Inhibitory Activities of Caffeoylquinic Acid Derivatives from Ilex

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Inhibitory Activities of Caffeoylquinic Acid Derivatives from Ilex Kudingcha C.J. Tseng on #-Glucosidase from Saccharomy cerevisiae Donglan Xu, Qingchuan Wang, Wenqin Zhang, Bing Hu, Li Zhou, Xiaoxiong Zeng, and Yi Sun J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 25 Mar 2015 Downloaded from http://pubs.acs.org on March 25, 2015

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Journal of Agricultural and Food Chemistry

Inhibitory Activities of Caffeoylquinic Acid Derivatives from Ilex Kudingcha C.J. Tseng on α-Glucosidase from Saccharomy cerevisiae

Donglan Xu, Qingchuan Wang, Wenqin Zhang, Bing Hu, Li Zhou, Xiaoxiong Zeng* and Yi Sun*

College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, P.R. China

*

Corresponding author. Fax: +86 25 84396791; E-mail address: [email protected] (X Zeng), [email protected] (Y Sun). 1

ACS Paragon Plus Environment


1

ABSTRACT

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Polyphenols and caffeoylquinic acid (CQA) derivatives (3-CQA, 4-CQA, 5-CQA,

3

3,4-diCQA, 3,5-diCQA and 4,5-diCQA) were prepared from Ilex kudingcha C.J.

4

Tseng and their effects and mechanisms on the activities of α-glucosidase from

5

Saccharomy cerevisiae were investigated in the present study. As results, the IC50

6

values for CQA derivatives were 0.16-0.39 mg/mL, and the inhibition mode of CQA

7

derivatives was non-competitive. Based on the data of fluorescence spectroscopy and

8

circular dichroism spectroscopy, the binding constants and number of binding sites

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were calculated to be 106-108 M-1 and 1.42-1.87, respectively. CQA derivatives could

10

bind to the enzyme mainly through hydrophobic interaction, altering the

11

microenvironment and molecular conformation of the enzyme, thus decreasing the

12

catalytic activity. To our knowledge, this is the first report on α-glucosidase

13

inhibitory mechanism by CQA derivatives from I. kudingcha and the findings

14

suggest a potential use of kudingcha as functional foods for prevention and treatment

15

of diabetes and related symptoms.

16

Keywords: Kudingcha; α-Glucosidase; Caffeoylquinic acid; Inhibitory activity;

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Interaction

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INTRODUCTION

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Diabetes mellitus, a major health problem associated with a number of complications

20

such as diabetes-associated stroke, obesity, cancer and cardiovascular diseases, is a

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metabolic disorder characterized by hyperglycaemia and glucose intolerance.1-3 It

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has been reported that the postprandial state is an important contributing factor for

23

the development of atherosclerosis, and high postprandial plasma glucose

24

concentrations are associated with an increased risk of the development of type 2

25

diabetes and metabolic syndrome.4-6 Controlling the postprandial hyperglycemia,

26

therefore, is an effective way to mitigate the illnesses and treat diabetes. One

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approach for the control of postprandial hyperglycemia is to retard or suppress the

28

absorption of glucose in the intestine. While in mammals, the dietary carbohydrates

29

are hydrolyzed by enzymes such as α-amylase and α-glucosidase (α-D-glucoside

30

glucohydrolase, EC 3.2.1.20). α-Glucosidase, located at the brush-border surface

31

membrane of intestinal cells, is the key enzyme catalyzing the final step in the

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digestive process of carbohydrates that liberate glucose. Hence, α-glucosidase

33

inhibitors can delay carbohydrate digestion and reduce the rate of glucose absorption,

34

resulting in reduced postprandial plasma glucose levels and suppressed diabetes.6,7

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For example, acarbose and voglibose, two of the clinically approved drugs for the

36

treatment of type 2 diabetes, have been demonstrated to act by this mechanism.8,9

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However, the usage of these drugs may be associated with some undesirable side

38

effects, the most commonly observed being weight gain, hyperglycemia and

39

gastrointestinal disturbances.10,11 Thus, recently more attentions have been paid on

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40

other effective and safe α-glucosidase inhibitors from natural ingredients or herbal

41

extracts, which have been used medicinally for many years for the prevention of

42

metabolic disorders such as diabetes and obesity.12-16

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Kudingcha, a bitter tea of Chinese origin, has been used as folk medicine for

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more than 2000 years. The main species for the production of kudingcha in China

45

are Ilex kudingcha, I. latifolia and I. cornuta,17,18 which belong to the same genus as

46

mate (I. paraguariensis).19 I. kudingcha is rich in triterpenoids, phenolic acids,

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flavonoids, essential oils and other active substances and shows obvious anti-oxidant,

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anti-inflammatory, anti-tumor, anti-microbial, hepatoprotective and hypoglycemic

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activities.18 In addition, the major phenolic compounds in leaves of I. kudingcha C.J.

