evaporated under vacuum at 45°C until dry. The dried powder was dissolved by

120

adding 20 mL dimethylsulphoxide.

121

Total α-Glucosidase Inhibitory Assay

122

Evaluation of α-glucosidase inhibitory potency was based on a previous methodwith

123

some modifications 22. 100 µL of 1 U/mL α-glucosidase mixed with 100 µL of extract

124

dilutions (1, 5, 15, 25, 40 and 50 µg/mL) was incubated at 37°C for 10 min. Instead of

125

the extract dilutions, 100 µL of phosphate buffer (PBS buffer, 0.01 M, pH = 6.8) was

126

used as an enzyme control, and 100 µL of 0.01 M phosphate buffer acted as the

127

extract control. Next, 100 µL of a p-NPG (5 mM in PBS buffer) was added to the

128

above mixture. The mixtures was incubated at 37°C for 20 min and terminated by

129

adding 500 µL of a 1 M Na2CO3 solution. Mixture absorbance was determined at 405

130

nm in a 96-well plates. The enzyme inhibition activity was calculated using the

131

following Eq. 1:

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α-Glucosidase inhibitory potency (%) =

 A1 - A0）（（ - B1 - B0）   ×100 A1 - A0  

(Eq. 1)

133

where A1, A0, B1, and B0 represent the absorbance of the blank test group (containing

134

PBS buffer and enzyme), the blank control group (containing PBS buffer only), the

135

sample test group (containing sample extracts, PBS buffer, and enzyme), and the

136

sample control group (containing sample extracts and PBS buffer), respectively.

137

Bio-Affinity Ultrafiltration (BAUF) Conditions

138

The screening procedure was conducted based on the previous reports, with slight

139

modifications

140

dimethylsulphoxide. α-Glucosidase was dissolved in 10 mM PBS buffer (pH = 6.8).

141

100 µL of 2.0 mg/mL sample extracts were reacted with 200 µL of α-glucosidase (1

142

U/mL, 5 U/mL, 10 U/mL) at 37°C for 30 min. An inactivated α-glucosidase

143

(incubated at 100°C for 10 min) was used as the blank group in the same way. The

144

reaction mixtures were ultra-filtered through centrifugal filter devices in different

145

sizes (10, 30, and 50 kDa) and centrifuged at 10,000×g for 10 min to intercept the

146

α-glucosidase-ligand complexes at room temperature. The unbound components in

147

the complexes were washed three times using 200 µL of 10 mM PBS buffer (pH = 6.8)

148

by centrifugation. Afterward, the α-glucosidase-ligand complexes were incubation

149

with 70% ACN for 10 min, the ligands were released from the complexes by

150

centrifuged at 10,000×g for 10 min, which was repeated twice. Then the combined

151

filtrates were evaporated under vacuum at 37°C until dry. Finally, the dryness were

152

re-dissolved in 200 µL of 70% ACN and analyzed by the HPLC-ESI-TOF/MS.

16,17

. Briefly, the tested GLT extracts were dissolved in 5%

7



153

HPLC-ESI-TOF/MS Analysis

154

The Agilent 1200 HPLC system was fitted with a Zorbax Eclipse Plus C18 column

155

(250 mm × 4.6 mm, 5 µm, Agilent, USA) and an ultra-high resolution micro TOF-QII

156

mass spectrometer with 20,000 FWHM of mass resolution (maXis, Bruker, Billerica,

157

MA, USA). An binary solvent was consisted of 0.1% formic acid-H2O (v/v) as solvent

158

A and acetonitrile as solvent B with the following gradient program: 0-5 min with

159

15% B; 5-10 min with 15 to 20% B; 10-20 min with 20 to 25% B; 20-30 min with 25

160

to 35% B; 30-40 min with 35 to 50% B; 40-55 min with 80% B; 45-50 min with 15%

161

B. The injection volume was 20 µL. The flow rate was maintained at 0.8 mL/min and

162

the UV detection wavelength was performed by scanning from 200 to 600 nm at 30°C

163

column temperature. Conditions for MS operation were based on our previously work

164

23

165

fragmentation and some reference data 23,24.

