CN102228676A - Heparolysate injection pharmaceutical composition - Google Patents
Heparolysate injection pharmaceutical composition Download PDFInfo
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- CN102228676A CN102228676A CN201110189767XA CN201110189767A CN102228676A CN 102228676 A CN102228676 A CN 102228676A CN 201110189767X A CN201110189767X A CN 201110189767XA CN 201110189767 A CN201110189767 A CN 201110189767A CN 102228676 A CN102228676 A CN 102228676A
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Abstract
The invention discloses a Heparolysate injection pharmaceutical composition. The pharmaceutical composition is prepared from Heparolysate, vitamin C, potassium aspartate and magnesium aspartatse injection and polyethylene glycol 200. Low vitality stimulation indexes and limited treatment effects of the Heparolysate injection are overcome. The invention also discloses a preparation method of the pharmaceutical. The preparation method which has the characteristic of simple technology is suitable for industrialization production.
Description
Technical field
The present invention relates to a kind of pharmaceutical composition for the treatment of hepatitis, liver cirrhosis disease, further provided a kind of injection of hydrolyzed peptide of liver pharmaceutical composition, belong to the biochemical pharmacy technical field.
Background technology
Existing " injection of hydrolyzed peptide of liver " is China's prescription class medicine, intramuscular injection, the auxiliary treatment that is used for diseases such as chronic hepatitis, liver cirrhosis is to extract the aseptic aqueous solution that contains polypeptide class, nucleic acid class, amino acids material that makes through enzyme hydrolysis by the liver of healthy cattle, pig.Hydrolyzed peptide of liver can promote protein synthesis, reduces protein and decomposes, and promotes the propagation and the regeneration of normal liver cell, and the hepatocyte injury of tetrachloro-methane induction is had the better protect effect, reduces glutamate pyruvate transaminase, promotes pathological tissues to recover; But this medicine is still very limited for hepatitis, liver cirrhosis treatment of diseases effect, measure according to activity of hepatocyte auxin algoscopy (mtt assay) in the national drug standards, injection of hydrolyzed peptide of liver has the vigor of external promotion human hepatocyte growth, but the vigor stimulation index is for only being about 2.9.
Summary of the invention
The present invention discloses a kind of injection of hydrolyzed peptide of liver pharmaceutical composition, and the vigor stimulation index that has overcome " injection of hydrolyzed peptide of liver " is lower, the shortcoming that therapeutic effect is limited.The present invention also provides the preparation method of this medicine, and technology is simple, is applicable to suitability for industrialized production.
Injection of hydrolyzed peptide of liver compositions provided by the invention is characterized in that being made by following raw material:
Concentration is hydrolyzed peptide of liver solution 1000ml, vitamin C 2 ~ 5g, aspartic acid magnesium injection liquid 20 ~ 30ml, the Macrogol 200 10 ~ 20g of 9.2 ~ 12.2mg/ml.
Described aspartic acid magnesium injection liquid is that every 1ml contains L-Aspartic Acid 85mg: potassium 11.4mg:4.2mg.
The invention provides injection of hydrolyzed peptide of liver preparation of compositions method, comprise the steps:
1, hydrolyzed peptide of liver solution
The liver of cattle or pig, after removing connective tissue, by the parts by weight of 0.83 ~ 0.85:1 with after water for injection mixes, use high-speed tissue mashing machine's homogenate, be warming up to 37 ~ 40 ℃, transferring pH value with HCL is 2.0 ~ 2.5, presses 3g/ and rises the adding pepsin, hydrolysis 10 ~ 15 hours, it is 6.2 ~ 6.4 that hydrolyzed solution is regulated pH value with NaOH, adds 1g/ and rises kieselguhr, 90 ℃, heated 30 minutes, small-sized filter press filter pressing, with 0.45 μ m microporous filter membrane filtration, 8000 molecular weight ultrafilter membrane ultrafiltration get hydrolyzed peptide of liver solution, measure content of peptides, adding injection water adjustment solution peptide concentration is 9.2 ~ 12.2mg/ml;
2, the hydrolyzed peptide of liver solution 1000ml for preparing in step 1, the vitamin C that adds 2 ~ 5g, the aspartic acid magnesium injection liquid that adds 20 ~ 30ml, the Macrogol 200 that adds 10 ~ 20g, 121 ℃ of autoclavings are 20 minutes after the packing, through be up to the standards injection of hydrolyzed peptide of liver compositions final drug preparation.
