**Hypercholesterolemia as a Risk Factor for Catheterization-Related Cerebral Infarction — A Literature Review and a Summary of Cases**

Yusuke Morita, Takao Kato and Moriaki Inoko

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59282

#### **1. Introduction**

Hypercholesterolemia is one of the major risk factors of atherosclerotic disease. Treatment with statins reduces serum levels of low-density lipoprotein cholesterol, and attenuates athero‐ sclerotic plaque formation in the carotid artery, coronary artery, and thoracic aorta.

During diagnostic angiography or interventions for coronary artery diseases, catheterization can lead to devastating complications, including stroke. Previous studies reported stroke rates of 0.1–0.4% [1-9]. Catheterization-related acute stroke is associated with high in-hospital mortality and prevalence of overall major complications [5,10,11].

Because only new neurological complications are classified as stroke, clinically unapparent cerebral embolisms are not taken into account. Asymptomatic cerebral infarction is thought to be related to the incidence of symptomatic cerebral infarction, cognitive decline, and demen‐ tia [12], and may thus represent a significant complication of catheterization procedures.

Here, we summarize our review of published literature, present two case studies in our cardiovascular center, and summarize our data on hypercholesterolemia and catheterizationrelated stroke.

## **2. A review of published literature on hypercholesterolemia and catheterization-related stroke**

Previously published data on catheterization-related acute stroke is summarized in Figure 1. Previous studies have reported stroke rates of 0.1– 0.4% [1-9], and rates of stroke and

transient ischemia attack (TIA) after catheterization do not appear to have decreased over time (Figure 1). Much of the published data is limited to stroke. Stroke is defined as cerebral infarction or hemorrhage with a neurological deficit lasting >24 h; TIA is a neurological deficit lasting <24 h.

**Figure 1.** Rates of stroke and transient ischemic attack (TIA) after catheterization have not reduced overtime [1-9].

Magnetic resonance imaging (MRI) can be used to detect asymptomatic cerebral infarction related to catheterization. Diffusion-weighted MRI (DW-MRI), in particular, represents a highly sensitive tool for detecting acute cerebral ischemic lesions [13,14]. New lesions appear as focal high-intensity cerebral lesions on diffusion-weighted imaging (DWI) and have a low signal on apparent diffusion coefficient (ADC) maps [15].

Several prospective studies of silent cerebral infarction have been performed in a small number of patients. Bendszus et al. used DW-MRI before and after angiography of cerebral vessels to assess embolic events in 100 consecutive angiographies (66 diagnostic and 34 interventional) in 91 patients [16]. In their report, published in *The Lancet* in 1999, they showed that 23% of procedures caused silent embolic cerebral infarction: 42 bright lesions were observed in 23 patients after 23 procedures (17 diagnostic, six interventional), in a pattern consistent with embolic events, and in the absence of any new neurological deficit. More contrast medium, a longer fluoroscopy time, more frequent additional catheters, and having more vessels that were difficult to approach were the risks of silent embolic cerebral infarction. Patients' mean age did not differ between patients with lesions and those without lesions, but their mean age was relatively young (around 50 years old) compared to patients who are receiving cardiac catheterization.

Lund et al. monitored cerebral microemboli during catheterization in 42 unselected patients using multifrequency transcranial Doppler alongside cerebral DW-MRI and neuropsycholog‐ ical assessments. Measurements were taken on the days before and after catheterization [17]. Their report, published in the *European Heart Journal* in 2005, showed that new cerebral lesions were present in 15.2% of transradial catheterization patients, but in none of the transfemoral catheterization patients. These lesions were significantly associated with a higher number of solid microemboli and a longer fluoroscopy time. Approximately 80% of patients were male and had hyperlipidemia or were statin users; approximately 40% of patients had a previous history of myocardial infarction.

transient ischemia attack (TIA) after catheterization do not appear to have decreased over time (Figure 1). Much of the published data is limited to stroke. Stroke is defined as cerebral infarction or hemorrhage with a neurological deficit lasting >24 h; TIA is a neurological

**Figure 1.** Rates of stroke and transient ischemic attack (TIA) after catheterization have not reduced overtime [1-9].

signal on apparent diffusion coefficient (ADC) maps [15].

Magnetic resonance imaging (MRI) can be used to detect asymptomatic cerebral infarction related to catheterization. Diffusion-weighted MRI (DW-MRI), in particular, represents a highly sensitive tool for detecting acute cerebral ischemic lesions [13,14]. New lesions appear as focal high-intensity cerebral lesions on diffusion-weighted imaging (DWI) and have a low

Several prospective studies of silent cerebral infarction have been performed in a small number of patients. Bendszus et al. used DW-MRI before and after angiography of cerebral vessels to assess embolic events in 100 consecutive angiographies (66 diagnostic and 34 interventional) in 91 patients [16]. In their report, published in *The Lancet* in 1999, they showed that 23% of procedures caused silent embolic cerebral infarction: 42 bright lesions were observed in 23 patients after 23 procedures (17 diagnostic, six interventional), in a pattern consistent with embolic events, and in the absence of any new neurological deficit. More contrast medium, a longer fluoroscopy time, more frequent additional catheters, and having more vessels that were difficult to approach were the risks of silent embolic cerebral infarction. Patients' mean age did not differ between patients with lesions and those without lesions, but their mean age was relatively young (around 50 years old) compared to patients who are receiving cardiac

Lund et al. monitored cerebral microemboli during catheterization in 42 unselected patients using multifrequency transcranial Doppler alongside cerebral DW-MRI and neuropsycholog‐ ical assessments. Measurements were taken on the days before and after catheterization [17]. Their report, published in the *European Heart Journal* in 2005, showed that new cerebral lesions

deficit lasting <24 h.

122 Hypercholesterolemia

catheterization.

In 2005, Karen et al., using MRI before and after catheterization procedures, reported focal cerebral infarction without any symptoms in 15% of 48 patients [18]. In this prospective study, definitive conclusions could not be drawn owing to the small sample size, but procedure duration appeared to predict cerebral infarction following catheterization. Patients with cerebral infarction were also found to have a history of smoking, hyperlipidemia, hyperten‐ sion, and obesity.

It is often difficult to acquire MR images before and after procedures in prospective studies. In 2011, Kojuri et al. reported in *BMC Cardiovascular Disorders* the prevalence of retinal emboli after diagnostic and therapeutic catheterization during retinal examination: 6.3% of 300 patients and only 1 patient developed vision disorder [19].

Patients who undergo aortic stenosis (AS) procedures that involve crossing of the aortic valve carry a high risk of cerebral infarction. In 2003, Omran et al. reported in the *Lancet* that 22% of 101 AS patients undergoing retrograde catheterization of the left ventricle had new lesions on MRI, and 3 patients had symptomatic cerebral infarction [20]. A total of 152 patients with AS undergoing cardiac catheterization were randomized to receive catheterization with or without retrograde passage of the aortic valve in a ratio of 2:1. An additional 32 patients without AS were also assessed as healthy controls. In patients without retrograde passage of the aortic valve, and in healthy controls, there was no MRI or clinical evidence of cerebral embolism.

Although these studies used relatively small sample sizes, the incidence of asymptomatic cerebral infarction following catheterization, as assessed by MRI or other methods, appears to remain relatively high. Following improvements in catheter design (rendering them more slender) and in techniques, catheterization-related cerebral infarctions were expected to decrease, though this may be counterbalanced by the increased risk profile of patients who undergo catheterization.

### **3. Two cases of asymptomatic and symptomatic cerebral infarction related to catheterization**

Here we present two cases of cerebral infarction with and without symptoms which was related to cardiac catheterization.

Case 1: A 73-year-old man with hyperlipidemia, hypertension and diabetes underwent percutaneous coronary intervention (PCI) via the right radial artery. An initial diagnostic procedure was performed using 4F catheter; a 6F catheter was subsequently used to perform PCI for the left anterior descending artery (LAD). Due to the tortuosity of the LAD, the procedure time was 58 min and 275 ml contrast medium was used. MRI was performed 5 days later for MR angiography to detect carotid and intra-cranial lesions. MRI was not performed prior to the procedure; however, new lesions appeared as focal high-intensity cerebral lesions on DWI (Figure 2A) and gave a low signal on ADC maps (Figure 2B). ADC maps represent a useful tool for detecting acute ischemic infarct lesions [16].

Case 2: A 69-year-old woman with hyperlipidemia and diabetes underwent diagnostic catheterization for left ventricular systolic dysfunction of unknown etiology, with 45% ejection fraction. On the day after catheterization, the patient complained of dizziness. DW-MRI revealed a spotty high-intensity signal (Figure 2C).

**Figure 2.** Magnetic resonance imaging (MRI) in two cases of asymptomatic and symptomatic cerebral infarction relat‐ ed to catheterization. (A) Diffusion-weighted MR image of Case 1. (B) Apparent diffusion coefficient (AMC) map of Case 1. (C) DW-MR image of Case 2.

## **4. A single center retrospective analysis of catheterization-related cerebral infarction**

We next present the summarized results of a single center analysis of catheterization-related cerebral infarction in our cardiovascular center. A total of 84 patients who had undergone 1237 consecutive catheterizations with follow-up MRI within 14 days, between 2010 and 2011, were retrospectively analyzed. Of these, 10 symptomatic patients underwent MRI to check for cerebral infarction. The remaining 74 patients were asymptomatic, and underwent MRI for preliminary assessment before coronary artery bypass graft (35%), valvular surgery (18%), or aortic repair (6.8%). MRI revealed cerebral infarction in 5 out of 10 symptomatic patients (50%), and in 22 out of 74 asymptomatic patients (29.7%). Patient background characteristics are presented in Tables 1 and 2. In univariate analysis, the prevalence of dyslipidemia, the number of atherosclerotic risk factors, the number of catheters used, the procedure time, urgent settings, and reasons for intervention differed significantly between patients with and without cerebral infarction (Tables 1 and 2). Dyslipidemia and the number of catheters used were identified as predictive of catheterization-related cerebral infarction by multivariate analysis (odds ratio [OR], 4.66; 95% CI, 1.32−20.2; P=0.02 and OR, 2.04; 95% CI 1.02−4.35; P=0.04, respectively). Overall, the rate of asymptomatic catheterization-related cerebral infarction detected by DW-MRI was high (29.7%). This may be partially due to selection bias because MRI was performed in candidates for cardiac surgery for atherosclerotic disease. The rate of catheterization-related symptomatic ischemic stroke recorded in this study (0.24%) is roughly equivalent to those reported in previous studies [1-9].


**Table 1.** Baseline characteristics

procedure time was 58 min and 275 ml contrast medium was used. MRI was performed 5 days later for MR angiography to detect carotid and intra-cranial lesions. MRI was not performed prior to the procedure; however, new lesions appeared as focal high-intensity cerebral lesions on DWI (Figure 2A) and gave a low signal on ADC maps (Figure 2B). ADC maps represent a

Case 2: A 69-year-old woman with hyperlipidemia and diabetes underwent diagnostic catheterization for left ventricular systolic dysfunction of unknown etiology, with 45% ejection fraction. On the day after catheterization, the patient complained of dizziness. DW-MRI

**Figure 2.** Magnetic resonance imaging (MRI) in two cases of asymptomatic and symptomatic cerebral infarction relat‐ ed to catheterization. (A) Diffusion-weighted MR image of Case 1. (B) Apparent diffusion coefficient (AMC) map of

useful tool for detecting acute ischemic infarct lesions [16].

revealed a spotty high-intensity signal (Figure 2C).

124 Hypercholesterolemia

Case 1. (C) DW-MR image of Case 2.


**Table 2.** Procedural characteristics

#### **5. Conclusions**

Despite improvements in procedural techniques and catheter design, patients undergoing catheterization remain at greater risk of atherosclerosis. While ischemic or hemorrhagic stroke is the most debilitating complication of such procedures, conferring significant comorbidity and mortality, asymptomatic cerebral infarction, which has been associated with cognitive decline, is also a significant complication. Intervention against atherosclerotic risk factors is needed along with careful procedural planning, in order to reduce rates of catheterizationrelated cerebral infarction. Hypercholesterolemia is one of risks of catheterization-related cerebral infarction.

#### **Acknowledgements**

This work is supported from the AstraZeneca Research Grant and Grant from the Tazuke Kofukai Reseach Institute.

#### **Author details**

**Infarction group No infarction group P value**

(n=27) (n=57)

No. of catheters used 2.85±1.1 2.12±0.9 0.003 Catheter size, F 4.44±0.8 4.25±0.6 0.3 Contrast volume, ml 128±55 120±55 0.48 Fluoroscopy time, min 22.7±14 17.1±12 0.04 LV angiogram, % 48.2 43.9 0.71 Aortic angiogram, % 11.1 14 0.71 Urgent, % 22.2 7 0.04 IABP, % 3.7 0 0.14

Diagnostic, % 74.1 91.2 0.04 Interventional, % 25.9 8.8 0.04

Radial or brachial, % 81.5 80.7 0.93 Femoral, % 18.5 19.3 0.93

Despite improvements in procedural techniques and catheter design, patients undergoing catheterization remain at greater risk of atherosclerosis. While ischemic or hemorrhagic stroke is the most debilitating complication of such procedures, conferring significant comorbidity and mortality, asymptomatic cerebral infarction, which has been associated with cognitive decline, is also a significant complication. Intervention against atherosclerotic risk factors is needed along with careful procedural planning, in order to reduce rates of catheterizationrelated cerebral infarction. Hypercholesterolemia is one of risks of catheterization-related

This work is supported from the AstraZeneca Research Grant and Grant from the Tazuke

Purpose of procedure

126 Hypercholesterolemia

**Table 2.** Procedural characteristics

**5. Conclusions**

cerebral infarction.

**Acknowledgements**

Kofukai Reseach Institute.

Approach site

Yusuke Morita, Takao Kato\* and Moriaki Inoko

\*Address all correspondence to: takao-kato@kitano-hp.or.jp

Cardiovascular Center, the Tazuke Kofukai Medical Research Institute, Kitano Hospital, Ogimachi, Kita-ku, Osaka, Japan

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[9] Hoffman SJ, Routledge HC, Lennon RJ, Mustafa MZ, Rihal CS, Gersh BJ, Holmes DR Jr, Gulati R. Procedural factors associated with percutaneous coronary intervention-

[10] Hoffman SJ, Holmes DR Jr, Rabinstein AA, Rihal CS, Gersh BJ, Lennon RJ, Bashir R, Gulati R. Trends, predictors, and outcomes of cerebrovascular events related to per‐ cutaneous coronary intervention: a 16-year single-center experience. JACC Cardio‐

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[12] Vermeer SE, Prins ND, den Heijer T, Hofman A, Koudstaal PJ, Breteler MM. Silent brain infarcts and the risk of dementia and cognitive decline. N Engl J Med. 2003 Mar

[13] Fiebach JB, Schellinger PD, Jansen O, Meyer M, Wilde P, Bender J, Schramm P, Jüttler E, Oehler J, Hartmann M, Hähnel S, Knauth M, Hacke W, Sartor K. CT and diffusionweighted MR imaging in randomized order: diffusion-weighted imaging results in higher accuracy and lower interrater variability in the diagnosis of hyperacute ische‐

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**Role of Phytochemicals and Statin on Hypercholesterolemia**

## **Medicinal Values of Selected Mushrooms with Special Reference to Anti-Hypercholesterolemia**

Choong Yew Keong

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59424

#### **1. Introduction**

#### **1.1. Hypercholesterolemia**

Hypercholesterolemia is a known major risk factor in the development of athereosclerosis [29, 32, 51]. This circumstance is caused by internal homeostasis due to foods consumed. The hypercholesterolemia may be related to high cholesterol diet or regular saturated fatty acids intake [42]. The incidence of Chronic Heart Disease (CHD) remains high despite blood pressure being controlled in hypertensive patients. Thus, in hypercholesterolemia pa‐ tient's LDL (Low Density Lipoprotein) concentration increases, and the lipoprotein is more aged and more susceptible to oxidative modifications than LDL from healthy subjects [50]. These patients have been diagnosed with disability of LDL excreation and very low LDL receptor activity. The most potent inhibitors of cellular cholesterol synthesis are inhibitors of 3-hydroxy-3-methyglutaryl coenzyme A (HMG-CoA) reductase and consequently elevated the cellular LDL receptors synthesis, resulting in significant reduction of plasma LDL levels. [66].

#### *1.1.1. Relation of hypercholesterolemia with atherosclerosis*

Atherosclerosis is the disease caused by accumulation of foam cells originated from the monocytes which are transformed into macrophages that engulf excessive oxidized lipopro‐ tein cholesterol. There was an increased foam cell formation which leads to intimal thickening after migration of smooth muscle cells to the intima and lamellar calcification under the endothelium. Finally a typical plaque characterized (Voet and Voet, 1990). The lumen of the arteries was narrowed and high blood pressure induced. The formation of plaque occurrs internally and raises the risk of cardiovascular diseases (CVDs) and strokes while remaining asymptomatic.

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Cholesterol is an important component and needed in development of metabolism cell, but the excess of cholesterol content in serum could be problematic. Preclinical and clinical studies have shown that high cholesterol diet is regarded as a main factor in the development of hypercholesterolemia, atherosclerosis and ischemic heart disease [14]. The cholesterol content in hypercholesterolemia cases produced extremely high risk agents such as oxygen free radicals in serum as well as erythrocytes, platelets and endothelial cells. The elevation of total serum cholesterol and LDL cholesterol along with generation of reactive oxygen species (ROS) play a key role in the development of coronary artery disease and atherosclerosis.

In term of mechanism, these vascular problems of atherosclerosis, hypercholesterolemia and hypertension are all correlated to each other. The formation of plaque is associated with a period of time and relies on homeostasis of each individual in dealing with good cholesterol (HDL: high-density lipoprotein) and bad cholesterol (LDL).

