CN112641922A - Preparation method of compound bone peptide injection - Google Patents
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Abstract
The invention belongs to the technical field of medicines, and particularly relates to a preparation method of a compound bone peptide injection. The preparation method provided by the invention comprises the following steps: preparing bone peptide extract and Scorpio extract, mixing bone peptide extract and Scorpio extract, performing acid treatment, alkali treatment and active carbon treatment on the mixed solution, filtering, adding additive, and further processing. The invention innovates the production process, acid protein and alkali protein are processed in the step of preparing the bone peptide extracting solution; the step of preparing the scorpion extract is carried out with acid treatment and neutral filtration, and finally the additive is added, the bone peptide extract prepared by the invention has high medicament stability, solves the difficulty of reduced clarity in long-term storage, and has polypeptide substances of more than or equal to 30mg/ml and total nitrogen of more than or equal to 10 mg/ml; scorpion extract: the polypeptide substance is more than or equal to 0.5mg/ml, the total nitrogen is more than or equal to 0.25mg/ml, and the product is far superior to similar products.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a preparation method of a compound bone peptide injection.
Background
The bone peptide preparation is prepared by refining bone active substances extracted from fresh or frozen pig or fetal calf limb bones by high-tech biotechnology, contains various bone growth factors, various inorganic elements, trace elements and compound peptide active substances required by bone repair, can regulate bone metabolism, and has anti-inflammatory and analgesic effects. Is mainly used for treating proliferative osteoarticular diseases, rheumatism, rheumatoid arthritis and the like in clinic and can promote fracture healing. The product of the ossotide preparation comprises ossotide injection, ossotide tablets and the like, is on the market for many years and is popular with doctors and patients.
The effective components of the compound bone peptide injection are bone polypeptide and scorpion polypeptide. Through separation research, the bone polypeptide contains Bone Morphogenetic Protein (BMP), beta-transforming growth factor (TGF-beta), Fibroblast Growth Factor (FGF) and other bone growth factors, and the bone growth factors have synergistic effect and can quickly promote bone healing; the scorpion polypeptide contains active molecules such as scorpion venom analgesia (SAP) and scorpion venom anti-inflammatory peptide (SPP), and has good anti-inflammatory, analgesic and detumescence effects on common orthopedic wounds, Rheumatoid Arthritis (RA), Osteoarthritis (OA), and the like. Clinical research proves that the compound bone peptide injection has obviously better curative effect than the common bone peptide injection, has definite curative effect on neck, shoulder and leg pain, hyperosteogeny, osteoporosis, cartilage necrosis, bone healing and the like, and has the advantages of convenient use, no toxic or side effect and the like.
Chinese patent CN1939343B discloses a bone peptide sodium chloride solution injection and a preparation process thereof, which effectively controls the content of high molecular impurities of the injection under long-term storage, but still has the problem of low extraction efficiency of the bone peptide content.
Chinese patent CN108926703A discloses a preparation process of bone peptide glucose injection, which solves the problem that the medicine is easy to be polluted, but the clarity problem is not solved in the aspect of long-term storage.
Few enterprises which have compound bone peptide injection approval documents and are produced and marketed in China have the annual demand of 1 hundred million, and the existing compound bone peptide injection has obvious clinical reaction effect and moderate price, but has the problems of reduced clarity after long-term storage, and simultaneously needs to further improve the content of effective components of the bone peptide extract and the scorpion extract.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a preparation method of a compound ossotide injection, which effectively solves the problem of the reduction of clarity of the compound ossotide injection during long-term storage, and particularly obviously increases the contents of active ingredients of an ossotide extract and a scorpion extract.
