CN1290574C - Injection of donkey hide gelatin and its preparation - Google Patents
Injection of donkey hide gelatin and its preparation Download PDFInfo
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- CN1290574C CN1290574C CNB2004100227435A CN200410022743A CN1290574C CN 1290574 C CN1290574 C CN 1290574C CN B2004100227435 A CNB2004100227435 A CN B2004100227435A CN 200410022743 A CN200410022743 A CN 200410022743A CN 1290574 C CN1290574 C CN 1290574C
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- injection
- corii asini
- colla corii
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- hydrolysis
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Abstract
The present invention relates to a donkey hide gelatin injection which is prepared from effective components of the liquid of polypeptides, amino acids and microelements decomposed from donkey hide gelatin, does not contain anaphylactogens, meets the quality requirements of human injection medication, and has a formulation of venous administration. The present invention is mainly applied to patients unsuitable for oral administration, and weak patients, such as patients whose wounds are not healed after gastrointestinal tract partial excision, and patients after malignant tumor chemotherapy. The present invention treats adaptive diseases of hematogenesis and leukocyte increase, has the functions of promoting wound healing, enhancing an immune function, etc. The present invention also discloses a method for preparing the injection.
Description
Technical field:
The Colla Corii Asini injection that the present invention relates to is the novel form of Colla Corii Asini, and this dosage form and preparation method thereof belongs to pharmacy and biomedicine field.
Background technology:
Colla Corii Asini is a nourishing Chinese medicine, the effect of YIN nourishing is arranged, enriching blood, and the skin moistening that nourishes blood makes skin moist, high resilience, hair is pitch-black, and appearance is pleased color.Pai Chu-yi once used " the hot spring neatly is washed lipoid " to describe that the beautiful flesh flower of Yang Yuhuan holds in " song of everlasting sorrow ".According to textual criticism " thick soup of donkey-hide gelatin " is exactly one of normal medicinal diet for beautifying of eating of Yuhuan.
Modern medicine finds that the Colla Corii Asini main component is the peptide class and the aminoacid of collagen protein and hydrolysis thereof, simultaneously, also contains the abundant trace element relevant with keeping bioenzyme activity.Therefore, Colla Corii Asini has following effect:
1, promotes hemopoietic function, can obviously improve erythrocyte and content of hemoglobin, iron deficiency anemia and hemorrhagic anemia are had significant curative effect.
2, the aminoacid of collagen protein composition is provided, promotes wound healing.
3, can improve immunity, the obviously effect of enhancing human body immunity function to old, weakness due to chronic disease, easily catch a cold etc. fine treatment preventive effect is arranged, can be used as the paramedicines because of immunologic hypofunction associated diseases and radiotherapy, patients undergoing chemotherapy simultaneously.
4, promoting the absorption and the storage of calcium, is the active drug of prevention, treatment osteoporosis.
5, Colla Corii Asini has and improves anoxia enduring, fatigue proof effect, to the athlete of the student of the person that is not engaged in mental work for a long time, nervous study, large amount of exercise relieving fatigue rapidly, regains one's strength, and makes it energetic.
6, tangible anastalsis is arranged, therefore to spit blood, have blood in stool, hemorrhages such as epistaxis, hematuria have better curative effect.
7, menoxenia, antenatal care, postpartum recuperating there is unique effect.
But present commercially available Colla Corii Asini product is oral formulations, and to patient that can not oral administration, the dosage form that Colla Corii Asini need be prepared into intravenously administrable could be used.
Existing intravenous nutrition medicine is the nutritional labeling of single kind, and for example the composition of amino acid is 18 seed amino acids, and its aminoacid ratio neither the amino acid needed optimal proportion of wound healing, does not have the polypeptide composition; Micro-element injection is the trace element that 6 to 10 kinds of human bodies need.
Technology contents
Purpose of the present invention just provides a kind of new ass hide gelatin preparation of intravenously administrable.
The objective of the invention is to be achieved through the following technical solutions.
The main component of Colla Corii Asini is collagen protein, peptide class, aminoacid, soybean oil, trace element and other impurity.Wherein collagen protein and long peptide have antigenicity, are not suitable for direct injection and use.Soybean oil and other impurity also need to remove.