Today, strategies and remedies are available to combat CVDs. Even though changing life style with appropriate dietary intake remains the first line of defence advocated by the healthcare workers, drug treatment is still widely used because of the rapid effect especially in treating severe cases [7]. Hence, most research today focuses on screening and identifying compounds exhibiting anti-hypercholesterolemic properties.

To date, several cholesterol lowering medications were discovered and used singly or in combination to lower the LDL cholesterol and triglycerides, a type of fats in the blood that also increases the risk of atherosclerosis. Concomitantly increase of HDL cholesterol often offers protection from CVDs [21]. These types of drug include, bile acid binding resins, cholesterol absorption inhibitor, combination cholesterol absorption inhibitor and statins such as ezeti‐ mibe-simvastatin, fibrates, niacin and omega-3 fatty acids. However, almost all the antihypercholesteromia drugs have been reported as having various adverse effects [46]. Several side effects such as constipation, nausea, diarrhea, stomach pain, cramps, muscle soreness, pain and weakness are reported; while more severe side-effects such as facial and neck flushing, nausea, vomiting, diarrhea, gout, high blood sugar etc. are also known. The side effects of statin and niacin are similar to each other. In addition, adverse drug reactions are always encountered in multiple diseases treated with a number of drugs. If hypercholestero‐ lemia is accompanied by other diseases, these diseases may have an impact on the response of the body to anti-hypercholesterolemia drugs and the metabolic processes of the body may be affected negatively. Later on, increased dosages may be required, which in turn would only worsen the cholesterol medication drugs. The search for cholesterol lowering medication has now turned to complementary traditional medicine. However, the traditional use of herbs in lowering cholesterol is often not verified scientifically. On the other hand, even if proven effective, such herbs should be investigated on their mechanisms of action.

#### *1.1.2. Mushrooms as medicinal-functional food against hypercholesterolemia*

Medicinal mushrooms have been scientifically proven to be safe, efficacious, and novel antihypercholesterolemia therapeutic agents of natural source. An abundance of scientific research and studies on medicinal mushrooms or edible mushrooms shed light on them as functional food due to their broad spectrum of therapeutic efficacy beside culinary demand [80].

Cholesterol is an important component and needed in development of metabolism cell, but the excess of cholesterol content in serum could be problematic. Preclinical and clinical studies have shown that high cholesterol diet is regarded as a main factor in the development of hypercholesterolemia, atherosclerosis and ischemic heart disease [14]. The cholesterol content in hypercholesterolemia cases produced extremely high risk agents such as oxygen free radicals in serum as well as erythrocytes, platelets and endothelial cells. The elevation of total serum cholesterol and LDL cholesterol along with generation of reactive oxygen species (ROS)

In term of mechanism, these vascular problems of atherosclerosis, hypercholesterolemia and hypertension are all correlated to each other. The formation of plaque is associated with a period of time and relies on homeostasis of each individual in dealing with good cholesterol

Today, strategies and remedies are available to combat CVDs. Even though changing life style with appropriate dietary intake remains the first line of defence advocated by the healthcare workers, drug treatment is still widely used because of the rapid effect especially in treating severe cases [7]. Hence, most research today focuses on screening and identifying compounds

To date, several cholesterol lowering medications were discovered and used singly or in combination to lower the LDL cholesterol and triglycerides, a type of fats in the blood that also increases the risk of atherosclerosis. Concomitantly increase of HDL cholesterol often offers protection from CVDs [21]. These types of drug include, bile acid binding resins, cholesterol absorption inhibitor, combination cholesterol absorption inhibitor and statins such as ezeti‐ mibe-simvastatin, fibrates, niacin and omega-3 fatty acids. However, almost all the antihypercholesteromia drugs have been reported as having various adverse effects [46]. Several side effects such as constipation, nausea, diarrhea, stomach pain, cramps, muscle soreness, pain and weakness are reported; while more severe side-effects such as facial and neck flushing, nausea, vomiting, diarrhea, gout, high blood sugar etc. are also known. The side effects of statin and niacin are similar to each other. In addition, adverse drug reactions are always encountered in multiple diseases treated with a number of drugs. If hypercholestero‐ lemia is accompanied by other diseases, these diseases may have an impact on the response of the body to anti-hypercholesterolemia drugs and the metabolic processes of the body may be affected negatively. Later on, increased dosages may be required, which in turn would only worsen the cholesterol medication drugs. The search for cholesterol lowering medication has now turned to complementary traditional medicine. However, the traditional use of herbs in lowering cholesterol is often not verified scientifically. On the other hand, even if proven

effective, such herbs should be investigated on their mechanisms of action.

Medicinal mushrooms have been scientifically proven to be safe, efficacious, and novel antihypercholesterolemia therapeutic agents of natural source. An abundance of scientific research

*1.1.2. Mushrooms as medicinal-functional food against hypercholesterolemia*

play a key role in the development of coronary artery disease and atherosclerosis.

(HDL: high-density lipoprotein) and bad cholesterol (LDL).

exhibiting anti-hypercholesterolemic properties.

134 Hypercholesterolemia

The original term "functional food" has been defined by Martirosyan (1992) as "a natural or processed food that contains known biologically-active compounds which when in defined quantitative and qualitative amounts provides a clinically proven and documented health benefit, and thus, an important source in the prevention, management and treatment of chronic diseases of the modern age".

Medicinal Mushrooms (macrofungi), mostly members of the class Basidiomycetes fulfil the requirement of functional foods. Recently, they have become increasingly attractive as functional foods for their potential beneficial effects on human health. Hence, the food industry is especially interested in cultivating these mushrooms. The wild edible mushrooms have gained their reputation as health food due to their geographical origin in natural unpolluted environment. Nonetheless the cultivation of medicinal mushrooms using modern technology and the quality of the extracted product are crucial factors determining them as functionalmedicinal food.

A plethora of potent therapeutic components in medicinal mushrooms such as fibers, phytos‐ terols, saponins, polyphenols, flavanoids, terpenes and polysaccharides confer antihypercho‐ lesterolemic, antioxidant and anti-atherosclerotic properties [80].

The intensive study of [33] reported the hypocholesterolemic effect of the mushroom fruiting bodies and several types of these extracts exhibited different mechanisms of action, such as impairing dietary cholesterol absorption or inhibiting the endogenous cholesterol metabolism. Other reports showed that medicinal mushrooms are rich in chitin (dietary fibre) and specific β-glucans which may inhibit cholesterol absorption by increasing the faecal excretion of bile acids and reducing the amount of serum LDL-cholesterol [19, 28].

Among the most studied mushroom species are *Tricholoma giganteum* (giant mushroom), *Marasmius androsaceus* (horsehair parachute mushroom), *Grifola frondosa* (maitake mushroom), *Pleurotus* species (oyster mushroom), *Lentinula edodes* (shiitake mushroom), *Ganoderma lucidum* (reishi or lingzhi mushroom), *Sparassis crispa* (cauliflower mushroom), *Pholiota adiposa* (black tiger's paw mushroom), *Sarcodon aspratus* (yellow cap mushroom), *Hypsizygus marmoreus* (shimeji/buna shimeji mushroom), *Flammulina velutipes* (enoki mushroom), *Hericium erinaceus* (lion's mane mushroom), and *Agaricus bisporus* (button mushroom) [65].

The objectives of this article are to review the possible anti-hypercholesterolemic mechanisms of some putative bioactive compounds extracted from well known medical mushrooms particularly *G. lucidum*.

#### **2. Selective mushrooms as anti-hypercholesterolemia agent**

*Ganoderma* species are Basidiomycetes belonging to Polyporaceae (or Ganodermataceae) of Aphyllophorales. They differ from the ordinary mushrooms and are categorized in the order Agaricales in that they have pores rather than gills on the surface of the fruiting bodies. *G. lucidum* has been reported to have multi-beneficial values and concerted medicinal effects in the treatment of various diseases. The recent application of modern analytical techniques has, in a number of cases, provided a scientific basic for these earlier empirical observation. Thus, many putatively effective compounds are isolated and identified from *G. lucidum*. The pharmacological activities of *G. lucidum* have been attributed mainly to its polysaccharides and triterpenes [24].

#### **2.1.** *Ganoderma* **polysaccharides**

As fungal wall constituents, bioactive polyglycans (polysaccharides), such as β-glucans in *G. lucidum*, are found in all parts of the mushroom, including the mycelium (Bartnicki-Garcia, 1968). Fungal polyglycans can also be secreted into the growth medium and become extrac‐ ellular (Buck *et al*., 1968). Bioactive polyglycans in *G. lucidum* comprise neutral polysaccharides (β-1,3, β-1,6 homo D-glucan), acidic glucan and polyglycan [60], protein-bond heteroglucan [58], arabinoxyoglucan, a highly branched heteroglucan [61], with a heteroglycan with β-(1,4) core [62], and peptidoglycan: ganoderan A, B, C [35] in the fruiting body [84], β-D glucan [72] and lucidan, a protein-bond heteroglycan [44], as well as other polyglycans in the mycelia which have not been characterized.

In fact, *Ganoderma* glucans, β-(1,3), β-(1,6)-D-glucan is blocked by basic unit of β-(1,3)-Dglucopyronan which consists of 1-15 units of β-(1,6) monoglucosyl side chains (Mizuno, 1991). Numerous reports show that β-(1,3), β-(1,6)-D-glucans with molecular weight of 10<sup>4</sup> -106 Daltons exhibit antitumor activity (Mizuno, 1991). It seems that the higher the molecular weight, the more effective anti-hypercholesterolemia activity. Anti-hypercholesterolemia activity is also linked to the frequency of polysaccharide branching which varies during different stages of mycelial growth. Different extraction and purification processes yield a variety of bioactive glycans. Identification of these large and highly complex bioactive *Ganoderma* polysaccharides, whose precise structures have not been elucidated, involved expensive process.

#### **2.2.** *Ganoderma* **Triterpenes**

Triterpenes are relatively simple molecules which are easy to isolate and quantify. They can be used as a measure of the quality of different *Ganoderma* samples [24] (Stavinoha, 1995). Twenty or so bioactive triterpenes have been isolated from *G. lucidum* although over one hundred with known chemical compositions and molecular configurations have been reported to occur in *G. lucidum*.

Triterpenes are produced in the fruiting body. They can also be induced in the mycelial mat on solid medium (Nishitoba *et al.*, 1987) or in the still liquid culture of late stationary phase [86]. Limited amount of triterpene is formed in the mycelial pellets of liquid shaking culture [76]. It is said that strains producing basidiocarps with a light yellow underside may contain a high amount of triterpenes in their caps. Such observation has been used to grade commercial *Ganodema* fruiting bodies in Asia [37]. [75] found that the highest concentration of *Ganoderma* triterpenes was in the spore scrapings obtained from the underside of the mushroom in the 1-2 mm tube region (the hymenial layer). Only 18-58 mg of bioactive triterpenes were obtained from 1000 g of *Ganoderma tsugae* basidiocarps, while 4.5% (w/v) of crude ethanol extract was obtained from the sample [77]; thus, Stavinoha *et al*. (1993) used spore scrapings from the mushroom underside instead of the whole mushroom for extracting bioactive triterpenes.

Agaricales in that they have pores rather than gills on the surface of the fruiting bodies. *G. lucidum* has been reported to have multi-beneficial values and concerted medicinal effects in the treatment of various diseases. The recent application of modern analytical techniques has, in a number of cases, provided a scientific basic for these earlier empirical observation. Thus, many putatively effective compounds are isolated and identified from *G. lucidum*. The pharmacological activities of *G. lucidum* have been attributed mainly to its polysaccharides

As fungal wall constituents, bioactive polyglycans (polysaccharides), such as β-glucans in *G. lucidum*, are found in all parts of the mushroom, including the mycelium (Bartnicki-Garcia, 1968). Fungal polyglycans can also be secreted into the growth medium and become extrac‐ ellular (Buck *et al*., 1968). Bioactive polyglycans in *G. lucidum* comprise neutral polysaccharides (β-1,3, β-1,6 homo D-glucan), acidic glucan and polyglycan [60], protein-bond heteroglucan [58], arabinoxyoglucan, a highly branched heteroglucan [61], with a heteroglycan with β-(1,4) core [62], and peptidoglycan: ganoderan A, B, C [35] in the fruiting body [84], β-D glucan [72] and lucidan, a protein-bond heteroglycan [44], as well as other polyglycans in the mycelia

In fact, *Ganoderma* glucans, β-(1,3), β-(1,6)-D-glucan is blocked by basic unit of β-(1,3)-Dglucopyronan which consists of 1-15 units of β-(1,6) monoglucosyl side chains (Mizuno, 1991). Numerous reports show that β-(1,3), β-(1,6)-D-glucans with molecular weight of 10<sup>4</sup>

Daltons exhibit antitumor activity (Mizuno, 1991). It seems that the higher the molecular weight, the more effective anti-hypercholesterolemia activity. Anti-hypercholesterolemia activity is also linked to the frequency of polysaccharide branching which varies during different stages of mycelial growth. Different extraction and purification processes yield a variety of bioactive glycans. Identification of these large and highly complex bioactive *Ganoderma* polysaccharides, whose precise structures have not been elucidated, involved

Triterpenes are relatively simple molecules which are easy to isolate and quantify. They can be used as a measure of the quality of different *Ganoderma* samples [24] (Stavinoha, 1995). Twenty or so bioactive triterpenes have been isolated from *G. lucidum* although over one hundred with known chemical compositions and molecular configurations have been reported

Triterpenes are produced in the fruiting body. They can also be induced in the mycelial mat on solid medium (Nishitoba *et al.*, 1987) or in the still liquid culture of late stationary phase [86]. Limited amount of triterpene is formed in the mycelial pellets of liquid shaking culture [76]. It is said that strains producing basidiocarps with a light yellow underside may contain a high amount of triterpenes in their caps. Such observation has been used to grade commercial *Ganodema* fruiting bodies in Asia [37]. [75] found that the highest concentration of *Ganoderma* triterpenes was in the spore scrapings obtained from the underside of the mushroom in the


and triterpenes [24].

136 Hypercholesterolemia

expensive process.

to occur in *G. lucidum*.

**2.2.** *Ganoderma* **Triterpenes**

**2.1.** *Ganoderma* **polysaccharides**

which have not been characterized.

The bitter taste of *G. lucidum* as a traditional Chinese medicine or tonic is attributed to its highly oxygenated polar triterpenes [54]. Triterpenes as secondary metabolites are more strain specific in *G. lucidum* [64]. High temperature and prolonged oxidation should be avoided during extraction to retain intact structures of these volatile compounds [37].

### **3. Mechanism on different biomedical application of** *Ganoderma lucidum*

There are many studies on *G. lucidum* worldwide due to its superior therapeutic value in tackling many types of diseases. However the mechanisms involved have yet to be clarified and fully understood. This review is undertaking to specify the anti-hypercholesterolemia mechanisms of different *G. lucidum* medicinal compounds.

Excretion of second metabolism products of the mushroom are used for self protection in extremely severe environment condition. The second metabolism products of *G. lucidum,* which included β-glucans and triterpenes have been known to possess a broad spectrum of health benefits from disease prevention and maintenance of health to the regulation or treatment of chronic as well as acute life threatening illness [16]. The anti-hypercholesterolemia therapeutics of *G. lucidum* extracts involved several types of mechanisms.

#### **3.1. Mechanism of inhibitory effect of ganoderic acid on HMG-CoA reductase**

Previous research by [85] focused on the oxygenated lanostanoid triterpenes isolated from *G. lucidum*. The pure isolated triterpenes taste bitter and some are cytotoxic. Their unique chemical structures have been studied in detail. The derivatives of these terpenes type compounds (Figure 1) were obtained through chemical conversion during inhibition of cholesterol biosynthesis.

These mushroom triterpenes inhibited histamine release from rat mast cells. Compound VI with 7-oxo and 15 α-hydroxy groups at 40 μM showed highly potential inhibition of cholesterol synthesis from [24,25-3H]-24,25-dihydrolanosterol (18 μM). This encouraging result was obtained by testing 24, 25-dihydrolanosterol on rat hepatic subcellular 10,000 xg supernatant fraction. The triterpene involved is ganoderic acid C methyl ester. Its derivative is synthesized by a complicated reaction included the yield of tri β-methoxyethoxymethyl ether (MEM) derivative under Wolff-Kishner condition to allow the 7-oxo-11-deoxo derivative further decarboxylation and deprotection of the hydroxyl group. The whole structure of compound VI has no functional group in the side chain and has both 7-oxo and 15 α-hydroxy groups on the same skeleton and showed potent inhibitory effect compared with other derivatives with carboxyl groups at the side chain.

Compound I and II showed the other derivatives with oxo group at C-23 and decarboxyl compounds at the side chain had moderate inhibitory effects. Derivatives of compond IV and V has carboxyl group at C-25 in the side chain showed almost no inhibitory effect. These results provided an excellent clue and fundamental of specific side of triterpenes on the discovery of other *G. lucidum* bioactive triterpenes in anti-hypercholesterolemic study. and V has carboxyl group at C-25 in the side chain showed almost no inhibitory effect. These results provided an excellent clue and fundamental of specific side of triterpenes on

the discovery of other *G. lucidum* bioactive triterpenes in anti-hypercholesterolemic study.

has both 7-oxo and 15 α-hydroxy groups on the same skeleton and showed potent

Compound I and II showed the other derivatives with oxo group at C-23 and decarboxyl

compounds at the side chain had moderate inhibitory effects. Derivatives of compond IV

inhibitory effect compared with other derivatives with carboxyl groups at the side chain.

compound I with ethereal diazomethane. Decarboxylated compounds IV and V were (Source: [84]

10 synthesized by the reaction of compound I and III with lead tetraacetate in the presence of **Figure 1.** Ganoderic acid B methyl ester (compound II) was obtained by treating compound I with ethereal diazome‐ thane. Decarboxylated compounds IV and V were synthesized by the reaction of compound I and III with lead tetraa‐ cetate in the presence of cupric acetate containing a drop of pyridine in refluxing benxene. In the case of V derived from III, the 7β-hydroxyl group was further oxidized to the carbonyl group.

