Keywords

1 Introduction

Cardiovascular disease (CVD) remains the principal cause of death in both developed and developing countries accounting for roughly 25% of all deaths worldwide per year. High-fat diet, abnormalities in lipoproteins, diabetes, overweight, sedentary lifestyle, smoking, and genetic factors contribute to the risk of CVD including atherosclerosis and stroke [1]. Lifestyle changes including diet have been found to reduce the risk for premature CHD by 82% [2], whereas practices related to nutrition alone may reduce the risk by 60% [3]. Recently the American Heart Association Nutrition Committee [4] outlined diet and lifestyle goals for cardiovascular disease risk reduction. Interventions with nutrients include nutritious foods and beverages, functional foods, and dietary supplements. Dietary supplements deliver a concentrated form of a presumed bioactive ingredient (nutraceutical) from a food, in a nonfood matrix (usually in a tablet or capsule form), to enhance health in dosages that exceed those that can be obtained from normal food. The nutraceutical ingredients cover a wide range of chemical entities which include the polyphenols, phytoestrogens, organosulfur compounds, peptides, and vitamins. The bioactive compounds like quercetin, catechin, resveratrol, diosgenin, sulforaphane, lycopene, S-allylcysteine, hydroxytyrosol, and tocotrienol are generally of plant origin and are of interest to combat dyslipidemia and in turn reduce the risk of cardiovascular diseases. This review summarizes the findings of recent studies on the efficacy and mechanism of popular cholesterol-lowering dietary supplements and nutraceuticals.

2 Cholesterol, Cardiovascular Disease, and Its Management

Cholesterol has acquired an unsavory reputation for many years due to the strong correlation between the level of blood total cholesterol (TC) and the incidence of coronary heart disease (CHD). Mammals, including humans, require cholesterol for normal metabolism. However, cholesterol is not essentially required in the diet because humans are capable of synthesizing it. CHD induced by atherosclerosis is the main cause of mortality in humans. Elevated levels of plasma TC and LDL-C are the major risk factors for atherosclerosis, whereas a high concentration of plasma HDL-C and a low ratio of TC to HDL-C are protective against CHD [5]. In the United States, 30% of the adult population have levels of blood TC higher than 240 mg/dL, whereas in China 19% of the total population have abnormal blood lipids [6, 7]. Cholesterol-lowering agents can be classified into six major types: HMG-CoA reductase inhibitors, LDL receptor activators, acyl CoA:cholesterol acyltransferase (ACAT) inhibitors, cholesterol–bile acid absorption inhibitors, CETP inhibitors, and PPAR agonists.

HMG-CoA reductase inhibitors. Inhibition of cholesterol synthesis is the most efficient way to reduce serum cholesterol level. Cholesterol synthesis is a multienzyme pathway in which HMG-CoA reductase mediates the rate-limiting step. The discovery of the statin class of drugs (simvastatin and pravastatin) was a significant advance in the treatment of severe hypercholesterolemia. These drugs inhibit HMG-CoA reductase in the liver. However, side effects are associated with the use of these inhibitors, including rashes and gastrointestinal symptoms [8].

LDL receptor activators. Efficient removal of plasma LDL-C is essential for maintaining plasma cholesterol level in a healthy range. Removal of LDL-C from the blood is mediated by receptor-dependent and receptor-independent mechanisms. The former accounts for up to 60–80% of LDL clearance while the latter is responsible for 20–40% cholesterol clearance from the blood. Expression of LDL receptor is a function of cellular free cholesterol. When the cellular cholesterol decreases, the LDL receptor gene is transactivated. In contrast, sufficient cellular free cholesterol downregulates the LDL receptor gene. Theoretically, upregulation of LDL receptor will lead to a lower level of blood cholesterol [9].

ACAT inhibitors. Two major forms of ACAT, namely ACAT1 and ACAT2, have been identified in mammals. In humans, ACAT2 is important in cholesterol absorption. Reduced absorption of dietary cholesterol can lead to a lower level of blood cholesterol. Intestinal ACAT2 is the primary enzyme responsible for the intracellular esterification of cholesterol. ACAT2 plays an important role in the absorption of cholesterol in the small intestine, before cholesterol is incorporated into CM [10]. In the liver, this enzyme is partially responsible for the assembly of very low-density lipoproteins (VLDL) prior to secretion into the blood [11]. TG-rich VLDL particles derived from the liver are transformed into cholesterol-rich LDL after the removal of their TG by peripheral tissues. Inhibition of ACAT activity, therefore, lowers the plasma cholesterol level by decreasing cholesterol absorption in the intestine and VLDL production in the liver.

Bile acid absorption inhibitors. Bile acids are the major metabolites of cholesterol. Bile acid absorption inhibitors are known as bile acid sequestrants. They bind bile acids in the intestine, prevent their reabsorption, and generate an insoluble complex with bile acids that are excreted in the feces. The increased excretion of bile acids leads to an increase in the synthesis of bile acids from cholesterol in the liver. The lowered level of hepatic cholesterol increases the expression of LDL receptors, which remove the cholesterol from the circulation and decrease the LDL level in the blood. Ingestion of bile acid inhibitors is usually associated with upregulation of CYP7A1 encoding cholesterol 7R-hydroxylase in bile acid synthesis.

