CN110339336A - A kind of preparation method of cervus and cucumis polypeptide ejection preparation - Google Patents
A kind of preparation method of cervus and cucumis polypeptide ejection preparation Download PDFInfo
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Abstract
The invention belongs to biomedicine technical fields, and in particular to a kind of preparation method of cervus and cucumis polypeptide ejection preparation, this method are as follows: polypeptide, the sodium alginate that polypeptide, the melon seed extracted by deer bone extracts are prepared；Studies have shown that present invention process is simple, it is suitble to industrialized production, removes activated carbon process method, meet the regulation of Conformance Assessment；Auxiliary material using sodium alginate as preparation guarantees formulation products stability, further, obtains the more outstanding product of quality；Ejection preparation of the invention meets requirement of ejection preparation routine, such as endotoxin, content uniformity etc., has higher potential safety；Pharmaceutical adjunct dosage is few, and product cost is lower, reduces the drug cost of patient；Invention formulation passes through stability test, content of peptides, free aminoacid content, polymer substance content hardly increase or increase it is smaller, with better safety.
Description
Technical field
The invention belongs to biomedicine technical fields, and in particular to a kind of cervus and cucumis polypeptide ejection preparation.
Background technique
Cervus and cucumis polypeptide is mentioned respectively by the bone of animal in deer family sika deer and the dry mature seed of cucurbitaceous plant muskmelon
It is made after taking, mainly contains self-bone grafting polypeptide biotic factor, muskmelon seed extract, a variety of free amino acids and organic calcium, phosphorus
Equal ingredients.
Using deer bone and muskmelon seed as the pharmaceutical preparation of raw material, mainly cervus and cucumis polypeptide ejection preparation, said preparation have adjusting
Bone metabolism, stimulated osteoblastic proliferation, the effect for promoting new bone formation are also adjusted calcium phosphorus metabolism, increase bone doped calcium, prevent
Osteoporosis, and have the effects that anti-inflammatory, analgesia, antirheumatic, it is widely used in clinic.Clinical application show with deer bone and
Muskmelon seed is that the ejection preparation of raw material is that treatment nonunion, osteoporosis, rheumatism, rheumatoid arthritis, wrist are navicular
The active drug of ischemic necrosis of bone and soft tissue injury etc., in having a extensive future for orthopaedics.
Chinese patent CN02125357.9 is disclosed using deer bone and muskmelon seed as raw material and is prepared injection, the patented method
Are as follows: deer bone and muskmelon seed are extracted respectively, acid tune pH value, adjusting PH with base value, ultrafiltration is prepared into injection again after combined extract
Preparation；Chinese patent CN200410013602.7 discloses deer bone and muskmelon seed is that raw material is prepared into ejection preparation, the patent
Method are as follows: deer bone extracts, acid adjusts pH value, adjusting PH with base value, ultrafiltration, and muskmelon seed extraction, ultrafiltration, combined extract are prepared into note
Penetrate agent；Chinese patent 200510108157.7 discloses the preparation method of cervus and cucumis polypeptide injection, this method are as follows: and deer bone extracts,
By ultrafiltration three times, muskmelon seed extraction, ultrafiltration three times, combined extract is prepared into injection；Chinese patent
CN200910167155.3 discloses the preparation method of cervus and cucumis polypeptide injection, this method are as follows: respectively mentions deer bone and muskmelon seed
It takes, acid adjusts pH value, ultrafiltration after heating, centrifugation, adjusting PH with base value, centrifugation, pH value, three step ultrafiltration, ultrafiltrate is adjusted to be prepared into injection；
Chinese patent CN201210328858.1 discloses the preparation method of cervus and cucumis polypeptide injection, this method are as follows: after deer bone extracts, warp
Ultrafiltration three times is crossed, after muskmelon seed extracts, by ultrafiltration three times, combined extract is prepared into ejection preparation.Although the above method can
To obtain cervus and cucumis polypeptide injection, but the content of invalid components in cervus and cucumis polypeptide, therefore, above-mentioned preparation side are not all fully considered
Method remains to be discussed.
