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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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ABSTRACT
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Dietary polyphenols have shown hypolipidemic effects by reducing triglyceride
3
absorption. The mechanisms may involve modifying fat emulsion during digestion in
4
the gastrointestinal and suppressing the lipase during hydrolysis in the small intestine.
5
In vivo study, LSOPC decreased total serum triglyceride, total cholesterol, and
6
elevated high-density lipoprotein level in hyperlipidemic rat model. In addition,
7
LSOPC suppressed de novo lipogenesis-related genes expression. In vitro study,
8
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
12
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
14
LSOPC-enriched emulsion partly ascribed to the inhibition of lipase by LSOPC.
15
Key words: LSOPC; submicrometer emulsion; hypolipidemic; digestion; FFA release
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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
26
metabolic diseases are responsible for the majority of deaths in western countries. The
27
elevated rate of obesity and diabetes also occurred in developing countries because of
28
the acquisition of a westernized lifestyle3. A healthy, balanced diet containing
29
polyphenols could be a promising dietary strategy in regulating lipid metabolism.
30
Dietary lipid digestion undergoes several complicated steps before absorption by
31
the mucosa of the small intestine. Following the intake of fats, a slight hydrolysis
32
occurs by lingual lipase and subsequently travels to the stomach. When food is
33
present, the stomach becomes active. The churning in the stomach propelled by its
34
strong muscle disperses the fat into small droplets4. Still, little fat digestion by gastric
35
lipase takes place in the stomach. When lipid enters the small intestine, it triggers the
36
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
39
products of fat digestion, mostly monoglycerides, fatty acids, and very little glycerol,
40
move directly into bloodstream or form micelles and then are absorbed by
41
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
48
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
53
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,
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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
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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)
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to an organic phase (containing peanut oil and non-ionic surfactant) while
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magnetically stirring this system (DF-101S magnetic stirrer, Wuhan Keer Instrument
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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
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continuing stirring this system. The aqueous phase was slowly added to the organic
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phase. In our study, the experiments were conducted using standard conditions: (1) oil
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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).
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To avoid multiple scattering effects, samples were diluted before the particle size
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measurements. The calculation of particle size was followed by the difference
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between dispersive medium and sample using Mie theory. In our study, d43 was used
103
to represent the particle size.
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Temperature and pH Stability. The effects of temperature and pH on particle size
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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
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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
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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
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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
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solution were prepared. DPPH solution was prepared in ethanol, 2 mL 0.2 mmol/L of
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this solution was mixed with 2 mL the sample solutions. The mixture was incubated
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for 30 min in the dark at room temperature. Centrifuge the samples at 13,000 r/min
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for 20 min (Beckman Microfuge 20R, Shanghai, China) before the absorbance was
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read at 517 nm.
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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
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ferricyanide. To reduce ferricyanide into ferrocyanide, the mixture was incubated at
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50 ℃
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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
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acid reactive substances (TBARS). In the first step, emulsion with LSOPC and TBHQ
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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
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v/v), then were centrifuged at 1,000×g for 2 min. 200 μL of upper layer solution
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was mixed with 2.8 mL methanol/butanol mixture (2:1 v/v), 15 μL ammonium
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thiocyanate and (3.94 mol/L) and 15 μL Fe2+ solution (0.132 mol/L BaCl2 and 0.144
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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%
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(w/v) trichloroacetic acid (TCA), 0.375% (W/V ) thiobarbituric
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hydrochloric acid (0.25 mol/L) was added to 1.0 mL emulsion and mixed, followed
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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
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min (Microfuge 20R, Beckman Coulter, Shanghai, China) and filtered with the 1.2
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μm filter membrane. The absorbance of the filtrate was determined at 532 nm. The 8
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standard
curve
was
calculated
from
the
TBARS
reaction
with
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1,1,3,3-tetraethoxypropane. The results were expressed as μmol of malonaldehyde
157
(MDA).
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The Interaction of LSOPC-Enriched Emulsion and BSA. The fluorescence
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spectra were recorded with a RF5301 spectrofluorometer (Shimadzu Scientific
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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
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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
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mixture was centrifuged at 13,000 r/min for 20 min (Beckman Microfuge 20R,
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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
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300 and 450 nm. The changes in fluorescence emission intensity were measured after
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exactly 1 min of adding each sample to the protein solution. Emission of LSOPC
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enriched emulsion or solution without BSA was subtracted to correct background
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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
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measuring size particles and zeta potential.
