**1.10.1 Regulation of cholesterol by natural products**

In this section of book the regulation of cholesterol by natural products, tea, policosanol and garlic is discussed.

### **1.10.1.1 Tea and tea compounds**

Epidemiological studies have indicated that tea consumption is associated with a lower risk of cardiovascular disease. This decreased risk is attributed to the ability of tea to lower serum cholesterol levels, and several clinical studies have demonstrated that black tea can lower serum total- and LDL-cholesterol (Davies et al., 2003; Maron et al., 2003). Green tea has been shown to be hypocholesterolemic in animal studies, with the bulk of evidence indicating that tea polyphenols reduce the absorption of dietary and biliary cholesterol and promote its fecal excretion (Koo and Noh, 2007)..

Feeding studies have been equivocal on the ability of green tea extract to inhibit cholesterol synthesis. Although a recent study by Bursill and colleagues (Bursill et al., 2007) showed a decrease in serum lathosterol (an indicator of whole body cholesterol synthesis) in rabbits fed a green tea extract, a similar study with rats by these investigators (Bursill and Roach, 2007) was unable to demonstrate a decrease in this serum sterol, despite significant reductions in hepatic cholesterol levels and an increase in LDL receptor expression. A feeding study by Chan *et al.* (Chan et al., 1999) was similarly unable to demonstrate an effect of green tea extract on hepatic HMG-CoA reductase activity. Measuring cholesterol synthesis *in vivo* is difficult, whereas *in vitro* studies are more tractable. In this regard, Gebhardt and colleagues reported that several common polyphenols (luteolin, quercetin) were able to decrease cholesterol synthesis when added to cultured hepatocytes or hepatoma cell cultures (Gebhardt, 2003). This inhibition appeared to occur at the level of HMG-CoA reductase. Tea polyphenols (Abe et al., 2000), as well as the simple polyphenol resveratrol have been shown to directly inhibit squalene monooxygenase, a rate-limiting downstream enzyme in cholesterol synthesis. Two studies by Bursill and colleagues (Bursill et al., 2001; Bursill and Roach, 2006) demonstrated an increase in HMG-CoA reductase and LDL-receptor mRNA in HepG2 cells incubated with green tea extract or its principal component, epigallocatechin gallate (EGCG), and a decrease in cellular lathosterol, indicating that cholesterol synthesis was inhibited in treated cells (Singh et al., 2009a). Together, these studies suggest that green tea polyphenols are inhibitory to cholesterol synthesis by inhibiting HMGCoA reductase (Singh et al., 2009a). Moreover, the effect of black tea extract, which consists predominantly of a diverse mixture of polymerized polyphenols termed theaflavins and thearubigins, has not been examined, despite the recent clinical evidence that black tea can modestly reduce serum cholesterol levels.

#### **1.10.1.2 Decrease in cholesterol level by policosanol**

Policosanol, a mixture of very long-chain alcohols isolated from sugarcane, at doses of 10 to 20 mg/day has been shown to lower total and LDL cholesterol by up to 30%, equivalent to low-dose statin therapy (Gouni-Berthold and Berthold, 2002). In both short-term (≤12-week) and long-term (up to 2-year) randomized, placebo-controlled, double-blind studies, policosanol lowered LDL-cholesterol in normocholesterolemic patients by an average of 33%, and in hypercholesterolemic patients by 24% (for review, see (Gouni-Berthold and Berthold, 2002; Varady et al., 2003). In normocholesterolemic patients, policosanol caused a small and generally insignificant increase in high-density lipoprotein-cholesterol, whereas in seven clinical studies of dyslipidemic patients high-density lipoprotein-cholesterol was increased by an average of 17%. Policosanol is also effective in rabbits and monkeys, where it lowers blood cholesterol and reduces the development of atherosclerotic plaques (Wang et al., 2003), but it was found not to be effective in hamsters (Wang et al., 2003).

The major components of policosanol are the primary alcohols octacosanol (C28; ~60%), triacontanol (C30; 12–14%), and hexacosanol (C26; 6–12%), with lesser amounts of other alcohols with chain lengths of 24 to 34 carbons (Singh et al., 2006) . The product has no evident toxicity and is available over-the-counter in many outlets. The active component(s) has not been established, but it has been shown that very long-chain alcohols can undergo oxidation to fatty acids with subsequent peroxisomal β-oxidation, which also yields chain-shortened metabolites (Singh et al., 1987). D-003, a mixture of very long-chain saturated fatty acids, also purified from sugarcane, similarly lowers LDL and total cholesterol in normocholesterolemic patients (Castano et al., 2005) and in normocholesterolemic and casein-induced hypercholesterolemic rabbits, and a more rapid onset of effects suggests that oxidation of policosanols to very long-chain fatty acids may be necessary for their hypocholesterolemic actions (Menendez et al., 2001; Menendez et al., 2004). Several studies have demonstrated that policosanol inhibits cholesterol synthesis in laboratory animals and cultured cells, and it is thought that this is the principal mechanism by which it lowers blood cholesterol levels. In the latter study, policosanol did not affect the incorporation of [14C]mevalonate into cholesterol, indicating that policosanol was acting at or above mevalonate synthesis. However, policosanol did not inhibit HMG-CoA reductase (mevalonate synthase) when added to cell lysates, arguing against a direct interaction with this enzyme. The ability of policosanol to prevent the up-regulation of HMG-CoA reductase activity in these cells in response to lipid-depleted media suggested that policosanol suppresses HMG-CoA reductase synthesis or enhances enzyme degradation. Similar results were obtained with D-003 (Menendez et al., 2001), although neither study measured HMG-CoA reductase protein levels. Our studies explored that policosanol and identify the active component(s) of this natural product inhibits cholesterol synthesis by inhibiting HMGCoA reductase enzyme (Singh et al., 2006).

