In Vitro Research On The Anti-tumor Activity Of Various Biologic Polypeptides And The Purification And Structural Identification Of The Polypeptides Of Tilapia

Aim: To study the anti-tumor activity of some biologically active peptides likepolypeptides of Gekko japonicus,Cordyceps militaris, Grifola frondosa andtilapia on tumour cells in vitro like human breast carcinama cell(MCF-7cell),human pulmonary adeno carcinoma cell(A549cell), human gastric tumor(SGC7901),human esophageal carcinoma (Eca109), human colon carcinoma cell(HT29),humanliver carcinoma (Bel7402). Then to separate and purify the effectiveconstituent of the polypeptides of tilapia,and trying to find the most simpleuseful part with anti-tumor activity.Finally to determinate its structure.Methods: To test the anti-proliferative effect of the biologic polypeptidesagainst tumor cell by MTT assay.At the same time,the survival rate of normalcells were determined by this method.Utilizing Sephadex G-50and SephadexLH-20gel columns to separate the polypeptides of tilapia,the mobile phaseis water.Using protein purifier to received each single chromatographicpeak,the UV detector set to280nm and the mobile buffer for25mmol/L Tris-HCland150mmol/L NaCl.The isolated polypeptides were analyzed by High efficiencyliquid chromatography, the mobile phase is methanol-water (10:90); the finalpure polypeptide was purified with silica gel column chromatography,thebeginning buffer is water with one percent acetic acid,in order to replacethe impurities(sodium chloride and tris); Using MAILD-TOF-MS and NMR todetect the structure of the pure polypeptides. Results: To test the inhibition rate of the biologic polypeptides on humanesophageal carcinoma (Eca109)by MTT assay,the results showed that when thedose of the polypeptides of Gekko japonicu was1μg/mL,the inhibition rate was(42.83±4.88)%;the rate of polypeptides of cordyceps militaris on10μg/mLwas (38.34±0.54)%;the the inhibition rate of Grifola frondosaan on5μg/mLwas (42.78±4.10)%and the rate of the high dose group(the original fluid-water1:1) of polypeptides of tilapia was (86.78±2.72)%.To test the inhibition rateof the biologic polypeptides on human breast carcinama cell(MCF-7cell),theresults showed that when the dose of the polypeptides of Gekko japonicu was1μg/mL,the inhibition rate was (50.15±2.16)%; the rate of polypeptides ofcordyceps militaris on10μg/mL was (54.11±3.92)%;the inhibition rate ofGrifola frondosaan on100μg/mL was (63.13±2.27)%and the rate of the highdose group(the original fluid-water1:1) of polypeptides of tilapia was (91.55±2.37)%.To test the inhibition rate of the biologic polypeptides on humangastric tumor(SGC7901),the results showed that when the dose of thepolypeptides of Gekko japonicu was0.1μg/mL,the inhibition rate is (21.97±5.04)%; the rate of polypeptides of cordyceps militaris on1μg/mL was (35.32±8.37)%;the the inhibition rate of Grifola frondosaan on100μg/mL is(18.68±2.73)%.To test the inhibition rate of the biologic polypeptides onhuman pulmonary adeno carcinoma cell(A549cell)by MTT assay,the resultsshowed that when the dose of the polypeptides of Gekko japonicu was50μg/mL,the inhibition rate is (35.57±2.64)%;the rate of polypeptides ofcordyceps militaris on5μg/mL was (38.29±5.52)%;the the inhibition rate ofGrifola frondosaan on10μg/mL was (20.18±6.58)%and the rate of the high dosegroup(the original simple-water1:1) of polypeptides of tilapia was (90.07±0.69)%.To test the inhibition rate of the biologic polypeptides on humanliver carcinoma (Bel7402),the results showed that when the dose of thepolypeptides of Gekko japonicu was5μg/mL,the inhibition rate was (46.02±6.48)%;the the inhibition rate of Grifola frondosaan on100μg/mL was (44.52±5.37)%.To test the inhibition rate of the biologic polypeptides onhuman colon carcinoma cell(HT29),the results showed that when the dose of thepolypeptides of Grifola frondosaan was50μg/mL,the inhibition rate was(19.7±7.97)%;other biological peptides had no obvious inhibitory effecton it.To test the survival rate of the biologic polypeptides on normal cell(ratbone marrow stem cells) by MTT assay,the results showed that when the doseof the polypeptides of Gekko japonicu was100μg/mL,the survival rate was(114.08±4.88)%;the rate of polypeptides of cordyceps militaris on10μg/mLwas (60.11±6.79)%;the the survival rate of Grifola frondosaan on100μg/mLwas (61.50±15.25)%and the rate of the middle dose group(the originalfluid-water1:3) of polypeptides of tilapia was (104.65±21.48)%.To test thesurvival rate of the biologic polypeptides on normal cell(rat renal glomerulusmesangial cells) by MTT assay,the results showed that when the dose of thepolypeptides of Gekko japonicu was100μg/mL,the survival rate was (62.18±4.46)%;the rate of polypeptides of cordyceps militaris on10μg/mL was(31.99±1.73)%;the the survival rate of Grifola frondosaan on100μg/mL was(166.19±7.73)%and the rate of the middle dose group(the original fluid-water1:3) of polypeptides of tilapia was (101.23±11.46)%.The inhibition rate ofpure polypeptides of tilapia on the human pulmonary adeno carcinoma cell is（17.49±13.26）%，and The structure of it is: Conclusion: Traditional Chinese medicine is one of the marrows of our countryand already has enough research.To screen the drugs have the correspondingeffects(stasis-dissolving drugs,heating-clearing and detoxicating drugsetc.) is easier than blindly selection. The four kinds of biological peptidesselected in this project have broad-spectrum antitumor activity and theinhibition has certain selectivity,this satisfies the basic condition toproduce new drugs.The antitumor effect of the polypeptides of tilapia hasgood reproducibility and have linear correlation with concentration,at thesame time,it has no significant inhibitory effect on normal cells.So thefurther separation and purification was tried on this polypeptide.The purepeptide had certain antitumor activity,this provides some theoretical supportfor the development of new drugs in the future.