Chemical Compositions, Antiobesity, and Antioxidant Effects of

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Food and Beverage Chemistry/Biochemistry

The chemical compositions, anti-obesity and antioxidant effects of proanthocyanidins from lotus seed epicarp and lotus seed pot Jialing Cao, Xiuliang Yu, zeyuan deng, Yao Pan, Bing Zhang, Rong Tsao, and Hongyan Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05137 • Publication Date (Web): 17 Nov 2018 Downloaded from http://pubs.acs.org on November 18, 2018

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Journal of Agricultural and Food Chemistry

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The chemical compositions, anti-obesity and antioxidant effects

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of proanthocyanidins from lotus seed epicarp and lotus seed pot

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Jialing Cao†, Xiuliang Yu†, Zeyuan Deng†,‡, Yao Pan†, Bing Zhang†, Rong Tsao§,

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Hongyan Li1*,†

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† State

Key Laboratory of Food Science and Technology, University of Nanchang,

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Nanchang 330047, Jiangxi, China

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‡ Institute

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for Advanced Study, University of Nanchang, Nanchang 330031, Jiangxi, China

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§

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Guelph Research and Development Centre, Agriculture and Agri-Food Canada, 93 Stone Road West, Guelph, Ontario, N1G 5C9 Canada.

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*

Corresponding author. Tel.: +86 791 88314447-8226; fax:+86 791 88304402 E-mail address: [email protected] 1

ACS Paragon Plus Environment


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ABSTRACT

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Lotus seed epicarp (LSE) and lotus seed pot (LSP) were characterized and a total of

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5 and 7 proanthocyanidins (PAs) were identified in purified LSE and LSP extract,

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respectively. Purified LSE and LSP PAs significantly suppressed the body weight and

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weight gain of white adipose tissue (WAT) and decreased the WAT cell size in

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high-fat diet induced obese mice regardless of the daily food intake. LSE or LSP

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administration significantly lowered the serum leptin level and improved the serum

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and liver lipid profiles (including TC, TG, HDL-C, LDL-C levels), increased

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activities of antioxidant enzymes (SOD, GST) and GSH concentration, and

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suppressed lipid peroxidation in hepatic tissue. LSP PAs was generally more effective

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than LSE PAs. Both extracts ameliorated obesity, insulin resistance and oxidative

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damage in obese mice, suggesting they are good candidates for value-added

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functional food and nutraceutical ingredients.

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KEY WORDS: Lotus seed epicarp; Lotus seed pot; Proanthocyanidins; HPLC-MS/MS;

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Anti-obesity

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

INTRODUCTION

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Lotus (Nelumbo nucifera, Gaertn) is a widely cultivated aquatic plant in China,

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Japan, India, Thailand, South Korea, North America and Australia. The edible parts of

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lotus including seeds, leaves, stamens and roots are usually consumed and their

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nutritional value widely researched1, 2. However, during processing, the inedible parts,

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such as lotus seed epicarp (LSE), seed stem and lotus seed pot (LSP) are discarded.

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LSE and LSP are sometimes used as traditional medicines in China to modulate

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immunity, to reduce the serum total cholesterol and triglyceride contents, and to

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protect the liver from damages3, 4. LSP has been reported to contain proanthocyanidins

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(PAs), which showed great antioxidant, anticancer, hypolipidemic and memory

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improvement properties5-8. Recently, the LSP PAs were reported to be effective

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against ageing and Alzheimer’s disease9. Extract of LSE was also reported to have

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antioxidative and anti-obesity effects in a cell model10.

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PAs are oligomers and polymers of polyhydroxyflavan-3-ol units. The monomers

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are linked at 4-8 and 4-6 positions of the flavonol structure, giving linear or

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sometimes branched chains of higher degree of polymerization11. PAs of fruits and

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vegetables possess various health beneficial activities. Desaccharized PAs from

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Chinese bayberry was reported to effectively reduce the body weight of high-fat

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diet-induced obese rat and improve the levels of serum TC (total cholesterol), TG

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(triglycerides), AST (aspartate transaminase), ALT (alanine transaminase), ALP

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(alkaline phosphatase), LDL-C (low-density lipoprotein-cholesterol) and HDL-C 3



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(high-density lipoprotein-cholesterol), biochemical markers of many clinical diseases

