North American Ginseng & Auricularia sp. mushroom aq. extract have beneficial effect on cyclophosphamide induced immunosuppression in mice Kyakulaga A. Hassan, Ed Lui OGIRC Journal club, October 2012 Department of Physiology & Pharmacology University of Western Ontario, London, Canada 10/30/2012 1
Background of the study Natural sourced medicine is currently in vogue Major cause of skepticism had been the lack of experimental data A plethora of scientific information is now available Change in the attitude among scientists regarding the pharmaceutical potential of traditional medicine The focus is now on use of these traditional medicines to solve medical predicaments 10/30/2012 2
Background Cont d Medicinal use of Ginseng is documented in the old Chinese pharmacopoeia Stimulation of CNS, immune system, cardio-protection antidiabetic) Mushrooms are another significant source of compounds with significant immunomodulatory effects. Auricularia sp. mushrooms are used as food or medicine by local communities in Uganda Antitumor, immunomodulatory and antibacterial activities among its pharmacological effects 10/30/2012 3
The problem Immune system is a potential target of chemical exposure ( chemotherapeutic agents) Major constraint of cancer chemotherapy Traditional medicines with immunomodulatory properties could be of significant importance in chemotherapy induced immunosuppression 10/30/2012 4
Hypothesis Aqueous extracts of Auricularia sp and N. A ginseng have a protective and/or restorative effect on cyclophosphamide induced immunosuppression in mice 10/30/2012 5
Specific objectives: To determine the effects of extracts on; 1. Splenocyte and bone marrow cellularity in cyclophosphamide immunosuppressed Balb C mice 2. Selected circulating cytokines in cyclophosphamide immunosuppressed Balb C mice 3. Antibody production towards SRBC antigen in cyclophosphamide immunosuppressed Balb C mice 2. Phagocytic index as measured by carbon clearance test in cyclophosphamide immunosuppressed Balb C mice 10/30/2012 6
Methods: Extraction 500g dry mushroom powder Dissolved in 1L distilled water & boiled for 1hr (100 C) 4kg dried Ginseng powder Dissolved in 16L distilled water & boiled for 5hrs (40 C) Excess water removed by rotary evaporator under vacuum at 40 C Lyophilized Cooled & filtered through Whattman filter paper. Lyophilized (Yield=10% or 50g) Extract dissolved in water (10mg/mL). The 4 volumes of 95% ethanol added to ppt polysaccharide. Centrifuged & pellet lyophilized
Cyclophosphamide induced immunosuppression in mice Male albino Balb C mice were weighted and injected with 300mg/kg (i.p) cyclophosphamide (CYP). 10/30/2012 8
Study design Day 0: 300mg/kg CYP (i.p) Day 5: SRBC antigen (i.p) Day 10: Sacrifice Pre-treatment Post treatment Treatments: a. saline b. 50mg/kg ginseng ps c. 100mg/kg ginseng ps d. 100mg/kg Auricularia e. 50mg/kg Auricularia f.50mg/kg Levamisole Bio-assays: day 10 Splenocyte counts Bone marrow Phagocytic index HA titers Cytokines (IL-10, IL-6, TNF, IL-1) 10/30/2012 9
Methods: Bio-assays 1. Splenocyte cellularity and proliferation Spleens quickly removed & placed into cold PBS Mashed to obtain single cell suspension. Washed 3 times in PBS and RBCs were removed using 3%acetic acid in PBS, Cells suspended in 10 % FCS RPMI 1640 media supplemented with benzylpenicillin 100 U/mL, streptomycin 100μg/mL. The number of viable cells counted in Neuber s chamber The cells were then seeded in 96-well culture plates with 10μg/well LPS for 72 hrs and cell proliferation was determined by MTT assay.
