Bull Vet Inst Pulawy 48, 453-459, 2004

INFLUENCE OF OXIDATED VEGETABLE OIL AND GARLIC EXTRACT UPON THE DEVELOPMENT

OF EXPERIMENTAL ATHEROSCLEROSIS IN RABBITS

JOLANTA ZALEJSKA–FIOLKA1, SŁAWOMIR KASPERCZYK1, ALEKSANDRA KASPERCZYK1, EWA BIRKNER1, EWA GRUCKA-MAMCZAR1,

BARBARA STAWIARSKA-PIĘTA2 AND ANNA SCHNEIDER1

1Department of Biochemistry in Zabrze, 2Department of Pathology in Sosnowiec, Medical University of Silesia, 40-006 Katowice, Poland

e-mail: jfiolka@slam.katowice.pl

Received for publication May 24, 2004.

Abstract

The aim of the study was to find out if the consumption of oxidised oils aggravates the undesired changes in the concentration of lipids and homocystein in blood and in the aorta endothelium region and whether the addition of garlic to diet results in anti-atherosclerotic action. The experiments were carried out on mixed breed rabbits divided into 3 groups. The body weight of the rabbits was checked once a week and every six weeks blood samples were taken. The concentration of homocystein, total cholesterol, HDL cholesterol, and triacylglyceroles were determined in blood. After the experiment was completed, aortas were dissected for histological examinations. It was found that oxidised rapeseed oil, administered to animals, caused the development of atherosclerotic alterations in the aorta wall region and increased the homocystein content in blood serum. The administration of garlic in diet inhibited atherosclerotic changes in the aorta wall and this seems to be related to the decreasing concentration of triacylglyceroles in blood serum. Key words: rabbits, garlic, vegetable oil, oxidation, atherosclerosis.

Diseases of the cardiovascular system still take a leading world-wide position, as regards incidence and mortality (1). Prevention and treatment of ischaemic heart disease is connected with a recommendation to consume vegetable fats and to reduce animal fat content in the diet (2). Research proved, though, that such prophylaxis is not risk-free (3). Vegetable fats contain polyunsaturated fatty acids, which are prone to oxidation. Frying in the same vegetable oil and increasing its temperature to 1800C-1900C several times results in a chemical reaction which is similar to that taking place during vegetable fat hardening in industrial manufacturing. A substantial administration of oxidised vegetable oils in diet may lead to aggravation of the free radical processes. It is believed that such a diet, similar to excessive consumption of products rich in cholesterol, results in the development of vascular lesions, leading

to atherosclerosis and then to diseases of the cardiovascular system (4, 5).

Research revealed also that high consumption of antioxidant substances reduces the risk of developing circulatory system diseases (6, 7). It would seem crucial to find out whether additions of antioxidant substances to food results in reducing the disadvantageous changes caused by the consumption of oxidised vegetable oils. One of such natural substances having an anti-oxidative character is garlic extract, the protective role of which in the oxidation of edible oils we have proven in our earlier studies (8, 9). Garlic extract also appears to have a multidimensional influence upon the organism. Among others, it causes a reduction in the concentration of lipids, which have been recognised as one of the risk factors of developing cardiovascular system diseases (10, 11). Numerous communications indicate homocystein to be the new, independent factor testifying to the development of atherosclerosis in early stages of the disease (1, 12, 13).

Having the above in mind, we aimed of our studies at the following questions:

1. Does the oxidation of edible oil in 1200C cause

changes in the content percentage of fatty acids?

2. Will the consumption of oils oxidised in 1200C aggravate the undesired changes in the concentration of lipids and homocystein in blood and cause sclerotic lesions in the aorta endothelium region?

3. Does the addition of garlic to diet result in anti-atherosclerotic action?

Material and Methods

Preparation of oil: „Kujawski” rapeseed oil (ZT „Kruszwica” S.A., Poland) was used in the study. The oil was oxidised for 7 d at 1200C. The oxidation

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process was controlled by determining the peroxide value, in accordance with the Norm of the Polish Standardisation Committee (PN – ISO 3960) (14), and the content of fatty acids by means of gas-chromatography (15).