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caused by obesity12. By inhibiting the digestive enzymes of carbohydrates and fats,

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highly polymeric PAs from seed shells of the Japanese horse chestnut exhibited

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anti-obesity effects in mice fed a high-fat diet13. PAs from grape seed also showed

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anti-obesity effects as manifested in reduced plasma cholesterol concentration, hepatic

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steatosis and abdominal fat content in rats14. PAs from grape seed can also curb

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obesity by restoring dyslipidemia resulting from a high-fat diet in rats and repressing

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genes modulating lipogenesis and VLDL (very low-density lipoprotein) assembling in

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the liver15. While PAs of lotus seed pot have been characterized in our previous

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study16, the phytochemical compositions especially the PAs from LSE have not been

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investigated. The in vivo anti-obesity activities of PAs of LSE and LSP also remain

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unknown.

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Therefore, the objectives of this paper were: (a) to characterize the chemical

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profiles of purified PAs in LSE and LSP by HPLC-QTOF-MS2. (b) to assess the in

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vivo anti-obesity and antioxidant effects of purified PAs from LSE and LSP in the

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C57BL mouse model.

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

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Plant materials and chemical reagents

MATERIALS AND METHODS

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LSE and LSP were obtained from Guangchang county, Jiangxi province, China.

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The raw materials were separately freeze dried (2XZ-2, Linhai, China), ground in a

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high speed disintegrator (FW80, Shanghai, China) to obtain fine powders.

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Ascorbic acid, gallic acid and catechin standards were obtained from Sigma

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Chemical Company (St. Louis, MO, USA). TC, TG, H-DLC, L-DLC,

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malondialdehyde (MDA), superoxide dismutase (SOD), glutathione transferase (GST),

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reduced glutathione (GSH) kits were purchased from Nanjing Jiancheng

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Biotechnology Company (Nanjing, China). Folin-Ciocalteau reagent, acetonitrile, and

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formic acid were obtained from Merck KGaA (Darmstadt, Germany). Water was

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purified using a Milli-Q system from Millipore (Bedford, MA, USA).

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PAs preparation and purification

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The powder (10 g) of LSE or LSP was extracted with 200 mL of 70% acetone (v/v)

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containing 1 g L-1 of L-ascorbic acid to avoid oxidation for 12 h. The extract was

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vacuum-dried to about 50 mL at 45 °C to rid the organic solvent. The aqueous layer

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was then evaporated under a stream of nitrogen (DSY-VI, Beijing, China).

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The above dried extracts were reinstituted in 10 mL water and extracted again with

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chloroform, diethyl ether and ethyl acetate in sequence (each for two times, and each

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time with 100 mL), to remove the lipids, flavonoids and chlorophylls of the samples,

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respectively. The remaining aqueous phase containing PAs were then loaded onto a

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AB-8 column (71.574 g after drying; Φ1.5 cm × 35 cm) and then purified by washing 5



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the column with 1000 mL water and 500 mL 10% (v/v) acetone in water to remove

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carbohydrates and other water-soluble components. The adsorbed PAs were recovered

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by eluting with 500 mL 70% acetone.

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HPLC-QTOF-MS/MS of purified PAs

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The PAs from LSE and LSP were analyzed by an Agilent 1260 HPLC system

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(Agilent Technologies, Shanghai, China). An ODS C18 column (250×4.6 mm, 5 μm)

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was used at 25 °C with a flow rate of 0.5 mL min-1 and injection volume of 10 μL.

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The mobile phase consisted of 0.2% formic acid in water (A) and 100% acetonitrile

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(B), and a gradient elution was carried out in 45 min under the following conditions:

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0-5 min, B 5-8%; 5-15 min, B 8-13%; 15-45 min, B 13-25%. The post-running time

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was 5 min. Peaks were monitored at 278 nm with a variable wavelength UV detection

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(VWD). The HPLC system was coupled to an orthogonal acceleration

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quadrupole-time-of-flight mass spectrometer (6538 Accurate-Mass QTOF LC/MS

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system; Agilent Technologies, USA). An orthogonal ESI was operated in the negative

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ion/multiple reaction monitoring (MRM) mode. The optimum values of the source

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parameters were as follows: capillary voltage, +4.0 kV; drying gas flow, 10.0 L/min;

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drying gas temperature, 350°C; nebulizing gas pressure, 40 psi. Deprotonated

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molecular ions [M-H]− were selected as precursor ions and subjected to MS/MS

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analysis. The collision energy was set at 20 eV, and the fragmentor voltage was set at

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135 V, using nitrogen as collision gas. PAs peaks were quantified using a (+)-catechin 6


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external standard calibration curve17 with concentrations ranging from 0 to 0.25 mg

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ml-1.

