Oligomer procyanidins from lotus seedpod (LSOPC) regulates lipid

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Bioactive Constituents, Metabolites, and Functions

Oligomer procyanidins from lotus seedpod (LSOPC) regulates lipid homeostasis partially by modifying fat emulsification and digestion Xiaopeng Li, Ya Chen, Shuyi Li, Mo Chen, Juan Xiao, Bijun Xie, and Zhida Sun J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01469 • Publication Date (Web): 04 Apr 2019 Downloaded from http://pubs.acs.org on April 4, 2019

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Journal of Agricultural and Food Chemistry

Oligomer procyanidins from lotus seedpod (LSOPC) regulates lipid homeostasis partially by modifying fat emulsification and digestion Xiaopeng Li†,⊥; Ya Chen†,⊥; Shuyi Li§; Mo Chen#; Juan Xiao‡; Bijun Xie†; Zhida Sun†* †

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

430070, China § College

of Food Science and Engineering, Wuhan Polytechnic University, Wuhan, 430023,

China #College

of Informatics, Huazhong Agricultural University, Wuhan, 430070, China

‡College

of Food Science and Technology, Hainan university, Haikou, 570228, China

⊥These

authors contributed equally to this work.

Correspondence author: Zhida Sun. Email:[email protected]; Tel.: +86-27-87283201

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ACS Paragon Plus Environment


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ABSTRACT

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Dietary polyphenols have shown hypolipidemic effects by reducing triglyceride

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absorption. The mechanisms may involve modifying fat emulsion during digestion in

4

the gastrointestinal and suppressing the lipase during hydrolysis in the small intestine.

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In vivo study, LSOPC decreased total serum triglyceride, total cholesterol, and

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elevated high-density lipoprotein level in hyperlipidemic rat model. In addition,

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LSOPC suppressed de novo lipogenesis-related genes expression. In vitro study,

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LSOPC-enriched emulsion decreased the mean droplet size from 0.36 μm to 0.33 μm

9

and increased the viscosity of the emulsion. Moreover, LSOPC-enriched emulsion

10

improved the antioxidant properties. A digestion model was developed and showed

11

that the particle size of LSOPC-enriched emulsion increased in the oral cavity. But, an

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increase and then a significant drop of particle size was measured in the stomach and

13

small intestine. The free fatty acids (FFA) release rate was decreased in

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LSOPC-enriched emulsion partly ascribed to the inhibition of lipase by LSOPC.

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Key words: LSOPC; submicrometer emulsion; hypolipidemic; digestion; FFA release

16 17 18 19 20 21 22 2


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INTRODUCTION

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Lipid homeostasis is crucial in maintaining human health. A chronic lipid

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accumulation may lead to atherosclerosis, insulin resistance, and obesity1-2. These

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metabolic diseases are responsible for the majority of deaths in western countries. The

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elevated rate of obesity and diabetes also occurred in developing countries because of

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the acquisition of a westernized lifestyle3. A healthy, balanced diet containing

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polyphenols could be a promising dietary strategy in regulating lipid metabolism.

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Dietary lipid digestion undergoes several complicated steps before absorption by

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the mucosa of the small intestine. Following the intake of fats, a slight hydrolysis

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occurs by lingual lipase and subsequently travels to the stomach. When food is

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present, the stomach becomes active. The churning in the stomach propelled by its

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strong muscle disperses the fat into small droplets4. Still, little fat digestion by gastric

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lipase takes place in the stomach. When lipid enters the small intestine, it triggers the

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release of bile5. Act as an emulsifier, bile's emulsifying action converts large fat into a

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small droplet, making fat digestion more efficient as they encounter lipase enzymes6.

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Most of the hydrolysis occurs in the small intestine by pancreatic lipase. The end

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products of fat digestion, mostly monoglycerides, fatty acids, and very little glycerol,

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move directly into bloodstream or form micelles and then are absorbed by

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enterocytes4. During these processes, lipid digestion and absorption are fundamental

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processes for maintaining the lipid and lipoprotein metabolism. Thus, any change of

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this complicated digestion steps may influence fat digestion and absorption.

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Procyanidins are a class of naturally existing polyphenolic compounds that are 3



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widely found in dairy foods, which makes them essential in the human diet. Studies

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have strongly suggested that procyanidins protect against cardiovascular disease

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(CVD)7-8. Although the antioxidant and anti-inflammatory properties of procyanidins

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play an important role in this protection, one of the mechanisms in which

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procyanidins exert cardiovascular protection is regulating lipid homeostasis9. Animal

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and human studies show that procyanidins not only reduce the plasma triglyceride

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(TG) and low-density lipoproteins (LDL) but also improve serum cholesterol

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profile10-11. Consumption cranberry juice containing procyanidins improved several

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risk factors of CVD in adults, including blood TGs12.

