Inhibitory Effect of Persimmon Tannin on Pancreatic Lipase and the

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Bioactive Constituents, Metabolites, and Functions

Inhibitory effect of persimmon tannin on pancreatic lipase and the underlying mechanism in vitro Wei Zhu, yang yang Jia, Jinming Peng, and Chun-mei Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00850 • Publication Date (Web): 28 May 2018 Downloaded from http://pubs.acs.org on May 28, 2018

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Journal of Agricultural and Food Chemistry

Inhibitory effect of persimmon tannin on pancreatic lipase and the underlying mechanism in vitro Wei Zhu1, Yangyang Jia1, Jinming Peng1, Chun-mei Li1, 2*

1

College of Food Science and Technology, Huazhong Agricultural University, Wuhan,

China, 430070

2

Key Laboratory of Environment Correlative Food Science (Huazhong Agricultural

University), Ministry of Education

*Corresponding author: Chunmei Li (Tel: 86-27-87282966; Fax: 86-27-87282966; E-mail: [email protected])

1

ACS Paragon Plus Environment


1

Abstract：Pancreatic lipase (PL) is a critical enzyme associated with hyperlipidemia

2

and obesity. Our previous study suggested that persimmon tannin (PT) was the main

3

component accounting for the anti-hyperlipidemic effects of persimmon fruits, but

4

the underlying mechanisms were unclear. In present study, the inhibitory effect of PT

5

on PL was studied and the possible mechanisms were evaluated by fluorescence

6

spectroscopy, circular dichroism (CD) spectra, isothermal titration calorimetry (ITC)

7

and molecular docking. PT had a high affinity to PL and inhibited the activity of PL

8

with the half maximal inhibitory concertation (IC50) value of 0.44 mg/mL in a

9

non-competitive way. Furthermore, molecular docking revealed that the hydrogen

10

bonding and pi-pi stacking was mainly responsible for the interaction. The strong

11

inhibition of PT on PL in gastrointestinal tract might be one mechanism for its

12

lipid-lowering effect.

13

Key words: Persimmon tannin (PT); A-ECG and EGCG dimers; Pancreatic lipase;

14

Fluorescence spectroscopy; Molecular docking; Obesity

15

16

17

18

19

2


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Introduction

21

Hyperlipidemia is characterized by an excessive level of lipid in the blood. A large

22

number of epidemiological and clinical studies have demonstrated that

23

hyperlipidemia is an important risk factor for the incidence of atherosclerosis, insulin

24

resistance, diabetes and obesity1, 2. Therefore, its prevention attracts worldwide

25

attention. It was shown that some kind of dietary such as the Mediterranean diet is

26

beneficial for its prevention3, 4. Compared to drugs such as orlistat which have many

27

side effects including flatulence, diarrhea and nausea, dietary factors with significant

28

lipid-lowering effects provide a more suitable strategy to manage hyperlipidemia and

29

its associated diseases. Therefore, novel dietary components with potent

30

anti-hyperlipidemic effects for the prevention of hyperlipidemia have attracted great

31

attention among researchers.

32

Generally, triglyceride is hardly absorbed directly by human intestine before it is

33

hydrolyzed by pancreatic lipase (PL). Therefore, inhibiting PL can effectively reduce

34

the triglyceride absorption in intestinal tract, thus preventing hyperlipidemia and

35

obesity5-7. Natural polyphenols have been received much attention because they are

36

commonly consumed and they have promising inhibitory effects on PL8-10. For

37

example, green tea (-)-epigallocatechin-3-gallate (EGCG) could inhibit PL activity11.

