Scientific Evidence-Based Study: The Efficacy of Antroquinonol (Antrodia camphorata)

Scientific Evidence-Based Study: The Efficacy of Antroquinonol Table of Contents I. Antioxidant Effects on the Brain...........................................................3 1. The Effects of Antroquinonol on Alzheimer's Disease (AD) Prevention 1. and the Maintenance of Cognitive Function ( a ) Antroquinonol Enhances Antioxidant Factor Nrf2 Expression ( b ) Antroquinonol Inhibits HDAC2 Expression ( c ) Antroquinonol Lowers Aβ Accumulation ( d ) Antroquinonol Alleviates Brain Glial Scars ( e ) Antroquinonol Improves Learning Ability and Memory

II. Effects on Cholesterol Reduction........................................................9 1. Prevention of Hyperlipidemia and Lowering Cholesterol ( a ) The Effects of Antroquinonol on Lowering Blood Lipids in Animal Models ( b ) The Effects of Antroquinonol on Increasing HDL-C Levels ( c ) The Effects of Antroquinonol on Atherosclerosis Prevention

III. Effects on Liver Protection................................................................16 1. Improve Chronic Hepatitis ( a ) The Effects of Antroquinonol on Liver Protection and Functional Improvement ( b ) The Efficacy of Antroquinonol on Liver Protection 2. Improve Fatty Liver and Liver Cirrhosis ( a ) The Effects of Antroquinonol on Fatty Liver Improvement ( b ) The Effects of Antroquinonol on Liver Fibrosis Improvement

IV. Kidney Protection and Anti-inflammatory Effects on IV. Autoimmune System..................................................................................22 1. The Effects of Antroquinonol on Lupus Nephritis Improvement ( a ) Effects on Nephritis Improvement ① Proteinuria Reduction ② Hematuria Prevention ③ CRE Reduction ④ BUN Reduction

2. Effects on FSGS Improvement ( a ) Alleviate Chemokine MCP-1 Accumulation ( b ) Alleviate Cytokine Accumulation ( c ) Protect Glomerulus 3. Working Mechanisms of Autoimmune Disease Improvement ( a ) Activity Inhibition on Oxidation and Inflammation-related ( a ) Transcription Factor NF-ÎşB ( b ) Activation of Antioxidant Transcription Factor Nrf2

V. Anti-Fatigue.....................................................................................34 1. Animal Studies Regarding the Effects of Antroquinonol on Exercise1. induced Fatigue Improvement 2. Human Studies Regarding the Effects of Antroquinonol on Exercise1. induced Fatigue Improvement

Scientific Evidence-Based Study: The Efficacy of Antroquinonol List of Figures Figure 1. The Mechanisms and Evidence-based Effects of Antroquinonol............2 Figure 2. Symptoms and Signs of Alzheimer's Disease............................................3 Figure 3. The Enhancement of Nrf2 Expression.......................................................5 Figure 4. Effects on HDAC2 Inhibition......... ............................................................5 Figure 5. Changes in Total Aβ Levels........................................................................6 Figure 6. Changes in Aβ42 Levels..............................................................................6 Figure 7. Reduction of Brain Glial Scar in Hippocampus........................................6 Figure 8. The Reduction of Glial Scar is in Proportion to the Consumption Figure 8. of Antroquinonol.........................................................................................7 Figure 9. Morris Water Maze (MWM) Test.............................................................7 Figure 10. Changes of Escape Latency.......................................................................8 Figure 11. Effects on Lowering TC............................................................................10 Figure 12. Effects on Lowering LDL-C.......................................................................11 Figure 13. Effects on Enhancing Hepatic LDLR.......................................................11 Figure 14. Effects on Increasing Blood HDL-C........................................................13 Figure 15. The Ratio of HDL-C/LDL-C is Increased................................................13 Figure 16. Effects on Atherosclerosis and Stenosis Prevention..............................14 Figure 17. Effects on Liver Function Index Improvement.......................................17 Figure 18. Effects on Lowering GPT.........................................................................18 Figure 19. Effects on Lowering GOT.........................................................................18 Figure 20. Deterioration of Liver Diseases...............................................................19 Figure 21. Effects on Fatty Liver Improvement.......................................................20 Figure 22. Effects on Liver Fibrosis Inhibition.......................................................21 Figure 23. Effects on Proteinuria Improvement......................................................25 Figure 24. Effects on Hematuria Prevention...........................................................26 Figure 25. Effects on Renal Function Maintenance (CRE)....................................26 Figure 26. Effects on Renal Function Maintenance (BUN)...................................27 Figure 27. The Accumulation of Chemokine MCP-1 in the Kidney.......................29 Figure 28. The Changes of Kidney MCP-1...............................................................29 Figure 29. The Accumulation of Cytokine IL-6 in the Kidney..............................29 Figure 30. The Changes of Cytokine IL-6 in the Kidney.........................................30 Figure 31. Effects on Glomerular Protection............................................................31 Figure 32. Effects on FSGS Prevention.....................................................................31 Figure 33. Effects on Inhibiting Inflammatory Transcription Factor (NFFigure 33. κB)............................................................................................................32 Figure 34. Effects on Activating Antioxidant Factor Nrf2.....................................33

Figure 35. Exercise Exhaustion Test on Rats...........................................................35 Figure 36. Effects on Exercise Duration Prolongation...........................................36 Figure 37. Effects on CPK Reduction.......................................................................36 Figure 38. Effects on LDH Reduction.......................................................................37 Figure 39. Effects on Uric Acid Reduction...............................................................37 Figure 40. Effects on Lactic Acid Reduction............................................................38 Figure 41. Effects on BUN Reduction.......................................................................38 Figure 42. The Recovery of Exercise-induced Fatigue (CPK Level).......................39 Figure 43. The Recovery of Exercise-induced Fatigue (Ammonia Level).............40

Scientific Evidence-Based Study: The Efficacy of Antroquinonol In order to understand the working mechanisms of Antrodia camphorata extracts in depth on individual systems and organs in human bodies and animals and to scientifically prove the superiority of Antrodia camphorata extracts as healthcare food, Golden Biotechnology Corporation (GBC) has consigned various multiannual Antrodia camphorata extracts-based human and animal studies to domestic and international academic institutes.

