Experimental Investigation On Anti-Tumor Activity Of Extraction From Shark Cartilage Combining With Polysaccharide From Se-Enriched Spirulina Platensis

Objective:To optimize the experimental proposal of ultrasonic waves (UWs)extraction for Polysaccharide from Se-enriched Spirulina Platensis (Se-PSP).Purify Se-PSP and determine the contents of saccharide and proteins, investigate the effects of Se-PSP on the growth of tumor cells in vitro.Methods: Finding the best way to abstract Polysaccharide from Spirulina Platensis using the method of cross design and ultrasonic waves.Crude Se-PSP was abstracted and purified with DEAE-cellulose chromatographic column and 8 kDa～10 kDa bag filter after deproteinization with trichloracetic.The contents of saccharide and protein were determined with antrone-sulphuric acid and coomassie brilliant blue G-250 chromatometry.Investigate the effects of different concentration of Se-PSP₂ on the growth of SKOV-3 cells,HepG-2 cells,BGC-803 cells,CNE cells and Tca-8113 cells with MTT chromatometry.Results:The maximum Polysaccharide from Spirulina Platensis was got under the pH 11 and 60℃with the UWs treated 60 min,four composition of Polysaccharide Se-PSP₁,Se-PSP₂,Se-PSP₃ and Se-PSP₄ with different molecular weight were got using the method of DEAE-cellulose chromatographic column.Se-PSP₂ was main composition of Se-PSP total saccharide(70.2%). Content of saccharide was 98.49%and protein was 0.45%after dislysis and purification.MTT trial indicated that inhibition ratio of Se-PSP total saccharide, Se-PSP₁,Se-PSP₂,Se-PSP₃ and Se-PSP₄ on growth of SKOV-3 cell was (33.393±5.437)%,(22.290±8.399)%,(44.881±4.878)%,(45.354±4.931)%and (46.203±4.455)%respectively when the concentration of Se-PSP₂ exceeded 10μg/ml.We could conclude that Se-PSP₂ noticeably inhibits the proliferation of SKOV-3,HepG-2,BGC-803 and Tca-8113 cells,had broad anti-tumor spectra which were dependent on different dosage from the MTT chromatometry inhibition experiment indicated.The rate of inhibition was(45.202±4.401)%on SKOV-3 ovarian cancer cells and(22.238±2.481)%on CNE nasopharyngeal carcinoma.Conclusions:The best abstraction enviroment for Polysaccharide from Spirulina Platensis was pH 11 and 60^Cwith the UWs treated 60 min in water bath,Se-PSP₂ was the main composition of Se-PSP total saccharide.The Se-PSP₂ could inhibit the growth of many tumor cell strains effectively and had broad anti-tumor spectra,which was going to be a miracle drug on treatment of malignant tumor. Objective:Finding a way to abstract the low molecule protein from shark Cartilage and confirming its effect of angiogenesis growth inhibition.Methods: The protein was extracted after dealing with the shark Cartilage through crashing,freezing,centrifuging to remove the sediment,ultrafiltrating and vacuum drying in order.The morphologic change of EVC304 cells were affected by low molecule protein from shark Cartilage after 72h under light microscope and electron microscope.The protein had the effect on the growth, adhesion and migration of endothelial cell in vitro and had the effect on the angiogenesis of embryonated egg s allantocherion too.Results:EVC304 cells was dys-growth under light microscope,round cells,small cells crenulating cells and integrated cells showed up,intracytoplasma particle increased,lucency was lower;The shape of cellular nucleus was irregular,mitotic figure significantly decreased under electron microscope;Low molecule protein from shark Cartilage could inhibit vascular endothelial cell growth when the concentration above 50μg/ml and inhibit the adhesion of vascular endothelial cell when the concentration above 5μg/ml.(The inhibitory ratio was(36.770±2.820)%, adhesion inhibitory ratio was(52.954±4.442)%and locomotory movement inhibitory ratio was(68.01±5.65)%when the concentration was 100ug/ml with the MTT method.The angiogenesis numerus of embryonated egg allantocherion was(18.750±3.671)and(7.167±1.529)in the negative group and low molecule protein group separately.Low molecule protein from shark Cartilage could inhibit effectively angiogenesis growth of embryonated egg allantocherion.The low molecule protein could inhibit the formation of angiogenesis of embryonated egg s allantocherion effectively too.Conclusions:The low molecule protein from shark Cartilage which got from freezing and thawing ultrafiltration method could inhibit the growth of angiogenesis by inhibiting the growth,adhesion and locomotory movement of endothelial cell,which was hope to be a new target drug to inhibit growth of tumor angiogenesis. Objective:Bolting the optimization compatible concentration of SC low molecular weight protein combining with Se-PSP and observing its effects of anti-tumor.Methods:The low molecule protein from shark Cartilage and Polysaccharide from Se-enriched Spirulina Platensis were mixed respectively according to quality to 20:1,10:1,5:1,2.5:1,1:1,1:2.5,1:5,1:10 and 1:20. The inhibition of different proportion mixture above on growth and adhesion function of human ovarian cancer SKV-3 cells in vitro and on the angiogenesis of embryonated egg's allantocherion was observed.Fifty Balb/c rats were selected and divided into 5 groups randomly.After having given the drugs for ten days,splenic lymphocytes was observed and cultured in vitro.The proliferation status of splenic lymphocyte which induced with con-A in rats of all experimental groups was observed with the improved method of MTT.Sixty C57BL/6 rats were divided into 6 groups randomly.Construct rats model carrying Lewis lung cancer,10 days after continuous injection,we calculated inhibition ratio on tumor growth.Results:The growth and adhesion function of SKOV-3 cells were both inhibited by SC low-molecular weight protein combining with Se-PSP,the inhibition was extremely noticeable with the compatibility proportion of 5:1,inhibition ratio were(51.108±4.618)%and (53.303±5.194)%respectively;The inhibitive formation of chickembryo chorioallantoic membrane(CAM)nascent blood vessels demonstrated that the angiogenesis numerus of embryonated egg's allantocherion in all experimental group were(6.02±0.748),(5.200±0.980)and(5.600±0.800)respectively with the compatibility concentration of 20:1,10:1 and 5:1.There was no significant difference among the three groups(P＞0.05),but the difference was noticeable compared with the rest compatibility groups(P＜0.05).The trial on proliferation of splenic lymphocytes of rats demonstrated that the proliferative index (1.831±0.035)of splenic lymphocytes in rats in the proportion 5:1 group was notably higher than that in SC low-molecular weight protein group(1.533±0.046)and Se-PSP group(1.624±0.043),which were higher than the group of Sodium Chloride(1.367±0.058)(P＜0.01).The SC low-molecular weight protein and Se-PSP could promote the proliferation of splenic lymphocytes of Balb/c rats.The effect would be enhanced when the two drugs were used together.The inhibition rate on the tumor growth in 5:1 group was higher than that in the SC low-molecular weight protein group and Se-PSP group significantly.Mixture with proportion at 5:1 Cyclophosphamide Group noticeably higher than the Cyclophosphamide group significantly(P＜0.05). There was a similar change in the SC low-molecular weight protein group and Se-PSP group compared to the negative control group.Conclusions:The compatibility mixing at 5:1 was the best composition according to results,which can inhibit growth of tumor,promote cell proliferation of splenic lymphocytes of rats,increase immunization and have the effects on sensitization with chemotherapeutic drugs.The compositon of low molecule protein from Shark Cartilage with Polysaccharide from Se-enriched Spirulina Platensis could be a new target drug which can inhibit the growth of tumor.