Chemical composition, antioxidant activities of polysaccharide from Pine needle (Pinus massoniana) and hypolipidemic effect in high-fat diet-induced mice

Accepted Manuscript Chemical composition, antioxidant activities of polysaccharide from Pine needle (Pinus massoniana) and hypolipidemic effect in high-fat diet-induced mice

Lulu Chu, Licong Yang, Lezhen Lin, Jing Wei, Na Wang, Meng Xu, Gaoxiang Qiao, Guodong Zheng PII: DOI: Reference:

S0141-8130(18)35239-5 https://doi.org/10.1016/j.ijbiomac.2018.12.082 BIOMAC 11241

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

1 October 2018 28 November 2018 8 December 2018

Please cite this article as: Lulu Chu, Licong Yang, Lezhen Lin, Jing Wei, Na Wang, Meng Xu, Gaoxiang Qiao, Guodong Zheng , Chemical composition, antioxidant activities of polysaccharide from Pine needle (Pinus massoniana) and hypolipidemic effect in high-fat diet-induced mice. Biomac (2018), https://doi.org/10.1016/j.ijbiomac.2018.12.082

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ACCEPTED MANUSCRIPT Chemical composition, antioxidant activities of polysaccharide from Pine needle (Pinus Massoniana) and hypolipidemic effect in high-fat diet-induced mice Lulu Chu, Licong Yang*, Lezhen Lin, Jing Wei, Na Wang, Meng Xu, Gaoxiang Qiao,

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Guodong Zheng*

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Jiangxi Key Laboratory of Natural Product and Functional Food, College of Food

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Science and Engineering, Jiangxi Agricultural University, Nanchang, 330045, China

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*Corresponding Author: Guodong Zheng, Jiangxi Key Laboratory of Natural Product and Functional Food, College of Food Science and Engineering, Jiangxi

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Agricultural University, Nanchang, 330045, China. E-mail: [email protected],

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Tel & Fax: 0086-791-83813863.

*Corresponding Author: Licong Yang, Jiangxi Key Laboratory of Natural

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Product and Functional Food, College of Food Science and Engineering, Jiangxi

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Agricultural University, Nanchang, 330045, China. E-mail: [email protected], Tel & Fax: 0086-791-83813863.

Abstract The aim of this study is to investigate hypolipidemic and antioxidant effects of Pine needle polysaccharide (PNP) from Pinus Massoniana in high-fat diet

ACCEPTED MANUSCRIPT (HFD)-induced mice. PNP could significantly improve the serum lipid levels (total cholesterol, triacylglycerols, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol), enhance the antioxidant enzymes levels (total antioxidant capability, superoxide dismutase, glutathione peroxidase, catalase), and decrease

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malondialdehyde (MDA) content in HFD-induced mice. PNP exhibited distinct

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antioxidant ability on the superoxide anions, 1, 1-diphenyl-2-picrylhydrazyl (DPPH)

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and ferric ion reducing antioxidant power (FRAP) in vitro. The average molecular weight (Mw) of PNP was 6.17×105 Da, and mainly of fucose, arabinose, galactose,

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glucose, galacturonic acid. These results suggested that PNP might be used as

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functional foods and natural drugs in enhancing antioxidant ability and alleviating the hyperlipidemia.

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Key word: Pine needle polysaccharide, Chemical composition, Antioxidant

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activities, Hypolipidemic effect

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Abbreviations: PNP, Pine needle (Pinus Massoniana) polysaccharide; HFD, high-fat diet; Mw, molecular weight; TC, total cholesterol; TG, triacylglycerols;

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HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; T-AOC, total antioxidant capability; SOD, superoxide dismutase; GSH-PX, glutathione peroxidase; CAT, catalase; MDA, malondialdehyde; FRAP, ferric ion reducing antioxidant power; ml/kg bw/d, ml/kg body weight/day; mg/kg bw/d, mg/kg body weight/day 1. Introduction

ACCEPTED MANUSCRIPT The hyperlipidaemia is becoming a common chronic disease that cannot be ignored in daily life. It is universally accepted that the main causes of hyperlipidemia are high-energy diet and lipid metabolic disturbance with the improvement of living conditions. The clinical manifestation of hyperlipidemia are high levels of total

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cholesterol (TC), triacylglycerols (TG) and high-density lipoprotein cholesterol

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(LDL-C) and low level of low-density lipoprotein cholesterol (HDL-C), and it is

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closely linked in inducing fatty liver, atherosclerosis, cerebrovascular and cardiovascular diseases [1-3]. Many people are suffering from hyperlipidemia and its

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complications in worldwide, the most intuitive performance is obesity. Therefore,

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some effective and safe oral drugs are urgently needed to control and treat hyperlipidemia and its complications [4]. Many traditional drugs had been used to

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control and treat hyperlipidemia such as statins, fibrates, atorvastatin, nicotinic acid

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and probucol [5]. However, some studies indicated that these drugs have side effects and are expensive. Hence, the development of natural and low price lipid-lowering

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drugs has become a hot topic in the medical field [6].