Based on the study results published by GBC to date, Antrodia camphorata extracts have anti-cancer effects. For example, a current Phase II clinical trial targeting patients with non-small cell lung carcinoma (NSCLC) in the U.S. and Taiwan has shown that Antrodia camphorata extracts are equipped with low side effects but extensive therapeutic scopes. Therefore, Antrodia camphorata extracts are a highly anticipated area of study by scholars and experts from various circles.

Multiannual and extensive studies have uncovered that Antroquinonol from Antrodia camphorata manufactured by GBC is not limited to solely having anti-cancer effects. In fact, according to large-scale animal and human body efficacy studies, GBC has found that Antrodia camphorata not only maintains cardiovascular functions, protects the liver and kidneys from being damaged, and regulates the immune system, but it also has efficacies against physiological fatigue or improves physiological functions and measures. This brochure elaborates on evidence-based efficacy research of Antroquinonol in addition to its anti-cancer effects.

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Figure 1. The Mechanisms and Evidence-based Effects of Antroquinonol　

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I. Antioxidant Effects on the Brain 1. The Effects of Antroquinonol on Alzheimer's Disease (AD) 1. Prevention and the Maintenance of Cognitive Function There are 5.5 million elderly patients with dementia in Japan (according to data published by the Ministry of Health, Labour and Welfare in Japan, the number of elderly patients with dementia in Japan is 4.62 million as of June 2013, and 15% of them are over the age of 65). If we add onto that the 4 million elderly patients with mild cognitive impairment (MCI, also known as the “highrisk population of elderly dementia”), that totals to 9.5 million. Hence, it is urgent to uncover solutions to dementia. AD currently accounts for approximately 66% of dementia, making it the majority type of dementia. In addition, AD in males is higher than in females, and the population of AD is still growing relative to other types of dementia (approaching constant numbers without significant changes).

AD is caused by the massive accumulation of special proteins called Amyloid-Beta Peptides (Aβ) and Tau, consequently leading to brain atrophy and loss of body functions due to brain cell death or reduction. Prominent symptoms of AD consist of memory impairment, poor judgment, cognitive impairment and language disorders (refer to Figure 2 below).

Figure 2. Symptoms and Signs of Alzheimer’s Disease Because of the ageing population and changes of lifestyle, AD has become a major medical and societal issue in Japan and even around the world. There are currently 5.4 million people suffering from AD in the U.S. Based on statistics estimated by experts, the number of patients with AD will be over 16 million in 2050. The U.S. healthcare system spent 203 billion U.S. dollars in 2013 on medical expenses and social welfare for AD, and the cost is estimated to be above 1.2 trillion U.S. dollars in 2050.

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A large number of worldwide research institutes and large-scale pharmaceutical companies are focusing on novel drug R&D for AD. However, there is no significant breakthrough so far. The greatest challenge is to allow drugs to pass through the blood-brain barrier (BBB) in order to achieve therapeutic effects on the brain. BBB is a mechanism to limit substance exchange between blood/cerebrospinal fluid and the central nervous system such as the brain or spinal cord. Therefore, substances with molecular weights of more than 500 (e.g. proteins) or ions of a lipophobic nature may not be able to pass through the lipid bilayer of BBB. That is to say, most new drugs and antioxidants cannot pass the BBB and thus fail to achieve therapeutic or preventive effects in the brain against AD. In November 2015, GBC and the Institute of Brain Science at National Yang-Ming University jointly published an article regarding the effects of Antroquinonol, an indexed component from GBC Antrodia camphorata extracts, on AD prevention in Scientific Reports, a subset of the worldly prestigious academic journal “Nature," entitled “Antroquinonol Lowers Brain Amyloid-β Levels and Improves Spatial Learning and Memory in a Transgenic Mouse Model of Alzheimer's Disease."

The study used an AD transgenic mouse model to perform various experiments for 2 years. The results showed that the existence of dietary Antroquinonol in brain tissues proved its ability of BBB penetration and biosafety.

The results also showed that dietary supplements with Antroquinonol in AD transgenic mice prior to disease onset inhibited the expression of Nrf2 (a free radical) as well as HDAC2 (which preclude memory and learning).

Furthermore, not only did AD transgenic mice fed with dietary Antroquinonol have much fewer Aβ accumulated senile plaques (SPs) than the control group, but also the amount of Antroquinonol consumption was proportional to antiinflammatory effects on brain tissues.

In conclusion, Antroquinonol improves spatial learning and memory in a transgenic mouse model of Alzheimer's Disease.

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( a ) Antroquinonol Enhances the Antioxidant Factor Nrf2 Expression Antroquinonol enhances the antioxidant factor Nrf2 (NF-E2 related factor-2) expression and thus improves antioxidation in the brain.

Figure 3. The Enhancement of Nrf2 Expression

( b ) Antroquinonol Inhibits the HDAC2 Expression The inhibitory effects of dietary Antroquinonol on the HDAC2 (Histonedeacetylase 2) expression indicates that Antroquinonol can prevent HDAC2 from precluding memory and learning.