166

Principle Components Analysis (PCA)

167

MarkerLynx XS software (Waters, Milford, MA, USA) was used to regulate and

168

normalize the origin HPLC-ESI-TOF/MS data. The parameters were set as follow:

169

2% percent of peak baseline noise value, one second of peak width value at 10%

170

height, 100 counts of marker intensity threshold value, and 5% of a noise elimination

171

threshold value with the retention of isotopic peak. The mass tolerance value was set

172

at 4.0 ppm. Next, the obtained data were further analyzed using IBM SPSS 17.0

173

statistical software (Milford, MA, USA). PCA was performed to detect clustering

174

trends of samples (with α-glucosidase) and blank (with inactivated α-glucosidase).

. All compounds were identified by their mass spectra, distribution patterns of ion

8


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175

Calculation of Affinity Degree (AD) for Selected Potential α-Glucosidase

176

Inhibitors

177

Using the optimal bio-affinity ultrafiltration-HPLC method, the α-glucosidase

178

inhibitors from GLT extracts were selected based on the above described procedure.

179

According to variations in the peak areas before and after incubation with

180

α-glucosidase, the affinity degree (AD) was defined as the interaction ability between

181

the ligands and α-glucosidase. A higher affinity degree represented a stronger

182

inhibitory ability for α-glucosidase. The AD was calculated based on Eq. 2:

183

AD(%) =

A1 − A2 × 100% A0

(Eq. 2)

184

where A1, A2, and A0 represent the peak areas of selected compounds obtained from

185

incubations of the GLT extract with activated, inactivated and without α-glucosidase.

186

Statistical Analysis

187

All results presented in this paper were the average of three independent assays and

188

were expressed as the mean ± standard deviation (SD). The results were analyzed by

189

one-way analysis of variance (ANOVA). Significant differences were determined by

190

Duncan’s multiple range tests or by independent sample T-tests when necessary.

191

Samples with p < 0.05 were considered statistically significant in all cases. Statistical

192

analyses were conducted using IBM SPSS version 17.0 (SPSS Inc., Chicago, IL, USA)

193

software package for Windows.

194

RESULTS AND DISCUSSION

195

Total α-Glucosidase Inhibitory Potency of GLT extracts

9



196

Managing postprandial plasma glucose levels is important in the early management of

197

diabetes 22. Natural α-glucosidase inhibitors from medicinal plant extracts play an

198

important role in decreasing postprandial hyperglycemia 24,4,5. In this work, different

199

concentrations of GLT extracts were used to investigate total inhibitory activity

200

against α-glucosidase in vitro. Acarbose served as a positive control. As shown in

201

Figure 1AB, the GLT extracts showed remarkably higher α-glucosidase inhibitory

202

activity with an IC50 at 19.37 ± 0.21 µg/mL when compared with acarbose at 178.52 ±

203

1.37 µg/mL. The GLT extracts were confirmed to have potential anti-hyperglycemic

204

effects and were enriched in natural α-glucosidase inhibitory molecules. Consequently,

205

there is an urgent need to rapidly recognize and identify special natural α-glucosidase

206

inhibitors from GLT.

