Neutraceutical value of mushroom msd.ppt
- 1. NUTRACEUTICAL VALUE OF MUSHROOM SPECIES Presented by : Dr. Dinesh M S
- 2. Novel Medicinal Mushroom Blend Suppresses Growth And Invasiveness Of Human Breast Cancer Cells ( Jiahua Jiang and Daniel Sliva,2010) • In aim of the study was to evaluate the anticancer effects of the dietary supplement MycoPhyto Complex (MC). • MC demonstrates cytostatic effects and cell cycle arrest at the G2/M phase of highly invasive human breast cancer cells MDA-MB-231. • DNA-microarray analysis revealed that MC inhibits expression of cell cycle regulatory genes (ANAPC2,ANAPC2,BIRC5, Cyclin B1, Cyclin H, CDC20, CDK2, CKS1B, Cullin 1,E2F1, KPNA2, PKMYT1 and TFDP1). • MC also suppresses the metastatic behavior of MDA-MB-231 by the inhibition of cell adhesion, cell migration and cell invasion. • Conclusion: MC dietary supplement could have potential therapeutic value in the treatment of invasive human breast cancer.
- 3. Materials Used 1. MycoPhyto Complex (MC) : MC is a dietary supplement consisting of a blend of six varieties of mushroom mycelia from the species Agaricus blazei, Cordyceps sinensis, Coriolus versicolor, Ganoderma lucidum, Grifola frondosa and Polyporus umbellatus, and β-1,3-glucan isolated from the yeast, Saccharomyces cerevisiae. • Agaricus blazei (Brazil) is believed to treat diseases like atherosclerosis, hepatitis, hyperlipidemia, diabetes, dermatitis and cancer. Bioactive component- Polysaccharide Phytocomplex. • Cordyceps sinensis (high altitude (3500-5000 m) of Himalayas) has been used for the treatment of multiple disorders including respiratory, renal, hepatic, cardiovascular, immunologic, and nervous system, glucose metabolism, different inflammatory conditions and cancer. Bioactive component- Cordycepin. • Coriolus versicolor (year-round on logs, stumps, tree trunks, and branches) has been used in immunotherapy , induction of apoptosis and to suppress the metastatic effects of cancer cells. Bioactive component- structurally different protein-bound polysaccharide-K (PSK) and polysaccharopeptide (PSP). • Grifola frondosa (Maitake, Japan & korea) has anticancer activities, suppress tumor growth. Bioactive component- 1,3-β-glucan named grifolan L,1,3- branched-1,6-β-glucan.
- 4. Materials Used • Ganoderma lucidum (Asia) has anticancer effects suppress proliferation and metastatic effects of cancer cells. Bioactive component- Polysaccharides (mainly glucans and glycoproteins) and lanostanetype triterpenes (ganoderic acids, ganoderic alcohols and their derivatives). • Polyporus umbellatus (China) has been used for the urinary tract infection, as a diuretic, for the treatment of hepatitis B, in immunotherapy and it also has anti-inflammatory activity. Bioactive component- Polysaccharides, triterpenes (polyporusterones), ergostane-type ecdysteroids named polyporoids. • Supplied by EcoNugenics, Inc (Santa Rosa, CA, USA). • MC stock solution was prepared by dissolving MC in dimethylsulphoxide (DMSO) at a concentration 25 mg/ml and stored at 4˚C. 2. MDA-MB-231 cells : Human breast cancer cell line MDA-MB-231 was obtained from ATCC (Manassas, VA, USA) and was maintained in DMEM medium in the presence of penicillin (50 U/ml), streptomycin (50 U/ml), and 10% fetal bovine serum (FBS).
- 5. Experimental Procedure 1. Cell proliferation was determined by the tetrasolium salt method. Figure: Effect of MC on the growth of breast cancer cells. (A) Cell proliferation and (B) cell viability were determined. Data are the means ± SD of triplicate determinations. Similar results were obtained in at least two additional experiments. *p<0.05.
- 6. Experimental Procedure 2. The fractions of cells in different phase of the cell cycle (G0/G1, S, G2/M) are presented as a percentage of total cells analyzed. 3. The total was RNA isolated and fold change of gene expression was determined. 4. Invasion of MDAMB-231 cells treated with MC (0-0.5 mg/ml) was assessed. 5. The secretion of uPA was detected by Western blot analysis with anti-uPA antibody.