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Tseng are reported to be caffeoylquinic acid (CQA) derivatives (Figure 1, IUPAC

51

numbering system),20 including 3-CQA, 4-CQA, 5-CQA, 3,4-diCQA, 3,5-diCQA

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and 4,5-diCQA, which account for 93.8% of total content of polyphenols.21 CQA

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derivatives have been reported to have various biological functions such as

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anti-oxidant and anti-diabetic activities, anti-obesity, cancer suppression and

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inhibition of α-glucosidase and tyrosinase.18,21-29 Furthermore, it has been reported

56

that regular consumption of coffee is associated with a lower risk of type 2 diabetes

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mellitus,30 and 5-CQA in coffee, may contribute to the beneficial effects of coffee on

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type 2 diabetes mellitus.31 However, the inhibitory effects of CQA derivatives from

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kudingcha on α-glucosidase have never been reported before. As tea polyphenolic

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compounds strongly interact with proteins,32 CQA derivatives from kudingcha may

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exhibit potential interactions with α-glucosidase. This should inevitably result in the

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change of enzyme molecular configuration and lead to the loss of catalytic activity,

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thereby reducing carbohydrate digestibility and protecting against diabetes and

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obesity. Recently, we have reported the potent antioxidant activity of kudingcha

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polyphenols and isolated CQA derivatives from the leaves of I. kudingcha C.J.

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Tseng.22,33-35 In this study, therefore, the inhibitory effects against α-glucosidase of

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six kinds of CQA derivatives (3-CQA, 4-CQA, 5-CQA, 3,4-diCQA, 3,5-diCQA and

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4,5-diCQA) present in kudingcha made from the leaves of I. kudingcha C.J. Tseng

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were investigated. Furthermore, the potential mechanisms of interactions between

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α-glucosidase and CQA derivatives were characterized by fluorescence spectroscopy

71

and circular dichroism (CD) spectroscopy.

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MATERIALS AND METHODS

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Materials and Reagents. Kudingcha made from the leaves of I. kudingcha C.J.

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Tseng was obtained from Hainan Yexian Bio-Science Technology Co., Ltd. (Hainan,

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China), and the sample was ground by using a domestic blender, stored in sealed

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polyethylene bags and kept in a refrigerator at -20 ℃ until use. α-Glucosidase from

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Saccharomy cerevisiae (dialysis 48 h before use), 5-CQA and 4-nitrophenyl

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α-D-glucopyranoside (≥ 99%) were purchased from Sigma-Aldrich Chemical Co.

79

(St. Louis, MO, USA). Standards of 3-CQA, 4-CQA, 3,4-diCQA, 3,5-diCQA and

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4,5-diCQA (> 95%) were prepared from kudingcha according to our reported

81

methods.22,34,35 The solvents used for chromatographic purpose were grade of high

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performance liquid chromatography (HPLC), and all other reagents were of

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analytical reagent grade. 5



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Determination of Total Polyphenols Content and CQA Derivatives. The total

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polyphenols content was determined by Folin-Ciocalteu method according to the

86

reported procedure.22 The contents of CQA derivatives were measured by HPLC

87

with Agilent HPLC series 1100 (Agilent, Santa Clara, CA, USA) equipped a model

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G1379A degasser, a model G1311A quatpump, a model G1316A column oven, a

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model G1315B diode-array detector (DAD) and Chemstation software. The

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separation was completed on a TSK gel ODS-80TsQA column (4.6 × 250 mm, 5 µm,

91

Tosoh Corp., Tokyo, Japan) with a gradient mobile phase consisted of ultra-pure

92

water (A), methanol (B) and 1.0% formic acid (C, v/v) at a flow rate of 0.5 mL/min.

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Elution was performed with a linear gradient as follows: 0-45 min, A from 60 to 35%,

94

B from 20 to 45%, C 20%. The temperature of column oven was set at 40 ℃, the

95

injection volume was 20 µL, and CQA derivatives were detected at 326 nm with a

96

DAD. The chromatographic peaks were identified by comparing the retention times

97

and UV spectra of standards of CQA derivatives.

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Preparation of CQA Derivatives. The preparation of CQA derivatives was

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performed according to the reported methods22,33-35 with some modifications. Briefly,

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the kudingcha powder was extracted with water (10 for the ratio of extraction solvent

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to material, v/w) for 30 min at 95 ℃. After extraction, the extract was centrifuged at

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5000 g for 10 min, and the resulting insoluble residue was treated twice as described

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above. The combined supernatants were concentrated by a rotary evaporator (Tokyo

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Rikakikai Co., Ltd., Tokyo, Japan) and lyophilized (Labconco, Kansas, MO, USA)

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to afford the crude extract. The crude extract was dissolved in deinoized water and

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applied to a column (5 × 30 cm) of HP-20 macroporous resin. Then, the column was

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washed with 3 times bed volume of distilled water and eluted with 70% ethanol

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solution, respectively. The collected fractions (10 mL/tube) were analyzed by HPLC

109

as described above, and the factions containing CQA derivatives were combined,

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concentrated and freeze-dried, affording kudingcha polyphenols. The CQA

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derivatives (3-CQA, 4-CQA, 5-CQA; 3,4-diCQA, 3,5-diCQA and 4,5-diCQA) were

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further isolated from the kudingcha polyphenols by HPLC with a semi-preparative

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HPLC column of YMC-Pack ODS-A (20 × 250 mm, 5 µm, YMC Co., Ltd., Kyoto,

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Japan) according to our reported methods.22,34,35 The desired fractions containing

115

3-CQA, 4-CQA, 5-CQA; 3,4-diCQA, 3,5-diCQA and 4,5-diCQA were combined,

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concentrated and lyophilized, respectively. The structures of CQA derivatives were

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confirmed by electrospray ionization-mass spectrometry (ESI-MS), HPLC and

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nuclear magnetic resonance (NMR) spectrometry.

