**7. Optimization of Dietary Therapy**

Cellular model can be used not only to test drugs but foodstuffs as well. We have investigat‐ ed an anti-atherosclerotic, i.e. therapeutic, causing regression of atherosclerosis, and antiatherogenic, i.e. preventive activity of certain mushroom species and sea products.

Previously we have shown that alcohol and water extracts from 20 Korean mushroom spe‐ cies cause anti-atherosclerotic and anti-atherogenic activity in cell culture [59]. Thirteen of the 20 extracts tested were anti-atherosclerotic in culture, i.e. they caused a decrease in the cellular cholesterol and/or inhibited proliferation of atherosclerotic cells. Ten of 20 tested ex‐ tracts displayed anti-atherogenic activity in addition to anti-atherosclerotic effects. Four mushroom species were chosen for the study of anti-atherosclerotic effects ex vivo. Cultiva‐ tion of atherosclerotic cells during 24 hrs in the presence of serum from healthy subjects who had had mushroom meals resulted in a 21-30% decrease in the cellular cholesterol level, i.e. caused anti-atherosclerotic effect [59]. The atherogenic serum obtained from atherosclerotic patients after dietary mushroom consumption partly (30-41%) lost its ability to increase the cellular cholesterol content [59]. Thus, tested mushrooms exhibited anti-atherosclerotic and anti-atherogenic effects on ex vivo model.

Among sea products mollusk and krill meat were tested [36]. Specifically, the patients were given canned meat of a mollusk belonging to genus *Buccinum*. Two hours after a single diet‐ ary load the patient's blood serum acquired marked anti-atherosclerotic properties. The ad‐ dition of this serum to cultured atherosclerotic cells led to a fall in intracellular cholesterol level [36]. Four hours later the anti-atherosclerotic properties of the serum became even more prominent.

Patients of another group had an initially atherogenic serum causing more then 2-fold in‐ crease in cholesterol content of cells derived from normal intima. These patients received a single dietary load of Antarctic krill meat. Two hours later the atherogenicity of their blood serum decreased and four hours later it was practically absent [36]. Thus, krill meat exhibits a preventive anti-atherogenic action on arterial cells.

The results obtained suggest that the krill meat can be employed in diets aimed at the pre‐ vention of atherosclerosis. To develop a dietary therapy based on the krill meat, the effective dose and proper regimen should be established. As the first step to develop a dietary thera‐ py, the below outlined study was undertaken to determine the effective dose.

The patients' blood sera were analyzed for atherogenicity. Patients whose blood serum had an atherogenic potential were included in the study. The blood was collected from each pa‐ tient before, 2 and 4 hrs after a dose of krill meat. This protocol was repeated on the next day with another dose of krill meat. Blood serum samples were added to a culture of subendo‐ thelial cells isolated from uninvolved human aortic intima, and intracellular cholesterol ac‐ cumulation was assessed in each case. Anti-atherogenic activity of krill meat was evaluated by the ability to reduce serum atherogenicity which manifested in cholesterol accumulation in cultured cells (Figure 2). The dose-effect dependence was revealed by comparing the effi‐ cacy of two doses. The efficacy of each dose was evaluated by the analysis of at least 6 sera obtained from different patients. In can be seen from that the krill meat elicits an anti-athe‐ rogenic effect at a dose of 10-20 g, half maximum effect was reached at a dose of 30 g and the maximum effect - at a dose of 50 g.

therogenic, however, 28 days of regular propranolol therapy led to the emergence of athero‐ genicity revealed even before the drug administration [65]. Thus, a single dose of betablockers brings about temporary atherogenicity of the serum. Prolonged therapy with betablockers leads to the emergence of stable atherogenic properties of patients' blood serum.