#### **1.10.1.3 Inhibition of cholesterol biosynthesis by garlic**

Garlic is rich in sulfur-containing compounds, principally S-allylcysteine and alliin, the latter of which is rapidly metabolized when garlic is crushed and alliinase is released. The highly reactive sulfenic acid that is formed from alliin condenses to allicin, which then rapidly recombines to various di- and tri-sulfides, depending on conditions. Ultimately these compounds are believed to yield allyl mercaptan and allyl methyl sulfide, which can react with cellular components or be eliminated on the breath. The organosulfur compounds formed in garlic are highly reactive with other sulfhydryl compounds, including cysteines found in proteins, and it is likely that the chemical modification of enzyme-sulfhydryls is responsible for the purported therapeutic effects of garlic. The question of which compounds are most important to the therapeutic effects of garlic remains unresolved, although several studies have shown that the diallyl disulfides, allyl mercaptan, and S-

and long-term (up to 2-year) randomized, placebo-controlled, double-blind studies, policosanol lowered LDL-cholesterol in normocholesterolemic patients by an average of 33%, and in hypercholesterolemic patients by 24% (for review, see (Gouni-Berthold and Berthold, 2002; Varady et al., 2003). In normocholesterolemic patients, policosanol caused a small and generally insignificant increase in high-density lipoprotein-cholesterol, whereas in seven clinical studies of dyslipidemic patients high-density lipoprotein-cholesterol was increased by an average of 17%. Policosanol is also effective in rabbits and monkeys, where it lowers blood cholesterol and reduces the development of atherosclerotic plaques (Wang et

The major components of policosanol are the primary alcohols octacosanol (C28; ~60%), triacontanol (C30; 12–14%), and hexacosanol (C26; 6–12%), with lesser amounts of other alcohols with chain lengths of 24 to 34 carbons (Singh et al., 2006) . The product has no evident toxicity and is available over-the-counter in many outlets. The active component(s) has not been established, but it has been shown that very long-chain alcohols can undergo oxidation to fatty acids with subsequent peroxisomal β-oxidation, which also yields chain-shortened metabolites (Singh et al., 1987). D-003, a mixture of very long-chain saturated fatty acids, also purified from sugarcane, similarly lowers LDL and total cholesterol in normocholesterolemic patients (Castano et al., 2005) and in normocholesterolemic and casein-induced hypercholesterolemic rabbits, and a more rapid onset of effects suggests that oxidation of policosanols to very long-chain fatty acids may be necessary for their hypocholesterolemic actions (Menendez et al., 2001; Menendez et al., 2004). Several studies have demonstrated that policosanol inhibits cholesterol synthesis in laboratory animals and cultured cells, and it is thought that this is the principal mechanism by which it lowers blood cholesterol levels. In the latter study, policosanol did not affect the incorporation of [14C]mevalonate into cholesterol, indicating that policosanol was acting at or above mevalonate synthesis. However, policosanol did not inhibit HMG-CoA reductase (mevalonate synthase) when added to cell lysates, arguing against a direct interaction with this enzyme. The ability of policosanol to prevent the up-regulation of HMG-CoA reductase activity in these cells in response to lipid-depleted media suggested that policosanol suppresses HMG-CoA reductase synthesis or enhances enzyme degradation. Similar results were obtained with D-003 (Menendez et al., 2001), although neither study measured HMG-CoA reductase protein levels. Our studies explored that policosanol and identify the active component(s) of this natural product inhibits

al., 2003), but it was found not to be effective in hamsters (Wang et al., 2003).

cholesterol synthesis by inhibiting HMGCoA reductase enzyme (Singh et al., 2006).

Garlic is rich in sulfur-containing compounds, principally S-allylcysteine and alliin, the latter of which is rapidly metabolized when garlic is crushed and alliinase is released. The highly reactive sulfenic acid that is formed from alliin condenses to allicin, which then rapidly recombines to various di- and tri-sulfides, depending on conditions. Ultimately these compounds are believed to yield allyl mercaptan and allyl methyl sulfide, which can react with cellular components or be eliminated on the breath. The organosulfur compounds formed in garlic are highly reactive with other sulfhydryl compounds, including cysteines found in proteins, and it is likely that the chemical modification of enzyme-sulfhydryls is responsible for the purported therapeutic effects of garlic. The question of which compounds are most important to the therapeutic effects of garlic remains unresolved, although several studies have shown that the diallyl disulfides, allyl mercaptan, and S-

**1.10.1.3 Inhibition of cholesterol biosynthesis by garlic** 

alk(en)yl cysteines are effective inhibitors of cholesterol synthesis in cells (Gebhardt and Beck, 1996; Liu and Yeh, 2000; Singh and Porter, 2006). Similarly, the enzyme targets that mediate the effects of garlic have not been identified.

Our studies with hepatoma cells in which cholesterol and intermediates are radiolabeled and identified by coupled gas chromatography–mass spectrometry reveal that garlic causes the accumulation of sterol 4α-methyl oxidase substrates and that an allyl disulfide or allyl sulfhydryl group is necessary for inhibition by garlic-derived compounds (Singh and Porter, 2006).
