Inhibitory Activities of Caffeoylquinic Acid Derivatives...

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Inhibitory Activities of Caeoylquinic Acid Derivatives from Ilex kudingcha C.J. Tseng on αGlucosidase from Saccharomyces 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, Peoples Republic of China ABSTRACT: Polyphenols and caeoylquinic acid (CQA) derivatives (3-CQA, 4-CQA, 5-CQA, 3,4-diCQA, 3,5-diCQA, and 4,5-diCQA) were prepared from Ilex kudingcha C.J. Tseng, and their eects and mechanisms on the activities of α-glucosidase from Saccharomyces cerevisiae were investigated in the present study. As results, the IC 50 values for CQA derivatives were 0.160.39 mg/mL, and the inhibition mode of CQA derivatives was noncompetitive. On the basis of uorescence spectroscopy and circular dichroism spectroscopy data, the binding constants and number of binding sites were calculated to be 10 6 10 8 M 1 and 1.421.87, respectively. CQA derivatives could bind to the enzyme mainly through hydrophobic interaction, altering the microenvironment and molecular conformation of the enzyme, thus decreasing the catalytic activity. To the authorsknowledge, this is the rst report on α-glucosidase inhibitory mechanism by CQA derivatives from I. kudingcha, and the ndings suggest a potential use of kudingcha as functional foods for the prevention and treatment of diabetes and related symptoms. KEYWORDS: kudingcha, α-glucosidase, caeoylquinic acid, inhibitory activity, interaction INTRODUCTION Diabetes mellitus, a major health problem associated with a number of complications such as diabetes-associated stroke, obesity, cancer, and cardiovascular diseases, is a metabolic disorder characterized by hyperglycemia and glucose intoler- ance. 13 It has been reported that the postprandial state is an important contributing factor for the development of atherosclerosis, and high postprandial plasma glucose concen- trations are associated with an increased risk of the develop- ment of type 2 diabetes and metabolic syndrome. 46 Controlling postprandial hyperglycemia, therefore, is an eective way to mitigate the illnesses and treat diabetes. One approach for the control of postprandial hyperglycemia is to retard or suppress the absorption of glucose in the intestine. While in mammals, the dietary carbohydrates are hydrolyzed by enzymes such as α-amylase and α-glucosidase (α-D-glucoside glucohydrolase, EC 3.2.1.20). α-Glucosidase, located at the brush-border surface membrane of intestinal cells, is the key enzyme catalyzing the nal step in the digestive process of carbohydrates that liberate glucose. Hence, α-glucosidase inhibitors can delay carbohydrate digestion and reduce the rate of glucose absorption, resulting in reduced postprandial plasma glucose levels and suppressed diabetes. 6,7 For example, acarbose and voglibose, two of the clinically approved drugs for the treatment of type 2 diabetes, have been demonstrated to act by this mechanism. 8,9 However, the use of these drugs may be associated with some undesirable side eects, the most commonly observed being weight gain, hyperglycemia, and gastrointestinal disturbances. 10,11 Thus, recently more attention has been paid to other eective and safe α-glucosidase inhibitors from natural ingredients or herbal extracts, which have been used medicinally for many years for the prevention of metabolic disorders such as diabetes and obesity. 1216 Kudingcha, a bitter tea of Chinese origin, has been used as a folk medicine for more than 2000 years. The main species for the production of kudingcha in China are Ilex kudingcha, Ilex latifolia, and Ilex cornuta, 17,18 which belong to the same genus as mate (Ilex paraguariensis). 19 I. kudingcha is rich in triterpenoids, phenolic acids, avonoids, essential oils, and other active substances and shows obvious antioxidant, anti- inammatory, antitumor, antimicrobial, hepatoprotective, and hypoglycemic activities. 18 In addition, the major phenolic compounds in leaves of I. kudingcha C.J. Tseng are reported to be caeoylquinic acid (CQA) derivatives (Figure 1, IUPAC numbering system), 20 including 3-CQA, 4-CQA, 5-CQA, 3,4- diCQA, 3,5-diCQA, and 4,5-diCQA, which account for 93.8% of total content of polyphenols. 21 CQA derivatives have been reported to have various biological functions such as antioxidant and antidiabetic activities, antiobesity, cancer Received: January 22, 2015 Revised: March 25, 2015 Accepted: March 25, 2015 Published: March 25, 2015 Figure 1. Structures of caeoylquinic acids (IUPAC numbering system). Article pubs.acs.org/JAFC © 2015 American Chemical Society 3694 DOI: 10.1021/acs.jafc.5b00420 J. Agric. Food Chem. 2015, 63, 36943703

Transcript of Inhibitory Activities of Caffeoylquinic Acid Derivatives...