The present invention becomes not have antigenic polypeptide and aminoacid with the collagen hydrolysate of Colla Corii Asini, keeps healthy trace elements with household, and according to the requirement of human body with injection, is prepared into and can offers the Colla Corii Asini injection that the human vein injection is used.The inventor has carried out the research of hydrolysising condition to the Colla Corii Asini hydrolysis, and the product that obtains has been carried out zoopery, has investigated safety and effectiveness, optimizes preparation technology on this basis, has prepared the Colla Corii Asini injection, and zoopery proves that it is safe and effective.
Colla Corii Asini injection of the present invention contains polypeptide, aminoacid and trace element, polypeptide wherein and aminoacid are collagen hydrolysates, and the latter is the important structure composition of wound healing, contained trace element, particularly zinc element also is of value to the recovery of wound healing and physical function.And middle the present invention also studies the preparation method of Colla Corii Asini injection.
Colla Corii Asini injection of the present invention is mainly used in the patient that is not suitable for during the oral administration and the patient of whole body situation weakness, the wound patient of serious vomiting etc. behind the patient, chemotherapy of healing not as yet after the gastrointestinal surgery for example, indication and donkey-hide gelatin oral preparation are basic identical, comprise hemopoietic, leukocyte increasing, promotion wound healing, enhance immunity function, promote body recovery etc.
Description of drawings
Fig. 1 pH is to the influence of papain hydrolysis
Fig. 2 temperature is to the influence of papain hydrolysis
Fig. 3 enzyme-to-substrate is than (E/S) influence to papain hydrolysis
Fig. 4 response time is to the influence of papain hydrolysis
The following sample soft ionization of Fig. 5 complex enzyme hydrolysis hydrolyzed solution 5KD mass spectrum
The specific embodiment
Below in conjunction with example the present invention is further specified.
Experimental example 1 Colla Corii Asini hydrolysising experiment:
One, raw material and medicine
Subtilisin:
Papain:
Ultra-filtration centrifuge tube: Millipore company product, molecular cut off are 5KD, 10KD
Two, experiment and method
1, enzyme digestion reaction
Accurately take by weighing a certain amount of Colla Corii Asini powder and be dissolved in the phosphate buffer, be stirred to colloid.After being adjusted to certain pH, adding a certain amount of protease and be hydrolyzed.The constant of stirring and maintenance pH interrupted in hydrolysis.After hydrolysis finishes, 100 ℃ of heating of hydrolyzed solution 5min cessation reaction.
2, ultrafiltration segmentation
Hydrolyzed solution is used two metafiltration paper successively, 0.45 μ micropore filter paper, 0.22 μ micropore filter paper filtering.Filtering back solution is 5 with molecular cut off respectively, and it is centrifugal that 10KD ultra-filtration centrifuge tube (Millipore company product) carries out ultrafiltration, and it is partly following to collect 5KD and 10KD.
3, protein degree (DH) is measured
Get the centrifugal liquid of an amount of hydrolyzed solution, with the quick titration measuring free amino nitrogen of formaldehyde fixed (FAN) content, determination of total nitrogen content adopts the semimicro Kjeldahl.
4, average peptide chain length
Peptide molecule average chain length (PCL) ≈ 100/DH (%)
Three, result and discussion
1, the enzymolysis optimal condition determines
(1) papain (P60) optimal condition determines
The influence of pH
Every 0.5h measures a pH in the hydrolytic process, and maintenance pH's is constant.
Enzymatic hydrolysis condition: concentration of substrate 0.1g/ml, enzyme-to-substrate is than being 20mg/g albumen, 55 ℃ of reaction temperatures, response time 4h.
As seen from Figure 1, the pH of the optimum hydrolysis Collagenase of papain is 5.0～6.0, is lower than 5.0 or be higher than 6.0 enzymatic activitys and descend.It is more that particularly pH is lower than enzyme activity decline in 4 o'clock.
Temperature Influence
Hydrolysising condition: concentration of substrate 0.1g/ml, enzyme-to-substrate react pH5.0, response time 4h than 20mg/g albumen.
The proteic optimum hydrolysis temperature of Fructus Chaenomelis is 55 ℃ as seen from Figure 2, surpasses 60 ℃ of enzymes and begins to take place degeneration, and enzymolysis efficiency reduces.
Enzyme-to-substrate is than the influence of (E/S)
Hydrolysising condition: concentration of substrate 0.1g/ml, reaction pH5.0,55 ℃ of reaction temperatures, response time 4h.
Papain hydrolysis Colla Corii Asini the suitableeest proteic enzyme-to-substrate is than being 20mg/g albumen as seen from Figure 3, and when the enzyme-to-substrate ratio surpassed ratio, the degree of hydrolysis increase was all not obvious.Consider economy, we select the enzyme-to-substrate ratio for use is that 15～20mg/g albumen is as optimum hydrolysising condition.
