ÓÄÊ 636.087.7:577.125

HYPOLIPIDEMIC EFFECT OF ZOSTERIN — PECTIN FROM SEA GRASS Zostera marina L.

E.I. Khasina1, A.S. Krivonogova2

The authors investigated the hypolipidemic action of zosterin — pectin from sea grass Zostera marina L. at the high fatty and high carbohydrate rations in laboratorial experiment on rats of the Vistar line. In the conditions of alimentary hyperlipidemia zosterin promotes to the reduction of content of total cholesterol and blood triglycerides, overall lipids and cholesterol in liver tissues and skeletal muscle of animals, also promotes to the increase of cholesterol part in the fraction of high density lipoproteins and to the decrease of atherogenic indices.

Key words: hyperlipidemia, pectin, zosterin, cholesterol, triglicyrides, total lipids, atherogenic index.

 

Nutritional diseases in animals are usually associated with an excess or deficiency in the diet of physiologically important substances. Metabolic disturbances lead to many diseases, including obesity, diabetes, gallstone disease, anemia and osteodystrophy (1-4). Most of these pathologies are accompanied by hyperlipidemia, which, in turn, provokes atherosclerosis in coronary, cerebral, ventral and renal arteries (5, 6). This all suggests the actual task of veterinary - prevention and treatment of diseases with pathogenesis caused by hyperlipidemia different etiology.

Pectins are used in medicine as effective lipid-lowering correctors of lipid metabolism. This group of bioactive substances is applied in veterinary medicine too. Pectins modify the synthesis and dynamics of bile acids, they exhibit lipid-lowering, immunostimulating, anti-carcinogenic and antidiarrhoeal effects, stimulate the production of mucus in the respiratory and gastrointestinal systems, provide skin hydration, increase satiety, are effective antidotes for poisoning by heavy metals and radionuclides (7).

Pectin isolated from the sea grass Zostera is adequately studied by its physical characteristics and chemical composition, it has many pharmacological properties and is widely used in prophylactic medicine (8).

The purpose of this study - to evaluate zosterin as a lipid-lowering preparation for treatment violations of lipid metabolism caused by high-energy diet.

Technique. The preparation was tested on rats of the line Vistar - mature males with initial weight 180-190 g (7 individuals in a group for each variant of a diet) kept in vivarium under standard conditions. Previously, all animals used in the experiment were given the fat-free basic diet (BD) during a week (“Laboratorkorm”, Russia) in order to level individual differences in initial content of lipids. Then the control groups of animals (I and V) were fed a regular BD. Nutritional hyperlipidemia was induced by introduction into BD of 2% cholesterol, 5% lard and 0,25% cholic acid (high-fat diet - HFD: groups II, III and IV) or 20% sucrose (high-carbohydrates diet - HCD: groups VI, VII and VIII) for 30 days without or with addition of zosterin (groups III and VII) or the reference lipid-lowering preparation (groups IV and VIII). Zosterin was provided by “Vostokfarm” (Vladivostok, Russia). Zosterin isolated from the sea grass Zostera marina L. has following physicochemical characteristics: molecular weight - 62 000 Da, the content of galacturonan - 74,8%, etherification degree - 5,7%. The preparation was administered to rats intragastrally as 2% solution in a dose of 100 mg/kg in 1 hour before feeding during all the process of hyperlipidemia development. The reference preparation - the statin Liprimar (atorvastatin, “Pfizer”, USA) - was per orally given to animals at the dose of 20 mg/kg as powder wrapped in a fish-skin Euthanasia of rats was performed according to the requirements of the European Convention for protection of vertebrate animals used for experimental and other scientific purposes (86/609/EEC).

Blood serum was analyzed to determine total cholesterol (TC), cholesterol – the fraction of high density lipoprotein (HDL-C) and triglycerides (TG) using diagnostic kits (“Biocon”, Germany). Atherogenity index (AI) was calculated as the ratio: the difference between TC and HDL-C to HDL-C (9). Total lipids were measured gravimetrically in liver tissue and skeletal muscle (quadriceps), TC - from the reaction of Liebermann-Burkhard after extraction with Blur mixture.