Inhibitory Activities of Caffeoylquinic Acid Derivatives from Ilexkudingcha C.J. Tseng on α‑Glucosidase from SaccharomycescerevisiaeDonglan 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, People’s Republic of China

ABSTRACT: Polyphenols and caffeoylquinic acid (CQA) derivatives (3-CQA, 4-CQA, 5-CQA, 3,4-diCQA, 3,5-diCQA, and4,5-diCQA) were prepared from Ilex kudingcha C.J. Tseng, and their effects and mechanisms on the activities of α-glucosidasefrom Saccharomyces cerevisiae were investigated in the present study. As results, the IC50 values for CQA derivatives were 0.16−0.39 mg/mL, and the inhibition mode of CQA derivatives was noncompetitive. On the basis of fluorescence spectroscopy andcircular dichroism spectroscopy data, the binding constants and number of binding sites were calculated to be 106−108 M−1 and1.42−1.87, respectively. CQA derivatives could bind to the enzyme mainly through hydrophobic interaction, altering themicroenvironment and molecular conformation of the enzyme, thus decreasing the catalytic activity. To the authors’ knowledge,this is the first report on α-glucosidase inhibitory mechanism by CQA derivatives from I. kudingcha, and the findings suggest apotential use of kudingcha as functional foods for the prevention and treatment of diabetes and related symptoms.

KEYWORDS: kudingcha, α-glucosidase, caffeoylquinic acid, inhibitory activity, interaction

■ INTRODUCTION

Diabetes mellitus, a major health problem associated with anumber of complications such as diabetes-associated stroke,obesity, cancer, and cardiovascular diseases, is a metabolicdisorder characterized by hyperglycemia and glucose intoler-ance.1−3 It has been reported that the postprandial state is animportant contributing factor for the development ofatherosclerosis, and high postprandial plasma glucose concen-trations are associated with an increased risk of the develop-ment of type 2 diabetes and metabolic syndrome.4−6

Controlling postprandial hyperglycemia, therefore, is aneffective way to mitigate the illnesses and treat diabetes. Oneapproach for the control of postprandial hyperglycemia is toretard or suppress the absorption of glucose in the intestine.While in mammals, the dietary carbohydrates are hydrolyzed byenzymes such as α-amylase and α-glucosidase (α-D-glucosideglucohydrolase, EC 3.2.1.20). α-Glucosidase, located at thebrush-border surface membrane of intestinal cells, is the keyenzyme catalyzing the final step in the digestive process ofcarbohydrates that liberate glucose. Hence, α-glucosidaseinhibitors can delay carbohydrate digestion and reduce therate of glucose absorption, resulting in reduced postprandialplasma glucose levels and suppressed diabetes.6,7 For example,acarbose and voglibose, two of the clinically approved drugs forthe treatment of type 2 diabetes, have been demonstrated to actby this mechanism.8,9 However, the use of these drugs may beassociated with some undesirable side effects, the mostcommonly observed being weight gain, hyperglycemia, andgastrointestinal disturbances.10,11 Thus, recently more attentionhas been paid to other effective and safe α-glucosidaseinhibitors from natural ingredients or herbal extracts, whichhave been used medicinally for many years for the preventionof metabolic disorders such as diabetes and obesity.12−16

Kudingcha, a bitter tea of Chinese origin, has been used as afolk medicine for more than 2000 years. The main species for

the production of kudingcha in China are Ilex kudingcha, Ilexlatifolia, and Ilex cornuta,17,18 which belong to the same genusas mate (Ilex paraguariensis).19 I. kudingcha is rich intriterpenoids, phenolic acids, flavonoids, essential oils, andother active substances and shows obvious antioxidant, anti-inflammatory, antitumor, antimicrobial, hepatoprotective, andhypoglycemic activities.18 In addition, the major phenoliccompounds in leaves of I. kudingcha C.J. Tseng are reported tobe caffeoylquinic acid (CQA) derivatives (Figure 1, IUPACnumbering system),20 including 3-CQA, 4-CQA, 5-CQA, 3,4-diCQA, 3,5-diCQA, and 4,5-diCQA, which account for 93.8%of total content of polyphenols.21 CQA derivatives have beenreported to have various biological functions such asantioxidant and antidiabetic activities, antiobesity, cancer

Received: January 22, 2015Revised: March 25, 2015Accepted: March 25, 2015Published: March 25, 2015

Figure 1. Structures of caffeoylquinic acids (IUPAC numberingsystem).

Article

pubs.acs.org/JAFC

© 2015 American Chemical Society 3694 DOI: 10.1021/acs.jafc.5b00420J. Agric. Food Chem. 2015, 63, 3694−3703

suppression, and inhibition of α-glucosidase and tyrosi-nase.18,21−29 Furthermore, it has been reported that regularconsumption of coffee is associated with a lower risk of type 2diabetes mellitus,30 and 5-CQA in coffee may contribute to thebeneficial effects of coffee on type 2 diabetes mellitus.31

However, the inhibitory effects of CQA derivatives fromkudingcha on α-glucosidase have never been reported before.As tea polyphenolic compounds strongly interact withproteins,32 CQA derivatives from kudingcha may exhibitpotential interactions with α-glucosidase. This should inevitablyresult in the change of enzyme molecular configuration andlead to the loss of catalytic activity, thereby reducingcarbohydrate digestibility and protection against diabetes andobesity. Recently, we have reported the potent antioxidantactivity of kudingcha polyphenols and isolated CQA derivativesfrom the leaves of I. kudingcha C.J. Tseng.22,33−35 In this study,therefore, the inhibitory effects against α-glucosidase of sixkinds of CQA derivatives (3-CQA, 4-CQA, 5-CQA, 3,4-diCQA,3,5-diCQA, and 4,5-diCQA) present in kudingcha made fromthe leaves of I. kudingcha C.J. Tseng were investigated.Furthermore, the potential mechanisms of interactions betweenα-glucosidase and CQA derivatives were characterized byfluorescence spectroscopy and circular dichroism (CD) spec-troscopy.