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Enzyme Inhibitory Activities Assay. The inhibitory activity against α-glucosidase

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was measured according to the reported method26 with some modifications. Briefly,

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the reaction mixture contained 8 µL of test sample with different concentration, 30

122

µL of 2.5 mM 4-nitrophenyl α-D-glucopyranoside as substrate, 20 µL of enzyme

123

solution (0.2 U/mL) and 102 µL of 0.1 M phosphate buffer saline (pH 6.9). The

124

reaction mixture was incubated at 37 ℃ for 15 min. The reaction was then

125

terminated by the addition of 80 µL of 0.2 M Na2CO3 solution. The increase in

126

absorbance

127

α-D-glucopyranoside was monitored with a microplate reader (BioTek Instruments,

at

405

nm

due

to

enzymatic

hydrolysis

7


of

4-nitrophenyl


128

Inc., Winooski, VT, USA). All samples were analyzed in triplicate. The inhibition

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percentage was calculated using the following formula:

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Inhibition rate (%) = [((Ac+ - Ac-) – (As - Ab)) / (Ac+ - Ac-)] × 100

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Where Ac+, Ac-, As and Ab are defined as the absorbances of 100% enzyme activity

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(only the solvent with the enzyme), 0% enzyme activity (only the solvent without

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enzyme), the test sample (with the enzyme) and a blank (only the sample),

134

respectively. The effective concentration that could inhibit 50% of α-glucosidase

135

activity is defined as IC50.

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Assay of Inhibitory Pattern of CQA Derivatives on α-Glucosidase Activity. The

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effects of CQA derivatives on α-glucosidase activity were investigated with

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increasing concentrations of enzyme substrate (4-nitrophenyl α-D-glucopyranoside,

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from 3.0 × 10-4 to 1.5 × 10-3 M) in the presence or absence of CQA derivatives. Then,

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the inhibition type was determined by Lineweaver-Burk plot analysis according to

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Michaelis-Menten kinetics.

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Fluorescence Measurement. General Procedure. A quantitative analysis of the

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potential interaction between CQA derivative and α-glucosidase was performed by

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fluorometric titration as follows: 3.0 mL solution containing appropriate

145

concentration of α-glucosidase was titrated by the addition of different concentration

146

of 5-CQA, 3,4-diCQA, 3,5-diCQA or 4,5-diCQA solution. All samples were

147

incubated at fixed temperature (293 or 310 K) for 0.5 h. Then, the fluorescence

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spectra were recorded with F-7000 Fluorescence Spectrophotometer (Hitachi

149

High-Technologies Corp., Tokyo, Japan) by using a 1.0 cm quartz cell and at

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excitation wavelength of 280 nm, and the emission spectra were recorded from 300

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to 450 nm. Spectral resolution for both excitation and emission was 5 nm. The

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three-dimensional (3D) fluorescence spectra of enzyme in absence and presence of

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CQA derivatives were obtained by recording the excitation and the emission spectra

154

in the range of 200-600 nm with an interval of 5 nm, respectively.

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Analysis of Fluorescence Quenching Constant and Thermodynamic Parameters.

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The fluorescence quenching data were analyzed via the modified Stern-Volmer

157

equation:36

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lg[( F0 − F ) / F ] = lg K a + n lg[Q ]

159

Here, F0 and F are the fluorescence intensities of the enzyme in the absence and

160

presence of quencher, [Q] is the quencher concentration, Ka is the binding constant

161

and n is the number of the binding sites per enzyme. The values of n and Ka can be

162

calculated by the slope and intercept with the plots of lg (F0-F)/F against lg [Q].37

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Considering that the enthalpy change (∆H) does not vary significantly over the

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temperature range, it can be considered as a constant. ∆H and entropy change (∆S)

165

can be calculated using the Van’t Hoff equation:

166

ln K = -

∆H ∆S + RT R

167

ln (K 2 / K1 ) = (1 / T1 − 1/ T2 )∆H / R

168

∆G = ∆H − T∆S

169

where R is the gas constant and T is the experimental temperature, K is the binding

170

constant at the corresponding T, and ∆G is the free energy. Then ∆H, ∆S and ∆G of

171

interaction can be calculated from the equations above.

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CD Measurement. The CD spectra of α-glucosidase and its complexes with CQA

173

derivatives were recorded with use of a Jasco-810 spectrophotometer (JASCO Corp.,

174

Tokyo, Japan) at room temperature. The spectra were measured in far-UV region

175

(190-250 nm) with a path length of 1.0 mm, a scan speed of 50 nm/min and a

176

response time of 4 s. In addition, three scans were accumulated for each spectrum.

177

All working solutions were prepared with 20 mM phosphate buffer (pH 6.9). The

178

enzyme concentration was set at 0.2 mg/mL (3 × 10-6 M) and the complexes were

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prepared by mixing the enzyme with CQA derivative (5-CQA or 3,5-diCQA) at a

180

molar ratio of 1:0.5, 1:1 and 1:2 (enzyme to CQA derivative). The resulting CD data

181

were analyzed by a curvefitting programme software CDPro using CONTIN,

182

SELCON and CDSSTR methods, as described by Sreerama and Woody38 to obtain

183

the secondary structural contents of α-glucosidase.