The influence of hydrolysis time
Hydrolysising condition: concentration of substrate 0.1g/ml, reaction pH5.0,55 ℃ of reaction temperatures, enzyme-to-substrate is than being 20mg/g albumen.
As seen from Figure 4,2 speed at one hour rating of enzymolysis pro-are very fast, culminate behind 4h, and the time, degree of hydrolysis remained unchanged substantially above behind the 4h.Drawing the proteic optimum condition of papain hydrolysis Colla Corii Asini by above experiment is concentration of substrate 0.1g/ml, reaction pH5.0, and 55 ℃ of reaction temperatures, enzyme-to-substrate is than 20mg/g albumen, response time 4h.
Secondary enzymolysis is for the influence of papain hydrolysis degree.
After the first time, enzymolysis finished, in reaction system, add enzyme-to-substrate and carry out the secondary hydrolysis than for the proteic papain of 10mg/g, FAN brings up to 2.86mg/ml by 2.28mg/ml behind the reaction 2h.The hydrolysis of repeated experiments proof secondary can improve degree of hydrolysis 20.28～22.35%.
(2) subtilisin, papain separate and the enzymolysis degree of complex enzyme hydrolysis relatively
Bacillus subtilis enzymolysis optimal condition has many pieces of reported in literature, and the enzymolysis optimal condition is, concentration of substrate 0.1mg/ml, and reaction pH7.5,55 ℃ of reaction temperatures, enzyme-to-substrate is than being 20mg/g albumen, response time 4h.
Two kinds of enzymes of table 1 are to the comparison of Colla Corii Asini Proteolytic enzyme effect
| The kind of enzyme | pH | Temperature (℃) | Enzyme-to-substrate is than (E/S) (mg/s albumen) | FAN (mg/ml) |
| Papain subtilisin Fructus Chaenomelis+hay hay+Fructus Chaenomelis | 5.0 7.5 5.0+7.5 7.5+5.0 | 55 55 55 55 | 20 20 20+20 20+20 | 1.32 1.41 1.52 2.59 |
Experiment is found, can effectively improve hydrolysis efficiency by complex enzyme hydrolysis, and particularly earlier with subtilisin hydrolysis reuse papain hydrolysis, comparable independent employing papain is separated and improved 96.2% hydrolysis degree.This may be because subtilisin has stronger non-specificity, collagen protein had more a plurality of restriction enzyme sites, and adopting the subtilisin hydrolysis that the papain restriction enzyme site that was wrapped in collagen protein triple helix inside is originally come out earlier, feasible papain is thereafter separated has higher efficient.
2, the peptide link analysis of enzymolysis sample after the ultrafiltration
Papain hydrolysis liquid:
The following sample F AN of 5KD is 0.98mg/ml, and total nitrogen content is 2.42mg/ml, and hence one can see that, and the average peptide chain length is 2.47.The FAN of the following sample of 10KD is 1.07mg/ml, and total nitrogen content is 4.38mg/ml, and hence one can see that, and the average peptide chain length is 4.09.
The subtilisin hydrolyzed solution:
The following sample F AN of 5KD is 1.06mg/ml, and total nitrogen content is 2.44mg/ml, and hence one can see that, and the average peptide chain length is 2.31.The FAN of the following sample of 10KD is 1.10mg/ml, and total nitrogen content is 4.35mg/ml, and hence one can see that, and the average peptide chain length is 3.95.Improve 7-8% than papain hydrolysis degree, this may give the credit to novel subtilases and have higher non-specificity, has more point of contact for the collagen protein in the Colla Corii Asini.
The compound protein enzyme hydrolyzate:
The following sample F AN of 5KD is 2.196mg/ml, and total nitrogen content is 2.53mg/ml, and hence one can see that, and the average peptide chain length is 1.15.The FAN of the following sample of 10KD is 2.74mg/ml, and total nitrogen content is 4.87mg/ml, and hence one can see that, and the average peptide chain length is 1.78.Improve 124.08-147.30% than papain hydrolysis degree, this may give the credit to novel subtilases and have higher non-specificity, has more point of contact for the collagen protein in the Colla Corii Asini.Mass Spectrometer Method is found, the small peptide of mainly forming in the 5KD sample in the compound protein enzymolysis liquid based on the 2-6 amino acid residue, and maximum molecular weight is 3101.9, approximately contains 25.85 amino acid residue (see figure 5)s.The following sample of 10KD, the mainly small peptide of forming based on 2-13 amino acid residue.