■ MATERIALS AND METHODSMaterials and Reagents. Kudingcha made from the leaves of I.

kudingcha C.J. Tseng was obtained from Hainan Yexian Bio-ScienceTechnology Co., Ltd. (Hainan, China), and the sample was ground byusing a domestic blender, stored in sealed polyethylene bags, and keptin a refrigerator at −20 °C until use. α-Glucosidase from Saccharomycescerevisiae (dialysis 48 h before use), 5-CQA, and 4-nitrophenyl α-D-glucopyranoside (≥99%) were purchased from Sigma-AldrichChemical Co. (St. Louis, MO, USA). Standards of 3-CQA, 4-CQA,3,4-diCQA, 3,5-diCQA, and 4,5-diCQA (>95%) were prepared fromkudingcha according to our reported methods.22,34,35 The solventsused for chromatographic purpose were of high-performance liquidchromatography (HPLC) grade, and all other reagents were ofanalytical reagent grade.Determination of Total Polyphenols Content and CQA

Derivatives. The total polyphenols content was determined byusing the Folin−Ciocalteu method according to the reportedprocedure.22 The contents of CQA derivatives were measured byHPLC with an Agilent HPLC series 1100 (Agilent, Santa Clara, CA,USA) equipped with a model G1379A degasser, a model G1311Aquatpump, a model G1316A column oven, a model G1315B diodearray detector (DAD), and Chemstation software. The separation wascompleted on a TSK gel ODS-80TsQA column (4.6 × 250 mm, 5 μm,Tosoh Corp., Tokyo, Japan) with a gradient mobile phase consistingof ultrapure water (A), methanol (B), and 1.0% formic acid (C, v/v) ata flow rate of 0.5 mL/min. Elution was performed with a lineargradient as follows: 0−45 min, A from 60 to 35%, B from 20 to 45%, C20%. The temperature of column oven was set at 40 °C, the injectionvolume was 20 μL, and CQA derivatives were detected at 326 nm witha DAD. The chromatographic peaks were identified by comparing theretention times and UV spectra of standards of CQA derivatives.Preparation of CQA Derivatives. The preparation of CQA

derivatives was performed according to the reported methods22,33−35

with some modifications. Briefly, the kudingcha powder was extractedwith water (10 for the ratio of extraction solvent to material, v/w) for30 min at 95 °C. After extraction, the extract was centrifuged at 5000gfor 10 min, and the resulting insoluble residue was treated twice asdescribed above. The combined supernatants were concentrated by arotary evaporator (Tokyo Rikakikai Co., Ltd., Tokyo, Japan) andlyophilized (Labconco, Kansas, MO, USA) to afford the crude extract.The crude extract was dissolved in deionized water and applied to acolumn (5 × 30 cm) of HP-20 macroporous resin. Then, the column

was washed with 3 times bed volume of distilled water and eluted with70% ethanol solution, respectively. The collected fractions (10 mL/tube) were analyzed by HPLC as described above, and the fractionscontaining CQA derivatives were combined, concentrated, and freeze-dried, affording kudingcha polyphenols. The CQA derivatives (3-CQA, 4-CQA, 5-CQA; 3,4-diCQA, 3,5-diCQA, and 4,5-diCQA) werefurther isolated from the kudingcha polyphenols by HPLC with asemipreparative HPLC column of YMC-Pack ODS-A (20 × 250 mm,5 μm, YMC Co., Ltd., Kyoto, Japan) according to our reportedmethods.22,34,35 The desired fractions containing 3-CQA, 4-CQA, 5-CQA; 3,4-diCQA, 3,5-diCQA, and 4,5-diCQA were combined,concentrated, and lyophilized, respectively. The structures of CQAderivatives were confirmed by electrospray ionization−mass spectrom-etry (ESI-MS), HPLC, and nuclear magnetic resonance (NMR)spectrometry.

Enzyme Inhibitory Activities Assay. The inhibitory activityagainst α-glucosidase was measured according to the reportedmethod26 with some modifications. Briefly, the reaction mixturecontained 8 μL of test sample with different concentrations, 30 μL of2.5 mM 4-nitrophenyl α-D-glucopyranoside as substrate, 20 μL ofenzyme solution (0.2 U/mL), and 102 μL of 0.1 M phosphate buffersaline (pH 6.9). The reaction mixture was incubated at 37 °C for 15min. The reaction was then terminated by the addition of 80 μL of 0.2M Na2CO3 solution. The increase in absorbance at 405 nm due toenzymatic hydrolysis of 4-nitrophenyl α-D-glucopyranoside wasmonitored with a microplate reader (BioTek Instruments, Inc.,Winooski, VT, USA). All samples were analyzed in triplicate. Theinhibition percentage was calculated using the formula

= − − −

− ×+ −

+ −

A A A A

A A

inhibition rate (%) [(( ) ( ))

/( )] 100c c s b

c c

where Ac+, Ac−, As, and Ab are defined as the absorbances of 100%enzyme activity (only the solvent with the enzyme), 0% enzymeactivity (only the solvent without enzyme), the test sample (with theenzyme), and a blank (only the sample), respectively. The effectiveconcentration that could inhibit 50% of α-glucosidase activity isdefined as IC50.