Figure 4. Effects on HDAC2 Inhibition

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( c ) Antroquinonol Lowers Aβ Accumulation

Figure 5. Changes in Total Aβ Levels

Figure 6. Changes in Aβ42 Levels

As described in the above Figure 5, Antroquinonol significantly lowered total Aβ levels in the brain tissues of AD transgenic mice. In addition, as described in the above Figure 6, the amount of Antroquinonol consumption is proportional to the inhibition of Aβ42 in brain tissues of AD transgenic mice.

Aβ42 is easily solidified in the brain and promotes Tau protein precipitation, thus becoming one of the key proteins leading to the pathogenesis of AD. ( d ) Antroquinonol Alleviates Brain Glial Scars

Figure 7. Reduction of Brain Glial Scar in Hippocampus

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Figure 8. The Reduction of Glial Scars is in Proportion to the Consumption of Antroquinonol

Based on the results shown in Figure 7 and Figure 8, glial scars in the hippocampus of the brain in both groups (low and high-dose Antroquinonol consumption) were found to have decreased. Moreover, the expression of the glial fibrillary acidic protein was also decreased.

( e ) Antroquinonol Improves Learning Ability and Memory This study used the Morris Water Maze (refer to Figure 9) to test the memory functions of mice.

The latency of finding invisible platforms underwater for 5 consecutive days (escape latency) was recorded as a reference to evaluate the learning ability and memory of mice.

Figure 9. Morris Water Maze (MWM) Test 7

Figure 10. Changes of Escape Latency

Figure 10 showed the results of the Morris Water Maze Test for 5 consecutive days. AD transgenic mice that consumed a highdose of Antroquinonol had a significantly shorter escape latency than the control group, which proved that Antroquinonol improved memory functions of transgenic mice with AD.

Alzheimer’s Disease is a great challenge for human beings. Among current scientific and technological advances, no effective solutions or AD cures are available so far. However, by showing antioxidant effects on brain cells or inhibition of senile plaques formation or glial scars formation to avoid memory impairment or brain atrophy, Antroquinonol has displayed potentials for AD treatment. As a result, the consumption of Antroquinonol is expected to improve learning abilities and memory functions in patients with AD. By showing the effects of Antroquinonol, GBC stood out above nearly 100 global contestants and won the only Innovative Drug Award in a worldwide contest, QuickFire Challenge, jointly hosted by the Australian Government and the worldwide top pharmaceutical company Johnson & Johnson Innovation for the potential treatment of Alzheimer’s Disease in 2016.

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II. Effects on Cholesterol Reduction According to statistics from the Ministry of Welfare, cardiovascular and cerebrovascular-related diseases are the 2nd and 4th largest causes of deaths among the top ten mortalities in Japan in 2011. Because cardiovascular and cerebrovascular diseases are usually caused by atherosclerosis, the prevention of atherosclerosis is the ultimate strategy to protect cardiac and cerebral vessels.

Atherosclerosis refers to arterial wall thickening and hardening and the loss of elasticity, and thus results in poor circulation. Pathogenesis of atherosclerosis includes hyperlipidemia, hypertension, smoking, obesity, ageing, diabetes mellitus, gout, hypolipoproteinemia, genetic factors, stress and insufficient exercise, wherein hyperlipidemia is the most important risk factor.

Primary Laboratory Test Items for Hyperlipidemia: Total Cholesterol (TC), LDL, HDL Hyperlipidemia refers to diseases caused by excessive blood lipids; in other words, the levels of blood cholesterol and neutral fat (triglycerides) are greatly increased. The measures of TC, LDL (low-density lipoprotein cholesterol) or neutral fat appears excessive in dyslipidemia, wherein LDL is the most common index used for the risk assessment of cardiovascular diseases.

Total Cholesterol TC level is a required marker for investigating the signs and disease progression of atherosclerosis. Long-term increments of TC and triglycerides may easily lead to coronary atherosclerosis. In addition to familial hypercholesterolemia, factors leading to excessive levels of TC such as obesity, nephritis and biliary atresia may also result in secondary hypercholesterolemia.

There are 4 types of lipoprotein particles that consist of cholesterol and lipoproteins, including chylomicron, VLDL (Very low-density lipoprotein cholesterol), LDL (Low-density lipoprotein cholesterol) and HDL (High-density lipoprotein cholesterol).

LDL (Low-density lipoprotein cholesterol; bad cholesterol) LDL refers to the low-density lipoprotein that transports cholesterol generated by the liver to individual organs. Therefore, LDL is considered a major risk factor for atherosclerosis. Moreover, LDL is approximately two-thirds of TC, which often makes excessive TC attributable to excessive LDL. HDL (High-density lipoprotein cholesterol; good cholesterol) HDL is responsible for recycling accumulated cholesterol (usually resulting in atherosclerosis) from blood vessel walls to the liver, and consequently prevents atherosclerosis by clearing up blood vessels.

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1. Prevention of Hyperlipidemia and Lowering Cholesterol ( a ) The Effects of Antroquinonol on Lowering Blood Lipids in Animal Models GBC and the School of Nutrition and Health Sciences at Taipei Medical University have jointly conducted a study called “The Effects of Antroquinonol on Lowering Blood Lipids in Animal Models.” The study results have proven the following effects: ( 1 ) Dietary Antroquinonol did not affect animal growth or result in malnutrition; ( 2 ) Consumption of Antroquinonol for 28 consecutive days significantly reduced ( 2 ) blood cholesterol; ( 3 ) Antroquinonol improved liver functions of hamsters with hyperlipidemia.