3, aminoacid is formed mutation analysis before and after the proteolysis
Protein content in the Colla Corii Asini is about about 80%, contains 18 seed amino acids (comprising 7 kinds of essential amino acids), its content is respectively lysine 2.63%, histidine 0.59%, arginine 4.42%, threonine 3.21%, serine 1.86%, glutamic acid 0.59%, proline 6.52%, glycine 13.36%, alanine 0.12%, valine 0.84%, methionine 0.20%, leucine 1.92%, isoleucine 0.91%, tyrosine 0.29%, phenylalanine 1.59% etc.But its free aminoacid content and kind are all less, wherein only contain 8 seed amino acids, and total amount only is the 1%-2% of total protein content.Bring up to 15 kinds through free amino acid kind behind the complex enzyme hydrolysis, content has increased 32-55 doubly, can directly be utilized by human body.And a large amount of 2-3 peptides also can directly directly be utilized by human body institute in the complex enzyme hydrolysis hydrolysate.
4, micronutrient levels analysis before and after the enzymolysis
Contain multiple metallic element such as K in the Colla Corii Asini, Na, Ca, Mg, Fe, Cu, Al, Mn, Zn, Cr, Pt, Pr, Mo, Sr etc. discover that previously the multiple pharmacological effect of Colla Corii Asini is relevant with its contained trace element.Ferrum, elements such as copper can be treated some nutritional anemia to be proved to be, other various trace elements then with miscellaneous body in enzyme confidential relation is arranged.We have adopted the ultrafiltration centrifugation technique behind enzymolysis, only macromolecular substances is held back.Promptly eliminated the proteic antigenicity of Colla Corii Asini, and do not destroyed the original various trace elements of Colla Corii Asini and form.We are to Zn before and after the ultrafiltration, Cu, and Fe, Mn, Zn/Cu detects, and finds that metal ion is not trapped (seeing Table 2) when ultrafiltration
Trace elements determination ug/g before and after table 2 ultrafiltration in the Colla Corii Asini)
| Sample | Zn | Cu | Fe | Mn | Zn/Cu |
| Before the ultrafiltration | 18.13 | 1.04 | 71.36 | 5.29 | 17.4 |
| After the ultrafiltration | 18.17 | 0.99 | 71.33 | 5.20 | 16.8 |
By above experiment, optimum enzymolysis process is: earlier carry out enzymolysis with subtilisin, reaction condition is, concentration of substrate 0.1g/ml, and reaction pH7.5,55 ℃ of reaction temperatures, enzyme-to-substrate is than 20mg/g albumen, response time 4h.In reactant liquor, add enzyme-to-substrate after enzymolysis finishes for the first time again than the proteic papain of 20mg/g, reaction pH5.0,55 ℃ of reaction temperatures, response time 4h, reaction finishes back 100 ℃ of heating 5min cessation reactions.Enzymolysis solution is used two metafiltration paper successively, 0.45 μ micropore filter paper, 0.22 μ micropore filter paper filtering.Filtering back solution is 5 with molecular cut off respectively, and it is centrifugal that 10KD ultra-filtration centrifuge tube (Millipore company product) carries out ultrafiltration, and it is partly following to collect 5KD and 10KD.
Experimental example 2 Colla Corii Asini hydrolyzed solution Cavia porcellus systemic anaphylaxis reaction tests
One, purpose
Observe the situation of Colla Corii Asini hydrolyzed solution, investigate the said preparation safety the reaction of Cavia porcellus systemic anaphylaxis.
Two, material and method
1, animal subject
24 of healthy guinea pigs, body weight 240～300g, the male and female dual-purpose is provided by Sichuan Academy of Medical Sciences institute of lab animals, laboratory animal occupancy permit number 019.
2, be subjected to the reagent thing
10KD and 5KD Colla Corii Asini hydrolysis injection (lyophilizing) are faint yellow loose sprills, specification: the 250mg/ bottle, and lot number: 040213, provide by the Yaoyou Science and Technology Development Co., Ltd., Chendu City.
3, reagent and equipment
Sodium chloride injection, lot number: 040328, the concentric pharmaceutical Co. Ltd of the strange power in Sichuan Province produces; Disposable syringe, lot number: 040218, thing rich medical apparatus and instruments company limited in Xinjin, Chengdu is produced; 1% ovalbumin is by the autogamy of Yaoyou Science and Technology Development Co., Ltd., Chendu City pharmacological room.
