Study On Preparation And Biological Activities Of Low Molecular Weight Squid Ink Polysaccharides

Background:Polysaccharides are important components of all living organisms.They play important roles in regulating cell growth,controlling cell division and maintaining normal metabolism of life.In recent years,the structures and activities of many marine polysaccharides have been revealed and marine polysaccharides have become an important part of the "Blue Drug Storehouse".Squid ink polysaccharides are one kind of important marine biological polysaccharides,which show good potential in anti-cancer,anti-oxidation,anti-mutation,immune regulation,chemotherapeutic protection and so on.Sepiella maindroni is one of the most productive economic cephalopods along the coast of China.In 2004,the polysaccharide of Sepiella maindroni ink(SIP)was first extracted by our group.In the following studies,the structure and properties of this polysaccharide and the sulfated derivative were further studied.Purpose:The biological activities of polysaccharides are closely related to their molecular weight.In general,low molecular weight polysaccharides have higher permeability and bioavailability in organisms compared to large one,and may have new activities.Therefore,the purpose of this study is to degrade SIP and obtain low molecular weight fragments,and to explore whether the biological activities of low molecular weight squid ink polysaccharides(LMWSIPs)are higher than undegraded one.Methods:(1)High performance gel permeation chromatography(HPGPC)and ultraviolet spectrophotometry at OD232nm were used to detect whether SIP could be degraded by common glycosidases.(2)Trifluoroacetic acid(TFA)was selected to degrade SIP,and the optimum degradation conditions were screened by high performance thin layer chromatography(HPTLC)method.(3)Sephadex G-50 was used to isolate and purify SIP degradation products.The molecular weight of SIP degradation products was determined by HPGPC and matrix assisted laser desorption ionization-time of flight mass spectrometry(MALDI-TOF MS).(4)The degradation products were grouped and named according to their molecular weight distribution.LMWSIP-1,LMWSIP-2 and LMWSIP-3 were obtained.The structural differences between LMWSIPs and SIP were analyzed by IR and 1H NMR.(5)The anti-proliferative activity and difference on tumor cells of LMWSIPs and SIP was detected by MTT assay and the activity difference was compared.(6)The apoptosis inducing activity and difference on tumor cells of LMWSIPs and SIP were detected by flow cytometry and the activity difference was compared.(7)The anti-metastasis activity on tumor cells of LMWSIPs and SIP were detected by scratch method and the activity difference was compared.(8)The anti-oxidation activity and difference of LMWSIPs and SIP were evaluated by DPPH radical(·DPPH)scavenging experiment,OH radical(·OH)scavenging experiment,diammonium 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonate)(ABTS)method and ferric ion reducing anti-oxidation power(FRAP)method.(9)The activities in stimulating splenic lymphocyte proliferation by LMWSIPs and SIP were detected by CCK-8 method,as well as the activities on ConA-induced splenic lymphocyte proliferation.Results:(1)SIP could not be degraded by common glycosidases,such as hyaluronidase,α-amylase,β-amylase,chitinase,cellulose and hemicellulase.(2)When the concentration of SIP in aqueous solution was 2 mg/mL,the optimum acid degradation conditions were 0.5 mol/L TFA,80℃ and 3.5 h.(3)LMWSIPs with molecular weight distribution ranging from 1.8 kDa to 9.4 kDa were obtained by TFA degradation.Three fractions of samples with molecular weight distribution ranging from 7 kDa to 9 kDa,5 kDa to 7 kDa and 3 kDa to 5 kDa were obtained and named as LMWSIP-1,LMWSIP-2 and LMWSIP-3,respectively.HPGPC results showed that the purities of the fractions purified by Sephadex G-50 gel column chromatography were high.The molecular weight determination results of MAIDI-TOF MS and HPGPC for the samples were similar.(4)Compared with SIP,LMWSIP-1 and LMWSIP-2 had the same primary structure,but the degree of polymerization decreased.Compared with SIP,the primary structure of LMWSIP-3 changed obviously,and it may be that the polar groups such as carboxyl and hydroxyl groups fell off from monosaccharide units.(5)At a certain concentration,the activities of LMWSIP-2 in anti-proliferation,promoting apoptosis and inhibiting migration of tumor cells were significantly higher than those of SIP and other degradation products.(6)The·DPPH and·OH scavenging capacities and total anti-oxidation capacities of LMWSIPs did not change significantly compared with SIP.(7)SIP,LMWSIP-1 and LMWSIP-2 significantly promoted splenic lymphocyte proliferation at various concentrations,but LMWSIP-3 only played this role at high concentrations.In a certain concentration range,LMWSIP-2 promoted the proliferation of splenic lymphocyte significantly higher than SIP and other degradation products.(8)There were no significant differences between LMWSIPs and SIP in the promotion of ConA-induced splenic lymphocyte proliferation at different concentrations,and LMWSIPs significantly enhanced ConA-induced splenic lymphocyte proliferation of mice at high concentrations.Conclusions:The method of preparing LMWSIPs was established,and the anti-tumor,anti-oxidation and immunoregulatory activities of LMWSIPs were preliminarily studied.It was found that when the molecular weight of LMWSIPs was above 5 kDa,the primary structure of LMWSIPs did not change compared with SIP,only the degree of polymerization changed.When the molecular weight of LMWSIPs was below 5 kDa,the primary structure of polysaccharides was destroyed,and the structure of monosaccharide residues might also be destroyed.At certain concentrations,the anti-tumor and immunopotenting activities of LMWSIPs from 5 kDa to 7 kDa were stronger than those of SIP and other LMWSIPs.There was no significant difference in anti-oxidation activity between LMWSIPs and SIP.The results of this study suggest that SIP can not only directly act on tumor cells,but also enhance the immune function to achieve anti-tumor effect,but the specific mechanism needs to be further revealed.In addition,the molecular weight of SIP has close relations to its bioactivity.The results of this study provides a basis for further research and development of SIP.