| 摘要 |
FIELD: medicinal industry. ^ SUBSTANCE: invention relates to a method for isolation of biologically active substance from mammalian cartilage and to preparing a medicinal formulation for parenteral administration. Agent is made as a medicinal formulation for parenteral administration and represents peptide complex with the content of low-molecular fraction from 70% to 90%, molecular mass of its peptide components in the range 75-846 Da and concentration of polypeptides 2.5-2.9 mg/ml. Agent is prepared from cartilage of calves (age is 12 months, not above) or pigs by extraction with acetic acid in the presence of zinc chloride. Proposed method for preparing agent from cartilage of calves (age is 12 months, not above) or pigs involves tissue freezing at temperature -40C (not less), keeping at temperature -20-22C for 2 months (not less) followed by milling and addition of 3% acetic acid solution in the volume ratio = 1:5 at temperature 20 5C. Extraction is carried out at constant stirring up to preparation homogenous suspension and then 1% zinc chloride solution is added to suspension in the volume ratio = 50:1. Mixture is cooled at constant stirring up to temperature 7-16C, stirred for 1 h in each 4 h settling for 48 h. Extract is separated from inert substances by separating and acetone is added to extract in the volume ratio = 1:5 and kept at temperature 3-5C for 4 h. Formed homogenized deposit is precipitated with acetone repeatedly twice (not less) and deposit containing active substance is washed out on Nutch filter with two-fold volumes of acetone cooled to temperature 7-16C up to preparing light-gray deposit. Deposit is rubbed through metallic sieve, dried, dissolved in distilled water at room temperature and constant stirring up to the concentration of polypeptides 2.5-2.9 mg/ml. Solution is centrifuged, filtered and subjected for ultrafiltration treatment in device at anti-pressure 1.0 kgf/cm2 (not above) through materials with retaining capacity 15000 Da. Glycocol is added to ultrafiltrate up to its final concentration 10-20 mg/ml at pH = 5.6-6.6, solution is subjected for sterilizing filtration under pressure 2.0 kgf/cm2 (not above), poured into ampoules in volume 2 ml and subjected for autoclaving at temperature 120C for 8 min under atmosphere pressure 1.1 kgf/cm2. Invention provides optimal technology in isolation of peptide complex with the content of low-molecular fraction from 70% to 90%, molecular mass of peptide components in the range from 75 to 846 Da and preparing aqueous extract with the concentration of polypeptides 2.5-2.9 mg/ml. Invention provides both purification of the end product from impurities and to enhance its yield. The isolated substance differs from the known substances prepared early from mammalian raw by molecular mass of peptide components, absence of toxicity and apyretic properties based on the complete removing impurities. Proposed substance can be used in medicine as an agent normalizing functions of cartilage tissue. ^ EFFECT: improved preparing method, valuable medicinal properties of agent. ^ 3 cl, 1 tbl, 1 dwg, 4 ex |