4, method
Cavia porcellus is divided into Colla Corii Asini hydrolyzed solution 5KD and 10KD group, 1% ovalbumin (normal saline dilution) positive controls, 4 groups of normal saline negative control group, 6 every group, male and female dual-purpose at random by body weight.Respectively tried Cavia porcellus and all tried thing accordingly by 0.5ml/ every other day intramuscular injection, continuous 3 times, 14 days, 21 days each group are respectively got 3 Cavia porcelluss and are subjected to test solution 1ml/ from this group of right crus of diaphragm back of the body outside intravenous injection behind the 1st drug administration by injection respectively, according to 2000 editions regulations of the Pharmacopoeia of the People's Republic of China, observe after the administration that Cavia porcellus has or not anaphylaxiss such as perpendicular hair, sneeze, retch, cough, dyspnea, hello sound, tic, collapse or death in 15 minutes.Estimate by table 3 grading standard.
Table 3 Cavia porcellus systemic anaphylaxis grading standard
| | Reaction symptom | |
| 0 1 2 3 4 | No significant reaction has only and slightly grabs nose, trembles or perpendicular hair has cough several times, grab nose, tremble or erect hair and repeatedly or continuously cough, with spasm, tic, gatism, shock deaths such as dyspnea, spasm, tics |
Three, result
The reaction test of Cavia porcellus systemic anaphylaxis the results are shown in Table 4.
Table 4 Cavia porcellus systemic anaphylaxis irritation progression
| Tried thing | 14 days | 21 days |
| Normal saline group Colla Corii Asini hydrolyzed solution 5KD group Colla Corii Asini hydrolyzed solution 10KD organizes 1 | 0 0 0 3.3 | 0 0 0 3.7 |
Colla Corii Asini hydrolyzed solution 5KD and 10KD group does not have in 15 minutes behind 14 days, 21 days intravenously administrables after the 1st administration respectively and grabs nose, trembles or symptoms of allergic such as perpendicular hair, anaphylaxis average level number average is 0, and prompting Colla Corii Asini hydrolyzed solution 5KD, 10KD do not have systemic anaphylaxis to Cavia porcellus.
Conclusion: Colla Corii Asini hydrolyzed solution 5KD, 10KD Cavia porcellus systemic anaphylaxis are negative, and no anaphylaxis meets 2000 editions regulations of the Pharmacopoeia of the People's Republic of China.
The research of experimental example 3 Colla Corii Asini hydrolysis injection (lyophilizing) blood tonification effects
One, purpose
Observe Colla Corii Asini hydrolysis injection (lyophilizing) to the administration of hemorrhagic anemia rats by intraperitoneal injection after, to the influence of erythrocyte number in the blood and content of hemoglobin.
Two, material and method
1, is subjected to reagent
10KD and 5KD Colla Corii Asini hydrolysis injection (lyophilizing) are faint yellow loose sprills, specification: the 250mg/ bottle, and lot number: 040213, provide by the Yaoyou Science and Technology Development Co., Ltd., Chendu City.
E-jiao hematinic ointment is tan semifluid, specification: the 200g/ bottle, and lot number: 040124, four directions, Henan Province pharmaceutcal corporation, Ltd produces.
Amino Acid Compound Injection (15AA) is colourless or almost colourless clear liquid, specification: 250ml: 20g (total amino acids), and lot number: 040223, Zhejiang Province Jimin Pharmaceutical Co., Ltd produces.
Colla Corii Asini hydrolysis injection (lyophilizing), e-jiao hematinic ointment, facing the time spent is the test desired concn with the normal saline dilution all.
2, laboratory animal
The SD rat, body weight 180～200g, totally 100, male and female half and half are provided by Sichuan Academy of Medical Sciences institute of lab animals, laboratory animal occupancy permit numbers 119.
3, instrument
Full-automatic blood counting instrument, M-4000, Germany produces.
4, method
Get 90 of healthy SD rats, male and female half and half cause anemia with 5ml disposable syringe heart blood-letting 5ml before the test, treat that cutting tail after the animal blood volume is stablized gets blood, counting RBC and Hb next day.Other get 10 not the blood-letting healthy rat as the normal control group, the same counting RBC and Hb.Grouping and dosage see Table 5.Administration every day 1 time, continuous 14 days, cut tail respectively in d7 and d14 and get blood, survey RBC and Hb.
