The Pharmacological Activities And The Structural Identification Of Phellinus Igniarius Polysaccharides

Objective The therapeutic effects of Phellinus igniarius polysaccharides on liver cancer H22 tumor-bearing mice and collagen-induced arthritis(CIA)rats were research using the modem pharmacology.By utilizing the method of separation and purification,Phellinus igniarius polysaccharides were separated and purified.Structures of uniform compositions were identified using the modern analysis method in order to provide experiments and theoretical basis.Methods(1)The method of water extract and alcohol precipitation was used to extract Phellinus igniarius polysaccharides.60 Kunming mice were randomly divided into 6 groups,normal group,model group,control group(cyclophosphamide,CTX),Phellinus igniarius polysaccharides low-dose(100 mg/kg),medium-dose(200 mg/kg)and high-dose(400 mg/kg)groups.The mice were inoculated with 0.2 mL H22 cells subcutaneously in the left armpit expect the blank group to establish H22-bearing mice model.Drugs were administrated for consecutive 15 days.All mice were sacrificed and tumors were dissected and weighed to calculate the tumor-inhibition rates.Thymuses and spleens were dissected and weighed to calculate thymus index and spleen index.The levels of IL-2,IL-6,TNF-? were determined by ELISA.The proliferation of T and B lymphocytes were determined by MTT assay.The tumors were removed for sections and observed under light microscope.The expression of Bax and Bcl-2 protein were measured by immunohistochemical method.(2)60 Wistar rats were randomly divided into 6 groups,normal group,model group,control group(celecoxib),Phellinus igniarius polysaccharides low-dose(100 mg/kg),medium-dose(200 mg/kg)and high-dose(400 mg/kg)groups.Except the blank group,the rest groups were inflammatory in the right rear foot to establish collagen-induced arthritis(CIA)model.The paw volume was measured to determine arthroncus degree during administration.Ankle pathological sections were observed under microscope.The levels of IL-17,TNF-?,IL-1?,IL-10 were determined by ELISA.(3)Uniform components of Phellinus igniarius polysaccharides were got by separation of DEAE Sepharose Fast Flow anion exchange resin and purification of Sephacryl S-300 High Resolution gel resin.UV scanning was measured their purity.High performance liquid(HPLC)united refractive index detector was to determine the molecular weight.After trifluoroacetic acid(TFA)hydrolysis,reduction by sodium borohydride(NaBH4)and acetylation with acetic anhydride,the composition of monosaccharides was analyzed on gas phase-mass spectrometry(GC-MS).Energy groups were analyzed by Infrared spectrum.After methylation reaction,TFA hydrolysis,reduction by NaBH4 and acetylation with acetic anhydride,the connection was analyzed on GC-MS.Results(1)The inhibitory rate of various dosage groups were:control group,79.36%;Phellinus igniarius polysaccharides low-dose,26.71%;Phellinus igniarius polysaccharides medium-dose,34.48%;Phellinus igniarius polysaccharides low-dose,56.65%.Compared to model group,tumor weights of Phellinus igniarius polysaccharides high-dose,medium-dose group and positive group were significantly decreased(P<0.05);no significant differences of the thymus index and spleen index(P>0.05);in addition to TNF-? in Phellinus igniarius polysaccharides low-dose group were no significant difference,IL-6,IL-2 and TNF-? in each dosage groups were significantly increased(P<0.05).In T lymphocyte proliferation experiment,compared with model group,absorbance(A)value of Phellinus igniarius polysaccharides high-dose group was increased significantly(P<0.05).In the B lymphocyte proliferation experiments,A value in each dose group were higher than that of model group,but only the high-dose group had significant difference(P<0.05).H22 tumor cells were arranged loosely with swelling and degeneration in each dose of Phellinus igniarius polysaccharides group.Bax and Bcl-2 positive cells showed a diffuse distribution in H22 tumor cells.Combined of IPP software to calculate the average optical density(OD)value,Bax protein expression was weakly positive while the Bcl-2 shows strong positive in model group,Bax protein expression was strong while the Bcl-2 was weakly positive in positive group and each dose of Phellinus igniarius polysaccharides group,and had significant difference with model group(P<0.05).(2)Compared to model group,positive group and each dose of Phellinus igniarius polysaccharide could decrease the degree of arthroncus(P<0.05),improve joint tissue pathological situation,decrease the levels of TNF-?,IL-1?,IL-17(P<0.05)and increase the level of IL-10(P<0.05)significantly.(3)Two components P1A1 and P1B were got after separation of DEAE Sepharose Fast Flow anion exchange resin and purification of Sephacryl S-300 High Resolution gel resin.UV scanning showed that the two components were uniform component.The molecular weights of P1A1 58313,P1B 27365 were determined by HPLC united refractive index detector.GC-MS analysis of monosaccharide composition,known P1A1 is composed of five known monosaccharide rhamnose,arabinose,glucose,mannose,galactose and three speculated compositions of methyl-?-D-glucopyranoside,methyl-a-D-glucopyranoside,methyl-?-D-ma-nnopyranoside;P IB is composed of three monosaccharide rhamnose,arabinose,glucose and methyl-?-D-mannopyranoside.Infrared spectrum scanning results showed that the P1A1 which was belonged to pyranoside and containned ?-and ?-glycosidic bond was a polymerization sugar made up of monosaccharide containing acid residues;P1B which was belonged to pyranoside and containned ?-glycosidic bond was was a polymerization sugar made up of monosaccharide not containing acid residues.P1A1 had four connection modes,respectively:Glcp(1?,?4)Glcp(1?,?6)Glap(1?,?3,6)Manp(1?;P1B also had four connection modes,respectively:Glcp(1?,?4)Glcp(1?,?6)Glap(1?,?3)Manp(1?.Conclusion(1)Phellinus igniarius polysaccharides could through regulating the body's immune organs,significantly increasing the concentration of IL-2,IL-6 and TNF-? in serum,promoting the proliferation of T,B lymphocytes in splenocyte,increasing the expression of Bax protein and depressing the expression of Bcl-2 protein,increasing the number of necrosis tumor cell,to achieve the effect of inhibiting tumor.(2)Phellinus igniarius polysaccharides may be through inhibiting the expression of inflammatory cytokines TNF-?,IL-1?,IL-17 and increasing the expression of inflammation suppression factor IL-10 to therapy CIA rats.(3)Further study on the Phellinus igniarius polysaccharides structure was provided some experimental basis by utilizing the method of separation and purification of Phellinus igniarius polysaccharides and using the modern analysis method for the structure identification of uniform components P1A1 and P1B.