38

Apple oligomeric procyanidins and berry polyphenols were also proved to inhibit the

39

activity of PL and triglyceride absorption6, 12. Persimmon (Diospyros kaki L.) is

3



40

cultivated widely in China, Korea and Japan, and it is traditionally used for many

41

medicinal purposes and are related to various health benefits such as anti-oxidant,

42

anti-inflammation and anti-obesity13-15. High molecular weight persimmon tannin

43

(PT) is a highly galloylated, A-linked tannin contained in persimmon fruit, and it is

44

proved to be responsible for the numerous beneficial effects of persimmon16-18. Our

45

previous study suggested that PT is the main component accounting for the

46

anti-hyperlipidemic effects of persimmon fruits17, 19, but the underlying mechanisms

47

are unclear. Considering PT is highly polymerized and it could be hardly absorbed in

48

the small intestine, we proposed that PT might exert its lipid-lowering effect as

49

unabsorbable, complex structures with binding properties that can have local effects

50

in gastrointestinal tract. Therefore, the aim of the study was to evaluate the effects of

51

PT on the inhibition of PL and the possible inhibitory mechanisms. Fluorescence

52

spectroscopy, CD spectra, ITC study and molecular docking approaches were

53

applied to characterize the inhibitory effect and mechanisms. The results would shed

54

a light on the anti-hyperlipidemic mechanism of PT and persimmon fruit as well as

55

related persimmon foodstuffs.

56

Materials and methods

57

Chemicals

58

Porcine pancreatic lipase (100-400 U/mg, Type II, Sigma product L3126) was

59

purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). All other solvents and 4


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60

reagents were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai,

61

China) and were of analytical grade.

62

PT and dimers preparation

63

Mature and fully colored fruit of the astringent persimmon (Diospyros kaki Niuxin)

64

was harvested in late November from an orchard in Shan’xi province (China). After

65

harvest, fruit was held at 100 oC for 5 min to inactivate polyphenol oxidase, and then

66

stored deep frozen at -20 oC. The freezing persimmon fruit was cut into slices,

67

methanol extraction combined with macroporous adsorptive resin, lyophilized and

68

then powdered to prepare PT according to our previous reports. PT was

69

characterized by MALDI-TOF, thiolysis-HPLC-ESI-MS and NMR7. The mean

70

degree of polymerization of PT was estimated to be 26 by thiolysis. The proposed

71

structure was identified in our earlier papers7, 20 and was shown in Fig. 1A. The total

72

polyphenols content in PT was 98.7% on a mass basis by Folin–Ciocalteu method21.

73

The characteristic structural elements of PT: epicatechin-3-gallate-(4β→8, 2β→O→

74

7)-epicatechin-3-gallate

75

epigallocatechin-3-gallate-(4β → 8, 2β → O → 7)-epigallocatechin-3-gallate (A-type

76

EGCG dimer, Fig.1C) were separated from persimmon tannin as the method we

77

previously reported22 and were further purified by medium-pressure and

78

high-pressure preparative HPLC. Their purity and identity were confirmed by HPLC

79

and mass spectrometry. The purity of A-type ECG dimer and EGCG dimer were

(A-type

ECG

dimer,

5


Fig.

1B),


80

analyzed by HPLC and calculated to be 96% and 95% using procyanidin A2 as the

81

standard.

82

PL activity inhibition

83

The PL activity was determined by measuring the release rate of oleic acid from

84

triolein using spectrophotometry according to previous method23. Briefly, 21 g of

85

triolein (the substrate) and 100 mL of 4% polyvinyl alcohol (PVA) (the emulator)

86

were completely mixed on a vortex shaker for 5 min to form PVA-oil stock emulsion

87

with a concentration of 0.21 g/mL. 0.05 mL of various concentrations of PT (0.4–2.0

88

mg/mL), 0.05 mL of 0.75 mg/mL PL solution (0.1 mM PBS, pH 7.4) and 1.9 mL

89

PVA-oil substrate emulsion were incubated. After incubating the mixture at 37 °C

90

for30 min, 4 mL of toluene was added to terminate the reaction and extract the

91

generated oleic acid. After the mixture was centrifuged at 4000 r/min for 10 min),

92

and the upper toluene organic layer was taken out and colored by 1 mL of Cu2+ (5%

93

copper acetate, pH 6.1) for 15 min. Then the mixture was centrifuged (4000 r/min 10

94

min) again and the absorbance of the supernatant was measured at 710 nm with a

95

spectrophotometer (Hitachi, Tokyo, Japan). The PL activity was quantified by the

96

amount of oleic acid released. Kinetic parameters such as inhibition constant (Ki),

97

Vmax and inhibition mode were determined from Lineweaver-Burk and Dixon plots.