#### *3.1.1. Mechanism*

Figure 2 showed the statins as HMG-CoA reductase inhibitor drug playing its key role in the pathway of blocking the biosynthesis mevalonate from the HMG-CoA. The mechanism involved the statin by interrupting the structure of HMG-CoA reductase binding to NADPH in HMG-CoA to produce mevalonate. Therefore the metabolic pathway that produces cholesterol and other isoprenoids has terminated.

cupric acetate containing a drop of pyridine in refluxing benxene. In the case of V derived

Figure 2 showed the statins as HMG-CoA reductase inhibitor drug playing its key role in

the pathway of blocking the biosynthesis mevalonate from the HMG-CoA. The

mechanism involved the statin by interrupting the structure of HMG-CoA reductase

binding to NADPH in HMG-CoA to produce mevalonate. Therefore the metabolic

from III, the 7β-hydroxyl group was further oxidized to the carbonyl group.

(Source: Yusuo komoda *et al*., 1989)

3.1.1 Mechanism

Figure 2 : Figure 2 showed the statins as HMG-CoA reductase inhibitor drug play its key (Source: [84]; (Akira Endo, 1971)

V has carboxyl group at C-25 in the side chain showed almost no inhibitory effect. These results provided an excellent clue and fundamental of specific side of triterpenes on the discovery of

These results provided an excellent clue and fundamental of specific side of triterpenes on

the discovery of other *G. lucidum* bioactive triterpenes in anti-hypercholesterolemic study.

Compound I Compound II

Compound IV

and V has carboxyl group at C-25 in the side chain showed almost no inhibitory effect.

has both 7-oxo and 15 α-hydroxy groups on the same skeleton and showed potent

Compound I and II showed the other derivatives with oxo group at C-23 and decarboxyl

compounds at the side chain had moderate inhibitory effects. Derivatives of compond IV

inhibitory effect compared with other derivatives with carboxyl groups at the side chain.

10

Figure 2 showed the statins as HMG-CoA reductase inhibitor drug playing its key role in the pathway of blocking the biosynthesis mevalonate from the HMG-CoA. The mechanism involved the statin by interrupting the structure of HMG-CoA reductase binding to NADPH in HMG-CoA to produce mevalonate. Therefore the metabolic pathway that produces

Figure I : Ganoderic acid B methyl ester (compound II ) was obtained by treating compound I with ethereal diazomethane. Decarboxylated compounds IV and V were synthesized by the reaction of compound I and III with lead tetraacetate in the presence of

**Figure 1.** Ganoderic acid B methyl ester (compound II) was obtained by treating compound I with ethereal diazome‐ thane. Decarboxylated compounds IV and V were synthesized by the reaction of compound I and III with lead tetraa‐ cetate in the presence of cupric acetate containing a drop of pyridine in refluxing benxene. In the case of V derived

Compound V Compound VI

other *G. lucidum* bioactive triterpenes in anti-hypercholesterolemic study.

Compound III

from III, the 7β-hydroxyl group was further oxidized to the carbonyl group.

cholesterol and other isoprenoids has terminated.

(Source: [84]

138 Hypercholesterolemia

*3.1.1. Mechanism*

role in the pathway of blocking the biosynthesis mevalonate from the HMG-CoA. Whereby ganoderic acid inhibit the cholesterol formation by competed with squalene oxido-cyclase at the last stage of cholesterol synthesis. **Figure 2.** Figure 2 showed the statins as HMG-CoA reductase inhibitor drug play its key role in the pathway of block‐ ing the biosynthesis mevalonate from the HMG-CoA. Whereby ganoderic acid inhibit the cholesterol formation by competed with squalene oxido-cyclase at the last stage of cholesterol synthesis.

11 (Source: Yusuo komoda *et al*., 1989; Akira Endo, 1971) Akira Endo and his group discovered statins in 1976, and these HMG-CoA reductase inhibitors showed competitive effect on inhibiting HMG reductase due to their very close molecular structure to HMG-CoA. The use of statins is able to reduce the blood cholesterol levels significantly as HMG reductase is the first committed enzyme in the sequence of cholesterol synthesis cumulative process. However, since mevalonic acid (MVA) is a common precursor for many isoprenoids, blocking of MVA formation may induce undesired side effects besides inhibiting sterol synthesis. More specific inhibition of cholesterol synthesis may be attained by inhibition at some later stage of cholesterol synthesis. In this case, lanosterol was chosen in the ganoderic acid test as it originally converts from squalene by squalene oxido-cyclase at the end of cholesterol synthesis (Figure 2).

#### *3.1.2. Comparison of structure of statins and ganoderic acid derivatives*

These renowned drugs have been studied for their functional anti-hypercholesterolemic mechanism as a model for preliminary comparison of undefined natural products based on the results of the laboratory and the spectroscopy elucidation of their structure. Therefore the highest percentage of similarity of both compound structures implied similar highest effec‐ tiveness. This theory has been validated when the structure of HMG-CoA and the binding site of competitor lovastatin drug in HMG-CoA reductase inhibition was compared (figure 3).

**Figure 3.** Comparison structure of HMG-CoA and the binding site of competitor lovstatin drug in HMG-CoA reduc‐ tase inhibition.

**Figure 4.** Comparative molecular structure of lavastatin as pharmaceutical drug and compound VI as triterpene deriv‐ ative of ganoderic acid.

The active side of lovastatin is circled in box, while the structure site of the ganoderic acid derivatives automatically refers to the site chain for comparison. The important difference is statin inhibited the earlier stage of cholesterol formation whereby ganoderic acid inhibited at the late stages of cholesterol formation. Figure (4a) showed that lavostatin has 5 carbons in aromatic ring when the reaction of carboxyl group and the hydroxyl group occurred. The active sites mostly rely on the double bond of oxide group and the beta hydroxyl group. The position of both active sites is separated and impossible to react intra-molecularly. Conversely the site chain of ganoderic acid derivatives (figure 4b) is aliphatic whereby the position of carboxyl group and double bond could possibly interact. Compound IV and V (in figure 1) has close double bond at C-23 and 24 and this contribute to instability. Structure of figure (VI) shows that it is a potent inhibitor but is surprisingly without any carboxyl and double bond at the site chain. Its competitor effect could be due to the interaction of C-7 and C-15 with the binding site of 24, 25-dihydrolanosterol. The binding site of ganoderic acids with 24, 25-dihydrolanos‐ terol is essentially similar to ganoderic acid and hence no side effect to the patient will result.

#### **3.2. Mechanism as angiotensin converting enzyme inhibitor in rennin angiotensionaldosterone system**

The other mechanism that could apparently be involved is the inhibition of angiotensin converting enzyme (ACE) in renin-angiotensin system. This system is indirectly related to hypercholesterolemia. Atherosclerosis is the main contributory factor in this case but it may result in high blood pressure due to the narrowing of lumen of blood arteries. Therefore the regulatory mechanism of blood pressure by vasoconstriction and vasodilation may attenuate the hypercholesterolemia impact.

[62] identified five novel lanostane triterpenes, namely ganoderal A; ganoderols A and B; ganoderic acids K and S in the methanolic extract. These compounds were tested for their ACE inhibitory effect by a modification of the method described by Friedland and Silverstein (1976) and expressed in terms of IC50 (the amount of samples needed to inhibit 50% of ACE activity). All the newly discovered lanostane triterpenes showed IC50 of the order of 10-5 M which is considered potent. However, the earlier reported ganoderic acid F (figure 5) achieved the highest inhibitory effect with IC50 of 4.7 x 10-6 M.

In 2012, Abdullah *et al.* reported that the hot water extract of *G. lucidum* exhibited the best ACE inhibitory effect compared to other culinary-medicinal mushrooms. Normally the hot water extract consists of polar compounds. It has been proposed that the multitudes of phenolic substances present in *G. lucidum* contributed to this inhibitory action [62]. In addition the anti-ACE activity of the hot water extract of *G. lucidum* was enhanced when the mushroom was grown on the germinated brown rice [35].

#### *3.2.1. Mechanism of Renin-Angiotensin System (RAS) of G. lucidum extract*

The RAS is important for the aldosterone hormone system in kidneys and lung. Consequently, the blood pressure and water (fluid) balance is regulated.

Briefly, when the blood volume is low in the circulation, this scenario would be sensed by the juxtaglomerular cells at the afferent arterioles of the renal glomeruli [11] in kidneys and concurrently activate the prorenin which converts to renin directly into circulation. Plasma rennin hydrolyzes its substrate, angiotensinogen released by the liver to produce a decapeptide known as angiotensin I, which is then rapidly converted to an octapeptide, angiotensin II, by a circulating angiotensin-converting enzyme found in the lungs. Angiotensin II is a potent vasoconstrictor peptide that stimulates the cells of the zona glomerulosa of adrenal cortex to produce aldosterone [10] and [41] which causes blood vessels to constrict, resulting in increased blood pressure. When the re-intake of sodium and water in the kidneys tubules is caused by Aldosterone, the body fluid eventually increases resulting in increase of blood pressure. [4].

#### *3.2.2. ACE inhibitor*

**Figure 3.** Comparison structure of HMG-CoA and the binding site of competitor lovstatin drug in HMG-CoA reduc‐

**Figure 4.** Comparative molecular structure of lavastatin as pharmaceutical drug and compound VI as triterpene deriv‐

The active side of lovastatin is circled in box, while the structure site of the ganoderic acid derivatives automatically refers to the site chain for comparison. The important difference is statin inhibited the earlier stage of cholesterol formation whereby ganoderic acid inhibited at the late stages of cholesterol formation. Figure (4a) showed that lavostatin has 5 carbons in aromatic ring when the reaction of carboxyl group and the hydroxyl group occurred. The active sites mostly rely on the double bond of oxide group and the beta hydroxyl group. The position of both active sites is separated and impossible to react intra-molecularly. Conversely the site chain of ganoderic acid derivatives (figure 4b) is aliphatic whereby the position of carboxyl group and double bond could possibly interact. Compound IV and V (in figure 1) has close double bond at C-23 and 24 and this contribute to instability. Structure of figure (VI) shows that it is a potent inhibitor but is surprisingly without any carboxyl and double bond at the site chain. Its competitor effect could be due to the interaction of C-7 and C-15 with the binding site of 24, 25-dihydrolanosterol. The binding site of ganoderic acids with 24, 25-dihydrolanos‐ terol is essentially similar to ganoderic acid and hence no side effect to the patient will result.

tase inhibition.

140 Hypercholesterolemia

ative of ganoderic acid.

Interrupting the RAS effectively control the constriction of blood vessels. When the arteries are confronted with the problem of narrowing lumen caused by the formation of plaque in hypercholesterolemia, the treatment of using ACE inhibitor or compound that blocks the activity of ACE could reduce some risk drastically via vasodilation. Most of the synthetic pharmaceutical drug for the treatment of hypertension has no curative effect, and in fact needs prolonged administration for congestive heart failure protection. These types of drugs are usually used in combination with other medication and are usually well-tolerated by most individuals. Nevertheless, side effects such as cough, headache, drowsiness, weak‐ ness, abnormal taste (metallic or salty taste), rash are very common. By testing the active ingredient(s) in *G. lucidum*, it is possible to identify ACE inhibitor(s) which may be devoid of side effect.

#### *3.2.3. Comparative structure of ACE inhibitor captopril and ganoderic acid F*

Captopril (figure 5) is a ACE inhibitor used for the treatment of hypertension and some types of congestive heart failure. Captopril plays its role in blocking the conversion of angiotensin I to angiotensin II. Both captopril and ganoderic acid F are highly active ACE inhibitor. In term of structure, captopril contains a side chain with 3 carbons with different functional groups and an aromatic ring formed with 4 carbons and a nitrogen atom as the main attachment to the side chain. The molecule is small with a molecular weight of 217 Daltons. In contrast, ganoderic acid F is a natural product. Its structure is huge with 7 carbons at the side chain attached to the main skeleton which contains 4 aromatic rings. Its molecular weight is 2.6 times more than captopril. Both compounds have the carboxyl, ketone and methyl functional groups except captopril which, in addition has a sulphate group at the tail. There is no sulphate and nitrogen atom in ganoderic acid F.

**Figure 5.** Comparative molecular structure of captopril as a commercial pharmaceutical drug and ganoderic acid F ex‐ tracted from *G. lucidum*.

Comparison of the molecular structure cannot really ascertain the effectiveness of both compounds in term of anti-hypertension or indirectly on anti-hypercholesterol activity. In addition, there are many other differences in term of their configurations and binding site of both compounds. Moreover, there could be many other factors influencing the mechanism of RAS system. As the comparison is based on the isolated and characterized *G. lucidum* lanostane triterpenes which have shown ACE-inhibitory activity, this wound provide some hints on the working structure. The effect of captopril on hypertension is rapid but comes with side effect. While ganoderic acid F is a natural product, experimental studies and clinical trials are still needed. The findings from this present review contrasted the conclusion by [8] that the compounds responsible for anti-hypertensive activity have molecular weights of more than 1,000,000 Daltons, based on their in vivo data in Spontaneously Hyoertensive rats.

#### **3.3. Mechanism of inhibition of oxidative damage**

activity of ACE could reduce some risk drastically via vasodilation. Most of the synthetic pharmaceutical drug for the treatment of hypertension has no curative effect, and in fact needs prolonged administration for congestive heart failure protection. These types of drugs are usually used in combination with other medication and are usually well-tolerated by most individuals. Nevertheless, side effects such as cough, headache, drowsiness, weak‐ ness, abnormal taste (metallic or salty taste), rash are very common. By testing the active ingredient(s) in *G. lucidum*, it is possible to identify ACE inhibitor(s) which may be devoid

Captopril (figure 5) is a ACE inhibitor used for the treatment of hypertension and some types of congestive heart failure. Captopril plays its role in blocking the conversion of angiotensin I to angiotensin II. Both captopril and ganoderic acid F are highly active ACE inhibitor. In term of structure, captopril contains a side chain with 3 carbons with different functional groups and an aromatic ring formed with 4 carbons and a nitrogen atom as the main attachment to the side chain. The molecule is small with a molecular weight of 217 Daltons. In contrast, ganoderic acid F is a natural product. Its structure is huge with 7 carbons at the side chain attached to the main skeleton which contains 4 aromatic rings. Its molecular weight is 2.6 times more than captopril. Both compounds have the carboxyl, ketone and methyl functional groups except captopril which, in addition has a sulphate group at the tail. There is no sulphate and

**Figure 5.** Comparative molecular structure of captopril as a commercial pharmaceutical drug and ganoderic acid F ex‐

Comparison of the molecular structure cannot really ascertain the effectiveness of both compounds in term of anti-hypertension or indirectly on anti-hypercholesterol activity. In addition, there are many other differences in term of their configurations and binding site of both compounds. Moreover, there could be many other factors influencing the mechanism of RAS system. As the comparison is based on the isolated and characterized *G. lucidum* lanostane triterpenes which have shown ACE-inhibitory activity, this wound provide some hints on the working structure. The effect of captopril on hypertension is rapid but comes with side effect. While ganoderic acid F is a natural product, experimental studies and clinical trials are still needed. The findings from this present review contrasted the conclusion by [8] that the compounds responsible for anti-hypertensive activity have molecular weights of more than

1,000,000 Daltons, based on their in vivo data in Spontaneously Hyoertensive rats.

*3.2.3. Comparative structure of ACE inhibitor captopril and ganoderic acid F*

of side effect.

142 Hypercholesterolemia

nitrogen atom in ganoderic acid F.

tracted from *G. lucidum*.

Extracts prepared from either mycelial or fruiting body of *G. lucidum* have been accorded a prominent role as a source of natural antioxidants [57]. The antioxidant activity of these extracts was found mainly correlated with their polysaccharide content as well as with their phenolic content [21]. Several scientific reports [26, 38, 45] have proven that the mechanism involved is direct inhibition of the process of oxidation at the cell membrane of the host. The reactive compounds react directly with the free radicals and neutralize the oxidation effect in reducing oxi LDL which forms the key precursor in cardiovascular diseases included hypercholestero‐ lemia. The physiological effects of these extracts was shown to depend on the strain and the nature of cultivation [65].

(1 → 6) or (1 → 3)-β-D-glucans from *G. lucidum* are reportedly potential drugs against oxidation. These substances seem to enhance the activity of the immune system, but there is no accepted mechanism of action nor agreement on the parameters which influence the activity (Werner *et al*., 1997). Therefore, glucans with different structures and/ or varying molar mass were characterized by spectroscopy, spectrometry coupled with size-exclusion chromatography in order to obtain the molar mass distribution and to gain an idea of the structure in solution. The activity of polysaccharides is determined by their conformation, composition and size [13]. Additionaly, polysaccharides may contribute to the oxidation properties, depending on their molecular structure, the sugar unit and conformation in whole. [78]

Many synthetic chemicals such as phenolic compounds are found to be strong radical scanvengers but they usually have side effects [33]. Most of the 2,2-diphenyl-1-picrylhydra‐ zyl (DPPH) scavenging activities of *G. lucidum* crude extract were compared with those of the well-known antioxidants such as vitamin C. At all concentrations tested, the *G. lucidum* polysaccharides exhibited a dose-dependent DPPH radical-scavenging activity. [19]

#### *3.3.1. The mechanism of scavenging DPPH radicals*

Crude hot water extract of *G. lucidum* showed its maximum scavenging activity (94.8%) at 2.5 mg/mL. However a declining tendency was also noted with higher concentrations (till 10 mg/ mL). This could be caused by limitation of solubility, and increased hydrogen bonding once the concentration of polysaccharide is increased. [43] reported that the concentration of available hydroxyl groups is responsible for the scavenging ability of polysaccharides. [56] supported the direct relationship between monosaccharide composition and conformation of side chains with scavenging ability of polysaccharides. In this case, the scavenging of DPPH radicals is conducted by the arabinose linked by 1, 4 linkages of the side-chain and glucose linked to the 1,6 glycosidic linkage respectively [57].