Cellular model can be used not only to test drugs but foodstuffs as well. We have investigat‐ ed an anti-atherosclerotic, i.e. therapeutic, causing regression of atherosclerosis, and anti-

Previously we have shown that alcohol and water extracts from 20 Korean mushroom spe‐ cies cause anti-atherosclerotic and anti-atherogenic activity in cell culture [59]. Thirteen of the 20 extracts tested were anti-atherosclerotic in culture, i.e. they caused a decrease in the cellular cholesterol and/or inhibited proliferation of atherosclerotic cells. Ten of 20 tested ex‐ tracts displayed anti-atherogenic activity in addition to anti-atherosclerotic effects. Four mushroom species were chosen for the study of anti-atherosclerotic effects ex vivo. Cultiva‐ tion of atherosclerotic cells during 24 hrs in the presence of serum from healthy subjects who had had mushroom meals resulted in a 21-30% decrease in the cellular cholesterol level, i.e. caused anti-atherosclerotic effect [59]. The atherogenic serum obtained from atherosclerotic patients after dietary mushroom consumption partly (30-41%) lost its ability to increase the cellular cholesterol content [59]. Thus, tested mushrooms exhibited anti-atherosclerotic and

Among sea products mollusk and krill meat were tested [36]. Specifically, the patients were given canned meat of a mollusk belonging to genus *Buccinum*. Two hours after a single diet‐ ary load the patient's blood serum acquired marked anti-atherosclerotic properties. The ad‐ dition of this serum to cultured atherosclerotic cells led to a fall in intracellular cholesterol level [36]. Four hours later the anti-atherosclerotic properties of the serum became even

Patients of another group had an initially atherogenic serum causing more then 2-fold in‐ crease in cholesterol content of cells derived from normal intima. These patients received a single dietary load of Antarctic krill meat. Two hours later the atherogenicity of their blood serum decreased and four hours later it was practically absent [36]. Thus, krill meat exhibits

The results obtained suggest that the krill meat can be employed in diets aimed at the pre‐ vention of atherosclerosis. To develop a dietary therapy based on the krill meat, the effective dose and proper regimen should be established. As the first step to develop a dietary thera‐

The patients' blood sera were analyzed for atherogenicity. Patients whose blood serum had an atherogenic potential were included in the study. The blood was collected from each pa‐ tient before, 2 and 4 hrs after a dose of krill meat. This protocol was repeated on the next day

py, the below outlined study was undertaken to determine the effective dose.

atherogenic, i.e. preventive activity of certain mushroom species and sea products.

**7. Optimization of Dietary Therapy**

196 Current Trends in Atherogenesis

anti-atherogenic effects on ex vivo model.

a preventive anti-atherogenic action on arterial cells.

more prominent.

We believe that this approach will be useful in the development and optimization of antiatherosclerotic and anti-atherogenic diet therapies.

**Figure 2.** Antiatherogenic effect of krill meat on blood serum of atherosclerotic patients

Blood serum atherogenicity was determined using primary culture of cells derived from un‐ involved intima of the human aorta.

#### **8. Natural Products for Anti-Atherosclerotic Therapy**

Anti-atherogenic effects of dietary products promote the development of anti-atherosclerotic therapy on the basis of natural products. Atherosclerosis develops over many years, so the anti-atherosclerotic therapy should be long-term or even lifelong therapy. For such a longterm therapy conventional medicine might not work. Drugs based on natural products can be a good alternative.

We have tested numerous natural products' extracts to reveal their effects on blood athero‐ genicity, i.e., a capacity to prevent intracellular cholesterol accumulation caused by athero‐ genic blood sera from patients. Table 3 presents effective natural products only. As a fact, among tested agents were revealed not only anti-atherogenic but also pro-atherogenic and neutral. Among antiatherogenic natural products the most effective was garlic.

anti-atherogenic-related (preventive) effects. Garlic inhibits ACAT and stimulates CEH, thus displaying a direct influence on synthesis and degradation of cholesteryl esters in the cell.

