5. GCP Safety

Isoflavones have been consumed daily for thousands of years as a component of soybeans and soybean products in Asian countries, especially Japan. Isoflavones can be considered safe for human consumption because they have been consumed daily in the human diet for centuries. Similarly, basidiomycete mushrooms, such as Lentinus edodes and Ganoderma lucidum, have also been consumed and used in folk medicine for thousands of years. Yet, to further clarify the safety of GCP in humans, an oral single-dose toxicity study and a one-month toxicity study were done in mice.

A) Single dose toxicity study on GCP

Animals: The GCP treated group consisted of two groups of 7 weeks old ddY mice (8 male and 8 female). The control group consisted of 5 male and 5 female mice. The animals were kept at a constant temperature (24±2°C) and humidity (55±20%), under a 12 hour light/dark cycle.

Administration of GCP: GCP was suspended in tap water and orally administered at a dose of 15g/kg body weight (25ml GCP solution/kg body weight). Mice in the control group were administered an equal volume of tap water.

GCP was administered to the mice p.o. in a practicable maximum dose. No deaths occurred within the 24 hours after administration of GCP (Table 1). The observation period lasted for one month and no unusual changes related to the administration of GCP were observed.

Results No toxicity found in acute toxicity test.

B) One-month toxicity study of GCP

Animals: 3 groups of 10-15 male and female mice (7 week old ddY mice) were used in the study. The animals were kept at constant temperature (24 ± 2ºC) and humidity (55 ± 20%) under a 12 hour light/dark cycle.

Administration of GCP: The mice received AIN-76 standard powder diet with a 0.06 % and 1.5 % content of GCP for 4 weeks. The control group was fed the standard powder diet without GCP. Mice were given free access to feed and water.

At the end of the experimental period, the mice were killed and whole blood and serum were obtained and examined. The numbers and differentiation of whole blood cells were counted and organs were weighed. The serum concentration of total cholesterol, triglycerides, blood urea nitrogen (BUN), glucose, glutamic oxaloacetic transaminase (GOT), bilirubin, albumin and alkaline phosphatase (ALP) were measured with detection kits. The serum protein was determined using Lowry’s method.

Statistics Analysis: All the results were expressed by meanｱSE. Comparison between different groups were analyzed by ANOVA and Fisher’s PLSD test.

Results Changes in weight gain, organ weights, food consumption, and other blood, urine parameters were tested. No abnormalities were found.

**The number of red blood cells, white blood cells, and platelets in peripheral blood

Further more, we established a standard range of GCP on their hygienic parameters.

C) Human Monitor Test

26 May-23 June, 2000

Animals: 27 healthy volunteers were tested (14 males and 13 females).

The GCP group: 7 males and 7 females ingested 2g of GCP per day at 8 AM. The blood samples were collected and analyzed on day 0, 14, and 28.

The GCP+AHCC group: 7 males and 6 females ingested 2g GCP per day at 8 AM, and 3g per day of AHCC at 8 PM. The blood samples were collected and analyzed on day 0, 14, and 28.

Biological parameters measured: 1.) Genistein concentration in serum and in urine, and 2.) Biochemical parameters of blood and blood cell counts: ALP, GOT, GPT,γ-GTP, uric acid, creatine, blood sugar, total cholesterol, triglyceride, HDL- cholesterol, BUN and WBC, RBC, Hematocrit, and PLT.

A) Genistein concentration in serum and in urine

The GCP + AHCC group had similar results to the GCP solo group for genistein concentrations both in serum and in urine. The levels increased gradually during the four weeks of administration, suggesting that AHCC does not influence the absorption or metabolism of GCP in humans.

B) Biological Parameters in Blood

No abnormal parameters could be found during the 4 weeks in both the GCP group or the GCP + AHCC group.

Summary:

The results of the single dose toxicity study showed the LD₅₀ of GCP is more than 15g/kg body weight, which means GCP is a non-toxic substance. In the one-month toxicity study which fed mice with GCP, there was no effects on weight, caloric intake, and weights of the liver, kidney or other organs, (even in the 1.5% GCP diet group). Furthermore, there were no changes found in lipids, liver and kidney function, or blood glucose levels. Whole blood cell counts in all the groups were in normal ranges, and eosinophils were up-regulated in GCP-treated groups. In summary, GCP is safe as a nutrition supplement.