207

Optimal Conditions of Bio-Affinity Ultrafiltration

208

In the present work, α-glucosidase was selected as the drug target (receptor), and the

209

potential bioactive compounds from GLT extracts were considered the ligands. The

210

ligand-receptor complexes formed by interactions between α-glucosidase and the

211

ligands were separated by centrifugal ultrafiltration. A schematic of the

212

BAUF-HPLC-ESI-TOF/MS assay method is shown in Fig. 2. PCA was used as a

213

screening tool to explore profile changes in the metabolome in GLT extracts before

214

and after reactions with α-glucosidase. To determine the optimal affinity ultrafiltration

215

conditions, the concentrations of enzymes and sizes of centrifugal ultrafiltration filter

216

devices in affinity ultrafiltration assays were investigated17. Fig. 3A shows the effects

217

of α-glucosidase concentration on its metabolomic profile. The differences between

10


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218

the metabolomic profiles of the GLT extract samples (with an activated α-glucosidase

219

group) and blanks (with an inactivated α-glucosidase group) were more evident with

220

increasing enzyme concentration. The degrees of separation in the PCA analysis

221

corresponded with α-glucosidase inhibitory ability. The results indicated that 10 U/mL

222

of α-glucosidase showed the largest differences between the samples and blanks.

223

Hence, 10 U/mL of α-glucosidase was selected as the optimal inhibitory concentration

224

for the following experiments.

225

In the bio-affinity ultrafiltration assays, membrane filter size is a very important

226

factor for separating α-glucosidase ligands. Fig. 3B shows the effects of molecular

227

membrane filter size on the metabolomic profiles of GLT extract samples and blanks.

228

The results showed that 30 kDa membrane filters resulted in a good separation

229

performance between samples and blanks. Ma et al. (2017) reported that two types of

230

complexes

231

α-amylase-inhibitors) in membrane filters during the separation process

232

work, because a 10 kDa filter was not suitable for separating the two types of

233

complexes, the retention of α-glucosidase-inhibitor complexes was dominant for 30

234

kDa filters, and the retention of the two types of complexes was weak for 50 kDa

235

filters. Therefore, 30 kDa filters were used for the formed ligand-receptor complexes.

236

In conclusion, 10 U/mL of α-glucosidase and 30 kDa membrane filters were selected

237

as the optimal bio-affinity ultrafiltration conditions used in the following experiments.

238

Separation of Potential α-Glucosidase Inhibitors from GLT Extract

exist

with

different

retention

rates

11


(macromolecule

and

17

. In this


239

After incubating with α-glucosidase and optimal bio-affinity ultrafiltration, the bound

240

ligands in the GLT extracts were released by 70% acetonitrile solution and analyzed

241

by HPLC-TOF/MS. The 11 bioactive compounds incubated with α-glucosidase in the

242

GLT extracts showed higher bio-affinity ability when compared with the inactivated

243

control group. These results indicated that these 12 constituents showed specific

244

binding toward α-glucosidase. Therefore, these 12 constituents were considered major

245

potential α-glucosidase ligands (Fig. 4). It is worth noting that the major

246

α-glucosidase ligands in GLT extracts were clearly described for the first time.

247

Based on variations in peak areas before and after incubation with α-glucosidase,

248

the affinity degree (AD) of 12 potential inhibitors with α-glucosidase are listed in

249

Table 1. Compound 12 possessed the greatest affinity degree (18.86 ± 0.28%),

250

followed by compounds 3 (8.54 ± 0.15%), 8 (7.47 ± 0.09%), 7 (6.56 ± 0.13%), 4

251

(5.32 ± 0.02%), 6 (5.18 ± 0.08%), 5 (4.96 ± 0.11%), 2 (4.75 ± 0.14%) 10 (3.93 ±

252

0.07%), 11 (3.15 ± 0.02%), 9 (2.31 ± 0.11%) and 1 (1.07 ± 0.03%). As expected, there

253

were very obvious differences in the ADs among the selected compound.

254

Theoretically, the differences among the ADs may be due to different competitive

255

binding relationships between the bioactive constituents with α-glucosidase.