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Researches also showed that polyphenols have inhibitory effects on lipase in

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vitro13-14, suggesting that fat digestion may be inhibited when lipid enter our body

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together with polyphenols. Procyanidins are also known as condensed tannins, and

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their main constitutive units are (+)-catechin, (−)-epicatechin, (+)-gallocatechin and

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(−)-epigallocatechin 3 gallate. LSOPC are linked through a C4—C8 or C4—C6 bond.

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The structure (aromatic hydrophobic rings, hydrophilic hydroxyl groups) suggests that

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procyanidins may have surface active properties, and thus stabilize emulsions15.

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Therefore, changing in the emulsion droplet size and inhibition of lipase may have a

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significant role in regulating fat digestion and stability. However, there is still limited

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information on the mechanism of how LSOPC may influence fat emulsion,

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absorption, and the mechanism.

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To gain understanding the mechanism of LSOPC in regulating lipid homeostasis in

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vitro and in vivo, an oil in water emulsion system mimicking the physiological state in 4


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the small intestine was prepared and hypolipidemic effects were studied. This study

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mainly focuses on the effects in LSOPC on changes of fat emulsification, antioxidant

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activity, and fat digestion. Meanwhile, the assessments of blood lipid profiles and

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lipid metabolism were tested.

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MATERIALS AND METHODS

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Materials. Oil phase, peanut oil, was kindly provided by Oil Crops Research

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Institute, Chinese Academy of Agricultural Sciences. Surfactants (Tween 80 and Span

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20) and bile salt were bought from Sinopharm Chemical Reagent Co., Ltd. DPPH,

75

lipase from porcine pancreas (typeⅡ), pancreatin, uric acid, mucin, pepsin, and BSA

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were provided by Sigma-Aldrich (Shanghai, China). p-Nitrophenyl palmitate (pNPP)

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was bought form Aladdin company (Shanghai, China). The TG, TC, and HDL kits

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were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

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Isolation of Lotus Seed Oligomeric Procyanidins (LSOPC). The frozen lotus

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seed of Nelumbo nucifera Gaertn. (Number 2 Wuhan plant) was extracted to obtain

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LSOPC according to our laboratory method16. Lotus seed procyanidins was extracted

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by ethyl acetate to obtain the LSOPC, including 10.9% (+)-catechin, 9.1%

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(-)-epicatechin,53.6% dimer, 19.5% trimer and 1.9% tetramer by LC-MS analysis17.

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Its purity was 99.35 ± 0.79% compared to that of commercial grape seed procyanidins

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measured by Butanol-HCl assay. The LSOPC is stable during the storage of -18 ℃.

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Emulsion Preparation. Emulsions formation was prepared using a spontaneous

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emulsification method according to previous literature with some minor

88

modifications18. Spontaneous emulsification was conducted by addition of an aqueous 5



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phase with LSOPC dissolved (0.035%, 0.055% and 0.1% in pH 7.0 phosphate buffer)

90

to an organic phase (containing peanut oil and non-ionic surfactant) while

91

magnetically stirring this system (DF-101S magnetic stirrer, Wuhan Keer Instrument

92

Co., LTD, China). For preparing the organic phase, 12 g surfactant mixture (Tween

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80: Span 20=3:1) was mixed and heated to 50 ℃, then adding 4 g peanut oil while

94

continuing stirring this system. The aqueous phase was slowly added to the organic

95

phase. In our study, the experiments were conducted using standard conditions: (1) oil

96

content of 4% (w/w), surfactant content 12% (w/w), and aqueous content of 84%

97

(w/w). (2) magnetic stirrer speed of 400 r/min at 40 ℃ for 20 min.

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Particle Size and Zeta Potential Measurements. The particle size was measured

99

by Mastersizer 2000 particle size analyzer equipped with He/Ne laser (Malvern, UK).

100

To avoid multiple scattering effects, samples were diluted before the particle size

101

measurements. The calculation of particle size was followed by the difference

102

between dispersive medium and sample using Mie theory. In our study, d43 was used

103

to represent the particle size.

104

Temperature and pH Stability. The effects of temperature and pH on particle size

105

were investigated by incubating the emulsion at different temperature (4-80 ℃) for

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0-72 h or preparing the aqueous phase using different pH phosphate buffer prior to

107

measurement of particle size.