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Polysaccharides play an important role in the growth and development of organisms [7-9]. Especially, plant polysaccharides have a wide range of biological activities and desirable functional properties. They have attracted widespread attention from researchers in the food and biomedical fields worldwide [10-12]. A large amount of literature reported that natural plant polysaccharides have the potential to control and treat hyperlipidemia as a new type of reducing lipid drug, such as Yang et al. had explored antihyperlipidemic activities of polysaccharide

ACCEPTED MANUSCRIPT fraction from Cyclocarya paliurus, Xu et al. had testified the hypolipidemic effect of mycelia zinc polysaccharides by Pleurotus eryngii var. tuoliensis [13-14] and so on. In recent years, the research on Pine needle polysaccharide has gradually increased. It had been reported that Pine needle polysaccharide from Cedrus deodara had some

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biological activities, such as antioxidant, and antibacterial activity and cell protection

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[15-17]. Pinus Massoniana, widely cultivated in China, is a tree species of economic

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importance. The immune regulation effect of pollen polysaccharide from Pinus

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massoniana was reported by some literature [18-20].

However, the study about hypolipidemic and antioxidant effects of Pine needle

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polysaccharide (PNP) from Pinus Massoniana in vivo was few reported. In this work, PNP was extracted from Pine needle of Pinus Massoniana. The effects of PNP on

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body weight, serum lipids, antioxidant enzyme activities and hypolipidemic were

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evaluated in high-fat diet (HFD)-induced mice. Concurrently, chemical composition

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and antioxidant activities of PNP were determined in vitro. 2. Materials and method

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2.1 Materials and reagents Pine needle of Pinus Massoniana was collected from tree specimen garden of Jiangxi Agricultural University, Jiangxi, China. The Pine Needles were air-dried and milled into fine powder using a high-speed disintegrator at Key Laboratory of Food Science and Engineering, Jiangxi Agricultural University.

ACCEPTED MANUSCRIPT The standard monosaccharideincluding fucose (Fuc), rhamnose (Rha), arabinose (Ara), galactose (Gal), glucose (Glu), xylose (Xyl),mannose (Man), fructose (Fru), galacturonic acid (Gala) and glucuronic acid (Glca) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). DEAE-cellulose was purchased

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from Yuanye Bioengineering Institute (Shanghai, China). The NaCl, phenol, sulfuric

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acid, galacturonic acid, ferrous sulfate (FeSO4), ferric chloride (FeCl3),

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2,2-diphenyl-1-picrylhydrazyl (DPPH), ascorbic acid, coomassie brilliant blue-G250, bovine serum albumin (BSA), potassium bromide (KBr, spectrum pure grade)

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purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). The

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different Mw (T-10, T-40, T-70, T-500 and T-2000) of dextrans were purchased from Pharmacia Biotech Co. (Uppsala, Sweden). Lard oil, cholesterol and

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methylthiouracil were obtained from Xilong Chemical Co., Ltd., (Shantou, China).

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The assay kits of total antioxidant capability (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), catalase (CAT), malondialdehyde (MDA), TC,

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TG, LDL-C and HDL-C were purchased from Nanjing Jiancheng Bioengineering

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Institute (Nanjing, China).Water used in the experiment was purified by Milli-Q water purification system (Millipore, Bedford, MA, USA). All other chemicals and reagents used in the experiments were of analytical reagent grade. 2.2 Extraction and purification of PNP The polysaccharide was extracted from Pine needle of Pinus Massoniana according to Xie et al. with a slightly modified [21]. Briefly, the pretreated Pine needle powder was fully submerged with 95% ethanol for 12 h to remove some

ACCEPTED MANUSCRIPT interference substances, such as monosaccharide, disaccharides, oligosaccharides, lipids, pigments and polyphenols. The soaked residue was dried and then extracted three times by hot water at 95 °C with a ratio of 1:20 (w/v) for 2 h. Subsequently, the aqueous extract was concentrated to 20% of the initial solution using a rotary