256

Identification of Potential α-Glucosidase Inhibitors

257

The 12 compounds from the GLT extracts with different affinity degrees on

258

α-glucosidase were identified by comparing their HPLC retention times and m/z

259

fragmentation patterns of quasi-molecular ions based on reference data 25. The HPLC

260

retention time, the corresponding MS/MS data and the identification of the labeled

12


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261

peaks are shown in Table 1. As shown in Table 1 and Fig. 4, compound 1 was

262

identified as gallic acid based on its UV/Vis absorption spectrum (215 nm and 270 nm)

263

and the main [M+H]+ ion at m/z 171.1221. Compound 2 was determined to be

264

L-epicatechin according to the UV/Vis absorption spectrum (256 nm and 280 nm) and

265

the main ions [M+H]+ ion at m/z 291.3121. Compound 3 was likely procyanidin B3

266

based on the parent [M+H]+ ions at m/z 579.1520 [M+H]+ and the [M-C15H10O7]+ ion

267

at m/z 462.3567 and the [C15H10O7+H]+ ion at m/z 303.0512. Compounds 4 and 5

268

were two isomers from the parent [M+H]+ ion at m/z 465.3610 that produced two

269

main ions fragmentations at 303.0501 [C15H10O7+H]+ and 163.1221 [M-C15H10O7]+.

270

Compounds 4 and 5 were identified as hyperoside and isoquercitrin after comparing

271

with the standards, respectively. Three isomers of quercetin glucoside (compounds 6,

272

7, and 8) were characterized by the parent ion m/z 435.0901, which produced two

273

main ions at 303.0501 and 133.2510. Wang et al. (2017)25 reported that compounds 6,

274

7

275

quercetin-3-O-α-L-arabinopyranoside and avicularin. Compound 9 can be identified

276

as quercitrin based on the parent ion m/z 449.0984 [M+H]+ that produced two main

277

ions at 303.0510 [C15H10O7+H]+ and 146.1037 [M-C15H10O7]+. Compound 10 was

278

likely kaempferol-3-arabofuranoside based on the parent ion m/z 419.0984 [M+H]+,

279

which produced the two main ions at 287.0563 [C15H10O6+H]+ and 133.2036

280

[M-C15H10O6]+. Compound 11 was likely a flavonoid compound based on the UV/Vis

281

absorption spectrum (210 nm, 284 nm and 355 nm) and the parent ion m/z 573.1625

and

8

were

likely

quercetin-3-O-β-D-xylopyranoside,

13



Page 14 of 30

282

[M+H]+, which produced the two main ions at 315.0721 and 259.0975. Compound 12

283

was likely quercetin based on the main ion at 303.0501 [C15H10O7+H]+.

284

Structure-Activity

285

α-Glucosidase

286

Phenolics and flavonoid compounds are known secondary metabolites of plant

287

matrices and have shown well pharmacological effects, such as anti-diabetic,

288

anti-oxidant, anti-inflammatory, and anticancer activities

289

compounds structure according to substituent groups largely leads to their differences

290

in bio-activities 30. The chemical structures of the identified potential α-glucosidase

291

inhibitors from the GLT extracts are shown in Fig. 5. As shown in Table 1, three

292

representative compounds, quercetin with an AD at 18.86%, procyanidin B3 at 8.56%

293

and avicularin at 7.47%, exhibited notably stronger affinity to α-glucosidase than the

294

other selected compounds. The IC50 values of the compound were: quercetin (IC50 =

295

4.51 ± 0.71 µg/mL), L-epicatechin (IC50 = 45.56 ± 0.11 µg/mL), procyanidins B3

296

(IC50 = 28.67 ± 5.81 µg/mL), hyperoside (IC50 = 55.31 ± 4.17 µg/mL), isoquercitrin

297

(IC50 = 42.94 ± 3.11 µg/mL), quercetin-3-O-α-L-arabinopyranoside (IC50 = 41.81 ±

298

5.12 µg/mL), quercetin-3-O-β-D-xylopyranoside (IC50 = 44.78 ± 2.62 µg/mL),

299

avicularin (IC50 = 21.84 ± 3.82 µg/mL), quercitrin (IC50 = 43.27 ± 2.17 µg/mL),

300

kaempferol-3-O-arabofuranoside (IC50 = 58.19 ± 3.32 µg/mL) and gallic acid (IC50 =