Use of Natural Products for Direct Anti-Atherosclerotic Therapy

http://dx.doi.org/10.5772/52967

199

Further investigations of garlic anti-atherosclerotic effects included ex vivo study and ani‐ mal model study. Both types of studies confirmed the in vitro effects of garlic. Finally, we have developed a drug on the basis of garlic powder and carried out atherosclerosis regres‐

The AMAR study (Atherosclerosis Monitoring and Atherogenicity Reduction) was designed to estimate the effect of two-year treatment with time-released garlic-based drug Allicor on the progression of carotid atherosclerosis in asymptomatic men in double-blinded placebocontrolled randomized clinical trial. The primary outcome was the rate of atherosclerosis progression, measured by high-resolution B-mode ultrasonography as the increase in caro‐

Atherosclerosis affects most vascular beads, and noninvasive imaging of superficial arteries by ultrasound has been recognized as a surrogate measure of overall atherosclerotic burden in numerous studies. Extracoronary atherosclerotic lesions can be quickly and safely evalu‐ ated in the carotids, femoral arteries, and the abdominal aorta. The grade of atherosclerosis in extracoronary sites correlates with a greater number of standard risk factors and, more importantly, with greater cardiac risk [77]. Of the peripheral arterial surrogates, carotid atherosclerosis has been most closely correlated with coronary artery disease [78-82]. Pe‐ ripheral arterial ultrasonography is regarded to be a sensitive tool for the detection of early atherosclerosis and may be useful in assessing response to therapy. Thickening of the inti‐ ma-media of the arterial wall is the earliest detectable anatomic change in the development and progression of atherosclerosis. High-resolution B-mode ultrasonography is widely used for noninvasive quantification of carotid IMT as a measure of subclinical atherosclerosis [83]. Carotid IMT is believed to be a marker of generalized atherosclerosis and is predictive of clinical cardiovascular events [79,81,84-88]. Thus, ultrasound imaging of intima-media thickening in carotid arteries served as a means of monitoring atherosclerosis during Allicor long-term treatment. Indeed, Allicor significantly reduced carotid arterial intima-media thickness compared to baseline and the placebo group. In Allicor recipients, a significant in‐ crease in the IMT in one or both carotid arteries was observed in 30 (32.2%) patients, and was significantly reduced in 44 (47.3%) patients. In 8 patients (8.6%) there were no signifi‐ cant IMT changes in either the carotid artery, and in the remaining 11 patients (11.8%) diver‐ gent changes were observed, i.e. IMT increased in one carotid artery and decreased in the other. IMT lesion progression was observed in 50 (48.5%) placebo cases, and decreased sig‐ nificantly in one or both arteries in 31 (30.1%) patients. Stable situation was observed in 11 (10.7%), and divergent changes occurred in the remaining 7 (6.8%) patients. The difference in the IMT changes between Allicor and placebo recipients was statistically significant (Pear‐

**9. Atherosclerosis Regression Clinical Studies of Natural Products**

tid intima-media thickness (IMT) of the far wall of common carotid arteries.

This finding may explain direct anti-atherosclerotic effects of garlic.

sion clinical study of this drug.


**Table 3.** Anti-atherogenic effects of natural products

We extended investigation of the in vitro effect of garlic extract on lipids of cultured hu‐ man aortic cells. We have earlier shown that lipid accumulation in human aortic cells is accompanied by stimulation of other cellular manifestation of atherosclerosis, namely: prolif‐ eration and extracellular matrix synthesis [41,63]. Thus, investigation of garlic action on cellular lipid parameters is closely related to the study of the mechanism of garlic anti-atherosclero‐ sis effect.