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evaporator original at 55 °C under vacuum, and proteins were dislodged by Sevag

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method [22]. Afterwards, Sevag reagent was removed by evaporator at 45°C and the

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filtrate was precipitated with four times its volume of 95% (v/v) ethanol at 4°C overnight. The precipitate was centrifuged at 8400 g for 10 min by a high speed

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centrifuge. After that, the precipitate was further dissolved in ultrapure water,

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dialyzed (molecular weight (Mw) cut-off 8000-14000 Da), frozen and freeze-dried. Finally, the crude polysaccharide was obtained.

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The crude polysaccharide was further purified by using a DEAE-cellulose

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column (80 × 5.0 cm), and eluted through stepwise gradient of NaCl solution (0, 0.1, 0.2, 0.3 and 0.4 M) with a flow rate of 1.5 mL/min. After dialyzing and freeze-dried,

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five purified polysaccharides were obtained, respectively. The eluted component by

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deionized water was named PNP which accounted for more than 90% in all purified samples. Hence, PNP was the main research object in our experiment. 2.3 Chemical composition and monosaccharide components of PNP The total sugar of PNP was determinate through the method of phenol-sulfuric acid using glucose as the standard and the protein content was determined by the method of Bradford analysis using bovine serum albumin (BSA) as the standard. The

ACCEPTED MANUSCRIPT molecular weight of PNP was measured by high-performance liquid gel permeation chromatography

(HPGPC)

on

an

Agilent

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high-performance

liquid

chromatography (HPLC) system equipped with series of SHODEX KS-804 and KS-802 columns (8 mm × 300 mm) and a refractive index detector [22].

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Monosaccharide components of PNP was measured with a high performance anion

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exchange chromatography (HPAEC) system equipped with pulsed amperometric

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detection (PAD), a CarboPacTM PA10 column (2.0 mm × 250 mm) and dionex

2.4 Antioxidant activities of PNP in vitro

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ICS-2500 system [23].

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2.4.1 Scavenging activity assay of DPPH radical

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The DPPH radical scavenging capacity of PNP was evaluated by the method

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of Du et al. with few modifications [24]. Briefly, a fresh 0.1 mM DPPH solution (95% ethanol dissolved) was prepared and PNP samples were dissolved in ultra-pure water

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with the concentration of 20, 40, 80, 160, 320, 640 and 1280 μg/mL, respectively. Then, 2 mL PNP and 2 mL DPPH solution were fully mixed to react for 30 min in

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dark, the absorbance was measured at 517 nm and ascorbic acid was used as the positive control and 95% ethanol was the blank control. The DPPH radical scavenging ability was calculated as follows: DPPH• free radical scavenging rate (%) = [1-(A2-A1)/A0] × 100% Where A0 is the absorbance of the blank (95% ethanol without adding the samples or positive control), A1 is the absorbance of the samples (without DPPH

ACCEPTED MANUSCRIPT solution), and A2 is the absorbance of the mixture with both the samples and DPPH solution. 2.4.2 Scavenging activity assay of superoxide anions radical

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The scavenging activity for superoxide anions radical was performed with the method previously reported method of Lin et al [23]. Briefly, PNP was dissolved in

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ultra-pure water and then 0.5 mL PNP solution and 5 mL Tris-HCl buffer solution

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(pH 8.2, 50 mM/L) was incubated in water bath at 25 °C for 20 min. Immediately,

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adding 0.1 mL preheated pyrogallol (3 mM/L) and shaking it to make mixed liquid fully reacting for 3 min, then the absorbance of mixed liquid was performed once

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every 30 s at 325 nm for 5 min. Ascorbic acid was selected as a positive control. The

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scavenging activity for superoxide anions radical was calculated as follows:

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Superoxide anion free radical scavenging rate (%) = (1-A1/A0) × 100% Where A1 is the absorbance of the sample, A0 is the absorbance of the water

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without the sample.