301

348.63 ± 2.93 µg/mL) (Fig. 6). Quercetin and procyanidins B3 showed the highest

302

inhibitory effects on α-glucosidase. By contrast, gallic acid had the lowest inhibitory

303

capacity. In previous studies, the structure-activity relationships have been known to

Relationships

between

Phenolics/Flavonoids

14


and

26-29

. The diversity of

Page 15 of 30


304

involve the following characteristics: (1) flavonoid hydroxylation improved their

305

inhibitory effects on α-glucosidase; (2) flavonoid glycosylation reduced their

306

inhibitory activity; and (3) procyanidins exhibited strong inhibitory activity against

307

α-glucosidase 26,27. Because quercetin possessed a high number of hydroxy groups,

308

especially 3’- and 4’-dihydroxyl groups in the B-ring and 3-OH in the C-ring, which

309

are important structures that contribute toward inhibiting α-glucosidase activity 31.

310

Wang et al. (2010) also confirmed that the glycosylation of quercetin significantly

311

reduced the inhibitory activity against α-glucosidase

312

replaced by different glycosides, the ability for flavonol glycosides to inhibit

313

α-glucosidase activity was significantly lower than that of quercetin. Procyanidin B3

314

(catechin dimer) differed from other natural polyphenols due to their polymeric nature

315

26,27

316

inhibitory activity against carbohydrate hydrolysis enzymes and could be used as lead

317

compounds for the development of antidiabetic therapeutics

318

showed the lowest affinity degree to α-glucosidase, which was consistent with the

319

reports described by Xiao et al. (2013) 26. This may have been due to the number and

320

structure of OH groups in the phenolic compounds. In this study, quercetin and

321

procyanidin B3 were primarily responsible for the anti-hyperglycemic effects of GLT,

322

which also further verified the established screening method. Additionally, the

323

structure-activity relationships revealed that the hydroxylation of flavonoids improved

324

the α-glucosidase inhibitory effect, and the glycosylation of hyroxyl groups on

325

flavonoids

21

. Because 3-hydroxy was

. Hakamata et al. (2006) reported that procyanidin isomers showed stronger

decreased

the

inhibitory

effect.

15


32

. However, gallic acid

Consequently,

the


326

BAUF-HPLC-ESI-TOF/MS method could be used as a valuable high-throughput

327

screening platform for the rapid screening of natural α-glucosidase inhibitors from

328

complex medicinal plant extracts.

329

AUTHOR INFORMATION

330

Corresponding author:

331

20-39380663; E-mail: [email protected]

332

Funding

333

The work was supported by the Science and Technology Project of Guangdong

334

Province, China (2016A020210011 and 2017B020207003) and the Special fund for

335

Agricultural Science and Technology Research Project of Jiangmen City, China

336

(20150160008347)

337

Notes

338

The authors declare no competing financial interest

339

ABBREVIATIONS USED

340

GLT, guava leaves tea; DM, dried mass; PCA, principal components analysis; IDF,

341

International Diabetes Federation; AACE , American Association of Clinical

342

Endocrinologists; BAUF-HPLC-TOF/MS, bio-affinity ultrafiltration and high

343

performance liquid chromatography time-of-flight coupled with mass spectrometry;

344

AD, Affinity degree

345

References

346

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*

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16


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(31) Xu, H. Inhibition kinetics of flavonoids on yeast alpha-glucosidase merged with docking simulations. Protein Peptide Lett. 2010, 17, 1270–1279. (32) Hakamata, W.; Nakanishi, I.; Masuda, Y.; Shimizu, T.; Higuchi, H.; Nakamura, Y.;

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Saito, S.; Urano, S.; Oku, T.; Ozawa, T.; Ikota, N.; Miyata, N.; Okuda, H.;

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Fukuhara, K. Planar catechin analogues with alkyl side chains: Apotent

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6524–6525.