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PAs and flavonoid contents of purified LSE and LSP extracts

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Total contents of PAs. Total PAs contents of the purified extracts were

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measured using the vanillin-sulfuric acid method. In brief, diluted samples (0.5 mL),

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vanillin (2.5 mL, 30 mg mL-1) and sulfuric solution (2.5 mL, 30%) were mixed and

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allowed to react for 20 min at room temperature until the absorbance reached 500 nm

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as monitored with a spectrophotometer (722G, Hitachi Instruments Inc., Shanghai,

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China). The results were expressed as (+)-catechin equivalents in milligrams per

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milliliter of LSE and LSP extract. The linearity range of the calibration curve was 0.1

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to 0.5 mg mL-1 (R2 = 0.9956). Total PAs was also converted to dry matter-based using

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the following equation:

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(1) Yield of PAs (mg g-1)=(V*ρ*n)/(w*1000)

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Where ρ is the concentration of PAs from the calibrated regression equation (mg

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mL-1); V is the total volume of extraction solution (mL); n is the dilution factor; w is

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the weight of dried raw material (g); The yield is milligrams PAs per gram dry LSE

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(or LSP).

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Total flavonoid contents. The purified extracts or quercetin standard (1 mL), 5%

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(w/w) NaNO2 (0.7 mL) and 30% (v/v) ethanol (10 mL) were mixed for 5 min, and

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then 10% AlCl3 (w/w, 0.7 mL) was added and allowed to react at room temperature 7



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for 6 min. The reaction was stopped by adding 5 mL of 1 M NaOH. The reaction

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mixture was then diluted to 25 mL with 30% (v/v) ethanol for further measurement 10

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min after. The absorbance of the solution was measured at 430 nm with a

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spectrophotometer (722G, Hitachi Instruments Inc., Shanghai, China)18. The total

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flavonoid content was extrapolated using a standard curve of quercetin (0.1-0.5 mg

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ml-1, R2=0.9933). The results were expressed as quercetin equivalents in microgram

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per milliliter of LSE and LSP extract, and the expression of the content in the results

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was also expressed as a percentage of flavonoid content in the unit extract.

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Animal experiment

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Animal treatment. Seventy C57BL/6j mice (6 weeks old, male) were obtained

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from Slac Laboratory Animal Co. Ltd (Hunan, China). Before the experiment, the

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mice were acclimatized and fed with a commercial standard diet for one week. Mice

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were randomly divided into seven groups as follows: (1) normal diet (ND), (2)

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high-fat diet (HFD), (3) high-fat and low LSE diet (HFD + LSE 25), (4) high-fat and

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high LSE diet (HFD + LSE 100), (5) high-fat and low LSP diet (HFD + LSP 25), (6)

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high-fat and high LSP diet (HFD + LSP 100), (7) high-fat and positive control diet

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(HFD + Xuezhikang Capsule (XZK)). The ND diet contained 50.8% corn starch, 24.2%

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casein, 11.9% sucrose, 5% lard, 4% mineral mixture, 2% vitamin mixture, 2% gelatin

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and 0.1% DL-methionine. The HFD contained 20% fat (lard), 10% protein, 0.15%

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cholesterol and 69.85% ND diet. The purified PAs from LSE (or LSP) was dissolved 8


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in distilled water, and was administered by gavage to mice daily at doses of 25 mg

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PAs kg-1 and 100 mg PAs kg-1 body weight (BW) for 60 days in HFD + LSE (or LSP)

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25, HFD + LSE (or LSP) 100 groups. Food intakes of the mice were recorded daily,

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and their body weights were monitored twice a week throughout the experimental

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period. The food efficiency ratio (FER) = [body weight gain (g)/diet consumed (g)] ×

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100.