CETP inhibitors. A decreased level of plasma HDL-C and an increased level of plasma LDL-C (e.g., the ratio of LDL-C/HDL-C) have been associated with an increased incidence of heart disease. CETP inhibitors prevent the transfer of cholesteryl ester from HDL to TG-rich lipoproteins in exchange for TG, which has the ability to increase the HDL-C level [12].

PPAR agonists. Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that control the expression of genes involved in carbohydrate and lipid metabolism. Activation of PPARα (e.g., by fibrates) stimulates the uptake and catabolism of fatty acids, promotes lipoprotein lipase-mediated lipolysis, and enhances HDL synthesis, resulting in reductions in plasma TGs and increases in plasma HDL-C [13]. Modulation of PPARγ (e.g., by thiazolidinediones) reduces insulin resistance and enhances peripheral glucose utilization [14]. Hence, current agents that affect nuclear receptors include PPARα and -γ agonists, while in development are newer PPARα, -γ, and -δ agonists, as well as dual PPARα/γ and “pan” PPARα/γ/δ agonists to manage dyslipidemia and associated metabolic events and combat the risk of CHD.

3 Atherosclerosis

Atherosclerosis remains the major cause of age-related disease and death in the world. Many epidemiological clinical genetic and animal studies have indicated that it results from interaction between multiple genetic and environmental factorial process in which both elevated plasma cholesterol levels and proliferation of smooth muscle cells play a central role. Monocytes, macrophages, and T cells attracted to the site of injury produce inflammatory cytokines, chemokines, and reactive oxygen species [15–19].

Reactive oxygen species (ROS) are implicated in the pathogenesis of a wide variety of human diseases and atherosclerosis. Oxygen-free radicals and their byproducts that are capable of causing oxidative damage, collectively referred to as active or reactive oxygen species, may be cytotoxic when produced in excess. NADPH oxidase is a key enzyme in the generation of ROS that is expressed by many cell types found both in vessel wall and in blood, has been implicated in the pathogenesis of hypercholesterolemia [20–22]. Studies suggest that at moderately high concentrations, certain forms of ROS such as H2O2 may act as signal transduction messengers [23]. Especially important, although not so extensively studied is the role played by vascular smooth muscle cells (VSMC) in the process of formation of the atherosclerotic plaque. A family of genes, the scavenger receptors (CD 36 is the most important one), recognizes and internalizes modified lipoproteins, making them susceptible to degradation [24]. These cells can accumulate oxidized LDL through their scavenger receptors in an uncontrolled manner leading to formation of the so-called foam cells. There is also an increasing body of evidence showing that VSMC apoptosis is involved in the pathogenesis of atherosclerosis [25]. For example, apoptotic VSMCs are present in human atherosclerotic lesions. Recent investigations have demonstrated that simultaneous treatments with IFN-γ and TNF-α and/or IL-β can trigger apoptosis in cultured human and rat VSMC. Oxidatively modified LDL can induce apoptosis in VSMC. Nevertheless, the mechanisms whereby apoptosis of VSMC is triggered still remain largely unknown. Previous studies have shown that H2O2 is effective in stimulating the in vitro growth of several cell types. Also human and rat VSMC have been reported to undergo DNA synthesis in response to H2O2 stimulation. Convicting evidence has been presented to show that intracellular H2O2 can act as a signaling molecule or as a second messenger involved in many cellular functions such as oxidant-induced stress apoptosis and proliferation [26]. H2O2 is generated when cells are stimulated with cytokines and growth factors. Intracellularly generated H2O2 produces its effects through the activation of tyrosine kinase or MAP kinase since catalase or N-acetylcysteine blocks PDGF-induced tyrosine phosphorylation and MAPK activation. It has been also reported that H2O2 increases CD36 expression [24, 25].

4 Cholesterol-Lowering Nutraceuticals

Although several factors play an important role in the metabolism of cholesterol, there is no doubt that plasma TC, LDL-C, and HDL-C levels are influenced profoundly by diets. In general, plasma TC is raised by dietary cholesterol and saturated and trans fatty acids and lowered by monounsaturated and polyunsaturated fatty acids. In recent years, there has been considerable interest in the potential for using natural food components as functional foods to treat hypercholesterolemia, especially for patients whose cholesterol level is marginally high (200–240 mg/dL) and does not warrant the prescription of cholesterol-lowering drugs. Clinical trials of dietary approaches to lowering LDL-cholesterol levels have been reported to be as effective as statin medication. A combination of foods like soy, plant sterols, almonds, and viscous fibers could reduce LDL-cholesterol levels by 20%, and work better together than independently [27]. Enriched margarines were used as the source of the plant sterols; the fiber came from oats, barley, okra, and eggplant; and the soy proteins came from soy milk and tofu. There is now a consensus about recommending the Mediterranean diet pattern for the prevention of coronary heart disease (CHD) since it has a striking effect on survival. Furthermore, the Mediterranean diet appears to be effective at reducing atherosclerosis and the risk of fatal complications (i.e., sudden cardiac death and heart failure) of atherosclerosis. Finally, unlike drug therapies, no harmful side effect has been reported following the adoption of this dietary pattern. Many micro- and macronutrients characteristic of the Mediterranean diet interact in a synergistic way to induce states of resistance to chronic diseases [28]. The extra-nutritional constituents known as “bioactive compounds” are naturally occurring in small quantities in plant products and lipid-rich foods [29]. They exhibit a significant role in reducing the risk of CHD by decreasing the total cholesterol, LDL-C, TG, LDL oxidation, cholesterol absorption or by increasing the HDL-C and antioxidant status [30]. The bioactive compounds like quercetin, catechin, resveratrol, diosgenin, sulforaphane, lycopene, S-allylcysteine, hydroxytyrosol, and tocotrienol are of interest to combat dyslipidemia and in turn reduce the risk of cardiovascular diseases, which are discussed in the following sections.

