**9. Cholesterol-lowering natural products**

#### **9.1. Plant sterols/ stanols**

**8.4. Bile acid sequestrant**

196 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

and the bulkiness of resins [64].

**8.5. Fibrates**

Bile acid sequestrant or bile binding anion (Chloride) exchange resins that is effective in reducing total cholesterol and LDL cholesterol levels. The primary and direct action of the bile acid sequestrants is to bind to bile acids in the gut and thus interrupt the enterohepatic recirculation of bile acids [63]. Three key enzyme are affected by bile acid sequestrant, which are phosphatidic acid phosphatase, cholesterol 7-alpha-hydroxylase and HMG-CoA reduc‐ tase. Activation of phosphatidic acid phosphatase promotes hepatic triglyceride synthesis, induces secretion of triglyceride-rich VLDL particles and consequently increases plasma triglyceride levels. The activation of hepatic cholesterol 7-alpha-hydroxylase promotes the conversion of intracellular cholesterol to bile acids. The decrease in intracellular cholesterol stores, in turn, increases LDL receptor expression on hepatocyte membranes and consequently, increases receptor-mediated fractional catabolism of LDL or LDL uptake by liver cells. Reduction of intracellular cholesterol may also increase the synthesis of cholesterol through activation of HMG-CoA reductase. The potential loss of the bile acid sequestrant's cholesterollowering efficacy can be overcome by adding HMG-CoA reductase inhibitor (statins). Finally, bile acid sequestrants promote apoprotein AI synthesis and tend to raise high-HDL cholesterol levels, primarily by increasing plasma HDL-2 concentrations. Three drugs in this class are synthetic cholestyramine, colestipol, colesevelam. The side effect profile of the bile acid sequestrants is tolerable, with most complaints related to effects on the gastrointestinal tract

Fibrates are primarily effective for the treatment of hypertriglyceridemia or mixed hyper‐ lipidemia by stimulating the peroxisomal β-oxidation pathway. Their main action is to lower plasma triglyceride levels, but they also reduce total and LDL cholesterol concen‐ trations and induce a moderate increase in HDL cholesterol. Fibrates act by stimulating the activity of peroxisome proliferator-activated receptor (PPAR)-α, a member of the PPAR subfamily of nuclear receptors [65]. It controls the transcription of regulatory genes of fatty acids and cholesterol metabolism. It inhibits the synthesis and secretion of triglycerides by the liver and a stimulation of the degradation of triglyceride-rich lipopro‐ teins. This increased clearance of triglycerides results from a stimulation of the expres‐ sion of lipoprotein lipase and a decreased expression and concentration of apolipoprotein-CIII, an inhibitor of lipoprotein lipase activity. PPAR-α activation modifies the expression of several key genes controlling HDL cholesterol metabolism and reverse transport of cholesterol [65]. Several fibrate drugs such as ciprofibrate, bezafibrate, fenofi‐ brate, and gemfibrozil has revolutionized lipid-lowering but research has shown the pro‐ longed use of some of these drugs like clofibrate and ciprofibrate causes peroxisome proliferation leading to hepatomegaly and tumor formation in the liver of rodents [66].

A recent report demonstrated that between the periods 1988-1994 and 1999-2002, mean total cholesterol and mean LDL cholesterol declined in American adults. Coincidently, during this time there also was an increase in the percentage of adults receiving lipid-lowering medica‐ tions. However, among adults not receiving lipid-lowering medications, trends in lipids were Plant sterols/ stanols, naturally occurring in foods of plant origin, perform similar biological functions to cholesterol, and contain a similar chemical structure. They differ from cholesterol only in the presence of either an extra methyl or ethyl group. Absorption efficiency for plant sterols in humans is less (2-5%) than that of cholesterol (60%). The most common forms are unsaturated plant sterols β-sitosterol, campesterol, and stigmasterol and the saturated sitostanol and campestanol [68]. Plant sterols reduce the absorption of both dietary and biliary cholesterol from the intestinal tract by 30%–50% [69]. The exact mechanism is yet fully elucidated while it is generally assumed that the presence of increased quantities of plant sterols in the gut lowers the micellar solubility of cholesterol, therefore lowering the amount of cholesterol available for absorption [69]. Intake of phytosterol and stanols at an average of 2 g/day has been shown to lower low density lipoprotein cholesterol (LDL-C) by 10-15%. The effect appeared to be peaked at intakes of 2 g/d, with little additional benefit being achieved at intakes higher than 2.5 g/d. The recommended daily intake of phytosterols is 2 g/d in humans [70]. Plant sterols and their derivatives reduce plasma cholesterol levels independently from the mRNA expression of ABCG5 and ABCG8 transporters [71]. Food products such as margarine, milk, yoghurt, and cereal products enriched with plant sterols/stanols are promot‐ ed as functional foods to help lower serum cholesterol levels. Human and animal studies have shown that plant sterol and stanol esters are non-toxic [72]. There have been concerns raised over the reduced absorption of some fat soluble vitamins from the use of plant sterols. For example, plant sterols and stanols have been shown to reduce β-carotene, α-carotene, and vitamin E levels by around 25%, 10%, and 8%, respectively [72]. Used alone in the diet, or as an adjuvant to drug therapy, or in combination with other functional food components, plant sterols/stanols-enriched products are generally effective at reducing serum total and LDL-C [72], and thus the most popular natural ingredient in cholesterol-lowering product market.