Assay of Inhibitory Pattern of CQA Derivatives on α-Glucosidase Activity. The effects of CQA derivatives on α-glucosidase activity were investigated with increasing concentrationsof enzyme substrate (4-nitrophenyl α-D-glucopyranoside, from 3.0 ×10−4 to 1.5 × 10−3 M) in the presence or absence of CQA derivatives.Then, the inhibition type was determined by Lineweaver−Burk plotanalysis according to Michaelis−Menten kinetics.

Fluorescence Measurement. General Procedure. A quantitativeanalysis of the potential interaction between CQA derivative and α-glucosidase was performed by fluorometric titration as follows: 3.0 mLof solution containing appropriate concentration of α-glucosidase wastitrated by the addition of different concentration of 5-CQA, 3,4-diCQA, 3,5-diCQA, or 4,5-diCQA solution. All samples wereincubated at fixed temperature (293 or 310 K) for 0.5 h. Then, thefluorescence spectra were recorded with an F-7000 fluorescencespectrophotometer (Hitachi High-Technologies Corp., Tokyo, Japan)by using a 1.0 cm quartz cell and at an excitation wavelength of 280nm, and the emission spectra were recorded from 300 to 450 nm.Spectral resolution for both excitation and emission was 5 nm. Thethree-dimensional (3D) fluorescence spectra of enzyme in the absenceand presence of CQA derivatives were obtained by recording theexcitation and the emission spectra in the range of 200−600 nm withan interval of 5 nm, respectively.

Analysis of Fluorescence Quenching Constant and Thermody-namic Parameters. The fluorescence quenching data were analyzedvia the modified Stern−Volmer equation:36

− = +F F F K nlg[( )/ ] lg lg[Q]0 a

Here, F0 and F are the fluorescence intensities of the enzyme in theabsence and presence of quencher, [Q] is the quencher concentration,Ka is the binding constant, and n is the number of the binding sites per

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enzyme. The values of n and Ka can be calculated by the slope andintercept with the plots of lg(F0 − F)/F against lg[Q].37

Considering that the enthalpy change (ΔH) does not varysignificantly over the temperature range, it can be considered as aconstant. ΔH and entropy change (ΔS) can be calculated using thevan’t Hoff equation

= − Δ + ΔK

HRT

SR

ln

= − ΔK K T T H Rln( / ) (1/ 1/ ) /2 1 1 2

Δ = Δ − ΔG H T S

where R is the gas constant, T is the experimental temperature, K is thebinding constant at the corresponding T, and ΔG is the free energy.Then ΔH, ΔS, and ΔG of interaction can be calculated from theequations above.CD Measurement. The CD spectra of α-glucosidase and its

complexes with CQA derivatives were recorded with the use of aJasco-810 spectrophotometer (JASCO Corp., Tokyo, Japan) at roomtemperature. The spectra were measured in the far-UV region (190−250 nm) with a path length of 1.0 mm, a scan speed of 50 nm/min,and a response time of 4 s. In addition, three scans were accumulatedfor each spectrum. All working solutions were prepared with 20 mMphosphate buffer (pH 6.9). The enzyme concentration was set at 0.2mg/mL (3 × 10−6 M), and the complexes were prepared by mixing theenzyme with CQA derivative (5-CQA or 3,5-diCQA) at molar ratiosof 1:0.5, 1:1, and 1:2 (enzyme to CQA derivative). The resulting CDdata were analyzed by curve-fitting program software CDPro usingCONTIN, SELCON, and CDSSTR methods, as described bySreerama and Woody,38 to obtain the secondary structural contentsof α-glucosidase.Statistical Analysis. Data were expressed as the mean ± standard

deviation (SD) of triplicates. The IC50 value was calculated from linearregression analysis. Any significant difference was determined by one-way analysis of variance (ANOVA) followed by t test for multiplecomparisons at P < 0.05 level (SPSS Statistics 20.0, IBM, Armonk, NY,USA).