4.1 Polyphenols

Phenolic compounds, commonly referred to as polyphenols, are present in all plants and, thus, are in the diet. There are more than 8,000 phenolic structures that have been identified that vary structurally. More than 10 classes of polyphenols have been defined on the basis of chemical structure [31]. The flavonoids are the most common polyphenolic compounds present in plant food. The vast majority of plant phenolics are simple phenols and flavonoids. Although polyphenols are present in virtually all plant foods, their levels vary enormously among diets depending on the type and quantity of plant foods in the diet. For example, some plant foods and beverages that are particularly rich in polyphenols are red wine, apple and orange juices, and legumes. The primary phenols in cereals and legumes are flavonoids, phenolic acids, and tannins. The major polyphenols in wine include phenolic acids, anthocyanins, tannins, and other flavonoids. The most abundant phenolic compound in fruits is flavonol. Nuts are rich in tannins [32]. Olive oil contains both phenolic acids and hydrolyzable tannins. The predominant flavonoid in onions is quercetin glycoside, whereas in tea and apples it is quercetin-3-rutinoside. Several population studies have reported an inverse association between flavonoid intake and risk of coronary disease [32–35]. Much of the epidemiologic evidence suggests that flavonoids have a protective effect against coronary mortality. For those studies that have reported an association, putative mechanisms of action include inhibition of LDL oxidation [36] and inhibition of platelet aggregation and adhesion [37].

The cholesterol-lowering activity of tea catechins has been extensively investigated. Tea, derived from the leaves of Camellia sinensis, is the world’s most popular and widely consumed beverage. Four green tea catechin (GTC) derivatives, namely (–)-epicatechin (EC), (–)-epicatechin gallate (ECG), (–)-epigallocatechin (EGC), and (–)-epigallocatechin gallate (EGCG), have been extensively studied for their wide range of biological and pharmacological properties. GTCs have been shown to lower plasma cholesterol in several animal models and to alter cholesterol metabolism favorably in cell cultures. Although the mechanisms responsible for the cholesterol-lowering activity of GTCs are not yet fully understood, some evidence suggests that they reduce the level probably by the following mechanisms. First, they upregulate the LDL receptor mediated by activation of SREBP-2 [38, 39]. It has been claimed that EGCG was the active ingredient, which was able to increase LDL receptor activity by 3-fold and protein by 2.5-fold when it was incubated with HepG2 liver cells [40]. In rats fed a diet containing 2% GTCs, LDL receptor-binding activity and protein were increased by 2.7- and 3.4-fold, respectively [41]. Similarly, LDL receptor activity and protein could be upregulated by 80 and 70%, respectively, in rabbits fed a hypercholesterolemic-GTC diet [42]. Second, GTCs reduce the plasma cholesterol level by increasing fecal bile acid and cholesterol excretion. In hamsters fed a 0.1% cholesterol diet, GTCs not only decreased both plasma TC and TG but also increased excretion of both neutral and acidic sterols [43]. A similar effect was observed in rats fed tea extracts [44]. Third, GTCs have been shown to inhibit cholesterol synthesis in rabbits [42] but not in rats [44]. Data from studies of the cholesterol-lowering effects of GTCs in humans are not consistent. Epidemiological observations indicate that tea consumption is associated with reduced levels of plasma TC and LDL-C in Japanese [45] and Norwegian subjects [46]. One study demonstrated that theaflavin-rich tea extract at a dose of 375 mg a day effectively reduced TC and LDL-C in mild-to-moderate hypercholesterolemia subjects [47]. Another study found that GTCs were able to attenuate the postprandial increase in plasma TG following a fat load [48]. However, some studies did not observe a favorable effect. The results from a cross-sectional study did not demonstrate that drinking green tea was associated with changes in any of the lipid levels [49]. One study found that consumption of 900 mL of green or black tea/day by 45 volunteers for 4 weeks did not affect serum lipid concentration [50]. As the evidence for the cholesterol-lowering activity of tea in humans is mixed, further additional clinical randomized, double-blind crossover studies are needed to clarify the issue.