98

Substrate solution without PL was used as the blank. Each experiment was

99

conducted three times and data were expressed as mean±SD.

6


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Fluorescence spectroscopy measurement

101

The effects of PT, A-ECG dimer and A-EGCG dimer on the tryptophan fluorescence

102

spectra of PL were recorded on an F-4600 fluorescence spectrometer (Hitachi, Tokyo,

103

Japan)24. PL was prepared to be 2.0×10-6 M in PBS (0.1 M, pH 7.4), and PT or

104

dimers were in DMSO. An aliquot of PL solution in the absence (use DMSO as

105

control) or presence of PT or dimers (0, 10, 20, 40, 60×10-6 M) was incubated at 37

106

°C for 45 min. The 2-D fluorescence emission spectra were recorded in a 1 cm

107

quartz cell at λex=280 nm. The excitation and emission band widths were 5 nm. The

108

emission spectra were recorded from 300 to 400 nm. The 3-D fluorescence spectra

109

were recorded continuously at the wavelength of Ex/Em=200-600 nm.

110

The emission intensity was corrected for “inner optical filter effect” according the

111

equation25:

112

Fcorr =Fm×10(Aex+Aem) /2

113

Where Fcorr and Fm are the corrected and measured fluorescence, respectively. Aex

114

and Aem are the absorbance value at the excitation (280 nm) and emission (350 nm)

115

wavelength, separately. The intensity of fluorescence used in this study was the

116

corrected fluorescence intensity.

117

The fluorescence quenching mode was analyzed using Stern-Volmer equation and

118

the binding constant was determined from the following formula26:

7



119

F0/F=1+Ksv [Q] =1+τ0Kq [Q]

120

Where F0/F is the intensity ratio in the absence or presence of quencher (PT or

121

dimers), [Q] is the concentration of PT or dimers，τ0 is the average life of the

122

emissive excited state of PL (about 10-8 s). Kq is the quenching rate constant, Ksv is

123

the dynamic quenching constant. The Ksv is determined from the slope of

124

Sterm-Volmer plots.

125

According to the value of Kq, we could know the quenching mode was static

126

(complex formation) or dynamic (molecule collision). For the static quenching

127

interaction, the apparent binding constant (Ka) between small molecule and protein

128

and the number of binding sites (n) can be calculated from the static quenching

129

equation27:

130

Log [(F0-F) /F] =log Ka+ nlog [Q]

131

Circular Dichroism (CD) study

132

CD spectra were performed by Jasco-810 spectrophotometer (JASCO, Tokyo, Japan)

133

in cells of 1.0 mm path length28. The PL was prepared as a solution of 2.0×10-6 M in

134

PBS ((0.1 M, pH 7.4). PT, ECG dimer and EGCG dimer were prepared as a stock

135

solution of 20 mM in DMSO. 2 mL of PL solution and 2 µL of PT or dimers were

136

mixed. After incubation the mixture at 37 °C for 30 min, the spectra of samples were

137

measured and recorded from 190 to 250 nm. Three scans were conducted for each

138

spectrum. The SELCON3 method in DICHROWEB was applied to analyze the 8


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secondary structure of PL.

140

Isothermal Titration Calorimetry (ITC) study

141

ITC studies were carried out with MicroCal Auto-ITC200 calorimeter (Malvern, UK)

142

at 37 °C according to previous study28. The sample cell and syringe of the

143

calorimeter was washed by working buffer for 2 h before use. PT or dimers of 20

144

µM was set as titrate and PL solution of 0.4 mM was set as titrant. In all, total 20

145

injections of PT and dimers solution were titrated into PL solution at 3 min interval

146

with stirring at 1000 rpm/min. The volume of PT of dimers was 2 µL in each

147

injection. The control experiments were set that the PBS was titrant and PT or

148

dimers suspension was titrate. The raw data were integrated and normalized by use

149

of Origin ver7.0 (MicroCal Inc.).