■ RESULTSInhibitory Activity of Kudingcha Polyphenols against

α-Glucosidase. The effect of kudingcha polyphenols, partiallypurified polyphenols from the crude extract of I. kudingcha C.J.Tseng by chromatography of HP-20 macroporous resin, on α-glucosidase from S. cerevisiae was investigated. As shown inFigure 2A, the kudingcha polyphenols exhibited stronginhibitory activity against α-glucosidase. The IC50 value wasdetermined to be 0.42 mg/mL. To further explore theinhibition type of kudingcha polyphenols against α-glucosidase,the kinetic reaction of α-glucosidase was investigated withdifferent concentrations of enzyme. As shown in Figure 2B, itwas found that the line obtained from the kinetic reaction plotpassed through the origin point (0, 0) of the coordinate,indicating that the inhibition type of kudingcha polyphenols onα-glucosidase was reversible inhibition. As for irreversibleinhibition, the inhibitor binds with enzyme covalently andforms a stable complex, making the enzyme inactivated. Theenzyme will exhibit its activity only when a certain amount ofenzyme is added to the reaction system. Therefore, the plot ofenzyme concentration versus reaction velocity does not passthrough the origin of the coordinate, whereas for reversibleinhibition, which is characterized by the existence ofequilibrium between enzyme and inhibitor, the plot may passthrough the origin.39,40 It is obvious that kudingchapolyphenols are reversible inhibitors of α-glucosidase.As shown in Figure 3A, the kudingcha polyphenols contained

3-CQA (3.2 ± 0.15%), 4-CQA (2.1 ± 0.09%), 5-CQA (8.4 ±

0.13%), 3,4-diCQA (11.8 ± 0.25%), 3,5-diCQA (23.2 ±1.61%), and 4,5-diCQA (25.6 ± 1.52%). Because the kudingchapolyphenols showed potent α-glucosidase inhibitory activity asmentioned above, CQA derivatives, the main polyphenols in I.kudingcha C.J. Tseng, were isolated by semipreparative HPLCfrom kudingcha polyphenols (78.4% for total polyphenolcontent determined by the Folin−Ciocalteu method). Thepurified CQA derivatives, with purity >95% (Figure 3B), wereconfirmed to be 3-CQA, 4-CQA, 5-CQA, 3,4-diCQA, 3,5-diCQA, and 4,5-diCQA by HPLC, ESI-MS, and 1H NMRthrough comparison with the reported data.22,34,35,41−43

Inhibitory Activities of CQA Derivatives against α-Glucosidase. The resulting CQA derivatives from I. kudingchaC.J. Tseng were further examined for their inhibition on α-glucosidase by evaluating their IC50 values. The results revealedthat all of the CQA derivatives had inhibitory activity against α-glucosidase in a dose-dependent manner (Figure 4A). At aconcentration of 1.0 mg/mL, α-glucosidase was almostinhibited (98%) by CQA derivatives. The IC50 values for 3-CQA, 4-CQA, 5-CQA, 3,4-diCQA, 3,5-diCQA, and 4,5-diCQAwere determined to be 0.39, 0.34, 0.30, 0.27, 0.27, and 0.16 mg/mL, respectively. Notably, diCQA derivatives with doublecaffeoyl moieties exhibited higher inhibition activities thanCQA derivatives with a single caffeoyl moiety, indicating thatthe addition of caffeoyl moiety significantly increased theinhibitory ability.To characterize the inhibition pattern of α-glucosidase by

CQA derivatives, the enzyme reactions were examined withincreasing concentrations of substrate without or with a fixedconcentration of inhibitor. As an example, Figure 4B shows theLineweaver−Burk double-reciprocal plots of α-glucosidasekinetics with 4,5-diCQA as the inhibitor. It is obvious thatthe Vmax for α-glucosidase decreased while the Km (0.68 mM)remained unchanged in the presence of 4,5-diCQA, and theother CQA derivatives showed similar trends. Thus, theinhibition of α-glucosidase by CQA derivatives was non-competitive, in which the inhibitor and the substrate shouldbind simultaneously with the enzyme.

Fluorescence Spectra. Fluorescence spectroscopy and CDspectroscopy were used to explore the interactions between α-glucosidase and CQA derivatives. As the inhibitory activity of

Figure 2. Inhibitory effect (A) and inhibition type (B) of kudingchapolyphenols on α-glucosidase.

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diCQA derivatives was higher than that of CQA derivativeswith a single caffeoyl moiety, 5-CQA, 3,4-diCQA, 3,5-diCQA,and 4,5-diCQA were used for further studies of interactionmechanisms.Fluorescence spectroscopy is used to measure the interaction

between a biomacromolecule and a small molecule ligand.44 Ina protein molecule, Trp has the most powerful activity to emitfluorescence and the intrinsic fluorescent intensity may changedepending on the impact of the interaction between the proteinand another molecule.36 It contains 18 Trp residues in α-glucosidase (obtained from the Brookhaven Protein Data Bank,PDB ID: 1 VAD). Therefore, it is possible to use quenching ofthe intrinsic Trp fluorescence in enzyme to study theinteractions between CQA derivatives and α-glucosidase.Under our measurement condition (i.e., from 300 to 450

nm), all fluorescence emissions from CQA derivatives, buffer,and other reagents were so weak that their impact could beignored. As shown in Figure 5, the fluorescence intensities of α-glucosidase decreased gradually with the addition of CQAderivatives at 37 °C, and the maximal absorption peak (near332 nm) was red-shifted; thus, the CQA derivatives interactedwith the enzyme (data at 20 °C not shown). As a result, themaximum emission wavelength was shifted from 332 to 360 nmfor the addition of 5-CQA, from 332 to 352 nm for 3,4-diCQA,from 331 to 356 nm for 3,5-diCQA, and from 332 to 356 nmfor 4,5-diCQA, respectively. These changes suggested that theinteractions between the CQA derivatives and α-glucosidase ledto a polarity variation for Trp residues in the enzyme and madethe microenvironment change from hydrophilic to hydro-phobic, thus resulting in greater exposure of Trp residues andunfolding of protein structure.45,46