 Statistical processing of data was performed using Statistica 6.0, significance of differences was assessed by Student's t-test. Data were presented as mean (M) and standard error of the mean (m) (10).

Results. The diet with high contents of fat and cholesterol (HFD) contributed to development of hyperlipidemia in animals (Table 1). This diet caused in serum the content of TC increased by 169%, TG - 50%. At the same time, a significant decrease (23%) was found in cholesterol content in anti-atherogenic lipoproteins (HDL-C) providing excretion of cholesterol from the body by it transformation in the liver into bile acids. As the result, the rats fed HFD demonstrated quite high AI (6,9-fold excess over the norm) indicating  high risks of atherosclerotic disease.

Zosterin was found to prevent the development of hypercholesterolemia, hypertriglyceridemia, it reduced HDL-C and raised AI. Against the background of zosterin, total cholesterol and triglycerides levels in serum were significantly lower (respectively 51 and 21%), HDL - higher (14%), and AI - 1,5 times lower than in rats with fat load not receiving the preparation. Therapeutical effects of zosterin were less pronounced compared with Liprimar, however, the data (Table 1) indicate reliable lipid-lowering effects of this pectin.

The diet enriched with carbohydrates (Table 1) has led to the lipid spectrum of blood serum corresponded to dyslipidemia: the content of total cholesterol and triglycerides increased by, respectively, 54 and 87%, HDL-C - decreased by 25%, AI exceeded control in 3,7 times.

Zosterin has shown the expressed hypolipidemic and antiatherogenic effects under HCD conditions: the contents of TC and TG in blood serum reduced by 32 and 43%, HDL-C - increased by 12%, AI decreased in 1,9 times compared with parameters of animals fed HCD during a month. At the high-carbohydrates diet, Liprimar caused greater effects than Zosterin (Table 1).

1. The lipid spectrum in blood serum of rats the line Vistar fed the diets with high contents of fat (HFD) and carbohydrates (HCD) against the background of Zosterin and Liprimar supplements (M±m)

Groups of animals          (dietary variants)

TC, mmol/l

HDL-C, mmol/l

TG, mmol/l

AI, conv. units

I (control)

1,38±0,08

0,78±0,04

0,99±0,06

0,76±0,04

II (HFD)

3,72±0,22*

0,60±0,02*

1,49±0,07*

5,25±0,23*

III (HFD + Zosterin)

3,01±0,15**

0,71±0,02**

1,28±0,09

3,44±0,25**

IV (HFD + Liprimar)

2,36±0,14**

0,74±0,03**

1,16±0,05**

2,36±0,22**

V (control)

1,32±0,06

0,80±0,05

0,91±0,06

0,66±0,04

VI (HCD)

2,03±0,12*

0,60±0,05*

1,70±0,11*

2,43±0,12*

VII (HCD + Zosterin)

1,61±0,09**

0,70±0,04

1,31±0,06**

1,29±0,06**

VIII (HFD + Liprimar)

1,43±0,06**

0,77±0,03**

1,03±0,06**

0,85±0,04**

Note. TC – total cholesterol, HDL-C – high density lipoproteins – cholesterol, TG – triglycerides, AI – atherogenity index.

* Ð < 0,05 when comparing the groups I-II and V-VI.

** Ð < 0,05 when comparing the groups II-III and IV, as well as VI-VII and VIII.

When feeding the high-fat diet, Zosterin prevented dyslipidemia (violation of lipid metabolism in animal tissues) (Table 2). The group of rats given HFD combined with Zosterin showed lower contents of total lipids in the liver and skeletal muscles - respectively, 23 and 33% lower than in the group not fed the preparation. At the same time, Zosterin caused hypocholesterolemic effects: in these groups of animals, TC contents in the liver and skeletal muscles differed at, respectively, 33 and 28%.