Below experiment shows that the external promotion human hepatocyte growth of medicine of the present invention vigor greatly improves:
Experimental technique: the promoting hepatocyte growth factor solution national drug standards: activity of hepatocyte auxin algoscopy (mtt assay, standard numbering: WS-10001-(HD-0860)-2002).
Test specimen
1.1 test sample:
Original formulation: hydrolyzed peptide of liver solution contains polypeptide 11 mg/ml.
Vitamin C: the solution that contains vitamin C 50 μ g among the every 1ml that makes with the RPMI-1640 culture fluid.
Motassium magnessium aspartate: the solution that contains L-Aspartic Acid 25.5 μ g, potassium 3.42 μ g, magnesium 1.26 μ g among the every 1ml that makes with the RPMI-1640 culture fluid.
Embodiment 1 preparation;
Embodiment 2 preparations;
Embodiment 3 preparations;
Embodiment 4 preparations;
1.2 reference substance: Hepatocyte Growth-Promtting Factors For Injection, specification are 20mg
2. material
2.1 instrument: CO
2Incubator, horizontal desk centrifuge, microplate reader, knockout plate machine, Tissue Culture Flask, Tissue Culture Plate, disposable aspiration needle filter.
2.2 reagent:
Sodium chloride, potassium chloride, disodium hydrogen phosphate,anhydrous, potassium dihydrogen phosphate, RPMI-1640 MEDIUM, sodium bicarbonate, hydrochloric acid, sodium hydroxide, calf serum, trypsin, EDTA disodium, MTT, dimethyl sulfoxide, human liver cell strain.
Method
3.1 the preparation of reagent:
3.1.1 0.01mol/L phosphate buffer (pH7.3) is got sodium chloride 8.0g, potassium chloride 0.2g, sodium hydrogen phosphate (Na2HPO4) 1.15g and potassium dihydrogen phosphate 0.2g, add ultra-pure water 1000ml dissolving after, autoclaving.
3.1.2 after the RPMI-1640 culture fluid is got sodium bicarbonate 2.00g and RPMI-164010.0g and added the about 800ml dissolving of ultra-pure water, regulate pH value to 6.9, add ultra-pure water and be diluted to 1000ml, filtration sterilization with the 1mol/L hydrochloric acid solution.
3.1.3 10% calf serum culture fluid is got above-mentioned RPMI-1640 culture fluid 900ml, adds the newborn calf serum 100ml of deactivation.0.25% trypsin-Calcium Disodium Versenate Digestive system is got 0.25g trypsin and 0.02g Calcium Disodium Versenate, after adding 0.01mol/L phosphate buffer 1 00ml dissolving, regulate pH value to 7.2, filtration sterilization ,-20 ℃ of preservations with 5.6% sodium bicarbonate.
3.1.4 tetrazolium bromide (MTT) solution is got MTT50mg, add 0.01mol/L phosphate buffer 1 0ml dissolving after, filtration sterilization (be stored in cold place, use and be no more than for two weeks)
3.2 assay method:
3.2.1 it is an amount of that test sample is got in the preparation of need testing solution, makes the solution that contains polypeptide 100 μ g among every 1ml with the RPMI-1640 culture fluid.
3.2.2 algoscopy: cultivate the SMMC-7721 cell to increased logarithmic phase with 10% calf serum culture fluid, with 0.25% trypsin-Calcium Disodium Versenate Digestive system digestion, add 10% calf serum culture fluid and be diluted among every 1ml and contain the individual cell in (2.5 * 10＜4 〉) ~ (5 * 10＜4 〉), with above-mentioned cell suspension bed board on 96 porocyte culture plates, every hole 100 μ l, wherein stay 3 holes to add 10% calf serum culture fluid, 100 μ l as blank, put 37 ℃, cultivate in the 5% carbon dioxide saturation vapour incubator and made it adherent in 3 ~ 4 hours.The test sample group, every hole adds need testing solution 100 μ l, every batch of test sample is all done 3 holes, cell matched group and the every hole of blank group add RPMI-1640 culture fluid 100 μ l respectively, put 37 ℃, cultivated 48 hours in the 5% carbon dioxide saturation vapour incubator, finish to cultivate preceding 4 hours taking-up culture plates, culture fluid is removed in suction, every hole adds 0.01mol/L phosphate buffer (pH7.3) and washes once, and then in every hole, add above-mentioned phosphate buffer (pH7.3) 100 μ l and MTT solution 20 μ l, continue to cultivate.After cultivating end, the sucking-off culture fluid, every hole adds 100 μ l dimethyl sulfoxide, shakes up, and the wavelength place with 550nm on microplate reader measures its trap A value respectively.