Animals. The experiments were carried out on mixed breed rabbits, with the initial body weight of 2500g ± 50g. The animals after initial selection (lipid parameters) were divided into 3 groups, six rabbits in each.

Experimental design. The rabbits of group I were fed fresh rapeseed oil, group II – oxidised rapeseed oil and group III – oxidised rapeseed oil and garlic (Allium sativum L). Garlic was chopped and homogenized, then 1 g of the homogenized garlic was added to the feed. The detailed experimental design and fodder content were presented in Table 1. The experiment lasted 24 weeks.

The body weight gain in the rabbits was checked once a week and every six weeks blood samples were taken from the central auricular vein, in the amount of 5 ml each time. After the experiment was completed, the animals were anaesthetised and blood was taken from their hearts for biochemical examination. After lethal anaesthesia, their aortas were dissected for histological examinations.

Biochemical and histological examina-tions. Serum concentration of homocystein was determined with the liquid chromatography (HPLC) method by Kuo (16). The contents of total cholesterol, HDL cholesterol,

and triacylglyceroles were determined by enzymatic methods, using tests by Alpha Diagnostics (Germany). Histopathological examination of the aortas was performed in the Department of Pathology of the Medical University of Silesia in Sosnowiec. The aortas was fixed in 10% formalin and paraffin sections stained with haematoxylin and eosin (H-E) and frozen sections stained with Sudan III for neutral fats (17) were prepared. Colour microphotographs were taken with the Docuval microscope equipped with the photo device (Carl Zeiss Jena).

Statistical analysis. Software Statistica PL was used for statistical analysis. For comparison of changes between particular groups of animals the test ANOVA by Kruskal-Wallis and test U by Mann-Whitney were used.

Results

The results of the investigations are presented in Tables 2 and 3, as well as in Figs 1 through 8. As can be seen from Table 1, the rapeseed oxidized for 7 d at 1200C showed an increased content of palmitic acid by 27% and oleic acid by 21% and a decreased content of linolic acid by 63% and linolenic acid by 92%. At the same time, the peroxide value (PN) increased 39 times (Table 3).

Table 1 Experimental design and fodder composition

Group I – control (C) Group II – experimental (O) Group III – experimental (OG)

Fodder composition Protein Fats Saccharide Protein Fats Saccharide Protein Fats Saccharide

21% 34% 45% 21% 34% 45% 21% 34% 45% Fresh rapeseed oil

(12 g/200 g of fodder, accounted for in total fat content)

Oxidised rapeseed oil (12 g/200 g of fodder, accounted for in

total fat content)

Oxidised rapeseed oil (12 g/200 g of fodder, accounted for in total

fat content) and 1 g of garlic

Table 2

Percentage content of fatty acids in rapeseed oil before and after its oxidation for 7 d at 1200C Content of fatty acids (%) in rapeseed oil

Fatty acids before oxidation after oxidation Palmitic acid 16:0 5.6±0.06 7.1±0.06

Oleic acid 18:1 71.4±0.10 86.5±0.10 Linolic acid 18:2 15.5±0.06 5.8±0.10

Linolenic acid 18:3 7.4±0.21 0.6±0.08 ± standard deviation (SD); all results are averages of 4 analyses

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Table 3

Peroxide value (PV) in rapeseed oil before and after oxidation

Rapeseed oil

Before oxidation After oxidation 4.5±0.25 181.24±0.10

± standard deviation (SD); all results are averages of 4 measurements The concentration of homocystein in blood

serum increased temporarily (the 12th week of the experiment) by 20-25% in groups receiving oxidised oil and oxidised oil with garlic, and at the end of the experiment no changes in the concentration of that parameter were observed in comparison with control (Fig.1). In the OG group, in week 24 of the experiment, a statistically significant (P<0.010) drop in triacylglyceroles concentration was noted (by 35%) in comparison with the group administered oxidised oil only (group O). At the same time no difference in the concentration of that parameter was noted in comparison with control (Fig.2). The concentration of total cholesterol and HDL cholesterol revealed no statistically significant differences throughout the experiment (Figs 3 and 4). The body weight of animals in the

experimental groups did not differ from that of animals in control during the experiment (Fig.5).