184

Statistical Analysis. Data were expressed as mean ± standard deviation (SD) of

185

triplicates. The IC50 value was calculated from linear regression analysis. Any

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significant difference was determined by one-way analysis of variance (ANOVA)

187

followed by t test for multiple comparisons at P < 0.05 level (SPSS Statistics 20.0,

188

IBM, New York, USA).

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RESULTS

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Inhibitory Activity of Kudingcha Polyphenols against α-Glucosidase. The

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effect of kudingcha polyphenols, partially purified polyphenols from the crude

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extract of I. Kudingcha C.J. Tseng by chromatography of HP-20 macroporous resin,

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on α-glucosidase from S. cerevisiae was investigated. As shown in Figure 2A, the 10


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kudingcha polyphenols exhibited strong inhibitory activity against α-glucosidase.

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The IC50 value was determined to be 0.42 mg/mL. To further explore the inhibition

196

type of kudingcha polyphenols against α-glucosidase, the kinetic reaction of

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α-glucosidase was investigated with different concentrations of enzyme. As shown

198

in Figure 2B, it was found that the line obtained from the kinetic reaction plot was

199

passed through the origin point (0, 0) of the coordinate, indicating that the inhibition

200

type of kudingcha polyphenols on α-glucosidase was reversible inhibition. As for

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irreversible inhibition, the inhibitor binds with enzyme covalently and forms a stable

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complex, making the enzyme inactivated. The enzyme will exhibit its activity only

203

when a certain amount of enzyme is added to the reaction system. Therefore, the plot

204

of enzyme concentration versus reaction velocity does not pass through the origin of

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the coordinate. While for reversible inhibition, which is characterized by the

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existence of equilibrium between enzyme and inhibitor, the plot may pass through

207

the origin.39,40 It is obvious that kudingcha polyphenols are reversible inhibitors of

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α-glucosidase.

209

As shown in Figure 3A, the kudingcha polyphenols contained 3-CQA (3.2 ±

210

0.15%), 4-CQA (2.1 ± 0.09%), 5-CQA (8.4 ± 0.13%), 3,4-diCQA (11.8 ± 0.25%),

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3,5-diCQA (23.2 ± 1.61%) and 4,5-diCQA (25.6 ± 1.52%). Since the kudingcha

212

polyphenols showed potent α-glucosidase inhibitory activity as mentioned above,

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CQA derivatives, the main polyphenols in I. Kudingcha C.J. Tseng, were isolated by

214

semi-preparative HPLC from kudingcha polyphenols (78.4% for total polyphenol

215

content determined by the Folin-Ciocalteu method). The purified CQA derivatives,

11



216

with purity > 95% (Figure 3B), were confirmed to be 3-CQA, 4-CQA, 5-CQA,

217

3,4-diCQA, 3,5-diCQA and 4,5-diCQA by HPLC, ESI-MS and 1H NMR through

218

comparison with the reported data.22,34,35,41-43

219

Inhibitory Activities of CQA Derivatives against α-Glucosidase. The resulting

220

CQA derivatives from I. Kudingcha C.J. Tseng were further examined their

221

inhibition on α-glucosidase by evaluating their IC50 values. The results revealed that

222

all the CQA derivatives had

223

dose-dependent manner (Figure 4A). At a concentration of 1.0 mg/mL,

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α-glucosidase was almost inhibited (98%) by CQA derivatives. The IC50 values for

225

3-CQA, 4-CQA, 5-CQA, 3,4-diCQA, 3,5-diCQA and 4,5-diCQA were determined to

226

be 0.39, 0.34, 0.30, 0.27, 0.27 and 0.16 mg/mL, respectively. Notably, diCQA

227

derivatives with double caffeoyl moieties exhibited higher inhibition activities than

228

CQA derivatives with a single caffeoyl moiety, indicating that the addition of

229

caffeoyl moiety significantly increased the inhibitory ability.

inhibitory activity against α-glucosidase in a

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In order to characterize the inhibition pattern of α-glucosidase by CQA

231

derivatives, the enzyme reactions were examined with increasing concentrations of

232

substrate without or with a fix concentration of inhibitor. As an example, Figure 4B

233

shows the Lineweaver-Burk double reciprocal plots of α-glucosidase kinetics with

234

4,5-diCQA as the inhibitor. It is obvious that the Vmax for α-glucosidase decreased

235

while the Km (0.68 mM) remained unchanged in the presence of 4,5-diCQA, and the

236

other CQA derivatives showed similar trends. Thus, the inhibition of α-glucosidase

237

by CQA derivatives was non-competitive, in which the inhibitor and the substrate

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should bind simultaneously with the enzyme.

239

Fluorescence Spectra. Fluorescence spectroscopy and CD spectroscopy were

240

used to explore the interactions between α-glucosidase and CQA derivative. As the

241

inhibitory activity of diCQA derivative was higher than CQA derivative with a single

242

caffeoyl moiety, 5-CQA, 3,4-diCQA, 3,5-diCQA and 4,5-diCQA were used for

243

further studies of interaction mechanisms.