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Rheological Property of LSOPC-Enriched Emulsion. The rheological

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characteristic of the emulsion was monitored with AR2000ex controlled strain

110

rheometer with Peltier plate system (TA Instrument, New Castle, USA)19. 6


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Steady-state flow curves were obtained using parallel plates (60 mm diameter and 1.0

112

mm gap) at 25 ℃ in the shear rate ranging 0.1–100 s-1.

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The Distribution of LSOPC in Emulsion System. The emulsion system was kept

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at 25 ℃ for 1-13 days. First, the distribution of LSOPC was conducted by

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centrifugation of LSOPC-enriched emulsion in the condition of 12,000 g at 4 ℃ for

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30 min to separate aqueous phase. Then, the content of LSOPC was measured using

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Butanol-HCl assay.

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Scavenging DPPH Free Radical and Reducing Power Determination.

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DPPH· scavenging activity was determined using LSOPC-enriched emulsion and

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LSOPC solution. First, different concentrations of LSOPC-enriched emulsion and

121

solution were prepared. DPPH solution was prepared in ethanol, 2 mL 0.2 mmol/L of

122

this solution was mixed with 2 mL the sample solutions. The mixture was incubated

123

for 30 min in the dark at room temperature. Centrifuge the samples at 13,000 r/min

124

for 20 min (Beckman Microfuge 20R, Shanghai, China) before the absorbance was

125

read at 517 nm.

126

The reducing power of LSOPC enriched emulsion and solution was determined

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according to the previously published method20. The different concentration of

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emulsion and solution 1.0 mL were mixed with 1.0 mL 2.5% w/v potassium

129

ferricyanide. To reduce ferricyanide into ferrocyanide, the mixture was incubated at

130

50 ℃

131

trichloroacetic acid followed by centrifugation at 13,000 r/min for 10 min (Beckman

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Microfuge 20R, Shanghai, China). 2.0 mL of the upper layer solution was collected

for 20 min. The reaction was stopped by adding 5.0 mL 10% (m/v)

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and mixed with 2.0 mL distilled water and 0.5 mL 0.1% ferric chloride (w/v). The

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absorbance of the mixed solution was read at 700 nm.

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Antioxidant Activity of LSOPC in Oil-in-Water Emulsions. The lipid

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peroxidation inhibition was determined by peroxide value (POV) and thiobarbituric

137

acid reactive substances (TBARS). In the first step, emulsion with LSOPC and TBHQ

138

were prepared. Then put the emulsion in a glass tube at 60 ℃ for oxidation. Samples

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for POV and TBARS were collected at intervals of 3 days throughout 12 days.

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POV was determined according to previous literature. 0.3 g samples were collected

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into a centrifuge tube and mixed with 1.5 mL isooctane/ isopropanol mixture (3:1

142

v/v), then were centrifuged at 1,000×g for 2 min. 200 μL of upper layer solution

143

was mixed with 2.8 mL methanol/butanol mixture (2:1 v/v), 15 μL ammonium

144

thiocyanate and (3.94 mol/L) and 15 μL Fe2+ solution (0.132 mol/L BaCl2 and 0.144

145

mol/L FeSO4 v/v) for 20 min. The mixture solution was filtered with 0.22 μm filter

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membrane and was read at 510 nm.

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TBARS was detected according to Tan's method21. 2.0 mL solution containing 15%

148

(w/v) trichloroacetic acid (TCA), 0.375% (W/V ） thiobarbituric

149

hydrochloric acid (0.25 mol/L) was added to 1.0 mL emulsion and mixed, followed

150

by 100 ℃ water bath for 20 min to accelerate the formation of a pink pigment

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generating form the reaction with oxidation products malondialdehyde. Afterward, the

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mixture was cooled rapidly to room temperature, centrifuged at 5,000 r/min for 10

153

min (Microfuge 20R, Beckman Coulter, Shanghai, China) and filtered with the 1.2

154

μm filter membrane. The absorbance of the filtrate was determined at 532 nm. The 8


acid (TBA) and

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standard

curve

was

calculated

from

the

TBARS

reaction

with

156

1,1,3,3-tetraethoxypropane. The results were expressed as μmol of malonaldehyde

157

(MDA).

158

The Interaction of LSOPC-Enriched Emulsion and BSA. The fluorescence

159

spectra were recorded with a RF5301 spectrofluorometer (Shimadzu Scientific

160

Instruments, Japan). The fluorescence spectra were recorded under thermostated

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conditions (25 ℃ and 35 ℃), using 5 nm excitation and 5 nm emission slit widths.