The cervus and cucumis polypeptide preparation of list marketing at present is mainly Allium ovalifolium hand-mazz and cervus and cucumis polypeptide freeze drying powder injection.Specially
Sharp CN1579542A discloses Cervus and Cucumis Polypeptide for injection and its preparation process, is prepared into freeze-dried powder by polypeptides matter and excipient
Injection；Patent CN1742778A discloses a kind of Cervus and Cucumis Polypeptide for injection pharmaceutical composition and preparation method thereof, it contains deer bone
Extract, Semen Melo extract and one or more excipient are made.However the peptide matters in Allium ovalifolium hand-mazz are easily poly-
Synthetic macromolecule, alkali therein and tannin can accelerate this polymerization reaction, therefore Allium ovalifolium hand-mazz is easy during preservation
Polymerization causes Clinical allergy reaction to increase, while being unfavorable for storing for a long time；The freeze drying powder injection of cervus and cucumis polypeptide was placed for a long time
There is also the problems of stability difference for journey, and the content of effective component is caused to reduce, and bioavilability reduces, and affect clinical application.
Summary of the invention
For these reasons, in order to solve the deficiencies in the prior art, applicant obtains a kind of new deer by studying for many years
The preparation method of melon polypeptide ejection preparation, polypeptide, the sodium alginate that this method uses the polypeptide extracted by deer bone, melon seed to extract
Preparation.There is better stability using the cervus and cucumis polypeptide ejection preparation that preparation method of the present invention obtains.
To achieve the above object, technical scheme is as follows:
A kind of preparation method of cervus and cucumis polypeptide ejection preparation, should be the preparation method comprises the following steps: polypeptide, the melon seed that deer bone extracts extract
Polypeptide and sodium alginate weight ratio be 1.5-2.5:0.7-0.8:0.070-0.10；Sodium alginate is taken, water for injection is added,
Dissolution completely, is heated to 30-40 DEG C, and the polypeptide that deer bone extracts and the polypeptide that melon seed extracts are added at such a temperature, keeps 30-
40 DEG C of stirring 10-20min, are cooled to room temperature, filtering, filtrate
The weight ratio of polypeptide, the polypeptide that melon seed extracts and sodium alginate that wherein deer bone extracts is 2:0.75:0.083.
Wherein deer bone extract polypeptide the preparation method comprises the following steps:
Deer bone is taken, marrow is removed, is crushed, the compound protease and deer bone weight of deer bone weight 1.5%-2.5% is added in weighing
The water for injection of 2~4 times of amount, compound protease is the pepsin and papain that weight ratio is 1.85-2.35:1；40℃
~50 DEG C are extracted 1~3 hour, filtering, and filtrate is filtered, filtrate sodium citrate tune with citric acid tune pH to 4.5-6.0, standing
0.1~0.3 times of pancreatin of deer bone weight is added in pH to 8.5~9.0, and 40 DEG C~50 DEG C hydrolyze 1~3 hour, and filtering will filter
It is 6.5-7.5 that liquid citric acid, which modulates pH value, successively with interception 100,000 ultrafiltration system of molecular weight, interception 10,000 ultrafiltration system of molecular weight
System, interception 8,000 ultrafiltration unit of molecular weight, carry out ultrafiltration, retain permeate, and freeze-drying obtains the polypeptide of deer bone extraction.
Wherein melon seed extract polypeptide the preparation method comprises the following steps:
Melon seed is taken, is crushed, the compound protease and melon seed weight 2 of melon seed weight 1.5%-2.5% is added in weighing
~4 times of water for injection, compound protease are the pepsin and papain that weight ratio is 1.85-2.35:1；40 DEG C~
50 DEG C are extracted 1~3 hour, filtering, and filtrate is stood with citric acid tune pH to 4.5-6.0, are filtered, and filtrate is with sodium citrate tune pH
To 8.5~9.0,0.1~0.3 times of pancreatin of melon seed weight is added, 40 DEG C~50 DEG C hydrolyze 1~3 hour, filtering, filtrate
It is 6.5-7.5 with citric acid modulation pH value, successively with interception 100,000 ultrafiltration system of molecular weight, interception 10,000 ultrafiltration system of molecular weight
System, interception 8,000 ultrafiltration unit of molecular weight, carry out ultrafiltration, retain permeate, freeze-drying, obtain melon seed extract to obtain it is more
Peptide.
This hair has following beneficial technical effects:
1, simple process is suitble to industrialized production, removes activated carbon process method, meets the regulation of Conformance Assessment.
2, the auxiliary material using sodium alginate as preparation guarantees formulation products stability, further, it is more excellent to obtain quality
Elegant product.
3, ejection preparation of the invention meets requirement of ejection preparation routine, such as endotoxin, content uniformity etc., has
Higher potential safety.
4, pharmaceutical adjunct dosage is few, and product cost is lower, reduces the drug cost of patient.
5, invention formulation passes through stability test, and content of peptides, free aminoacid content, polymer substance content are almost
Do not increase or increase it is smaller, have better safety.
