EFFECT OF SILYMARIN AND SILIBININ-PHOSPHATIDYLCHOLINE COMPLEX ON PLASMA AND LIPOPROTEIN CHOLESTEROL, AND OXIDATION OF LDL IN RATS FED ON HIGH CHOLESTEROL DIET SUPPLEMENTED WITH CURRANT OIL

This work was aimed to investigate the currant oil-induced modulation of antihypercholesterolemic and LDL antioxidant effects of silymarin (SM), the extract from Silybum marianum L. containing silibinin as the main flavonolignan, and of better bioavailable silibinin-phosphatidylcholine complex (SPC) in rats fed on high-cholesterol (HC) high-fat diet. Feeding of rats on HC-diet supplemented with 10 % of currant oil (from Ribes nigrum L.) containing polyunsaturated fatty acids (PUFA, 61.1% of n–6 and 15.4 % of n–3) and lower amounts of saturated (SFA, 7.7%) and monounsaturated (MUFA, 14.3%) fatty acids caused a significant decrease in plasma cholesterol associated with a mild decrease in VLDL-C and an increase in HDL-C, when compared to rats fed on HC-diet with 10 % of lard fat containing low amounts of PUFA (7.7% of n–6 and 0.7 % of n–3) and higher amounts of SFA (42.7%) and MUFA (47.5%). However, currant oil feeding led to the increased oxidizability of LDL. Silymarin, but not SPC, was effective in prevention of development of dietary induced hypercholesterolemia in the both dietary fats with slightly better result in diet containing the currant oil. On the other hand, SPC supressed more effectively than silymarin LDL oxidizability. The results suggest that antihypercholesterolemic effect of SM in rats fed on HCdiet is improved by dietary currant oil, but the currant oil induces an increased oxidizability of LDL. This can be supressed by improvement of bioavailability of silibinin, as shown here for silibinin-phosphatidylcholine complex.


INTRODUCTION
Saturated fatty acids and cholesterol are two dietary constituents well-established as serum cholesterol-raising nutrients 1 .In human, a 15 % higher LDL-cholesterol can increase the risk of coronary heart disease by about 15 %-45 % 2 .Substitution of dietary saturated fats by oils containing high amounts of polyunsaturated fatty acids has beneficial effects on hypercholesterolemia 1 .On the other hand, high intake of polyunsaturated fatty acids can induce formation of oxidatively modified LDL, which are proposed as risk factor involved in the socalled "oxidation hypothesis" of atherosclerosis [3][4][5] .Since the type of dietary fat can inversely modulate plasma cholesterol level and sensitivity of LDL to oxidation, requirements of dietary antioxidants seem to be mandatory in the case of diets rich in PUFA 6 .
Flavonoids are large group naturally occuring antioxidant polyphenols and they have received considerable attention during the last decade 7 .In human the flavonoids contribute greatly to the dietary sources of antioxidants and their intake decreased the risk of coronary heart disease as shown in Zutphen and other studies [8][9][10] .Silymarin is the standardized extract of flavonolignans from medicinal plant Silybum marianum (L.) Gaertn., Asteraceae, containing as the main compound flavonolignan silibinin.Silymarin is used in supportive therapy of liver diseases and its cytoprotective activity is believed to be based on antioxidant properties 11 .Recently, we have shown that silymarin (but not silibinin) prevents the development of dietary induced hypercholesterolemia in rats 12 , and both silymarin and silibinin possess LDL antioxidant activity in vitro 13 .However, in vivo effects of silibinin are limited by its low bioavailability, which can be improved by complexation of silibinin with phosphatidylcholine 14 .
In this study the rats were fed on high cholesterol diet and effects of silymarin and silibinin: phosphatidylcholine complex on changes of plasma lipoprotein levels and oxidizability of LDL induced by dietary PUFA -rich currant oil were investigated.
Animals.Female Wistar rats (160-170 g b.w.) were kept in standard laboratory conditions with free access to water and high cholesterol diet containing 1 % of cholesterol and 10 % of lard fat or currant oil (from the seeds of Ribes nigrum, L.) for 3 weeks.Silymarin (1% w/ w) or silibinin:phosphatidylcholine complex (0.1 % w/ w) were administered as dietary supplements in high cholesterol diets.Neither amounts of consumed diet nor body weight were affected due to the dietary interventions.After overnight fasting the blood was taken from aortic bifurcation under ether anesthesia.The blood was collected into Na 2 EDTA (mg.ml -1 ) and NaN 3 (0.1 mg.ml -1 ).
Analysis.Plasma was separated by centrifugation for 20 min at 2500 x g.Plasma lipoproteins (VLDL, LDL, HDL 2 and HDL 3 ) were isolated by sequential ultracentrifugation in density gradient 15 , cholesterol was measured enzymatically using Bio-La-Test.LDL oxidation was evaluated as Cu 2+ -induced formation of malondialdehyde measured by thiobarbituric acid reactive substances assay 16 .Composition of fatty acids in lard fat and currant oil was analyzed by gas chromatography 17 as previously described 18 and relative content of individual fatty acids was expressed as a percentage of individual peak areas of total sum peak areas.Statistical analysis was performed using Student's t-test.

