CC BY
33
БИОХИМИЯ И БИОТЕХНОЛОГИЯ
УДК 664:577.1
Changqing Tong, Xinyao Li, Chengcheng Cai, Xuan Shi, Wei Li
College of Food Science and Engineering, Dalian Ocean University,
116023, Dalian, China
HEPATOPROTECTIVE AND LIPID-LOWERING EFFECT OF AN APPLE VINEGAR BEVERAGE WITH OYSTER POLYSACCHARIDES
The hepatoprotective and lipid-lowering effects of apple vinegar with oyster polysaccharides (OPAV) were investigated. The protective effect of OPAV on acute ethanol-induced liver injury and high-fat diet were investigated in BALB/c mice. Treatment with OPAV decreased serum aspartate aminotransferase (AST), serum alanine aminotransferase (ALT), and malondialdehyde aldehyde (MDA) levels and increased superoxide dismutase (SOD) and alcohol dehydrogenase (ADH) activity in an acute ethanol-induced liver injury model. Treatment with OPAV decreased serum total cholesterol (TC) and triglycerides (TG), and liver tissue TC and TG levels, and it increased high-density lipoprotein cholesterol (HDL-C) and excrement TC and fat content in a high-fat diet model.
Key words: apple vinegar, oyster polysaccharides, hepatoprotective, lipid-lowering effect.
Чанцин Тун, Синьяо Ли, Чэнчэн Цай, Сюань Ши, Вэй Ли
Колледж пищевых наук и инженерии, Даляньский океанологический университет,
116023, Далянь, КНР
ГЕПАТОПРОТЕКТОРНОЕ И ГИПОЛИПИДЕМИЧЕСКОЕ ДЕЙСТВИЕ НАПИТКА ИЗ ЯБЛОЧНОГО УКСУСА С ПОЛИСАХАРИДАМИ УСТРИЦ
Были исследованы гепатопротекторное и гиполипидемическое действие яблочного уксуса с устричными полисахаридами. Защитный эффект при остром повреждении печени, вызванном этанолом, и диете с высоким содержанием жиров был исследован на белых мышах. Лечение с использованием яблочного уксуса с устричными полисахаридами снижало уровни аспартатами-нотрансферазы, аланинаминотрансферазы и малонового диальдегида сыворотки крови, а также повышало активность супероксиддисмутазы и алкогольдегидрогеназы при остром поражении печени, вызванном этанолом. Лечение с помощью яблочного уксуса с устричными полисахаридами снижало общий уровень холестерина и триглицеридов в сыворотке крови и уровень холестерина и триглицеридов в тканях печени, а также повышало содержание холестерина липопротеинов высокой плотности (ЛПВП-Х), способствовало выведению холестерина и жира в модели диеты с высоким содержанием жиров.
Ключевые слова: яблочный уксус, устричные полисахариды, гепатопротекторное и гиполипи-демическое действие.
Introduction
Excessive consumption of ethanol leads to fat accumulation, inflammation in the liver, cirrhosis and hepatocellular carcinoma [1]. Ethanol abuse is associated with serious health problems of the liver. High-fat food also can also potentially damage the liver and cause the accumulation of a large amount of fat in hepatocytes [2, 3]. Numerous studies have focused on identifying the effects of protective agents on ethanol-induced liver injury and lowering the hepatic lipid levels [4-14]. Therefore, therapies that prevent alcoholic liver disease may also be beneficial in the later stages of the disease.
Oysters are used as food products and have been used as traditional medicinal shellfish for a long time [15]. Oysters are rich in protein and polysaccharides. In recent years, the polysaccharides present in oysters have become the focus of intense interest because of their various bioactivities. A water-soluble polysaccharide (CGPS-1) with hepatoprotective effects was isolated from Crassostrea gigas [16]. Unfortunately, data regarding the use of CGPS-1 in food products are limited.
Vinegar is used as a condiment, and because of its physiological effects, it also has traditional medicinal uses, including promoting recovery from exhaustion [17], regulating blood glucose [18] and blood pressure [19], and exerting antioxidant activity [20]. Vinegar can be made from various sources. Fruit vinegar beverages are produced by fermenting fruits, including apples, grapes, strawberries, pears and hawthorn berries, as the main raw material [21-25]. Fruit vinegar beverages have often been considered to have physiological health benefits [26].