#### *3.3.2. Inhibition of Lipid Peroxidation (LPO)*

LPO is regarded as one of the basic mechanisms of cellular damage caused by free radicals [68]. The relationship between LPO and hypercholesterolemia is well recognized. A cholesterol rich diet results in increased LPO by the induction of free radical production [3]. Hypercholestero‐ lemia and lipid peroxidation are believed to be critically involved in development of atheroscle‐ rosis [9].

*G. lucidum* extract expressed the same pattern in prevention of linoleic acid peroxidation. In such experiment, the antioxidant activity of *G. ludicum* extract increased from 0.1 to 10.0 mg/mL and reached a plateau of 77.2−77.3% at 10.0−20.0 mg/mL. [56] measured the inhibition of lipid peroxidation by conjugated diene method. They concluded that strong relationship existed between monosaccharide ratio and antioxidant activity. Antioxidant activity increased with increasing concentrations of mannose and rhamnose,whereas the activity decreased with corresponding increases in concentration of arabinose and glucose.

#### *3.3.3. Reducing power*

*G. lucidum* polysaccharide extract exerted a high potential in hydrogen-donating ability in the ferric–reducing antioxidant power assay [57]. This extract showed promising reducing power gradually when the concentration was increased from 0.1 to 0.5 mg/mL and achieved a relative stable level of 3.2−3.4 at 5.0−20.0 mg/mL. The effectiveness of *G. lucidum* extract in reducing power test was obvious when compared to ascorbic acid tested as positive control which had a reducing power of 3.5 at 20.0 mg/mL.

[70] reported xanthan and methylcellulose showed hardly any hydrogen donating activity compared with the very huge activity of ascorbic acid. [56] also reported that weak relationship was found between monosaccharide composition and reducing ability. In fact the nonpolysaccharide components of the *Ganoderma* extract play the main role in reducing power. These components could react with free radicals and eventually stabilize and block chain reactions.

#### *3.3.4. Chelating ability on ferrous ions*

The molecular masses of the polysaccharide fractions are important for the chelating ability [43]. The ferrous ions chelating ability of polysaccharides extract of *G. lucidum* is 11.0−64.6% at 0.1−20 mg/mL and achieved a maximum of 68.9 % at 10 mg/mL. A mole number of poly‐ saccharide is required to chelate a mole number of ferrous (Fe2+) ions. The absolute chelating power is inversely related with the mean molecular mass, showing that higher molecular weight of polysaccharide exhibits higher chelating ability. However, this rule excludes amylopectine and starch which have no chelating effect despite their higher molecular weight. Therefore glycoside linkage of β-D-glucan exhibited higher ranking in chelating ability compared to α-D-glucan.

#### **3.4. Mechanism of inflammation — Hepatoprotective effect**

Total triterpenes extract from *G. lucidum* have been tested on two different experimental liver injury mice models induced by carbon tetrachloride and D-galactosamine [69]. In this test model, the extract showed inhibition of liver triglyceride and serum alanine aminotransferase levels significantly. Such result is encouraging when compared to a known reference sub‐ stance, malotilate for this form of protective effects. Both superoxide dismutases (SOD) activity and the glutathione content have been antagonized and decreased by *G. lucidum* extract. This corresponds to the reduction of malondialdehyde content in the carbon tetrachloride and Dgalactosamine liver-injured mice. (Andréia *et al.,* 2013).

These data indicated that peptides and ganoderic acids which have been isolated from *G. lucidum* have a powerful protective effect against liver damage induced by carbon tetrachloride and D-galactosamine. The increased activity of free radical scavenging enzymes could be related to the hepatoprotective effects and, thus, enhancing the effectiveness of anti-oxidation.

However the other experiments revealed administration of *G. lucidum* polysaccharides dosedependently significantly enhanced antioxidant enzymes activities in the serum of rats fed with polysaccharides compared to model group [73]. Another report reported that rats fed with ergosterol-rich and nicotinic acid-rich extract had significantly higher serum glutathione peroxidase and SOD activities (Andréia, 2013).

The liver is the main organ of detoxification and is the site of metabolic conversion of endog‐ enous and exogenous compounds. Another major function of the liver is to synthesize bile acids from cholesterol and to secrete these compounds from the hepatocytes into the intestine, thereby generating bile flow and facilitating dietary fat emulsification and absorption [82]. The studies on hepatoprotective effect of *G. lucidum* relied on many types of substrates. Since the liver is the main organ of metabolism, many biochemical pathway are related, thus more than one substrates are involved in the respective mechanism.

#### *3.4.1. Mechanism of hepatoprotective effect of Ganoderma extract*

*G. lucidum* extract expressed the same pattern in prevention of linoleic acid peroxidation. In such experiment, the antioxidant activity of *G. ludicum* extract increased from 0.1 to 10.0 mg/mL and reached a plateau of 77.2−77.3% at 10.0−20.0 mg/mL. [56] measured the inhibition of lipid peroxidation by conjugated diene method. They concluded that strong relationship existed between monosaccharide ratio and antioxidant activity. Antioxidant activity increased with increasing concentrations of mannose and rhamnose,whereas the activity decreased with

*G. lucidum* polysaccharide extract exerted a high potential in hydrogen-donating ability in the ferric–reducing antioxidant power assay [57]. This extract showed promising reducing power gradually when the concentration was increased from 0.1 to 0.5 mg/mL and achieved a relative stable level of 3.2−3.4 at 5.0−20.0 mg/mL. The effectiveness of *G. lucidum* extract in reducing power test was obvious when compared to ascorbic acid tested as positive control which had

[70] reported xanthan and methylcellulose showed hardly any hydrogen donating activity compared with the very huge activity of ascorbic acid. [56] also reported that weak relationship was found between monosaccharide composition and reducing ability. In fact the nonpolysaccharide components of the *Ganoderma* extract play the main role in reducing power. These components could react with free radicals and eventually stabilize and block chain

The molecular masses of the polysaccharide fractions are important for the chelating ability [43]. The ferrous ions chelating ability of polysaccharides extract of *G. lucidum* is 11.0−64.6% at 0.1−20 mg/mL and achieved a maximum of 68.9 % at 10 mg/mL. A mole number of poly‐ saccharide is required to chelate a mole number of ferrous (Fe2+) ions. The absolute chelating power is inversely related with the mean molecular mass, showing that higher molecular weight of polysaccharide exhibits higher chelating ability. However, this rule excludes amylopectine and starch which have no chelating effect despite their higher molecular weight. Therefore glycoside linkage of β-D-glucan exhibited higher ranking in chelating ability

Total triterpenes extract from *G. lucidum* have been tested on two different experimental liver injury mice models induced by carbon tetrachloride and D-galactosamine [69]. In this test model, the extract showed inhibition of liver triglyceride and serum alanine aminotransferase levels significantly. Such result is encouraging when compared to a known reference sub‐ stance, malotilate for this form of protective effects. Both superoxide dismutases (SOD) activity and the glutathione content have been antagonized and decreased by *G. lucidum* extract. This

corresponding increases in concentration of arabinose and glucose.

*3.3.3. Reducing power*

144 Hypercholesterolemia

reactions.

a reducing power of 3.5 at 20.0 mg/mL.

*3.3.4. Chelating ability on ferrous ions*

compared to α-D-glucan.

**3.4. Mechanism of inflammation — Hepatoprotective effect**

Whatever substrates are involved, the basic of this mechanism is promoting the release of SODs into the blood stream. Kurt and Stefan (2014) reported that plasma clearance of human extracellular-superoxide dismutase C (EC-SOD C) in rabbits was initiated in the liver which contained the most 125I-EC-SOD C, followed by kidney, spleen, heart, and lung. This scenario shows that almost all 125I-EC-SOD C in the organs was deposited on endothelial cell surfaces and was not associated with any other tissue cell surfaces, or present within the cells. [47].

Pathology studies on the hepatocytes that the SODs are a family of metalloenzymes which need mineral copper as integral component. About 50−80% copper absorption is maximal in the duodenum and may be absorbed from the stomach. Within the intestinal mucosal cells, copper can react with metallothionein, a sulfhydryl group-rich protein that binds copper through the formation of mercaptide bonds. Factors affecting copper absorption include gender, the chemical form and certain dietary constituents.

The rich and multi nutrient ingredient of ganoderma extract has provided sufficient natural supplement in enhancing the liver metabolism. Besides beta-glucan, coumarin, mannitol, and alkaloids, triterpenes isolated from *G. lucidum* included ganodenic acid which have a molecular structure similar to steroid hormones and ganoderol, ganoderenic acid, ganoderiol, ganoder‐ manontriol, lucidadiol, and ganodermadiol which is believed to stimulate the metabolism of liver to achieve the detoxifying effect. Combination of different substrates and components also provoke the synergic in term of efficacy. It is interesting that phytochemistry profile of *G. lucidum* includes 18 types of amino acids and more than 10 minerals which could be the vital ingredient of 50−80 % cupper and other minerals absorbed from duodenum and stomach in men.

There are three types of SODs in eukaryotic cells catalysing the same reaction. They are copper and zinc-containing SOD (CuZnSOD) exists in cytosol, an manganese-containing SOD (MnSOD) which is encoded in the nucleus, synthesized in the cytosol and imported posttranslationally into the mitochondrial matrix, and an extracellular CuZnSOD.

Basically 90% of the cell's oxygen is consumed by MnSOD at the mitochondria matrix. Thus mitochondria are sensitive to oxidative damage, especially inducible by environment oxida‐ tive stress. Most probability due to mitochondria are lack of histones and an efficient DNA repair [12].

The SODs are playing their role in the initial stage of cellular anti-oxidant defense by the dismutation of the superoxide radical into hydrogen peroxide and molecular oxygen. Super‐ oxide is the one-electron reduction product of molecular oxygen.

There are two possible equations of SOD-catalysed dismutation of superoxide where the oxidation state of the metal cation oscillates between n and n+1. The half-reactions could be written as :

$$\begin{aligned} \text{M}^{\text{(n+1)+}}\text{-SOD} + \text{O}\_2\text{-} &\rightarrow \text{Mn}^+\text{-SOD} + \text{O}\_2 \\\\ \text{M}^{\text{n+}}\text{-SOD} + \text{O}\_2\text{-} &+ 2\text{H}^+ \rightarrow \text{M}^{\text{(n+1)+}}\text{-SOD} + \text{H}\_2\text{O}\_2 \end{aligned}$$

The H2O2 will be disproportionating to H2O and O2 by catalase which is excreted from hepatocytes to complete the oxygen radical detoxification process [12]

The mechanisms of the hepatoprotective effects of *G. lucidum* have been largely undefined. However accumulating evidences suggest most of the identified substrates of *G. lucidum* extract are able to enhance the release of SOD or other anti-oxidant enzymes from the cytosolic and extracellular which have potential hepatoprotective effect. They play their role in modu‐ lation of hepatic phase I and II enzymes, modulation of nitric oxide production, and mainte‐ nance of hepatocellular calcium homeostasis [81].

#### **3.5. Mechanism on immunostimulation with** *Ganoderma* **polysaccharides**

Many bioactive components in *G. lucidum* are biological response modifiers which stimulate the host's own defense system [54] by evoking favorable immune responses. The cell surface is the *Ganoderma* bioactivity target site in terms of receptors and membrane alternation. In contrast to starch and a number of other naturally occurring polysaccharides, bioactive *Ganoderma* glycans are not degraded into their component sugars in animals or humans. Thus, such fungal polysaccharides are able to produce therapeutic effects. Starch, on the other hand, is decomposed enzymatically into its component sugar, glucose, and can be used as an energy source [37].

Of great interest is the discovery of β-D-glucan receptors on the surface of a number of white blood cells (leukocytes, monocytes, macrophages, natural killer (NK) cells, and other lympho‐ cytes) in animals and humans [17, 22]. The broad stimulatory effects of *Ganoderma* β-glucans, via transduced cell surface receptors in the immune system, lead to the release of cytokines and lymphokines (cell mediators), such as IL-1, IL-2, IL-4 (interleukins), interferon and TNF (tumor necrosis factor). Many immune parameters are improved, e.g., increase of T-cell functions and antibody production. Potential benefits and low toxicity make *Ganoderma* polysaccharides desirable for boosting the immune system of patients undergoing chemothe‐ raphy, radiation therapy or during recovery from major surgery [17]. Immunomodulatory effects of *Ganoderma* polysaccharides may also be useful in atherosclerosis, hypercholestero‐ lemia and heart failure prevention.

ingredient of 50−80 % cupper and other minerals absorbed from duodenum and stomach in

There are three types of SODs in eukaryotic cells catalysing the same reaction. They are copper and zinc-containing SOD (CuZnSOD) exists in cytosol, an manganese-containing SOD (MnSOD) which is encoded in the nucleus, synthesized in the cytosol and imported post-

Basically 90% of the cell's oxygen is consumed by MnSOD at the mitochondria matrix. Thus mitochondria are sensitive to oxidative damage, especially inducible by environment oxida‐ tive stress. Most probability due to mitochondria are lack of histones and an efficient DNA

The SODs are playing their role in the initial stage of cellular anti-oxidant defense by the dismutation of the superoxide radical into hydrogen peroxide and molecular oxygen. Super‐

There are two possible equations of SOD-catalysed dismutation of superoxide where the oxidation state of the metal cation oscillates between n and n+1. The half-reactions could be



The H2O2 will be disproportionating to H2O and O2 by catalase which is excreted from

The mechanisms of the hepatoprotective effects of *G. lucidum* have been largely undefined. However accumulating evidences suggest most of the identified substrates of *G. lucidum* extract are able to enhance the release of SOD or other anti-oxidant enzymes from the cytosolic and extracellular which have potential hepatoprotective effect. They play their role in modu‐ lation of hepatic phase I and II enzymes, modulation of nitric oxide production, and mainte‐

Many bioactive components in *G. lucidum* are biological response modifiers which stimulate the host's own defense system [54] by evoking favorable immune responses. The cell surface is the *Ganoderma* bioactivity target site in terms of receptors and membrane alternation. In contrast to starch and a number of other naturally occurring polysaccharides, bioactive *Ganoderma* glycans are not degraded into their component sugars in animals or humans. Thus, such fungal polysaccharides are able to produce therapeutic effects. Starch, on the other hand, is decomposed enzymatically into its component sugar, glucose, and can be used as an energy



translationally into the mitochondrial matrix, and an extracellular CuZnSOD.

oxide is the one-electron reduction product of molecular oxygen.

M(n+1)+

<sup>M</sup>n+-SOD + O2

nance of hepatocellular calcium homeostasis [81].

hepatocytes to complete the oxygen radical detoxification process [12]

**3.5. Mechanism on immunostimulation with** *Ganoderma* **polysaccharides**


men.

146 Hypercholesterolemia

repair [12].

written as :

source [37].

[64] reported activation of T-cell by administering *Ganoderma* polysaccharides orally. Stimu‐ lation of cytokines [15] and activation of cytotoxic NK cells [83] by *Ganoderma* polysaccharides were subsequently reported. The major polysaccharide fraction, β-1,3 glucan has been shown to exhibit a wide-based adjuvant stimulatory activity on macrophages and T-cells, leading to IL-1 [39], IL-2 [15, 87], and TNF production [88] which play a role in antitumor immune surveillance [49]. *Ganoderma* has been reported to have some effects on immunofunctions which can stimulate the activity of acid phosphatase and β-glucuronidase of peritoneal macrophages in mice (Liu, 1993). It is also observed that there is a significant increase in plague forming cell and agglutination titer of anti-sheep erythrocytes (SRBC) antibody as well as the activity of γ interferon of mice [52]. The activity of crude *Ganoderma* extract on NK cells was of specific interest. Firstly, the effectiveness of a water soluble *Ganoderma* polysaccharide fraction derived from mycelium was shown to enhance splenic NK activity in normal mice when administered via intraperitoned, intravenous or oral route. The fraction also restored depressed NK cytotoxicity in tumor-bearing mice [83]. Secondly, the capacity of *Ganoderma* polysaccharide in activating macrophages [89] and lastly the polysaccharides markedly enhanced the cytotoxicity of T-lymphocytes [52]. The last two activities are established tumoricidal affector pathways of the host immune system.

#### **3.6. Mechanism —** *Ganoderma lanostane***-type triterpenes as potent Farnesoid-X-Receptor (FXR) agonists**

[79] reported the application of *in silico* tools for the identification of natural products, namely *Ganoderma lanostane*-type triterpenes as potent FXR agonists. Intriguingly, three lanostanes secondary metabolites from *G. lucidum*, that is, ergosterol peroxide, ganodermanontriol, and ganoderiol F, dose-dependently induced FXR in the low micromolar range in a reporter gene assay.