244

Fluorescence spectroscopy is used to measure the interaction between a

245

biomacromolecule and a small molecule ligand.44 In a protein molecule, Trp has the

246

most powerful activity to emit fluorescence and the intrinsic fluorescent intensity

247

may change depending on the impact of the interaction between the protein and

248

another molecule.36 It contains 18 Trp residues in α-glucosidase (obtained from the

249

Brookhaven Protein Data Bank, PDB ID: 1 VAD). Therefore, it is possible to use

250

quenching of the intrinsic Trp fluorescence in enzyme to study the interactions

251

between CQA derivatives and α-glucosidase.

252

Under our measurement condition (i.e., from 300 to 450 nm), all fluorescence

253

emissions from CQA derivatives, buffer and other reagents were so weak that their

254

impact could be ignored. As shown in Figure 5, the fluorescence intensities of

255

α-glucosidase decreased gradually with the addition of CQA derivatives at 37 ℃,

256

and the maximal absorption peak (near 332 nm) was red shifted, thus the CQA

257

derivatives interacted with the enzyme (data at 20 ℃ not shown). As results, the

258

maximum emission wavelength was shifted from 332 to 360 nm for the addition of

259

5-CQA, 332 to 352 nm for 3,4-diCQA, 331 to 356 nm for 3,5-diCQA and 332 to 356

13



260

nm for 4,5-diCQA, respectively. These changes suggested that the interactions

261

between the CQA derivatives and α-glucosidase led to a polarity variation for Trp

262

residues in enzyme and made the microenvironment change from hydrophilic to

263

hydrophobic, thus resulting in a more exposure of Trp residues and unfolding of

264

protein structure.45,46

265

The 3D fluorescence spectra have been applied to investigate the characteristic

266

conformational change of protein in recent years.47 The 3D fluorescence contour

267

maps of α-glucosidase and the mixtures with four CQA derivatives in different

268

concentration (0.01 and 0.025 mg/mL) are shown in Figure 6. The 3D spectra

269

showed two distinct peaks in enzyme marked 1 (λex / λem: 280 nm / 330 nm) and 2

270

(λex / λem: 230 nm / 330 nm). Peak 1 corresponded to the characteristics of Trp and

271

Tyr residues and peak 2 provided spectral properties of the polypeptide backbone

272

which occur mainly due to the presence of the π–π* and n–π* transitions

273

respectively.48,49 The two peaks were quenched by the addition of CQA derivative in

274

a dose-dependent manner. The corresponding parameters (Table 1) indicated that the

275

fluorescence intensity decreased with addition of CQA derivatives, following a red

276

shift of 10 nm in each of peaks 1 and 2. It was also found that the quenching effect

277

showed a sequence as diCQA > 5-CQA, which was in accordance with the results of

278

fluorescence experiments. These effects might be due to hydrophobic interactions

279

between the aromatic ring and the hydrophobic moieties present, therefore inducing

280

conformational changes in α-glucosidase molecule.14

281

Fluorescence Binding Constant and Binding Site. To obtain the binding

14


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constant (Ka) and the number of binding site per enzyme (n) between CQA

283

derivative and α-glucosidase, the fluorescence quenching data were analyzed

284

according to the modified Stern–Volmer equation by using the Scatchard plots

285

(Figure 5). The results are summarized as shown in Table 2. The results

286

demonstrated that Ka was in order of diCQA > 5-CQA, indicating that introduction

287

of the second caffeoyl moiety into CQA to from diCQA enhanced the binding

288

affinity to the enzyme. As compared with tea polyphenol–protein interactions with

289

Ka from 1.0 × 104 to 1.0 × 105 M-1,50-52 the Ka values (106 to 108 M-1) for interactions

290

of CQA derivatives and α-glucosidase were quite high, indicating comparatively

291

strong ligand–protein interactions.

292

Thermodynamic Parameters and Binding Mode. The interactions between

293

phenolic compounds and biomolecules may involve electrostatic interactions, van

294

der Waals interactions, hydrophobic forces, hydrogen bonds and so on. According to

295

∆H and ∆S data, the model of interaction between quencher and a protein molecule

296

can be concluded.53 More specifically, if ∆H > 0 and ∆S > 0, the main force would

297

be hydrophobic force; if ∆H < 0 and ∆S > 0, it would be electrostatic force; if ∆H
3,5-diCQA ≈ 3,4-diCQA > 5-CQA >

336

4-CQA > 3-CQA by comparing their IC50 values. The inhibitory activity of diCQA

337

was higher than that of CQA derivative with a single caffeoyl moiety, indicating that

338

the inhibitory activity of CQA derivative increases with increasing the number of

339

caffeoyl moieties. In addition, the inhibitory activity was affected by the substitution

340

position of the caffeoyl moiety on quinic acid. The substitution at the 5-position

341

exhibited relatively higher inhibitory activity than that at the 4-position, but it was

342

relatively higher than that at the 3-position (p < 0.05). As for the three diCQA

343

derivatives, 3,4-diCQA showed equal inhibitory activity to 3,5-diCQA (p > 0.05),

344

but it was weaker than 4,5-diCQA (p < 0.05). The differences in the α-glucosidase

345

inhibitory activities of CQA derivatives may be due to their differences in structures,

346

particularly the distribution of caffeoyl group and its position in the quinic acid

347

aromatic ring.26 Such results are in agreement with the conclusions of previous

17



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structure-activity relationship investigations by other researchers.16,23,57-59 For

