Preparation And Toxicological Safety Evaluation Of Soft Capsules Making Of Glycosaminoglycan From Pinctada Martensii Dunker

OBJECTIVE:To develop a soft capusule of CPG from Pinctada martensii Dunker with enhancingimmunity though preparation research，quality research，and toxicology research.METHOD:(1)With the dissolution rate of lemon yellow added in the capsule shell as the index，ratio between gelatin and glycerin,ratio between gelatin and water were investigated to testtheir influence on the solubility of capsule shell, we adopted orthogonal experiment tooptimize the formula of the capsule shell.(2)Taking the volume ratio of sedimentation and the fluidity as the evaluationindexes,we adopted Response Surface Methodology (RSM) to optimize the formula of thesoft capsule content. the response surface analysis was employed to investigate the influenceof three main factors, including the dosage of PEG400, the dosage of PEG6000and thedosage of propylene glycol.(3)The content of CPG in the soft capsules was determined with the Alcian blue.(4)According to“Chinese Pharmacopoeia”(2010),the content uniformity anddisintegration time were examined. According to “Inspection and Assessment Standard forHealth Food” microbial limit and heavy metal content of the soft capsules was also carriedout;According to"Stability test requests for Health Food ", Stability test of the soft capsuleswas carried out.(5)The methods of safety toxicology evaluation:①In the acute toxicity test, mice wereorally treated with CPG soft capsules content at the dose of26.6g/kg-1BW to observe thedeath or changes in behavior for14days.②Ames tests, reverse mutation assays ofSalmonella typhimurium strains TA97,TA98,TA100and TA102, were used for evaluatingthe mutagenesis of CPG soft capsules content. The number of revertant colonies was recordedafter treating Salmonellar strains with different doses of CPG soft capsules content(5000,1000,200,40,8μg/dish) for48hours.③In the micronucleus test of bone marrowcells, the mice were orally treated with CPG soft capsules content at the doses of6,3,1.5g/kg-1BW for detecting the micronucleus ratio of bone marrow cells.④In the sperm shapeabnormality test, the mice was treated with CPG soft capsules content at the same dose ofmicronucleus ratio tests, and recording the number of abnormality sperm of1000sperms. ⑤A30-day-feeding test was conducted in SD rats to assess the effects of CPG soft capsulescontent on behaviour, body-weight gain, food intake, food utilization ratio and haematologyparameters, blood biochemistries, organ weights and the ratio of organ weight and bodyweight，and the histopathologic examination.RESULTS:(1)According to orthogonal experiment,we confirmed the best formulation was: theratio of glycerol to gelatin was0.5:1,the ratio of water to gelatin was1.2∶1;the percentageof PEG400was7%. Under the optimization formulation,the dissolution rates of soft-gelatincapsule shell were5.8mg/min·cm~2.(2)According to formula optimization and RSM experiment,we confirmed the ratio ofCPG and PEG400was1:3,the ratio which thePEG6000, propylene glycol occupy CPG was10.6%,9.3%.The content fluidity was good,the stability of suspend was good.(3)The linear ranges of CPG was100-600μg/mL,linearregression equation wasY=0.0002x+0.0036,r~2=0.9959;the recovery was97.2%(RSD=0.58%);the sample solutionwas stable within30min(RSD=0.67%); the average content of CPG was33.21mg/pill.(4)The examining results of three batches of samples showed that the contentuniformity<±10%;the disintegration time was in1h.The microbial limit and the heavy metalcontent were in accordance with requirement;Through inspection on stability of theseproducts for3months, the key indicators were no great change.(5)The results of safety toxicology test:①The result of acute toxicity test showed that,The maximum tolerable dose (MTD) was larger than26.6g/kg BW. No animals were deadand no any other harmful symptoms were observed.②The mutagenicity of CPG soft capsulescontent was not found in the three mutation tests. All the results of Ames test, bone marrowcell micronucleus test in mice and sperm abnormality test in mice were negative.③Theresults of the30-days-feeding test showed that CPG soft capsules content has no significantinfluence on the weight gain, food utility ratio, food intake, haematology parameters, bloodbiochemical index, organ weight and the ratio of organ weight and body weight of the SDrats. And there were no obviously harmful changes on the main visceral organs of the ratsby observation of vivisection and histopathologic examination.CONCLUSION:The formula of the CPG soft capsules including the content and the shell selected waswell formed and stable.;the quality of soft capsules meeted the requirements of relatedstandards;the inspection on stability of preparation showed that it was stable;the results ofsafety toxicology evaluation were negative.