Specific embodiment
The following describes the present invention further through the description of specific embodiments, but it is to limit of the invention that this, which is not,
System, those skilled in the art's basic thought according to the present invention can make various modifications or improvements, but without departing from this
The basic thought of invention, is all within the scope of the present invention.
The present invention following test commissions Shen Yao (Xianghe) Science and Technology Ltd. completes.
Prepare embodiment
Embodiment 1
Take deer bone, remove marrow, crush, weighing, be added deer bone weight 1.5% compound protease and 2 times of deer bone weight
Water for injection, compound protease is the pepsin and papain that weight ratio is 1.85:1；40 DEG C are extracted 3 hours, mistake
Filter, filtrate are stood with citric acid tune pH to 4.5, filtering, and the 0.1 of deer bone weight is added with sodium citrate tune pH to 8.5 in filtrate
Pancreatin again, 40 DEG C hydrolyze 3 hours, filtering, are 6.5 by filtrate citric acid modulation pH value, successively use interception ten Wan Chao of molecular weight
Filter system, interception 10,000 ultrafiltration system of molecular weight, interception 8,000 ultrafiltration unit of molecular weight, carry out ultrafiltration, retain permeate, freezing
It is dry, obtain the polypeptide of deer bone extraction.
Wherein melon seed extract polypeptide the preparation method comprises the following steps:
Melon seed is taken, is crushed, the compound protease and 2 times of melon seed weight of note of melon seed weight 1.5% is added in weighing
It penetrates and uses water, compound protease is the pepsin and papain that weight ratio is 1.85:1；40 DEG C are extracted 3 hours, are filtered, filter
Liquid citric acid tune pH to 4.5 is stood, filtering, and 0.1 times of melon seed weight is added with sodium citrate tune pH to 8.5 in filtrate
Pancreatin, 40 DEG C hydrolyze 3 hours, filtering, and it is 6.5 that filtrate citric acid, which modulates pH value, successively with interception 100,000 ultrafiltration system of molecular weight
System, interception 10,000 ultrafiltration system of molecular weight, interception 8,000 ultrafiltration unit of molecular weight, carry out ultrafiltration, retain permeate, and freezing is dry
It is dry, it obtains melon seed and extracts to obtain polypeptide.
The preparation method of preparation: the polypeptide and the weight ratio of sodium alginate that polypeptide that deer bone extracts, melon seed extract be
1.5:0.7:0.070 takes sodium alginate, and water for injection is added, and dissolution completely, is heated to 30 DEG C, and deer bone is added at such a temperature and mentions
The polypeptide that the polypeptide and melon seed taken extracts, keeps 30 DEG C of stirring 20min, is cooled to room temperature, and filters, filtrate is through 0.22 μm of filter core
Filtering, being concentrated into content of peptides is 4.5mg/ml, filling, obtains injection 100.
Embodiment 2
The polypeptide that deer bone extracts the preparation method comprises the following steps: take deer bone, remove marrow, crush, deer bone weight is added in weighing
2.5% compound protease and 4 times of deer bone weight of water for injection, compound protease are the pepsin that weight ratio is 2.35:1
And papain；50 DEG C are extracted 1 hour, filtering, and filtrate is filtered, filtrate citric acid with citric acid tune pH to 6.0, standing
0.3 times of pancreatin of deer bone weight is added in sodium tune pH to 9.0, and 50 DEG C hydrolyze 1 hour, filtering, and filtrate citric acid is modulated pH value
It is 7.5, successively with interception 100,000 ultrafiltration system of molecular weight, interception 10,000 ultrafiltration system of molecular weight, interception 8,000 ultrafiltration of molecular weight
Unit carries out ultrafiltration, retains permeate, and freeze-drying obtains the polypeptide of deer bone extraction.
The polypeptide that melon seed extracts the preparation method comprises the following steps: take melon seed, crush, melon seed weight 2.5% is added in weighing
Compound protease and 4 times of melon seed weight of water for injection, compound protease are the pepsin and wood that weight ratio is 2.35:1
Melon protease；50 DEG C are extracted 1 hour, filtering, and filtrate is filtered, filtrate sodium citrate tune with citric acid tune pH to 6.0, standing
0.3 times of pancreatin of melon seed weight is added in pH to 9.0, and 50 DEG C hydrolyze 1 hour, filtering, and filtrate modulates pH value with citric acid and is
7.5, successively with interception 100,000 ultrafiltration system of molecular weight, interception 10,000 ultrafiltration system of molecular weight, interception 8,000 ultrafilter of molecular weight
Group carries out ultrafiltration, retains permeate, and freeze-drying obtains melon seed and extracts to obtain polypeptide.