The technical scheme of the invention is as follows:
a preparation method of a compound bone peptide injection comprises the following specific steps:
s1: preparing bone peptide extract and scorpion extract;
s2: mixing the bone peptide extract prepared in the step S1 with the scorpion extract to obtain a mixed solution, sequentially carrying out acid treatment, alkali treatment and activated carbon treatment, and filtering to obtain a treated solution;
s3: adding an additive into the treated solution obtained in the step S2, mixing, stirring uniformly, washing and drying an ampoule bottle, and filling;
s4: sterilizing, detecting leakage, detecting by lamp, packaging, and making into final product;
further, the method for preparing the bone peptide extract in step S1 includes:
1) pulverizing pig limbs bone, adding water at 105-120 deg.C and 1-2kg/cm2Extracting under pressure for 2 times, each for 1-2 hr, mixing the extractive solutions, and filtering;
2) standing and cooling the filtrate, adding petroleum ether for extraction, removing upper-layer grease, concentrating the residual filtrate to 1/7-1/6 of the original volume, adding ethanol into the obtained concentrated solution to ensure that the volume content of the ethanol in the filtrate is 65-75%, standing for 18-20h, filtering, and distilling the filtrate;
3) adjusting pH of the filtrate obtained in step 2) with hydrochloric acid, removing acid protein, filtering, adjusting pH with sodium hydroxide, removing alkali protein, filtering with 0.2 μm filter element, filtering with ultrafiltration membrane with pore size of 15nm, and removing pyrogen to obtain bone peptide extract.
Further, the mass ratio of the pig limb bones used for preparing the bone peptide extracting solution and the scorpions used for preparing the scorpions extracting solution in the step S1 is 70-100: 1.
Further, the mass of the petroleum ether added in the step 2) is 1/5 of the mass of the filtrate.
Further, the step 2) distills the filtrate to 1/3-1/5 of original volume.
Further, the pH of the filtrate obtained in the step 2) is adjusted to 3-5 by using hydrochloric acid in the step 3), and the pH is adjusted to 7-9 again by using sodium hydroxide.
Further, the preparation method of the scorpion extract in the step S1 includes:
drying the scorpion in an oven at 80 ℃, crushing, sieving by a 100-mesh sieve, and leaching for 2 times by using a solvent, wherein the volume of the solvent is 2-3ml/g of the weight of the scorpion;
② combining leaching liquor extracted twice, filtering, distilling to 1/6 of the original volume to obtain concentrated solution;
③ hydrochloric acid is added into the concentrated solution to adjust the pH value to 4 to 6, neutral filter paper is used for filtration, and then high pressure steam sterilization is carried out to obtain the scorpion extract.
Further, the solvent of the step (i) is an ethanol aqueous solution with the volume fraction of 60%.
Further, in step S3, the additives are: l-serine and proline.
Further, the mass relationship between the additive in the step S3 and the treated solution obtained in the step S2 is as follows: 95% of the treated solution, 2% of L-serine and 3% of proline.
Compared with the prior art, the invention has the following advantages:
(1) according to the invention, acid protein and alkali protein are further processed in the step of preparing the bone peptide extracting solution, and acid treatment and neutral filtration are performed in the step of preparing the scorpion extracting solution, so that the problem of reduced clarity of the compound bone peptide injection during long-term storage is effectively solved.
(2) The invention innovates the additive of the compound bone peptide injection, the additive used in the invention comprises L-serine and proline, and researches show that the additive can enhance the overall stability of the preparation by being mixed with the bone peptide extract obtained by extraction and the scorpion extract.
(3) The invention further defines the specific parameters of the preparation process, and ensures the content of the effective components while solving the problem of the clarity reduction of the compound bone peptide injection during long-term storage, so that the polypeptide substances and the total nitrogen of the bone peptide extract prepared by the invention are more than or equal to 30mg/ml and more than or equal to 10 mg/ml; scorpion extract: the polypeptide substance is more than or equal to 0.5mg/ml, the total nitrogen is more than or equal to 0.25mg/ml, the polypeptide substance is far superior to similar products, and the polypeptide substance is at the domestic advanced level.
Detailed Description
The present invention will be further explained by way of specific embodiments in the form of examples. The scope of the above-described subject matter of the present invention is not limited to the following examples.
Example 1 preparation of bone peptide extract
The preparation method of the bone peptide extracting solution comprises the following steps:
1) pulverizing pig limbs bone, adding 10 times of water, heating at 105 deg.C and 1kg/cm2Extracting under pressure for 2 times, each for 1 hr, mixing the extractive solutions, and filtering;
2) standing and cooling the filtrate, adding petroleum ether with the mass of 1/5 of the filtrate for extraction, removing upper layer grease, concentrating to 1/7 of the original volume, adding ethanol into the concentrated solution to enable the volume content of the ethanol in the filtrate to be 65%, standing for 18h, filtering, and distilling the filtrate to 1/3 of the original volume;
3) adjusting pH of the filtrate obtained in step 2) to 5 with hydrochloric acid, removing acid protein, filtering, adjusting pH to 7 with sodium hydroxide, removing alkali protein, filtering with 0.2 μm filter core, filtering with ultrafiltration membrane with 15nm pore size, and removing pyrogen to obtain bone peptide extract.