451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 20


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468

Figure captions

469

Figure 1. The half-maximal inhibitory concentrations (IC50) of the guava leaves tea

470 471 472

extract (A) and the positive control acarbose (B) on α-glucosidase in vitro Figure 2. A schematic diagram of BAUF-HPLC-ESI-TOF/MS assay to screen for potential α-glucosidase inhibitors from guava leaves tea extract

473

Figure 3. The effect of α-glucosidase concentration (A) and the size of membrane

474

filter (B) on its PCA scores plot before and after its reaction with α-glucosidase

475

by centrifugal ultrafiltration (Samples-1U indicates sample reaction with 1 U/mL

476

α-glucosidase through 30 kDa the membrane filter, Blanks-1U indicates sample

477

reaction with the inactivated 1 U/mL α-glucosidase by 30 kDa the membrane

478

filter; Samples-5U indicates sample reaction with 5 U/mL α-glucosidase through

479

30 kDa the membrane filter, Blanks-5U indicates sample reaction with the

480

inactivated 5 U/mL α-glucosidase by 30 kDa the membrane filter; Samples-10U

481

indicates sample reaction with 10 U/mL α-glucosidase by 30 kDa the membrane

482

filter, Blanks-10U indicates sample reaction with the inactivated 10 U/mL

483

α-glucosidase by 30 kDa the membrane filter; Samples-10K indicates sample

484

reaction with 10 U/mL α-glucosidase by 10 kDa the membrane filter,

485

Samples-30K indicates sample reaction with 10 U/mL α-glucosidase by 30 kDa

486

the membrane filter, Samples-50K indicates sample reaction with 10 U/mL

487

α-glucosidase by 50 kDa the membrane filter, Blanks-10K indicates sample

488

reaction with 10 U/mL inactivated α-glucosidase by 10 kDa the membrane filter,

489

Blanks-30K indicates sample reaction with 10 U/mL inactivated α-glucosidase

490

by 30 kDa the membrane filter, Blanks-50K indicates sample reaction with 10

491

U/mL inactivated α-glucosidase by 50 kDa the membrane filter; 1-3 indicate

492

three parallel tests)

493

Figure 4. The HPLC chromatograms (280 nm) of the chemical constituents in the

494

guava leaves tea extract obtained by ultrafiltration. The blue solid line represents

495

HPLC profiles of guava leaves tea extract without ultrafiltration; the red and

496

black lines represent HPLC profiles of guava leaves tea extract with activated

497

and inactivated α-glucosidase by ultrafiltration, respectively. IS, internal 21



498 499 500

standards (p-hydroxycinnamic acid) Figure 5. The chemical structures of the selected potential α-glucosidase inhibitors in the guava leaves tea extracts

501

Figure 6. The half-maximal inhibitory concentrations (IC50) of the screened

502

individual α-glucosidase inhibitor from guava leaves tea extract. Different letters

503

(a-f) means statistically significant differences at p < 0.05.

504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 22


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522 523 524 Peak No.

Retention

Table 1 Identification of potential α-glucosidase inhibitors from GLT extract using HPLC-ESI-TOF/MS method and their bio-affinity degrees. Notes: “ND” Not identified; SD, Standard deviation; AD, Affinity degree λmax (nm)

time (min)

Molecular ion

MS (m/z)

Mw

Formula

Compounds

(m/z)

Affinity degree

Reference

(AD, ± SD %) +

1

3.73

215, 270

169.2101[M+H]

2

14.05

256, 280

291.0876[M+H]+ +

171.1221 291.0876

170

C7H6O5

Gallic acid

1.07 ± 0.03

Standard

290

C15H14O6

L-epicatechin

4.75 ± 0.14

Standard

578

C30H26O12

Procyanidin B3

8.54 ± 0.15

Standard

3

17.31

254,354

579.1520 [M+H]