The beneficial effects of polyphenols in red wine are relatively well established. In many countries, a high intake of saturated fats strongly correlates with a high risk of coronary heart disease, but not in France and some regions where wine consumption is high. This paradox has been attributed to the anti-LDL oxidation activity and anti-atherogenic effect of wine polyphenols [51]. The polyphenols present in grape and its seed are mainly hydroxycinnamic acid, resveratrol, flavonols, anthocyanins, catechins, and proanthocyanidins [52]. In addition, favorable modification of lipoproteins by decreasing the LDL-C/HDL-C ratio and LDL-C oxidation has also been claimed to be responsible for the reduced risk of coronary heart disease associated with moderate consumption of red wine [53]. The hypocholesterolemic activity of grape polyphenols has been demonstrated in rats [54] and hamsters [55]. Although they could not lower plasma lipids, grape polyphenols were capable of attenuating atherosclerosis in rabbits [56] and ovariectomized guinea pigs [57]. In postmenopausal women, lyophilized grape powder decreased plasma LDL cholesterol and apolipoproteins B and E [58]. When the diets of both healthy subjects and hemodialysis patients were supplemented with red grape juice, there was a significant decrease in LDL-C and apolipoprotein B-100 concentration and an increase in the concentration of HDL-C and apolipoprotein A-I [59]. In other studies on human subjects, purple grape juice or grape polyphenols did not affect the plasma cholesterol level but were able to reduce the susceptibility of LDL to oxidation and improve the endothelial function [60, 61]. Several mechanisms have been proposed to explain the cholesterol-lowering activity of grape polyphenols. One possibility is that grape polyphenols increase fecal bile acids and reduce cholesterol absorption, as rats given diets containing 2% grape monomer and polymer of anthocyanins had a greater output of fecal acidic and neutral sterols [54]. Indeed, expression of the key enzyme controlling bile acid synthesis, CYP7A1, was upregulated in rats given grape seed polyphenol extract [62]. A second possibility is that the cholesterol-lowering activity of grape polyphenols is mediated by regulating expression of LDL receptor. When HepG2 cells were incubated with dealcoholized wine extract, the mRNA of LDL receptor gene was significantly increased [63]. In HepG2 and HL-60 cells, red grape juice increased the level of the active form of SREBP, and this was accompanied by greater mRNA expression of LDL receptor [64].

4.2 Phytoestrogens

Phytoestrogens are compounds found in plants and have weak estrogenic activity by binding to estrogen receptor and initiating some estrogen-dependent transcription. Phytoestrogens have been claimed to have benefits for heart, bone, breast, and general menopausal health [65]. There is some evidence to suggest that phytoestrogens can reduce blood cholesterol level by inhibiting cholesterol synthesis and increased expression of the LDL receptors [66]. Major classes of phytoestrogens include isoflavones, flavones, flavanones, comestans, lignans, and stilbenes [67]. Resveratrol has been recognized as a phytoestrogen since it possesses structural similarities with estrogenic compounds and may exert some biological activities through estrogen receptors. In some studies, resveratrol has been shown to bind to estrogen receptors as an agonist [68]. Isoflavones have been extensively studied and are mainly found in soybean (genistein, daidzein, glycitein, and their glycosides). Soy isoflavones are the most consumed phytoestrogens in humans. Extensive research has focused on the role of soy phytoestrogens on the plasma cholesterol level in animals [69, 70]. Soy phytoestrogens are hypocholesterolemic in most animal studies. The hypocholesterolemic activity of soy phytoestrogens is mediated by their stimulating effect on LDL receptor like natural estrogen. Dietary isoflavones have been shown to reduce plasma cholesterol and atherosclerosis in C57BL/6 mice but not in LDL receptor-deficient mice [71]. In HepG2 cells, incubation with isoflavonoids, formononetin, biochanin A, and daidzein caused significant elevations in LDL receptor activity [72]. This stimulating effect on LDL receptor is probably mediated by its effect on SREBP-2, which regulates expression of both LDL receptor and HMG-CoA reductase. In phytoestrogen-treated HepG2 cells, a mature form of SREBP-2 was increased, as were LDL receptor and HMG-CoA reductase [73]. The results of randomized clinical trials in humans, however, have been inconsistent. Some clinical trials have shown that soy phytoestrogens reduce plasma TC and LDL-C in hypercholesterolemic subjects [74, 75]. However, it has been demonstrated that soy isoflavones had no dose-dependent effect [76] or had no significant effect on blood cholesterol [77]. In normocholesterolemic subjects, some clinical trials demonstrated that consumption of soy protein with a high isoflavone content significantly decreased plasma LDL-C compared with the same soy intake with a low isoflavone intake [78, 79]. In contrast, other trials found no difference in blood cholesterol between a high-isoflavone and a low-isoflavone diet [80, 81]. To evaluate more precisely the effect of soy isoflavones on plasma TC and LDL-C concentrations, Zhuo et al. [82] performed a meta-analysis of eight clinical trials, concluding that with the same soy protein intake, a high-isoflavone diet had greater hypocholesterolemic activity than a low-isoflavone diet and that soy isoflavones had an LDL-C lowering effect independent of soy protein. However, Zhan and Ho [83] performed a meta-analysis of 23 randomized control trials and found that soy protein containing isoflavones significantly reduced plasma TC, LDL-C, and TG and increased HDL-C, whereas tablets containing extracted isoflavones had no effect on these parameters. In this regard, one study found soy protein fortified with isoflavones had greater LDL-C lowering activity than soy protein with isoflavone removed [84]. Further studies are needed to investigate the interaction of soy isoflavones with soy protein or the synergistic action of these two components in their contribution to the hypocholesterolemic activity of soy products.