Example 2 preparation of bone peptide extract
The preparation method of the bone peptide extracting solution comprises the following steps:
1) pulverizing pig limbs bone, adding 10 times of water, heating at 120 deg.C and 2kg/cm2Extracting under pressure for 2 times, each for 2 hr, mixing the extractive solutions, and filtering;
2) standing and cooling the filtrate, adding petroleum ether with the mass of 1/5 of the filtrate for extraction, removing upper layer grease, concentrating to 1/6 of the original volume, adding ethanol into the concentrated solution to enable the volume content of the ethanol in the filtrate to be 75%, standing for 20h, filtering, and distilling the filtrate to 1/5 of the original volume;
3) adjusting pH of the filtrate obtained in step 2) to 3 with hydrochloric acid, removing acid protein, filtering, adjusting pH to 9 with sodium hydroxide acid, removing alkali protein, filtering with 0.2 μm filter core, filtering with ultrafiltration membrane with pore of 15nm, and removing pyrogen to obtain bone peptide extract.
Example 3 preparation of bone peptide extract
The preparation method of the bone peptide extracting solution comprises the following steps:
1) pulverizing pig limbs bone, adding 10 times of water, heating at 110 deg.C and 2kg/cm2Extracting under pressure for 2 times, each for 2 hr, mixing the extractive solutions, and filtering;
2) standing and cooling the filtrate, adding petroleum ether with the mass of 1/5 of the filtrate for extraction, removing upper layer grease, concentrating to 1/6 of the original volume, adding ethanol into the concentrated solution to enable the volume content of the ethanol in the filtrate to be 75%, standing for 18h, filtering, and distilling the filtrate to 1/4 of the original volume;
3) adjusting pH of the filtrate obtained in step 2) to 4 with hydrochloric acid, removing acid protein, filtering, adjusting pH to 8 with sodium hydroxide, removing alkali protein, filtering with 0.2 μm filter core, filtering with ultrafiltration membrane with 15nm pore size, and removing pyrogen to obtain bone peptide extract.
Example 4 preparation of Scorpion extract
The preparation method of the scorpion extract comprises the following steps:
putting the scorpion in an oven at 80 ℃ for drying, crushing, sieving by a 100-mesh sieve, and leaching for 2 times by using a solvent, wherein the volume of the used solvent is 2ml/g of the weight ratio of the scorpion, and the solvent is an ethanol water solution with the volume fraction of 60%;
② combining leaching liquor extracted twice, filtering, distilling to 1/6 of the original volume to obtain concentrated solution;
③ hydrochloric acid is added into the concentrated solution to adjust the pH value to 6, neutral filter paper is used for filtration, and then high pressure steam sterilization is carried out to obtain the scorpion extract.
Example 5 preparation of Scorpion extract
The preparation method of the scorpion extract comprises the following steps:
putting the scorpion in an oven at 80 ℃ for drying, crushing, sieving by a 100-mesh sieve, and leaching for 2 times by using a solvent, wherein the volume of the used solvent is 3ml/g of the weight ratio of the scorpion, and the solvent is an ethanol water solution with the volume fraction of 60%;
② combining leaching liquor extracted twice, filtering, distilling to 1/6 of the original volume to obtain concentrated solution;
③ hydrochloric acid is added into the concentrated solution to adjust the pH value to 4, neutral filter paper is used for filtration, and then high pressure steam sterilization is carried out to obtain the scorpion extract.
Example 6 preparation of Scorpion extract
The preparation method of the scorpion extract comprises the following steps:
putting the scorpion in an oven at 80 ℃ for drying, crushing, sieving by a 100-mesh sieve, and leaching for 2 times by using a solvent, wherein the volume of the used solvent is 3ml/g of the weight ratio of the scorpion, and the solvent is an ethanol water solution with the volume fraction of 60%;
② combining leaching liquor extracted twice, filtering, distilling to 1/6 of the original volume to obtain concentrated solution;
③ hydrochloric acid is added into the concentrated solution to adjust the pH value to 5, neutral filter paper is used for filtration, and then high pressure steam sterilization is carried out to obtain the scorpion extract.