349

example, the α-glucosidase inhibitory activities of 3,4-diCQA, 3,5-diCQA and

350

4,5-diCQA, separated from flower buds of Tussilago farfara L., were higher than

351

that of 5-CQA.57 For porcine pancreas α-amylase isozyme I, diCQA derivative

352

exhibited higher inhibitory activity than mono-CQA derivatives and feruloylquinic

353

acids.58

354

3,4-di-O-caffeoylquinate (3,4-diCQM) and 3,5-diCQM were isolated from Lonicera

355

fulvotomentosa HSU et S. C. CHENG and their interactions with bovine serum

356

albumin (BSA) were determined, their binding constants with BSA ranked in the

357

following order: 3,4-diCQM > 3,5-diCQM≈3,4-diCQA > 3,5-diCQA > 5-CQA

358

(IUPAC numbering system: 4,5-diCQM > 3,5-diCQM≈4,5-diCQA > 3,5-diCQA >

359

5-CQA).59 It has also been reported that diCQA derivatives with double caffeoyl

360

moieties had higher antioxidant capacity as compared to CQA derivative with a

361

single caffeoyl moiety, indicating the addition of caffeoyl moiety significantly

362

increased the antioxidative ability.23 Furthermore, the inhibitory types of 3-CQA,

363

4-CQA, 5-CQA, 3,4-diCQA, 3,5-diCQA and 4,5-diCQA from I. Kudingcha C.J.

364

Tseng for α-glucosidase were found to be non-competitive in the present study. This

365

is the first report on the inhibitory kinetics of the enzyme of six CQA derivatives

366

from kudingcha although the α-glucosidase inhibitory activities of several CQA

367

derivatives from other herbs were investigated before.

Five

CQA

derivatives

(5-CQA,

3,4-diCQA,

3,5-diCQA,

methyl

368

Some polyphenols have a strong ability to interact with digestive enzymes on the

369

basis of interactions between polyphenols and the protein, which reduces food

18


Page 19 of 42


370

digestibility.60,61 Thus, we explored the interactions between the CQA derivative and

371

α-glucosidase by fluorescence spectroscopy and CD spectroscopy for the first time.

372

As for fluorescence spectra, the decrease of fluorescence intensities and a red shift of

373

maximal absorption peak were observed, indicating a conformational change in

374

α-glucosidase. From the fluorescence analysis, the CQA derivative-α-glucosidase

375

binding was achieved by hydrophobic interactions of the interior hydrophobic

376

groups of enzyme and then might be stabilized by hydrogen bonding between polar

377

groups (-OH, -SH and -NH group) of the enzyme with the -OH groups of CQA

378

derivative. It has been reported that the integy moment of hydrophobicity descriptors

379

(vsurf_ID4 and vsurf_ID7) in 5-CQA derivatives were contributed to the inhibitory

380

activity of α-glucosidase from Bacillus stearothermophilus or S. cerevisiae. And the

381

requirement of the hydrophilic properties on the van der Waals surface of the

382

molecules by properly aligned polar and aromatic/hydrophobic regions for all highly

383

active and less active compounds was confirmed by pharmacophore analysis.28,62

384

The binding abilities of CQA derivatives were found to be quite high with a binding

385

constant from 106 to 108 M-1, and the order were diCQA derivative > CQA derivative

386

with a single caffeoyl moiety, which is in accordance with the results of enzyme

387

inhibition. Interactions between small molecules and enzymes may alter the

388

conformation of the enzyme.63,64 In our present study, the changes in secondary

389

structures of α-glucosidase with addition of 5-CQA or 3,5-diCQA were demonstrated

390

by using CD spectroscopy. Thus, CQA derivatives may have a binding effect on

391

α-glucosidase, changing the polarity and molecule conformation of enzyme,

19



392

resulting in partial loss of the enzyme activity. These structural changes of

393

α-glucosidase finally caused the inhibition of enzyme activity.

394

In conclusion, six kinds of CQA derivatives, 3-CQA, 4-CQA, 5-CQA,

395

3,4-diCQA, 3,5-diCQA and 4,5-diCQA separated from I. Kudingcha C.J. Tseng,

396

were demonstrated to be effective non-competitive α-glucosidase inhibitors. The

397

diCQA derivatives exhibited relatively higher inhibition activity than CQA

398

derivatives with a single caffeoyl moiety. Furthermore, the fluorescence spectra

399

suggested that the interaction was spontaneous, and hydrophobic force might be

400

primarily responsible for the interaction. The CD spectra revealed that the change in

401

protein conformation occurred due to the binding of α-glucosidase molecule to

402

5-CQA or 3,5-diCQA. All the present results suggested that the CQA derivatives

403

from I. Kudingcha C.J. Tseng could be physiologically useful for suppressing

404

postprandial hyperglycemia and therefore may be developed as functional foods for

405

the prevention or treatment of diabetes and obesity.

406

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α-Glucosidase inhibitory effect by the flower buds of Tussilago farfara L. Food

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beans and cinnamate derivatives on the activity of porcine pancreas α-amylase

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isozyme I. Food Chem. 2011, 127, 1532-1539.

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components caffeoylquinic acid derivatives in Chinese medicines with bovine

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11-24.

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Liu, Z. G.; He, S. H.; Gao, K. P.; He, Z. D. Phenylpropanoid glycoside

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inhibition of pepsin, trypsin and α-chymotrypsin enzyme activity in Kudingcha

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leaves from Ligustrum purpurascens. Food Res. Int. 2013, 54, 1376-1382.