Table 5 test grouping and administration
| Group | Number of animals (n) | Dosage | Route of administration | The administration volume |
| Normal group model group e-jiao hematinic ointment group Amino Acid Compound Injection group 10KD donkey-hide gelatin water is high, and to separate in the parenteral solution high (freeze-drying) group of separating in the parenteral solution of the low 5KD donkey-hide gelatin water of (freeze-drying) group low | 10 10 10 10 10 10 10 10 10 10 | Physiological saline physiological saline 0.67g/ml 0.08g/ml 0.08g/ml 0.04g/ml 0.02g/ml 0.08g/ml 0.04g/ml 0.02g/ml | ip ip ig ip ip ip ip ip ip ip | 0.5ml/100g 0.5ml/100g 1ml/100g 0.5ml/100g 0.5ml/100g 0.5ml/100g 0.5ml/100g 0.5ml/100g 0.5ml/100g 0.5ml/100g |
Three, result
10KD and 5KD Colla Corii Asini hydrolysis injection (lyophilizing) to the influence of RBC and Hb in the hemorrhagic anemia rat blood see Table 6, table 7.
The influence of RBC in the table 6 pair hemorrhagic anemia rat blood (x ± s)
| Group | Dosage | RBC(10 12/L) | ||
| Before the medicine | d7 | d14 | ||
| Normal group model group e-jiao hematinic ointment group amino acid 10KD donkey-hide gelatin water is high, and to separate in the parenteral solution high (freeze-drying) group of separating in the parenteral solution of the low 5KD donkey-hide gelatin water of (freeze-drying) group low | - - 0.67g/ml 0.08g/ml 0.08g/ml 0.04g/ml 0.02g/ml 0.08g/ml 0.04g/ml 0.02g/ml | 7.44±0.25 5.72±0.41 ** 5.68±0.22 * 5.77±0.45 ** 5.87±0.13 ** 5.72±0.77 ** 5.70±0.23 ** 5.81±0.07 ** 5.65±0.34 ** 5.73±0.72 ** | 7.55±0.59 5.73±0.32 ** 7.30±0.28 ▲▲ 6.92±0.72 ▲ 7.42±0.14 ▲▲ 7.38±0.29 ▲▲ 7.33±0.75 ▲▲ 7.40±0.65 ▲▲ 7.35±0.27 ▲▲ 7.32±0.12 ▲▲ | 7.61±0.37 7.12±0.47 7.57±0.22 7.41±0.11 7.59±0.29 7.47±0.36 7.52±0.44 7.58±0.55 7.41±0.32 7.50±0.74 |
Annotate: compare P＜0.01 with the normal control group; With model group comparison ▲ P＜0.05, ▲ ▲ P＜0.01.
The influence of Hb in the table 7 pair hemorrhagic anemia rat blood (x ± s)
| Group | Dosage | Hb(g/100ml) | ||
| Before the medicine | d7 | d14 | ||
| Normal group model group e-jiao hematinic ointment group Amino Acid Compound Injection group 10KD donkey-hide gelatin water is high, and to separate in the parenteral solution high (freeze-drying) group of separating in the parenteral solution of the low 5KD donkey-hide gelatin water of (freeze-drying) group low | - - 0.67g/ml 0.08g/ml 0.08g/ml 0.04g/ml 0.02g/ml 0.08g/ml 0.04g/ml 0.02g/ml | 12.85±0.22 9.37±0.17 ** 9.46±0.25 ** 9.42±0.57 ** 9.39±0.26 ** 9.41±0.58 ** 9.40±0.12 ** 9.45±0.21 ** 9.43±0.16 ** 9.39±0.56 ** | 12.73±0.12 10.10±0.22 11.97±0.32 △ 11.31±0.27 △ 11.96±0.16 △ 11.87±0.42 △ 11.62±0.11 △ 11.87±0.22 △ 11.83±0.17 △ 11.62±0.34 △ | 12.92±0.27 11.83±0.33 12.36±0.26 12.03±0.42 12.53±0.37 12.40±0.42 12.21±0.22 12.47±0.64 12.39±0.36 12.17±0.11 |
Annotate: compare with the normal control group
*P＜0.05,
*△ P＜0.05 is compared with model group in P＜0.01.
Conclusion: by table 6, table 7 result as seen, RBC and Hb obviously reduce after the rat heart blood-letting, are in the hemorrhagic anemia state, give the treatment of 10KD and 5KD Colla Corii Asini enzyme water injection (lyophilizing) after, the comparison of d7 and model group has utmost point significant difference to rising RBC; To rising Hb, has significant difference.Illustrate that this medical instrument has blood tonification effect.