Example 7 preparation of Compound ossotide injection
A preparation method of a compound bone peptide injection comprises the following specific steps:
s1: preparing bone peptide extract according to example 1 and Scorpio extract according to example 4, wherein the mass ratio of pig limb bone used for preparing bone peptide extract and Scorpio used for preparing Scorpio extract is 70: 1;
s2: mixing the bone peptide extract prepared in the step S1 with the scorpion extract to obtain a mixed solution, sequentially carrying out acid treatment, alkali treatment and activated carbon treatment, and filtering to obtain a treated solution;
the acid treatment method comprises the steps of adjusting the pH value of the mixed solution to 4 by using hydrochloric acid, heating the mixed solution at the temperature of 95 ℃ for 1 hour, refrigerating the mixed solution at the temperature of 0 ℃ for 18 hours, and filtering the refrigerated solution;
the alkali treatment method comprises the steps of adjusting the pH value of the mixed solution to 8 by using sodium hydroxide, heating the mixed solution at the temperature of 95 ℃ for 1 hour, refrigerating the mixed solution at the temperature of 0 ℃ for 18 hours, and filtering the refrigerated solution;
the activated carbon treatment method comprises adjusting pH of the mixed solution to neutral with hydrochloric acid, heating at 95 deg.C for 1h, refrigerating at 0 deg.C for 18h, filtering, adding activated carbon, adding 1g of activated carbon per 100ml of the mixed solution, and stirring at 70 deg.C for 45 min;
s3: adding an additive into the treated solution obtained in the step S2, mixing, stirring uniformly, washing and drying an ampoule bottle, and filling;
the mass relationship between the additive and the treated solution obtained in the step S2 is as follows: 95% of the treated solution, 2% of L-serine and 3% of proline;
s4: sterilizing, detecting leakage, inspecting by lamp, packaging, and making into final product.
Example 8 preparation of Compound ossotide injection
A preparation method of a compound bone peptide injection comprises the following specific steps:
s1: preparing bone peptide extracting solution according to the embodiment 2 and preparing scorpion extracting solution according to the embodiment 5, wherein the mass ratio of the pig limb bones used for preparing the bone peptide extracting solution to the scorpions used for preparing the scorpion extracting solution is 100: 1;
s2: mixing the bone peptide extract prepared in the step S1 with the scorpion extract to obtain a mixed solution, sequentially carrying out acid treatment, alkali treatment and activated carbon treatment, and filtering to obtain a treated solution;
the acid treatment method comprises the steps of adjusting the pH value of the mixed solution to 4 by using hydrochloric acid, heating the mixed solution for 2 hours at the temperature of 95 ℃, refrigerating the mixed solution for 18 hours at the temperature of 0 ℃, and filtering the cooled mixed solution;
the alkali treatment method comprises the steps of adjusting the pH value of the mixed solution to 9 by using sodium hydroxide, heating the mixed solution for 2 hours at the temperature of 95 ℃, refrigerating the mixed solution for 18 hours at the temperature of 0 ℃, and filtering the cooled mixed solution;
the activated carbon treatment method comprises adjusting pH of the mixed solution to neutral with hydrochloric acid, heating at 95 deg.C for 2h, refrigerating at 0 deg.C for 18h, filtering, adding activated carbon, adding 4g of activated carbon per 100ml of the mixed solution, and stirring at 70 deg.C for 45 min;
s3: adding the additive into the treated solution obtained in the step S2, mixing, stirring uniformly, washing and drying an ampoule bottle, and filling;
the mass relationship between the additive and the treated solution obtained in the step S2 is as follows: 95% of the treated solution, 2% of L-serine and 3% of proline;
s4: sterilizing, detecting leakage, inspecting by lamp, packaging, and making into final product.