#### **9.2. Soy products**

Soy foods have been consumed for centuries in Asian countries. Consumption of soy foods contribute to lower incidences of coronary heart diseases, atherosclerosis, type 2 diabetes, and decreased risk of certain types of carcinogenesis such as breast and prostate cancers [73]. Animal and human studies have also shown that consumption of soy protein or associated isoflavones has beneficial impacts such as lowering liver or blood triglyceride, total and LDL cholesterol levels, increasing HDL cholesterol and the ratio of HDL/LDL cholesterol [73, 74]. Soy protein regulates SREBP-1 expression by modulating serum insulin concentration, thus preventing the development of fatty liver [75]. Isoflavones are major soy phytoestrogens present in soy foods and Genistin, daidzin, and glycitein are the main soy isoflavones (Xiao 2008). Soy and soy bioactive components are well-tolerated and the adverse effects reported are gastrointestinal symptoms (e.g., diarrhea), followed by menstrual complaints (e.g., prolonged periods, amenorrhea) headache, dizziness, and musculoskeletal complaints [76].

#### **9.3. Dietary fibre**

Dietary fibre is one of the most studied dietary components associated with cardiovascular benefits. It is a complex of non-digestible carbohydrates and lignin that are intrinsic and intact in plants and are resistant to digestion and absorption in the small intestine. Dietary fibre can modulate body weight and promotes beneficial physiological effects such as laxation, reduc‐ tion in blood cholesterol and postprandial blood glucose [77]. Traditionally, dietary fibre has been classified on the basis of its solubility in water (soluble or insoluble). Foods rich in fibre need to be chewed longer, leading to an increase in the time needed to eat and the feeling of satiety. Fibres which make up viscous solutions also delay the passage of food from the stomach to duodenum and contribute to an increase in satiety and a decrease in energy consumption [78]. In the intestine, the incorporation of fibre in food may complicate the interaction between digestive enzymes and their substrates, thus slowing down the absorption of nutrients[77]. The hypocholesterolemic action of fibre is partly mediated by a lower absorption of intestinal bile acid because the interruption of the enterohepatic bile acid circulation, thus increasing faecal bile acid loss, and its de novo synthesis in liver. The physicochemical properties of soluble fibre result in important modifications in volume, bulk and viscosity in the intestinal lumen, which will alter metabolic pathways of hepatic cholesterol and lipoprotein metabolism, also resulting in lowering of plasma LDL cholesterol [79]. Dietary fibre increases the enzymatic activity of cholesterol-7-α-hydroxylase, contributing to a higher depletion of hepatic cholesterol but increased endogenous cholesterol synthesis. However, there is an increase in the number of LDL receptors and in the recruitment of the esterified cholesterol from the circulating LDL particles. Several types of soluble dietary fibre such as pectin, glucomannan, psyllium can decrease plasma total cholesterol and LDL cholesterol. Epidemiological evidences showed a stronger association of cardiovascular protection with soluble fibre than insoluble fibre. Insoluble fibre such as that from wheat or cellulose has not been reported to have any significant effect on blood cholesterol [78]. Consumption of too much high fibre foods that have not been cooked can cause side effects of abdominal bloating and gas.

#### **9.4. Flaxseed lignans**

Flax seed is the richest source of natural lignans, with secoisolariciresinol diglucoside (SDG) being the principal lignan compound. Flaxseed or flaxseed meal have cardioprotective properties and can suppress atherosclerosis by virtue of its antioxidant properties due to the presence of flaxseed lignans. Lignan reduces serum triglycerides and LDL and raises HDL cholesterol. In addition, flaxseed oil possesses anti-inflammatory properties and reduces platelet aggregation as well [80]. Flaxseed lignans along with soy isoflavones are phytoestro‐ gens commonly consumed in the human diet.

