1．Introduction
*Huang Xingshu,* Zhu Yongping, Shen Lingling**
In China fungi have been consumed as food traditional drug for more than three thousand years. Among various fungi Grifola frondosa has attracted our attention since 1990. As a food it is delicious and nutritive, consisting all kinds of nutrients. In the eighties of last century non-nutrient components of fungus were reported to have various biological activities including anticancer effect.
We developed protocol for extracting hot water soluble active components from maitake (Grifola frondosa) produced locally. Chemical analysis showed that the extract (GFE) contained about 33% grifolan which is the main active component exerting immune enhancement effect in vivo. The raw dry maitake contained about 3.3% grifolan. We demonstrated that hot water extract of Grifola frondesa (GFE) exerted immune-enhancement and anticancer effect in tumor-bearing mice. In addition, GFE exerted antimutagenic effect to chemical mutagens--benz(a)pyrene and cyclophosmide, i.e. prevention of DNA damage by mutagenic chemicals.
We carried out clinical trial with GFE on 157 cancer patients. It has been showed that it
Enhanced curative effect of chemotherapy and radiotherapy; in addition GFE counteracted the
the side effects of anticancer drugs and radiotherapy. The mechanism involved host-mediated
immune-enhancement and inducing apoptosis in cancer cells. GFE as a new traditional
Chinese drug, entirely different from conventional chemotherapeutic drug. The basic principle
of Chinese Traditional Medicine in the treatment of cancer is “fuzheng quxie” (扶正祛邪)
and “fuzheng guben” (扶正固本) which are approximated to “immunological homeostasis”
in western medicine. There are many biological studies published recently on the mechanism
___________________________________________________________________________
l Professor in Toxicology Department , Zhejiang University School of Medicine, Hangzhou 310031 China
l Rmit University, Australia.
of therapeutic effect of GFE support the concept in traditional Chinese medicine.
Since 1995 as a tonic medicine, GFE capsule was approved by the Ministry of Health. And. 2002 is was registered by SFDA (China), as a supplementary drug (traditional Chinese medicine) to chemotherapy or radiotherapy for cancer patients. The trade name is Maitake 麦特消/灰树花胶囊. The certificate number was 国药 B20020023. It is marketing as an OTC drug in China.
2．The Immune-modulation Effect of GFE on
S180 tumor-bearing Mice (Abstract)
Shen Linling, Huang Xingshu
The immunomodulation effect and antitumor effect of Grifola frondosa Extract (GFE) were studied in tumor-bearing NIH mice. The phagocytic rate and phagocytosis index of peritoneal macrophages in GFE 1000 mg/kg gropu were markely higher than that of the control group (P<0.05 and P<0.01), so did the antibody capacity of spleen T cells (P<0.05). when CYP was used with GFE the inhibition on delayed type hypersensitivity (DTH) and on IL-2 production by CYP were antagonized. IL-2 is ant to activate macrophges and natural killing cells which attack cancer cells.
We found exclusively that GFE increased significantly the diameter of Peyer’s Patch. This revealed that GFE stimulated proliferation of macrophages in Peyer’s patches (Table 1) and this increased phagocytic rate and phagocytesis index of peritoneal-macrophages. When associate this effect with chemotherapy which impairs immune responses in GALT, GFE may counteract the immune impairment by chemotherapy.
The results suggested that host-mediated immune-modulation effect played and important role in the anticancer-effect of GFE in mice.
From these finding the mechanism of antitumor effect of GFE may involve both homoral and cell-mediated immune responses.
Table 1 Effect of PFE on Ileum Peyer’s patch of S-180 bearing mice
|
GFE (mg/kg)
|
N
|
Diameter of Peyer’s patches (%)
|
|
≥2.5 mm
|
<2.5 mm
|
Total
|
|
NS (control)
|
19
|
35
|
65
|
100
|
|
500
|
15
|
65*
|
35
|
100
|
|
1000
|
18
|
70**
|
30
|
100
|
|
2000
|
17
|
62*
|
38
|
100
|
* compared with control P<0.05, ** P<0.01
3．Anticancer Effect and Mechanism of
Grifola frondosa Extract in Mice
Shen Linling, Huang Xingshu
The in-vivo immune-enhancement and anticancer effect of Grifola frondosa hot water extract obtained from the fruit bodies of Grifola frondosa has been studied in mice model with transplanted tumor. The inhibition to solid sarcoma-180 growth and the immune response in S180-bearing mice by oral administration, and the effect of combining GFE with cyclophosphamide (CYP, an anticancer drug) were also examined.
Anticancer experiment in mice: S180 tumor cells of 1×107/ml were implanted into ht axilla of mice. 24 h after transplantation 10 ml/kg of GFE in various doses (500, 1000, and 2000 mg/kg) was administered orally on alternated days with 20 successive days, and the control group was given distilled water. CYP 20 mg/kg was administered intraperitoneally to the treated groups every 4 days for 5 times, and the control group was given normal saline. After completion of administration, the mice were sacrificed and the solid tumors were extirpated, weighted and the inhibition rates of tumor growth were calculated.