The effect of the blended foods or food components on biological systems is greater than or different from the corresponding effects of the individual food or food components [27]. This study establishes the possibility of formulating a 'liver-healthy' functional apple vinegar beverage using hepatoprotective CGPS-1. Blending apple vinegar with oyster polysaccharide CGPS-1 (OPAV) could enhance its physiological effects. In this report, we describe the potential hepato-protective and lipid-lowering effects of OPAV. The hepatoprotective effect of OPAV was investigated using an ethanol-induced (acute) hepatocyte toxicity mouse model. The protective effect against ethanol-induced liver injury was assessed by measuring alanine aminotransferase (ALT), aspartate aminotransferase (AST), malondialdehyde (MDA), and trace glutathione (GSH) levels and superoxide dismutase (SOD) activity. The lipid-lowering effect of OPAV was investigated using a fatty liver mouse model and assessed by measuring low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), total cholesterol (TC), triglycerides (TG) and fat content and by evaluating histopathological changes.
Materials and methods
Materials
Apple vinegar was purchased from Henan Zhengxin Tianyuan Food Co., Ltd. (Xindeng, China). Oyster polysaccharides (CGPS-1) were isolated and separated according to the method described by Shi et al. (17). Tiopronin was purchased from Henan Xinyi Pharmaceutical Co., Ltd. (Xinxiang, China). Simvastatin was purchased from Harbin Pharmaceutical Group Sanjing Pharmaceutical Co., Ltd. (Suihua, China). Assay kits for alcohol dehydrogenase (ADH), ALT, AST, MDA, SOD, LDL-C, HDL-C, TC and TG were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The male Kunming mice were purchased from Dalian Medical University Animal Center (Dalian, China).
Preparation of OPAV
To prepare the OPAV, the 1 % yeast (W/V) was added into the oyster polysaccharides solution and cultured at 30 °C for 30 min. After culturing, the apple vinegar beverage was added to the fermentation solution and centrifuged at 4000 x g for 15 min. The resulting supernatant was the OPAV. The general OPAV composition used in this study is given in Table 1. The OPAV components consisted of water, carbohydrate, sodium, total acid, malic acid, citric acid and lactic acid were 948.65, 47.00, 0.20, 3.28, 0.56, 0.29 and 0.02 g/kg, respectively.
Animals and treatment protocol
Six- to eight-week-old BALB/c male mice were purchased from the Laboratory Animal Center of Dalian Medical University (China) and maintained in a well-ventilated room with free access to sterile standard mouse chow and water. The mice were housed under standard husbandry conditions (22 ± 2 °C, 60-70 % relative humidity and 12-h light-dark cycle). The animals were handled according to the rules and regulations of the Institutional Animal Ethics Committee of Dalian Ocean University, China. The high-fat diet consisted of 77.6 % standard laboratory diet, 10 % egg yolk, 10 % pig fat, 2 % cholesterol and 0.4 % sodium cholate and was prepared by our laboratory.
Table 1
General composition of the apple vinegar beverage (g /kg)
Component Amount
Water 948,65
Carbohydrate 47,00
Lipid 0
Protein 0
Sodium 0,20
Total acid 3,28
Malic acid 0,56
Citric acid 0,29
Lactic acid 0,02
The intoxication time, potential hepatoprotective effect and lipid-lowering effect of OPAV were evaluated using the following three treatment protocols:
a) BALB/c male mice were randomly divided into five groups (ten animals per group). After fasting for 24 h, the mice were gavaged with 52 % aqueous ethanol solution (10, 12.5, 15, 17.5 and 20 mL/kg body weight [b.w.] oral dose [p.o.]). The intoxicated time is defined as the disappearance time of the righting reflex.