Experimental example 4 Colla Corii Asini hydrolysis injections are to the influence of trauma in rat wound healing and Fn Fiberonectin level
One, purpose
Observation is subjected to the influence of reagent thing to Fn Fiberonectin level in trauma in rat wound healing time and blood and the granulation tissue, understands the curative effect of this medicine to the treatment wound.
Two, material and method
1, is subjected to reagent
10KD and 5KD Colla Corii Asini hydrolysis injection (lyophilizing) are faint yellow loose sprills, specification: the 250mg/ bottle, and lot number: 040213, provide by the Yaoyou Science and Technology Development Co., Ltd., Chendu City.
E-jiao hematinic ointment is tan semifluid, specification: the 200g/ bottle, and lot number: 040124, pharmaceutcal corporation, Ltd provides by the four directions, Henan Province.
Amino Acid Compound Injection (15AA) is colourless or almost colourless clear liquid, specification: 250ml: 20g (total amino acids), and lot number: 040223, by Zhejiang Province Jimin Pharmaceutical Co., Ltd.
Colla Corii Asini hydrolysis injection (lyophilizing), e-jiao hematinic ointment, facing the time spent is the test desired concn with the normal saline dilution all.
2, experimental animal
The SD rat, body weight 180～200g, totally 190, male and female half and half are provided by Sichuan Academy of Medical Sciences institute of lab animals, laboratory animal occupancy permit numbers 119.
3, reagent and instrument
Reagent: rat Fn reference serum, the anti-Mus Fn of rabbit antiserum, Huaxi Medical Univ's Pathological Staff Room provides (lot number: 040217); Agarose, reagent three factories in Shanghai produce (lot number: 031025); Barbital hydrochloric acid electrophoretic buffer, sample diluting liquid, the preparation of biochemical teaching and research room of Huaxi Medical Univ.
Instrument: electrophresis apparatus, microsyringe, constant temperature shaking bath, high speed centrifuge, electronic balance, electric grinding device.
4, method
Behind the conventional raising of the rat 3d, slough back wool with 8% sodium sulfide, modeling behind the 3d.With 4% pentobarbital sodium (4mg/100g body weight) intraperitoneal injection of anesthesia, cut off back part of animal one circular holostrome skin with shears, reach fascia deeply, hemostasis, wound diameter 3.0cm, the single cage in back of waiting to wake up is raised, and test grouping and administration see Table 1.Every component is 2 batches, and a collection of administration every day 1 time is healed fully until wound, relatively its wound healing time; Behind another batch medication 7d, femoral artery is got blood 5ml and is put to death, and separation of serum is gone up sample with rat blood serum dilution back, the row rocket electrophoresis, and with.Get the wound surface granulation tissue simultaneously and the skin physiological normal saline washing is clean, blot, take by weighing 0.2g, shred, add normal saline 2ml, place ice bath, electric grinding is made homogenate, and high speed centrifugation is got the direct electrophoresis of supernatant, also, measure Fn Fiberonectin content in serum and the tissue respectively simultaneously with reference standard serum drawing standard curve.
Table 8 test grouping and administration
| Group | Number of animals (n) | Dosage | Route of administration | The administration volume |
| Normal group model group e-jiao hematinic ointment group Amino Acid Compound Injection group 10KD donkey-hide gelatin enzyme is high, and to separate in the parenteral solution high (freeze-drying) group of separating in the parenteral solution of the low 5KD donkey-hide gelatin enzyme of (freeze-drying) group low | 10 20 20 20 20 20 20 20 20 20 | - - 0.67g/ml 0.08g/ml 0.08g/ml 0.04g/ml 0.02g/ml 0.08g/ml 0.04g/ml 0.02g/ml | ip ip ig ip ip ip ip ip ip ip | 0.5ml/100g 0.5ml/100g 1ml/100g 0.5ml/100g 0.5ml/100g 0.5ml/100g 0.5ml/100g 0.5ml/100g 0.5ml/100g 0.5ml/100g |
Three, result
Result of the test sees Table 9, table 10.