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Prepared the emulsion containing 0-0.1% LSOPC and 0-0.1% LSOPC solution. BSA

163

concentration was kept constant (4 µM) in Tris-Hcl buffer (pH 7.4). LSOPC enriched

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emulsion was added to BSA solution, and keep at 25 ℃ or 35 ℃ 10 min. The

165

mixture was centrifuged at 13,000 r/min for 20 min (Beckman Microfuge 20R,

166

Shanghai, China) and supernatant was collected for measuring fluorescence intensity.

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The excitation wavelength was 280 nm, and emission spectra were recorded between

168

300 and 450 nm. The changes in fluorescence emission intensity were measured after

169

exactly 1 min of adding each sample to the protein solution. Emission of LSOPC

170

enriched emulsion or solution without BSA was subtracted to correct background

171

fluorescence.

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Effect of LSOPC on Digestion in vitro in Emulsion System. The in vitro

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digestion model used in this study was a modification of those described

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previously22-23. Stimulated oral, gastric and intestinal digestion juice components were

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summarized in Table1. The stability of emulsion in simulated gastrointestinal medium

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was determined by subjecting the 10 g LSOPC enriched emulsion to 10 g simulated 9



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oral juice at pH 6.8 in the presence of α-amylase and mucin for 5 min, followed by

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simulated gastric digestion (10 g) at pH 2.0 in the presence of pepsin for 2 h and

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simulated intestinal digestion (30 g) in the presence of pancreatin-lipase-bile mixture,

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pH 7.0 at 37 ℃for 2 h. After each phase of incubation, samples were collected for

181

measuring size particles and zeta potential.

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Free Fatty Acid (FFA) Release. The FFA release test was monitored according to

183

the previously published work with some modifications22. At the simulated intestinal

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digestion phase, free fatty acid is released from the emulsion when lipase interacts

185

with triacylglycerols. To maintain the pH of intestinal digestion juice at 7.0, 0.1 mol/L

186

NaOH was titrated into the mixture. The volume of NaOH added to the emulsion was

187

recorded during this process and was used to calculate the concentration of free fatty

188

acids generated by lipolysis.

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FFA (%) =

CNaOH * VNaOH * MTG 2 * mTG

×100%

190

where CNaOH is the concentration of NaOH, VNaOH is the volume of sodium

191

hydroxide required to neutralize the FFA produced, MTG is the molecular mass of the

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triacylglycerol (g/mol), MTG is the total mass of triacylglycerol oil (peanut oil) present

193

in the digestion cell.

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Surface and maps of components and surfactants. The structures of the

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molecular used in our study were drew using ChemDraw software (version 10.0). All

196

of the molecular surface analysis was operated in MOE software (version 2014). Red

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represents the hydrophilic site, while blue stands for the lipophilic site (White for

198

neutral). AlogP values of all the molecular were also obtained from MOE software 10


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using the LigX tools. The higher value of AlogP, the easier the molecular dissolves in

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the fat.

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Measurement of lipase activity. The lipase inhibition assay was measured

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according to the previous study with some modifications24. p-Nitrophenol palmitate

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(pNPP) was used as the substrate which was hydrolyzed by lipase to p-nitrophenol

204

(pNP). In brief, 450 μL 0.05 M sodium phosphate buffer (pH 7.6) was mixed with 50

205

μL pNPP in isopropanol (0.01 M) and 20 μL 0.1 mg/mL LSOPC in centrifuge tube. 5

206

μL of lipase enzyme solution (0.5 mg/mL) was added and incubated at 37 ℃ for 10,

207

20, 30, 45 min. 200 μL of the reaction mixture was taken and added to 96 well plates.

208

The absorbance was measured at 410 nm.

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Lipase inhibition%=[1 ― ( ATest ― Acontrol )] × 100%

210

ALSOPC ― Ablank

where A

LSOPC

is the absorbance of the mixture of LSOPC, pNPP solution, and

211

enzyme solution; A blank is the absorbance of the mixture of the LSOPC sample, pNPP

212

solution without enzyme; A

213

LSOPC), pNPP solution and enzyme; Acontrol is the absorbance of the mixture of

214

buffer, pNPP solution without enzyme.

test

is the absorbance of the mixture of buffer (instead of

215

Animal study. Seventy SD rats were obtained from laboratory animal center,

216

Huazhong University of Science and Technology. All the animal procedures were

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performed in accordance with the Chinese legislation on the use and care of

218

laboratory animals and were approved by the ethics committee of Huazhong

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University of Science and Technology. After 7 days of acclimatization, rats were

220

divided into two groups: negative control (n=15) and positive control (n=55). The 11