b) BALB/c male mice were randomly divided into six groups of twelve animals each: (1) Group 1, treated with saline as a negative control; (2) Group 2, treated with saline and given 52 % (v/v) ethanol [12.5 mL/kg b.w. p.o. for 6 days]; (3) Group 3, treated with tiopronin (50 mg/kg b.w. p.o.) + 52 % (v/v) ethanol (12.5 mL/kg b.w. p.o.) for 6 days as a positive control; (4) Group 4, treated with OPAV (5 mL/kg b.w. p.o.) + 52 % (v/v) ethanol (12.5 mL/kg b.w. p.o.) for 6 days; (5) Group 5, treated with OPAV (10 mL/kg b.w. p.o.) + 52 % (v/v) ethanol (12.5 mL/kg b.w. p.o.) for 6 days; and (6) Group 6, treated with OPAV (15 mL/kg b.w. p.o.) + 52 % (v/v) ethanol (12.5 mL/kg b.w. p.o.) for 6 days. Thirty minutes after the last treatment, the mice were sacrificed, and their blood and liver were collected immediately.
c) BALB/c male mice were randomly divided into six groups of twelve animals each: (1) Group 1, treated with saline as a negative control; (2) Group 2, treated with saline and given a high-fat diet for 30 days; (3) Group 3, treated with simvastatin (7 mg/kg b.w. p.o.) + high-fat diet for 30 days as a positive control; (4) Group 4, treated with OPAV (5 mL/kg b.w. p.o.) + high-fat diet for 30 days; (5) Group 5, treated with OPAV (10 mL/kg b.w. p.o.) + high-fat diet for 30 days; and (6) Group 6, treated with OPAV (15 mL/kg b.w. p.o.) + high-fat diet for 30 days. Sixteen hours after the last treatment, the mice were sacrificed, and their blood and liver were collected immediately.
Assays of ALT, AST, MDA, SOD and GSH in mice serum
The blood of b) was kept at room temperature for 1 h and then centrifuged at 1500 x g for 10 min to obtain serum. The ALT, AST, MDA, and GSH levels and the SOD activity were measured using kits according to the manufacturer's instructions.
Assays of lipids in serum and liver
The TC, TG, LDL-C and HDL-C levels in serum of c) were measured using kits according to the manufacturer's instructions.
Lipids were extracted from liver tissue according to a method reported by Ding et al.29 The contents of liver TC and TG were determined using kits.
Assays of TC and fat in excrement
The excrement of mice from c) was weighed after freeze-drying. The excrement fat content was determined via Soxhlet extraction. The excrement TC content was measured using a kit.
Histopathological examination
The liver tissues of c) were fixed in 10 % phosphate-buffered neutral formalin, dehydrated in graded (50-100 %) ethanol and embedded in paraffin. The sections were cut, stained with hematoxylin and eosin and examined using a light microscope (Novel XYL-1, Ningbo Yongxin Optics Co., Ltd., China).
Statistical analysis
The values are presented as the means ± SD. The data were evaluated by one-way analysis of variance followed by Duncan's multiple-range tests. P-values less than 0.05 and 0.01 were regarded as significant and very significant, respectively.
Results and discussion
Protective effect of OPAV against ethanol-induced acute liver injury in mice
As shown in Table 2, after 52 % ethanol feeding, the ethanol-induced intoxication status of mice occurred at ethanol doses of 12.5, 15.0, 17.5 and 20.0 mL/kg. The intoxication rates of mice at the ethanol doses of 12.5, 15.0, 17.5 and 20.0 mL/kg were 50 %, 80 %, 100 % and 100 %, respectively. The intoxication times of mice at the ethanol doses of 12.5, 15.0, 17.5 and 20.0 mL/kg were 68.6, 62.38, 18.35 and 9.90 min, respectively. Ethanol-induced mortality occurred at the ethanol doses of 15.0, 17.5 and 20.0 mL/kg. The mortality of mice at the ethanol doses of 15.0, 17.5 and 20.0 mL/kg were 20 %, 30 % and 60 %, respectively. Thus, only the ethanol dose of 12.5 mL/kg was used in the ethanol-induced acute liver injury model.