#### **9.5. Polyunsaturated fatty acids and omega-3 fatty acids**

cholesterol levels, increasing HDL cholesterol and the ratio of HDL/LDL cholesterol [73, 74]. Soy protein regulates SREBP-1 expression by modulating serum insulin concentration, thus preventing the development of fatty liver [75]. Isoflavones are major soy phytoestrogens present in soy foods and Genistin, daidzin, and glycitein are the main soy isoflavones (Xiao 2008). Soy and soy bioactive components are well-tolerated and the adverse effects reported are gastrointestinal symptoms (e.g., diarrhea), followed by menstrual complaints (e.g., prolonged periods, amenorrhea) headache, dizziness, and musculoskeletal complaints [76].

198 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

Dietary fibre is one of the most studied dietary components associated with cardiovascular benefits. It is a complex of non-digestible carbohydrates and lignin that are intrinsic and intact in plants and are resistant to digestion and absorption in the small intestine. Dietary fibre can modulate body weight and promotes beneficial physiological effects such as laxation, reduc‐ tion in blood cholesterol and postprandial blood glucose [77]. Traditionally, dietary fibre has been classified on the basis of its solubility in water (soluble or insoluble). Foods rich in fibre need to be chewed longer, leading to an increase in the time needed to eat and the feeling of satiety. Fibres which make up viscous solutions also delay the passage of food from the stomach to duodenum and contribute to an increase in satiety and a decrease in energy consumption [78]. In the intestine, the incorporation of fibre in food may complicate the interaction between digestive enzymes and their substrates, thus slowing down the absorption of nutrients[77]. The hypocholesterolemic action of fibre is partly mediated by a lower absorption of intestinal bile acid because the interruption of the enterohepatic bile acid circulation, thus increasing faecal bile acid loss, and its de novo synthesis in liver. The physicochemical properties of soluble fibre result in important modifications in volume, bulk and viscosity in the intestinal lumen, which will alter metabolic pathways of hepatic cholesterol and lipoprotein metabolism, also resulting in lowering of plasma LDL cholesterol [79]. Dietary fibre increases the enzymatic activity of cholesterol-7-α-hydroxylase, contributing to a higher depletion of hepatic cholesterol but increased endogenous cholesterol synthesis. However, there is an increase in the number of LDL receptors and in the recruitment of the esterified cholesterol from the circulating LDL particles. Several types of soluble dietary fibre such as pectin, glucomannan, psyllium can decrease plasma total cholesterol and LDL cholesterol. Epidemiological evidences showed a stronger association of cardiovascular protection with soluble fibre than insoluble fibre. Insoluble fibre such as that from wheat or cellulose has not been reported to have any significant effect on blood cholesterol [78]. Consumption of too much high fibre foods that have not been cooked can cause side effects of abdominal bloating

Flax seed is the richest source of natural lignans, with secoisolariciresinol diglucoside (SDG) being the principal lignan compound. Flaxseed or flaxseed meal have cardioprotective properties and can suppress atherosclerosis by virtue of its antioxidant properties due to the presence of flaxseed lignans. Lignan reduces serum triglycerides and LDL and raises HDL

**9.3. Dietary fibre**

and gas.

**9.4. Flaxseed lignans**

An old assumption regarding fatty acids and their effects on atherosclerosis was that monounsaturated fatty acids were neutral, saturated fatty acids were bad, and polyunsa‐ turated fatty acids were good. However, a study conducted by Scott Grundy and Fred Mattson in 1985 turned the "world of monounsaturated fatty acids" around[81]. They demonstrated that diets rich in saturated fatty acids caused a high LDL cholesterol/HDL cholesterol ratio, and that substitution of monounsaturated fatty acid for saturated fatty acids reduced LDL cholesterol but did not reduce HDL cholesterol. Consequently the LDL cholesterol/HDL cholesterol ratio was the lowest with monounsaturated fatty acids, given that polyunsaturated fatty acids reduced HDL cholesterol as well as LDL cholester‐ ol. A similar phenomenon was observed in a 5-year study in male African green mon‐ keys[82]. In the monkey studies, average HDL cholesterol was 50 mg/dl in the polyunsaturated fatty acids group versus 86 and 81 mg/dl in the saturated fatty acids and monounsaturated fatty acids groups, respectively. Average plasma LDL cholesterol concentrations in the polyunsaturated fatty acids and monounsaturated fatty acids-fed monkeys were 157 and 167 mg/dl, respectively (no significant difference between them) versus 257 mg/dl in the saturated fatty acids-fed animals.