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2.4.3 Ferric ion reducing antioxidant power (FRAP) assay The method of Tang et al with minor modifications was used to evaluate the reducing power of PNP [25]. In short, the FRAP working solution was prepared to mix acetate buffer (pH 3.6, 300 mmol/L), 2, 4, 6-Tris (2-pyridyl)-s-triazime (TPTZ) solution (10 mmol/L) and FeCl3 solution (20 mmol/L) with a volume ratio 10:1:1. Afterwards, 450 μL FRAP working solution and 15 μL sample solution was mixed with reaction in a water bath at 37 °C for 10 min. The absorbance was determined at

ACCEPTED MANUSCRIPT 593 nm, and ascorbic acid was selected as a positive control. The concentration of FeSO4 was indirectly expressed as with equivalent antioxidant activity of sample. 2.5 Animal experiment

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High-fat emulsion was prepared according previous literature [26]. The oil phase mainly contained 25 g liquid lard oil, 10 g cholesterol, 1 g methylthiouracil

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and 25 mL tween-80. The water phase principally contained 30 mL distilled water,

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20 mL propylene glycol and 2 g sodium deoxycholate. Before animal administration,

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the oil phase and the aqueous phase were freshly mixed to get a high-fat emulsion.

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Fifty female ICR mice (SPF grade, weight 20 ± 2 g) were purchased from Hunan Silaike Laboratory Animal Co., Ltd., (Changsha, China). The living

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conditions of mice were in animal cages with room temperature (24 ± 2 °C), relative

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humidity 50 ± 5% and 12 h light and 12 h dark cycle. The padding of animals was changed every three day and freely access to water and food. All animal experiments

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procedures were strictly performed on Guide for the Care and Use of Laboratory Animals and approved by the Animal Care and Use Committee of the Jiangxi

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Agricultural University (JXAUA01) and all efforts are made to minimize the suffering of animal. After feeding adaptively for seven days, all mice were randomly assigned to five groups (n = 10, normal control, HFD, HFD+200, 400, 800 mg/kg bw/d PNP, respectively) and body weight was weighed and recorded every day (Sch.1). Normal control was received 10 mL/kg bw/d ultra-pure water, HFD and HFD+200, 400, 800

ACCEPTED MANUSCRIPT mg/kg, bw/d PNP were severally perfused 10 mL/kg bw/d high-fat emulsion with a gavage at 8:00 am. Intermittently, Normal control and HFD were severally received 10 mL/kg bw/d ultra-pure water, and HFD+200, 400, 800 mg/kg bw/d PNP were perfused PNP solution (200, 400 and 800 mg/kg bw/d, respectively) at 18:00 pm.

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The feeding process continued for 8 weeks.

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After 8 weeks, mice were fasting over 12 h. Then the mice were sacrificed and

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the serum was collected by a centrifuge (5800 × g) for 10 min at 4 °C. The liver and

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abdominal adipose were obtained and weighed, respectively. Serum and liver were preserved in the -80 °C refrigerator for further biochemical analysis.

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2.5.1 Determination of serum lipid

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The levels of TC, TG, LDL-C and HDL-C in serum were measured by

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corresponding commercial kits [3].

2.5.2 Antioxidant activity of PNP in vivo

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The indexes of T-AOC, SOD, GSH-PX, CAT and MDA in serum and liver

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were measured by using commercial kits [14]. 2.6 Statistical analysis All experiment dates were expressed as means ± SD (standard deviation). The dates of mice were analyzed by using SPSS software version 21(IBM software, NY, USA). Differences between experimental groups were carried out pass t-test and P < 0.05 was deemed as a significant difference.

ACCEPTED MANUSCRIPT 3. Result 3.1 Chemical composition of PNP analysis As illustrated in Table 1, the total sugar and protein content of PNP were about

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80.63 % and 6.35 %, respectively. The average Mw of PNP was about 6.17×105 Da. The monosaccharide composition of PNP was mainly contained of fucose, arabinose,

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galactose, glucose and galacturonic acid with the molar ratio of 4.59: 7.77: 62.24:

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23.74: 0.65.

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3.2 The analysis of antioxidant activities of PNP in vitro

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The experiments of superoxide anion, DPPH and FRAP were frequently used to determine the antioxidant capacities of natural products in vitro. As shown in Fig.