The preparation method of preparation: the polypeptide and the weight ratio of sodium alginate that polypeptide that deer bone extracts, melon seed extract be
2.5:0.8:0.10；Sodium alginate is taken, water for injection is added, dissolution completely, is heated to 40 DEG C, and deer bone is added at such a temperature and mentions
The polypeptide that the polypeptide and melon seed taken extracts, keeps 40 DEG C of stirring 20min, is cooled to room temperature, and filters, filtrate is through 0.22 μm of filter core
Filtering, being concentrated into content of peptides is 4.5mg/ml, filling, obtains injection 100.
Embodiment 3
The polypeptide that deer bone extracts the preparation method comprises the following steps: take deer bone, remove marrow, crush, deer bone weight is added in weighing
2.0% compound protease and 3 times of deer bone weight of water for injection, compound protease are the pepsin that weight ratio is 2.05:1
And papain；40 DEG C~50 DEG C are extracted 1~3 hour, filtering, and filtrate is filtered, filtrate with citric acid tune pH to 5.5, standing
With sodium citrate tune pH to 8.8,0.2 times of pancreatin of deer bone weight is added, 45 DEG C hydrolyze 2 hours, filtering, by filtrate citric acid
Modulating pH value is 7.0, successively with interception 100,000 ultrafiltration system of molecular weight, interception 10,000 ultrafiltration system of molecular weight, interception molecular weight
8000 ultrafiltration units carry out ultrafiltration, retain permeate, and freeze-drying obtains the polypeptide of deer bone extraction.
Melon seed extract polypeptide the preparation method comprises the following steps:
Melon seed is taken, is crushed, the compound protease and 3 times of melon seed weight of note of melon seed weight 2.0% is added in weighing
It penetrates and uses water, compound protease is the pepsin and papain that weight ratio is 2.05:1；45 DEG C are extracted 2 hours, are filtered, filter
Liquid citric acid tune pH to 5.0 is stood, filtering, and 0.2 times of melon seed weight is added with sodium citrate tune pH to 8.8 in filtrate
Pancreatin, 45 DEG C hydrolyze 2 hours, filtering, and it is 7.0 that filtrate citric acid, which modulates pH value, successively with interception 100,000 ultrafiltration system of molecular weight
System, interception 10,000 ultrafiltration system of molecular weight, interception 8,000 ultrafiltration unit of molecular weight, carry out ultrafiltration, retain permeate, and freezing is dry
It is dry, it obtains melon seed and extracts to obtain polypeptide.
The preparation method of preparation: the weight ratio of polypeptide, the polypeptide that melon seed extracts and sodium alginate that deer bone extracts is 2:
0.75:0.083；Sodium alginate is taken, water for injection is added, dissolution completely, is heated to 35 DEG C, and deer bone is added at such a temperature and extracts
Polypeptide and melon seed extract polypeptide, keep 35 DEG C of stirring 15min, be cooled to room temperature, filtering, filtrate is through 0.22 μm of filter core mistake
Filter, being concentrated into content of peptides is 4.5mg/ml, filling, obtains injection.
Embodiment 4
The polypeptide that deer bone extracts the preparation method comprises the following steps: take deer bone, remove marrow, crush, deer bone weight is added in weighing
1.8% compound protease and 3.5 times of deer bone weight of water for injection, compound protease are the stomach cardia that weight ratio is 1.95:1
Enzyme and papain；45 DEG C are extracted 2.5 hours, filtering, and filtrate is filtered, filtrate Chinese holly with citric acid tune pH to 5.5, standing
0.25 times of pancreatin of deer bone weight is added in rafter acid sodium tune pH to 9.0, and 45 DEG C hydrolyze 1.5 hours, filtering, by filtrate citric acid
Modulating pH value is 6.5, successively with interception 100,000 ultrafiltration system of molecular weight, interception 10,000 ultrafiltration system of molecular weight, interception molecular weight
8000 ultrafiltration units carry out ultrafiltration, retain permeate, and freeze-drying obtains the polypeptide of deer bone extraction.