579.1520, 462.3567,

4

17.89

256, 351

465.3610 [M+H]+

303.0501, 163.1221

465

C21H20O12

Hyperoside

5.32 ± 0.02

Standard

256, 351

+

303.0501, 163.1221

465

C21H20O12

Isoquercitrin

4.96 ± 0.11

Standard

+

301.0512

5

18.09

465.3610 [M+H]

6

19.78

254, 359

435.0901 [M+H]

303.0490, 133,1412

434

C20H18O11

Quercetin-3-O-β-D-xylopyranoside

5.18 ± 0.08

Standard

7

20.41

254, 356

435.0930 [M+H]+

303.0509,133.2510

434

C20H18O11

Quercetin-3-O-α-L-arabinopyranoside

6.56 ± 0.13

Standard

253, 357

+

303.0511, 133.1526

434

C20H18O11

Avicularin

7.47 ± 0.09

Standard

+

449.1194, 303.0510,

449

C21H20O11

Quercitrin

2.31 ± 0.11

Standard

418

C20H18O10

Kaempferol-3-arabofuranoside

3.93 ± 0.07

Standard

572

C28H28O13

ND

3.15 ± 0.02

Unknown

302

C15H10O7

Quercetin

18.86 ± 0.28

Standard

8 9

21.29 22.05

262,391

435.0940 [M+H] 449.1194[M+H]

146.1037 10

25.76

257, 363

419.0984 [M+H]+

419.0984, 287.0563,

11

29.17

210, 284,

573.1624 [M+H]+

573.1624, 315.0721,

133.2036

355 12

32.78

254, 364

259.0975 +

303.0516 [M+H]

303.0516

525 526

23



A

B


Page 24 of 30

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Figure 2. A schematic diagram of BAUF-HPLC-ESI-TOF/MS assay to screen for potential α-glucosidase inhibitors from guava leaves tea extract 399x249mm (300 x 300 DPI)



Figure 3. The effect of α-glucosidase concentration (A) and the size of membrane filter (B) on its PCA scores plot before and after its reaction with α-glucosidase by centrifugal ultrafiltration (Samples-1U indicates sample reaction with 1 U/mL α-glucosidase through 30 kDa the membrane filter, Blanks-1U indicates sample reaction with the inactivated 1 U/mL α-glucosidase by 30 kDa the membrane filter; Samples-5U indicates sample reaction with 5 U/mL α-glucosidase through 30 kDa the membrane filter, Blanks-5U indicates sample reaction with the inactivated 5 U/mL α-glucosidase by 30 kDa the membrane filter; Samples-10U indicates sample reaction with 10 U/mL α-glucosidase by 30 kDa the membrane filter, Blanks-10U indicates sample reaction with the inactivated 10 U/mL α-glucosidase by 30 kDa the membrane filter; Samples-10K indicates sample reaction with 10 U/mL α-glucosidase by 10 kDa the membrane filter, Samples-30K indicates sample reaction with 10 U/mL α-glucosidase by 30 kDa the membrane filter, Samples-50K indicates sample reaction with 10 U/mL α-glucosidase by 50 kDa the membrane filter, Blanks-10K indicates sample reaction with 10 U/mL inactivated α-glucosidase by 10 kDa the membrane filter, Blanks-30K indicates sample reaction with 10 U/mL inactivated α-glucosidase by 30 kDa the membrane filter, Blanks-50K indicates sample reaction


Page 26 of 30

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with 10 U/mL inactivated α-glucosidase by 50 kDa the membrane filter; 1-3 indicate three parallel tests) 399x629mm (300 x 300 DPI)




Figure 4

Page 28 of 30

Page 29 of 30


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Page 30 of 30

Page 31 of 30


Graphical abstract 254x190mm (300 x 300 DPI)


Quickly Screening for Potential Î±-Glucosidase Inhibitors from Guava

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