RESULTS
Data presented in the Table 1 show that supplementation of high cholesterol diet with 10 % of currant oil leads to development of lower level of plasma cholesterol than supplementation of the diet with lard fat.This effect of currant oil is associated with a mild decrease in VLDL cholesterol and an increase in HDL cholesterol due to the HDL 2 cholesterol.In currant oil supplemented diet silymarin administration resulted in slightly lower level of plasma and VLDL cholesterol than in lard fat diet.Administration of silibinin: phophatidylcholine complex did not additionaly decrease the levels of plasma cholesterol in any dietary fat, but in currant oil supplemented diet mild positive changes in cholesterol distribution among lipoproteins appeared -a decrease in VLDL and LDL, and an increase in HDL due to HDL 2 .Currant oil induced an increase in Cu 2+ -oxidation of LDL.Silymarin slightly and silibinin: phosphatidylcholine complex significantly reduced LDL oxidizability in the both high fat diets.
Analysis of fatty acid composition of dietary fats -lard fat and currant oil (Table 2) demonstrates that the contents of myristic, palmitic, stearic and oleic acid are lower, and of linoleic, linolenic and octadekatetraenoic acid are higher in current oil than in lard fat.Table 3 summarizes types of fatty acids in lard fat and currant oil showing majority of polyunsaturated fatty acids in currant oil and majority of saturated and monounsaturated fatty acids in lard fat.Ratio PUFA/SFA in current oil is higher (almost 10 times) and ratio PUFA n-6/n-3 lower than in lard fat.

DISCUSSION
Feeding of rats for 3 weeks with high cholesterol diet containing 10 % of lard fat induced the development of higher hypercholesterolemia than the diet containing 10 % of currant oil.This can be related to the qualitative differences in fatty acid composition between the lard fat (majority of SFA and MUFA) and currant oil (majority of PUFA).An important role in prevention of degree of diet-induced hypercholesterolemia can be mainly ascribed to the higher content of n-6 PUFA in currant oil.It has been shown that n-6 PUFA lower plasma and LDL-cholesterol level when substituted for saturated fatty acids in the diet 1 .Antihypercholesterolemic effect of current oil was associated with a mild decrease in VLDL and an increase in HDL, both changes accepted as positive trends in distribution of cholesterol among lipoproteins.
In contrast to cholesterol lowering effect of currant oil there is an increased sensitivity of LDL to copperinduced oxidation, probably due to the higher content of n-6 and n-3 polyunsaturated fatty acids, which are prone to oxidative processes 19 .It has been suggested that high intake of PUFA may be disadvantageous, because the oxidative degradation of PUFA may lead to the formation of modified atherogenic LDL 3 .At present there is accumulating evidence that PUFA-rich diets increase the lipoprotein susceptibility to undergo oxidative modifications, while oleate-rich diets (and generaly MUFA-rich diets) produce lipoproteins more resistant to in vitro oxidation 6,[19][20][21] .Thus the high content of monounsaturated oleic acid (44.2 %) in lard fat could contribute to the relative resistance of LDL to oxidation after lard fat feeding.
In agreement with our previous results 12 silymarin administered as a 1 % dietary supplement in high-cholesterol, high-fat diets inhibited the development of dietinduced hypercholesterolemia in rats and positively affected cholesterol distribution in lipoprotein fractions (decrease in VLDL and LDL, increase in HDL).A combination of silymarin with current oil in high cholesterol diet led to the slightly lower level of plasma cholesterol than the combination of silymarin with lard fat suggesting the trend of adding effect of both cholesterol  lowering compounds.The finding that the better bioavailable silibinin-phosphatidylcholine complex 14 is not effective in prevention of diet-induced hypercholesterolemia supports our previous suggestion 12 that other constituent(s) of silymarin than silibinin may be responsible for antihypercholesterolemic activity of the extract.However, the supplementation of the both highcholesterol, high-fat diets with silibinin-phosphatidylcholine complex generated LDL more resistant to oxidation than 10 times higher concentration of silymarin in the diets.It suggests that LDL antioxidant activity of silibinin found in vitro 13 is determined in vivo by silibinin bioavailability.Therefore an improvement of biovailability of silibinin as a component of silymarin seems to be necessary to enable the LDL antioxidant activity in addition to antihypercholesterolemic effect of silymarin + currant oil combination.

Table 2 .
Fatty acid composition of dietary fats

Table 3 .
Saturated and unsaturated fatty acids of dietary fats