4.3 Phytosterols

Phytosterols are naturally occurring plant sterols that are present in the nonsaponifiable fraction of plant oils. Structurally, plant sterols are similar to cholesterol except that there are always some substitutions on the sterol side chain at the C24 position. They are not synthesized in humans, are poorly absorbed, and are excreted faster from the liver than cholesterol, which explains their low abundance in human tissues [85]. The primary plant sterols in the diet are sitosterol, stigmasterol, and campesterol. Typical consumption of plant sterols is approximately 200–400 mg/day. The most abundant plant sterol in Western diets is β-sitosterol. Studies with sitosterol or mixtures of plant sterols (approximately 1 g/day) have shown that they reduce serum cholesterol levels in humans by approximately 10% [86]. This discovery has resulted in subsequent research to evaluate the effects of sitosterol derivatives on cholesterol absorption and serum cholesterol levels. Sitostanol (a 5-beta saturated sitosterol) was shown to be more potent in reducing cholesterol absorption and serum cholesterol levels than sitosterol [87]. These findings provided the basis for the current era of research evaluating the effects of sitostanol and sitostanol esters from different plant oil sources. Special margarines are the primary food source of plant sterols/stanols. The plant sterol mixtures are derived from different oil sources, including pine tree wood pulp (tall oil), soybean oil, rice bran oil, and shea nut oil. The stanol/stanol ester margarine studies have fed approximately 2–3 g/day of stanols either as the free or esterified form in full-fat or lower fat margarines or mayonnaise. Typically, there is an approximate 10% reduction in total cholesterol and about a 14% decrease in LDL cholesterol and no change in HDL cholesterol or triglyceride levels [88–93]. With a reduced-fat spread (40% fat) providing 1.1 or 2.2 g/day of plant sterol esters, LDL cholesterol was reduced 7.6 and 8.1% beyond that achieved with a National Cholesterol Education Program Step 1 diet in subjects with mild-to-moderate hypercholesterolemia [94]. Thus, both plant stanol and sterol esters evoke a significant serum cholesterol-lowering response beyond that attained with a cholesterol-lowering diet. The cholesterol-lowering effects of stanol are due to the reduction in total and LDL cholesterol as a result of decrease in cholesterol absorption and an alteration of enzymes involved in cholesterol metabolism and excretion [95]. There is some emerging evidence that the sterols present in the unsaponifiable fraction of rice bran oil, oryzanols (a group of ferulate esters of triterpene alcohols and phytosterols), decrease plasma cholesterol levels [96] and that tocotrienols, another group of phytosterols present in rice bran oil, may have important antioxidant properties [97]. Further work is needed to evaluate the effects of rice bran oil to establish its efficacy as a source of plant sterols that lower CVD risk.

4.4 Organosulfur Compounds

Sulfur-containing phytochemicals of two different kinds are present in all Brassica oleracea (Cruciferae) vegetables. These are glucosinolates (GLSs, previously called thioglucosides) and S-methylcysteine sulfoxide (SMCSO). The two types of organosulfur phytochemicals found in all B. oleracea vegetables, GLS and SMCSO, or, more specifically, many of their metabolites show cardioprotective [98] and chemopreventive effect [99]. A recent clinical study with 12 healthy subjects has suggested that consumption of fresh broccoli sprouts (100 g/day) for a week reduced LDL and total cholesterol and increased HDL cholesterol [100]. Another related prospective study of 34,492 postmenopausal women in Iowa showed that broccoli was strongly associated with reduced risk of coronary heart disease [101]. The other commonly consumed vegetables in daily diet include the onions and garlic which contain organosulfur compounds such as allicin and S-allylcysteine and are widely regarded as a cholesterol-lowering functional food ingredient, and results from animal trials support this notion [102]. Garlic powder has been shown to suppress serum TC and TG in rats [103], whereas aged garlic extracts decreased TC by 15% and TG by 30% in rats fed a high-cholesterol diet [104]. Interestingly, raw garlic had a pronounced effect in reducing plasma TC and TG levels, whereas boiled garlic had little effect [105]. When garlic powder was added into the diet at 1% level, it not only decreased plasma TC and LDL-C but also increased HDL-C concentration in rabbits [106], thus lowering favorably the LDL-C/HDL-C ratio. Randomized controlled trials in humans have produced conflicting results. Stevinson et al. [107] conducted a meta-analysis of 13 trials and found that garlic was able to reduce blood cholesterol compared with the placebo. Similarly, in an analysis of 10 studies, Alder et al. [108] found that 6 trials demonstrated the cholesterol-lowering activity of garlic. However, some of these randomized controlled trials had methodological shortcomings, including short duration, lack of power analysis, and lack of the control of diet as a confounding variable. The underlying mechanism by which garlic and its active ingredients lower blood cholesterol has been investigated in cell culture and in animals. Principally, garlic inhibits HMG-CoA reductase. The water-soluble organosulfur compounds S-allylcysteine, S-ethylcysteine, and S-propylcysteine have been shown to reduce cholesterol synthesis by deactivating HMG-CoA reductase via enhanced phosphorylation, but did not change the levels of mRNA or the amount of the enzyme in cultured rat hepatocytes [109]. To a lesser extent, garlic also inhibits CETP activity and modifies the LDL-C/HDL-C ratio. The effect of garlic supplementation on CETP activity, together with its anti-atherosclerotic effect, has been studied in cholesterol-fed rabbits, and it was found that CETP activity was significantly reduced in the garlic-supplemented group compared to the control group. Thus, garlic not only reduced atherosclerosis lesions but also altered the ratio of LDL-C/HDL-C [106].

