CN1110915A

CN1110915A - Method for prepn. of cerebral proteolytic liquid-cerebral health injection

Info

Publication number: CN1110915A
Application number: CN94104516A
Authority: CN
Inventors: 徐康森; 朱炯; 孙磊; 杨昭鹏; 滕毓敏
Original assignee: CHINA RESEARCH CENTRE OF MEDICINE BIOLOGICAL PRODUCTS STANDARDIZATION
Current assignee: CHINA RESEARCH CENTRE OF MEDICINE BIOLOGICAL PRODUCTS STANDARDIZATION
Priority date: 1994-04-26
Filing date: 1994-04-26
Publication date: 1995-11-01
Anticipated expiration: 2014-04-26
Also published as: CN1087940C

Abstract

A method for extracting cerebroprotein hydrolysate from animal's brain, esp. pig's brain and the process for preparing injection of said cerebroprotein are disclosed.

Description

Method for prepn. of cerebral proteolytic liquid-cerebral health injection

The present invention relates to a kind of happy method of Cerebrolysin Vial one brain of from animal, particularly Medulla sus domestica, extracting, the invention still further relates to injection of described Cerebrolysin Vial and preparation method thereof.

Cerebrolysin Vial is that trade name is sold with Cerebrolysin by Austrian EBEWE pharmaceutical factory the earliest, wherein contains multiple human body essential amino acid and non essential amino acid, and to contain molecular weight be 10,000 or lower peptide.That although it is used is open already (referring to EP452299 etc.), its extracting method does not appear in the newspapers so far.

The object of the present invention is to provide a kind of method of from animal brain, especially Medulla sus domestica, extracting Cerebrolysin Vial (hereinafter referred is " brain is happy ").

Another object of the present invention provides the method for preparing the happy injection of brain.

Hereinafter will describe purpose of the present invention in detail.

Fig. 1 has summarized the extracting method of brain pleasure and has been prepared into the method for injection;

Fig. 2 is an import cerebrolysin vial reference substance peptide content HPLC chromatogram;

Fig. 3 is import cerebrolysin 201468 peptide content HPLC chromatograms;

Fig. 4 is import cerebrolysin 901724 peptide content HPLC chromatograms;

Fig. 5 is import cerebrolysin 912950 peptide content HPLC chromatograms;

Fig. 6 is the happy injection 930401 peptide content HPLC chromatograms of brain;

Fig. 7 is the happy injection 930402 peptide content HPLC chromatograms of brain;

Fig. 8 is the happy injection 930403 peptide content HPLC chromatograms of brain;

Fig. 9 is the pure product HPLC of cerebrolysin peptide II (MW5270) chromatogram;

Figure 10 is the pure product HPLC of cerebrolysin peptide I (MW1770) chromatogram.

The happy parenteral solution of brain as herein described prepares by following process:

(1) homogenate: with fresh pig brain decerebrate film fat, be cut into small pieces, homogenate in refiner repeats once, gets brain homogenate liquid;

(2) degreasing: brain homogenate liquid is poured in the cylinder with cover, is added while stirring cold acetone, then in 4 ℃ place 4 hours after, the supernatant that inclines repeats 2-3 time, and sediment is poured in the porcelain dish, puts fume hood and dries up, and pulverizes, and sieves, and gets the dried brain powder of degreasing;

(3) hydrolysis: get dried brain powder, enzyme-added liquid was put 37 ℃ of water bath heat preservations 8 hours, and is centrifugal then, discards precipitate, obtains hydrolyzed solution;

(4) concentrate: hydrolyzed solution is concentrated with rotary film evaporator;

(5) centrifugal: concentrated solution with freezing, at a high speed, high capacity centrifuge centrifugal (10000-20000rpm), get supernatant;

(6) ultrafiltration: the supernatant after centrifugal obtains the clear and bright solution of pale brown color through ultrafilter 10,000 molecular weight mwco membrane ultrafiltration;

(7) refining: as the solution after the ultrafiltration to be separated through DEAE-Sephadex A-25 column chromatography,,, and collect each main peak fraction, must make with extra care clear liquid with the ultraviolet spectrophotometer monitoring with Tris-HCl buffer and the buffer solution elution that contains NaCl;

(8) sterilization:, get aseptic parenteral solution with the refining clear liquid sterilization of gained.

Embodiment:

Get fresh Medulla sus domestica, decerebrate film fat is cut into small pieces, twice homogenate in refiner.Homogenate is poured in the cylinder with cover, added cold acetone while stirring, in 4 ℃ of placements 4 hours, the supernatant that inclines repeated twice of this process then.Precipitate is poured in the porcelain dish, is put in the fume hood and dry up, pulverize, sieve, the dried brain powder of defat.Add enzyme liquid in dried brain powder, insulation is 8 hours in 37 ℃ of water-baths, and is centrifugal then, discards precipitation, gets hydrolyzed solution.Use the rotary film evaporator concentrating hydrolysate.With concentrated solution in freezing, at a high speed, on the high capacity centrifuge centrifugal (15000rpm), get supernatant.Supernatant after centrifugal through ultrafilter 10,000 molecular weight mwco membrane ultrafiltration, is got the clear and bright solution of pale brown color.Gained solution separates through DEAE-Sephadex A-25 column chromatography, and method is as follows:

(1) preparation Tris-HCl buffer is as basic buffer; In the basic buffer of part, add NaCl, as eluent.