Histological examinations of aorta specimens revealed that the presence of fresh rapeseed oil in diet caused no histopathological changes in the aorta membrane region (Fig. 6), whereas the presence of oxidised rapeseed oil in diet caused focal thickening of the wall, with proliferation of myocytes in the aortic media in all animals examined (Fig. 7). In rabbits receiving oil diet supplemented with garlic, focal thickening of the internal layer of the aorta was noted only in 2 animals (Fig. 8).

Histochemical examination of aorta specimens stained with Sudan III revealed that the presence of fresh rapeseed oil and garlic with the oxidised oil in diet caused no changes in neutral fat content in the aorta membrane region (Fig. 10).

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Fig. 1. The concentration of homocysteine in blood serum at the start of the experiment, at its 12th week, and at the end (average ± SEM).

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Fig. 2. The concentration of triacylglyceroles in blood serum at the start of the experiment, at its 12th week, and at the end (average ± SEM).

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p=0.944ANOVAp=0.548

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Fig. 3. The concentration of total cholesterol in blood serum at the start of the experiment, at its 12th week, and at the end (average ± SEM).

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Fig. 4. The concentration of HDL-cholesterol in blood serum at the start of the experiment, at its 12th week, and at the end (average ± SEM).

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Fig. 5. Body weight of animals at the start of the experiment, at its 12th week, and at the end (average ± SEM).

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Fig. 6. Control group I. Rabbit aorta after 3-month administration of fresh rapeseed oil. H-E. x 150.

Fig. 7. Group II after 3-month administration of oxidised rapeseed oil. H-E. x 150.

Fig. 8. Group III after 3-month administration of oxidised rapeseed oil and garlic. H-E. x 150.

Fig. 9. Control group I. Rabbit aorta after 3-month administration of fresh rapeseed oil. Sudan III. x 150.

Fig. 10. Group II after 3-month administration of oxidised rapeseed oil. Sudan III. x 150.

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Discussion

It was noted that the oxidation of rapeseed oil caused a significant increase in the peroxide value (PN), as well as changes in the percentage content of fatty acids in the oil, which indicate the disadvantageous process taking place during the oxidation (Tables 2 and 3). We obtained similar results in our earlier in vitro studies (8). The changes in the content of fatty acids prove that the oxidation of rapeseed oil at 1200C lead to the saturation of polyunsaturated fatty acids. Due to the fact that numerous studies point to the diets rich in saturated fatty acids as diets inducing atherosclerotic lesions (4, 18, 19), it could then be assumed that diet enriched with rapeseed oil oxidised in such a way would cause the development of atherosclerotic lesions in the experimental model assumed.

The results of the research revealed that the supplementation of diet with oxidised rapeseed oil and oxidised rapeseed oil with garlic did not cause changes in the level of total cholesterol or HDL cholesterol in comparison with control. After 24 weeks of the experiment it was found that garlic added to oxidised rapeseed oil as part of fodder caused a decrease in the level of triacylglyceroles (TG) in comparison with the group receiving only oxidised rapeseed oil. It follows that addition of garlic reduces the level of TG compared to the group receiving fresh oil. It could be assumed then that garlic acted effectively in the initial phase of lipid accumulation.

No relative changes were observed throughout the experiment in animal body weight, in all experimental groups. Still, increased condition of the hair and claws was noted in the group receiving garlic, which may testify to its multi-directional influences.

Both the histopathological and histochemical examinations demonstrated that fresh rapeseed oil did not cause lesions in the aortas of experimental animals. The lesions were noted in animals given the oxidised rapeseed oil. The beneficial influence of garlic upon the animal aortas was confirmed. The results are in line with the reports of other authors (10, 11) that indicate the protective role of garlic in hypercholesterolaemia.