The 3D fluorescence spectra have been used to investigatethe characteristic conformational change of protein in recentyears.47 The 3D fluorescence contour maps of α-glucosidase

and the mixtures with four CQA derivatives in differentconcentrations (0.01 and 0.025 mg/mL) are shown in Figure 6.The 3D spectra showed two distinct peaks in enzyme marked 1(λex/λem: 280 nm/330 nm) and 2 (λex/λem: 230 nm/330 nm).Peak 1 corresponded to the characteristics of Trp and Tyrresidues, and peak 2 provided spectral properties of thepolypeptide backbone, which occur mainly due to the presenceof the π−π* and n−π* transitions, respectively.48,49 The twopeaks were quenched by the addition of CQA derivative in adose-dependent manner. The corresponding parameters (Table1) indicated that the fluorescence intensity decreased with theaddition of CQA derivatives, following a red shift of 10 nm ineach of peaks 1 and 2. It was also found that the quenchingeffect showed a sequence as diCQA > 5-CQA, which was inaccordance with the results of fluorescence experiments. Theseeffects might be due to hydrophobic interactions between thearomatic ring and the hydrophobic moieties present, thereforeinducing conformational changes in the α-glucosidase mole-cule.14

Fluorescence Binding Constant and Binding Site. Toobtain the binding constant (Ka) and the number of bindingsite per enzyme (n) between CQA derivative and α-glucosidase,the fluorescence quenching data were analyzed according to themodified Stern−Volmer equation by using the Scatchard plots(Figure 5). The results are summarized as shown in Table 2.The results demonstrated that Ka was in the order diCQA > 5-CQA, indicating that introduction of the second caffeoyl moietyinto CQA to form diCQA enhanced the binding affinity to theenzyme. As compared with tea polyphenol−protein inter-actions with Ka from 1.0 × 104 to 1.0 × 105 M−1,50−52 the Ka

values (106−108 M−1) for interactions of CQA derivatives andα-glucosidase were quite high, indicating comparatively strongligand−protein interactions.

Figure 3. HPLC chromatograms of the extract (A) and six purified caffeoylquinic acids (B) from Ilex kudingcha C.J. Tseng.

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Thermodynamic Parameters and Binding Mode. Theinteractions between phenolic compounds and biomoleculesmay involve electrostatic interactions, van der Waalsinteractions, hydrophobic forces, hydrogen bonds, and so on.According to ΔH and ΔS data, the model of interactionbetween quencher and a protein molecule can be concluded.53

More specifically, if ΔH > 0 and ΔS > 0, the main force wouldbe hydrophobic force; if ΔH < 0 and ΔS > 0, it would beelectrostatic force; if ΔH < 0 and ΔS < 0, it would be hydrogenbonding. According to the van’t Hoff equation, thethermodynamic parameters were calculated and the resultsare presented in Table 2. Notably, the ΔH and ΔS values wereboth positive, indicating that hydrophobic forces played a majorrole in the bindings between CQA derivative and α-glucosidase.Furthermore, the negative value of ΔG suggested that thebinding reaction was spontaneous.CD Spectra. CD spectroscopy was conducted to evaluate

the influence of 5-CQA and 3,5-diCQA on the secondarystructure of α-glucosidase in the far-UV CD range (from 250 to190 nm). As shown in Figure 7, the CD spectra of α-glucosidase were characterized mainly by two negative bands at208 and 222 nm, which are caused by a negative Cotton effectcharacteristic of helical structure.54,55 The CD spectra wereanalyzed by using the CDPro software package to afford thecontents of four secondary structures: α-helix, β-sheet, turns,and unordered in the absence and presence of 5-CQA or 3,5-

diCQA. As shown in Table 3, the secondary structureproportions for free α-glucosidase were α-helical, 21.8%; β-

Figure 4. (A) Inhibitory effects of caffeoylquinic acids against the α-glucosidase-catalyzed hydrolysis of 4-nitrophenyl α-D-glucopyranosideat 37 °C and pH 6.9. Each point represents the means of triplicateexperiments. (B) Lineweaver−Burk plots of the reaction of α-glucosidase in the presence and absence of 4,5-diCQA at theconcentration of 5 × 10−4 M.

Figure 5. Fluorescence emission spectra of α-glucosidase in thepresence of various concentrations of 5-CQA (A), 3,4-diCQA (B), 3,5-diCQA (C), and 4,5-diCQA (D). Conditions: T = 37 °C, λex = 280nm; α-glucosidase = 0.05 mg/mL; caffeoylquinic acid concentrationsof 0, 0.005, 0.01, 0.015, 0.02, 0.025, and 0.03 mg/mL (a−g).