4.5 Plant Proteins

A major interest for atherosclerosis prevention has been addressed to vegetable proteins, particularly soy proteins whose consumption has been shown to successfully reduce cholesterolemia in experimental animals [110, 111], as well as in humans with cholesterol elevations of genetic or non-genetic origin [112–116]. In addition, prospective observational studies, initially in vegetarians [117], then in Chinese women [118], and more recently in a large population in Japan [119], have shown a reduction of total and LDL cholesterol as well as of ischemic and cerebrovascular events with a daily soy protein intake of more than 6 g compared to less than 0.5 g/day. Beneficial effects of a soy protein-based diet were recently described also in Iranian women with the “metabolic syndrome” [120]. Besides improved lipid metabolism, these women also showed reduced inflammatory markers as well as reduced insulin resistance [121]. The cholesterol-reducing effect of soy proteins, potentially leading to a reduced cardiovascular risk, became the basis for the US Food and Drug Administration (FDA) approval of the health claim for the role of soy protein consumption in coronary disease risk reduction [122]. The numerous ensuing clinical studies were summarized in a meta-analysis [123] of 38 studies up to 1995, in both hypercholesterolemic and normolipidemic individuals. This meta-analysis confirmed that serum LDL-C concentrations are modified, dependent on baseline cholesterolemia, from a minimum of −7.7% in subjects with total cholesterol in the normal range (<200 mg/dL) up to −24% in clear-cut hypercholesterolemics. Rodent and in vitro studies have attempted to establish a link between the hypocholesterolemic effects of soy and the activation/depression of liver low-density lipoprotein receptors (LDL-R) [124, 125]. Animals on cholesterol/cholic acid dietary regimens with casein have a dramatic downregulation of liver LDL-R, and this effect is reversed in the presence of soy proteins. Two studies have addressed the potential of soy protein preparations to increase LDL-R expression in human beings. Studies on the mechanism whereby vegetable proteins may reduce cholesterolemia have clearly indicated that the intact soy protein per se is effective for cholesterol reduction, not a mixture of soy amino acids [126]. The identification of soybean components responsible for the hypocholesterolemic effect has received a significant contribution from the early clinical studies where soy protein products contained less than 0.15 mg/g isoflavones [127] versus contents of 2–3 mg/g very frequently encountered in most commercial soybean products. While initially the responsibility of these phytoestrogens in cholesterol reduction was suggested primarily on the base of studies in monkeys [128], a number of more recent reports have definitely concluded that dietary isoflavones make no contribution to the hypocholesterolemic action [129–131], including a clinical study performed on pure genistein [132]. A full understanding of the mechanism of action of soy protein has become vital for the selection of the most appropriate forms of soy for treating hypercholesterolemia. The major storage proteins of soybeans are 7S and 11S globulins: from early studies, the 7S globulin appeared to be primarily responsible for the hypocholesterolemic effects of soy protein preparations, whereas the 11S component appeared essentially inactive [133, 134]. Very recently a hypocholesterolemic protein sub-component has been pinpointed more precisely, i.e., by showing that the isolated 7S globulin subunit given to cholesterol-fed rats leads to a strong upregulation of liver LDL-R activity as well as to dramatic plasma cholesterol/TG reductions [135]. An interesting activity on hypertriglyceridemia and body weight was very recently shown by Kohno et al. [136] confirming prior data by Deibert et al. [137]. The remarkable effectiveness of four candies containing 5 g of 7S globulin, a very simple regimen, thus reinforces the recommendation to increase the intake of soy proteins for cardiovascular protection. Since proteins are hydrolyzed in the gastrointestinal tract, it is quite likely that the hypocholesterolemic soy components are peptides with less than 15 amino acids, considering their potential to be absorbed. Many animal feeding studies as well as in vitro studies have documented the effects of soy peptides on serum lipids and lipid metabolism. Two clinical trials [138, 139] have reported dramatic LDL-lowering effects.

