Determination Of Physiological Biochemical Compositions Of Oyster And Its Effect On Immune Regulation And Acute Toxicity In Mice

Objective To determine physiological biochemical compositions of Ostrea talienwhanensis Crosse and study its immune regulation and acute toxicity in normal mice based on the support of 863 project "the development of new drugs of traditional Chinese medicine based on the traditional marine medicine",providing experimental data for marine traditional Chinese medicine Ostrea talienwhanensis Crosse in medicinal evaluation.Methods Determination of physiological biochemical compositions of oyster: Gel electrophoresis analysis were used to determined molecular of polypeptide in oyster;LC-MS/MS method was used to measure amino acid sequence of prime polypeptide MLS-1 and other polypeptides in oyster;pectrophotometry was used to determine polypeptide content in oyster;automatic amino acid analyzer was used to determine the composion and content of the amino acids in oyster.Immunomodulatory effects of oysters on mice: ICR mice were randomly divided into control group,astragalus membranaceus-ligustrum lucidum tonic capsules(positive drug)group(3.9 g/kg)and the each dose of oyster(0.0625 g/kg,0.125g/kg,0.25 g/kg,0.5 g/kg,1.0 g/kg,2.0 g/kg)according to the weight of the mice.The carbon clearance test had no oyster(0.0625 g/kg,0.125 g/kg)group.Every rat received 0.2 ml/d according to the above dose by oral gavage addition to the mice of control group receiving 0.2 ml/d distilled water separately.The immune indexes of mice of each group were determined after 15 days: the clearance rate of carborn,MTT method and hemolytic plaque experiments were used to detect the phagocytosis of macrophage cell,the spleen lymphocyte proliferation and the formation of antibodies generated cells(B cell)respectively in mice;more over,the immune organ indexes of mice of each group were measured.Acute toxicity test: 40 ICR mice,female and male accounting for half respectively,were randomly divided into the control group and the oyster group according to the weight of the mice,20 mice in each group and male and female accounting for half respectively as well.According to the maximum tolerance dose method(MTD),the mice in oyster group received oysters for the concentration of 0.5412 g/ml and a volume of 0.8 ml by intragastric administration,2 times a day,the dosing interval time was 6 h,total dose was 43.3 g/kg.The mice in normal control were given 0.8 ml distilled water,2 times a day,the dosing interval time was 6 h.In the 14 days after administration,the general behavior,poisoning and death of the mice were observed and recorded.After 14 d,all the mice were killed and dissected,and the main tissues and organs of the mice were observed with naked eyes.Results Determination of physiological biochemical constituents of oyster: Molecular of polypeptide in oyster was 2 ~ 10 k D.The sequences of the prime MLS-1 and other peptide were: MLAILDQYSK;VIKFSDLPDP;SSTWEVQSLV;GEGTYGEIHK;AIHKESGEVV;AMKVLDSIHE;KIEEIEEEYR;VLHDLGNHPN;MPRFHGIYLK;PPTTGMDAE;WIAMELCGRG;SVTHLVRELI;GRGEALEEDL;IAHIIRETLK;ALQHLHKNHV;IHRDVKGHNI;LINDQGQIKL;IDFGVSGHLA;STLARKNTSV;GTPYWMAPEV;IACERQVEYN;YDIRCDIWSL;GITAIELADG;EPPLSDLHPM;RALFKIPRNP;QPQFANPDRW;SEDFKDFIAK;CLIKDFEVRP;FTHDLLRHPF;IAKLPDDTSQ;VNYLTGQDGS;NNTIFLSNI.Polypeptide content in oyster was 89.72%.Oyster included Asp?Thr?Ser?Glu?Gly?Ala?Cys?Val?Met?Ile?Leu?Tyr?Phe?Lys?His and Arg.Asp?Glu?Lys and Arg were main amino acid.Immunomodulatory effects of oysters on mice: Compared to control group,oyster of 0.25 g/kg,0.5 g/kg,1.0 g/kg,2.0 g/kg could obviously enhance the phagocytosis of macrophages(23.31%,27.75%,31.79%,27.75%)and promote the formation of antibodies generated cells in mice(17.71%,20.42%,18.50%,18.09%);oyster of 0.0625 g/kg,0.125 g/kg,0.25 g/kg,0.5 g/kg could observably promote the proliferation of lymphocyte in mice(53.66%,120.73%,132.93%,123.17%);the spleen index and thymus index weren't improved obviously(P>0.05).Acute toxicity test: MTD of mice for oyster by oral administration was more than 34.4 g/kg.Compared with the control,after 2 times by oral administration,the mice in oyster group showed no obvious abnormal change;after 14 days,all mice survived and the appearance,behavior,posture,eating,drinking,fur,respiration and excretion showed no abnormality in oyster group;the difference of the body weight and food intake of the mice between oyster group and control group was not significant(P>0.05);at the end of the observation period,viscera and tissues of dissected mice showed no obvious abnormal change by macroscopic observation.Conclusions Oyster contains polypeptides with a molecular weight of 2 ~ 10 k D and the content of 89.72% and could enhance immune function by enhancing non-specific immunity and specific immunity(cellular immunity,humoral immunity).MTD of the mice for oyster is greater than 43.3 g/kg(equivalent to adults with a dose of 4.7582 g/kg).In accordance with acute toxicity dose classification in appendix D of ? Technical specification for inspection and evaluation of health foods? in 2003 edition,oyster belongs to non-toxic grade.