Table 2
Ethanol dosage experiment in acute alcoholism BALB/c mice (means ± SD, n = 10)
Dose (mL/kg) Intoxication rate (%) Intoxication time (min) Mortality (%)
10,0 0 - 0
12,5 50 68,60±5,54 0
15,0 80 62,38±15,58 20
17,5 100 18,35±7,45 30
20,0 100 9,9±5,54 60
Acute ethanol consumption has been associated with ethanol-induced liver injury (1). The levels of AST and ALT after 6 days of treatment with 52 % (v/v) ethanol at the dose of 12.5 mL/kg b.w. p.o. are shown in Table 3. As shown in Table 3, treatment with 52 % (v/v) ethanol significantly elevated the release of AST and ALT. Treatment with the OPAV (5 mL/kg b.w. p.o., 10 mL/kg b.w. p.o. or 15 mL/kg b.w. p.o.) significantly reduced serum ALT (p < 0.01) and serum AST (p < 0.01) levels relative to the mice treated with 52 % (v/v) ethanol alone. Tiopronin (50 mg/kg b.w. p.o.) also significantly lowered serum AST (p < 0.01) and serum ALT (p < 0.01) levels compared with the ethanol-treated BALB/c mice (Table 3).
OPAV (5, 10 or 15 mL/kg b.w. p.o.) significantly decreased MDA (5, 10 or 15 mL/kg b.w. p.o.) levels and increased SOD and ADH activity (5, 10 or 15 mL/kg b.w. p.o.) compared with the ethanol group. Tiopronin (50 mg/kg b.w. p.o.) also significantly decreased MDA (p < 0.01) and increased SOD and ADH activity (p < 0.01) compared with the ethanol group (Table 3). MDA is a decomposition product of lipid hydroperoxides,30 and the SOD system and ADH protect hepatic membranes from free radical injury via a number of mecha-nisms.31 OPAV inhibited the ethanol-induced increase of hepatic MDA, suggesting that OPAV inhibits lipid peroxidation. Pretreatment with OPAV also elevated hepatic SOD and ADH activity in the mice with liver injury. The polysaccharides are well recognized for their potential to act as protective agents against common types of alcohol liver injury. OPAV contains many polysaccharides, particularly CGPS-1. This suggests that OPAV has potential hepatoprotective effects.
Table 3
Effect of OPAV on the levels of serum markers in BALB/c mice with ethanol-induced acute hepatic injury
Group ALT (U/L) AST (U/L) MDA (nmol/mg protein) SOD (U/mg protein) ADH (U/mg protein)
Control 32,09±6,85## 51,70±1,35## 31,74±1,78## 131,83±8,68## 131,73±10,06##
Ethanol 51,83±2,68** 83,73±4,99** 36,68±1,63** 89,94±3,99** 77,78±5,72**
Tiopronin + ethanol 45,49±0,60** 60,41±2,60## 31,16±0,55## 111,51±1,65**## 111,95±18,24*##
OPAV (5 mL/kg/day) + ethanol 44,82±4,31* 72,47±1,68** 32.26±0.84## 106.26±1.06**## 91,46±2,96**
OPAV (10 mL/kg/day) + ethanol 64,57±8,59**# 78.92±3.34** 33,19±1,15# 105.61±1.49**## 109,49±6,59**##
OPAV (15 mL/kg/day) + ethanol 56,84±2,86** 77,47±16,66** 29,84±1,90## 109,27±6,33**## 124,82±11,71##
-----—#-^
*p < 0.05 versus normal control group, **p < 0.01 versus normal control group. p < 0.05 versus model control group, ##p < 0.01 versus model control group. The results are the mean ± SD.
Lipid-lowering effect of OPAV
High-fat food also has the potential to cause liver damage and the accumulation of a large amount of fat in hepatocytes [2, 3]. Here, we investigated the effect of OPAV on the TC, TG, LDL-C, and HDL-C levels in the serum, TC and TG in the liver tissue, and TC and fat in the excrement of mice. The effect of simvastatin (7 mg/kg b.w. p.o.) on the TC, TG, LDL-C, HDL-C and fat levels was used as a positive control. After 30 days of high-fat diet treatment, the TC, TG and LDL-C levels in the serum were increased (6.14, 5.64 and 1.05 nmol/L, respectively), and the HDL-C levels (1.72 nmol/L) were decreased in the high-fat diet group compared with the control group, indicating that a high-fat diet leads to fat accumulation. However, the TC, TG and LDL-C levels were reduced, and the HDL-C level was increased in the high-fat diet group treated with simvastatin or OPAV (5, 10 or 15 mL/kg b.w. p.o.) compared with the high-fat diet group (Table 4).
