12. The Activity of GCP’s Various Fractions

GCP is a fermented product made of a combination of basidiomycetes and soybean isoflavones. GCP contains a rich content of isoflavone aglycone that seem to be responsible for induction of apoptosis and inhibition of angiogenesis. GCP also has a rich content of polysaccharides derived from basidiomycetes which seem to enhance immune response. To further observe GCP’s various properties, we extracted GCP fractions and detected their different functions.

A) Method of GCP fraction

GCP fractionation was done according to the following figure below. First, GCP was treated with ethanol to extract the isoflavones such as genistein. Next, water-soluble polysaccharides, amino acids, proteins, etc. were extracted with water. There were water insoluble polysaccharides such as fiber in ethanol and water insoluble fraction.

B) GCP’s Anti-Angiogenesis Effects in vivo: Rat Mesenteric-Window Assay

Methods:

Samples: ① GCP containing 116 mg/g of genistein, taken as 100 % extraction ratio; ② E: Ethanol soluble fraction containing 199 mg/g of genistein, extraction ratio = 48 %; ③ W: Water soluble fraction containing 11.8 mg/g of genistein, extraction ratio = 38.5 %; ④ EW: Ethanol and Water insoluble fraction containing 13.4 mg/g of genistein, extraction ratio = 5.5 %. The administration dosages of each fraction were adjusted according to the extraction ratio from GCP.

A modification of the rat mesenteric-window assay was used for investigation of anti-angiogenesis, angiogenesis on mesenteric windows of rats was induced by intraperitoneal injection of mast cell stimulation for a given period. 5 weeks old Sprague-Dawley rats were fed freely available rat diet. They were divided into five groups (♂3,♀3 per group). The control group, GCP group, E group, W group, EW group. The control group was fed with sample an un- supplemented diet; and the others were fed a supplemented diet with each sample. The dose of GCP group was 1.0g/kgBW, and the dose of the other groups depended on the yield (%) of each fraction, the dose of E group was 0.48g/kg, the dose of W group was 0.385g/kg, and EW group was 0.05g/kg. Compound 48/80 that induces angiogenesis was administered by intraperitoneal injection for a week. After all of the groups were fed each diet that was freely available for 10 days, the rats were sacrificed. Mesenteric windows whose vasculatures were visualized by the introduction of black ink were surgically removed. The vascularized areas on the mesenteric windows were calculated using the soft NIH imaging and each group was compared.

Results：

This figure and table show that the percentage of vascularized areas in mesenteric window from rats fed GCP was significantly decreased when compared with that of rats on sample un-supplemented diets (Inhibition rate = 37.5 %). E (Ethanol soluble) fraction appeared to have the strongest efficacy on anti-angiogenesis (Inhibition rate = 51.1 %). W (Water-soluble) fraction (Inhibition rate = 16.1 %), EW (Ethanol-Water insoluble) fraction (Inhibition rate = 11.4 %) have no statistically significant difference against Control. This result revealed GCP isoflavone rich fraction, mainly genistein might be responsible for the effect of anti-angiogenesis. But water-soluble fraction still have limited inhibitory effect even through it was not significant statistically. It suggested there might be some water-soluble components that have weak effect on inhibition of angiogenesis.

C) Anti-tumor activity of GCP Fractions

GCP exerted its cytotoxicity on cancer cells as described in chapter 6-1, whereby the IC 50 of GCP inhibits various tumor cell proliferations in a concentration range of 10-50 µg/ml. This IC50 was higher than that for purified commercial Genistein, which suggests that the cytotoxicity of GCP is not only from the genistein itself but also from non-genistein components in the total GCP – FD. In order to verify this assumption, we detected the effects of GCP and its fractions on tumor cell proliferation by using MTT assay.

Method: The tumor cell used in this assay was 3LL mouse lung cancer cell line. The contents of GCP and its fractions are shown in the table above. All the samples were dissolved in DMSO to form 1000 g/ml and 100 g/ml. The final DMSO concentration was less than 0.1%. The content of the genistein samples is as described earlier. The commercial genistein was taken as a positive control since it contained the same content as the genistein in GCP. The tumor cell suspensions were adjusted into 2 x10⁴/well and incubated at 37ºC for 24 hours in 96-well microplates. The samples were added into the cells in the concentration of 1000 µg/ml to 0.1 µg/ml consecutively. The cells were cultured for an additional 48 hours. The proliferation of the tumor cells was measured by MTT assay.

Result:

The figures show that the two strongest fractions were E–L3 and W–L3. These were consistent with their high content of genistein. Fraction E-L2 showed higher cytotoxicity to the tumor cells than did the high concentration of Genistein. These results suggest that genistein is the main component responsible for cytotoxicity, but other unknown components in Fraction E-L2 are also responsible for cytotoxicity.

D) Enhancement of immune response by GCP fractions

In this study, we investigated the cytokine production from the splenocytes of tumor bearing mice by treatment with GCP and its fractions.

Methods：

Mouse lung cancer cells 3LL (2x10⁴/mouse) were inoculated s.c. into syngeneic C57 mice (three mice per group) and administered orally with various samples for one week (the samples were: control = tap water; GCP = total GCP – FD, GCP water-soluble fraction; and GCP insoluble fraction for both water or EtOH, which was respected containing mainly polysaccharide derived from basidiomycetes). After one week of treatments, the splenocytes were removed sterilely and adjusted into 1x10⁶/ mL, and stimulated with Con-A (10 g/ml) for 48 hours, then collected the supernatants, the cytokines in the supernatants including IL-2, IFN-γ,and TNF-α were detected by ELISA assay.

Results：

As the results show in following figures, IFN-γ, IL-2, TNF-α production were GCP insoluble fractions ＞ GCP water soluble fraction ＞ total GCP-FD＞Control (only Con-A stimulated). Particularly in the GCP insoluble fraction, the immune enhancing activities were 7 times higher than the control for IFN-γ, 30 times higher for IL-2, and 11 times higher for TNF-α.

Summary: The results described above indicate that GCP insoluble fraction induced higher IFN-γproduction as well as other cytokines production. It strongly suggests basidiomycetes derived polysaccharide are involved in enhancing immune response. Furthermore, these results suggest that GCP is an anti-tumor substance due to its anti-angiogenesis activity, ability to induce apoptosis, and also its immune enhancing activity.