The polypeptide that melon seed extracts the preparation method comprises the following steps: take melon seed, crush, melon seed weight 1.8% is added in weighing
Compound protease and 2 times of melon seed weight of water for injection, compound protease are the pepsin and wood that weight ratio is 1.95:1
Melon protease；45 DEG C are extracted 1.5 hours, filtering, and filtrate is filtered, filtrate sodium citrate with citric acid tune pH to 4.5, standing
PH to 8.5 is adjusted, 0.15 times of pancreatin of melon seed weight is added, 45 DEG C hydrolyze 2 hours, and filtering, filtrate modulates pH with citric acid
Value is 6.5, successively thousand is surpassed with interception 100,000 ultrafiltration system of molecular weight, interception 10,000 ultrafiltration system of molecular weight, interception molecular weight 8
Filter group carries out ultrafiltration, retains permeate, and freeze-drying obtains melon seed and extracts to obtain polypeptide.
The weight ratio of polypeptide, the polypeptide that melon seed extracts and sodium alginate that deer bone extracts is 1.7:0.7:0.075；Take sea
Water for injection is added in mosanom, and dissolution completely, is heated to 35 DEG C, and polypeptide and melon seed that deer bone extracts are added at such a temperature
The polypeptide of extraction keeps 35 DEG C of stirring 15min, is cooled to room temperature, and filtering, filtrate is concentrated into polypeptide through 0.22 μm of filter element filtering
Content is 4.5mg/ml, filling, obtains injection.
Embodiment 5
The polypeptide that deer bone extracts the preparation method comprises the following steps: take deer bone, remove marrow, crush, deer bone weight is added in weighing
2.4% compound protease and 2.5 times of deer bone weight of water for injection, compound protease are the stomach cardia that weight ratio is 2.20:1
Enzyme and papain；45 DEG C are extracted 3 hours, filtering, and filtrate is filtered, filtrate citron with citric acid tune pH to 6.0, standing
0.25 times of pancreatin of deer bone weight is added in sour sodium tune pH to 9.0, and 45 DEG C hydrolyze 2.5 hours, filtering, by filtrate citric acid tune
PH value processed is 7.5, successively with interception 100,000 ultrafiltration system of molecular weight, interception 10,000 ultrafiltration system of molecular weight, interception molecular weight eight
Thousand ultrafiltration units carry out ultrafiltration, retain permeate, and freeze-drying obtains the polypeptide of deer bone extraction.
The polypeptide that melon seed extracts the preparation method comprises the following steps: take melon seed, crush, melon seed weight 2.5% is added in weighing
Compound protease and 3.5 times of melon seed weight of water for injection, compound protease be weight ratio be 2.25:1 pepsin and
Papain；45 DEG C are extracted 2.5 hours, filtering, and filtrate is filtered, filtrate citric acid with citric acid tune pH to 6.0, standing
0.25 times of pancreatin of melon seed weight is added in sodium tune pH to 9.0, and 45 DEG C hydrolyze 2 hours, and filtering, filtrate is modulated with citric acid
PH value is 7.5, successively with interception 100,000 ultrafiltration system of molecular weight, interception 10,000 ultrafiltration system of molecular weight, interception molecular weight 8,000
Ultrafiltration unit carries out ultrafiltration, retains permeate, and freeze-drying obtains melon seed and extracts to obtain polypeptide.
The preparation method of preparation: the polypeptide and the weight ratio of sodium alginate that polypeptide that deer bone extracts, melon seed extract be
2.4:0.8:0.095；Sodium alginate is taken, water for injection is added, dissolution completely, is heated to 40 DEG C, and deer bone is added at such a temperature
The polypeptide that the polypeptide and melon seed of extraction extract, keeps 40 DEG C of stirring 10min, is cooled to room temperature, and filters, and filtrate is filtered through 0.22 μm
Core filtering, being concentrated into content of peptides is 4.5mg/ml, filling, obtains injection 100.
Comparative example:
The polypeptide that deer bone extracts the preparation method comprises the following steps: take deer bone, remove marrow, crush, deer bone weight is added in weighing
2.0% compound protease and 3 times of deer bone weight of water for injection, compound protease are the pepsin that weight ratio is 2.05:1
And papain；40 DEG C~50 DEG C are extracted 1~3 hour, filtering, and filtrate is filtered, filtrate with citric acid tune pH to 5.5, standing
With sodium citrate tune pH to 8.8,0.2 times of pancreatin of deer bone weight is added, 45 DEG C hydrolyze 2 hours, filtering, by filtrate citric acid
Modulating pH value is 7.0, successively with interception 100,000 ultrafiltration system of molecular weight, interception 10,000 ultrafiltration system of molecular weight, interception molecular weight
8000 ultrafiltration units carry out ultrafiltration, retain permeate, and freeze-drying obtains the polypeptide of deer bone extraction.