The relevance of FXR as bile acid (BA) activated receptor was illustrated with regard to the treatment of atherosclerosis and its counter-regulatory role in immunity and inflammation. FXR exhibits a regulating function in many endogenous pathways. Its active site contains specific features which are well-characterized. These important characteristics have contrib‐ uted to the attractiveness of this nuclear receptor as an atypical drugable target for the development of novel therapaeutic agents which may be effective in the prevention and **15** 37 enhance splenic NK activityin

normal

secondary

bile acids are

**17** Figure 6.

**16** 14- 15

**15** 24 Nakashima et al (1979) reported Nakashima *et al.* (1979) reported

treatment of, including, the metabolic syndrome, dyslipidemia and atherosclerosis. Cheno‐ deoxycholic acid (CDCA) and other BA are natural ligands for FXR. **16** 8 as Bile Acid (BA) activated as bile acid (BA) activated

enhance splenic NK activity in

Intriguingly, three lanostanes

A dose-dependent FXR-inducing activity showed the EC50 of the most active lanostanes identified from *G. lucidum* activated FXR at even lower concentrations than control, the ranking from the most active is ergosterol peroxide (0.85l M), ganodermanontriol (2.5l M), ganoderiol F (5.0l M) and the control CDCA (16.8 lM). The tested *Ganoderma* compounds significantly decreased the levels of cholesterol 7α- hydroxylase (CYP7A1) mRNA. The degree of inhibition was similar to that induced by the positive control CDCA. **16** 13 therapaeutic agentswhich may be therapaeutic agents which may be chenodeoxycholic acid and other chenodeoxycholic acid (CDCA) and

#### *3.6.1. Comparative molecular structure of active lanostanes and CDCA* other BA are

normal

secondary

Four of the compounds have the basic molecular structure with 3 benzene rings and a penta ring, and the side chain is completely different (Figure 6). In term of number of carbon, ergosterol peroxide has the higher number of carbons at the side chain which is 4 carbons more than CDCA. The other two compounds contain same number of carbon. Ergosterol peroxide has 4 methyl groups at the side chain excluding hydroxyl group. The rest contain at least a hydroxyl group at the side chain with the possibility of intermolecular bond forming. Only ergosterol peroxide and ganoderiol F formed double bond at their side chain but at different position. This is important as double bond is favoured in binding and activating the compound. The position of double bond of ergosterol peroxide is ideal compared with ganoderiol F, because there is no other functional group beside the double bond. There is a carbonyl group at the side chain of CDCA compared with others. This interpretation is comparable with the possible mechanism of inhibition of HMG-CoA reductase by ganoderic acid derivatives, as the compound with carboxyl group at the side chain has lower inhibitory potential. In the component of aromatic ring, the position of carbon 7 determines the activity of the compound. In this case, ergosterol peroxide showed double bond within carbon 6 and 7. Ganodermanon‐ triol and ganoderiol F formed double bond at carbon 7 but within carbon 8 and both com‐ pounds have another double bond of which the configuration is not as stable as ergosterol peroxide and CDCA. The interesting point is, only ergosterol peroxide showed the binding of oxygen between carbon 5 and 8 which stablises the compound and equivalent the active side of double bond between carbon 6 and 7 in the ring. **16** 15 for FXR. (Ulrike Grienke et al., 2011) for FXR. **16** 22 levels of CYP7A1 mRNA levels of cholesterol 7αhydroxylase (CYP7A1) mRNA **16** 26 completely different. In term completely different (Figure 6). In term

**Figure 6.** Comparative molecular structure of chenodeoxycholic acid and three types of bioactive lanostanes extracted from *G. lucidum*.

5

#### *3.6.2. Mechanism*

treatment of, including, the metabolic syndrome, dyslipidemia and atherosclerosis. Cheno‐

enhance splenic NK activity in

Intriguingly, three lanostanes

A dose-dependent FXR-inducing activity showed the EC50 of the most active lanostanes identified from *G. lucidum* activated FXR at even lower concentrations than control, the ranking from the most active is ergosterol peroxide (0.85l M), ganodermanontriol (2.5l M), ganoderiol F (5.0l M) and the control CDCA (16.8 lM). The tested *Ganoderma* compounds significantly decreased the levels of cholesterol 7α- hydroxylase (CYP7A1) mRNA. The degree of inhibition

chenodeoxycholic acid (CDCA) and

Four of the compounds have the basic molecular structure with 3 benzene rings and a penta ring, and the side chain is completely different (Figure 6). In term of number of carbon, ergosterol peroxide has the higher number of carbons at the side chain which is 4 carbons more than CDCA. The other two compounds contain same number of carbon. Ergosterol peroxide has 4 methyl groups at the side chain excluding hydroxyl group. The rest contain at least a hydroxyl group at the side chain with the possibility of intermolecular bond forming. Only ergosterol peroxide and ganoderiol F formed double bond at their side chain but at different position. This is important as double bond is favoured in binding and activating the compound. The position of double bond of ergosterol peroxide is ideal compared with ganoderiol F, because there is no other functional group beside the double bond. There is a carbonyl group at the side chain of CDCA compared with others. This interpretation is comparable with the possible mechanism of inhibition of HMG-CoA reductase by ganoderic acid derivatives, as the compound with carboxyl group at the side chain has lower inhibitory potential. In the component of aromatic ring, the position of carbon 7 determines the activity of the compound. In this case, ergosterol peroxide showed double bond within carbon 6 and 7. Ganodermanon‐ triol and ganoderiol F formed double bond at carbon 7 but within carbon 8 and both com‐ pounds have another double bond of which the configuration is not as stable as ergosterol peroxide and CDCA. The interesting point is, only ergosterol peroxide showed the binding of oxygen between carbon 5 and 8 which stablises the compound and equivalent the active side

hydroxylase (CYP7A1) mRNA

**Figure 6.** Comparative molecular structure of chenodeoxycholic acid and three types of bioactive lanostanes extracted

5

deoxycholic acid (CDCA) and other BA are natural ligands for FXR.

secondary

normal

was similar to that induced by the positive control CDCA.

**15** 24 Nakashima et al (1979) reported Nakashima *et al.* (1979) reported

**16** 8 as Bile Acid (BA) activated as bile acid (BA) activated

148 Hypercholesterolemia

**16** 13 therapaeutic agentswhich may be therapaeutic agents which may be

**16** 22 levels of CYP7A1 mRNA levels of cholesterol 7α-

**16** 26 completely different. In term completely different (Figure 6). In

**15** 37 enhance splenic NK activityin

**16** 5 Intriguingly, five lanostanes

chenodeoxycholic acid and other

**16** 15 for FXR. (Ulrike Grienke et al., 2011) for FXR.

from *G. lucidum*.

normal

secondary

bile acids are

**17** Figure 6.

**16** 14- 15

of double bond between carbon 6 and 7 in the ring.

chenodeoxycholic acid chenodeoxycholic acid (CDCA)

term

*3.6.1. Comparative molecular structure of active lanostanes and CDCA*

other BA are

Farnesoid X receptor (FXR; NR1H4) plays a role in the pathogenesis of cardiovascular disease [28]. It is a ligand-induced transcriptional activator and is expressed at high level in liver, intestine, kidney, adrenal glands, and also in the vasculature. It targets the enterohepatic recycling and detoxification of BA. When activated, FXR translocates to the cell nucleus, forms a heterodimer with retinoid-X-receptor and binds to hormone response elements on DNA, which produces either repression or an up regulation of gene transcription. The resulting mechanisms are affected by antagonist or agonist character of the respective ligand [79].

On the other hand, both (*CYP7A1*) and sterol response element binding protein 1c (*SREBP1c*) have shown their expression by elevation of oxysterol-induced liver X receptor-α (LXR-α) activation. This may result in increased triglyceride and BA biosynthesis, whereby the FXRα-mediated induction of small heterodimer partner (SHP), will interrupt the activity of (LXRα) to induce *CYP7A1* and *SREBP1c*, and hence inhibits BA and lipogenesis synthesis. Supernumerary, FXR-α-mediated induction of intestinal mouse fibroblast growth factor 15 is a surrogate SHP-independent signal from the gut to the liver to inhibit BA biosynthesis [18].

#### **4. Conclusion**

These mechanisms are possibly valid due to the promising results by comparing the treated and the control group. The activity of isolated compounds mentioned herein is very convincing and the working mechanisms mentioned above are involved. However, it is likely more than one mechanism may interplay. The method of extracting the bioactive compounds or the fraction used is closely related to the anti-hypercholesterolemia effect. There are several factors that may influence the possible mechanisms. The effective dosage of *G. lucidum* extract have yet to be standardized in animal and human clinical trial. Although many reports showed the favourable effects of *G. lucidum* extract to patients in China, such reports are anecdotal. In the absence of case-control studies, these reports could not be validated. The duration of admin‐ istration of *G. lucidum* for its anti-hypercholesterolemia activity is also arguable. The mecha‐ nism responsible for the inhibition of cholesterol synthesis is also unclear, as the test subjects were provided with high cholesterol diet during the course of trial which did not reflect the real situation. Hence, to date, the effectiveness of *G. lucidum* on the hypercholesterolemia patients is still undefined. There is a lack of evidence to prove that *G. lucidum* successful dislodge the cholesterol plaque from the affected blood vessels. Whether other mechanisms are also playing a part in cholesterol lowering activity is still unclear. Another compounding factor is the different absorption rate of each test subject. In addition, the effectiveness data of *G. lucidum* acquired from testing in rats could not be applied to human, as the dosage is based on body weight. Moreover, the strain of *Ganoderma* used is also an important factor in determining the rate and type of mechanism involved, since different strain produces different contents in the extract. This becomes more prominent when the mushroom is harvested from different geographical regions.

Synthetic anti-hypercholesterolemia drug works by affecting an array of intermediate precur‐ sors which could be important for health. Therefore the side effect of such drug would be more severe than someone taking a mixture of active components present in *G. lucidum* since *G. lucidum* extract most probably exerts its inhibitory effects at the late stage of the pathway.

#### **Author details**

Choong Yew Keong

Address all correspondence to: yewkeong11@yahoo.co.uk

Phytochemistry Unit, Herbal Medicine Research Centre, Institute for Medical Research, 50588 Jalan Pahang, Kuala Lumpur, Malaysia

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**Hypercholesterolemia — Statin Therapy — Indications, Side Effects, Common Mistakes in Handling, Last Evidence and Recommendations in Current Clinical Practice**

Lucía Cid-Conde and José López-Castro

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59622

#### **1. Introduction**

#### **1.1. Pharmacology of statin**

It is known as statins a group of drugs used to lower cholesterol in patients suffering from hypercholesterolemia and who have therefore increased risk of developing atherosclerosis and have episodes cardiovascular disease. From the pharmacological point of view, fall within the HMG-CoA reductase. Enzyme inhibition is precisely this which justifies the reduction of certain fractions of cholesterol in the body and explains its importance: their positive inter‐ vention on cardiovascular risk factors, leading to numerous cardiovascular diseases, which are the leading cause of death in the developed world [1,2]. Despite its short history (less than forty years) are many studies that have been done on statins and hundreds of thousands of patients who have taken these drugs. This has given rise to an extensive knowledge of the characteristics of these drugs has led to the synthesis of new substances that improve the properties of the above statins; in this line part of pharmaceutical research is still moving. However, it has also given rise to broadly meet the real toxicological profile for each substance. The phase IV studies have revealed the risks of using these substances for long periods or in certain basal conditions, which has led, among other things, the withdrawal of any member of the family due to their increased incidence of severe adverse reactions. Because of the variability in origin, the pharmacokinetics of statins differ greatly, however, its pharmacody‐ namic similarities allowed their joint study group them because, in terms of mechanism of action and effects of statins, and especially, regarding the clinical consequences of its use, there

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

is an important congruence group, which has been widely studied (Table 1). All developed statins are used orally and absorbed by this route on a variable range from 30% of lovastatin to 35% of pravastatin, decreasing its absorption in the presence of food in the stomach. However, the changes in peak concentrations or the respective curves of assimilation have no impact on the final results in the modification of cholesterol levels, so it is generally advisable to take them at any time of day and in most cases with or without food. Also, there appears to be accumulation due to multiple doses, which is general consensus decision single dose. The recommendations do not drink grapefruit juice while being treated with statins is due to interference with the metabolism, not altered absorption. Generally, the bioavailability of the statins is low, ranging from 5% of lovastatin and 17% of pravastatin. Binding to plasma proteins is variable, but in general very high lines. Except 50% of pravastatin, all have a 95% binding to proteins. The tissue distribution is broad, crossing the blood-brain and placental barriers, even going to milk in lactating women. Liver specificity of these drugs is determined by its degree of lipophilicity and by the presence of some organic anion transporter proteins that allow more hydrophilic statins such as pravastatin and rosuvastatin, entering the hepatocyte [3]. Moreover, some statins may inhibit P-glycoprotein (multidrug resistance protein), a carrier protein of many drugs in the cell, which could predispose to drug interactions. [4]


**Table 1.** Pharmacological characteristics of statin

The metabolism of statins is liver, undergoing first pass metabolism. In most, there are differences in the metabolism regarding sex and age, but not enough to change the doses in the absence of other pathologies. It seems clear that are substrates of CYP450: lovastatin, simvastatin and atorvastatin are metabolized exclusively by CYP3A4, and fluvastatin does exclusively by 2C9. For rosuvastatin, only 10% use the CYP2C9 and 2C19. Pitavastatin has a low affinity for CYP2C9, so not a major metabolic pathway. Pravastatin is not metabolised by the cytochrome, but does so by enzymes present in the cytoplasm of hepatocytes. The metab‐ olites may be hydroxylated derivatives, omega or beta-oxidized methylated glucuronide. The pharmacological activity of the same is very variable. Thus, the range is wide, from lovastatin, simvastatin, which are really a pharmacologically inactive lactones and performing their pharmacological activity through its metabolites, to fluvastatin, which has virtually inactive metabolites. For the most part, excretion in feces is due to its poor absorption. According to each type of statins, renal excretion ranges from 2% to 20%.

Statins are inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. This enzyme catalyzes the conversion of HMG-CoA to mevalonate, which is a key metabolite in the biosynthesis of cholesterol. Blocking occurs due to the high structural resemblance with these drugs exhibit HMG-CoA. The affinity of the enzyme by statins is 1.000 to 10.000 times that of the natural substrate (Figure 1). Statin Therapy 3 biosynthesis of cholesterol. Blocking occurs due to the high structural resemblance with these drugs exhibit HMG-CoA. The affinity of the enzyme by statins is 1.000 to 10.000 times that of the natural

The blockade of hepatic cholesterol synthesis causes an activation of the regulatory proteins SREBP (sterol regulatory elements-binding proteins), which activate transcription of protein and thus result in higher expression of the LDL receptor gene and increased the number of functional receptors on hepatocytes [5]. Moreover, it has been shown that statins also produced inhibition associated antigen-1 with the function of lymphocytes (LFA-1: lymphocyte function-associated antigen-1) [6]. The LFA-1 is a glycoprotein integrin family expressed on the surface of leukocytes. When the LFA-

Figure 1. Biosynthesis of cholesterol **Figure 1.** Biosynthesis of cholesterol

substrate (Figure 1).

is an important congruence group, which has been widely studied (Table 1). All developed statins are used orally and absorbed by this route on a variable range from 30% of lovastatin to 35% of pravastatin, decreasing its absorption in the presence of food in the stomach. However, the changes in peak concentrations or the respective curves of assimilation have no impact on the final results in the modification of cholesterol levels, so it is generally advisable to take them at any time of day and in most cases with or without food. Also, there appears to be accumulation due to multiple doses, which is general consensus decision single dose. The recommendations do not drink grapefruit juice while being treated with statins is due to interference with the metabolism, not altered absorption. Generally, the bioavailability of the statins is low, ranging from 5% of lovastatin and 17% of pravastatin. Binding to plasma proteins is variable, but in general very high lines. Except 50% of pravastatin, all have a 95% binding to proteins. The tissue distribution is broad, crossing the blood-brain and placental barriers, even going to milk in lactating women. Liver specificity of these drugs is determined by its degree of lipophilicity and by the presence of some organic anion transporter proteins that allow more hydrophilic statins such as pravastatin and rosuvastatin, entering the hepatocyte [3]. Moreover, some statins may inhibit P-glycoprotein (multidrug resistance protein), a carrier

protein of many drugs in the cell, which could predispose to drug interactions. [4]

**Prodrugs** YES NO YES NO NO NO NO **Food and absorption**No influence ↓ ↑ ↓ ↓ No influence **Bioavailability** ≤5% 17% ≤5% 24% 14% 20% ≥30%

**Metabolism** CYP3A4 sulphation CYP3A4 CYP2C9 CYP3A4 CYP2C9

**Biliary excretion** 60% 70% 83% 95% - 90% - **Urinary excretion** 13% 20% 10% 5% <2% 30% 3% **Elimination half-life**2-3h 0.8h 1-4h 2.5h 20h 20h -

The metabolism of statins is liver, undergoing first pass metabolism. In most, there are differences in the metabolism regarding sex and age, but not enough to change the doses in the absence of other pathologies. It seems clear that are substrates of CYP450: lovastatin, simvastatin and atorvastatin are metabolized exclusively by CYP3A4, and fluvastatin does exclusively by 2C9. For rosuvastatin, only 10% use the CYP2C9 and 2C19. Pitavastatin has a

**Binding**

**barrier**

**to plasma proteins**

160 Hypercholesterolemia

**Crosses blood-brain**

**Table 1.** Pharmacological characteristics of statin

**PHARMACOLOGICAL CHARACTERISTICS OF STATIN**

Sinvastatin Pravastatin Lovastatin Fluvastatin Atorvastatin Rosuvastatin Pitavastatin

94% 50% >95% 98% 98% 88% -

YES NO YES NO NO NO YES

CYP2C9 CYP2C8 The blockade of hepatic cholesterol synthesis causes an activation of the regulatory proteins SREBP (sterol regulatory elements-binding proteins), which activate transcription of protein and thus result in higher expression of the LDL receptor gene and increased the number of functional receptors on hepatocytes [5]. Moreover, it has been shown that statins also produced inhibition associated antigen-1 with the function of lymphocytes (LFA-1: lymphocyte functionassociated antigen-1) [6]. The LFA-1 is a glycoprotein integrin family expressed on the surface of leukocytes. When the LFA-1 is activated by certain receptors, binds to the intracellular molecule-1 adhesion (ICAM-1 or CD-54) and stimulates the extravasation of leukocytes and activation of T lymphocytes. This means that the LFA-1 is a proinflammatory agent and its inhibition is beneficial in conditions such as rheumatoid arthritis and rejection of homograft. It was shown that statins and particularly, lovastatin, bind to a site of LFA-1 domain, lovastatin currently designated site. This is the molecular mechanism by which lovastatin, simvastatin and other statin lesser extent inhibit the LFA-1 [7]. This would be one of the anti-inflammatory mechanisms and hence possessing antiatherogenic statins.

