| The Lingzhi (in Chinease) mushroom or Reishi (in Japanese) mushroom encompasses several fungal species of the genus Ganoderma, and most commonly refers to the closely related species, Ganoderma lucidum (Leys.ex Fr.)Karst and Ganoderma sinense Zhao,Xu et Zhang,based on Chinese pharmacopeia description. Scientificaly, the Ganoderma species fall into the Kingdom Fungi, Phylum Basidiomycota, Class Agaricomycetes, Order Polyporales, Family Ganodermataceae, and Genus Ganoderma. Lingzhi mushroom enjoys special veneration in East Asia, where it has been used as a medicinal mushroom in traditional Chinese medicine for more than 2,000 years, making it one of the oldest mushrooms known to have been used medicinally. In traditional Chinese beliefs, Lingzhi or Ganoderma mushroom has specicific preferable effects such as benefiting the Qi (vital energy), tranquilizing the mind, strengthening healthy energy and consolidating the constitution, and is looked as a precious Chinese herbal medicine or a gem in the treasure house of Chinese medicine. Modern studies indicate that Ganoderma species have many effects, such as immune regulation, tumor inhibition, deferring of senility, liver protection, lowering of blood sugar, sedative and analgesic action, memory improvement, etc. The active constituents in Ganoderma mushroom are considered to be polysaccharides, triterpenes and peptides, which have attracted the most attention. Water extract of the mushroom contains primarily polysaccharides. However, ethanol extract consists of a variety of active ingredients in which triterpenes are of predominance, exhibiting lots of biological activities. Alcohol extract of Ganoderma lucidum or the ethanolic active fraction study revealed that ethanol extract improved the learning and memory ability in memory impairment of animal models, inhibited tumor cell growth in mice, and suppressed mouse resposes related to acute itching. Ganoderma triterpenoids also has specific anti-tumor effect. However, the fraction of Ganoderma lucidum obtained by ethanol extraction and then followed by chloroform fractionation as a whole has not been studied, and the immune regulatory function and anti-tumor effect are not very clear. The present study explored the immunomodulatory and anti-tumor effects and the underlying mechanisms of the chloroform fraction of Ganoderma lucidum.Objectives:1,To prepare Chloroform Fraction of Ganoderma lucidum (CFGL) with procedures in which the fruiting body of Ganoderma lucidum was first extracted with ethanal and the ethanal extract was then followed by chloroform fractionation.2,To study the effects of CFGL on proliferative activity of mouse splenocytes induced by concanavalin A (ConA) and lipopolysaccharides (LPS). To study the effects of CFGL on the swelling degree in delayed type hypersensitivity (DTH), the phagocytic function of monocytes/macrophages and the level of serum hemolysin induced by RRBC(rabbit red blood cells) immunization in normal mice in vivo.3,To study the effects of CFGL on the swelling degree in DTH, the phagocytic function of monocytes/macrophages and the level of serum hemolysin induced by RRBC immunization in immunosuppressed mice induced by cyclophosphamide (CTX) in vivo.4,To study the effects of CFGL on the swelling degree in DTH, the phagocytic function of monocytes/macrophages and the level of serum hemolysin induced by RRBC immunization in S180 bearing mice.5,To study the effect of CFGL on proliferative activity of S180 sarcoma cells and the tumor growth inhibition rate in S180 tumor-bearing mice in vivo.Methods:1. Coarse powder of Ganoderma lucidum was extracted for 1h by ethanol (95%) under reflux on a water bath at 70℃for two times and the two portions of ethanol extract solutions were combined and concentrated by vacunm evaporation at 50℃, fowllowed by drying at the constant temperature. Brown extract was collected and suspended in water, followed by petroleum ether and chloroform fractionation sequentially based on polarity difference. The organic reagents were recovered and the petroleum ether and chloroform fractions were vacuum dried.2. Mouse spleen cells were isolated and cultured in the presence of CFGL and Con A or LPS, which are T or B lymphocyte stimulant respectively. After cultured for 48h at 37℃, the cell metabolic or proliferative activity was assayed with MTT colorimetry.3. Fifty normal mice were randomly divided into 5 groups, including DMSO: olive oil (1:9) control group, positive control group and CFGL (200,100,50mg/kg) groups. Animals were gastrically administered with vehicle or investigated drugs once daily for 7 consecutive days. Mouse model of DTH was established by priming the mice with 1% dinitrofluorobenzene(DNFB) applied to the shaved abdomen skin and attacking on the right ear 7 days later.24 hours after attack, the weight of double ears was weighed to evaluate the influence of CFGL on the swelling of the ear tissues. 4. Fifty normal mice were randomly divided into 5 groups, including DMSO:olive oil(1:9)control group, positive control group and CFGL (200,100,50mg/kg) groups. Animals were gastrically administered with vehicle or investigated drugs once daily for 7 consecutive days. On the seventh days,2h after administration, the animals were first weighed and then injected with India ink (diluted with saline for 5 folds) via tail vein in a dose of 0.1mL/10g (body weight), respectively.2min (t1) and 12min (t2) after ink injection, 10μl of blood was collected with micro pipette (the tips were pre-wetted with heparin solution) from the orbital venous plexus of each animal and transferred to an EP tube containing 1mL of distilled water beforehand. The tubes were shaked homogeneously and then 100μl of the mixture from each tube was transferred into the wells of 96-well plates. The density (OD value) was determined with microplate reader at 450nm wavelength.5. Fifty normal mice were randomly