150

Molecular docking study

151

The docking program Accelrys Discovery Studio (Vers. 2.5) was used to explore the

152

probable interaction between dimers and lipase. The X-ray crystal structure of PL

153

(PDB

154

(http://www.rcsb.org/pdb)29. The structures of ECG and EGCG dimer were created

155

with the Cambridge Soft ChemBioOffice Ultra (Version 14.0) and energy was

156

roughly minimized with a MM2 job. Subsequently, the structure was further

157

optimized by the Hartree-Fock calculations with the 6-31G (d, p) basis set HF/6-31G

158

(d, p)** of GAUSSIAN 09 code. The optimized conformations of A-ECG and

ID:1LPB)

was

retrieved

from

the

RCSB

9


Protein

Data

Bank


159

A-EGCG dimer were shown in Supplementary Fig. S1a, b. Before the docking

160

procedure, water molecules were removed from the crystal structure of PL and the

161

protein was cleaned including correct non-standard amino acids names and

162

incomplete residues, remove alternative conformations, and add hydrogens.

163

Discovery Studio LibDock module was applied to execute the docking. From the

164

docking results, the best scoring docked model which had the lowest docking energy

165

was selected to represent the most favorable binding mode of the compound

166

predicted by Discovery Studio.

167

Statistical analysis

168

All experiments were performed in triplicate, and the results were expressed as mean

169

value ± SD. Duncan’s test (p < 0.05) was applied to analyze the significance by

170

using SPSS Statistics software (Ver 19.0, SPSS Inc., Chicago, IL).

171

Results and discussion

172

Lipase inhibition

173

As shown in Table 1, PT exerted significant inhibitory effect on the PL activity with

174

the IC50 of 0.44 mg/mL. Enzyme kinetics study suggested that the inhibitory mode

175

of the PT towards PL belonged to the non-competitive type and the inhibitory

176

constant (Ki) was calculated to be 0.41 mg/mL. The inhibitory effect of PT on PL

177

was more potent than that of safflower extracts, chiisanoside and senna extract with

178

the IC50 of 0.56, 0.74 and 0.81 mg/mL, respectively30, 31, but it was less potent than 10


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orlistat (IC50=1.34 µg/mL), which was a novel clinical cholesterol lowering agent

180

and a positive control used in this study. Although PT is > 300 times less potent than

181

orlistat, the physiological likelihood of effectiveness of PT is possible. According to

182

the IC50 value, 880 mg PT was required to reach the gut (the volume of intestinal

183

juice is estimated to be 2000 mL). In fact, after 1 to 2 medium persimmon fruit

184

(200-300 g) was consumed daily by a person, the total amount of PT in the gut could

185

reach 1200-1800 mg (the content of PT in persimmon fruits is about 3% on dry

186

weight). Therefore, the physiological likelihood of effectiveness would be possible.

187

Although the PL used in this study derived from porcine, we analyzed the homology

188

of PL from porcine and human, and the sequence alignment between porcine

189

pancreatic lipase and human pancreatic lipase was shown in Supplementary Fig. S2.

190

Their sequence identity and similarity reached 86% and 93%, separately. The high

191

sequence homology suggested that PT could be expected to inhibit PL activity in

192

human gut.

193

Fluorescence spectroscopy

194

We used fluorescence spectroscopy to explore the interaction mode between PT and

195

PL. The fluorescence emission spectra of PL at various concentrations of PT

196

following the excitation at 280 nm was shown in Fig. 2. PL contains seven Trp

197

residues which give the intrinsic fluorescence of PL32, therefore, the change in the

198

intrinsic fluorescence intensity of PL can be applied to study interactions between PT

11



199

or dimers and PL. As shown in Fig. 2a, there was a clear fluorescence emission peak

200

near 351 nm, which belonged to Trp residues located at protein interior. With the