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A daily dose of 100 mg PAs kg-1 BW in mice was estimated as 1.2 g day-1 of LSE

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or 0.96 g day-1 of LSP for a 60 kg human (Human equivalent dose (mg-1 kg) = Animal

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does (mg-1 kg) Km ratio)19. It was speculated that this dose is sufficient to have an

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effect on animal model according to our preliminary experiment. At the end of the

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experiment, mice were anesthetized with 2% ether for 8 seconds, and then killed by

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breaking the spine after a 12 h fasting period. Blood samples were collected from the

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celiac artery. The serum was separated by 10 min of centrifugation (2000 rpm, 4 °C).

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Livers and visceral fat-pads from epididymal and perirenal regions were removed,

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rinsed with phosphate buffered saline (PBS), and weighed. All experiments were

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

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laboratory animals and were approved by the Experimental Animals Ethics

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Committee of Nanchang University (approval number: SCKY (XIANG) 2013-0004;

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expiry date: October 11, 2016).

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Morphological analysis of hepatic and adipose tissues. Liver and epididymal

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white adipose tissue (WAT) samples were removed from mice and fixed overnight 9



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with 40 g L−1 paraformaldehyde. Fixed tissues were embedded in paraffin, sliced into

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3 µm samples and stained with haematoxylin and eosin (H&E). The stained areas

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were viewed using an optical microscope (Olympus CX31, Tokyo, Japan) with a

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magnifying power of ×200.

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Biochemical analysis. Serum TG, TC, LDL-C and HDL-C concentrations were

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measured using commercial diagnostic kits (Nanjing, China). The atherogenic index

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(AI) was calculated as AI = (TC−HDL-C)/HDL-C20. Blood glucose levels were

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measured using a glucometer (Accu-Chek, Roche Diagnostics, Indianapolis, IN,

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USA). Insulin levels were measured with commercial kit from Youersheng

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Biotechnology Co., Ltd. (Wuhan, China). The homeostatic index of insulin resistance

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(HOMA-IR) was calculated as HOMA-IR = [fasting glucose (mmol L−1) × fasting

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insulin (µU mL−1)]/22.521. Hepatic tissues were extracted according to Folch et al22.

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Hepatic indexes (TG, C, LDL-C and HDL-C) were measured by the same kits used

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for the serum analysis.

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Serum AST, ALT and ALP activities. Degree of hepatic injury was evaluated by

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measuring enzyme activities of AST, ALT, and ALP in collected serum samples using

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a commercial assay kit (Jiancheng Biotechnology, Nanjing, China) according to the

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manufacturer’s instructions.

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Serum leptin level. Serum leptin levels were measured using a commercial ELISA

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kit specific for mouse (Invitrogen, Carlsbad, CA, USA). The optical density (OD) was

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determined by a microplate reader set to 450 nm. 10


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The antioxidant enzyme activities. In brief, 0.1 g hepatic tissue was weighed and

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mixed with 10 mL extraction solution (each enzyme has its specific extraction

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solution). Each extraction mixture was homogenized separately in an ice-bath and

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centrifuged (8500 rpm, 4 °C for 10 min). The supernatants were subjected to assays

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for superoxide dismutase (SOD), glutathione S-transferase (GST), total glutathione

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(GSH)

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Biotechnology, Nanjing, China) according to the manufacturer’s instructions.

and

malondialdehyde

(MDA))

using

commercial

kits

(Jiancheng

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Total protein determination. The protein content in hepatic tissue was also

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determined using a commercial protein assay kit (Beyotime, Haimen, China). Bovine

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serum albumin (BSA) was used as standard for generating the calibration curve.

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Statistical analysis

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All assays were performed in triplicate. The results were expressed as mean ± SD

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(standard deviation). The LC-MS data were acquired and analyzed by the software

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Mass Hunter Acquisition B.03.01, Qualitative Analysis B.03.01 and Quantitative

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Analysis B.03.02. Other data were analyzed by the SPSS statistical software, version

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18.0 (SPSS Inc., USA). Statistical analysis was carried out using the One-way

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analysis of variance (ANOVA) followed by Duncan’s multiple range tests to measure

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statistically different values on the level of significance at P

Chemical Compositions, Antiobesity, and Antioxidant Effects of

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