In the last few years, other legumes have attracted the attention of research. A very special case is lupin since its seeds contain up to 35–40% protein as soybean, but are completely devoid of isoflavones. Two main species are cultivated: white lupin (Lupinus albus) and narrow-leaf lupin (Lupinus angustifolius). Whole seeds of narrow-leaf lupin showed a remarkable cholesterol-lowering effect in pigs fed a cholesterol-rich diet, compared with casein [140]. The results of this study are probably due to several bioactive components, such as protein, soluble and insoluble fibers, phytosterols, and possibly others. Other studies were instead focused only on the protein. White lupin protein, evaluated in a rat model of hypercholesterolemia [141], indicated a substantial reduction of cholesterolemia, with moderate changes of TGs and no effect of glucose. The decrease in plasma TG concentrations appears to depend on a downregulation of liver sterol regulatory element-binding protein (SREBP)-1c [142], a transcription factor that regulates the expression of lipogenic enzymes. White lupin protein was also evaluated in a rabbit model of focal soft plaque generated in common carotid arteries [143]. In this model, carotid lesions are mostly constituted by extracellular lipids and macrophages, thus reflecting the main feature of the human arterial plaques defined as unstable, frequently associated to acute ischemic events [144]. Dietary treatment with lupin protein significantly reduced atherosclerosis development versus casein. White lupin protein isolate was also shown to significantly lower blood pressure in spontaneous diabetic and hypertensive rats [145], possible consequence of their high content of arginine which may lead to an increased NO production. A preliminary clinical study [146], based on a lupin beverage (daily lupin protein intake = 36 g), confirmed the beneficial activities observed in the animal models. Lupin contains also fiber that added to the diet provides favorable changes in lipid metabolism as shown in a clinical study [147]. Other legumes investigated in the rat model of hypercholesterolemia are pea [148, 149], chickpea [150], and faba bean [151]; all induced a significant decrease of plasma LDL-C. A comparative study of diets containing four different legumes, baked beans, marrowfat peas, lentils, and butter beans [152], showed effectiveness of all, but baked beans and butter beans were the most potent. The hypocholesterolemic activity was confirmed in the pig model of hypercholesterolemia: a study compared bean, pea, lentil, and butter bean [153], while another investigated only pea [154]. Very recently Winham and Hutchins [155] reported that baked bean consumption in moderately hypercholesterolemic adults (mean LDL cholesterol 138 mg/dL) was associated with a reduction of LDL cholesterolemia (5.4%) with no changes in HDL-C levels. In addition, grain legumes are nutritionally important because they are valuable sources of alpha-linolenic acid (ALA) which aids in cardioprotection. Although the clinical data on legumes different from soybean are still very scarce, the general impression is that their consumption may have a very favorable role in the prevention of dislipidemia. Unfortunately data on the other risk factors such as diabetes and hypertension are completely lacking.

4.6 Tocopherols

Among the factors which have been found to retard the development of atherosclerosis is the intake with food of a sufficient amount of vitamin E. An inverse association between serum vitamin E and coronary heart disease mortality has been demonstrated in epidemiological studies [156–159]. Some of the effects of α-tocopherol, which is the most active form of vitamin E, can be attributed to special properties of this compound such as the inhibition of smooth muscle cells proliferation, an important event during the progression of atherosclerosis, via inhibition of protein kinase C activity [160–162].

α-Tocopherol at concentrations of 50 μM inhibits rat A7r5 smooth muscle cell proliferation, while β-tocopherol is ineffective. The oxidized product of α-tocopherol, α-tocopherylquinone, is not inhibitory indicating that the effects of α-tocopherol are not related to its antioxidant properties [163]. δ-Tocopherol, α-tocopherol, and γ-tocopherol are within experimental error equally inhibitory [164]. On the other hand, it appears that the inhibition by β-tocopherol is 10-fold less potent relative to the other compounds. Tocotrienols, although possessing a greater antioxidant activity than tocopherols, inhibit cell proliferation to the same extent [164, 165]. Janero et al. [166] have shown in a series of 6-hydroxy-chroman-2-carbonitrile tocopherol derivatives whose antioxidant properties strongly depend on the nature and length of their side chains. These compounds were tested in smooth muscle cells (A7r5) and their relative potency in inhibiting cell proliferation was established [164].

Protein kinase C has been originally suggested to be regulated, at a cellular level, by α-tocopherol [164, 167–169]. A number of reports have subsequently confirmed this finding in different cell types, including monocytes, macrophages, neutrophils, fibroblasts, and mesangial cells [170–175]. The inhibitions by α-tocopherol of protein kinase C activity and of proliferation are parallel events in vascular smooth muscle cells. Inhibition is observed to occur at concentrations of α-tocopherol close to those measured in healthy adults [164, 167–169]. While α-tocopherol inhibits protein kinase C activity, β-tocopherol is ineffective. The inhibition by α-tocopherol and the lack of inhibition by β-tocopherol of cell proliferation and protein kinase C activity show that the mechanism involved is not related to the radical scavenging properties of these two molecules, which are essentially equal [169]. Animal works have also confirmed the importance of protein kinase C inhibition by α-tocopherol in vivo showing that vitamin E protects development of atherosclerosis in cholesterol-fed rabbits by inhibiting vascular smooth muscle protein kinase C activity [176–178].

