Ganoderma lucidum

Ganoderma lucidum of LakzwamLakzwam is een merkwaardige en mooi glanzende zwam, die wel uit gelakt hout lijkt gesneden. In Japan noemt men deze soort Reishi, hij wordt er al eeuwen gebruikt als gezondheidsmiddel en zelfs als geluksbrenger.Wetenschappelijk onderzoek lijkt de veelzijdige en wonderbaarlijke werking van deze paddestoel te bevestigen. Zo werd er in verschillende dierproeven zowel bij astma als bij eczeem een anti-allergische activiteit aangetoond.Waterige extracten van de Lakzwam verlaagden ook de glucoseconcentratie in het bloed bij ratten, waarmee het volksgebruik bij suikerziekte bevestigd werd.Daarbij werden in de paddestoel twee glycanen (Ganoderma A en B) gevonden die bloedsuikerverlagend werken. Verder zijn er ook stoffen gevonden die de aanmaak van cholesterol afremmen. En alsof dat nog niet genoeg is, werd er in Ganoderma ook een bloeddrukverlagende ACE-inhibitor (Angiotensin Converting Enzym) gevonden.

Le Ganoderma lucidum (GL) est un agent apoptoptique incontournable dans la lutte antitumorale. Il agit par de nombreuses voies (caspases, p38 mitogen-activated kinase [p38 MAPK], NF-kappaB, Akt/Erk, Bcl2, VEGF) et est utilisé avec succès dans les cancers digestifs, mammaires, prostatiques et les leucémies-lymphomes. Il possède des propriétés antivirales contre l’herpès ou l’hépatite B. C’est un excellent néphrohépatoprotecteur; propriété précieuse chez les patients qui subissent une chimiothérapie. Il s’impose dans le traitement du syndrome métabolique, grâce à ses effets sur le cholestérol, l’HTA ou la glycémie. Par ses propriétés antalgiques ou anti-inflammatoires dénuées d’effet indésirable, il soulage les patients atteints d’arthrose ou d’arthrite.

Ganoderma lucidum (GL) is an essential apoptotic agent against tumoral cells which action implies several pathways (caspases, p38 MAPK, NF-kappaB, Akt/Erk, Bcl2, VEGF). It is appropriately recommended in digestive, breast, prostatic or leucemic cancers. It possesses anti-viral properties against herpes or hepatitis B. Its excellent nephro-hepatoprotective profile enables to protect patients undergoing chemotherapy and receiving many medications. Because of its actions against hypertension, diabetes, cholesterol, it could be used in metabolic diseases. It is an anti-inflammatory and antalgic agent devoided of noxious effect.

Ganoderma lucidum onderzoek kanker

Fresh fruit bodies of Ganoderma lucidum were extracted with 70% ethanol at room temperature. The extract (GL) showed significant anti-angiogenic activity, which was detected using a chick embryo chorioallantoic membrane assay. GL significantly inhibited LPS-induced NO production in RAW 264.7 macrophages. These results support the anti-tumor effect of Ganoderma lucidum.

Author Keywords: Ganoderma lucidum; Anti-angiogenic activity; Nitric oxide; Folk medicine