28


Page 28 of 42

Page 29 of 42


590

(62) Moorthy, N. S. H. N.; Ramos, M. J.; Fernandes, P. A. Comparative structural

591

analysis of α-glucosidase inhibitors against difference species: A computational

592

study. Arch. Pharm. 2012, 345, 265-274.

593

(63) Wu, X. L.; He, W. Y.; Zhang, H. P.; Li, Y.; Liu, Z. G.; He, Z. D. Acteoside: a

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lipase inhibitor from the Chinese tea Ligustrum purpurascens kudingcha. Food

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Chem. 2014, 142, 306-310.

596

(64) Wu, X. L.; He, W. Y.; Li, Y.; Zhang, H. P.; Liu, Z. G.; Wang, W. P.; Ye, Y.; Cao, J.

597

J. Characterization of binding interactions of (-)-epigallocatechin-3-gallate from

598

green tea and lipase. J. Agric. Food Chem. 2013, 61, 8829-8835.

599

This work was supported by Grants-in-Aid for scientific research from the National

600

Natural Science Foundation of China (31171666) and a project funded by the

601

Priority Academic Program Development of Jiangsu Higher Education Institutions

602

(PAPD).

29



Page 30 of 42

603

Figure Captions

604

Figure 1. Structures of caffeoylquinic acids (IUPAC numbering system).

605

Figure 2. Inhibitory effect (A) and inhibition type (B) of kudingcha polyphenols on

606

α-glucosidase.

607

Figure 3. HPLC chromatograms of the extract (A) and six purified caffeoylquinic

608

acids (B) from Ilex Kudingcha C.J. Tseng.

609

Figure

610

α-glucosidase-catalysed hydrolysis of 4-nitrophenyl α-D-glucopyranoside at 37 ℃

611

and pH 6.9. Each point represents the means of triplicate experiments; (B)

612

Lineweaver-Burk plots of the reaction of α-glucosidase in the presence and absence

613

of 4,5-diCQA at the concentration of 5 × 10-4 M.

614

Figure 5. Fluorescence emission spectra of α-glucosidase in the presence of various

615

concentrations of 5-CQA (A), 3,4-diCQA (B), 3,5-diCQA (C) and 4,5-diCQA (D).

616

Conditions: T = 37 ℃, λex = 280 nm; α-glucosidase 0.05 mg/mL, caffeoylquinic

617

acid concentrations of 0, 0.005, 0.01, 0.015, 0.02, 0.025 and 0.03 mg/mL (a-g).

618

Figure 6. The three dimensional fluorescence spectra of α-glucosidase (A) and the

619

enzyme-CQA systems (5-CQA (B), 3,4-diCQA (C), 3,5-diCQA (D) and 4,5-diCQA

620

(E)).

621

Figure

622

enzyme-caffeoylquinic acid complexes. The free enzyme and enzyme-caffeoylquinic

623

acid complexes in phosphate buffer solution with a protein concentration of 3 × 10-6

624

M and caffeoylquinic acid concentrations of 0, 1.5 × 10-6, 3.0 × 10-6 and 6 × 10-6 M.

4.

7.

(A)

Inhibitory

Circular

effects

dichroism

of

caffeoylquinic

spectra

of

free

30


acids

against

α-glucosidase

the

and

Page 31 of 42


Table 1 Three-dimensional Fluorescence Spectral Characteristic Parameters of α-Glucosidase and Enzyme-Caffeoylquinic Acid Systems System α-Glucosidase (Enzyme) Enzyme-5-CQA Enzyme -3,4-diCQA Enzyme -3,5-diCQA Enzyme -4,5-diCQA

Peak 1 (nm)

Intensity

λex

λem

230

330

230

Peak 2 (nm)

Intensity

λex

λem

149.40

280

330

150.20

330

51.68

280

330

55.83

230

340

73.46

280

340

79.01

230

340

60.21

280

340

65.05

230

330

65.14

280

330

68.98

31



Page 32 of 42

Table 2 Binding Parameters and Relative Thermodynamic Variables for Caffeoylquinic Acid Derivatives

(5-CQA,

3,4-diCQA,

3,5-diCQA and

4,5-diCQA)

Binding

to

α-Glucosidase at Different Temperatures

Sample

Temperature (K)

5-CQA

293 310

3,4-diCQA

Ka (L mol-1)

n

∆G (kJ mol-1)

∆H (kJ mol-1)

∆S (J mol-1K-1)

5.53×106 1.19×107

1.42 1.50

-37.82 -41.99

34.04

245.26

293 310

7.93×107 6.31×108

1.70 1.87

-44.31 -52.22

92.13

465.56

3,5-diCQA

293 310

6.32×107 6.90×108

1.63 1.83

-43.76 -52.45

106.18

511.74

4,5-diCQA

293 310

2.18×107 4.73×107

1.53 1.62

-41.16 -45.55

34.41

257.92

32


Page 33 of 42


Table 3 Secondary Structure Contents of α-Glucosidase and Its Complexes with 5-CQA and 3,5-diCQA by Circular Dichroism Spectroscopy at Room Temperature α-Helix (%)

β-Sheet (%)

Turn (%)

Unordered (%)

