John C. Holliday1
Wang Ruwei, Ji Peijun, Li Shuifu, Xia Jinxing2
Xie Jianjun3
Zhan Hongpeng, Sun Huiling, Lei Lixing, Zhang Guoyong4
Li Songhua5
Yu Jin6

1 Aloha Medicinals, Maui, Hawaii
2 The National Medicinal Academy of Zhejiang Province, China
3 The Sino-Japanese Academy of Plant Resources in Lishui City, China
4 The People’s Hospital in Lishui City, China
5 Daogen Medical College in Japan, Jiangjin Guochi Internal Medicine Hospital Daogen ,Japan
6 The Chinese Medicine Hospital in Hangzhou City, China

Abstract: This paper reports on the clinic trial results of Immune–Assist Brand mushroom extract mixture for the treatment of Alcoholic Liver Disease and hyperlipidemia. Through this study the preparation method, the quality control standards, the medicinal function and the safety and toxicity study, we found that this preparation was both safe and effective, and shows great potential as a preventive and health care medicine for treating and curing the disease of alcoholic liver and hyperlipidemia. We found that this polysaccharide mixture could not only restrain the alcohol-induced damage to the liver cells, but also enhances the restoration of liver function and decreases blood lipids. The results were significant and safe. This compound shows great promise for use in clinical therapy.

Key words: Immune-Assist, polysaccharide, medicinal mushrooms, Alcoholic Liver Disease, Hyperlipidemia

Introduction: Alcohol is used widely throughout society, and often for medicinal purposes such as to stimulate the appetite, cure pain, and eliminate fatigue and as a disinfectant. But alcohol use is more often indicated in health problems, for example a wide range of acute and chronic diseases and behavioral dysfunction. Chronic alcoholism has become a global social problem. In America alone, an estimated 100,000 people die annually due to the abuse of alcohol. The economic burden in this one country is estimated to be about 100 billion dollars a year. Chronic alcoholism has become a familiar disease in many other countries as well. Because of this, many experts at home and abroad have been studying effective ways to restrain the damage to liver cells caused by chronic abuse of alcohol, and ways of treating or curing the fibrosis of the liver resultant of chronic alcohol abuse. In recent years, science has found that the polysaccharides extracted from edible mushrooms have various biologically active functions. The city of Lishui, which lies in the southwest of the Zhejiang province of China, has a rich resource of edible mushrooms, and a long history of the use of medicinal mushrooms. A thorough review of the literature and customs of this area has shown that these mushrooms can protect the liver, decrease blood lipids and improve immune function. According to this long history of folk-usage, a modern extraction method was developed to produce an effective medicinal mushroom polysaccharide combination. This mushroom extract combination is marketed in America under the brand name Immune-Assist Critical Care Formula by Aloha Medicinals Inc. The components of this formula are alcohol precipitated hot water extracts of Lentinula edodes, Grifola frondosa, Coriolus [Trametes] versicolor, Agaricus blazei, Ganoderma lucidum, Cordyceps sinensis, and the extra-cellular compounds derived from the spent culture broth of liquid-fermented Cordyceps sinensis. From April of 2001 to May of 2002, this research group used the Immune-Assist formula in the treatment of alcoholic liver disease and hyperlipidemia. The tablets used in this study were 500 mg each of active ingredients containing 400 mg mixture of very complex polysaccharides, mainly (d)beta-glucans of differing structures, primarily 1↔3 main chain structure with 1↔6 side branching. With more than 200 differing polysaccharide structures, there are many other polysaccharides present besides these prototypical and well understood mushroom-derived immunomodulator compounds. The PRC government research authorization number granted to this project was 99-118. The primary research results are as follows.

1) Formula and preparation method
1.1Formula: Each tablet contains 500 mg mixture of active ingredients consisting of equal parts of the following: Lentinula edodes polysaccharides (Lentinan), Grifola frondosa polysaccharides (Maitake D-Fraction), Trametes versicolor protein-bound polysaccharides (PSK and PSP), Cordyceps sinensis Polysaccharides and exo-polysaccharides, Agaricus blazei Polysaccharide and Ganoderma lucidum polysaccharides. These polysaccharides were extracted from full spectrum mycoproducts grown by sterile tissue culture using a proprietary two step extraction and purification process consisting of extraction with hot water, repeated 4 times at 98-99 degrees C, concentration of the water extract portion under reduced pressure, then addition of 4 times the volume of pharmaceutical grade ethanol, which causes the purified polysaccharides, protein bound polysaccharides and heteropolysaccharides to precipitate from the solution, thus separating the alcohol soluble portion. The precipitated polysaccharide compounds are then collected and spray dried. This purified protein-polysaccharide complex is blended with pharmaceutical binders and excipients to manufacture tablets as per usual protocols used in tableting.