Methods: Bio-assays 2. Bone marrow isolation The femur of each mouse was removed and immersed in PBS under sterile conditions Epiphyses were cut using a blade & marrow flushed using a syringe & a 25 gauge needle Cell suspension was washed 3 times using PBS & then incubated with 3% acetic acid to lyse RBCs The cells were then suspended in RPM1 medium and counted using Neuber s chamber MTT assay was performed to determine cell viability after 48 hrs
Bio-assays 1. SRBC Haemagglutination test: 1. blood samples were collected by cardiac puncture & allowed to clot to obtain serum. 2. 100 μl of serum was mixed with 100 μl PBS and serially diluted (10 times) 3. 100 μl of each dilution was mixed with 100 μl of SRBC suspended in PBS 4. Incubated for 1 hr and assessed visually for agglutination 5. Antibody titer was expressed as min serum volume required to cause visible agglutination. 2.Carbon clearance assay: 1.Mice were injected with colloidal carbon (10μl/g body weight) via the tail vein. 2. Blood was collected at 0 and 15mins after carbon injection. 3. The blood was suspended in 0.1% Sodium carbonate 4. Absorbance measured at 660nm using a spectrophotometer. Phagocytic index = lngod1-lnod2/t2-t1 10/30/2012 12
Results 2. Splenocyte and bone marrow cellularity There was a considerable reduction in the size of spleens from saline treated mice. Cell counts per spleen and bone marrow was significantly greater in the extract treated mice as compared to the control mice (Figure 2) Antibody titers & Phagocytic index: Mice treated with saline (control) had decreased antibody levels and had lower phagocytic indices as compared to those treated with ginseng, Auricularia & levamisole (fig 3a, b). Ginseng polysaccharide had the highest stimulatory effect at 100mg/kg followed by Levamisole hydrochloride. Both ginseng and Auricularia had higher antibody levels & Phagocytic indices significantly greater than control (saline). 10/30/2012 13
Fig 2a. Splenocyte MTT viability Fig 2b. Bone marrow cell viability 0.6 0.5 OD values 0.4 0.2 OD values 0.4 0.3 0.2 0.1 0.0 Saline Gin 50 Gin 100 Aur 50 Aur 100 Lev 50 Groups 0.0 Saline Gin 50 Gin 100 Aur 50 Aur 100 Lev 50 Groups 4 Fig 2c. Spleen cell counts 5 Fig 2d. Bone cell counts/femur Cell counts(x10 6 ) 3 2 1 Cell counts(x10 6 ) 4 3 2 1 0 Saline Gin 50 Gin 100 Aur 50 Aur 100 Lev 50 Groups 0 Saline Gin 50 Gin 100 Aur 50 Aur 100 Lev 50 Groups Fig 2. Effect of ginseng polysaccharide (group I &II), Auricularia aq extract (group III&IV) and Levamisole hydrochloride (group V) on (a) Splenocyte proliferation, (b) bone marrow cell viability (c)spleen cell counts and (d) bone marrow cell counts in cyclophosphamide immunosuppressed mice. Data are mean SEM, p<0.05 10/30/2012 14
Fig 3a. SRBC Haemagglutination test Fig 3b. Phagocytic index Antibody titer (min serum/100µl) 3 2 1 0 Saline Gin 50 Gin 100 Aur 50 Aur 100 Lev 50 Groups Phagocytic index 0.020 0.015 0.010 0.005 0.000 Saline Gin 50Gin 100Aur 50Aur 100Lev 50 Groups Fig 3. Effect of gingseng polysaccharide and Auricularia aq. Extracts on (a) Sheep red blood cell haemoagglutination and (b) phagocytic index in cyclophosphamide immunosuppressed mice. Data are mean SEM, represents p-values <0.05 10/30/2012 15
Discussion Immunostimulation is important in immunosuppression Natural products modulate humoral or cell mediated immune response or both. Important in treatment of chemotherapy induced immunosuppression Animal testing is paramount for safety and efficacy The spleen & bone marrow are integral organs of the immune system The Carbon clearance test indirect measure of phagocytosis SRBC heamagglutination assay (HA)- humoral immune response 10/30/2012 16
Discussion cont d A single dose (300mg/kg) of CYP--reduction in spleen & bone marrow cellularity in mice. Both HA and phagocytic index were severely reduced Both ginseng & Auricularia had protective and restorative effect on CYP Bone marrow cellularity and Splenocyte cellularity were greatly enhanced in extract treated mice Levamisole is limited by side effects-cns excitation, agranulocytosis Ginseng more potent than Auricularia 10/30/2012 17
Conclusion Ginseng polysaccharide and Auricularia sp aq. extract could have a beneficial effect on chemotherapy induced immunosuppression 10/30/2012 18
END Thank you 10/30/2012 19