Suppressive impact of anethum graveolens consumption on biochemical risk factors of atherosclerosis in hypercholesterolemic rabbits.

BACKGROUND: We aimed to determine the effects of Anethum graveolens (Dill) powder on postprandial lipid profile, markers of oxidation and endothelial activation when added to a fatty meal.
METHODS: In an experimental study, 32 rabbits were randomly designated into four diet groups: normal diet, high cholesterol diet (1%), high cholesterol diet plus 5% (w/w) dill powder and high cholesterol diet plus lovastatin (10 mg/kg, bw). The concentrations of glucose, total cholesterol (TC), low-density lipoproteins-cholesterol (LDL-C), alanine aminotransferase (alt), aspartate aminotransferase (ast), fibrinogen, factor VII, apolipoprotein B (ApoB), nitrite and nitrate were measured in blood samples following 15 h of fasting and 3 h after feeding.
RESULTS: Concurrent use of A. graveolens powder or lovastatin significantly decreased ALT, TC, glucose, fibrinogen and LDL-C values in comparison with hypercholesterolemic diet group (P < 0.05). Consumption of A. graveolens or lovastatin did not change factor VII, ApoB, nitrite and nitrate levels significantly in comparison with hypercholesterolemic diet group. Intake of A. graveolens significantly decreased serum AST compared to hypercholesterolemic diet.
CONCLUSIONS: A. graveolens might have some protective values against atherosclerosis and that it significantly affects some biochemical risk factors of this disease. Our findings also confirm the potential harmful effects of oxidized fats and the importance of dietary polyphenols in the meal.

INTRODUCTION

Hyperlipidemia promotes atherosclerosis, which is a syndrome affecting arterial blood vessels, a chronic inflammatory response in the walls of arteries, caused largely by the accumulation of macrophage white blood cells and promoted by low-density lipoproteins (LDL) without adequate removal of fats and cholesterol from the macrophages by functional high-density lipoproteins (HDL). It is caused by the formation of multiple plaques within the arteries.[1]

Atherosclerosis is a chronic disease that remains asymptomatic for a long time.[2] Upon formation, intraluminal thrombi can occlude arteries outright (i.e., coronary occlusion), but more often they detach, move into the circulation and eventually occlude smaller downstream branches causing thromboembolism. Apart from thromboembolism, atherosclerotic lesions can cause complete closure of the arteries. Interestingly, chronically expanding lesions are often asymptomatic until the artery stenosis is so severe that the blood supply to downstream tissue (s) is insufficient and results in ischemia.[3]

Various, physiological, anatomic and behavioral risk factors for atherosclerosis are known. Diabetes or impaired glucose tolerance, dyslipoproteinemia, high serum concentration of LDL and/or very low density lipoprotein (VLDL), Low serum concentration of functioning HDL, LDL:HDL ratio greater than 3:1, elevated serum C-reactive protein concentrations, elevated lipid peroxidation, are among the most important modifiable risk factors of atherosclerosis.[456]

Hyperlipidemia and lipid peroxidation are more than others implicated in induction of atherosclerosis. As humans are predominantly in a postprandial state, attention has been paid, recently, on postprandial hyperlipidemia and its abnormalities on the contributing factors leading to atherosclerosis.[789]

Evidence suggests that postprandial hypertrigly-ceridemia is a risk factor for cardiovascular disease (CVD)[10] and that it has a negative effect on endothelial function in normal subjects.[71112]

It has also shown that medicinal plants, rich in antioxidants, may ameliorate hyperlipidemia,[131415] other than reduction of diabetic or atherosclerosis complications.[16] These plants mostly include phenolic and flavonoid components with antioxidant activities.[15]

It has also been shown that flavonoid-containing food such as fruits, vegetables and tea are protective against CVD through their free radical-scavenging quality.[17] As it was mentioned scientists mostly attribute these effects to flavonoids potential role in decreasing blood lipid levels and therefore changing the postprandial state.[1018]

Dill is a short-lived perennial herb and is the sole specie of the genus Anethum. Its seeds contain 3% oil, carotene, flandrenin, limonene and tannin. In traditional Iranian medicine, Dill has been used as sedative, carminative, antispasmodic, lactogogue, diuretic and home remedy for hyperlipidemia.[19]

It has been found that other than glucose, dill significantly may reduce triglyceride, total cholesterol (TC), VLDL, LDL-cholesterol (LDL-C) and increases HDL-cholesterol (HDL-C) in diabetic rats. These effects have also been attributed to antioxidant contents of dill.[20] It seems that if dill reduces postprandial hyperlipidemia, it would be beneficial to use in these patients. In this study, therefore, we assessed the effects of Anethum graveolens powder on postprandial risk hyperlipidemia and some other factors leading to atherosclerosis in rabbits, fed a high cholesterol diet.