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1, both antioxidant activities of PNP and ascorbic acid expressed dose-dependent

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with the concentration of PNP and ascorbic acid. At the concentration of 1.28 mg/mL, the superoxide anion radical scavenging effect of PNP and ascorbic acid were 83.34%

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and 99.15%, with their half inhibition concentration (IC50) values were about 0.04

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and 0.01 mg/mL (Fig. 1A). DPPH scavenging activities of PNP and ascorbic acid severally reached 90% and 95.87% when the concentration was 1.28 mg/mL and IC50 values were 0.21 and 0.06 mg/mL, respectively (Fig. 1B). Synchronously, the FRAP values of PNP and ascorbic acid were reached 1365.72 and 1433.48 μM at concentration of 1.6 mg/mL (Fig. 1C). In conclusion, the antioxidant activities of PNP were positive correlation with the concentration of samples. 3.3 Effects of PNP on body weight, liver weight, and abdominal adipose weight

ACCEPTED MANUSCRIPT The body weight, liver weight and abdominal adipose weight of mice can be observed in Table 2. At the end of the experiment, the body weight in HFD group had a prominent increase compared with normal control (P < 0.05). Body weight and abdominal adipose weight in 800 mg/kg bw/d PNP was significantly lower than

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HFD (P < 0.05). The liver weight in each group was no significant difference.

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3.4 Effects of PNP on serum lipid

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The level of serum lipid was exhibited in Fig. 2. Compared with HFD, the

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serum lipid level had an obvious improvement by a gavage administration using PNP. Especially, the levels of TC, TG and LDL-C of 800 mg/kg bw/d PNP had a

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increased by 19.11% (P < 0.05).

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significant decline by 36.44%, 31.91%, 34.67% and HDL-C was significantly

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3.5 Effects of PNP on antioxidant enzyme activities in vivo 3.5.1 The analysis of antioxidant enzyme activities in serum

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As shown in Fig. 3, treatment with PNP of HFD-induced mice could

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dramatically elevate antioxidant enzyme activities in serum. Compared with HFD, the T-AOC, SOD and GSH-PX activities in HFD+PNP were significantly increased and the CAT level of 800 mg/kg bw/d PNP was obviously increased by 35.70% (P < 0.05). The level of MDA in serum was explored in Fig. 5A, the MDA level in HFD+ PNP had a significant reduction than HFD (P < 0.05). 3.5.2 The analysis of antioxidant enzyme activities in liver

ACCEPTED MANUSCRIPT Fig. 4 was shown the activities of antioxidant enzyme in liver. The T-AOC, SOD, GSH-PX, CAT activities of the 800 mg/kg bw/d PNP in liver were obviously increased by 22.56%, 28.79%, 46.84%, 28.15% compared with those in HFD (P < 0.05) after feeding with PNP. As shown in Fig. 5B, the level of MDA in HFD+PNP

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were significantly reduced than HFD (P < 0.05).

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4. Discussion

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With the ingestion of high-fat food, more and more people were suffered from

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hyperlipidemia in daily life. Documented literature had reported that hyperlipidemia may cause a variety of diseases, such as atherosclerosis and cardiovascular disease

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[27]. It was generally believed that the reduction of TC and TG levels were more effective in preventing and treating on hyperlipidemia. It had been reported that

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LDL-C was the most important transporter of TC [28], and superfluous LDL-C was easily accumulated and oxidized in blood vessels with accelerating the formation of

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foam cells and plaques [29]. Conversely, HDL-C could transport cholesterol in tissues and blood vessels to liver by the “reverse cholesterol transport” pathway and

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eliminated it by bile acids and excretion [30-31]. In this study, after treatment with PNP, these levels of TC, TG, HDL-C and LDL-C in mice were distinctly relieved compared with HFD and it is worth mentioning that the indicators levels (TC, TG, HDL-C and LDL-C) of 800 mg/kg bw/d PNP were close to normal control. Compared with Cyclocarya paliurus and mycelia zinc polysaccharides [13-14], PNP showed more positive effect on reducing serum lipid levels in high-dose group. Concurrently, the decrease of abdominal adipose content in HDF-induced mice with

ACCEPTED MANUSCRIPT feeding PNP can directly reflect the improvement of lipid metabolism. These may be an important advantage for PNP to improve and alleviate hyperlipidemia. According to studies, high-fat diets maybe produce vast free radicals and destroy the antioxidant defense system in mice [32]. Literature had reported that

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reactive oxygen species (ROS) could oxidize LDL-C to form ox-LDL-C which was

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closely related to the formation of hyperlipidemia [33]. During enzymatic and

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non-enzymatic antioxidant systems, the main endogenous antioxidant enzymes

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including SOD, CAT and GSH-PX was a protective mechanism which can prevent oxidative stress and cell damage [34]. SOD could catalyzesu peroxide into hydrogen