According to Figure 11, the consumption of Antrodia camphorata extracts containing Antroquinono l significantly reduces blood TC levels.

Figure 11. Effects on Lowering TC

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As described in Figure 12, the consumption of Antrodia camphorata extracts containing Antroquinonol significantly reduces blood LDL.

Figure 12. Effects of Lowering LDL-C a further cellular study investigating the potential 　　In mechanisms of Antroquinonol lowering blood lipids, we have found that GBC Antrodia camphorata extracts greatly enhanced the LDLR (lowdensity lipoprotein receptor) gene expression in hepatic cells. As a result, Antroquinonol could lower blood cholesterol because the LDLR could reabsorb excessive LDL by conjugating circulatory LDL.

As shown in Figure 13, animals fed with Antroquinonol displayed the hepatic LDLF gene expression several times as much as the control group.

Figure 13. Effects of Enhancing Hepatic LDLR

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What is LDLR? LDLR refers to low-density lipoprotein receptors, which can carry several ligands including LDL and remove those from cells. It also has signal transduction functions, wherein the most important one is to transport LDL to the liver. ( b ) The Effects of Antroquinonol on Increasing HDL-C Levels Based on the aforementioned study entitled “The Effects of Antroquinonol on Lowering Blood Lipids in Animal Models”, GBC and the Development Center for Biotechnology in Taiwan jointly conducted a study called “The Effects of Antroquinonol on Increasing HDL-C in Animal Models” for further investigation. The study used hamsters with hyperlipidemia (established through high-fat chow gavage) as animal models and randomly allocated them into three groups: High-Dose Antroquinonol (High-Antro), Low-Dose Antroquinonol (Low-Antro) and the control group.

Whether hamsters consumed Antroquinonol or not, the body weights and liver weights of hamsters fed with high-fat chow were significantly increased. Hence, the consumption of Antroquinonol is rather safe for experimental animals. In addition to LDL-C reduction, an increased level of HDL-C (good cholesterol) was also observed during the study.

Because LDL-C was decreased and HDL-C was inversely increased, the ratio of HDL-C/LDL-C (the inverse of the atherosclerosis index) was in proportion to the amount of Antroquinonol consumption. Considering the greater the atherosclerosis index, the higher the incidence of atherosclerosis occurs, and vice versa. From the atherosclerosis index point of view, Antroquinonol not only can regulate cholesterol levels, but also is expected to improve atherosclerosis.

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As shown in Figure 14, the consumption of Antroquinonol significantly increased HDL-C relative to the control group.

Figure 14. Effects of Increasing Blood HDL-C

As shown in Figure 15, the ratio of HDL-C/ LDL-C is proportional to the amount of Antroquinonol consumption.

Figure 15. The Ratio of HDL-C/LDL-C is Increased

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( c ) Effects on Antroquinonol on Atherosclerosis Prevention There are no particular symptoms in the early stage of atherosclerosis, but some complications may occur during the progression of the disease. For example, at the initial stage of cerebrovascular atherosclerosis, symptoms such as headaches, vertigo or amnesia may appear; but at an advanced stage of vasculature occlusion, severe symptoms including hemorrhagic and ischemic strokes or loss of consciousness may occur. As to cardiac coronary atherosclerosis, chest pain may take place when climbing stairs.

Also, if the sclerotic change of coronary artery remains and eventually establishes into angina, myocardial necrosis, or infarction may develop due to the complete interruption of coronary circulation.

GBC and National Defense Medical Center have jointly conducted an atherosclerosis study called “The Platform of Carotid Artery Ligation and Sclerosis in Rats”. The levels of rats' carotid arterial intimal hyperplasia were observed after the consumption of Antrodia camphorata extracts containing Antroquinonol for 28 consecutive days. The study results showed that the levels of rats' carotid arterial intimal hyperplasia in the Antroquinonol consumption group was significantly less than those in the control group. The following figure taken under 400x microscopy directly proved that Antroquinonol could effectively inhibit arterial intimal hyperplasia and thus maintain good circulation. (Refer to Figure 16 below.)

Figure 16. Effects on Atherosclerosis and Stenosis Prevention

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In addition, the inflammatory index CRP (C-reactive protein) closely related to cardiac diseases was also significantly decreased due to the consumption of Antroquinonol.

Based on the results of the above study, the consumption of Antroquinonol is expected to achieve the following healthcare effects on the cardiovascular system: 1. To induce massive LDLR gene expression and thus reduce the threats of bad 1. cholesterol (LDL-C) against vessel walls.

2. To increase the levels of good cholesterol (HDL-C), improve atherosclerosis 2. index and decrease the risks of atherosclerosis.

3. To decrease arterial intimal hyperplasia that may lead to atherosclerosis.

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III. Effects on Liver Protection The liver is a silent organ; patients may not be aware of it even if it has developed cirrhosis or hepatocellular carcinoma (HCC). The fundamental protection method for the liver is to reduce inflammation in the liver.

If abnormal liver functions continue for 3 months or longer, it could be chronic hepatitis. Chronic hepatitis (esp. viral hepatitis) is often caused by the Hepatitis B Virus (HBV) or Hepatitis C Virus (HCV). In addition, acute hepatitis induced by alcohol abuse or a poor immune system could also become chronic.

Most patients with chronic hepatitis do not have symptoms, or usually have systems such as general fatigue, feeling tired easily, poor appetite, abdomen distention, vomiting, chest discomfort, jaundice, brown urine, etc.