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normal diet for negative control group (LAD 2001, crude protein ≥ 18%, fat ≥5%,

222

fiber ≤ 5%) and model diet for positive control group (TP0800, 20% sucrose, 15%

223

lard oil, 1.2% cholesterol and 0.2% sodium cholate) were supplied by Trophic Animal

224

Feed High Tech Co. Ltd, China. Two weeks later, blood samples of the tails from

225

non-fasting rats were collected and TC, TG, and LDL were tested. Then positive

226

control group was randomly divided into 4 groups (positive control, 0.05 g/kg·bw

227

LSOPC, 0.1 g/kg·bw LSOPC, 0.15 g/kg·bw LSOPC), and each group contains 10

228

rats.

229

LSOPC dissolved in distilled water, and negative control and positive control were

230

treated distilled water. After the end of the experiment (28 days), non-fasting tail

231

blood samples were collected and stored in -80 ℃ prior to analysis.

The rats in 0.05, 0.1 and 0.15 g/kg·bw LSOPC group were administrated

232

All 10 male ICR mice (20±2 g) were bought from Experimental Animal Center of

233

Disease Prevention and Control of the Hubei Province (Wuhan, China). The animals

234

were kept five per cage and allowed access to diet (TP 23302) and water. The animal

235

facility was maintained at room temperature 25±2 ℃. The composition of TP 23302

236

was listed in supplementary Table 3. After 1 week of acclimatization, mice were

237

divided to two groups: normal control (NC, n=5) and LSOPC group (NC + LSOPC,

238

n=5). LSOPC group were administrated by gavage with LSOPC at a dose of 150

239

mg/kg·bw, while normal group was treated with water. 6 weeks later, all mice were

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fasted overnight and sacrificed. The livers of mice were immediately removed and

241

stored at -80 ℃.

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Quantitative Real-Time RT-PCR. Total RNA from livers were homogenized and 12


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extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to

244

manufactory’s instructions, and cDNA was synthesized using a first-strand cDNA

245

synthesis kit (GeneCopoeia,Rockville, MD, USA). The reaction mixture was

246

incubated as follows: 25 ℃ 5 min, 50 ℃ 15 min，85 ℃ 5 min，4 ℃ 10 min.

247

Diluted cDNA (1:4) were subjected to quantitative RT-PCR amplification using the

248

SYBR Green PCR Master Mix (Toyobo) according to manufacturer's protocol. The

249

reaction mixtures were incubated at 95 ℃ for 10 min, followed by 40 cycles of

250

incubation at 95 ℃ for 30 s, then 60 ℃ for 30 s. The sequences of the primers used

251

in this study are shown in supplementary Table 4. The expression of mRNA values

252

was calculated using the threshold cycle value (CT). For each sample, ΔCT sample was

253

calculated through analyzing the difference between CT value of the target gene and

254

that of reference gene (NC group). The relative expression levels were estimated by

255

analyzing the ΔΔCT (ΔCT sample-ΔCT reference) and using the 2-△△Ct method.

256

Statistical analysis. The data were analyzed by using SPSS 16 software, according

257

to Student's t-test (between 2 groups) or analysis of variance (ANOVA, more than 2

258

groups; p < 0.05 or p < 0.01). All results are expressed as the mean ± standard error.

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RESULTS

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Hypolipidemic Effects of LSOPC in Animal Study. No significant changes of

261

TG, TC, and HDL in the negative control group during the period of the experiment

262

(Table 2). In the positive control group, TG and TC were dramatically increased from

263

0.76 ± 0.28 to 1.32 ± 0.15, and 1.88 ± 0.36 to 3.89 ± 1.10, respectively. Moreover, the

264

HDL level was decreased to 0.57 mmol/L. After 4 weeks of LSOPC treatment, all 13



265

LSOPC treated groups distinctly lowered blood TC, TG and elevated HDL level in

266

rats (Table 2). A 29.5% reduction in TG, 23.9% decrease in TC and 35% elevation in

267

HDL were observed in 0.05% LSOPC group compared to that of the positive control

268

group (Table 2). In Figure 1 A, the genes expression of sterol regulatory

269

element-binding protein 1 c (SREBP-1c), fatty acid synthase (FAS), acetyl-CoA

270

carboxylase 1 (ACC1), peroxisome proliferator-activated receptor gamma (PPARγ),

271

cluster of differentiation 36 (CD36) were inhibited by 24%, 23%, 20%, 21% and

272

15%, respectively, after 6 weeks intake of LSOPC (p

Oligomer procyanidins from lotus seedpod (LSOPC) regulates lipid

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