Melon seed extract polypeptide the preparation method comprises the following steps:
Melon seed is taken, is crushed, the compound protease and 3 times of melon seed weight of note of melon seed weight 2.0% is added in weighing
It penetrates and uses water, compound protease is the pepsin and papain that weight ratio is 2.05:1；45 DEG C are extracted 2 hours, are filtered, filter
Liquid citric acid tune pH to 5.0 is stood, filtering, and 0.2 times of melon seed weight is added with sodium citrate tune pH to 8.8 in filtrate
Pancreatin, 45 DEG C hydrolyze 2 hours, filtering, and it is 7.0 that filtrate citric acid, which modulates pH value, successively with interception 100,000 ultrafiltration system of molecular weight
System, interception 10,000 ultrafiltration system of molecular weight, interception 8,000 ultrafiltration unit of molecular weight, carry out ultrafiltration, retain permeate, and freezing is dry
It is dry, it obtains melon seed and extracts to obtain polypeptide.
After taking deer bone extracting solution and muskmelon seed extracting solution to thaw, it is mixed to prepare according to content of peptides weight ratio 2:0.8
Cervus and cucumis polypeptide extracting solution, is added active carbon, filtering, and the ultrafiltration membrane ultrafiltration of filtrate interception molecular weight 8000 retains permeate, thoroughly
Liquid is crossed through 0.22 μm of filter element filtering, being concentrated into content of peptides is 4.5mg/ml, it is filling, obtain injection 100.
[determining content of peptides] is accurate to measure sample 1ml, sets in 25ml measuring bottle, 0.1mol/L sodium hydroxide solution is added to dilute
It is measured to scale as test solution according to forint phenol measuring method.
Forint phenol measuring method
Reagent alkaline copper test solution takes sodium hydroxide 10g, sodium carbonate 50g to add water 400ml to make to dissolve, as solution A；Take winestone
Sour potassium 0.5g, adds water 50ml to make to dissolve, and separately takes copper sulphate 0.25g, and water 30ml is added to make to dissolve, and two liquid is mixed, as second liquid.
Before use, merge two liquid of first, second, and add water to 500ml.
The preparation of maneuver reference substance solution takes bovine serum albumin(BSA) reference substance, and water is added to be made in every 1ml containing the molten of 0.3mg
Liquid.
Defined method preparation under each kind item is shone in the preparation of test solution.
The preparation precision of standard curve measures reference substance solution 0.0ml, 0.1ml, 0.3ml, 0.5ml, 0.7ml, 0.9ml,
It sets in tool plug test tube respectively, respectively adds water to 1.0ml, then be separately added into alkaline copper test solution 1.0ml, shake up, it is each that the examination of forint phenol is added
Liquid (taking the stock solution 1 → 16 in forint test solution) 4.0ml, mixes immediately, sets in 55 DEG C of water-baths accurate response 5 minutes, set cold water
Bath 10 minutes is surveyed at the wavelength of 650nm according to UV-VIS spectrophotometry (two IV A of annex of China's coastal port)
Determine trap；Using No. 0 pipe as blank.Regression equation is calculated with corresponding trap with reference substance solution concentration.
Measuring method precision measures test solution 1.0ml, the method under the preparation of sighting target directrix curve, from " addition alkalinity
Copper test solution " rise, measure in accordance with the law, from the content of regression equation calculation polypeptide, and multiplied by extension rate to get.
[free amine group assays]
Precision measures amino acid reference substance solution and 200 μ L of test solution, is set in a 1ml centrifuge tube respectively, and precision adds
Enter 20 μ l of nor-leucine inner mark solution (1mg/ml), 100 μ l of 0.1mol/L phenyl isothiocyanate (PITC) acetonitrile solution be added,
100 μ l of 1mol/L triethylamine acetonitrile solution mixes, is placed at room temperature for 1 hour, and 400 μ l n-hexanes are added, and oscillation is placed 10 minutes,
Lower layer's solution is taken, is filtered with 0.45 μm of filter.
Chromatographic condition: chromatographic column: Agela XBP-C18Column 250mm*4.6mm, 5 μm；Mobile phase: A liquid: 0.1mol/L acetic acid
Sodium (pH6.5)/acetonitrile=93/7, B liquid: acetonitrile/water=4/1；Detection wavelength: 254nm；Column temperature: 43 DEG C；Sample volume: 2 μ L.