Example 9 preparation of Compound ossotide injection
A preparation method of a compound bone peptide injection comprises the following specific steps:
s1: preparing bone peptide extract according to example 3 and Scorpio extract according to example 6, wherein the mass ratio of pig limb bone used for preparing bone peptide extract and Scorpio used for preparing Scorpio extract is 80: 1;
s2: mixing the bone peptide extract prepared in the step S1 with the scorpion extract to obtain a mixed solution, sequentially carrying out acid treatment, alkali treatment and activated carbon treatment, and filtering to obtain a treated solution;
the acid treatment method comprises the steps of adjusting the pH value of the mixed solution to 4 by using hydrochloric acid, heating the mixed solution for 2 hours at the temperature of 95 ℃, refrigerating the mixed solution for 18 hours at the temperature of 0 ℃, and filtering the cooled mixed solution;
the alkali treatment method comprises the steps of adjusting the pH value of the mixed solution to 8 by using sodium hydroxide, heating the mixed solution for 2 hours at the temperature of 95 ℃, refrigerating the mixed solution for 18 hours at the temperature of 0 ℃, and filtering the cooled mixed solution;
the activated carbon treatment method comprises adjusting pH of the mixed solution to neutral with hydrochloric acid, heating at 95 deg.C for 2h, refrigerating at 0 deg.C for 18h, filtering, adding activated carbon, adding 3g of activated carbon per 100ml of the mixed solution, and stirring at 70 deg.C for 45 min;
s3: adding the additive into the treated solution obtained in the step S2, mixing, stirring uniformly, washing and drying an ampoule bottle, and filling;
the mass relationship between the additive and the treated solution obtained in the step S2 is as follows: 95% of the treated solution, 2% of L-serine and 3% of proline;
s4: sterilizing, detecting leakage, inspecting by lamp, packaging, and making into final product.
Comparative example 1 preparation of bone peptide extract
The preparation method of the bone peptide extracting solution comprises the following steps:
1) pulverizing pig limbs bone, adding 10 times of water, heating at 110 deg.C and 2kg/cm2Extracting under pressure for 2 times, each for 2 hr, mixing the extractive solutions, and filtering;
2) standing and cooling the filtrate, adding petroleum ether with the mass of 1/5 of the filtrate for extraction, removing upper layer grease, concentrating to 1/6 of the original volume, adding ethanol into the concentrated solution to enable the volume content of the ethanol in the filtrate to be 75%, standing for 18h, filtering, and distilling the filtrate to 1/4 of the original volume;
unlike example 3, comparative example 1 does not have step 3).
Comparative example 2 preparation of Scorpion extract
The preparation method of the scorpion extract comprises the following steps:
putting the scorpion in an oven at 80 ℃ for drying, crushing, sieving by a 100-mesh sieve, and leaching for 2 times by using a solvent, wherein the volume of the used solvent is 3ml/g of the weight ratio of the scorpion, and the solvent is an ethanol water solution with the volume fraction of 60%;
② combining leaching liquor extracted twice, filtering, distilling to 1/6 of the original volume to obtain concentrated solution;
unlike example 6, comparative example 2 has no step (c).
Comparative example 3 preparation of Compound ossotide injection
A preparation method of a compound bone peptide injection comprises the following specific steps:
s1: preparing bone peptide extract according to comparative example 1 and preparing scorpion extract according to comparative example 2, wherein the mass ratio of the pig limb bones used for preparing the bone peptide extract to the scorpions used for preparing the scorpion extract is 80: 1;
s2: mixing the bone peptide extract prepared in the step S1 with the scorpion extract to obtain a mixed solution, sequentially carrying out acid treatment, alkali treatment and activated carbon treatment, and filtering to obtain a treated solution;
the acid treatment method comprises the steps of adjusting the pH value of the mixed solution to 4 by using hydrochloric acid, heating the mixed solution for 2 hours at the temperature of 95 ℃, refrigerating the mixed solution for 18 hours at the temperature of 0 ℃, and filtering the cooled mixed solution;
the alkali treatment method comprises the steps of adjusting the pH value of the mixed solution to 8 by using sodium hydroxide, heating the mixed solution for 2 hours at the temperature of 95 ℃, refrigerating the mixed solution for 18 hours at the temperature of 0 ℃, and filtering the cooled mixed solution;
the activated carbon treatment method comprises adjusting pH of the mixed solution to neutral with hydrochloric acid, heating at 95 deg.C for 2h, refrigerating at 0 deg.C for 18h, filtering, adding activated carbon, adding 3g of activated carbon per 100ml of the mixed solution, and stirring at 70 deg.C for 45 min;
s3: adding the additive into the treated solution obtained in the step S2, mixing, stirring uniformly, washing and drying an ampoule bottle, and filling;
the mass relationship between the additive and the treated solution obtained in the step S2 is as follows: 95% of the treated solution, 2% of L-serine and 3% of proline;
s4: sterilizing, detecting leakage, inspecting by lamp, packaging, and making into final product.
Different from example 9, the method for extracting the bone peptide extract and the scorpion extract in comparative example 3 is different.