METHODS

Plant preparation

A. graveolens was purchased from the local market in Shahrekord, Iran. The genus and species were authenticated by a botanist in the Medical Plants Research Centre of Shahrekord University of Medical Sciences and a voucher specimen was deposited in the institution herbarium (number 236). The plants were dried, powdered and anthocyanins, flavonoids and phenolic contents were measured for standardization as follows.

Measurement of total anthocyanins

A total of anthocyanins were assayed by spectrophotometeric method at 535 nm.[21] A total of 3 g of the sample was weighed in a 50-mL centrifuge tube and 24 mL of acidified ethanol (ethanol and HCl 1.0 N, 85:15, v/v) was added. The solution was mixed and adjusted to pH 1 with 4 N HCl. The resulting solution was shaken for 15 min, readjusted to pH 1 if necessary and the solution was shaken for an additional 15 min. The tube was centrifuged for 15 min, at 27,200 × g and the supernatant was poured into a 50-mL volumetric flask and made up to volume with acidified ethanol. Cyanidin 3-glucoside was used as a standard pigment. A series of cyanidin 3-glucoside standard solutions was prepared at 0-0.02 mmol (0-27 mg/3 mL). Absorbance was read at 535 nm against a reagent blank.

Measurement of total flavonoids

The amount of flavonoid components in the Anethum extract was evaluated by colorimetric method.[22] Thus, 0.5 mL of rutin (standard flavonoid compound) or garlic extract was added to a flask containing 1.5 mL of methanol, 2.8 mL of distilled water, 0.1 mL of 1 M potassium acetate, 0.1 mL of 10% aluminum chloride and left at room temperature for 30 min. The reaction absorbance was measured at 415 nm after preparing a standard curve from rutin solutions at concentrations of 25-500 ppm in methanol. The experiment was repeated in triplicate. The total flavonoid components were expressed in terms of rutin equivalents (in mg/g).

Measurement of total phenolic compounds

The amount of total phenolic components in the Anethum extract was determined colorimetrically using the Folin-Ciocalteu reagent.[2324] in brief, 5 mL of gallic acid (standard phenolic compound) or Anethum extract was mixed with Folin-Ciocalteu reagent diluted with distilled water (1:10) and aqueous Na2CO3 (4 mL, 1 M). The mixture was left to stand for 15 min and the total phenolic compounds were determined by colorimetry at 765 nm. A standard curve was prepared from different concentrations (0-250 mg/L) of gallic acid in methanol: water (50:50, vol/vol). The experiment was repeated in triplicate and the total phenolic values were expressed in in mg/g of gallic acid equivalent.

Treatment of rabbits

This study was approved by the ethics committee of Shahrekord University of Medical Sciences. 32 male white New Zealand rabbits weighing 2010 ± 275 g were obtained from Razi Vaccine and Serum Research Institute in Iran. They were kept in an air-conditioned room for 2 weeks and allowed water and standard rabbit chow containing 16% protein, 4-5% vegetable fat, 13% fiber, 1.4% calcium, 0.8% phosphor, 0.5% cysteine and 0.5% tryptophan.[25]

The rabbits were randomized into four groups of eight and fed a diet as follows: group I: normal diet, Group II: diet containing 1% cholesterol, Group III: a high cholesterol diet containing 5% dill powder and Group IV: high cholesterol (1%) plus lovastatin (10 mg/kg bw).

After fasting for 12-15 h, venous blood samples were taken to obtain baseline data. Venous blood samples were also obtained 3 h after the diet to determine the acute effects of A. graveolens.[10]

Measurement of biochemical factors in rabbits

Serum and plasma samples were collected by centrifuging blood samples at 3500 rpm for 20 min. Fibrinogen and factor VII values were obtained through the plasma and the serum was used for other biomarker measurements.

Serum TC, LDL-C, apolipoprotein B (ApoB), alanine aminotransferase (alt), aspartate aminotransferase (ast) and serum glucose were determined using standard enzymatic kits (Pars Azmoon Co, Iran) and an auto-analyzer (Hitachi 902, Japan). Factor VII was measured using clotting time, in the presence of the STA-Neoplastine reagent of a system in which all factors were present, constant and in excess except factor VII, which is derived from the sample being tested (Diagnostic Stago, French). The serum levels of nitrite and nitrate were measured using a colorimetric assay kit (R and D Systems, USA), which involves the Griess reaction.

Statistical analysis

Results were analyzed using Instat 3 software. To investigate the biochemical results and comparison of the experimental groups, Kruskal-Wallis and Dunn tests were used and P < 0.05 was considered statistically significant.

RESULTS

Determination of some physiochemical factors in A. graveolens

Analyzing A. graveolens factors showed that the amounts of total anthocyanins, flavonoids and phenolic compounds in the extract were 94 mg/100 g, 62 mg/100 g (equivalent to gallic acid) and 95 mg/100 g (equivalent to gallic acid), respectively.