4.7 CD36 and α-Tocopherol

CD36 is a multifunctional membrane receptor and a cell adhesion molecule expressed by platelets, monocytes/macrophages and capillary endothelial cells, adipocytes, human cardiac and skeletal muscles, and at very low levels in the liver [179–182]. It has been shown that mice knockouts for the apolipoprotein E exhibited a marked decrease in atherosclerotic lesions if CD36 gene was made inactive [183]. CD36 scavenger receptor has been shown to be expressed in cultured human aortic smooth muscle cells and macrophages; treatment with α-tocopherol (at physiological concentration) downregulates CD36 expression by reducing its promoter activity [187]. A similar phenomenon has been also shown in human monocyte-derived macrophages where α-tocopherol decreases CD36 expression by inhibition of tyrosine kinase [183, 185, 186]. Also in rat liver, it has been shown that vitamin E regulates the scavenger receptor CD36, the coagulation factor IX, the hepatic gamma-glutamylcysteinyl synthetase, and the 5-alpha-steroid reductase type 1 gene at the transcriptional level [186].

Common models of atherosclerosis indicate in the formation of foam cells an early event in the onset of atherosclerosis. Thus, the inhibition of foam cell formation by vitamin E may be important in the prevention of the disease. A central role of CD36 in atherogenesis in a mouse model has been described by Febbraio et al. [188]. This group has shown that targeted disruption of the class B scavenger receptor CD36 protects against atherosclerotic lesion development in mice. In an in vivo model it has been shown that CD36 scavenger receptor is highly expressed in hypercholesterolemic rabbits and that vitamin E downregulates its expression without changing serum cholesterol level significantly. The significance of this finding rests on the fact that vitamin E appears to have a role in the prevention of atherosclerosis. This study also indicates that non-genetic manipulation of the CD36 expression product by vitamin E administration to rabbits on a high-cholesterol diet results in a diminution of CD36 expression and of foam cell formation [189].

Animal model studies cannot be transferred to human situations. However, the following considerations can also be made. As indicated by the propensity to atherosclerosis created in the mouse by the knock out of the ApoE gene (and the protection by additional CD36 knockout), also the rabbit may have a genetic background that makes it highly sensitive to the effect of vitamin E on the expression of CD36 and possibly of the SR-A, scavenger receptors [188, 190]. The notion that small intervention studies, conducted with a more homogeneous population, have shown a significant effect of vitamin E suggests that investigations on individuals having a homogeneous genetic background, especially referred to ApoE and other polymorphisms, may lead to a better understanding of the discrepancies among human studies and with the animal ones [191]. The second and more precise paradigm has been that of the identification of some antioxidants provided with different and more specific functions. One of the most important consequences of this concept is that through specific recognition interactions more precise and site-directed events could take place in a cell. This point to the uniqueness of some natural compounds, whose combination of antioxidant and non-antioxidant properties cannot be imitated or substituted for by simple synthetic antioxidants.

4.8 Other Dietary Products

Very recently also fish proteins have been considered. Animal studies with fish proteins versus casein have suggested a potential hypocholesterolemic activity [192, 193]. Similar to soy, fish proteins increased liver LDL-R and SREBP-2 mRNA concentrations and significantly reduced cholesterolemia. Different from vegetable proteins, however, an HDL-C reduction was noted, albeit with an increased mRNA expression for apo AI. At present, however, little evidence has come from studies in humans, where at best, fish intake has been linked to a hypotriglyceridemic activity [194].

5 Conclusion

Cholesterol-lowering nutraceuticals and functional foods play an important role in reducing the risk of coronary heart disease by improving the plasma lipoprotein profile. More and more attention is now being paid to combined atherogenic dyslipidemia which typically is presented in patients with type-2 diabetes and metabolic syndrome. This mixed dyslipidemia (or “lipid quartet”)—hypertriglyceridemia, low high-density lipoprotein (HDL) cholesterol levels, a preponderance of small, dense LDL particles, and an accumulation of cholesterol-rich remnant particles—emerged as the greatest “competitor” of LDL cholesterol among lipid risk factors for cardiovascular disease. Plant-derived nutraceuticals exhibit varied lipid-lowering effects due to the presence of a number of bioactive compounds. However, the action mechanisms for favorable modification of plasma lipids vary with individual nutraceuticals and functional foods. In addition to the nutraceuticals and foods discussed above, almond, fish oil, flaxseed, black rice, licorice, lycopenes, olive, and ginseng oil have also been claimed to possess cholesterol-lowering activity. Future studies could profitably focus on the interaction of the active ingredients with the expression of the genes involved in cholesterol metabolism. The synergistic effects of nutraceuticals on the regulation of blood cholesterol at more than one metabolic site should be tested to develop effective cholesterol-lowering functional foods and translated to the human needs.