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Free Fatty Acid (FFA) Release. The FFA release test was monitored according to
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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
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with triacylglycerols. To maintain the pH of intestinal digestion juice at 7.0, 0.1 mol/L
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NaOH was titrated into the mixture. The volume of NaOH added to the emulsion was
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recorded during this process and was used to calculate the concentration of free fatty
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acids generated by lipolysis.
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FFA (%) =
CNaOH * VNaOH * MTG 2 * mTG
×100%
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where CNaOH is the concentration of NaOH, VNaOH is the volume of sodium
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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
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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
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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
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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
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(pNP). In brief, 450 μL 0.05 M sodium phosphate buffer (pH 7.6) was mixed with 50
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μL pNPP in isopropanol (0.01 M) and 20 μL 0.1 mg/mL LSOPC in centrifuge tube. 5
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μL of lipase enzyme solution (0.5 mg/mL) was added and incubated at 37 ℃ for 10,
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20, 30, 45 min. 200 μL of the reaction mixture was taken and added to 96 well plates.
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The absorbance was measured at 410 nm.
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Lipase inhibition%=[1 ― ( ATest ― Acontrol )] × 100%
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ALSOPC ― Ablank
where A
LSOPC
is the absorbance of the mixture of LSOPC, pNPP solution, and
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enzyme solution; A blank is the absorbance of the mixture of the LSOPC sample, pNPP
212
solution without enzyme; A
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LSOPC), pNPP solution and enzyme; Acontrol is the absorbance of the mixture of
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buffer, pNPP solution without enzyme.
test
is the absorbance of the mixture of buffer (instead of
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Animal study. Seventy SD rats were obtained from laboratory animal center,
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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
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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
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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%,
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fiber ≤ 5%) and model diet for positive control group (TP0800, 20% sucrose, 15%
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lard oil, 1.2% cholesterol and 0.2% sodium cholate) were supplied by Trophic Animal
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Feed High Tech Co. Ltd, China. Two weeks later, blood samples of the tails from
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non-fasting rats were collected and TC, TG, and LDL were tested. Then positive
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control group was randomly divided into 4 groups (positive control, 0.05 g/kg·bw
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LSOPC, 0.1 g/kg·bw LSOPC, 0.15 g/kg·bw LSOPC), and each group contains 10
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rats.
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LSOPC dissolved in distilled water, and negative control and positive control were
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treated distilled water. After the end of the experiment (28 days), non-fasting tail
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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
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All 10 male ICR mice (20±2 g) were bought from Experimental Animal Center of
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Disease Prevention and Control of the Hubei Province (Wuhan, China). The animals
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were kept five per cage and allowed access to diet (TP 23302) and water. The animal
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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
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divided to two groups: normal control (NC, n=5) and LSOPC group (NC + LSOPC,
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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
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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
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manufactory’s instructions, and cDNA was synthesized using a first-strand cDNA
245
synthesis kit (GeneCopoeia,Rockville, MD, USA). The reaction mixture was
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incubated as follows: 25 ℃ 5 min, 50 ℃ 15 min,85 ℃ 5 min,4 ℃ 10 min.
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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
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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
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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
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to Student's t-test (between 2 groups) or analysis of variance (ANOVA, more than 2
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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
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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
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0.76 ± 0.28 to 1.32 ± 0.15, and 1.88 ± 0.36 to 3.89 ± 1.10, respectively. Moreover, the
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HDL level was decreased to 0.57 mmol/L. After 4 weeks of LSOPC treatment, all 13
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LSOPC treated groups distinctly lowered blood TC, TG and elevated HDL level in
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rats (Table 2). A 29.5% reduction in TG, 23.9% decrease in TC and 35% elevation in
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HDL were observed in 0.05% LSOPC group compared to that of the positive control
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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),
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cluster of differentiation 36 (CD36) were inhibited by 24%, 23%, 20%, 21% and
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15%, respectively, after 6 weeks intake of LSOPC (p