201

increased concentration (0-60 µM) of PT, the fluorescence intensity of PL was

202

considerably decreased, which was mainly caused by the microenvironment changes

203

of PL due to protein–polyphenol interaction. From 3D fluorescence spectrum of PL

204

(Supplementary Fig. S3), it was clearly observed that PL had two absorption peaks

205

at excitation wavelength of 350 nm (Fig. S3a, the red rectangle), with the addition

206

of PT, the fluorescence emission intensity of PL decreased (Fig. S3b, c). Because the

207

structure of PT is very complex, to study the interaction mechanism between PL and

208

PT is really challenging. To understand the possible mechanism by which PT

209

inhibited PL more fully and the structural requirements of PT for the inhibition, we

210

began with the characteristic structural elements of PT. The two characteristic

211

structural units of PT (A-ECG dimer and A-EGCG dimer) were subsequently used to

212

explore the interaction with PL. From Fig. 2b, c, we could see that 0-60 µM dimers

213

also caused a significant decrease in the fluorescence intensity of PL. Especially for

214

A-ECG dimer, it caused a similar degree of decrease in the fluorescence intensity as

215

that of PT. The 3D fluorescence spectrum of PL with dimers (Fig. S3d-h) revealed

216

that dimers decreased the absorption peaks at excitation wavelength of 350 nm,

217

which was consistent with the effect of PT. Even though the PT, A-ECG dimer and

218

A-EGCG dimer exhibited different quenching effects on PL, the absorption

219

characteristics of PT and dimers with PL was very similar, indicating that PT and 12


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dimers might interact with PL in the same mode. The fluorescence quenching

221

parameters for the interactions of PT and dimers with PL were presented in Table 2.

222

The Stern–Volmer plots (Supplementary Fig. S4a, b) for the quenching of PL by

223

PT or dimers showed that they all exhibited a good linear relationship within the

224

studied concentrations, suggesting a single type of quenching, either static or

225

dynamic quenching occurred in the formation of polyphenol–PL complex33. The

226

values of Kq calculated from the plots of linear equation (F0/F vs. [Q]) were 1.09,

227

5.23, 2.04 ×1012 L/(mol·s) for PT, A-ECG and EGCG dimers, separately, which were

228

all

229

macromolecule-participating quenching rate constant in dynamic quenching34.

230

Therefore, we concluded that the process of quenching is not the dynamic quenching

231

induced by the collision of molecules, but the static quenching by forming a complex.

232

We then calculated the binding constant (Ka) of PT to be 6.84×104 L/mol by the

233

slope value of the regression curve based on static quenching plots (Supplementary

234

Fig. S4c). The number of binding sites for the PT-PL complex was approximately

235

equal to 1, suggesting the presence of a single class of PT binding on PL, which was

236

agreement with the results obtained from enzyme kinetics study. As for A-ECG and

237

EGCG dimers, the calculated Ka were 4.04, and 2.77×104 L/mol, separately, and the

238

binding sites were also about 1. PT has a higher Ka value than either dimer,

239

indicating the higher affinity of PT for PL than that of the dimers.

240

CD studies

higher

than

the

maximal

value

(2.0×1010

13


L/(mol·s))

for


241

The effects of PT and dimers on the secondary structure of PL were evaluated by CD

242

spectroscopy. The percentage of the PL secondary structural elements derived from

243

the spectra were listed in Table 3. We found on complexation of PL with PT, the

244

α-helix content of PL increased from 18 to 67%, the β-sheet content decreased by

245

about 20%, and the unordered structure content decreased from 38 to 27%,

246

suggesting that PT disrupted the PL conformation severely. While adding the same

247

concentration of A-ECG dimer or A-EGCG dimer into PL solution, an increase of

248

α-helix and a decrease of β-sheet was also observed, but the influence of dimers on

249

the secondary structure of PL was less potent than that of PT. These results suggested

250

that the binding of PT and dimers to PL caused conformational changes of the

251

enzyme which was consistent with the result of fluorescence study. The quenching of

252

PL fluorescence with PT and dimers revealed a change in polarity of the fluorophore

253

environment and the CD study also demonstrated the conformation alteration after

254

binding PT or dimers.