A direct influence of garlic on atherosclerosis is discussed [70-73]. The anti-atherosclerotic effect of garlic has been attributed to its hypolipidemic activity. Experimental and clinical data have clearly demonstrated that garlic reduces blood cholesterol and LDL levels [74,75]. The cholesterol lowering effect of garlic results from inhibition of hepatic hydroxymethyl‐ glutaryl coenzyme A (HMG-CoA) [76]. In contrast to these studies, we examined not the hy‐ perlipidemic but the direct anti-atherosclerotic-related and anti-atherogenic-related effects of garlic, i.e., the ability of garlic to act directly on atherosclerotic process in the vessel wall. To investigate anti-atherosclerotic-related (therapeutic) effect we used smooth muscle cells cultured from atherosclerotic plaques of human aorta. To study anti-atherogenic-related (preventive) effect we imitated atherogenesis in primary cultures of smooth muscle cells de‐ rived from grossly uninvolved human aortic intima by adding atherogenic blood serum of patients with angiographically assessed coronary atherosclerosis. Garlic decreased triglycer‐ ide, cholesteryl ester and free cholesterol contents of cells cultured from atherosclerotic pla‐ que and prevented atherogenic serum-induced accumulation of these lipids in cells cultured from grossly normal aorta, i.e., elicited direct anti-atherosclerotic-related (therapeutic) and anti-atherogenic-related (preventive) effects. Garlic inhibits ACAT and stimulates CEH, thus displaying a direct influence on synthesis and degradation of cholesteryl esters in the cell. This finding may explain direct anti-atherosclerotic effects of garlic.

We have tested numerous natural products' extracts to reveal their effects on blood athero‐ genicity, i.e., a capacity to prevent intracellular cholesterol accumulation caused by athero‐ genic blood sera from patients. Table 3 presents effective natural products only. As a fact, among tested agents were revealed not only anti-atherogenic but also pro-atherogenic and

> Atherogenicity decrease, %

neutral. Among antiatherogenic natural products the most effective was garlic.

*Spirulina platensis* 51 *Allium cepa* 21 *Beta vulgaris* 31 *Triticum vulgaris* 70 *Glycyrrhiza glabra* 55 *Salsola collina* 11 *Allium sativum* **77** *Pinus sylvestris* 52

We extended investigation of the in vitro effect of garlic extract on lipids of cultured hu‐ man aortic cells. We have earlier shown that lipid accumulation in human aortic cells is accompanied by stimulation of other cellular manifestation of atherosclerosis, namely: prolif‐ eration and extracellular matrix synthesis [41,63]. Thus, investigation of garlic action on cellular lipid parameters is closely related to the study of the mechanism of garlic anti-atherosclero‐

A direct influence of garlic on atherosclerosis is discussed [70-73]. The anti-atherosclerotic effect of garlic has been attributed to its hypolipidemic activity. Experimental and clinical data have clearly demonstrated that garlic reduces blood cholesterol and LDL levels [74,75]. The cholesterol lowering effect of garlic results from inhibition of hepatic hydroxymethyl‐ glutaryl coenzyme A (HMG-CoA) [76]. In contrast to these studies, we examined not the hy‐ perlipidemic but the direct anti-atherosclerotic-related and anti-atherogenic-related effects of garlic, i.e., the ability of garlic to act directly on atherosclerotic process in the vessel wall. To investigate anti-atherosclerotic-related (therapeutic) effect we used smooth muscle cells cultured from atherosclerotic plaques of human aorta. To study anti-atherogenic-related (preventive) effect we imitated atherogenesis in primary cultures of smooth muscle cells de‐ rived from grossly uninvolved human aortic intima by adding atherogenic blood serum of patients with angiographically assessed coronary atherosclerosis. Garlic decreased triglycer‐ ide, cholesteryl ester and free cholesterol contents of cells cultured from atherosclerotic pla‐ que and prevented atherogenic serum-induced accumulation of these lipids in cells cultured from grossly normal aorta, i.e., elicited direct anti-atherosclerotic-related (therapeutic) and

**Table 3.** Anti-atherogenic effects of natural products

sis effect.

198 Current Trends in Atherogenesis

Further investigations of garlic anti-atherosclerotic effects included ex vivo study and ani‐ mal model study. Both types of studies confirmed the in vitro effects of garlic. Finally, we have developed a drug on the basis of garlic powder and carried out atherosclerosis regres‐ sion clinical study of this drug.