divided into 5 groups, including DMSO'. olive oil (1:9) control group, positive control group and CFGL (200,100,50mg/kg) groups. Animals were gastrically administered with vehicle or investigated drugs once daily for 7 consecutive days. On the next day after treatment, each mouse was then administered intraperitoneally with 20% RRBCs in a total volume of 0.2 ml. On the sixth day after priming, the serum samples of the animals were collected for haemolysin assay.6. The immunosuppressed mice were induced by cyclophosphamide (CTX).Sixty mice were randomly divided into 6 groups, including DMSO:olive oil (1:9)control group,CTX group, positive control group and CFGL (200,100,50mg/kg)+CTX groups. Cellular immunity was assessed with the mouse DTH model induced by DNFB, Phagocytosis of mononuclear macrophages was determined by the method of carbon particle clearance test, and humoral immunity was evaluated with the production of specific antibody (haemolysin) against rabbit red blood cells.7. Mouse models bearing S180 solid tumor were established. DMSO: olive oil (1:9) control group, positive control group and CFGL (200,100,50mg/kg) groups were assigned. Cellular immunity was assessed with the mouse DTH model induced by DNFB, Phagocytosis of mononuclear macrophage was determined by the method of carbon particle clearance test, and humoral immunity was evaluated with the production of specific antibody (haemolysin) against rabbit red blood cells.8. Cell metabolic or proliferative activity of S180 tumor was determined with methylthiazolyl tetrazolium(MTT) colorimetry assay.9. Mouse models bearing S180 solid tumor were established. DMSO : olive oil (1:9) control group, positive control group and CFGL (200,100,50mg/kg) groups were assigned and the animals were treated once daily for 7 consecutive days. Solid tumors were isolated and weighed, and tumor inhibition rate was calculated.10. SPSS 13.0 statistical software was used for statistical analysis. Two sample comparison was carried out with independent sample t test (Independent-Sample T Test). For more than one sample mean comparison, ANOVA (One-Way ANOVA) was applied after testing of homogeneity of variance. If variance was of homogeneity, LSD method for pairwise comparison between groups was adopted. When variance of heterogeneity, Welch test was applied at first and then DunnettT3 method for pairwise comparisons between groups was employed. Significant level was set atα= 0.05 and P<0.05 was considered statistically significant. Results:1. CFGL at high, medium and low concentrations (100,50,25μg/ml) could significantly inhibit the Con A-induced proliferative response of mouse spleen cells (metabolic activity) with statistical significance (P=0.000,P=0.000,P=0.026) compared with ConA group and inhibit the LPS-induced proliferative response statistically significant (P=0.000,P=0.002,P=0.028) compared with LPS in a concentration dependent manner within a given range.2. CFGL at high, medium and low concentrations (100,50,25μg/ml) had no effects on normal mice ear edema reaction induced with DTH and mononuclear macrophage phagocytosis. However high and medium doses (200,100 mg/kg) could significantly increase the level of serum hemolysin in normal mice (P=0.002, P=0.031).3. CFGL (200, 100mg/kg) remarkably increased the swelling degree in DTH in immunosuppressed mice induced by CTX in vivo (P=0.000, P=0.033) and enhanced celluar immunity function. CFGL (200,100mg/kg) remarkably promoted the serum clearance of carbon particles in immunosuppressed mice induced by CTX in vivo (P=0.001, P=0.019), indicating an increase in phagocytic function of macrophages. CFGL (200,100 mg/kg) increased the level of serum hemolysin induced by RRBC immunization in immunosuppressed mice (P=0.000,P=0.001) indicating an enhancement in humoral immunity.4. CFGL (200,100mg/kg) remarkably increased the swelling degree in DTH in tumor bearing mice in vivo(P=0.001,P=0.011) and enhanced celluar immune function. CFGL (200, 100mg/kg) remarkably promoted the serum clearance of carbon particles in tumor bearing mice in vivo (P=0.001,P=0.015), indicating an increase in phagocytic function of macrophages. CFGL (200,100mg/kg) increased the level of serum hemolysin induced by RRBC immunization in tumor bearing mice (P=0.000,P=0.000), indicating an enhancement in humoral immunity.5. CFGL (100,50,25μg/ml) significantly inhibited the metabolic activity of mouse S180 cells (P=0.000,P=0.000,P=0.000); CFGL (200p.g/ml) at high dose significantly inhibited mouse S180 solid tumor growth (P=0.001).Conclusion:1. Chloroform Fraction of Ganoderma lucidum (CFGL) can inhibit proliferation of mouse T and B lymphocytes in vitro.2. CFGL has no effect on cellular immunity and phagocytic activity of macrophages in vivo, but can potentiate mouse humoral immune response.3. The experimental model of immunosuppressed mice induced by cyclophosphamide (CTX) was established successfully.4. CFGL has the capacity of enhancement of immune function in immunosuppressed mice induced by CTX in vivo. It markedly potentiates DTH, elevates the corrected phagocytic index A, promotes the phagocytic function of macrophages, and increases the level of serum hemolysin in immunosuppressed mice, suggesting that CFGL is able to enhance the function of cellular and humoral immunity in immunosuppressed mice induced by CTX in vivo.5. CFGL has the capacity of enhancement of immune function in tumor bearing mice in vivo, including potentiation of DTH, elevation of corrected phagocytic index A, promotion of phagocytic function of macrophages, and increase of level of serum hemolysin in tumor bearing mice. It is suggested that CFGL is able to enhance the function of cellular and humoral immunity in tumor bearing mice in vivo.6. CFGL can inhibit the proliferation of tumor cells in vitro and inhibit the growth of S180 solid tumor in vivo. |
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