#### **2. Effects of statins**

The consequences of the inhibition of HMG-CoA may be grouped into two groups:


By inhibiting HMG-CoA reductase inhibitors, statins interfere with the formation of isopre‐ noids from mevalonate. [10] Isoprenoids are molecules such as farnesyl pyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP), derived from the metabolism of mevalonate, which serve as lipid-tags for the posttranslational modification of a variety of proteins, including the gamma subunit of G proteins and small GTP unidoras proteins. As a result, the prenylation of the G proteins (Rho, Rac, Ras and Rab Rac1) is reduced. Prenylation of these molecules is necessary for anchoring to the cell membrane and thus to exercise their mecha‐ nism of action related to migration, differentiation and cell proliferation. Generally, stimulate and inhibit proinflammatory pathways useful mechanisms for endothelial homeostasis. Through these potential effects on cellular proteins, statins may have a number of antiather‐ osclerotic and antithrombotic properties, such as inhibiting the growth of smooth muscle cells, cell adhesion, platelet activation and secretion of C-reactive protein among other. The meva‐ lonic acid, may also act directly by inhibiting the synthesis of nitric oxide (NO) in a process dependent transferase inhibiting genilgeranil. NO is an essential molecule for proper function and vasodilatation of endothelium. To this should be added the effects resulting from inhibi‐ tion of LFA-1, which in turn significantly impacting on endothelial function in blood vessels. These pleiotropic effects are constant source of research, since they can extend the usage profile of statins. Moreover, these drugs maintain and improve endothelial function to increase the bioavailability of NO, which is synthesized by the enzyme NO synthase (eNOS). NO is the principal regulator of the homeostasis of the arteries and endothelium-dependent vasodila‐ tion. The functions are, among others, inhibiting proinflammatory mechanisms and act as an antioxidant on lipoproteins [12].

Statins preserve and increase the bioavailability of NO in several ways:


The blockade of hepatic cholesterol synthesis causes an activation of the regulatory proteins SREBP (sterol regulatory elements-binding proteins), which activate transcription of protein and thus result in higher expression of the LDL receptor gene and increased the number of functional receptors on hepatocytes [5]. Moreover, it has been shown that statins also produced inhibition associated antigen-1 with the function of lymphocytes (LFA-1: lymphocyte functionassociated antigen-1) [6]. The LFA-1 is a glycoprotein integrin family expressed on the surface of leukocytes. When the LFA-1 is activated by certain receptors, binds to the intracellular molecule-1 adhesion (ICAM-1 or CD-54) and stimulates the extravasation of leukocytes and activation of T lymphocytes. This means that the LFA-1 is a proinflammatory agent and its inhibition is beneficial in conditions such as rheumatoid arthritis and rejection of homograft. It was shown that statins and particularly, lovastatin, bind to a site of LFA-1 domain, lovastatin currently designated site. This is the molecular mechanism by which lovastatin, simvastatin and other statin lesser extent inhibit the LFA-1 [7]. This would be one of the anti-inflammatory

mechanisms and hence possessing antiatherogenic statins.

observed in many studies intervention [9].

The consequences of the inhibition of HMG-CoA may be grouped into two groups:

**a. Derivatives of interaction on cholesterol metabolism**: Lower levels of total cholesterol and LDL, substances closely related to atherosclerosis and increased cardiovascular risk. Density decreases LDL particles, increasing the size of these, leading to decreased atherogenesis [8]. Apolipoprotein B also falls substantially during treatment with statins. In addition, some statins modestly increase cHDL and reduces plasma triglycerides. As a result of these changes, the ratio of total cholesterol to HDL cholesterol and the ratio of LDL and HDL cholesterol are reduced. We have considered the combination of fibrate and enhancer preventing cardiac of statins, especially having no competitive metabolism

**b. Pleiotropic effects:** In addition to its effects on the lipid profile, statins have other beneficial cardiovascular effects, especially on the arterial wall, known as pleiotropic effects which explain the additional benefit not attributable to the reduction in cLDL

By inhibiting HMG-CoA reductase inhibitors, statins interfere with the formation of isopre‐ noids from mevalonate. [10] Isoprenoids are molecules such as farnesyl pyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP), derived from the metabolism of mevalonate, which serve as lipid-tags for the posttranslational modification of a variety of proteins, including the gamma subunit of G proteins and small GTP unidoras proteins. As a result, the prenylation of the G proteins (Rho, Rac, Ras and Rab Rac1) is reduced. Prenylation of these molecules is necessary for anchoring to the cell membrane and thus to exercise their mecha‐ nism of action related to migration, differentiation and cell proliferation. Generally, stimulate and inhibit proinflammatory pathways useful mechanisms for endothelial homeostasis. Through these potential effects on cellular proteins, statins may have a number of antiather‐

**2. Effects of statins**

162 Hypercholesterolemia

pathways.


The hypolipidemic action itself inherently reduces oxidative stress. However, of statins have their own antioxidant mechanisms that inhibit the production of superoxide anion radical. Superoxide is synthesized by NADPH oxidase, an enzyme which can be activated by the action of membrane receptor of angiotensin II, type I (R-1) receptors. Statins block the R-AT1 and also inhibit the phosphorylation of the NADPH oxidase, inactivating it [13].

Statins also block RhoA, one of the mediators of smooth muscle proliferation. The smooth muscle proliferation is a central phenomenon in the pathogenesis of vascular lesions, including post-angioplasty restenosis, transplant atherosclerosis and occlusion of the coronary vein grafts [14].

Atherosclerosis is a strong inflammatory component characterized by the presence of mono‐ cytes, macrophages and T cells in the plate. This process is induced by proinflammatory cytokines, free radicals and NO deficiency. Statins increase the bioavailability of NO and inhibit several proinflammatory cytokines [1].

A marker of inflammation and predictor of coronary heart disease risk is C-reactive protein (PCR). It is considered that PCR is also proinflammatory as joining the cLDL of the atherom‐ atous, activates complement plate and induces the expression of inhibitor-1, plasminogen activator (PAI-1), reduces the expression of eNOS and increases the expression of adhesion molecules [15]. Therefore, it is valid to assume that the decrease in plasma CRP levels might be beneficial. Large studies with statins, as the AFCAPS/TexCAPS showed reduced blood PCR. For its anti-inflammatory action, statins increase the stability of the atheromatous plaque, and much of the reduction in coronary events attributable to the mechanism. Preclinical studies demonstrated that statins reduce the accumulation of macrophages in the atheromatous plaque and inhibit metalloproteinase production by activated macrophages. Metalloprotei‐ nases are capable of degrading proteins support and are therefore partly responsible for the accident plaque with thrombus formation [16].

Clinically the effects of statins lead to a reduction in cardiovascular risk, through the following mechanisms:


Then offer recommendations for clinical practice for the treatment of hypercholesterolemia in adults and reduce the risk of atherosclerotic cardiovascular disease, which includes coronary heart disease, cerebrovascular disease, peripheral artery disease and other atherosclerotic probable origin.

#### **3. Indications of statin in clinical practice**

Statins are indicated as an adjunct to diet to reduce elevated total cholesterol, LDL cholesterol, apolipoprotein B and triglycerides; and to increase HDL cholesterol in patients with:


Also present clear indication in cardiovascular prevention [17]:

Primary prevention of coronary events: in hypercholesterolemic patients without clinical evidence of coronary heart disease.


Secondary prevention of cardiovascular events: in patients with clinical evidence of cardio‐ vascular disease.


molecules [15]. Therefore, it is valid to assume that the decrease in plasma CRP levels might be beneficial. Large studies with statins, as the AFCAPS/TexCAPS showed reduced blood PCR. For its anti-inflammatory action, statins increase the stability of the atheromatous plaque, and much of the reduction in coronary events attributable to the mechanism. Preclinical studies demonstrated that statins reduce the accumulation of macrophages in the atheromatous plaque and inhibit metalloproteinase production by activated macrophages. Metalloprotei‐ nases are capable of degrading proteins support and are therefore partly responsible for the

Clinically the effects of statins lead to a reduction in cardiovascular risk, through the following

Then offer recommendations for clinical practice for the treatment of hypercholesterolemia in adults and reduce the risk of atherosclerotic cardiovascular disease, which includes coronary heart disease, cerebrovascular disease, peripheral artery disease and other atherosclerotic

Statins are indicated as an adjunct to diet to reduce elevated total cholesterol, LDL cholesterol,

Primary prevention of coronary events: in hypercholesterolemic patients without clinical

**3.** Reduce the risk of cardiovascular mortality with no increase in death from non-cardio‐

Secondary prevention of cardiovascular events: in patients with clinical evidence of cardio‐

apolipoprotein B and triglycerides; and to increase HDL cholesterol in patients with:

accident plaque with thrombus formation [16].

**1.** Directly decreasing cholesterol levels.

**3. Indications of statin in clinical practice**

**3.** Homozygous familial hypercholesterolemia.

**1.** Reduce the risk of myocardial infarction.

Also present clear indication in cardiovascular prevention [17]:

**2.** Reduce the risk of myocardial revascularization procedures.

**3.** Stabilizing atherosclerotic plaque. **4.** Preventing thrombus formation.

**1.** Primary hypercholesterolaemia.

evidence of coronary heart disease.

**2.** Mixed dyslipidemia.

vascular causes.

vascular disease.

**2.** Improving endothelial function and inflammatory response.

mechanisms:

164 Hypercholesterolemia

probable origin.


The latest evidence recommends an individualized approach (tailored treatment approach) identified four risk groups associated with therapeutic strategy (Figure 2). Groups that benefit from the use of statin therapy in moderate intensity: LDL reduction of 30-49% or high intensity, LDL reduction of >49% both demonstrate reduction cardiovascular risk (RCV) [17].


#### **3.1. Types of statin therapy**


The main therapeutic strategies are (Table 2),

**Table 2.** Low, moderate and high-intensity statin therapy categories in treating patients with varying risks.

Statin therapy of high, moderate and low intensity according to studies is classified:


\* FDA approved but not tested in randomized controlled trials.

#### **3.2. Specific risks**


#### **3.3. Treatment strategies**


166 Hypercholesterolemia Statin Therapy 9 Hypercholesterolemia — Statin Therapy — Indications, Side Effects, Common Mistakes in Handling, Last Evidence… http://dx.doi.org/10.5772/59622 167

> 4. *Risk throughout life:* Are yet tracking data on >15 years, safety, reduction of atherosclerotic cardiovascular disease when statins are used for periods >10 years and

**Figure 2.** Statin therapy for atherosclerotic cardiovascular disease prevention

Figure 2. Statin therapy for atherosclerotic cardiovascular disease prevention

**4.- SIDE EFFECTS OF STATIN AND CLINICAL MANAGEMENT** 

treatment in individuals <40 years.

#### **4.1- ADVERSE EFFECTS OF STATINS 4. Side effects of statin and clinical management**

#### effect is <10%, similar to that of patients taking placebo, and less than 1% are serious side effects **4.1. Adverse effects of statins**

**1.** *Statin therapy in high-intensity studies*: Atorvastatin 40-80 mg or rosuvastatin 20-40 mg.

pitavastatin 2 to 4 mg.\*

**3.2. Specific risks**

and ethnicity.

**3.3. Treatment strategies**

disease.

strategy is advocated today.

individuals <40 years.

**6.** Ankle-brachial index <0.9.

mg, fluvastatin 20-40 mg and \*Pitavastatin 1 mg.

**1.** Primary prevention with LDL ≥ 160 mg/dL.

degree relative or <65 years female.

**2.** Genetic testing hyperlipidemias.

\* FDA approved but not tested in randomized controlled trials.

**4.** C-reactive protein levels of high sensitivity (hs-CPR) >2 mg /L,

polypharmacy, which may be necessary to achieve a specific goal.

**2.** *Statin therapy in moderate intensity*: Atorvastatin 10-20 mg, rosuvastatin 5-10 mg, simvas‐ tatin 20-40 mg (higher doses are not recommended by the incidence of adverse effects), pravastatin 40-80 mg, lovastatin 40mg, fluvastatin XL 80 mg, \*Fluvastatin 40 mg bid and

**3.** *Statin therapy in low-intensity*: Simvastatin 10 mg, \* Pravastatin 10 mg-20 mg, lovastatin 20

**3.** Family history of premature cardiovascular disease with onset <55years in a male first-

**5.** Coronary artery calcium (CAC) score ≥300 Agatston units or ≥ 75th percentile for age, sex

**1.** *Treat for goals*: It is the strategy most used in the past 15 years, but with 3 problems: Current randomized clinical trials do not specify what the best goal. The magnitude of the further reduction of the atherosclerotic cardiovascular disease than is obtained with a lower cholesterol goal another is unknown. Does not account the possible adverse effects of

**2.** *"Lower is better":* This approach does not take into account the possible adverse effects of polypharmacy with an unknown magnitude reduction in atherosclerotic cardiovascular

**3.** *Dealing with the level of atherosclerotic cardiovascular disease* (currently preferred): Consider both benefits in reducing cardiovascular risk and adverse effects of statin therapy. Hence the 4 groups that benefit, as previously mentioned, with the exception of use in individuals undergoing hemodialysis or heart failure with NYHA functional class III-IV out. This

**4.** *Risk throughout life:* Are yet tracking data on >15 years, safety, reduction of atherosclerotic cardiovascular disease when statins are used for periods >10 years and treatment in

(Table 3). In general, statins are well tolerated and the dropout rate in clinical trials as a result of any adverse effect is <10%, similar to that of patients taking placebo, and less than 1% are serious side effects (Table 3).

In general, statins are well tolerated and the dropout rate in clinical trials as a result of any adverse


**Table 3.** Differences in adverse effects between statins

**1.** *Myotoxicity*: The most serious adverse effect associated with the muscular condition, which can range from myalgia (proximal muscle pain and/or weakness with a value of creatine kinase (CK) normal or slightly increased) to more severe forms, such as myopathy (pain and/or weakness over the presence of very high CK, usually >10 times normal) or rhabdomyolysis (serious muscle condition, with muscle weakness and pain, the presence of very high CK, myoglobinuria and renal failure) [18]. In general, the most common disorder is myalgia without elevated CK. Special mention should be cerivastatin with‐ drawal from the market, since it is the statin showed higher number of severe myopathy. The rate of fatal rhabdomyolysis associated with cerivastatin use at least 15 times higher than that produced by other statins, and was associated with the use of high doses of the drug (0.8 mg/day) or when coadministered with gemfibrozil [19].

The drugs and clinical conditions that increase the risk of myopathy are,

	- **•** Fibrates.

**DIFFERENCES IN ADVERSE EFFECTS BETWEEN STATINS**

6% yes Simvastatin/atorvastatin

1% yes Simvastatin/atorvastatin

**Diabetes** 9% - - -

drug (0.8 mg/day) or when coadministered with gemfibrozil [19].

**a.** Chronic diseases (renal failure and diabetes).

**b.** Multiple drugs.

**c.** Surgical interventions. **d.** High doses of statins.

The drugs and clinical conditions that increase the risk of myopathy are,

2% - - -

0.6% yes Fluvastatin/others Pitavastatin

**1.** *Myotoxicity*: The most serious adverse effect associated with the muscular condition, which can range from myalgia (proximal muscle pain and/or weakness with a value of creatine kinase (CK) normal or slightly increased) to more severe forms, such as myopathy (pain and/or weakness over the presence of very high CK, usually >10 times normal) or rhabdomyolysis (serious muscle condition, with muscle weakness and pain, the presence of very high CK, myoglobinuria and renal failure) [18]. In general, the most common disorder is myalgia without elevated CK. Special mention should be cerivastatin with‐ drawal from the market, since it is the statin showed higher number of severe myopathy. The rate of fatal rhabdomyolysis associated with cerivastatin use at least 15 times higher than that produced by other statins, and was associated with the use of high doses of the

**Differences (favor/ against)**

Pravastatin/atorvastatin Pravastatin/rosuvastatin

Simvastatin/fluvastatin Pravastatin/atorvastatin Pravastatin/fluvastatin Rosuvastatin/atorvastatin Rosuvastatin/fluvastatin

**Statistically significant overall difference against**


Atorvastatin Fluvastatin

**Dose-response relationship**

**Adverse effects Incidence**

**Abandonment adverse impact**

168 Hypercholesterolemia

**Significantly relevant myalgia**

**Relevant significantly increased transaminases**

**Relevant significantly increased CPK** **versus placebo**

**Table 3.** Differences in adverse effects between statins


#### **4.2. Absolute contraindications to statins**


#### **4.3. Relative contraindications to statins (but you can take a special medical supervision is required)**


#### **6.** Alcoholism.

#### **7.** Concomitant weak inhibitors of CYP3A4 (Table 4)


**Table 4.** Other drug that interact with statin

#### **4.4. Recommendations to prevent adverse effects with statin use**

Select the dose and type of statin, according to the type of patient cardiovascular risk and potential adverse effects.

Use moderate intensity therapy if the patient has renal or hepatic dysfunction including unexplained persistent transaminase elevations, history of muscular disorders or intolerance to statin use, ALT elevations >3 times the upper limit, concurrent use of medication known interactions with statin, age greater than 75 years, history of hemorrhagic stroke event, asian ancestry. This behavior can substantially reduce adverse events with statin use. The use of simvastatin is not recommended at doses of 80 mg/day because of the risk of toxicity. Liver function tests should be performed before starting statin therapy, with dose changes with the change of drug. During treatment should be monitored signs and symptoms of muscle toxicity.

Whenever a statin prescribing or replaced by another is important to analyze the other coadministered drugs due to the risk of clinically relevant interactions. In the absence of abnormal liver function and potential interactions with coadministered drugs, statins are all interchangeable. In the presence of inducing drugs or inhibitors of CYP3A4, fluvastatin, pravastatin and rosuvastatin are interchangeable. In the presence of inducers or inhibitors of CYP2C9 drugs, statins are all interchangeable, except fluvastatin. In the presence of drugs inducing activad or inhibitors of P-gp, fluvastatin and rosuvastatin are interchangeable. In the presence of inducers or inhibitors of OATP1B1 drugs, is not recommended therapeutic interchange of pravastatin and rosuvastatin. For all other statins, the exchange must be made with caution.