2) Quality control and verification standard
2.1 Character: The tablet is granular, light brown with a characteristic taste and smell.
2.2 Differentiation and verification:
(1) To 1.0 g of the ground tablets add 2 mol/L solution of hydrochloric acid, then dissolve both. Next add ninhydrin 2 mg, slowly heating so the solution changes from deep blue to light blue.
(2) To 1.0 g of the ground tablets add 20ml water to dissolve. To 5ml of this solution add silver nitrate 2.5ml and a black deposition of silver appears.
2.3 This product should measure up to all the medicine rules in the first addendum of “The Codex in the People’s Republic of China” (Edition 1).
2.4 Shelf life experiment: Divide the tablets into 3 groups and store them under normal temperature. Periodically perform character differentiation, assay the solubility in water, and differentiate with computer aided analytical equipment GC, FTIR and HPLC spectroscopy to assure a minimum of 90% conformance with datum as-manufactured. Confirm sterility with SPC method for total CFU count, yeast and mold and e-coli in January, February, March, June, and December. All the datum accords with the related rules in the first addendum of “The Codex in the People’s Republic of China” (Edition 1).

3) Toxicity study (presided over by Dr. Li Songhua at Daogen Medical College, Japan )
3.1 Acute toxicity experiment: 20 baby mice (20±1g each, half male half female), administer P.O. a solution of the polysaccharide tablet 3 times / 24 hours (500 mg for each mouse every time), total dose is 75g/kg/day. Maintain this dosage for 7 days and otherwise feed according to normal. At the end of the seven days all the mice are healthy and none show any signs of toxicity or abnormality. This short-term overdosage is approximately 835 times the normal recommended human adult dosage on a mg/kg basis.
3.2 Long-term toxicity experiment: 80 healthy adult mice were administered the solution P.O. at 10g/kg/day for 90 days. At 90 days, the mice are sacrificed and all organs assayed for signs of toxicity. The mice showed no abnormal characteristics and the tissues show no toxicity changes. This confirms that this product has little toxicity for long-term administration.

Clinical Research

4.1 Objectives and methods
Subjects: The experimental group includes 48 cases of Alcoholic Liver Disease (some of whom also have Hyperlipidemia), all the cases are patients confined in the analytical facility and all have abnormal indexes after medical examination. The patients are all male and their ages are from 31 to 70, with an average age of 54. Among these cases, there are 26 cases of alcoholic fatty liver, 11 cases of alcoholic hepatitis, 10 cases of alcoholic fibrosis of liver and one case of hepato-cirrhosis. The diagnosis all comply with well accepted medical standards. The control group consists of 17 cases that are patients in the analytical facility at the same time. The subjects in the control group have no obvious differentiation to those of experimental group for age, history of drinking alcohol or state of illness according to statistical methods. (All patients came from Lishui City, PRC. Four cooperative medical units referred both groups of patients).

Methods: The experimental group is treated with Immune-Assist, 3 times a day, 2 tablets per dose for the full 13-month period. The comparison group is treated with vitamin E, 400 IU, 3 times a day for the same period.

4.2 Result
4.2.1 HA and LN: Research the anti-fibrosis function of the Immune-Assist tablets according to the analytical changes of blood serum HA and LN. The result are shown in Table 1 and Graph 1A.

Table 1 Analysis of blood serum HA and LN between experiment group and comparison group

Group	HA (µg/L)	LN (µg/L)
Experimental group	Before treatment 296.81±23.36	176.30±12.37
	After treatment 102.29±19.83	97.45±17.50
Control group	Before treatment 276.12±25.20	181.27±13.51
	After treatment 206.31±21.56	156.17±16.79

The content changes of blood serum HA and LN before and after curing (X±SD)
The comparisons to before treatment for the same group are P<0.01, after treatment for the same group are *P<0.001

Graph 1A Changes in Blood Serum HA and LN

These result shows that the content of blood serum HA and LN decreased apparently during and after treatment for the experimental group, but not so obviously with the control group. According to the comparison of HA and LN of the experimental group and those of control group after treatment, there is a great differentiation. The lesser changes in blood serum shown by the control group can be entirely attributed to the cessation of alcohol consumption by that group during the duration of the test, while the greater improvement noted in the experimental group must be due to other factors. That is to say that this Immune-Assist product has great functionality for anti-fibrosis and protection of liver in these advanced liver dysfunction cases.

