Preparation Of Antler Protein In Lujiaofang And Its Protective Effect On Rabbit Chondrocytes

Objective: Osteoarthritis is a degenerative disease of the elderly.The "Lu Jiao Wan " in "Yan Shi Ji Sheng Fang" is a classical prescription for treating osteoarthritis.However,taking large dosage of pills is inconvenient for patients.The "Lu Jiao Fang " composition reference from " Lu Jiao Wan " prescription.To support product development and application in future,the research has studied the extraction process and enzyme hydrolysis process to improve antler protien oral bioavailability with intestinal absorption dynamics study,and provide rabbit articular chondrocytes efficiency data for treatment OA.Methods: Deionized water was used to extract total protein in antlers,and the protein content was taken as the index.L9?34?orthogonal test was used to extract the antler protein with the factors of extraction time,extraction time and amount of water.The best enzyme from acid protease,pepsin,trypsin,alkaline protease,neutral protease,papain,flavourzyme was chosen to hydrolysis antler protein respectively,and hydrolysis degree and Antioxidant effects were used as index.Alkaline protease and flavourzyme were used for protein hydrolysis process optimization,a Box-Behnken design was carried out to investigate the effects of p H value,hydrolysis temperature and the ratio of flavourzyme and alkaline protease with hydrolysis degree and O₂^-· inhibition as double index.The isolated everted intestine model was used to measure the absorption of free-amino acids,hydroxyproline and glycine within 120 min,the cumulative osmotic quantity and absorption rate constant were calculated for evaluating the absorption of antler protein in intestine.The experiment monolayer culture New Zealand rabbit primary chondrocytes in vitro and treated with fhydrogen Peroxide and interleukin-1?as Osteoarthritis cell model.The antler protein hydrolysate and Lu Jiao Fang were used to stimulated model cell respectively to study the effect on cell proliferation,cell viability and the content of type ? collagen?COL??and glycosaminoglycan?GAG?.Results:?1?The optimal extraction process was as follow: extracted 4 times with 10-folds of water for 2h each time.?2?The hydrolysate of flavourzyme got the highest hydrolysis degree 33.21±9.29% after enzymatic hydrolysis 330 min;The antler protein hydrolysate of flavourzyme got the best activity of hydroxyl radical scavenging action also,and the IC50 was 1.04 mg/m L;The hydrolysate of alkaline protease got the best activity of O₂^-· inhibition,and the IC50 was 1.87 mg/m L?3?The optimal hydrolysis condition of compound enzyme was obtained by single factor experiments and response surface optimization.The results showed that optimum hydrolysis condition was as follows: p H 7.5,hydrolysis temperature of 51.61 ?,the ratio of flavourzyme and alkaline protease was 7:3.?4?Compared with antler protein,the hydrolysates could significantly improve the absorption in the small intestine?P <0.01?.?5?The concentration of 10?g/m L antler protein hydrolysate and Lu Jiao Fang both could significantly promote IL-1?-induced chondrocytes proliferation?P<0.05?.protein hydrolysate and Lu Jiao Fang both showed great inhibtory effect on the H2O₂^-induced viability decreaseing?P<0.05?.The concentration of 100?g/m L antler protein hydrolysate and Lu Jiao Fang both could significantly?P<0.05?improve the content of COL?in IL-1?-induced chondrocytes an the content of GAG in the culture medium.Conclusion:?1?The result of intestinal absorption dynamics experimental improved,the antler protein hydrolysates could significantly improve the absorption of free-amino acids,hydroxyproline and glycine.?2?The antler protein hydrolysates and "Lu Jiao Fang " both show significantly inhibtory effect on H₂O₂ or IL-1?-induced chondrocytes damage.