Comparative example 4 preparation of Compound ossotide injection
A preparation method of a compound bone peptide injection comprises the following specific steps:
s1: preparing bone peptide extract according to example 3 and Scorpio extract according to example 6, wherein the mass ratio of pig limb bone used for preparing bone peptide extract and Scorpio used for preparing Scorpio extract is 80: 1;
s2: mixing the bone peptide extract prepared in the step S1 with the scorpion extract to obtain a mixed solution, sequentially carrying out acid treatment, alkali treatment and activated carbon treatment, and filtering to obtain a treated solution;
the acid treatment method comprises the steps of adjusting the pH value of the mixed solution to 4 by using hydrochloric acid, heating the mixed solution for 2 hours at the temperature of 95 ℃, refrigerating the mixed solution for 18 hours at the temperature of 0 ℃, and filtering the cooled mixed solution;
the alkali treatment method comprises the steps of adjusting the pH value of the mixed solution to 8 by using sodium hydroxide, heating the mixed solution for 2 hours at the temperature of 95 ℃, refrigerating the mixed solution for 18 hours at the temperature of 0 ℃, and filtering the cooled mixed solution;
the activated carbon treatment method comprises adjusting pH of the mixed solution to neutral with hydrochloric acid, heating at 95 deg.C for 2h, refrigerating at 0 deg.C for 18h, filtering, adding activated carbon, adding 3g of activated carbon per 100ml of the mixed solution, and stirring at 70 deg.C for 45 min;
s3: adding the additive into the treated solution obtained in the step S2, mixing, stirring uniformly, washing and drying an ampoule bottle, and filling;
the mass relationship between the additive and the treated solution obtained in the step S2 is as follows: 95% of the treated solution, 5% of L-serine;
s4: sterilizing, detecting leakage, inspecting by lamp, packaging, and making into final product.
Unlike example 9, the additive added in step S3 did not include proline.
The invention will now be described in further detail in connection with the following experiments:
test 1, determination of Polypeptides and Total Nitrogen content in bone peptide extract and Scorpio extract
Test subjects: the extracts obtained in examples 1 to 6.
The test method comprises the following steps:
1. the content determination of the polypeptide substance adopts high performance liquid chromatography, and the chromatographic conditions are as follows: the high performance liquid chromatography (Agilent1100) has chromatographic column of C18 (Saimerfi 17X26-102130-3VB), mobile phase of methanol and potassium dihydrogen phosphate (50:50), column temperature of 30 deg.C, detection wavelength of 254nm, and flow rate of 0.6 ml/min.
2. Total nitrogen content was measured using a total nitrogen meter (KN580 full-automatic Kjeldahl apparatus).
And (3) test results: as shown in table 1.
TABLE 1 determination of the Polypeptides and Total Nitrogen content
As can be seen from Table 1, the improvement of the method for preparing the bone peptide extract and the scorpion extract ensures the content of the effective components, so that the polypeptide substances in the bone peptide extract prepared by the invention are more than or equal to 30mg/ml, and the total nitrogen is more than or equal to 10 mg/ml; scorpion extract: the polypeptide substance is more than or equal to 0.5mg/ml, and the total nitrogen is more than or equal to 0.25mg/ml, which proves that the method is beneficial to the treatment of the bone peptide extract and the scorpion extract.
Test 2, measurement of clarity in accelerated test
Test subjects: compound bone peptide injection prepared in examples 7-9 and comparative examples 3-4.
The test method comprises the following steps: according to the second stability test guideline of Chinese pharmacopoeia, the sample is placed for 6 months under the conditions of 40% + -2 ℃ and 75% + -5% relative humidity, and is sampled at the end of 1, 3 and 6 months respectively, and the clarity of each sample is detected by using a clarity detector (Tianjin optical YB2 type).
Clarity-qualified range: 1-2.2.
And (3) test results: as shown in table 2.