Our study revealed a temporary increase in homocystein concentration in the groups of animals receiving oxidised oil and oxidised oil with garlic. Perhaps longer oxidation of oil in higher temperatures would result in a more substantial increase in homocystein concentration and more profound progression of atherosclerotic lesions.

Acknowledgments: The work has been

reviewed by the Ethic Committees and obtained the consent of the Academic Animal Ethic Committees in Katowice.

References 1. Brzósko Sz., Hryszko T., Małyszko J., Myśliwiec M.:

Homocysteina – nowy czynnik ryzyka rozwoju

miażdżycy i jej powikłań. Pol Merk Lek 2001, 10, 55-56.

2. Applenwhite T.H.: Trans-isomers, serum lipids and cardiovascular disease: Another point of view. Nutr Rev 1993, 51, 344-352.

3. Felton C.V., Crook D., Davies M.J., Oliver M.F.: Dietary polyunsaturated fatty acids and composition of human aortic plaques. Lancet 1994, 334, 1195-1196.

4. Naruszewicz M., Woźny E., Mirkiewicz E., Nowicka G., Szostak W.: The effect of thermally oxidised soya bean oil on metabolism of chylomicrons. Increased uptake and degradation of oxidizes chylomicrons in cultured mouse macrophages. Atherosclerosis 1987, 66, 45-53.

5. Naruszewicz M.: Do oxidized fats in human nutrition constitute a risk factor of atheromatosis? Czyn Ryz 1996, 2-3, 11-14.

6. Pałgan K., Sienkiewicz W.: The protective role of flavonoids in cardiovascular diseases. Czyn Ryz 1998, 1, 48-55.

7. Bok S.H., Shin Y.W., Bae K.W., Jeong T.S., Kwon Y.K., Park Y.B., Choi M.S.: Effects of naringin and lovastatin on plasma and hepatic lipids in high-fat and high-cholesterol fed rats. Nutr Res 2000, 20, 1007-1015.

8. Zalejska-Fiolka J.: The influence of garlic extract on oxidation process of edible oils. Riv Ital Sost Grasse 2001, 6, 343-346.

9. Zalejska-Fiolka J.: The influence of garlic extract on oxidation process of edible oils - part II. Riv Ital Sost Grasse 2002, 10, 365-368.

10. Adler A., Holub B.J.: Effect of garlic and fish-oil supplementation on serum lipid and lipoprotein concentrations in hipercholesterolemic men. Am J Clin Nutr 1997, 65, 445-450.

11. Stevinson C., Pittler M.H., Ernst E.: Garlic for treating hypercholesterolemia. Ann Intern Med 2000, 133, 420-429.

12. Palasik W.: Homocysteine – risk factor for ischemic stroke. Post N Med 2001, 3-4, 74-80.

13. Prątnicka M.: Postępy w diagnostyce hiperhomocysteinemii. Diagn Lab 2003, 39, 83-91.

14. Polish Standardisation Committee. Animal and vegetable oils and fats – determination of peroxide value. Edited by Norm Alfa–Wero, Warsaw, 1996.

15. Mol J., Rostański K., Szulik J.: The Oenothera species evaluation by gas chromatography. Riv Ital Sost Grasse 2001, 1, 35-40.

16. Kuo K., Still R., Cale S., McDowell I.: Standardization (external and internal) of HPLC assay for plasma homocysteine. Clin Chem 1997, 43, 1653-1655.

17. Zawistowski S.: Technika histologiczna, histologia oraz podstawy histopatologii. Edited by PZWL, Warsaw, 1986, pp. 108-122

18. Staprans I., Rapp J.H., Pan X.M., Hardman D.A., Feingold K.R.: Oxidized lipids in the diet accelerate the development of fatty streaks in cholesterol-fed rabbits. Arterioscler Thromb Vasc Biol 1996, 16, 533-538.

19. Staprans I., Rapp J.H., Pan X.M., Kim K.Y., Feingold K.R.: Oxidized lipids in the diet are a source of oxidised lipid in chylomicrons of human serum. Arterioscler Thromb Vasc Biol 1994, 14, 1900-1905.