- 7. Results and discussion MC demonstrates cytostatic effect on the highly invasive breast cancer cells. • Chemically synthesized chemotherapeutic agents have demonstrated activity in the metastatic breast cancer setting but some of these chemotherapeutic drugs have undesirable toxic side effects. MC (natural complexes) is a unique blend of mushrooms demonstrated significant anticancer activities. 1. To determine if the effect of MC on cancer cells is cytotoxic or cytostatic, the cell viability after 24, 48 and 72 h of MC treatment was evaluated. MC (mg/ml) G0/G1 S G2/M 0 72.8 ± 0.8 24.9 ± 1.0 3.1 ± 0.5 0.25 73.5 ± 2.2 22.6 ± 1.3 3.8 ± 0.7 0.5 71.4 ± 2.3 16.4 ± 2.7a 12.2 ± 0.5 Effect of MC on cell cycle distribution
- 8. Results and discussion • The viability of MDA-MB-231 cells was not affected by the MC treatment suggesting that the MC inhibits growth of breast cancer cells through its cytostatic effect. • MC induces cell cycle arrest at G2/M phase. 2. To check whether MC-induced cell cycle arrest is associated with the expression of specific cell- cycle regulatory genes, MDA-MB-231 cells was treated with MC and DNA-microarray analysis was performed. Figure: Effect of MC on the expression of cell cycle regulatory genes. MDA-MB-231 cells were treated with (A) control - 0 mg/ml MC or (B) 0.5 mg/ml MC for 24 h and RNA prepared and DNA-microarray analysis performed.
- 10. Results and discussion • MC down-regulated expression of ANAPC2, ANAPC2, BIRC5, Cyclin B1, Cyclin H, CDC20, CDK2, CKS1B, Culin 1, E2F1, KPNA2, PKMYT1 and TFDP1. • Some of the functional proteins (e.g., ANAPC2, ANAPC2 and CDC20) form the anaphase promoting complex/cyclosome (APC/C) inhibition of which can induce cell cycle arrest at G2/M phase. • Inhibition of survivin (product of gene BIRC5) and CDC28 (product of gene CKS1B) was also associated with cell cycle arrest at G2/M phase. • G2/M cell cycle arrest is controlled by Cyclin B1 and Cyclin H (CDK7), and inhibitors of CDK2 were developed as G0/G1 and G2/M inhibitors for cancer therapy. • Down-regulation of transcription factors E2F1 and DP-1 (product of gene TFDP1) and Myt-1 protein (product of gene PKMYT1) was also linked to cell cycle arrest at G2/M phase in cancer cells. • Therefore, the induction of cell cycle arrest of breast cancer cells by MC was associated with the down-regulation of expression of genes involved in G2/M phase.
- 11. Results and discussion 3. Effect of MC on the invasive behavior of breast cancer cells was studied. • Breast cancer cells express integrin receptor αvβ3, and the interaction of αvβ3 with the extracellular matrix (ECM) protein vitronectin is involved in the adhesion of MDA-MB-231 cells to ECM. • In addition, αvβ3 and vitronectin form a complex with urokinase plasminogen activator (uPA) and its receptor uPAR, and this whole complex activates the intracellular signaling responsible for cell adhesion and migration. • To investigate if MC affects adhesion of invasive breast cancer cells, MDAMB- 231 cells were pretreated with MC (0-0.5 mg/ml) for 24 h and their adhesion to vitronectin was determined. • Adhesion of MDA-MB-231 cells to vitronectin was markedly suppressed by the MC treatment. (Fig: A) • The effect of MC on migratory potential of breast cancer cells was evaluated in MDA-MB-231 cells pretreated with MC (0-0.5 mg/ml) for 1 h and cell migration was determined after additional 4 h of incubation. As expected, MC also noticeably suppressed migration of breast cancer cells in a dose dependent- manner. (Fig: B) 4. Effect of MC on cell invasiveness was evaluated. • MDA-MB-231 cells were plated on the Matrigel-coated filters in the presence of MC (0-0.5 mg/ml) and the amount of cells invaded through Matrigel counted after 24 h of incubation. • MC markedly inhibited invasion of MDAMB- 231 cells in a dose-response manner. (Fig: C)
- 12. Results and discussion Figure: Effect of MC on the secretion of uPA and expression of CXCR4 and COX-2 in breast cancer cells. (A) MDA-MB-231 cells were treated with MC (0-0.5 mg/ml) for 24 h, and the secretion of uPA detected in conditioned media with anti-uPA antibody by Western blot analysis. The results are representative of three separate experiments. (B) MDA-MB-231 cells were treated as described in (A), and the expression of CXCR4, COX2 and β-actin determined. The results are representative of three separate experiments. Figure: Effect of MC on invasive behavior of breast cancer cells. (A) Cell adhesion. MDA-MB-231 cells were treated with MC (0- 0.5 mg/ml) for 24 h and cell adhesion to vitronectin determined. Each bar represents the mean ±SD of three experiments. *p<0.05; (B) Cell migration. Cell migration was determined after 5 h of incubation in the presence of MC (0-0.5 mg/ml) in Boyden Chambers. Each bar represents the mean ±SD of three experiments.*p<0.05; (C) Cell invasion. Cell invasion was determined after 24 h of incubation in the presence of of MC (0-0.5 mg/ml) in Boyden Chambers coated with Matrigel. Each bar represents the mean ±SD of three experiments. *p<0.05.