The ultimate status of chronic hepatitis is liver cirrhosis, wherein liver tissues become too hard and deformed to perform important functions (e.g. detoxification) after repeated inflammation, necrosis and regeneration. Moreover, the possibility of liver cirrhosis becoming HCC is high enough to be noted specifically.

1. Improve Chronic Hepatitis ( a ) Effects on Liver Protection and Functional Improvement A business consignment of GBC to Taiwan Agricultural Chemicals and Toxic Substances Research Institute from the Council of Agriculture (TACTRI/COA) regarding “A Functional Study Investigating the Effects of Antrodia camphorata Extracts Containing Antroquinonol on Liver Protection” used carbon tetrachloride (CCL4) induced chronic hepatitis animal models to investigate the effects of Antrodia camphorata containing Antroquinonol on liver protection.

The study results showed that liver function indexes (the activities of hepatic enzymes) such as ALT (GOT) and AST (GPT) were significantly lower in feeding group that consumed Antrodia camphorata containing Antroquinonol for 8 consecutive weeks than in the control group (refer to Figure 16 below), which meant that Antrodia camphorata containing Antroquinonol improved chronic hepatitis.

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Figure 17. The Effects of Liver Function Index Improvement ( b ) The Efficacy of Antroquinonol on Liver Protection The aforementioned study, “The Effects of Antroquinonol on Lowering Blood Lipids in Animal Models,” jointly conducted with the School of Nutrition and Health Sciences at Taipei Medical University also appraised the effects of Antroquinonol on liver functions in animal models.

The study results showed that the liver function indexes such as GOT and GPT were significantly improved depending on the amount of Antroquinonol consumption.

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As described in F i g u r e 18, the live r function index (GPT) of rats fed with Antrodia camphorata ext ra cts contain ing Antroquinonol was significantly decreased.

Figure 18. The Effects of Lowering GPT

As described in F i g u r e 1 9 , the liver function index (GOT) of rats fed with Antrodia camphorata extracts containin g Antroquinonol was significantly decreased.

Figure 19. Effects on Lowering GOT

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2. Improve Fatty Liver and Liver Cirrhosis Fatty liver refers to neutral fat and cholesterol accumulation in the liver and when the liver is surrounded by fat due to overeating. The fatty liver covered by neutral fat and cholesterol is a risk factor for various lifestyle-related diseases that may lead to atherosclerosis.

Between genders, the incidence of fatty liver in males is approximately twice as much as in females. In addition, the predilection age is between 30~70 years, wherein age 40 for males and age 40 and older (middle-aged and older) for females is highest. A recent investigation showed that nearly 20%~30% of adults in Japan had fatty livers: approximately 1 in 3 adult males and 1 in 5 adult females, which in total were about twice as much as 10 years ago.

Fatty liver can be improved by changing one's lifestyle, and is thus considered a minor illness among all liver diseases. However, without changing lifestyles, liver functions may be degenerated and fatty liver may evolve into chronic hepatitis, liver cirrhosis or ultimately HCC (refer to the following figure).

Figure 20. Deterioration of Liver Diseases ( a ) The Effects of Antroquinonol on Fatty Liver Improvement GBC and Stelic Institute & Co., Inc. jointly conducted a study to investigate the effects of Antroquinonol on fatty liver improvement in rats with nonalcoholic steatohepatitis (NASH). The NASH rat model was established by feeding rats with high-fat chow. The study results showed that fat tissue (indicated by a red stain) accumulation in liver (fatty liver) was improved significantly after feeding with mixed chow containing Antroquinonol for 4~6 weeks (see Figure 21 below).

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Figure 21. Effects on Fatty Liver Improvement As described in Figure 21, red spots referred to fat adhesion areas. The study results showed that fat adhesion was significantly improved in groups fed with Antrodia camphorata extracts containing Antroquinonol relative to the control group.

The working mechanisms of reducing fatty liver are the same as those described in cellular experiments; that is to say, GBC Antrodia camphorata extracts could decrease blood lipid levels, reduce fat accumulation in the liver, improve fatty liver/liver cirrhosis, prevent HCC and improve hyperlipidemia by greatly increasing the expression of the LDLR (low density lipoprotein receptor, which promotes LDL-C metabolism) in the liver while simultaneously transporting circulating LDL back to the liver in order to achieve good health management of the liver and cardiovascular system. ( b ) The Effects of Antroquinonol on Liver Fibrosis Improvement GBC and Stelic Institute & Co., Inc. not only used an experimental model (rats with NASH) to verify the effects of Antroquinonol on fatty liver improvement, but also applied the same animal model for a longer period to induce liver fibrosis.

As the name suggests, liver fibrosis refers to increased connective tissues in liver. When the fibrotic changes occur on a larger scale, it is called cirrhosis.

Most patients with liver fibrosis have only mild general fatigue; almost no significant symptoms are noted.

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Figure 22. The Effects of Liver Fibrosis Inhibition 　　As shown in Figure 22, red areas indicated stained liver fibrotic tissues. A few echo-free lesions were noted between fibrotic tissues in the liver. The results showed that liver fibrosis is milder in the group fed with Antroquinonol relative to the control group. In other words, Antroquinonol reduced liver fibrosis.

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IV. Kidney Protection and Anti-inflammatory Effects on Autoimmune System The so-called autoimmune disease refers to inflammatory disease and various symptoms caused by one's self-defense system attacking its own body. Immune reactions are the primary defense mechanisms against foreign substance invasion. When such a mechanism fails to distinguish between self and foreign matter, autoimmune diseases such as atopic dermatitis, Systemic Lupus Erythematosus (SLE), chronic rheumatoid arthritis and ankylosing spondylitis may occur, wherein SLE is one of the most typical autoimmune diseases.