TABLE 2 determination of clarity
| Number of groups | Example 7 | Example 8 | Example 9 | Comparative example 3 | Comparative example 4 |
| Clarity at the end of the month | 1.7 | 1.8 | 1.9 | 1.6 | 1.7 |
| 3 clarity at the end of the month | 1.5 | 1.5 | 1.6 | 1.1 | 1.3 |
| Clarity at the end of the 6 month | 1.3 | 1.4 | 1.4 | 0.7 | 0.9 |
As can be seen from Table 2, the compound bone peptide injection finally prepared in the examples has better clarity compared with the comparative examples, the comparative example 3 changes the extraction method of the bone peptide extract and the scorpion extract, the clarity of long-term storage is influenced, the comparative example 4 changes the composition of the additive, only single L-serine is taken as the additive, the clarity after long-term storage is obviously reduced, and if better effect is obtained, the L-serine and proline are required to be combined for use. Therefore, the clarity of long-term storage can be ensured to the maximum extent by improving the extraction methods of the bone peptide extract and the scorpion extract and preparing the bone peptide extract and the scorpion extract according to the additive composition provided by the invention, and the experimental data prove that the invention effectively solves the problem of clarity reduction caused by long-term storage, improves the storage time of the medicine and has higher medical value and economic value.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. It will be appreciated by those skilled in the art that changes may be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims.
Claims (9)
1. A preparation method of a compound bone peptide injection is characterized by comprising the following specific steps:
s1: preparing bone peptide extract and scorpion extract;
s2: mixing the bone peptide extract prepared in the step S1 with the scorpion extract to obtain a mixed solution, sequentially carrying out acid treatment, alkali treatment and activated carbon treatment, and filtering to obtain a treated solution;
s3: adding an additive into the treated solution obtained in the step S2, mixing, stirring uniformly, washing and drying an ampoule bottle, and filling;
s4: sterilizing, detecting leakage, detecting by lamp, packaging, and making into final product;
the preparation method of the bone peptide extracting solution in the step S1 comprises the following steps:
1) pulverizing pig limbs bone, adding water at 105-120 deg.C and 1-2kg/cm2Extracting under pressure for 2 times, each for 1-2 hr, mixing the extractive solutions, and filtering;
2) standing and cooling the filtrate, adding petroleum ether for extraction, removing upper-layer grease, concentrating the residual filtrate to 1/7-1/6 of the original volume, adding ethanol into the obtained concentrated solution to ensure that the volume content of the ethanol in the filtrate is 65-75%, standing for 18-20h, filtering, and distilling the filtrate;
3) adjusting pH of the filtrate obtained in step 2) with hydrochloric acid, removing acid protein, filtering, adjusting pH with sodium hydroxide, removing alkali protein, filtering with 0.2 μm filter element, filtering with ultrafiltration membrane with pore size of 15nm, and removing pyrogen to obtain bone peptide extract.
2. The method for preparing the compound bone peptide injection as claimed in claim 1, wherein the mass ratio of the pig limb bone used for preparing the bone peptide extracting solution and the scorpion used for preparing the scorpion extracting solution in the step S1 is 70-100: 1.
3. The method for preparing the compound ossotide injection of claim 1, wherein the amount of petroleum ether added in the step 2) is 1/5 of the amount of the filtrate.
4. The method for preparing the compound ossotide injection of claim 1, wherein the step 2) is to distill the filtrate to 1/3-1/5 of the original volume.
5. The method for preparing the compound ossotide injection of claim 1, wherein the pH of the filtrate obtained in the step 2) is adjusted to 3-5 by using hydrochloric acid in the step 3), and the pH is adjusted to 7-9 again by using sodium hydroxide.
6. The method for preparing the compound bone peptide injection as claimed in claim 1, wherein the scorpion extract solution prepared in the step S1 is prepared by the following steps:
drying the scorpion in an oven at 80 ℃, crushing, sieving by a 100-mesh sieve, and leaching for 2 times by using a solvent, wherein the volume of the solvent is 2-3ml/g of the weight of the scorpion;
② combining leaching liquor extracted twice, filtering, distilling to 1/6 of the original volume to obtain concentrated solution;
③ hydrochloric acid is added into the concentrated solution to adjust the pH value to 4 to 6, neutral filter paper is used for filtration, and then high pressure steam sterilization is carried out to obtain the scorpion extract.
7. The method for preparing the compound ossotide injection as claimed in claim 6, wherein the solvent of step (i) is an aqueous ethanol solution with a volume fraction of 60%.
8. The method for preparing the compound ossotide injection of claim 1, wherein the additives in step S3 are: l-serine and proline.
9. The method for preparing the compound ossotide injection of claim 8, wherein the mass relationship between the additive of step S3 and the treated solution obtained in step S2 is as follows: 95% of the treated solution, 2% of L-serine and 3% of proline.
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