Measurement biochemical factors in rabbit

Consumption of A. graveolens powder or lovastatin significantly decreased ALT, TC, glucose, fibrinogen and LDL-C values in comparison with hypercholesterolemic diet group [Table 1]. Concurrent use of A. graveolens or lovastatine did not change factor VII, ApoB, nitrite and nitrate levels significantly in comparison with hypercholesterolemic diet group. Intake of A. graveolens significantly decreased serum AST compared with hypercholesterolemic diet [Table 1].

Table 1

Comparison of LDL-C, factor VII, ApoB, TC, AST, ALT, glucose, nitrite, nitrate and fibrinogen values at the end of experiment

DISCUSSION

The association between postprandial triglyceride and atherosclerosis has been confirmed by various studies.[1026] Evidence suggests that postprandial lipoproteins induce the expression and release of endothelial mediators in vitro and that this metabolic syndrome is associated with humeral risk markers of endothelial origin.[27282930]

Recently, the importance of therapeutic plants has become the focus of many studies. Vegetables, fruits and their juices contain phenolic compounds, which have antioxidant properties and are protective against atherosclerosis.[3132]

In a study, the hypolipidemic effects of powdered dill and its special oil (which is the main constituent of this plant) was investigated in male rats fed a high cholesterol diet. The results of this study showed that adding powdered dill to rat's diet for 2 weeks significantly reduced TC, triglyceride and LDL-C and also significantly increased HDL-C levels.[33] In the present study, we assessed the effects of A. graveolens powder on postprandial risk factors leading to atherosclerosis in rabbits fed a high cholesterol diet. In this study, lovastatin was used to compare the effects of standard drug with dill. Statins reduce the cholesterol content from lipoporoteins by their hypercholestric effects and also deplete the amount of oxidable compounds.[32]

Lovastatin attaches to phospholipids on LDL surface and therefore prevents the diffusion of free radicals under oxidative pressure to the core of lipoproteins.[34]

By significantly reducing LDL-C, cholesterol, glucose, ALT, AST values in A. graveolens seems to have the potential to protect against coronary artery disease. It decreases LDL-C and cholesterol levels by either upregulating lipoprotein lipase, which hydrolyses triglycerides or reducing the hepatic VLDL production or by reducing the activity of two enzymes involved in cholesterol metabolism (3-hydroxy-3 methyl glutaryl coenzyme A reductase and/or acyl cholesterol acyl transferase). Acyl cholesterol acyl transferase catalyses the intracellular esterification of cholesterol and plays a role in cholesterol absorption and hepatic secretion of VLDL and ApoB.[35]

The glucose reducing effects of powdered dill can be due to the following:[3637]

The presence of flavonoids, which inhibits glucose absorption in the intestine

Flavonoids block the enzyme glucose 6 phosphatase and therefore reduce blood glucose levels. This enzyme mediates separation of phosphate from phosphorylated glucose and therefore releases glucose into the blood stream.

Nitric oxide (NO) is the most important vasodilator derived from the endothelium. NO produced by endothelial nitric oxide synthase (eNOS) has a significant role in the initiation of coronary vessel hemostasis, whereas NO produced by inducible nitric oxide synthase (iNOS) causes CVD. iNOS is an enzyme produced by various cells and mediated by cytokines in inflammatory conditions.[38394041424344] Anthocyanins reduce iNOS expression and increase the expression of eNOS and thus play a role in the equilibrium between iNOS and eNOS in different pathologic systems.[45] In the present study, its blood level was not changed, perhaps due to shortage of the experiment.

Studies indicate the role of vegetarian diets in the hemostasis of coagulation and fibrinolysis. These compounds are effective in reducing coagulation factors, increasing fibrinolysis and decreasing blood coagulation by reducing fibrinogen, inhibiting platelet aggregation and increasing prothrombin time.[46] In the present study consumption of A. graveolens powder, similar to lovastatin, significantly decreased fibrinogen compared with hypercholesterolemic diet group.

The results of this study indicate that consumption of A. graveolens can decreases the negative effects of postprandial cholesterol rich diet. Considering the price and the availability of A. graveolens, this plant seems to be an effective choice for preventing some of the risk factors of atherosclerosis.

CONCLUSIONS

A. graveolens decreases the postprandial atherosclerosis risk factors and might be beneficial in hyperchlesterolemic patients. Considering the price and availability of A. graveolens, this plant seems to be an effective choice for hyperlipidemia, especially for reducing postprandial risk factors of atherosclerosis. However, the mechanism of action and the structures of the active ingredients should be established. These are our focus for future studies. Further studies are also need to be performed to determine similar effects of A. graveolens in human.