Native α-Glucosidase

21.8

18.0

17.2

25.7

Glucosidase : 5-CQA=1:0.5

20.7

25.0

20.4

29.6

Glucosidase : 5-CQA=1:1

18.5

31.4

22.6

33.1

Glucosidase : 5-CQA=1:2

15.9

27.5

20.5

30.7

Glucosidase : 3,5-diCQA=1:0.5

21.1

21.6

18.8

27.6

Glucosidase : 3,5-diCQA=1:1

18.5

25.4

18.8

28.0

Glucosidase : 3,5-diCQA=1:2

16.4

31.0

21.6

32.1

Secondary structural content

33



O

R3

6

4

5

HOOC

R2 2

1

OH

Page 34 of 42

-

OH

O

3

R1

Compound

caffeoyl

R1

3-Caffeyolquinic acid (3-CQA) 5-Caffeyolquinic acid (5-CQA) 4-Caffeyolquinic acid (4-CQA) 3,4-Dicaffeyolquinic acid (3,4-diCQA) 3,5-Dicaffeyolquinic acid (3,5-diCQA) 4,5-Dicaffeyolquinic acid (4,5-diCQA)

caffeoyl OH OH caffeoyl caffeoyl OH

OH

R2 OH OH caffeoyl caffeoyl OH caffeoyl

Figure 1

34


R3 OH caffeoyl OH OH caffeoyl caffeoyl

Page 35 of 42


A Inhibition (%)

100 80 60 40 20 0 0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Concentration (mg/mL)

B Velocity (µmol/L/min) .

35

Without inhibitor

30

Kudingcha polyphenols

25 20 15 10 5 0 0

1

2

3

4

5

Concentration of enzyme (U/mL)

Figure 2

35


6

7


A

Page 36 of 42

3,5-diCQA

1600 1400

4,5-diCQA

Response (mAU)

1200 1000

3,4-diCQA 800

5-CQA 600 400

3-CQA

4-CQA

200 0 5

0

15

10

20

25

30

40

35

c

4-CQA

150

8 6 4 2

60

Response (mAU)

Response (mAU)

10

125 100 75 50

40

20

25 0

0

0 5

10

15

20

25

30

35

0

40

5

10

20

0

25

e

3,4-diCQA

5

0

12.5 10 7.5 5

0 15

20

25

30

35

25

4,5-diCQA

40 30 20 10

2.5

10

20

50

Response (mAU)

Response (mAU)

10

15

60

15

5

10

f

3,5-diCQA

20 17.5

15

0

5

Time (min)

32.535

31.693

d

20

15

Time (min)

Time (min)

38.447

0

Response (mAU)

5-CQA

80

175

3-CQA

12

Response (mAU)

b

200

22.015

a

16 14

21.631

B

14.973

Time(min )

0 0

5

10

15

20

25

30

Time (min)

Time (min)

Figure 3

36


0

5

10

15

20

Time (min)

25

30

35

40

Page 37 of 42


A

4,5-diCQA y = 0.2396x + 0.3443 R2 = 0.9804

1.4

1/V (µmol/L/min)-1

.

B

1.2 1

No Inhibitor y = 0.1903x + 0.2772 R2 = 0.9968

0.8 0.6 0.4 0.2 0

-3

-2

-1

-0.2 0

1

2

3

-0.4 1/[S] (mM)-1

Figure 4

37


4


A

0.8

y = 1.497x + 7.0763

0.6 lg[(F 0-F )/F ]

a g

Page 38 of 42

2

R = 0.9949

0.4 0.2 0 -0.2 -0.4 -0.6 -5.2

-5

-4.8

-4.6

-4.4

-4.2

lg[Q]

B

1.2

lg[(F0-F)/F]

g

y = 1.8722x + 8.8003

1

a

2

R = 0.9895

0.8 0.6 0.4 0.2 0 -0.2 -0.4 -5

-4.8

-4.6

-4.4 lg[Q]

38


-4.2

-4

Page 39 of 42


C lg[(F0-F)/F]

a g

1.2 1 0.8 0.6 0.4 0.2 0 -0.2 -0.4 -0.6 -5.2

y = 1.8385x + 8.8391 2

R = 0.9929

-5

-4.8

-4.6

-4.4

-4.2

lg[Q]

D

0.8

lg[(F0 -F)/F]

a g

y = 1.6151x + 7.6748

0.6

2

R = 0.9952

0.4 0.2 0 -0.2 -0.4 -0.6 -5.2

-5

-4.8

-4.6 lg[Q]

Figure 5

39


-4.4

-4.2


(A)

10 µg/mL

25 µg/mL

(B)

Excitation wavelength (nm)

(C)

(D)

(E)

Emission wavelength (nm)

Figure 6

40


Page 40 of 42

Page 41 of 42


Figure 7

41



Page 42 of 42

Extraction Purification

Response (mAU)

Graphic Abstract 1600 1400

3,5-diCQA 4,5-diCQA

1200 1000

3,4-diCQA

800 600

5-CQA

400 200

3-CQA

4-CQA

0 0

Kuding Tea

5

10

15

20

25

30

35

Time (min)

Kinetic analysis Interaction

Fluorescence quenching Mechanism

Circular dichroism spectroscopy

42


α-glucosidase inhibitory activities: diCQA > CQA

40

Inhibitory Activities of Caffeoylquinic Acid Derivatives from Ilex

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