(2) dress DEAE-Sephadex A-25 post (5 * 120cm), DEAE-Sephadex A-25 soaked 1-2 days with Tris-HCl basis buffer in advance, behind the dress post with basic buffer balance.

(3) solution that ultrafiltration is intact with suction pipe slowly add chromatographic column above, treat that sample infiltrates in the glue and add a little basic buffer, seal post then, start constant flow pump, the control flow velocity is 60ml/ hour, goes up sample 6 at every turn and restrains.

(4) with eluent and basic buffer, in the gradient former volumetric flask, carry out gradient elution.

(5) optical density and the absorption curve of managing number are measured and drawn to effluent through the uv-spectrophotometric instrument, collects each main peak fraction.

(6) separate the peptide components of this sample through the HPLC gel column, obtain both at home and abroad all not two peptide components of report, its content surpasses 90%.The gained fraction is made aseptic parenteral solution through sterilization.Analytical method is as follows:

One, sample: the big pharmaceutical factory of Austrian Yi Biwei lot number: 901724,912950.

This development type lot number: 930401,930402,930403.

Nat'l Pharmaceutical ﹠ Biological Products Control Institute's biochemical drug chamber cerebrolysin peptide I and the pure standard substance of peptide II HPLC

Two, method: HPLC gel filtration

1, reagent: dipotassium hydrogen phosphate, potassium dihydrogen phosphate, isopropyl alcohol.

2, instrument: Waters HPLC, 510 type pumps, 481 type detectors, 740 type data processors.

3, chromatographiccondition: mobile phase is 0.1M(PH=7) phosphate buffer, flow velocity 0.3ml/ branch;

Gel column: column temperature 30 ℃ ± 0.1, detect wavelength 280nm.

4, operational approach: get testing sample 10ul, sample introduction, the record analysis result calculates the degree of peptide.

Three, result

1, the HPLC chromatogram of import cerebrolysin vial and this developed product peptide components analysis;

(1), the chromatogram of the pure product of HPLC of peptide I and peptide II in the Cerebrolysin Vial;

(2), import cerebrolysin: (seeing figure) 901724 batches, 912950 batches;

(3), the happy injection of this developed product brain: (seeing figure) 930401,930402,930403 batches.

2, HPLC analyzes import cerebrolysin and the percentage composition of the happy injection peptide components of this developed product brain and the mathematical statistics of retention time.

The HPLC retention time of table 1 peptide I and peptide II and the mathematical statistics result of content

The result of table 2 peptide total amount and its ratio

Brief summary:

1, with the peptide components in HPLC gel column Protein60 separation cerebrolysin or the happy injection of brain.Get two main constituent peptide I and peptide II.These goods gained peptide I is consistent with import sample peptide I and its retention time of peptide II with the peptide II.Also consistent with pure peptide I and the peptide II standard substance of HPLC.Illustrate that this developed product and standard substance, its peptide I of external similar sample are identical with peptide II composition.(seeing chromatogram and table 1).

2, its percentage composition of peptide I is 12.86～15.59 in the import cerebrolysin, and its percentage composition of peptide II is 63.19～65.83.

Its percentage composition of peptide I is 18.11～18.29 in this developed product, and its percentage composition of peptide II is 74.00～74.33.

The main component of this developed product of presentation of results not only composition is identical with the import sample, and content is higher than similar import sample with purity.(seeing Table 1).

3, the total content of import cerebrolysin peptide is 77.38～79.66%, its peptide I/peptide II ratio 0.20～0.24.The total content of this developed product peptide is 92.11～92.62%, and its peptide I/peptide II ratio is 0.25.(seeing Table 2).

The total content that the result draws this developed product main component peptide has surpassed external product.

Happy injection of brain and import sample cerebrolysin vial are compared experiment, find that the result of its free amino acid is very consistent.Analysis result sees the following form 3:

The free amino acid analysis result of table 3 import cerebrolysin and the happy injection of this developed product brain

Claims

1, the preparation method of the happy injection of brain is characterized in that:

(1) with animal brain homogenate,

(2) use the acetone defat, drying, pulverize sieves,

(3) enzyme-added liquid is centrifugal after 8 hours in 37 ℃ of insulations in dry powder, discards precipitate, gets hydrolyzed solution,

(4) concentrating hydrolysate,

(5) with 10000-20000rpm freezing, at a high speed, centrifugal concentrated solution on the high capacity centrifuge, get supernatant and hold back ultrafiltration through ultrafilter 10,000 molecular weight,

(6) solution after the ultrafiltration separates through DEAE-Sephadex A-25 column chromatography, collects the main peak fraction, must make with extra care clear liquid,

(7) will make with extra care the clear liquid sterilization, get aseptic parenteral solution.