Test sample group 3 hole trap meansigma methodss (A＜[T] 〉-blank group 3 hole trap meansigma methodss (A＜[O] 〉)
Stimulation index=------------------------------
Matched group 3 hole trap meansigma methodss (A＜[R] 〉-blank group 3 hole trap meansigma methodss (A＜[O] 〉)
4. result of the test:
The injection of hydrolyzed peptide of liver vitality test
Conclusion: the external promotion human hepatocyte growth of combination formula medicine of the present invention vigor greatly improves.
In the present invention, vitamin C has stoped the destruction of active ingredient in the finished product high temperature sterilize in the hydrolyzed peptide of liver as antioxidant, and vitamin C itself also has the hepatocellular reparation of promotion and regenerated effect; Motassium magnessium aspartate is the medicine that improves liver function, can quicken the hepatocyte tricarboxylic acid cycle; Polyethylene Glycol has excellent biological compatibility and amphipathic, Macrogol 200 can make that the ruined polypeptide structure that has active hydrophobic group is stretched in the hydrolyzed peptide of liver raw material leaching process, also can stop the destruction of active ingredient in the finished product high temperature sterilize in the hydrolyzed peptide of liver, thus the function of performance hepatic cell growth promotion; Antioxidant vitamin C and cosolvent Macrogol 200 make also that the color and luster of finished product is better, stability improves greatly.Result of the test shows that the vigor stimulation index of the external promotion human hepatocyte growth of the present composition is far longer than the vigor stimulation index that hydrolyzed peptide of liver and vitamin C, the external routine tests of motassium magnessium aspartate promote human hepatocyte growth.
Good effect of the present invention is:With hydrolyzed peptide of liver solution and vitamin C, aspartic acid magnesium injection liquid and Macrogol 200 are organically in conjunction with compatibility, bring into play their synergism and be far longer than the independent effect sum of their each medicines, medicine can promote human hepatocyte growth external, promote the vigor stimulation index of human hepatocyte growth to significantly improve, measure according to activity of hepatocyte auxin algoscopy (mtt assay) in the national drug standards, present composition vigor stimulation index is about 5.2, be far longer than the vigor stimulation index 2.9 of folk prescription " injection of hydrolyzed peptide of liver ", can become a kind of new treatment hepatitis, the pharmaceutical composition of liver cirrhosis disease has the obvious curative effects effect.
The specific embodiment:
For the ease of understanding the present invention, especially exemplified by following 4 embodiment, its effect is understood that it is to explaination of the present invention but not to any type of restriction of the present invention.
Embodiment 1
1, hydrolyzed peptide of liver solution
The liver of cattle, after removing connective tissue, the parts by weight of pressing 0.85:1 are with after water for injection mixes, use high-speed tissue mashing machine's homogenate, be warming up to 37 ~ 40 ℃, transferring pH value with HCL is 2.0 ~ 2.5, presses 3g/ and rises the adding pepsin, hydrolysis 10 hours, it is 6.2 ~ 6.4 that hydrolyzed solution is regulated pH value with NaOH, adds 1g/ and rises kieselguhr, 90 ℃, heated 30 minutes, small-sized filter press filter pressing, with 0.45 μ m microporous filter membrane filtration, 8000 molecular weight ultrafilter membrane ultrafiltration get hydrolyzed peptide of liver solution, measure content of peptides, adding injection water adjustment solution peptide concentration is 9.2 ~ 12.2mg/ml.