The parameters of immunity of S180-bearing mice included: WBC count, phagocytosis by peritoneal macrophages, delayed type hypersensitivity response, antibody production capacity of spleen T cells, and interleukin-2 production and activity were measured. In addition, diameter of Peyer’s patches in intestinal mucosa was measured. The morphological changes were examined under microscope.
Experimental results showed that oral administration of GFE inhibited the growth of S180 sarcoma in mice. The inhibitory rates were 48.5% at the dose of 1000 mg/kg. The difference of the average of tumor weight between treated group and control group was significant (P<0.01) and the tumor growth inhibition rate was increased with the GFE doses. GFE in combination with CYP anticancer effect was increased significantly and inhibitory rate reaching 95.2%. The complete restraint of S180 tumor was observed in either CYP alone group or the combined group.
Table 1 Combined anticancer effect of CYP with GFE in mice*
|
Groups dose (mg/kg)
|
Number of mice
(initial / final)
|
Weight of cancer (g)
±s
|
Cancer inhibition
(%)
|
|
Control (distilled water)
|
10/10
|
2.72±1.58
|
|
|
GFE 1000
|
10/10
|
1.40±0.84
|
48.5
|
|
CYP 20
|
10/10
|
0.57±0.59
|
79.0
|
|
GFE 1000 + CYP 20
|
10/10
|
0.13±0.14**
|
95.2**
|
** P<0.01 when compare with control group.
The result of immune function test indicated that the phagocytosis rate and phagocytic index of peritoneal macrophages in the GFE treatment group were significantly higher than the control group (P<0.05 and P<0.01 respectively), so did the antibody production capacity of spleen T cells (P<0.05). GFE could antagonize the inhibition of WBC count and DTH suppression caused by CYP (P<0.01 and P<0.01 respectively) and IL-2 production was restored as well. There was significant difference between the combined group and CYP alone group (P<0.01). The phagocytic index of peritoneal macrophages was augmented when CYP was given in combination with GFE simultaneously (P<0.05). The results that GFE promoted both specific and nonspecific immune functions suggested that host-mediated immune-medulation effect played an important role in the antitumor effect of GFE.
GFE stimulated the gut-associated lymphoid tissue: the ileum Peyer’s patches are important source of immune cells and cytokines. We found that the number of Peyer’s patches with diameter >2.5 mm in S180-bearing mice were increased in GFE group ( table 1 in Part 2 ). The results support the assumption that phagocytic cells in the epithelium of the digestive tract together with the macrophages in Peyer’s patches exhibited phagocytic and antigen-presenting activities. Therefore, the activation of these cells by GFE resulted in increase production of IL-1 by macrophages in gut-associated lymphoid tissue. Then the IL-1 produced entered blood circulation and stimulated the immune system, such as the activation of T cell, B cell, NK, LAK, CTL, macrophages etc. it was reported that helper T cells, NK cells and macrophages exerted cytotoxicity to tumor cells and caused apoptosis.
The essential factors required for anticancer activity were beta-D-glycosyl linkages and high molecular weight (Adachi Y et al.,1989).
Takeyama et al. (1988) demonstrated that targeting of isotope-labeled grifolan to spleen and tumor masses in tumor bearing mice suggested a close relation between anticancer effect and the distribution of grifolan in mice.
GFE stimulated interferon-a production: GFE stimulated interferon-a which exerted anticancer effect and protected the animals from NDV (New Castle disease virus) (Shao Xiang et al.,1995).
The conclusions drew from the above studies are:
(1) GFE administered orally to NIH mice exerted definite anticancer effect and increase the cancer inhibition rate to 95.2% when combined with CYP (or other anticancer drugs).
(2) The mechanism of anticancer effect by GFE is host-mediated immune-modulation (increase immune cells , their activity, and cytokines) and killing cancer cells finally.
(3) GFE restored immune function of S180-bearing mice suppressed by CYP or other anticancer drugs so that augmented the cancer effect in total.
4．Toxicology and adverse reactions of GFE
Toxicology study:
(1)Mutagenicity tests: (a) Ames test revealed no mutagenicity; by contraries GFE exerted antimutagenicity. (b) Micronucleus test in mice revealed no mutagenicity; in contrast GFE exerted antimutagenic effect.
The results of these two tests were reported exclusively in the world. These strongly support the counter action of GFE on mutagenic toxicity of anticancer drugs.
(2) GFE exerted no cytotoxicity on 3T3 cells by Nelson Procedure.
(3) Oral LD50 of GFE in rat > 10000 mg/kg.
(4) Long-term toxicity test: times clinical dose fed mice for I month revealed no abnormal change.
Adverse reactions: No reported since 1995.
Interactions with drugs: Monitor blood glucose levels closely due to claims that maitake has hypoglycemic effects.
Interactions with food: No interactions are known to occur.
GFE is considered as GRAS (generally recognized as safe) by USFDA. In a word, GFE is a very safe traditional Chinese drug.