1 eluent gradient table of table
Different preparations are taken to measure according to amino acid assays, (L-aminobutanedioic acid, glutamic acid, serine, sweet ammonia containing free amino acid
It is acid, histidine, arginine, threonine, alanine, proline, tyrosine, valine, methionine, cystine, isoleucine, bright
Propylhomoserin, phenylalanine, lysine) total amount be no more than 500 μ g/ml.
[high molecular weight material] is measured according to high performance liquid chromatography (two V D of annex of Chinese Pharmacopoeia version in 2010).
Chromatographic condition and system suitability are with gel chromatographic columns (TSKgelG2000SW)；Mobile phase is acetonitrile-
Water-trifluoroacetic acid (35:65:0.1)；Flow velocity is 0.7ml/mim；Detection wavelength is 214nm.Number of theoretical plate presses ribonuclease A
Peak, which calculates, is not less than 2000.
The preparation of standard curve takes ribonuclease A (molecular weight 13700), actrapid monotard's (molecular weight 5808), chest respectively
Gland peptide α1(molecular weight 3108), growth hormone release inhibiting hormone (molecular weight 1638) in right amount, are made in every 1ml with mobile phase respectively containing the molten of 0.5mg
Liquid is as molecular weight standard solution.Take respectively 10 μ L of molecular weight standard solution inject liquid chromatograph, record chromatogram, repeat into
Sample, relative deviation of retention time are not greater than 2.0%.Using the retention time at each peak as abscissa, molecular weight logarithm is vertical sits
It is denoted as standard curve, carries out linear regression, related coefficient cannot be less than 0.99.
Measuring method takes 10 μ L of test sample, injects liquid chromatograph, records chromatogram, is calculated with area normalization method, for examination
The component that product middle-molecular-weihydroxyethyl is greater than 10000 dalton must not cross 5.0%.
Test method: stability test: embodiment 1-5 and comparative example 12 are respectively placed in 70 DEG C of high temperature, relative humidity 95%
With illumination according under the conditions of 6000Lx, indices are investigated respectively at the 10th day, sampling in 30 days, as a result see the table below 2:
2 different tests group preparation comparative test of table
Conclusion (of pressure testing): it is above-mentioned experiments have shown that, the preparation being prepared into using sodium alginate as pharmaceutic adjuvant, content of peptides is several
Constant, free aminoacid content, polymer substance changes of contents are smaller；And the polypeptide formulations of comparative example, content of peptides reduce
Comparatively fast, free histidine content and polymer substance content increase more, absolutely prove, after sodium alginate is added in the formulation, tool
There is better stability.
Claims (4)
1. a kind of preparation method of cervus and cucumis polypeptide ejection preparation, it is characterised in that should be the preparation method comprises the following steps: polypeptide, the sweet tea that deer bone extracts
The weight ratio of polypeptide and sodium alginate that melon seeds extracts is 1.5-2.5:0.7-0.8:0.070-0.10；Sodium alginate is taken, is added
Water for injection, dissolution completely, are heated to 30-40 DEG C, and the polypeptide and melon seed that the extraction of deer bone is added at such a temperature extract more
Peptide keeps 30-40 DEG C of stirring 10-20min, is cooled to room temperature, and filters, and filtrate is concentrated into polypeptide and contains through 0.22 μm of filter element filtering
Amount is 3-6mg/ml, filling, obtains injection.
2. a kind of cervus and cucumis polypeptide ejection preparation according to claim 1, wherein the polypeptide, melon seed of deer bone extraction extract
The weight ratio of polypeptide and sodium alginate is 2:0.75:0.083.
3. -2 described in any item a kind of cervus and cucumis polypeptide ejection preparations according to claim 1, the system for the polypeptide that wherein deer bone extracts
Preparation Method are as follows:
Deer bone is taken, marrow is removed, is crushed, the compound protease and deer bone weight 2 of deer bone weight 1.5%-2.5% is added in weighing
~4 times of water for injection, compound protease are the pepsin and papain that weight ratio is 1.85-2.35:1；40 DEG C~
50 DEG C are extracted 1~3 hour, filtering, and filtrate is stood with citric acid tune pH to 4.5-6.0, are filtered, and filtrate is with sodium citrate tune pH
To 8.5~9.0,0.1~0.3 times of pancreatin of deer bone weight is added, 40 DEG C~50 DEG C hydrolyze 1~3 hour, filtering, by filtrate
Citric acid modulate pH value be 6.5-7.5, successively with interception 100,000 ultrafiltration system of molecular weight, interception 10,000 ultrafiltration system of molecular weight,
8,000 ultrafiltration unit of molecular weight is intercepted, ultrafiltration is carried out, retains permeate, freeze-drying obtains the polypeptide of deer bone extraction.