1. Introduction

The fruit bodies of the mushroom Ganoderma lucidum (Fr.) Karst (Polyporaceae) have been used to treat a variety of diseases such as hepatitis, neurasthenia, deficiency fatigue, hypercholesterolemia, bronchitis, and cancer ([Lin et al., 1995 and Yang et al., 2000]). Water and methanol soluble substances isolated from Ganoderma lucidum contained antiviral activities, which were evaluated by the cytopathic effect inhibition assay and plaque reduction assay ([Eo et al., 1999]). The acidic protein-bound polysaccharide obtained from Ganoderma lucidum was identified to contain antiherpetic activities, which was enhanced in combination with acyclovir and interferons ([Oh et al., 2000 and Kim et al., 2000b]). Ganoderiol F and ganodermanontriol, isolated from a methanol extract of the fruit bodies of Ganoderma lucidum, were found to be active as anti-HIV-1 agents, and ganoderic acids and ganoderiols were moderately active inhibitors against HIV-1 protease ([El-Mekkawy et al., 1998]). Ganoderic acid of Ganoderma lucidum had potent beta-glucuronidase-inhibitory activity and hepatoprotective effect against CCl4-induced liver injury, indicating that beta-glucuronidase seems to be closely related to liver injury ([Kim et al., 1999]). Ganoderma lucidum extract induced the neuronal differentiation of PC12 cells and prevented nerve growth factor-dependent PC12 neurons from apoptosis ([Cheung et al., 2000]). These effects of Ganoderma lucidum might be mediated via the ras/extracellular signal-regulated kinase (Erk) and cAMP-response element binding protein (CREB) signaling pathways ([Cheung et al., 2000]). In this article, we report the anti-angiogenic activity of Ganoderma lucidum, a medicinal white-basidiomycete, and its inhibition of the inducible nitric oxide production.

2. Materials and methods

2.1. Microbiological materials

Fresh fruit bodies of Ganoderma lucidum were obtained from a local farm in Seoul, South Korea. The same were authenticated by Prof. Ki-Oug Yoo, Division of Life Sciences, Kangwon National University, Chuncheon, South Korea, and a voucher specimen of the mushroom was deposited in the herbarium of Division of Life Sciences, Kangwon National University, under the acquisition number of KNU51778.

2.2. Extraction

The fruit bodies of Ganoderma lucidum were ground under liquid nitrogen and extracted with 70% ethanol at room temperature. The ethanolic extract was evaporated in vacuo to give a solid material (yield: 2.8%).

2.3. Cell cultures

RAW 264.7, a mouse macrophage cell line, was obtained from American Type Culture Collection (Manassas, VA). Cells were cultured in phenol red-free DMEM containing 100 U/ml penicillin G, 100 small mu, Greekg/ml streptomycin, and 10% heat-inactivated FBS and were maintained at 37 °C in a humidified incubator containing 5% CO2.

2.4. Chorioallantoic membrane (CAM) assay

Anti-angiogenic activity was measured using chorioallantoic membrane assay as previously described ([Kim et al., 2000a]). The fertilized chicken eggs used in this study were kept in a humidified egg incubator at 37 °C. After 3.5-day incubation, about 2 ml of albumin was aspirated from the eggs with a 22-gauge needle through the small hole drilled at the narrow end of the eggs, allowing the small CAM and yolk sac to drop away from the shell membrane. The shell covering the air sac was punched out and removed by forceps, and the shell membrane on the floor of the air sac was peeled away. A sample-loaded Thermanox-coverslip was applied to the CAM surface of the 4.5-day-old chick embryo, which was then returned to the incubator. Two days later, an appropriate volume of a 10% fat emulsion (Intralipose, 10%) was injected using a 33-gauge needle into a 6.5-day-old embryo chorioallantois. The eggs were then observed under a microscope.

2.5. Assay for nitrite concentration

Accumulated nitrite (NO2-) in the media of cells was measured using an automated colorimetric assay based on the Griess reaction ([Murphy & Noack, 1994]). Cells were plated in 24-well culture plates at a density of 5×105 cells and incubated with LPS (1 small mu, Greekg/ml) plus IFN-small gamma, Greek (10 U/ml) in the presence or absence of GL for 24 h. The supernatants were reacted with the Griess reagent at room temperature for 10 min, and then NO2- concentration was determined by measuring the absorbance at 540 nm in a UV-Vis spectrophotometer. The standard curve was obtained using the known concentration of sodium nitrite.

2.6. Statistical analyses

The data were analyzed for statistical significance using Student's t-test. P values less than 0.05 were considered to be significant.