255

ITC studies

256

ITC is an attractive approach for studying interactions between bioactive compounds

257

and protein. It sensitively measures the enthalpy changes during ligand and protein

258

interaction in a calorimeter cell held. Additionally, ITC provides thermodynamic

259

properties of protein-ligand interactions by measuring the binding enthalpy

260

changes35. In this study, ITC was applied to determine the thermodynamic properties

14


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261

of the binding interaction of PT or dimers and PL. The results were shown in Fig. 3.

262

It was seen the interaction was typically exothermic. All the curves were typical of

263

enthalpy-driven protein-ligand interactions, with relatively decreasing exothermic

264

peaks and the number of available binding sites on PL upon PT or dimers addition.

265

As analysis listed in the Table 4 , The △G value was negative, indicating the

266

interaction was spontaneous. The Ka of PT was 6.22×104 L/mol, which was slightly

267

lower than the Ka calculated from fluorescence spectroscopy. The difference might

268

be due to the different sensitivity of the methods. The Ka of dimers were 3.98 and

269

2.65×104 L/mol, respectively. In all cases, the binding constants were higher than

270

1×103 L/mol, suggesting a strong interaction between PT and its structural units and

271

PL occurred. This result was in accordance with the data obtained from fluorescence

272

spectroscopy. The enthalpies were too low for covalent bond formation (200−400

273

kJ/mol), which suggested that the interaction of PT or dimers and PL was

274

non-covalent36. The partial immobilization of a protein and ligand occurs in an initial

275

step involving hydrophobic association, which results in a positive ∆S37.

276

Molecular docking studies

277

It is considered that the non-covalent binding is the main mode of the interaction

278

between polyphenols and protein driven by non-covalent forces such as hydrogen

279

bonds or electrostatic interaction. Because PT, A-ECG and EGCG dimers contain

280

various hydroxyl and galloyl groups, the polar phenolic groups might serve as donor

15



281

to form hydrogen bonds with polar groups of PL. And results from ITC study also

282

suggested that van der Waals interaction and hydrogen bonding could be formed

283

between PL and PT or dimers, and such non-covalent interaction might result in the

284

conformational change of PL as we observed in the CD study.

285

To confirm whether hydrogen bonds were responsible for the interaction, the

286

molecular docking approach was used. The docking results were presented in Fig. 4.

287

The A-ECG dimer was surrounded by 22 amino acid residues, including ALA-40,

288

LEU-41, ASN-88, LYS-239, ASP-247, ILE-248, ASP-249, GLU-253, GLY-254,

289

ASP-257, ARG-265, LYS-268, THR-271, GLY-330, ASP-331, ALA-332, SER-333,

290

ASN-334, PHE-335, ARG-337, LYS-367 and ASP-389 and hydrogen-bonding

291

interaction was observed with GLU-253, ASP-257, ARG-265, ALA-332, SER-333,

292

ARG-337 and LYS-367 residues of PL. Moreover, two additional pi-interaction was

293

observed between A-ECG dimer and LYS-268 and ARG-337 (Fig. 4A). Similarly,

294

A-EGCG dimer interacted with 13 amino acid residues, such as ILE-248, ASP-249,

295

GLU253, ARG-256, ARG-265, TYR-267, LYS-268, THR-271, ASP-272, ALA-332,

296

SER-333, ASN-334, and PHE-335. ASP-249, TYR-267, SER-333 residues could

297

form hydrogens bonds with EGCG dimer and one pi-π interaction between LYS-268

298

with EGCG dimer (Fig. 4B). These results indicated that the amino acid residues of

299

PL participated in hydrogen bonding and pi–π interaction with the phenolic

300

backbone and the galloyl moieties. The docking results revealed these extensive

301

hydrogen-bonding interactions might play an important role in the strong binding 16


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302

affinity of PT or dimers to lipase.