Table 4
Effects of OPAV on the levels of serum markers in BALB/c mice fed a high-fat diet
Group TC (mmol/L) TG (mmol/L) LDL-C (mmol/L) HDL-C (mmol/L)
Control 3,76±0,99 2,46±0,79 0,94±0,20 2,77±0,45
High-fat diet 6,14±0,70** 5,64±0,30** 1,05±0,14 1,72±0,39**
Simvastatin + high-fat diet 4,23±0,61## 2,98±0,41*## 1,00±0,19 2,37±0,44##
OPAV (5 mL/kg/day) + high-fat diet 4,70±0,41**## 3,09±0,56*## 1,05±0,17 2,29±0,47*##
OPAV (10 mL/kg/day) + high-fat diet 4,38±0,51*## 3,04±0,70*## 0,97±0,15 2,32±0,58*##
OPAV (15 mL/kg/day) + high-fat-diet 4,17±0,63## 3,04±0,55*## 0,95±0,15 2,37±0,48##
-----#-
*p < 0.05 versus normal control group, **p < 0.01 versus normal control group. p < 0.05 versus model control group, ##p < 0.01 versus model control group. The results are the mean ± SD.
In addition, after 30 days of high-fat diet treatment, the TC and TG levels in the liver tissue were increased (17.6 and 18.46 ^mol/g, respectively), and the TC and fat levels in the excrement were decreased (25.24 and 51.30 ^mol/g, respectively) in the high-fat diet group compared with the control group. The TC and TG levels in the liver tissue were reduced, and the TC and fat levels were increased in the high-fat diet group treated with simvastatin or OPAV (5, 10 or 15 mL/kg b.w. p.o.) compared with the high-fat diet group (Table 5).
Table 5
Effects of OPAV on the levels of serum markers in liver tissue and excrement in BALB/c
mice fed a high-fat diet
Group TC TG TC Fat
(^mol/g liver (^mol/g liver (^mol/g excrement) (mg/g excrement)
tissue) tissue)
Control 12,76±1,21## 12,58±4,00## 4,22±0,75## 12,73±0,84##
High-fat diet 17,60±1,66** 18,46±1,73** 25,24±5,87** 51,30±1,41**
Simvastatin+ high- 14,47±1,15## 15,56±1,40 69,21±3,64**## 77,10±2,04**##
fat diet
OPAV 16,95±1,33** 16,89±1,70** 66,63±3,57**## 88,00±5,37**##
(5 mL/kg/day) + high-fat diet
OPAV 16,21±2,44** 16,80±2,98** 86,84±0,44**## 96,9±2,69**##
(10 mL/kg/day) + high-fat diet
OPAV 15,93±0,78** 14,09±2,86## 95,81±7,68**## 98,57±3,29**##
(15 mL/kg/day) + high-fat diet
*p < 0.05 versus normal control group, **p < 0.01 versus normal control group. #p < 0.05 versus model control group, ##p < 0.01 versus model control group. The results are the mean ± SD.
The differences in mouse liver tissue slices from the control group, the high-fat diet group, the simvastatin + high-fat diet group and the various OPAV groups were also compared by optical microscopy. As shown in Fig. 1A, the liver sections from the controls showed normal hepatic cells, whereas abundant large fat vacuoles were observed in the high-fat diet group (Fig. 1B). In the simvastatin and OPAV (5, 10 and 15 mL/kg b.w. p.o.) groups, no fat vacuoles were found (Fig. 1C-F). These results suggest that OPAV prevents the high-fat diet from impairing hepatic function. A number of polysaccharides with preventive effects against fat-induced liver injury have been investigated [15, 28].
Acknowledgements
The work was supported by a grant from the Marine Public Welfare Research Project (201405017-03).
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Effect of OPAV on high-fat diet-fed BALB/c mice: A control; B high-fat diet; C simvastatin + high-fat diet; D OPAV (5 mL/kg/day) + high-fat diet; E OPAV (10 mL/kg/day) + high-fat diet; F OPAV (15 mL/kg/day) + high-fat diet. Original magnification 40*
Сведения об авторах: Чанцин Тун, Синьяо Ли, Чэнчэн Цай, Сюань Ши, Вэй Ли, e-mail: aisingioro@hotmail.com.