4. -2 described in any item a kind of cervus and cucumis polypeptide ejection preparations according to claim 1, the polypeptide that wherein melon seed extracts
The preparation method comprises the following steps:
Melon seed is taken, is crushed, the compound protease and melon seed weight 2~4 of melon seed weight 1.5%-2.5% is added in weighing
Water for injection again, compound protease is the pepsin and papain that weight ratio is 1.85-2.35:1；40 DEG C~50 DEG C
It extracts 1~3 hour, filtering, filtrate is stood with citric acid tune pH to 4.5-6.0, is filtered, and filtrate is with sodium citrate tune pH to 8.5
~9.0,0.1~0.3 times of pancreatin of melon seed weight is added, 40 DEG C~50 DEG C hydrolyze 1~3 hour, filtering, filtrate citron
Acid modulation pH value is 6.5-7.5, successively with interception 100,000 ultrafiltration system of molecular weight, interception 10,000 ultrafiltration system of molecular weight, interception
8,000 ultrafiltration unit of molecular weight carries out ultrafiltration, retains permeate, and freeze-drying obtains melon seed and extracts to obtain polypeptide.
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| CN110339335A (en) * | 2019-07-01 | 2019-10-18 | 哈尔滨誉衡制药有限公司 | A kind of cervus and cucumis polypeptide ejection preparation |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103040910A (en) * | 2012-12-17 | 2013-04-17 | 海南百思特医药科技有限公司 | Cervus and cucumis polypeptide liposome injection |
| CN104248653A (en) * | 2013-06-26 | 2014-12-31 | 哈尔滨誉衡药业股份有限公司 | Pharmaceutical composition prepared from deer bone and melon seed as raw materials and preparation thereof |
| CN104248652A (en) * | 2013-06-26 | 2014-12-31 | 哈尔滨誉衡药业股份有限公司 | Preparation method of cervus and cucumis polypeptide injection preparation |
| CN105688181A (en) * | 2014-11-28 | 2016-06-22 | 西藏誉衡阳光医药有限责任公司 | Injection preparation prepared from beer bones and muskmelon seeds |
| CN105687293A (en) * | 2014-11-28 | 2016-06-22 | 西藏誉衡阳光医药有限责任公司 | A preparing method of a Lugua polypeptide injection |
| CN105688182A (en) * | 2014-11-28 | 2016-06-22 | 西藏誉衡阳光医药有限责任公司 | A Lugua polypeptide injection |
| CN106036837A (en) * | 2016-06-03 | 2016-10-26 | 通化师范学院 | Formula and preparation method of microcapsules prepared from deer horns and pilose antlers of sika deers |
-
2019
- 2019-07-01 CN CN201910585619.6A patent/CN110339336A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103040910A (en) * | 2012-12-17 | 2013-04-17 | 海南百思特医药科技有限公司 | Cervus and cucumis polypeptide liposome injection |
| CN104248653A (en) * | 2013-06-26 | 2014-12-31 | 哈尔滨誉衡药业股份有限公司 | Pharmaceutical composition prepared from deer bone and melon seed as raw materials and preparation thereof |
| CN104248652A (en) * | 2013-06-26 | 2014-12-31 | 哈尔滨誉衡药业股份有限公司 | Preparation method of cervus and cucumis polypeptide injection preparation |
| CN105688181A (en) * | 2014-11-28 | 2016-06-22 | 西藏誉衡阳光医药有限责任公司 | Injection preparation prepared from beer bones and muskmelon seeds |
| CN105687293A (en) * | 2014-11-28 | 2016-06-22 | 西藏誉衡阳光医药有限责任公司 | A preparing method of a Lugua polypeptide injection |
| CN105688182A (en) * | 2014-11-28 | 2016-06-22 | 西藏誉衡阳光医药有限责任公司 | A Lugua polypeptide injection |
| CN106036837A (en) * | 2016-06-03 | 2016-10-26 | 通化师范学院 | Formula and preparation method of microcapsules prepared from deer horns and pilose antlers of sika deers |
Non-Patent Citations (2)
| Title |
|---|
| 王春霞等: ""海藻酸钠的综合应用进展"", 《食品与发酵科技》 * |
| 赵雨娜等: ""注射用鹿瓜多肽处方及工艺的研究"", 《医药与保健》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110339335A (en) * | 2019-07-01 | 2019-10-18 | 哈尔滨誉衡制药有限公司 | A kind of cervus and cucumis polypeptide ejection preparation |
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