#### **4.5. Analytical monitoring**

**6.** Alcoholism.

170 Hypercholesterolemia

**7.** Concomitant weak inhibitors of CYP3A4 (Table 4)

**antacids** ↓ absorption of statins

**Ion exchange resins** ↓ absorption of statins **colchicine** ↑ toxicity of colchicine

**Table 4.** Other drug that interact with statin

potential adverse effects.

with caution.

**anticoagulants** ↑ the anticoagulant effectiveness

**glibenclamide** ↑ plasma levels of glibenclamide

**4.4. Recommendations to prevent adverse effects with statin use**

**oral contraceptives** ↑ up to 30% in blood hormone levels

**OTHER DRUG THAT INTERACT WITH STATIN**

Select the dose and type of statin, according to the type of patient cardiovascular risk and

Use moderate intensity therapy if the patient has renal or hepatic dysfunction including unexplained persistent transaminase elevations, history of muscular disorders or intolerance to statin use, ALT elevations >3 times the upper limit, concurrent use of medication known interactions with statin, age greater than 75 years, history of hemorrhagic stroke event, asian ancestry. This behavior can substantially reduce adverse events with statin use. The use of simvastatin is not recommended at doses of 80 mg/day because of the risk of toxicity. Liver function tests should be performed before starting statin therapy, with dose changes with the change of drug. During treatment should be monitored signs and symptoms of muscle toxicity.

Whenever a statin prescribing or replaced by another is important to analyze the other coadministered drugs due to the risk of clinically relevant interactions. In the absence of abnormal liver function and potential interactions with coadministered drugs, statins are all interchangeable. In the presence of inducing drugs or inhibitors of CYP3A4, fluvastatin, pravastatin and rosuvastatin are interchangeable. In the presence of inducers or inhibitors of CYP2C9 drugs, statins are all interchangeable, except fluvastatin. In the presence of drugs inducing activad or inhibitors of P-gp, fluvastatin and rosuvastatin are interchangeable. In the presence of inducers or inhibitors of OATP1B1 drugs, is not recommended therapeutic interchange of pravastatin and rosuvastatin. For all other statins, the exchange must be made

Monitoring of CK at baseline in patients with or without a history of myopathy, have no solid evidence. It could only be recommended if the patient has muscle symptoms, weakness or fatigue. The only test that is fully justified, prior to initiating statin is the measurement of ALT. The liver function should be measured if the patient is suspected of hepatotoxicity statin use. With the same level of evidence is regular monitoring of blood glucose, the onset of diabetes mellitus associated with treatment [21].

#### **4.6. Attitude pain, muscle stiffness, weakness or muscle fatigue statin taker**

Clarify if the symptoms actually developed or intensified therapy. If there is a causal relation‐ ship apparent, and muscle symptoms are intolerable, discontinue medication. If rhabdomyol‐ ysis is suspected, measure CK-creatinine and urinalysis. If muscular symptoms are mild or moderate statin should be discontinued to reassess symptoms and assess whether the patient has conditions that increase risk of muscle symptoms (hypothyroidism, renal or hepatic dysfunction, polymyalgia rheumatica, steroid myopathy, vitamin D or primary) myopathies. If symptoms are resolved and there are no contraindications, restart the same statin at a lower dose. If symptoms relapse: start another statin at a lower dose and increase slowly. If after 2 months, the symptoms do not improve or CK levels do not decrease, consider alternative etiologies. If muscle symptoms persist after stopping statin or other clinical condition corre‐ spond to restart therapy [21].

In summary, the adverse events associated with statin therapy are uncommon. Statins are not associated with cancer risk, but on the contrary there is a greater chance of diabetes. Simvas‐ tatin and pravastatin appear to be safer and better tolerated than other statins [22]. It has not been able to show that there are real differences between the effects generic statin drug and reference mark to the modification of the lipid profile, nor in adverse reactions, assessed according to the elevation of transaminases and CPK [23,24].

#### **5. Conclusions**

Atherosclerotic cardiovascular disease is one of the most important public health problems of our time, both in Europe and in the rest of the world. Consistency of clinical care, incorporating new evidence and synthesis of recommendations from current practice is common task in various committees for clinical practice worldwide (Europe-ESC, American-AHA / ACC, British-NICE, Australian, Canadian...). This has generated discrepancies with the publication of the latest guidelines of the 2013 AHA/ACC compared to its European namesake 2011 ESC/ EAS. The innovation of greatest impact of the latest guidelines, has been the abandonment of the therapeutic strategy based on the target values LDL. The individualized strategy (tailored treatment approach) is recommended identifying four risk groups associated with therapeutic strategy. Are advised to use statins as well as healthy habits and lifestyle changes for all patients. The CK should not be measured routinely in patients on statins and there is no reason to monitor LDL levels.

### **Author details**

Lucía Cid-Conde1\* and José López-Castro2

\*Address all correspondence to: lucidcon@hotmail.com

1 Department of Pharmacy, Hospital Comarcal Valdeorras. SERGAS. O Barco de Valdeorras-Ourense, Spain

2 Department of Internal Medicine, Hospital Comarcal Valdeorras. SERGAS. O Barco de Valdeorras-Ourense, Spain

Conflict of interest Not have any conflict of interest relating to the information in this article.

#### **References**


[11] Denicola A, Batthyany C, Lissi E, Freeman BA, Rubbo H, Radi R. Diffusion of nitric oxide into low density lipoprotein. J Biol Chem 2002; 277(2): 932-6.

**Author details**

172 Hypercholesterolemia

Ourense, Spain

**References**

27-35.

2002; 110(3):285-8.

Valdeorras-Ourense, Spain

Lucía Cid-Conde1\* and José López-Castro2

\*Address all correspondence to: lucidcon@hotmail.com

1 Department of Pharmacy, Hospital Comarcal Valdeorras. SERGAS. O Barco de Valdeorras-

2 Department of Internal Medicine, Hospital Comarcal Valdeorras. SERGAS. O Barco de

Conflict of interest Not have any conflict of interest relating to the information in this article.

[1] Vaughan CJ, Gotto AM Jr., Basson CT. The evolving role of statins in the manage‐

[2] Torzewski J, Bowyer DE,Waltenberger J, Fitzsimmons C. Processes in atherogenesis:

[3] De Angelis G. The influence of statin characteristics on their safety and tolerability.

[4] Alonso Karlezi, RA, Mata Pariente N, Mata López P. Control de las hiperlipemias en

[5] Nissen S, Tuzcu M, Schoenhagen P, Brown BG, Ganz P, Vogel RA, et al.. Effect of in‐ tensive compared with moderate lipid-lowering therapy on progression of coronary

[6] Frohlich ED. Target organ involvement in hypertension: a realistic primise of preven‐

[7] Kallen, J. et al. Structural basis for LFA-1 inhibition upon lovastatin binding to the

[8] Packard C, Caslake M, Shepherd J. The role of small, dense low density lipoprotein

[9] Davignon J.. The cardioprotective effects of statins. Curr Atheroscler Rep 2004; 6(1):

[10] Liao JK.. Isoprenoids as mediators of the biological effects of statins. J Clin Invest.

atherosclerosis: a randomized controlled trial. JAMA 2004; 291(9):1071-80.

ment of atherosclerosis. J Am Coll Cardiol 2000; 35(1):1–10.

complement activation. Atherosclerosis 1997; 132(2):131–38.

la práctica clínica. Rev Esp Cardiol 2006; 6(G): 24 – 35.

tion and reversal. Me Clin North Am 2004; 88(1): 209-21.

(LDL): a new look. Int J Cardiol 2000; 74(Suppl 1): S17-22.

CD11a I-domain. J Mol Biol 1999; 292(1): 1–9.

Int J Clin Pract 2004; 58(10):945-55.


#### **Chapter 9**

## **Pleiotropic Effects of Statins**

### Sigrid Mennickent

[22] Bellosta S, Paoletti R, Corsini A. Safety of statins: focus on clinical pharmacokinetics

[23] Agón Banzo PJ, Ejea Arquillué MV. Estudio comparativo entre estatinas genéricas y no genéricas, en relación a su uso terapéutico, efectos farmacológicos y reacciones

[24] Coscollar Santaliestra. Estatinización o el discreto encanto del colesterol LDL. AMF

and drug interactions. Circulation 2004; 109 (23 Suppl 1):III50-7.

adversas [Tesis Doctoral]. Universidad de Zaragoza 2013.

2014; 10(1):59-60.

174 Hypercholesterolemia

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59202

#### **1. Introduction**

Atherosclerosis is a multi-factorial condition involving dyslipidemia that can result in cardiovascular disease.

The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors or statins are potent inhibitors of cholesterol biosynthesis, and most of the benefits of statin therapy are owing to the lowering of serum cholesterol levels. Reduction of LDL-cholesterol leads to upregulation of the LDL receptor and increased LDL clearance. Statins are the principal drugs in the primary and secondary prevention of coronary heart disease. Recent evidence also shows that more intensive lowering of LDL-cholesterol by statins is associated with greater clinical benefits. The mechanisms attributed to lipid lowering with statin therapy include atheromatous plaque stabilization, modification of the atherosclerosis progression and improved endothelial functions.

Statins reduce cardiovascular events in not only hypercholesterolemic but also normocholes‐ terolemic patients with coronary heart disease (CHD) or cardiovascular risks.

Moreover, clinical trials and clinical benefits have shown that statins' effects involved other pharmacological activities and not only changes in lipid levels. "Pleiotropic" effects of statins involve improving endothelial function, decreasing vascular inflammation and oxidative stress, and inhibiting the thrombogenic response. Moreover, some works shows statins' beneficial extrahepatic effects on the immune system, CNS, and bone. Many of these pleio‐ tropic effects are mediated by inhibition of isoprenoids, which serve as lipid attachments for intracellular signaling molecules. In particular, inhibition of small GTP-binding proteins, Rho, Ras, and Rac, whose proper membrane localization and function are dependent on isopreny‐ lation, may play an important role in mediating the pleiotropic effects of statins. By inhibiting the conversion of HMG-CoA to L-mevalonic acid, statins prevent the synthesis of the impor‐ tant isoprenoids mentioned above, and also farnesyl pyrophosphate (FPP) and geranylgeranyl

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

pyrophosphate (GGPP), which are precursors of cholesterol biosynthesis. Isoprenylated proteins can modify diverse cellular functions, therefore, statins have additional effects cholesterol-independent. Indeed, recent studies suggest that statins might be involved in immunomodulation, neuroprotection, and cellular senescence.

Therefore, statins might exert cholesterol-independent or "pleiotropic" effects through direct inhibition of these small GTP-binding proteins.

#### **2. HMG-CoA reductase inhibition**

The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors or Statins (Figure 1) are potent inhibitors of cholesterol biosynthesis, and most of the benefits of statin therapy are owing to the lowering of serum cholesterol levels. Because 60-70% of serum cholesterol is derived from hepatic synthesis and HMG-CoA reductase is the crucial rate-limiting enzyme in the cholesterol biosynthetic pathway, inhibition of this enzyme by statins results in a dramatic reduction in circulating low-density lipoprotein (LDL)-cholesterol. Reduction of LDL-cholesterol leads to up-regulation of the LDL receptor and increased LDL clearance. Moreover, statins increases HDL levels and decreases triglyceride levels. Statins are the principal drugs in the primary and secondary prevention of coronary heart disease for more than 25 million people at risk of cardiovascular disease worldwide. The Scandinavian Sim‐ vastatin Survival Study (4S) was the first randomized controlled trial to show significant risk reduction in cardiovascular mortality in patients with coronary-artery disease. Many studies,, such as 4S, Cholesterol and Recurrent Events (CARE), Long-term Intervention with Pravasta‐ tin in Ischemia Disease (LIPID), West of Scotland Coronary Prevention Study (WOSCOPS), Air Force/Texas Coronary Atherosclerosis Prevention Study (AFCAPS/TexCAPS), and the Heart Protection Study (HPS), have demonstrated the beneficial effects of statins in the prevention of cardiovascular disease. These works have shown significant risk reduction in cardiovascular mortality in patients with coronary artery disease, due to reduction in choles‐ terol levels and, therefore, reduction in atherosclerotic lesion development. The mechanisms attributed to lipid lowering with statin therapy include atheromatous plaque stabilization, modification of the atherosclerosis progression, and improved endothelial functions. The lowering of serum cholesterol levels is therefore thought to be the primary mechanism underlying the therapeutic benefits of statin therapy in cardiovascular disease. As 60-70% of serum cholesterol is obtained from hepatic synthesis mediated by HMG-CoA reductase, inhibition of this enzyme by statins is a principal way of reducing circulating low-density lipoprotein (LDL) cholesterol [1, 5, 7, 11, 10, 13, 16, , 24, 27, 30, 36, 39].

Statins in use today are: lovastatine, simvastatine, pravastatine, fluvastatine, athorvasta‐ tine and rosuvastatine. Lovastatine, simvastatine, and pravastatine are natural com‐ pounds, obtained from the fungi *Aspergillus terreus*, and the other statins are synthetic compounds [1, 22, 30].

Another statin used in the past was cerivastatine, but it produced some fatal adverse effect of rhabdomyolysis. Therefore, this drug was discontinued in 2000 [4].

**Fig. 1**: Chemical structure of statins (Sweetman, 2006; Mc Evoy, 2012). **Figure 1.** Chemical structure of statins [22, 30].

pyrophosphate (GGPP), which are precursors of cholesterol biosynthesis. Isoprenylated proteins can modify diverse cellular functions, therefore, statins have additional effects cholesterol-independent. Indeed, recent studies suggest that statins might be involved in

Therefore, statins might exert cholesterol-independent or "pleiotropic" effects through direct

The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors or Statins (Figure 1) are potent inhibitors of cholesterol biosynthesis, and most of the benefits of statin therapy are owing to the lowering of serum cholesterol levels. Because 60-70% of serum cholesterol is derived from hepatic synthesis and HMG-CoA reductase is the crucial rate-limiting enzyme in the cholesterol biosynthetic pathway, inhibition of this enzyme by statins results in a dramatic reduction in circulating low-density lipoprotein (LDL)-cholesterol. Reduction of LDL-cholesterol leads to up-regulation of the LDL receptor and increased LDL clearance. Moreover, statins increases HDL levels and decreases triglyceride levels. Statins are the principal drugs in the primary and secondary prevention of coronary heart disease for more than 25 million people at risk of cardiovascular disease worldwide. The Scandinavian Sim‐ vastatin Survival Study (4S) was the first randomized controlled trial to show significant risk reduction in cardiovascular mortality in patients with coronary-artery disease. Many studies,, such as 4S, Cholesterol and Recurrent Events (CARE), Long-term Intervention with Pravasta‐ tin in Ischemia Disease (LIPID), West of Scotland Coronary Prevention Study (WOSCOPS), Air Force/Texas Coronary Atherosclerosis Prevention Study (AFCAPS/TexCAPS), and the Heart Protection Study (HPS), have demonstrated the beneficial effects of statins in the prevention of cardiovascular disease. These works have shown significant risk reduction in cardiovascular mortality in patients with coronary artery disease, due to reduction in choles‐ terol levels and, therefore, reduction in atherosclerotic lesion development. The mechanisms attributed to lipid lowering with statin therapy include atheromatous plaque stabilization, modification of the atherosclerosis progression, and improved endothelial functions. The lowering of serum cholesterol levels is therefore thought to be the primary mechanism underlying the therapeutic benefits of statin therapy in cardiovascular disease. As 60-70% of serum cholesterol is obtained from hepatic synthesis mediated by HMG-CoA reductase, inhibition of this enzyme by statins is a principal way of reducing circulating low-density

immunomodulation, neuroprotection, and cellular senescence.

lipoprotein (LDL) cholesterol [1, 5, 7, 11, 10, 13, 16, , 24, 27, 30, 36, 39].

rhabdomyolysis. Therefore, this drug was discontinued in 2000 [4].

compounds [1, 22, 30].

Statins in use today are: lovastatine, simvastatine, pravastatine, fluvastatine, athorvasta‐ tine and rosuvastatine. Lovastatine, simvastatine, and pravastatine are natural com‐ pounds, obtained from the fungi *Aspergillus terreus*, and the other statins are synthetic

Another statin used in the past was cerivastatine, but it produced some fatal adverse effect of

inhibition of these small GTP-binding proteins.

**2. HMG-CoA reductase inhibition**

176 Hypercholesterolemia

#### **3. Pleiotropic effects of statins**

**3 PLEIOTROPIC EFFECTS OF STATINS** 

However, the overall clinical benefits observed with statin therapy appear to be greater than what might be expected from changes in lipid profile However, the overall clinical benefits observed with statin therapy appear to be greater than what might be expected from changes in lipid profile alone, suggesting that the beneficial effects of statins may extend beyond their effects on serum cholesterol levels.

alone, suggesting that the beneficial effects of statins may extend beyond their effects on serum cholesterol levels. Statins reduce cardiovascular events in not only hypercholesterolemic but also normocholesterolemic patients. Because of the effects of lipid lowering on atherosclerosis, statins reduce morbidity and mortality in patients with ischemic heart failure. Statins also improve heart function and survival in Statins reduce cardiovascular events in not only hypercholesterolemic but also normocholes‐ terolemic patients. Because of the effects of lipid lowering on atherosclerosis, statins reduce morbidity and mortality in patients with ischemic heart failure. Statins also improve heart function and survival in patients with non-ischemic heart failure. Indeed, statins improve neurohormonal imbalance and idiopathic dilated cardiomyopathy. Thus, the improvements in heart function by statins might be owing to cholesterol-independent mechanisms [2, 5, 7, 11, 13, 19, 22, 24, 29, 30, 32].

patients with non-ischemic heart failure. Indeed, statins improve neurohormonal imbalance and idiopathic dilated cardiomyopathy. Thus, Moreover, clinical trials and clinical benefits had shown that statins' effects involved other pharmacological activities and not only changes in lipid levels. Cholesterol-independent or "pleiotropic" effects of statins involve improving or restoring endothelial function, decreasing oxidative stress and vascular inflammation, enhancing the stability of atherosclerotic plaques, inhibiting the thrombogenic response, and lowering oxidative stress. Moreover, some works shows statins beneficial extrahepatic effects on the immune system, CNS, and bone.