The influences of dietary fatty acids on blood cholesterol profiles are also related to diet composition[83]. It is well accepted that the consumption of saturated fatty acids increases LDL cholesterol, whereas carbohydrates, monounsaturated fatty acids and polyunsaturated fatty acids do not. The effect of fatty acids on blood lipid profiles also depends on heath conditions. Among individuals who are insulin resistant, a low-fat, high-carbohydrate diet typically has an adverse effect on lipid profiles. In addition to lowering HDL cholesterol, it also increases triacylglycerols and LDL cholesterol. Consequently, a moderate fat diet in which unsaturated fatty acids replace saturated fatty acids and carbohydrates are not augmented is advised to lower LDL cholesterol[83].

Fish oil (marine n-3 fatty acids, eicosapentaenoic acid, and docosahexaenoic acid), whether from dietary sources or fish oil supplements, exhibit cardioprotective effects and reduce mortality due to cardiovascular diseases. Fish oil provides cell membrane stabilization, antiinflammatory, antiatherogenic effects and suppression of cardiac arrhythmias[84]. Omega-3 fatty acids lower moderately the blood pressure through primarily the improvement of vascular endothelial cell function whilst a multitude of mechanisms may be involved85. Polyunsaturated fatty acids lower triacylglycerols, which has also been important in cardio‐ protection and the management of insulin resistance and diabetes. The effect of omega-3 fatty acids on blood cholesterol is inconsistent effect. In general, omega-3 fatty acids do not offer a benefit of directly lowering blood cholesterol. Instead, in some studies fish oil has been found to cause a small rise in LDL-cholesterol; however a change in the LDL particle size from the smaller more atherogenic form to the larger less damaging particle size have been noted85.

Health Canada has recently reconsidered the classification of food products with disease risk reduction claims or therapeutic claims in light of clarified principles for the classification of foods at the Food-Natural Health Product interface (http://www.hc-sc.gc.ca/fn-an/labeletiquet/claims-reclam/assess-evalu/sat-mono-poly-fat-gras-eng.php). Health Canada has concluded that the results of the updated literature review are consistent with the 2002 report provided by the Institute of Medicine (IOM), which forms the basis of the US and Canada dietary guidance, on the replacement of saturated fat with unsaturated fat for blood cholesterol lowering. In other words, scientific evidence exists in support of the therapeutic claim linking the replacement of saturated fat with unsaturated fat to a reduction of blood cholesterol. It is stated that the claim is relevant and generally applicable to the Canadian population as a high proportion of the Canadian population is hyperlipidemic. It is allowed now by the Health Canada's to put therapeutic claim statements such as "Replacing saturated fats with polyun‐ saturated and monounsaturated fats (from vegetable oils) helps lower/reduce cholesterol" in vegetable oils and foods containing vegetable oils when specific conditions for the food carrying the claim are met.

#### **9.6. Olive oil**

Olive oil can reduce LDL and raise high-density lipoprotein cholesterol and decrease lipid damage due to oxidative stress. In addition, olive oil reduces inflammatory and thrombogenic status, endothelial dysfunction, and blood pressure[86].

#### **9.7. Green tea products**

Tea catechin-especially (-)-epigallocatechin-3-gallate-inhibits the expression of soluble adhesion molecules including vascular adhesion molecule-1 and intercellular adhesion molecule-1, endothelial cell inflammatory markers, decreased oxidized LDL, and prevents the development of atherosclerosis and[87].

Certainly, there are more natural products available, for instance a number of antioxidants and phenolic compounds, to lower blood cholesterol levels. However, the big challenges that the natural products have been facing in the past years lie in their relatively lower efficacies as compared with the cholesterol-lowering drugs. The apparent advantages of natural products are their better safety profiles. In order to promote the market share of cholesterol-lowering natural products in competing with the drugs, it is critical to develop new products that can offer better efficacies than the current natural products, without losing safety or introducing increased toxic or severe side effects. Considering that the majority of the current natural products fall into the same category of cholesterol absorption inhibitor, the future direction of research and development of cholesterol-lowering products, novel distinct pathways or targets should be focused. Herewith, we will provide some brief thoughts on new approaches that we believe worth to tackle into, with a hope that the new mind in the research and development direction and focus would help to discover and develop novel natural products with significantly improved cholesterol-lowering efficacy working through distinct mecha‐ nisms than the currently available natural products, without apparent side effects.