303

The catalytic site of PL was a SER-HIS-ASP trypsin-like catalytic triad with an

304

active serine being buried under a short helical fragment of a long surface loop38. It

305

was suggested that residues of SER-194, HIS-435, and ASP-320 were the catalytic

306

sites of porcine lipase39. Actually, it was reported that the clinical lipid-lowering drug

307

orlistat exert lipase inhibitory effect by binding with the catalytic residues SER-2340.

308

In contrast, our docking results showed that these residues did not surround ECG

309

dimer or EGCG dimer, indicating the binding mode was non-competitive which was

310

in agreement with the result of enzyme kinetics study.

311

As a crucial enzyme in hydrolysis of triglycerides, PL plays an important role in

312

blood lipid level. Polyphenols from green tea, oolong tea, berry and apple were

313

reported have the capacity to inhibit PL activity6, 12, 41, 42. However, the inhibitory or

314

binding potential of polyphenols on PL is highly related with the structure of

315

polyphenols. It was shown that highly polymeric proanthocyanidins from the seed

316

shells of the Japanese horse chestnut, cranberry and blueberry fruit exerted greater

317

inhibitory effect on PL than EGCG43. Phenols containing galloyl groups had higher

318

binding capacity to proteins44, because each galloyl group provided three hydroxyl

319

groups and a benzene ring, which could form hydrogen and hydrophobic bonds with

320

proteins. In addition, it was also observed that molecular size and flexibility could

321

influence the binding of polyphenols to proteins significantly45, 46. Gonçalves et al.

17



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322

(2010) suggested that the inhibition of procyanidin fractions on PL increased with

323

degrees of polymerization47. According to previous studies, higher molecular sizes

324

and more abundance of A-type bonds in polymeric proanthocyanidns are key

325

structural requirements of polyphenols for the potent PL inhibition activity which the

326

higher degree of polymerization, the stronger enzyme inhibition activity12, 48. Our

327

previous work showed that PT had a unique structure with highly polymeric (with

328

mean DP of 26), highly galloylated (about 72%) and doubly linked A-type

329

interflavan linkages besides the more common B-type interflavan bonds7. We

330

proposed the high degree of galloylation and polymerization as well as the A-type

331

interflavan bonds of PT might contribute significantly to its gastrointestinal lipase

332

inhibitory effect.

333

Although ECG and EGCG dimers have similar molecular weight and the same

334

number of galloyl groups, they showed different effects on PL. ECG dimer seemed

335

more effective in quenching PL fluorescence, and in altering PL secondary structures.

336

The difference might be due to the difference in the spatial configuration and

337

hydrophobicity property of the two compounds. This result suggested that except for

338

the degree of galloylation and polymerization, other structural characters such as

339

molecular size, hydrophobicity might also affect the binding of polyphenols to PL.

340

Beside the potent inhibition on PL, PT also inhibited starch digesting enzymes such

341

as α-amylase and α-glucosidase effectively49. Previous studies showed that the

342

digestion of carbohydrate may impact on the levels of blood lipid50, 18


51

. In the

Page 19 of 41


343

digestive tract, when the carbohydrate digestion is inhibited, the blood lipid level

344

would also be affected. The anti-digestive activity of PT on carbohydrate may partly

345

and indirectly contribute to its lipid-lowering effect of PT. Due to the very complex

346

structure of PT, studying the interaction mechanism between enzyme and PT is

347

challenging. Therefore, in exploring the possible mechanism by which PT inhibited

348

PL, we also included the characteristic structural elements of PT (A-type EGCG and

349

ECG dimer), which were proved to be the structTural requirements for the

350

interaction between PT and snake venom PLA252. In our previous studies, we found

351

the IC50 of PT on PLA2 was 0.88 mg/mL, while the values of these two dimers were

352

about 9.0 mg/mL. In our preliminary study, we observed similar tendency on PL.