2.  The contents of total lipids (TL) and total cholesterol (TC) in the liver and skeletal muscle of rats the line Vistar subjected to alimentary hyperlipidemia against the background of Zosterin and Liprimar supplements (M±m)

Groups of animals                (dietary  variants)

TL, mg/g

TC, umol/g

liver

muscle

liver

muscle

I (control)

48,0±2,84

10,6±0,76

9,6±0,69

2,8±0,13

II (HFD)

76,7±3,96*

15,7±0,91*

14,3±0,71*

4,0±0,20*

III (HFD + Zosterin)

65,9±3,22

12,2±0,76**

11,1±0,84

3,3±0,17**

IV (HFD + Liprimar)

56,9±3,19**

13,7±0,58

11,6±0,98**

3,0±0,12**

V (control)

51,7±4,00

11,7±0,81

8,3±0,69

2,5±0,23

VI (HCD)

84,8±4,26*

15,9±0,79*

11,9±0,86*

3,2±0,23*

VII (HCD + Zosterin)

70,3±2,90**

13,0±0,74**

10,5±0,68

2,6±0,16**

VIII (HFD + Liprimar)

65,0±2,84**

12,3±0,73**

9,6±0,60**

2,7±0,13

Note. HFD – high-fat diet, HCD – high-carbohydrates diet.

* Ð < 0,05 when comparing the groups I-II and V-VI.

** Ð < 0,05 when comparing the groups II-III and IV, as well as VI-VII and VIII.

 

The development in rats of dyslipidemia induced by high sucrose content in the diet was indicated by increased concentrations of total lipids in the liver and skeletal muscle (respectively, by 64 and 36%) and total cholesterol (by 43 and 29%) compared with control. Hypolipidemic action of Zosterin was manifested against such unfavorable nutritional factor as abnormally high daily doses of carbohydrates. The animals fed Zosterin demonstrated reliably lower TL contents in the liver and quadriceps, as well as TC content lower by, respectively, 18 and 26% than the animals not fed this preparation.

The shifts in lipid spectrum at hyperlipidemia induced by these nutritional factors correspond to those described in the literature (11, 12). Lipid-lowering effect of statins (in particular – Liprimar, or atorvastatin) it is well known (13, 14) and the authors don’t discuss it in detail. However, many authors reported about side effects of statins - diarrhea, lethargy, abdominal and muscle pain, hepatotoxicity (15).

The authors’ results suggest that Zosterin significantly reduced violations of lipid metabolism in animals fed both high-fat and high-carb diets. Previously, it has been shown that Zosterin decreases hypertriglyceridemia at liver lesions caused by ethanol, cadmium and carbon tetrachloride (16). S.Yu. Tolokonnikov (17) fed broiler chickens with the mixed fodder containing the Black Sea Zostera flour meal, which contributed to increased live weight of the bird and better quality of meat (reduced cholesterol content and increased content in muscle of unsaturated fatty acids).

Contemporary veterinary and medicine has lots of data about lipid-lowering action of pectins (18, 19). In particular, S.S. Khirug et al. (20) recommend using Amaranth and citrus pectins to produce low-cholesterol poultry products. In their experiments, pectins reduced cholesterol content in the blood of laying hens and in egg powder, and it also contributed to increased mass of eggs.

Hypolipidemic action of pectins isn’t completely clear yet, though there are some ideas. It has been found that pectins activate choleresis (21), increase the activity of cholesterol-7-a-hydroxylase - the enzyme affecting formation of primary biliary acids from cholesterol (22) while cholesterol is removed from the body mainly due to biliary excretion.

Reserves of Zostera are unlimited in the oceans and year-round available for harvesting. Along with other marine macrophytes, this sea grass should attract the attention of veterinarians and farmers as a raw material for effective bioactive substances and feed additives. Fresh and boiled Zostera is widely used in coastal countries for feeding cattle, horses, sheep, goats and waterfowl (23, 24).

Thus, these studies indicate feasibility of using Zosterin – the pectin from the sea grass Zostera marina L. - for treatment of some pathologies caused by alimentary hyperlipidemia in animals.

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1A.V. Zhirmundskii Institute of Marine Biology, RAS, Vladivostok 690041, Russia,
e-mail: inmarbio@mail.primorye.ru;
2Ural Research and Development Institute for Veterinary Medicine, RAAS, Yekaterinburg 620142, Russia,
e-mail: info@urnivi.ru

Received May 17, 2010