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sheet, 18%; turn, 17.2%; and unordered, 25.7%. With theaddition of 5-CQA or 3,5-diCQA to enzyme, the α-helixcontents decreased to 15.9 and 16.4%, whereas the β-sheetcontents increased to 27.5 and 31% and the unordered

structure contents increased to 30.7 and 32.1%, respectively.In addition, it was found that the difference was not significantfor the effects of 5-CQA and 3,5-diCQA on secondary structurecontents of α-glucosidase. The decrease in α-helical contents

Figure 6. Three-dimensional fluorescence spectra of α-glucosidase (A) and the enzyme−CQA systems (5-CQA (B), 3,4-diCQA (C), 3,5-diCQA(D), and 4,5-diCQA (E)).

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and increase in contents of unordered structure suggested thatthe binding of CQA derivatives to α-glucosidase induced theunfolding of protein structure, which is consistent with theresults obtained from fluorescence assay.

■ DISCUSSION

Natural α-glucosidase inhibitors from plant sources offer anattractive strategy to control postprandial hyperglycemia. Toour knowledge, this is the first report on α-glucosidaseinhibitory effects and mechanisms of CQA derivatives from I.kudingcha C.J. Tseng, although it has been used in China forthe treatment of diabetes and coronary heart diseases intraditional natural medicine for a long time.18

In our previous works, we investigated the potent antioxidantactivity of kudingcha polyphenols and purified and charac-terized the CQA derivatives present in I. kudingcha C.J.Tseng.22,34,35 As many plant polyphenols with relatively highantioxidant activity exhibit inhibitory effects on digestiveenzymes,26,56 we examined the inhibitory activities of CQAderivatives from I. kudingcha C.J. Tseng against α-glucosidasefrom S. cerevisiae and further determined the potentialinteraction mechanisms in the present study. As a result, theinhibitory activities of CQA derivatives were in the order 4,5-diCQA > 3,5-diCQA ≈ 3,4-diCQA > 5-CQA > 4-CQA > 3-CQA by comparison of their IC50 values. The inhibitory activityof diCQA was higher than that of CQA derivative with a singlecaffeoyl moiety, indicating that the inhibitory activity of CQAderivative increases with increasing number of caffeoyl moieties.In addition, the inhibitory activity was affected by thesubstitution position of the caffeoyl moiety on quinic acid.The substitution at the 5-position exhibited relatively higherinhibitory activity than that at the 4-position, but it wasrelatively higher than that at the 3-position (p < 0.05). As forthe three diCQA derivatives, 3,4-diCQA showed equalinhibitory activity to 3,5-diCQA (p > 0.05), but it was weakerthan 4,5-diCQA (p < 0.05). The differences in the α-

glucosidase inhibitory activities of CQA derivatives may bedue to their differences in structures, particularly thedistribution of caffeoyl group and its position in the quinicacid aromatic ring.26 Such results are in agreement with theconclusions of previous structure−activity relationship inves-tigations by other researchers.16,23,57−59 For example, the α-glucosidase inhibitory activities of 3,4-diCQA, 3,5-diCQA, and4,5-diCQA, separated from flower buds of Tussilago farfara L.,were higher than that of 5-CQA.57 For porcine pancreas α-amylase isozyme I, diCQA derivative exhibited higherinhibitory activity than mono-CQA derivatives and feruloyl-quinic acids.58 Five CQA derivatives (5-CQA, 3,4-diCQA, 3,5-diCQA, methyl 3,4-di-O-caffeoylquinate (3,4-diCQM), and 3,5-diCQM) were isolated from Lonicera fulvotomentosa Hsu et S.C. Cheng, and their interactions with bovine serum albumin(BSA) were determined; their binding constants with BSAranked in the following order: 3,4-diCQM > 3,5-diCQM ≈ 3,4-diCQA > 3,5-diCQA > 5-CQA (IUPAC numbering system:4,5-diCQM > 3,5-diCQM ≈ 4,5-diCQA > 3,5-diCQA > 5-CQA).59 It has also been reported that diCQA derivatives withdouble caffeoyl moieties had higher antioxidant capacity ascompared to CQA derivative with a single caffeoyl moiety,indicating the addition of caffeoyl moiety significantly increasedthe antioxidative ability.23 Furthermore, the inhibitory types of3-CQA, 4-CQA, 5-CQA, 3,4-diCQA, 3,5-diCQA, and 4,5-diCQA from I. kudingcha C.J. Tseng for α-glucosidase werefound to be noncompetitive in the present study. This is thefirst report on the inhibitory kinetics of the enzyme of six CQAderivatives from kudingcha, although the α-glucosidaseinhibitory activities of several CQA derivatives from otherherbs were investigated before.Some polyphenols have a strong ability to interact with

digestive enzymes on the basis of interactions betweenpolyphenols and the protein, which reduce food digestibil-ity.60,61 Thus, we explored the interactions between the CQAderivative and α-glucosidase by fluorescence spectroscopy and

Table 1. Three-Dimensional Fluorescence Spectral Characteristic Parameters of α-Glucosidase and Enzyme−CaffeoylquinicAcid Systems

peak 1 (nm) peak 2 (nm)

system λex λem intensity λex λem intensity

α-glucosidase (enzyme) 230 330 149.40 280 330 150.20enzyme−5-CQA 230 330 51.68 280 330 55.83enzyme−3,4-diCQA 230 340 73.46 280 340 79.01enzyme−3,5-diCQA 230 340 60.21 280 340 65.05enzyme−4,5-diCQA 230 330 65.14 280 330 68.98

