Studies On Analysis And Optimization Of Hydrolytic Condition Of Deer Placenta Component

Ancient books recorded that deers were regarded as auspicious objects by ancestors,and the superior qualities were usually used as medicine due to their precious medicinal values.CPIRF(cervine placental immune regulator factor),the natural peptides less than 10 kDa,had biological activities of anti-tumor,anti-fatigue and anti-oxidation.However,plenty of ingredients in deers placenta had not been exploited,which resulted in a waste of deer placenta.In order to solve the problem mentioned above,supernatant of deer placental homogenates(SDPH)after the extraction of CPIRF was used as raw material in this research.Enzymatic conditions were optimized based on the components of SDPH and then the activities and safeties of deers placenta double-enzyme solution(DPDS)were evaluated.The results were as follows:(1)Analysis of SDPH compositions:SDPH compositions were determined by the biochemical tests and HPLC.Peptide content was dominant with a concentration of 4.08±0.2 mg/ml,the polysaccharide content was 2.66±0.14 mg/ml,nucleic acid content was 0.11±0.03 mg/ml.Among estrogens,the content of ethinyl estradiol was 50.59±0.18 ?g/ml,while diethylstilbestrol was only 0.31±0.02 ?g/ml.(2)Optimization of hydrolysis condition:The orthogonal experiments including the factors of pH,enzyme activity,reaction temperature and hydrolysis time were studied so as to confirm the optimization condition of double-enzyme hydrolysis.Results suggested that hydrolysis conditions of pepsin were enzyme activity 5000 U,pH 2.5,reaction temperature 40?,hydrolyze time 3.5 h,and then pH was adjusted to 7.0 without inactivation of pepsin enzymatic activity,papain was added and the enzyme activity was 2500 U under the conditions of temperature 53?,hydrolyze time 2.5 h,while the degree of hydrolysis of DPDS was 46.30%.Compared with unhydrolyzed SDPH,polypeptide bands of dispersion of less than 2 KDa were found in DPDS.DPDS had typical absorption of protein by UV scanning.(3)Evaluations of biological activities and safeties for DPDS:The results of MTT assay showed that DPDS played an effective role in the inhibition of gastric cancer cells(823)and cervical cancer cells(Hela)in vitro.The growth inhibition of DPDS on gastric cancer cells(823)was superior to cervical carcinoma cells(Hela).In immune activity of DPDS,the proliferation rate of spleen lymphocytes in mice was obvious.After intragastric administration of DPDS,the phagocytic ability of mice macrophage was significantly enhanced.During anti-oxidation tests in vitro,DPPH radical scavenging rate and total reducing power were improved as the increasement of total protein concentration of DPDS.The total SOD activity of DPDS treated mice were significantly increased compared to the control group,while MDA content was notably lower than the control group.DPDS prolonged the burden swimming time as well as the content of hepatic glycogen,while serum urea content in mice was down.The safety evaluations of DPDS were investigated by the micronucleus test,the sperm teratogenicity test in mice and acute toxicity test.The amount of micronucleus in peripheral blood of mice and bone marrow in the DPDS group showed no significant difference compared with the control group.DPDS did not cause toxic effects on mice in the teratogenic rate of sperm teratogenicity test.The LD50 of DPDS was greater than 8 g/kg.In this study,enzymatic conditions of SDPH were optimized for a more rational development and utilization of deer placenta,taking full advantage of the most of the resources of deer placenta,which improved the bioavailability,functionality and achieved a high value-added utilization of deer placenta.