Statins might exert cholesterol-independent or pleiotropic effects by inhibiting the conversion of HMG-CoA to L-mevalonic acid and, in this manner, prevent the synthesis of important isoprenoids, such as farnesylpyrophosphate (FPP), geranylgeranyl pyrophosphate (GGPP) and ubiquinone, which are precursors of cholesterol biosynthesis and of lipid attachments for intracellular signaling molecules. In particular, inhibition of small GTP-binding proteins, Rho, Ras, and Rac, whose proper membrane localization and function are dependent on isopreny‐ lation, may play an important role in mediating the direct cellular effects of statins on the vascular wall. These isoprenylated proteins constitute approximately 2% of total cellular proteins, and isoprenylated proteins might control diverse cellular functions, as signal transduction, growth of vascular smooth muscle, apoptosis and in the regulation of the vascular activity of NAD(P) H oxidase.

Ras and Rho isoprenylation by statins, lead to increase of the amount to both compounds in the cytoplasm cells. As Rho is the more important target in the geranylgeranylation way, inhibition of Rho y de Rho-kinasa is an very important mechanism for the pleiotropic effects of statins at the vascular wall [8, 19, 25] (Figures 2 and 3).

Hypocholesterolemic effects of statins can be explained by hepatic HMG-CoA reductase inhibition, whereas the independent cholesterol effects can be found in all kinds of cells.

As isoprenylated proteins might control diverse cellular functions, we can explain that statins might have additional effects beyond lipid lowering [3, 4, 8, 17, 20, 23, 25, 37] (Li et al., 2002].

Indeed, recent studies suggest that statins might be involved in immunomodulation, neuro‐ protection, and cellular senescence [2, 5, 7, 11, 13, 18, 22, 24, 29, 30, 31, 32, 35].

Finally, statin therapy can be used for patients with autoimmune diseases, such as multiple sclerosis [26, 34, 35]. Furthermore, in a 6 month, randomized, double-blind placebo-controlled clinical trial, patients with rheumatoid arthritis who received atorvastatin showed a reduction in disease activity [21]. However, it is too early to predict whether these promising data can translate into clinical benefit by statins in patients with autoimmune disease.

The potential clinical implications of statin pleiotropy suggests that perhaps other biomarkers, in addition to lipid levels, should be used to gauge the full efficacy of statin therapy in patients with cardiovascular risks or that statin therapy may be effective in disease states, such as inflammatory conditions, ischemic stroke, or cancer, where elevated cholesterol levels have not been shown to be a strong epidemiological risk for these diseases [2, 5, 7, 8, 11, 12, 13, 19, 20, 22, 24, 25, 29, 30] (Vaughan et al., 2003).

Some clinical trials, such as MIRACL (Myocardial Ischemia Reduction with Aggressive Cholesterol Lowering), TNT (Treating to New Targets), PROVE IT-TIMI 22, and SPARCL (Stroke Prevention by Aggressive Reduction in Cholesterol Levels), have shown that statins, whenusedinhighdoses,canreducevascularrisksbetterthanwhenusedinlowdoses.Although high doses, adverse effects are relatively low, except atorvastatin 80-mg, that is associated with higher rates of elevated hepatic transaminase, and simvastatin 80-mg with higher rates of myopathy and rhabdomyolysis. A challenge today is to discover if high-dose statin therapy provides greater benefits due to lower cholesterol levels or due to statin pleiotropic effects [2, 5, 7, 11, 13, 18, 19, 22, 24, 29, 30, 31, 35] (Vaughan et al., 2003).

**Figure 2.** Biological actions of isoprenoids [19].

Statins might exert cholesterol-independent or pleiotropic effects by inhibiting the conversion of HMG-CoA to L-mevalonic acid and, in this manner, prevent the synthesis of important isoprenoids, such as farnesylpyrophosphate (FPP), geranylgeranyl pyrophosphate (GGPP) and ubiquinone, which are precursors of cholesterol biosynthesis and of lipid attachments for intracellular signaling molecules. In particular, inhibition of small GTP-binding proteins, Rho, Ras, and Rac, whose proper membrane localization and function are dependent on isopreny‐ lation, may play an important role in mediating the direct cellular effects of statins on the vascular wall. These isoprenylated proteins constitute approximately 2% of total cellular proteins, and isoprenylated proteins might control diverse cellular functions, as signal transduction, growth of vascular smooth muscle, apoptosis and in the regulation of the

Ras and Rho isoprenylation by statins, lead to increase of the amount to both compounds in the cytoplasm cells. As Rho is the more important target in the geranylgeranylation way, inhibition of Rho y de Rho-kinasa is an very important mechanism for the pleiotropic effects

Hypocholesterolemic effects of statins can be explained by hepatic HMG-CoA reductase inhibition, whereas the independent cholesterol effects can be found in all kinds of cells.

As isoprenylated proteins might control diverse cellular functions, we can explain that statins might have additional effects beyond lipid lowering [3, 4, 8, 17, 20, 23, 25, 37] (Li et al., 2002]. Indeed, recent studies suggest that statins might be involved in immunomodulation, neuro‐

Finally, statin therapy can be used for patients with autoimmune diseases, such as multiple sclerosis [26, 34, 35]. Furthermore, in a 6 month, randomized, double-blind placebo-controlled clinical trial, patients with rheumatoid arthritis who received atorvastatin showed a reduction in disease activity [21]. However, it is too early to predict whether these promising data can

The potential clinical implications of statin pleiotropy suggests that perhaps other biomarkers, in addition to lipid levels, should be used to gauge the full efficacy of statin therapy in patients with cardiovascular risks or that statin therapy may be effective in disease states, such as inflammatory conditions, ischemic stroke, or cancer, where elevated cholesterol levels have not been shown to be a strong epidemiological risk for these diseases [2, 5, 7, 8, 11, 12, 13, 19,

Some clinical trials, such as MIRACL (Myocardial Ischemia Reduction with Aggressive Cholesterol Lowering), TNT (Treating to New Targets), PROVE IT-TIMI 22, and SPARCL (Stroke Prevention by Aggressive Reduction in Cholesterol Levels), have shown that statins, whenusedinhighdoses,canreducevascularrisksbetterthanwhenusedinlowdoses.Although high doses, adverse effects are relatively low, except atorvastatin 80-mg, that is associated with higher rates of elevated hepatic transaminase, and simvastatin 80-mg with higher rates of myopathy and rhabdomyolysis. A challenge today is to discover if high-dose statin therapy provides greater benefits due to lower cholesterol levels or due to statin pleiotropic effects [2,

protection, and cellular senescence [2, 5, 7, 11, 13, 18, 22, 24, 29, 30, 31, 32, 35].

translate into clinical benefit by statins in patients with autoimmune disease.

vascular activity of NAD(P) H oxidase.

178 Hypercholesterolemia

20, 22, 24, 25, 29, 30] (Vaughan et al., 2003).

5, 7, 11, 13, 18, 19, 22, 24, 29, 30, 31, 35] (Vaughan et al., 2003).

of statins at the vascular wall [8, 19, 25] (Figures 2 and 3).

**Figure 3.** Regulation of the Rho GTPase cycle. Rho proteins cycle between a cytosolic, inactive GDP-bound state and a, membrane, active, GTP-bound state [35].

#### **3.1. Improve or restore endothelial function**

Hypercholesterolemia impairs endothelial function, and endothelial dysfunction is one of the earliest manifestations of atherosclerosis. When endothelial dysfunction appears, its principal sign is the impaired synthesis, release, and activity of endothelial-derived nitric oxide (NO). Endothelial NO inhibits several components of the atherogenic process, such as vascular relaxation and platelet aggregation, vascular smooth muscle proliferation, and endothelialleukocyte interactions. [9, 15, 17, 19, 28, 38].

Statins could restore endothelial function, in part, by lowering serum cholesterol levels. Indeed, statins increase endothelial NO production by action over NO synthase (eNOS). Also, statins restore eNOS activity in hypoxia condition. Statins also inhibit the expression of endothelin-1, a potent vasoconstrictor and mitogen [6, 14].

#### **3.2. Oxidative stress**

Oxidative stress is defined as tissue injury resulting from a disturbance in theequilibrium between the production of reactive oxygen species (ROS) also known as free radicals and antioxidant defense mechanisms [3].

ROS have been implicated in many disease states, including neurodegenerative disease like Alzheimer and Parkinson disease, atherosclerosis, inflammatory conditions, certain cancers, diabetes mellitus (DM), cataract in the eye, pulmonary, renal, heart diseases, and the process of aging.

NADPH oxidase is an important signaling mediator in the signaling pathway mediated alpha-1-AR, stimulating hypertrophy in adult rat cardiac myocytes. Moreover, recently had been identified calmodulin, Ras and Raf-1 as the upstream signaling molecules in this pathway. In addition, the role of NAD (P) H oxidase in the development of cardiac hypertrophy has been demonstrated. This indicated an interesting new direction in the research of alpha-1-AR signaling mechanisms.

Polyunsaturated lipid acids (PUFAS) in plasma are susceptible to the oxidation process mediated by EROs. This leads to the transformation of the native LDL (LDLn) to oxidative LDL (oxLDL). The oxLDLs does not bind to the LDLn hosts; however oxLDLs bind to the scavenger hosts in monocytes / macrophagues, endothelium and vascular smooth muscle cells, with the consequence of the increase of these compounds and the intracellular generation of the foam cells, an important sign of early atherosclerotic damage [2, 4, 6, 8, 9, 14, 15, 17, 18, 28, 29, 33, 38].

The proliferation of vascular smooth muscle cells is a central event in the pathogenesis of vascular lesions, including post-angioplasty restenosis, transplant arteriosclerosis and veinous graft occlusion.

Statins possesses antioxidant properties by reducing lipid generation and its peroxidation and ROS production by the vascular NAD (P) H oxidase pathway, the susceptibility of lipoproteins to oxidation both in vitro and in vivo, i.e. they decrease the LDL oxidation, especially simvas‐ tatine, pravastatine, and lovastatine [4, 6, 9, 14, 15, 17, 24, 27, 28, 33, 36, 38, 39], decrease the pro-oxidant effects of Angiotensin II and of Endothelin-1 (peptides that stimulate vasocon‐ striction and increase of vascular smooth muscle cells), decreasing the NAD(P)H oxidase activity and the generation of superoxide anion as in vascular cells as in phagocytes, and increase the vascular synthesis of nitric oxide. Moreover statins inhibit vascular SMC prolif‐ eration by arresting cell cycle between the G1/S phase transition, decrease inflammatory cytokines, C-reactive protein C, and adhesion molecules, stimulate NO release, and stimulate activated hosts by PPAR, therefore decrease plasma lipid peroxidation products [4, 9, 14, 15, 17, 24, 27, 28, 33, 36, 38].

#### **4. Statins and dementia**

**3.1. Improve or restore endothelial function**

leukocyte interactions. [9, 15, 17, 19, 28, 38].

antioxidant defense mechanisms [3].

**3.2. Oxidative stress**

180 Hypercholesterolemia

signaling mechanisms.

of aging.

29, 33, 38].

graft occlusion.

endothelin-1, a potent vasoconstrictor and mitogen [6, 14].

Hypercholesterolemia impairs endothelial function, and endothelial dysfunction is one of the earliest manifestations of atherosclerosis. When endothelial dysfunction appears, its principal sign is the impaired synthesis, release, and activity of endothelial-derived nitric oxide (NO). Endothelial NO inhibits several components of the atherogenic process, such as vascular relaxation and platelet aggregation, vascular smooth muscle proliferation, and endothelial-

Statins could restore endothelial function, in part, by lowering serum cholesterol levels. Indeed, statins increase endothelial NO production by action over NO synthase (eNOS). Also, statins restore eNOS activity in hypoxia condition. Statins also inhibit the expression of

Oxidative stress is defined as tissue injury resulting from a disturbance in theequilibrium between the production of reactive oxygen species (ROS) also known as free radicals and

ROS have been implicated in many disease states, including neurodegenerative disease like Alzheimer and Parkinson disease, atherosclerosis, inflammatory conditions, certain cancers, diabetes mellitus (DM), cataract in the eye, pulmonary, renal, heart diseases, and the process

NADPH oxidase is an important signaling mediator in the signaling pathway mediated alpha-1-AR, stimulating hypertrophy in adult rat cardiac myocytes. Moreover, recently had been identified calmodulin, Ras and Raf-1 as the upstream signaling molecules in this pathway. In addition, the role of NAD (P) H oxidase in the development of cardiac hypertrophy has been demonstrated. This indicated an interesting new direction in the research of alpha-1-AR

Polyunsaturated lipid acids (PUFAS) in plasma are susceptible to the oxidation process mediated by EROs. This leads to the transformation of the native LDL (LDLn) to oxidative LDL (oxLDL). The oxLDLs does not bind to the LDLn hosts; however oxLDLs bind to the scavenger hosts in monocytes / macrophagues, endothelium and vascular smooth muscle cells, with the consequence of the increase of these compounds and the intracellular generation of the foam cells, an important sign of early atherosclerotic damage [2, 4, 6, 8, 9, 14, 15, 17, 18, 28,

The proliferation of vascular smooth muscle cells is a central event in the pathogenesis of vascular lesions, including post-angioplasty restenosis, transplant arteriosclerosis and veinous

Statins possesses antioxidant properties by reducing lipid generation and its peroxidation and ROS production by the vascular NAD (P) H oxidase pathway, the susceptibility of lipoproteins to oxidation both in vitro and in vivo, i.e. they decrease the LDL oxidation, especially simvas‐ tatine, pravastatine, and lovastatine [4, 6, 9, 14, 15, 17, 24, 27, 28, 33, 36, 38, 39], decrease the

Recent epidemiological reports suggest that statins might be protective for Alzheimer's disease and for other types of dementia, as cerebrovascular disease. Alzheimer's disease is related to the effects of β-amyloid, and some experimental and clinical trials have shown that there is a pathophysiologic relation between β-amyloid and cholesterol levels. Statins, regardless of their brain availability, have been suggested to induce alterations in cellular cholesterol distribution in the brain. However, major studies are necessary to establish a relationship between statin therapy and Alzheimer disease [19].

#### **5. Clinical trials relationed to pleiotropic effects of statins**

HPS and ASCOT showed that the relative risk reduction by statin was independent of the treatment for lipid levels. Also, other studies suggest that the risk of myocardial infarctions in patients treated with statins is significantly lower compared to individuals with other choles‐ terol-lowering agents [18].

#### **6. Conclusions**

Statins reduce cardiovascular events in not only hypercholesterolemic but also normocholes‐ terolemic patients. Moreover, clinical trials and clinical benefits have shown that statins' effects involved other pharmacological activities and not only changes in lipid levels. Cholesterolindependent or "pleiotropic" effects of statins involve improving or restoring endothelial function, decreasing oxidative stress and vascular inflammation, enhancing the stability of atherosclerotic plaques, inhibiting the thrombogenic response, and lowering oxidative stress. Moreover, some works show that statins have a beneficial extrahepatic effects on the immune system, CNS, and bone.

Statins might exert cholesterol-independent or pleiotropic effects by inhibiting the conversion of HMG-CoA to L-mevalonic acid and, in this manner, prevent the synthesis of important isoprenoids, which are precursors of cholesterol biosynthesis and of lipid attachments for intracellular signaling molecules. Inhibition of Rho GTPases in vascular cellwalls by statins improves expression of atheroprotective genes and inhibition of vascular SMC proliferation.

#### **Author details**

Sigrid Mennickent\*

Address all correspondence to: smennick@udec.cl

Faculty of Pharmacy, University of Concepción, Concepción, Chile

#### **References**


[11] Hardman, J., & Limbird, L. (2006). *Las Bases Farmacológicas de la Terapéutica,* Mc Graw-Hill, Mexico.

intracellular signaling molecules. Inhibition of Rho GTPases in vascular cellwalls by statins improves expression of atheroprotective genes and inhibition of vascular SMC proliferation.

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[2] Arteaga, E., & Pollak, F. (2002). *Dislipidemias en la práctica clínica*, International Lipid

[3] Bendall, J.K., Cave, A.C., Heymes, C., Gall, N., & Shah, A.M. (2002). Pivotal role of gp91 (phox)-containing NADPH oxidase in angiotensin II-induced cardiac hypertrophy in

[4] Beltowski, J. (2005). Statins and Modulation of Oxidative Stress. *Toxicology Mechanism*

[5] Brody, T., Larner, J., & Minulman, K. (1994). *Human Pharmacology. Molecular to Clinical,*

[6] Carr, A.C., Mc Call, M.R., & Frei, B. (2000). Oxidation of LDL by mieloperoxidase and reactive nitrogen species: Reaction pathways and antioxidant protection. *Arterioscl.*

[7] Delgado, J., & Remers, W. (1998). *Wilson and Gisvolds. Textbook of Organic Medicinal and*

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[9] Fukumoto,Y.,Libby,P.,Rabkin,E.,Hill,C.C.,&Enomoto,M.(2001).Statinsalter smooth muscle cell accumulation and collagen content in established atheroma of watanabe

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*Pharmaceutical Chemistry*, Lippincott-Raven, Philadelphia, USA.

**Author details**

182 Hypercholesterolemia

Sigrid Mennickent\*

**References**

Address all correspondence to: smennick@udec.cl

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Information Bureau, Santiago de Chile.

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Faculty of Pharmacy, University of Concepción, Concepción, Chile