- 13. Results and discussion 5. Effect of MC on the levels of uPA was evaluated. • Conditioned media from MDA-MB-231 cells treated with MC (0-0.5 mg/ml) were collected and secretion of uPA was determined by Western blot analysis. • MC markedly decreased secretion of uPA from MDA-MB-231 cells.(Fig: A) • In order to evaluate whether MC affects expression of these proteins, whole cell extracts from MDA-MB-231 cells used for the determination of uPA (Fig: A) were subjected to Western blot analysis with specific antibodies against CXCR4 and COX-2, respectively. • MC treatment of MDA-MB-231 cells did not markedly affect expression of CXCR4 and COX-2 (Fig: B). Therefore, from data it was concluded that MC suppresses invasive behavior of breast cancer cells by the inhibition of secretion of uPA. • Therefore, it can be concluded that MC is a dietary supplement with the ability to suppress proliferation and invasive behavior of breast cancer cells. The biological effects of MC are probably mediated by the additive or synergistic effects of individual mushrooms. The mushroom based dietary supplement MC could have potential use in the treatment of invasive breast cancer.
- 14. Effect Of Oyster Mushroom On Glycemia, Lipid Profile And Quality Of Life In Type 2 Diabetic Patients (Agrawal RP, Chopra A, Lavekar GS, Padhi MM, Srikanth N, Ota S, Jain S, 2010) • The aim of the study was to evaluate efficacy of oyster mushroom (Pleurotus spp) on glycemic control, lipid profile and diabetic quality of life in type 2 diabetic patients. • After 3 months period blood sugar, HbA1c and blood pressure reduced. • Significant improvement in lipid profile was also observed in same groups. • Diabetes quality of life also improved significantly. No extra effect was observed due to supplementation of ajwain. • Conclusion: Oyster mushroom (Pleurotus spp) consumption appears to be effective in controlling glycemic control, lipid profile and diabetic quality of life.
- 15. Materials Used And Experimental Procedure 1. A total of 150 type 2 newly onset diabetic patients were recruited. • After one month of a stabilisation period 120 patients were selected having blood sugar levels 126-250 mg/dL. These 120 patients were randomly divided into three groups (n=40 in each group). Group 1 received biscuits of type A, group 2 received biscuits of type B and group 3 received biscuits of type C. 2. Standardisation was done of the oyster mushroom before clinical trials. 3. Dietary survey by 24 hour recall system was done. 4. Anthropometric parameters, fasting blood sugar and blood pressure were recorded weekly and HbA1c, lipid profile, diabetic quality of life questionnaire were performed initially as well as after 3 months. • After 3 months it was revealed that type A biscuits were ajwain biscuit, type B were ajwain + mushroom and type C were mushroom biscuits. Moist. Protein Fat CHO Fibre Ash Calories 90.2% 2.5% 0.2% 5.2% 1.3% 0.6% 35 Proximate composition of the Pleurotus sajor-ciju
- 16. Materials Used And Experimental Procedure 5. Subjects with a diagnosis of type 2 diabetes mellitus, fasting blood sugar levels greater than 126 but less than 250 mg/dL and a written consent showing willingness to participate in the study were included for further study. • Patients suffering from kidney disease, liver disease, arthritis, pulmonary tuberculosis, malabsorption, or alcoholism were excluded. 6. Anthropometric measurements were taken. • Height in cm and weight in kg with the use of a digital machine accessorised with a movable headboard. • Waist/hip ratio was estimated. • Blood pressure: by sphygmomanometer • Fasting blood sugar: by glucose oxidase method • Lipid profile: by auto analyzer • HbA1c: by DS5 Drew Scientific machine (ion exchange chromatography) • Diabetes quality of life questionnaire was assessed every month. 7. Statistical Analysis: • Data was presented as means ± SEM. Comparisons between baseline characteristics of each group were made by using ANOVA, confidential limit and correlation tests. At p value <0.05, differences were considered significant.
- 17. Results
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- 19. Results
- 20. Results and discussion After 3 month period, • The blood sugar levels were found reduced in the ajwain + mushroom group as well as in the mushroom group. • Systolic and Diastolic blood pressure was reduced in both the groups. • There was a significant effect on glycemic control (HbA1c) in both groups. • There was a significant reduction in lipid profile i.e. total cholesterol in both groups. • Diabetes quality of life improved significantly. • There was no significant change in BMI and waist hip ratio. • There was significant reduction in serum triglyceride and serum cholesterol. • The W/H ratio was slightly decreased in the ajwain + mushroom group and increased in the mushroom group but this change was not statistically significant. • Therefore, from the study it can be concluded that there is potential use of oyster mushroom for better glycemic control, positive effect on lipid profile and better quality of life.