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peroxide and hydrogen peroxide was catalytically reduced to water by CAT and GSH-PX for relieving oxidative stress [35]. Simultaneously, the T-AOC activity can

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reflect the ability of non-enzymatic antioxidant systems [36]. In addition, lipid

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peroxides were also considered as major markers of oxidative damage and the production of MDA in the liver was often accompanied by the occurrence of liver

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damage due to oxidative stress [37]. In this work, significant decreases of the levels

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of T-AOC, SOD, GSH-Px and CAT were observed in HFD-induced mice, indicating serious oxidative stress had developed mice. Synchronously, the increase of MDA also proved the occurrence of oxidative stress. The T-AOC, SOD, GSH-Px, CAT and MDA levels of PNP-treated had a significantly improvement compared with HFD. PNP could remit the oxidative stress in HFD-induced mice by promoting the activities of enzymes and non-enzymatic antioxidants. These results indicated PNP could offset part oxidative damage caused by hyperlipidemia for its antioxidant

ACCEPTED MANUSCRIPT capacity. Furthermore, it is well known that the antioxidant activities of polysaccharides may be closely related to the structure of polysaccharides such as monosaccharide composition and molecular mass [38]. In the present work, the monosaccharide

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compositions of PNP were analyzed. The results showed that two monosaccharide

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with the highest molar ratio in PNP were galactose and glucose, respectively. Lin et

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al had proved that galactose played important roles in keeping the antioxidant

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function [39]. Meanwhile, some literature reported that glucose was vital factors to alleviate the hyperlipidemia and its complications [40]. In this experiment, PNP

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showed evident hypolipidemic, and antioxidant effects, these results were similar to those mentioned of above literature. Therefore, the bioactivities and structural

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analysis of the PNP from Pine needle (Pinus Massoniana) will be beneficial for their

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5. Conclusions

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application in food and medicinal fields.

In this study, the Mw of PNP was about 6.17×105 Da, and the main

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monosaccharide compositions contained fucose, arabinose, galactose, glucose, galacturonic acid. PNP exhibited distinct hypolipidemic and antioxidant effects in HFD-induced mice. The results suggested that PNP could be used as functional foods and natural drugs in preventing the hyperlipidemia. Conflict of interest The authors declare that there are no conflicts of interest.

ACCEPTED MANUSCRIPT Acknowledgments The present study was kindly supported by National Natural Science Foundation of China (No. 81760157). The funders had no role in study design, data

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ACCEPTED MANUSCRIPT Table 1 Chemical composition of PNP. Pine needle polysaccharide

Content

Chemical composition (%, g/g)1 Total sugar Protein Monosaccharide composition (molar %)2 Fucose Arabinose Galactose Glucose Galacturonic acid Molecular weight Weight percentage

2

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4.59 7.77 62.24 23.74 0.65 6.17×105 Da

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1

80.63 % 6.35 %

Molar percentage

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All the data are expressed as mean ± SD and are the mean of three replicates.

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Table 2 Effect of PNP on body weight, liver weight and adipose weight of HFD-induced mice (g, n=10)a.

Groups

Initial body weight

Normal control

23.19±0.66

Final body weight

Body weight gain

31.63±1.41

7.44±0.82 #

Abdominal adipose weight

1.55±0.05

0.41±0.06

1.71±0. 09

1.29±0.18##

HFD

24.20±0.87

35.50±2.21

HFD +200mg/kg bw PNP

24.04±0.42

34.93±2.45#

10.89±1.02#

1.66±0.08

1.22±0.17##

HFD + 400mg/kg bw PNP

23.84±1.08

34.07±2.21#

10.23±0.74

1.61±0.11

1.05±0.19##

HFD + 800mg/kg bw PNP

24.09±0.89

32.84±1.54*

8.75±0.68*

1.58±0.05

0.92±0.20#*

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11.70±0.95

#

Liver weight

a

Values are expressed as means ± SD in each group.

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#, ## compared with normal control (p < 0.05, p < 0.01), *,** compared with HFD (p < 0.05, p < 0.01). Data within a column sharing the same super script letters are not significantly differ significantly between

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treatments.

ACCEPTED MANUSCRIPT Highlights

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Chemical composition of Pine needle polysaccharide (PNP) was analyzed for the first time. PNP exhibited a distinctly antioxidant activity in vivo and vitro.

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PNP shown obvious hypolipidemic effect in high-fat diet-induced mice.

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PNP might potentially be used for the anti-hyperlipidemic food ingredient.

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