2, the hydrolyzed peptide of liver solution 1000ml that obtains previously, the vitamin C that adds 5g, the aspartic acid magnesium injection liquid of adding 30ml, the Macrogol 200 of adding 15g, 121 ℃ of autoclavings are 30 minutes after the packing, through be up to the standards injection of hydrolyzed peptide of liver compositions final drug preparation.
Embodiment 2
1, hydrolyzed peptide of liver solution
The liver of cattle, after removing connective tissue, the parts by weight of pressing 0.85:1 are with after water for injection mixes, use high-speed tissue mashing machine's homogenate, be warming up to 37 ~ 40 ℃, transferring pH value with HCL is 2.0 ~ 2.5, presses 3g/ and rises the adding pepsin, hydrolysis 10 hours, it is 6.2 ~ 6.4 that hydrolyzed solution is regulated pH value with NaOH, adds 1g/ and rises kieselguhr, 90 ℃, heated 30 minutes, small-sized filter press filter pressing, with 0.45 μ m microporous filter membrane filtration, 8000 molecular weight ultrafilter membrane ultrafiltration get hydrolyzed peptide of liver solution, measure content of peptides, adding injection water adjustment solution peptide concentration is 9.2 ~ 12.2mg/ml.
2, the hydrolyzed peptide of liver solution 1000ml that obtains previously, the vitamin C that adds 2g, the aspartic acid magnesium injection liquid of adding 20ml, the Macrogol 200 of adding 10g, 121 ℃ of autoclavings are 30 minutes after the packing, through be up to the standards injection of hydrolyzed peptide of liver compositions final drug preparation.
Embodiment 3
1, hydrolyzed peptide of liver solution
The liver of cattle, after removing connective tissue, the parts by weight of pressing 0.85:1 are with after water for injection mixes, use high-speed tissue mashing machine's homogenate, be warming up to 37 ~ 40 ℃, transferring pH value with HCL is 2.0 ~ 2.5, presses 3g/ and rises the adding pepsin, hydrolysis 10 hours, it is 6.2 ~ 6.4 that hydrolyzed solution is regulated pH value with NaOH, adds 1g/ and rises kieselguhr, 90 ℃, heated 30 minutes, small-sized filter press filter pressing, with 0.45 μ m microporous filter membrane filtration, 8000 molecular weight ultrafilter membrane ultrafiltration get hydrolyzed peptide of liver solution, measure content of peptides, adding injection water adjustment solution peptide concentration is 9.2 ~ 12.2mg/ml.
2, the hydrolyzed peptide of liver solution 1000ml that obtains previously, the vitamin C that adds 5g, the aspartic acid magnesium injection liquid of adding 25ml, the Macrogol 200 of adding 20g, 121 ℃ of autoclavings are 30 minutes after the packing, through be up to the standards injection of hydrolyzed peptide of liver compositions final drug preparation.
Embodiment 4
1, hydrolyzed peptide of liver solution
The liver of cattle, after removing connective tissue, the parts by weight of pressing 0.85:1 are with after water for injection mixes, use high-speed tissue mashing machine's homogenate, be warming up to 37 ~ 40 ℃, transferring pH value with HCL is 2.0 ~ 2.5, presses 3g/ and rises the adding pepsin, hydrolysis 10 hours, it is 6.2 ~ 6.4 that hydrolyzed solution is regulated pH value with NaOH, adds 1g/ and rises kieselguhr, 90 ℃, heated 30 minutes, small-sized filter press filter pressing, with 0.45 μ m microporous filter membrane filtration, 8000 molecular weight ultrafilter membrane ultrafiltration get hydrolyzed peptide of liver solution, measure content of peptides, adding injection water adjustment solution peptide concentration is 9.2 ~ 12.2mg/ml.
2, the hydrolyzed peptide of liver solution 1000ml that obtains previously, the vitamin C that adds 3g, the aspartic acid magnesium injection liquid of adding 27ml, the Macrogol 200 of adding 13g, 121 ℃ of autoclavings are 30 minutes after the packing, through be up to the standards injection of hydrolyzed peptide of liver compositions final drug preparation.