3. Results and discussion

To investigate pharmacological actions of Ganoderma lucidum, a mushroom long used in the Oriental countries for a broad range of disorders, fresh fruit bodies of Ganoderma lucidum were extracted with 70% ethanol. Brownish solid material (GL) was obtained by evaporation to dryness under vacuum. Sugar composition in GL was analyzed as previously described ([Blakeney et al., 1983]). It is highly rich in Image-glucose residues (638.9 mg/100 g solid), and also relatively rich in Image-mannose and Image-galactose residues.

3.1. Anti-angiogenic activity

Anti-angiogenic therapy may be an important component of treatment regimens for cancer patients. Several endogenous angiogenesis inhibitors including angiostatin ([Gately et al., 1997]) and endostatin ( [Sasaki et al., 1998]) were identified. Angiogenesis inhibitors have been screened from various natural products. GL showed strong anti-angiogenic activity in the CAM assay, which is very useful for detecting in vivo angiogenesis ( Fig. 1). When 1.25, 2.5, 5, or 10 small mu, Greekg GL per egg was applied, the percentage inhibition of angiogenesis were found to be 47.1, 57.6, 64.7, or 67.1%, respectively (Fig. 1). This clearly indicates that the anti-angiogenic activity of GL is dose dependent. At a dose of 10 small mu, Greekg per egg, its anti-angiogenic activity is comparable to that of retinoic acid (1 small mu, Greekg per egg) used as a control. Propolis, used in Oriental medicine as an anti-inflammatory and anti-carcinogenic agent, was reported to exert anti-angiogenic activity detected in the CAM assay ([Song et al., 2002]). The anti-angiogenic activity of Ganoderma lucidum might be linked with its plausible anti-tumor activity.

3.2. Inhibition of nitric oxide production

Nitric oxide (NO) is synthesized from Image-arginine by nitric oxide synthase (NOS; [Kwon et al., 1990]). For the expression of inducible NOS (iNOS) gene expression, mammalian cells should be triggered by specific stimulants such as pro-inflammatory cytokines and bacterial lipopolysaccharide (LPS; [Chesrown et al., 1994]). Since iNOS-derived NO is involved in various pathological conditions such as autoimmune diseases ( [Singh et al., 2000]), iNOS inhibitors, which repress iNOS enzyme activity or gene expression in macrophages, have been screened from natural products. Inhibitory effect of GL was examined on LPS-induced NO production in RAW 264.7 macrophages ( Fig. 2). The accumulated nitrite, estimated by the Griess method, in the culture medium was used as an index for NO synthesis from these cells. After treatment with LPS plus IFN-small gamma, Greek for 24 h, nitrite concentration markedly increased about 40-fold (~40 small mu, GreekM). When cells were treated with various concentrations of GL, nitrite production induced by LPS plus IFN-small gamma, Greek was significantly inhibited at concentrations of 70 and 100 small mu, Greekg/ml in a concentration-dependent manner. The IC50 value of the extract is 78.89 small mu, Greekg/ml, which was obtained from Fig. 2. No effects on cell viability were observed in any concentrations of GL as determined by MTT assay (data not shown). This indicates that the Ganoderma lucidum extract reasonably inhibits LPS-induced NO production in macrophages, which corresponds with its anti-angiogenic activity. Recently, an iNOS inhibitor, NG-nitro-Image-arginine methyl ester, was reported to contain potent anti-angiogenic effects on plasmacytoma in a severe combined immunodeficient mouse model ([Uneda et al., 2003]), whereas potentiation of the NO signaling pathway, after irradiation, induced profound alterations in the phenotype of endothelial cells leading to tumor angiogenesis ( [Sonveaux et al., 2003]). The treatment of human umbilical vein endothelial cells with arsenic was found to lead to cell proliferation and activation which specifically enhances angiogenesis via the vascular endothelial growth factor (VEGF)-NOS signaling pathway ( [Kao et al., 2003]).

4. Conclusions

In this article, the Ganoderma lucidum extract was found to contain strong anti-angiogenic activity. It also has inhibitory activity on inducible nitric oxide production which supports its anti-angiogenic activity.

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