353

Theses results indicated that polymers were more potent on inhibiting the activitity

354

of enzymes than dimers. Although both A-EGCG dimer and A-ECG dimer did not

355

quench the fluorescence of PL as effective as PT (Fig. 2), and PT has a higher Ka

356

value than either dimer, the effects of A-ECG on PL secondary structure were similar

357

to that of PT. (Table 3). In addition, Although PT had a significant different structure

358

from the dimers and it seemed fetched that results from docking on the dimers gave

359

information on what the PT was doing, data from the enzyme kinetics study

360

demonstrated that the inhibition mode of PT against PL was non-competitive, fitting

361

with the docking results with the dimers well. These results suggested that the PT

362

and dimers might interact with PL in the same mode as that of the dimers. Therefore,

363

data from the characteristic structural dimers might provide some reference for better 19



364

understanding the interaction between PT and PL.

365

Generally, enzymes exert catalytic activity only in specific spatial conformations,

366

and when their conformations are altered, their catalytic activity will be greatly

367

influenced. Taken together, the data from the enzymatic kinetics, fluorescence

368

spectroscopy, CD and ITC studies proved that PT had a high affinity to PL and the

369

non-covalent bonding interaction between PT and PL through hydrogen bonds, pi-pi

370

stacking and electrostatic interaction could alter the molecular conformation of PL,

371

thus decreasing the catalytic activity of PL. As the potential inhibitory effect of PT

372

on lipid digestive enzymes, PT as a lipase inhibitor may have potency for the

373

treatment and prevention of obesity.

374

Supporting Information

375

The 3D conformation of A-type ECG and EGCG dimers and their binding with PL

376

and sequence alignment of porcine pancreatic lipase and human pancreatic lipase as

377

well as the 3D fluorescence quenching graphs, Stern-Volmer plots and static

378

quenching plots of PT and dimers.

379

Funding

380

This study was supported by the National Natural Science Foundation of China

381

(No.31571839).

382

Conflict of Interest

20


Page 20 of 41

Page 21 of 41


383

The authors have declared no conflicts of interest.

384

385

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552

553

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555

29



556

557

FIGURE CAPTIONS.

558

Fig. 1 The chemical structures of persimmon tannin (PT) and its characteristic

559

structural units A-type ECG dimer and EGCG dimer.

560

561

Fig. 2 Fluorescence quenching effect of PT and A-ECG dimer and A-EGCG dimer

562

on lipase fluorescence intensity, λex = 280 nm; lipase = 2 × 10−6 M; polyphenols

563

concentration increased (a-f) from 0 to 10, 20, 30, 40, and 60 (×10−6 M) at 37 °C.

564

565

Fig. 3 Results of isothermal titration calorimetry for (A) PT and (B) A-ECG dimer

566

and (C) A-EGCG dimer binding to lipase: (upper) raw data plot of heat flow against

567

time for the titration of PT or dimers into lipase; (below) plot of the total heat

568

released as a function of ligand concentration for the titration. The continuous black

569

line represented the best least-squares fit for the obtained data. The thermodynamic

570

parameters analyzed from ITC plots for PT or dimers binding to PL were listed in

571

the table.

572

573

Fig. 4 Best-docked conformations of A-ECG dimer−lipase (A) and A-EGCG

574

dimer-lipase (B) complexes. A-ECG dimer and A-EGCG dimer were shown in line 30


Page 30 of 41

Page 31 of 41


575

model (the white molecule) while lipase was shown in secondary structure model.

576

The amino acid residues thought to interact with A-ECG dimer and A-EGCG dimer

577

were shown as a 2-D representation by use of 2-D interaction diagram in Discovery

578

Studio. The violet circle represented the residues involved in hydrogen bonds or

579

electrostatic interactions. The green circle represented the residues involved in Van

580

der Waals interaction.

31



Table 1 Enzymatic Kinetic Parameters of PT against PL Parameters

Values

IC50 (mg/mL)

0.44±0.02

Inhibition type

Non-competitive

Vmax (µmol/(mL·min)) a

a

:

0 mg/mL PT

12.50±0.38a

0.40 mg/mL PT

5.26±0.23b

Vmax was determined at control, 0.4 mg/mL of PT, respectively. The values having different superscripts in the same column

are significantly different (p

Inhibitory Effect of Persimmon Tannin on Pancreatic Lipase and the

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