Claims (2)
1. injection of hydrolyzed peptide of liver pharmaceutical composition is characterized in that being made by following raw material:
Concentration is hydrolyzed peptide of liver solution 1000ml, vitamin C 2 ~ 5g, aspartic acid magnesium injection liquid 20 ~ 30ml, the Macrogol 200 10 ~ 20g of 9.2 ~ 12.2mg/ml;
Described aspartic acid magnesium injection liquid is that every 1ml contains L-Aspartic Acid 85mg: potassium 11.4mg:4.2mg.
2. the described injection of hydrolyzed peptide of liver preparation of compositions of claim 1 method comprises the steps:
1) hydrolyzed peptide of liver solution
The liver of cattle or pig, after removing connective tissue, by the parts by weight of 0.83 ~ 0.85:1 with after water for injection mixes, use high-speed tissue mashing machine's homogenate, be warming up to 37 ~ 40 ℃, transferring pH value with HCL is 2.0 ~ 2.5, presses 3g/ and rises the adding pepsin, hydrolysis 10 ~ 15 hours, it is 6.2 ~ 6.4 that hydrolyzed solution is regulated pH value with NaOH, adds 1g/ and rises kieselguhr, 90 ℃, heated 30 minutes, small-sized filter press filter pressing, with 0.45 μ m microporous filter membrane filtration, 8000 molecular weight ultrafilter membrane ultrafiltration get hydrolyzed peptide of liver solution, measure content of peptides, adding injection water adjustment solution peptide concentration is 9.2 ~ 12.2mg/ml;
2) in the hydrolyzed peptide of liver solution 1000ml of step 1 preparation, add the vitamin C of 2 ~ 5g, the aspartic acid magnesium injection liquid of adding 20 ~ 30ml, the Macrogol 200 of adding 10 ~ 20g, 121 ℃ of autoclavings are 20 minutes after the packing, injection of hydrolyzed peptide of liver compositions final drug preparation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110189767 CN102228676B (en) | 2011-07-07 | 2011-07-07 | Heparolysate injection pharmaceutical composition |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110189767 CN102228676B (en) | 2011-07-07 | 2011-07-07 | Heparolysate injection pharmaceutical composition |
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| CN102228676A true CN102228676A (en) | 2011-11-02 |
| CN102228676B CN102228676B (en) | 2013-01-30 |
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| CN 201110189767 Expired - Fee Related CN102228676B (en) | 2011-07-07 | 2011-07-07 | Heparolysate injection pharmaceutical composition |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0461261A1 (en) * | 1989-05-12 | 1991-12-18 | Otsuka Pharmaceutical Co., Ltd. | Oligopeptide mixture and composition containing the same |
| WO1994012524A1 (en) * | 1992-11-30 | 1994-06-09 | Celsus, Inc. | Protein hydrolysate derived from mucosal tissue |
| CN1824287A (en) * | 2005-12-21 | 2006-08-30 | 白求恩医科大学制药厂 | Preparation technology of liver hydrolytic peptide injection liquid |
| CN1903357A (en) * | 2005-07-27 | 2007-01-31 | 武汉同源药业有限公司 | Liver hydrolytic peptide for injection and prepn. method therefor |
| CN101091722A (en) * | 2007-06-29 | 2007-12-26 | 石海 | Injection of hydrolyzed peptide of liver |
-
2011
- 2011-07-07 CN CN 201110189767 patent/CN102228676B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0461261A1 (en) * | 1989-05-12 | 1991-12-18 | Otsuka Pharmaceutical Co., Ltd. | Oligopeptide mixture and composition containing the same |
| WO1994012524A1 (en) * | 1992-11-30 | 1994-06-09 | Celsus, Inc. | Protein hydrolysate derived from mucosal tissue |
| CN1903357A (en) * | 2005-07-27 | 2007-01-31 | 武汉同源药业有限公司 | Liver hydrolytic peptide for injection and prepn. method therefor |
| CN1824287A (en) * | 2005-12-21 | 2006-08-30 | 白求恩医科大学制药厂 | Preparation technology of liver hydrolytic peptide injection liquid |
| CN101091722A (en) * | 2007-06-29 | 2007-12-26 | 石海 | Injection of hydrolyzed peptide of liver |
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| CN102228676B (en) | 2013-01-30 |
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Granted publication date: 20130130 Termination date: 20160707 |