4.2.2 ALT and AST: Among 48 patients in the experimental group, 30 had abnormal levels of ALT, and 27 had abnormal levels of AST. After 4 weeks of treatment with Immune-Assist, the patients were analyzed and the following results were found: For the 30 abnormal cases of ALT, 10 cases had changed to normal (33.3%), 20 cases had no significant changes. For the 27 abnormal cases of AST, 7 cases changed to normal (25.9%), 20 cases had no significant changes.

After 4 weeks treatment for the control group, among 17 abnormal cases of ALT, 6 cases changed to normal (35.3%) and 6 abnormal cases of AST changed to normal (35.3%), 11 cases had no significant changes. For these two groups, there are no apparent differences.

These results suggest the changes of ALT and AST in the experimental group have no obvious differentiation to the control group before treatment and after treatment. This indicates that this product has no apparent effect for degradation of these enzymes.

4.2.3 Triglycerides and Cholesterol: Among 48 cases in the experimental group, after 4 weeks of treatment, for the 31 abnormal cases of triglyceride, 30 cases changed to normal (96.7%) and one case had no clear changes. Among 28 abnormal cases of cholesterol, 26 cases changed to normal (92.9%).

After 4 weeks treatment for the control group, among 16 abnormal cases of triglyceride, 6 cases changed to normal. (37.5%) Among 14 abnormal cases of cholesterol, 5 cases changed to normal (35.7%) and 9 cases have no apparently differentiation. Data shown in Graph 2 below

Graph 2- Hyperlipidemia results after 4 weeks treatment with Immune-Assist

These results suggests the changes of triglyceride and cholesterol in the experimental group have obvious differentiation to the control group before and after treatment. This indicates that this product has significant effect in reducing blood fat and cholesterol.

Efficacy Research in the Laboratory
The Hygienic Food College of the National Medicinal Academy of Zhejiang Province, PRC was entrusted to research the two main functions of this product to protect the liver and to reduce abnormal levels of blood fat. The Disease Control Center of Nanjing, China did the animal experimentation. All the results gathered suggest that the mushroom polysaccharide combination Immune Assist has apparent and profound function in decreasing the blood cholesterol and triglycerides, and at the same time has obvious effect for the chemical protection and repair of the damaged liver, but the effect on degradation of some of the liver enzymes was not as apparent. The above results have been reported to Health Bureau of China.

Discussion
In recent years the diseases of the Alcoholic Liver and high-blood fat have increased quickly. These diseases are not only a great economic burden for the family and society, but also have a great influence on the patients’ jobs and quality of life. It is very important for us to look for a reliable food or nutritional supplement to help prevent this alcohol damage and where possible to reliably treat the disease of Alcoholic Liver and the associated hyperlipidemia. According to the references [2-4], edible mushrooms have many good medicinal functions and bio-active compounds. They can improve the patient’s overall resistance and disease dormancy period, increase the appetite, ameliorate fatigue, regulate and enhance the patient’s immunity, etc. By consuming these mushrooms the body’s nonspecific immunity is enhanced, there is a measurable improvement in the secretion of IgA, an increase in the function of mononuclear-phagocytes and in the activity of the NK cells, regulation of the immune balance, resistance to the alcoholic damage of liver cell efficiency and acceleration of the restoration and regeneration of damaged liver tissue cells. This study shows that edible fungi have certain curative effect on chemically induced liver damage and in blood fat reduction. Edible fungi can play an important role in the clinical treatment for these conditions.

Reference:
1.Wang Qiqiu The Modern Analysis for Components of Chinese Medicine; Guiyang: Guizhou Scientific Publishing House, 1987; 270-27
2.Hileino H, Yoshxaha M, Suzuk Y, et al. Isolation and hypoglycemic activity of trichosons A.B.C.D. and E, glycans of trichos anthes kirilowii root, planta med, 1989; 55(4); 349
3.EA Boxle et al. Pharma pharmacol 1982; 34; 563
4.Li Guangzhou Research for Increasing Antineoplastic Function of lentinan, The Application of Medicine in China 2000, 17(5):354-355

Note 1 – The original Immune-Assist Critical Care Formula manufactured Aloha Medicinals Inc. is marketed in the form of capsules containing no inactive ingredients or additives, but for the purposes of this study it was decided to administer the compound in the form of tablets to make them indistinguishable from the placebo. The tablet exipients were normal pharmaceutical grade materials as used in normal tabl;eting priotocols. Otherwise, the formula used in this study was identical to that of the commercially available products.

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