Systemic Lupus Erythematosus (SLE) As the name suggests, the feature of SLE consists of erythema in the face and arthralgia. Furthermore, the overall organs may appear to have inflammation, wherein the autoimmune system is considered to be the primary cause of SLE. Currently there are still no cures available.

Symptoms such as fever, feeling tired easily or butterfly erythema in areas between the cheeks and nose may occur during acute onset of SLE. SLE requires long-term treatment, which is primarily based on symptom inhibition or anti-inflammation. Hence, patients are usually required to be on long-term immunosuppressants, anti-inflammatory agents or steroids to reduce inflammation.

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SLE and Nephritis SLE may induce inflammation in various general organs. The inflammatory symptoms and incidences are listed as follows: Clinical Symptoms

Incidence

Arthalgia or arthritis

≧ 90%

Round erythema on skin

70～80%

Pleurisy or pericarditis

40～50%

Lupus nephritis

40～50%

CNS diseases

20～60%

Leukocytosis

60%

Thrombocytosis

20%

Table 1. Various Symptoms of SLE The previous table showed that almost all organs may be attacked by the autoimmune system. Based on statistics, the incidence of lupus nephritis is up to 40~50％, which is also a complication with the worst prognosis among all symptoms.

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Primary Laboratory Tests for Nephritis: Blood in Urine, Proteinuria, Blood Urine Nitrogen, Creatinine Blood in Urine Erythrocytes are almost unfound in the urine of healthy people. Therefore, the existence of erythrocytes usually implies abnormalities in places urine passes through, including the kidneys, urinary tract or gallbladder.

Proteinuria Blood proteins will be filtered by the glomerulus in the kidneys; 99% of proteins will be recycled again back to blood by renal tubules, and only about 1% of proteins will be excreted along with urine. If any abnormalities occur in the glomerulus or renal tubules, massive proteins will be excreted along with urine and thus is called proteinuria. Under such circumstances, the incidences of kidney and urinary tract malfunctions or urinary tract infections may be increased. Moreover, the results of urine tests for proteinuria may appear abnormal due to hypertension-induced nephrosclerosis, nephritic syndrome or autoimmune disease.

BUN (Blood Urine Nitrogen) BUN is a categorical test for renal functions. Urea is the end-product of liver metabolism, and the combination of nitrogen and urea forms urine nitrogen.

If renal function deteriorates, the level of BUN will increase because toxins that are supposed to be excreted by the kidneys fail to be excreted along with urine and subsequently enter into the blood stream. Therefore, a significantly elevated level of BUN may indicate 25% or less normal renal functions; that is to say, the condition is rather severe.

CRE (Creatinine) CRE is the byproduct of muscle metabolism, which is filtered by the kidneys and excreted along with urine the same as BUN. When the level of CRE is greatly increased in the blood, it indicates problems in kidney excretion functions. Therefore, such a physiological parameter is usually tested for renal functions, glomerular screenings and disease follow-ups. Extremely high CRE levels may indicate renal parenchymal lesions caused by acute or chronic nephritis.

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1. The Effects of Antroquinonol on Lupus Nephritis Improvement A study called “The Anti-inflammatory Effects of Antroquinonol on the Auto-immune System,” jointly conducted by research teams from GBC, the Development Center for Biotechnology in Taiwan and National Defense Medical Center uncovered that Antroquinonol could improve lupus nephritis. As to the study verifying the anti-inflammatory and auto-immune effects of Antroquinonol, a rat model with lupus nephritis and focal segmental glomerulosclerosis (FSGS) was used.

The study results have proven the anti-inflammatory and immunoregulatory effects of Antrodia camphorata extracts containing Antroquinonol, which indicated that Antrodia camphorata extracts containing Antroquinonol could achieve dual effects including inflammation alleviation caused by autoimmune disease and kidney protection. ( a ) Effects on Nephritis Improvement The improvement of proteinuria and hematuria indicated that the overall inflammation of the kidneys had been controlled and improved. In addition, the improvement of CRE and BUN also indicated the recovery of kidney functions. ① Proteinuria Reduction As shown in Figure 23, the changes of proteinuria in rats fed with Antrodia camphorata extracts containing Antroquinonol were more stable than those in the control group and closer to the measures from the healthy group.

Figure 23. Effects on Proteinuria Improvement

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â‘Ą Hematuria Prevention As shown in Figure 24, rats with nephritis not fed with Antrodia camphorata extracts containing Antroquinonol began to show hematuria since Week 3, but those fed with Antrodia camphorata extracts containing Antroquinonol did not show mild hematuria until Week 5.

Figure 24. Effects on Hematuria Prevention

③ CRE Reduction As shown in Figure 25, CRE levels in rats f e d with Antrodia camphorata extracts containing Antroquinonol were far below those of the control group.

Figure 25. Effects on Renal Function Maintenance (CRE)

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â‘Ł BUN Reduction As shown in Figure 26, BUN levels in rats f e d with Antrodia camphorata extracts containing Antroquinonol were far below those of the control group.

Figure 26. The Effects of Renal Function Maintenance (BUN)

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2. Effects on FSGS Improvement If severe proteinuria continues, it may become nephrotic syndrome.

Nephrotic syndrome is a collective term of severe proteinuria and subsequent hypoalbuminemia. Several major symptoms such as hypoalbuminemia, severe proteinuria and eyelid/lower extremity edema caused by abnormal permeability of the glomerulus base membrane (GBM) may also appear.