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 temp (K) Ka (L mol−1) n ΔG (kJ mol−1) ΔH (kJ mol−1) ΔS (J mol−1K−1)

5-CQA 293 5.53 × 106 1.42 −37.82 34.04 245.26310 1.19 × 107 1.50 −41.99

3,4-diCQA 293 7.93 × 107 1.70 −44.31 92.13 465.56310 6.31 × 108 1.87 −52.22

3,5-diCQA 293 6.32 × 107 1.63 −43.76 106.18 511.74310 6.90 × 108 1.83 −52.45

4,5-diCQA 293 2.18 × 107 1.53 −41.16 34.41 257.92310 4.73 × 107 1.62 −45.55

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CD spectroscopy for the first time. As for fluorescence spectra,the decrease of fluorescence intensities and a red shift ofmaximal absorption peak were observed, indicating a conforma-tional change in α-glucosidase. From the fluorescence analysis,the CQA derivative−α-glucosidase binding was achieved by

hydrophobic interactions of the interior hydrophobic groups ofenzyme and then might be stabilized by hydrogen bondingbetween polar groups (−OH, −SH, and −NH groups) of theenzyme with the −OH groups of CQA derivative. It has beenreported that the integy moment of hydrophobicity descriptors(vsurf_ID4 and vsurf_ID7) in 5-CQA derivatives contributedto the inhibitory activity of α-glucosidase from Bacillusstearothermophilus or S. cerevisiae. The requirement of thehydrophilic properties on the van der Waals surface of themolecules by properly aligned polar and aromatic/hydrophobicregions for all highly active and less active compounds wasconfirmed by pharmacophore analysis.28,62 The binding abilitiesof CQA derivatives were found to be quite high with a bindingconstant from 106 to 108 M−1, and the order was diCQAderivative > CQA derivative with a single caffeoyl moiety, whichis in accordance with the results of enzyme inhibition.Interactions between small molecules and enzymes may alterthe conformation of the enzyme.63,64 In our present study, thechanges in secondary structures of α-glucosidase with theaddition of 5-CQA or 3,5-diCQA were demonstrated by usingCD spectroscopy. Thus, CQA derivatives may have a bindingeffect on α-glucosidase, changing the polarity and moleculeconformation of enzyme, resulting in partial loss of the enzymeactivity. These structural changes of α-glucosidase finally causedthe inhibition of enzyme activity.In conclusion, six kinds of CQA derivatives, 3-CQA, 4-CQA,

5-CQA, 3,4-diCQA, 3,5-diCQA, and 4,5-diCQA, separatedfrom I. kudingcha C.J. Tseng were demonstrated to be effectivenoncompetitive α-glucosidase inhibitors. The diCQA deriva-tives exhibited relatively higher inhibition activity than CQAderivatives with a single caffeoyl moiety. Furthermore, thefluorescence spectra suggested that the interaction wasspontaneous, and hydrophobic force might be primarilyresponsible for the interaction. The CD spectra revealed thatthe change in protein conformation occurred due to thebinding of the α-glucosidase molecule to 5-CQA or 3,5-diCQA.All of the present results suggested that the CQA derivativesfrom I. kudingcha C.J. Tseng could be physiologically useful forsuppressing postprandial hyperglycemia and therefore may bedeveloped as functional foods for the prevention or treatmentof diabetes and obesity.

■ AUTHOR INFORMATION

Corresponding Authors*(X.Z.) Fax: +86 25 84396791. E-mail: [email protected].*(Y.S.) Fax: +86 25 84396791. E-mail: [email protected].

FundingThis work was supported by Grants-in-Aid for scientificresearch from the National Natural Science Foundation ofChina (31171666) and a project funded by the PriorityAcademic Program Development of Jiangsu Higher EducationInstitutions (PAPD).

NotesThe authors declare no competing financial interest.

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Figure 7. Circular dichroism spectra of free α-glucosidase andenzyme−caffeoylquinic acid complexes: free enzyme and enzyme−caffeoylquinic acid complexes in phosphate buffer solution with aprotein concentration of 3 × 10−6 M and caffeoylquinic acidconcentrations of 0, 1.5 × 10−6, 3.0 × 10−6, and 6 × 10−6 M.

Table 3. Secondary Structure Contents of α-Glucosidase andIts Complexes with 5-CQA and 3,5-diCQA by CircularDichroism Spectroscopy at Room Temperature

secondary structuralcontent

α-helix(%)

β-sheet(%)

turn(%)

unordered(%)

native α-glucosidase 21.8 18.0 17.2 25.7glucosidase/5-CQA = 1:0.5 20.7 25.0 20.4 29.6glucosidase/5-CQA = 1:1 18.5 31.4 22.6 33.1glucosidase/5-CQA = 1:2 15.9 27.5 20.5 30.7glucosidase/3,5-diCQA =1:0.5

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glucosidase/3,5-diCQA =1:1

18.5 25.4 18.8 28.0

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16.4 31.0 21.6 32.1

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