Nephritic syndrome can be divided into idiopathic nephritic syndrome that is caused by idiopathic glomerular disease and secondary nephritic syndrome that is caused by secondary glomerular disease.

Among patients with idiopathic glomerular disease, 70~80% are middleaged and older adults. More than half of them have already had chronic kidney diseases. Therefore, they are prone to having kidney diseases due to increased age and gradual organ function deterioration. The recurrence rate within 5 years since the first onset is about 80~90%. On the other hand, the age of onset of secondary nephritic syndrome usually varies. For example, purpura nephritis often occurs in children, while diabetic nephropathy and lupus nephritis often occur in adults.

In the study investigating “The Anti-inflammatory Effects of Antroquinonol on the Auto-immune System,” not only was proteinuria and hematuria caused by nephritis improved or prevented, but it was also found that the cluster of neutrophils in the kidneys was reduced by Antroquinonol consumption. The results indicated that the levels of inflammatory factors such as cytokines generated by renal cells were also reduced due to the consumption of Antroquinonol.

A majority of patients with lupus nephritis may develop focal segmental glomerulosclerosis (FSGS). The same study also proved that Antrodia camphorata extracts containing Antroquinonol could effectively reduce the levels of chemokines such as MCP-1 and cytokines such as IL-6 caused by lupus nephritis, wherein chemokine MCP-1 induced tubulointerstitial nephritis, tubular atrophy and kidney fibrosis, and cytokine IL-6 caused FSGS. ( a ) Alleviate Chemokine MCP-1 Accumulation Figure 27 shows chemokine MCP-1 accumulation in the kidneys. Rats fed with Antrodia camphorata extracts containing Antroquinonol showed a significant reduction of MCP-1 accumulation, which originally caused tubular atrophy and kidney fibrosis.

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Figure 27. The Accumulation of Chemokine MCP-1 in Kidney As shown in Figure 28, the expression of MCP-1 was significantly inhibited by the consumption of Antroquinonol.

Figure 28. The Changes of Kidney MCP-1 ( b ) Alleviate Cytokine Accumulation

Figure 29. The Accumulation of Cytokine IL-6 in the Kidneys

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The effects of Antroquinonol on cytokine IL-6 accumulation were shown in Figure 29. IL-6, a cytokine causing FSGS, was affected by the consumption of Antroquinonol. In other words, IL-6 accumulation in rats fed with Antroquinonol was far lower than that of the control group.

As shown in Figure 30, the consumption of Antroquinonol inhibited the increased expression of IL-6.

Figure 30. The Changes of Cytokine IL-6 in the Kidneys Based on the experimental data from previous figures and tables, the deterioration levels of FSGS induced by chemokine MCP-1 and the destruction levels toward the kidneys caused by chemokine MCP-1 as well as cytokine IL-6 in rats fed with food containing Antroquinonol were both alleviated. That is to say, Antroquinonol was equipped with kidney protection effects.

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( c ) Protect the Glomerulus

Figure 31. The Effects of Glomerular Protection Figure 31 showed tissue biopsies of the glomerulus. Rats with FSGS caused by lupus nephritis had larger glomeruli on Day 7 and epithelium hyperplasia in the Bowman’s capsule on Day 21, which left the glomerulus with no filtration and recycle functions. However, rats with nephritis that were fed with Antrodia camphorata extracts containing Antroquinonol still had clear and normal sized glomeruli on Day 21, and no significant epithelium hyperplasia was found in the Bowman’s capsule.

As shown in Figure 32, An troquin onol significantly alleviated the deterioration of glomerulointerstitial nephritis-induced fibrosis.

Figure 32. Effects on FSGS Prevention 31

3. Working Mechanisms of Autoimmune Disease Improvement According to the study investigating “The Anti-inflammatory Effects of Antroquinonol on the Auto-immune System,” reasons to improve the aforementioned nephritis are derived from the anti-inflammatory and antioxidant effects of Antroquinonol.

The study proved that Antroquinonol not only could effectively inhibit the activity of inflammatory factor NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), but also could increase the expression of the Nrf2 (NFE2related factor 2) protein (which leads to free radical elimination and antioxidation).

NF-κB can be activated by stimulants such as stress, cytokines and UV light.

The main role of NF-κB is to initiate one of the transcription factors responsible for many physiological phenomena, including immune reactions, acute/chronic inflammatory reactions, cell proliferation and apoptosis. If control over NF-κB activity goes wrong, all kinds of autoimmune reactions and diseases may occur.

For example, inflammatory diseases such as Crohn’s Disease, Rheumatoid Arthritis, cancer, and septic shock are often related to constantly activated NFκB. Nrf2 is a transcription factor in normal cells against oxidative stress. Many studies have proven that the antioxidant effects of Nrf2 may inhibit cancer cell proliferation. ( a ) Activity Inhibition on Oxidation and Inflammation-related Transcription Factor NF-κB As described in Figure 33, the expression of NF-κB in t h e kidney cells of rats fed with Antroquinonol was significantly lower than that o f t h e control group.

Figure 33. Effects on Inhibiting Inflammatory Transcription Factor (NF-κB) 32

( b ) Activation of Antioxidant Transcription Factor Nrf2

As described in Figure 34, the expression of Nrf2 in kidney cells in rats fed with Antroquinonol was significantly higher than that of the control group.

Figure 34. Effects on Activating Antioxidant Factor Nrf2 　　The study results have been published on an international and peer-reviewed journal, which indicated that Antroquinonol had very powerful anti-inflammatory and anti-oxidant effects in order to restore normal cellular functions. Based on the study results, we can expect that Antroquinonol could be used to regulate excessive immune reactions, eliminate free radicals and reduce oxidative stress. Moreover, the anti-oxidative and anti-inflammatory effects of Antroquinonol could also be used on skin care, including the treatment for inflammation caused by acne and atopic dermatitis and anti-oxidative effects for anti-ageing therapy.

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V. Anti-Fatigue Due to increased health awareness, more and more people nowadays acknowledge the importance of exercise. Exercises including marathon training, jogging, biking and weight training are all very popular. Of course, Rome was not built in a day. All of these exercises are done gradually and with varying intensity. Hence, muscle fatigue is inevitable.

Generally speaking, high-intensity exercise or long-term continuous exercise may elevate the levels of metabolites such as creatinine phosphokinase (CPK) or the activities of enzymes such as lactate dehydrogenase (LDH) and thus lead to fatigue. However, these physiological parameters vary depending on the intensity and duration of exercise and reflect the levels of fatigue as well as muscle recovery.

CPK is an enzyme that plays an important role in skeletal and cardiac muscles during exercise. Therefore, the levels of muscle fatigue can be quantified by measuring the levels of CPK. Such an enzyme can be increased by strenuous exercise for several days.

LDH is one of the enzymes responsible for converting glucose into energy, and therefore it is usually utilized as an indicator of liver disease or myocardial infarction. It can also be used as an indicator of exercise intensity and physiological responses due to its prominent existence in skeletal muscles.

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1. Animal Studies Regarding the Effects of Antroquinonol on Exercise-induced Fatigue Improvement GBC, Yuanpei University of Medical Technology, National Chung Hsing University and National Hsinchu University of Education jointly conducted an exercise exhaustion-induced fatigue study using animal models. The purpose of this study was to compare post-exercise exhaustion levels of fatigue indicators such as CPK, LDH, lactic acid, uric acid and BUN in rats fed with Antrodia camphorata mycelia containing Antroquinonol relative to the control group (which was not fed with chow containing Antroquinonol). Rats were randomly allocated into the control group, low-dose Antroquinonol (lowAntro), medium-dose Antroquinonol (medium-Antro) and high-dose Antroquinonol (high-Antro) subgroups, with each group containing 20 rats.

After 4 weeks of intervention (placebo, control low-Antro, medium-Antro and high-Antro), all rats performed exercise exhaustion tests to compare the effects of Antroquinonol between groups.

Figure 35. Exercise Exhaustion Test on Rats Post-exercise exhaustion blood test samples in rats were collected and analyzed. The results showed that individual fatigue indicators were significantly elevated along with exercise exhaustion. The comparison results between experimental groups and the control group are shown in the following figure:

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As described in Figure 36, exercise durations between groups were dose-depe nden t to the amount of Antrodia camphorata consumption.

Figure 36. Effects on Exercise Duration Prolongation

As shown in Figure 37, the increment levels of CPK induced by exercise exhaustion were inhibited depending on the doses of Antrodia camphorata consumption.

Figure 37. Effects on CPK Reduction

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As shown in Figure 38, the increment levels of LDH induced by exercise exhaustion was inhibited depending on the doses of Antrodia camphorata consumption.

Figure 38. Effects on LDH Reduction

As shown in Figure 39, the increment levels of uric acid induced by exercise exhaustion were inhibited depending on the doses of Antrodia camphorata consumption.

Figure 39. Effects on Uric Acid Reduction

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As shown in Figure 40, the increment levels of lactic acid induced by exercise exhaustion were inhibited depending on the doses of Antrodia camphorata consumption.

Figure 40. Effects on Lactic Acid Reduction

As shown in Figure 41, the increment levels of BUN induced by exercise exhaustion were inhibited depending on the doses of Antrodia camphorata consumption.

Figure 41. Effects on BUN Reduction 　　Based on the aforementioned results, the increment and accumulation levels of exercise exhaustion generated wastes such as CPK, LDH, uric acid, lactic acid and BUN in rats fed with Antrodia camphorata mycelia containing Antroquinonol were inhibited significantly relative to the placebo group, which indicated that Antrodia camphorata mycelia containing Antroquinonol had effects on fatigue recovery.

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2. Human Studies Regarding the Effects of Antroquinonol on Exercise-induced Fatigue Improvement GBC, Yuanpei University of Medical Technology, National Hsinchu University of Education and Taipei Veterans General Hospital jointly conducted a study investigating “The Anti-Fatigue Effects of Antrodia camphorata Containing Antroquinonol after Exercise Exhaustion in Athletes.” The study recruited a total of 15 healthy athletes as subjects for the study. The postexercise exhaustion blood test samples of athletes consuming a consecutive week of Antrodia camphorata containing Antroquinonol were collected for analyzing CPK and ammonia levels relative to those of the control group. The results showed that the increment of CPK and ammonia levels in the DE group (dietary Antroquinonol with exercise) was significantly inhibited, which proved that Antroquinonol expedited fatigue recovery in the human body. The study results were also published in the scientific conference of the Olympic Games at Beijing in 2008.

As shown in Figure 42, the CPK level in the DE group (dietary Antroquinonol with exercise) recovered faster than the PE group (placebo with exercise).

Figure 42. The Recovery of Exercise-induced Fatigue (CPK Level)

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As shown in Figure 43, the ammonia level in the DE group (dietary Antroquinonol with exercise) recovered faster than the PE group (placebo with exercise).

Figure 43. The Recovery of Exercise-induced Fatigue (Ammonia Level)

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