The influence of table 9 pair wound healing time (x ± s)
| Group | Dosage | Wound healing time (my god) |
| Model group e-jiao hematinic ointment group Amino Acid Compound Injection group 10KD donkey-hide gelatin enzyme is high, and to separate in the parenteral solution high (freeze-drying) group of separating in the parenteral solution of the low 5KD donkey-hide gelatin enzyme of (freeze-drying) group low | - 0.67g/ml 0.08g/ml 0.08g/ml 0.04g/ml 0.02g/ml 0.08g/ml 0.04g/ml 0.02g/ml | 28.6±3.4 24.1±2.3 * 26.2±3.2 22.6±2.7 ** 23.1±2.8 ** 24.3±3.5 * 22.1±3.2 ** 23.6±4.3 ** 24.7±3.2 * |
Annotate: compare with model group
*P＜0.05,
*P＜0.01.
The influence of Fn in table 10 pair serum and the granulation tissue (x ± s)
| Group | Dosage | Fn(μg·ml -1) | |
| Serum | Granulation tissue | ||
| Normal control group skin model group e-jiao hematinic ointment group | - - 0.67g/ml | 220.7±52.9 427.1±73.4 573.6±64.1 * | Do not detect 21.6 ± 4.6 38.7 ± 5.7 * |
| Amino Acid Compound Injection group 10KD donkey-hide gelatin enzyme is high, and to separate in the parenteral solution high (freeze-drying) group of separating in the parenteral solution of the low 5KD donkey-hide gelatin enzyme of (freeze-drying) group low | 0.08g/ml 0.08g/ml 0.04g/ml 0.02g/ml 0.08g/ml 0.04g/ml 0.02g/ml | 506.9±77.2 612.7±92.5 ** 598.1±73.4 ** 532.6±62.4 * 631.9±73.5 ** 602.7±63.5 ** 537.7±87.4 * | 30.6±3.9 67.3±4.5 ** 58.7±2.3 ** 38.5±4.2 * 66.7±5.3 ** 55.1±3.7 ** 40.6±5.2 * |
Annotate: compare with model group
*P＜0.05,
*P＜0.01.
Conclusion: Fn Fiberonectin is the macromole glycoprotein that a kind of molecular weight is about 450K, has special affinity with collagen, participates in the overall process of wound healing, all plays an important role in many links of wound repair.Above result of the test shows, 10KD and 5KD Colla Corii Asini hydrolysis injection (lyophilizing) obviously shorten the trauma in rat wound healing time, Fn Fiberonectin content obviously raises in blood and the granulation tissue, compare with model group, have significant difference and utmost point significant difference, and good more with the increase effect of dosage.
Experimental example 5 Colla Corii Asini hydrolysis injection are to mouse immune organ weight's influence
One, purpose
Mice gives 10KD and 5KD Colla Corii Asini hydrolysis injection (lyophilizing) through lumbar injection repeatedly, observes the influence of this medicine to the mouse immune organ weights.
Two, material and method
1, is subjected to the reagent thing
10KD and 5KD Colla Corii Asini hydrolysis injection (lyophilizing) are faint yellow loose sprills, specification: the 250mg/ bottle, and lot number: 040213, provide by the Yaoyou Science and Technology Development Co., Ltd., Chendu City.
E-jiao hematinic ointment is tan semifluid, specification: the 200g/ bottle, and lot number: 040124, pharmaceutcal corporation, Ltd provides by the four directions, Henan Province.
Amino Acid Compound Injection (15AA) is colourless or almost colourless clear liquid, specification: 250ml: 20g (total amino acids), and lot number: 040223, by Zhejiang Province Jimin Pharmaceutical Co., Ltd.
Colla Corii Asini hydrolysis injection (lyophilizing), e-jiao hematinic ointment, facing the time spent is the test desired concn with the normal saline dilution all.
2, experimental animal
Kunming mouse, body weight 18～22g, totally 90, male and female half and half are provided by Sichuan Academy of Medical Sciences institute of lab animals, laboratory animal occupancy permit numbers 119.
3, instrument
Electronic balance.
4, method
Mice is divided into 9 groups at random, 10 every group, male and female half and half.Grouping and dosage see Table 11.Pressed table 1 successive administration 14 days, 1h after the last administration takes off cervical vertebra with mice and puts to death, and opens abdominal cavity and thoracic cavity and wins spleen and thymus respectively, and scales/electronic balance weighing calculates organ index.
Table 11 test grouping and administration
| Group | Number of animals (n) | Dosage | Route of administration | The administration volume |
| Normal group e-jiao hematinic ointment group Amino Acid Compound Injection group 10KD donkey-hide gelatin water is high, and to separate in the parenteral solution high (freeze-drying) group of separating in the parenteral solution of the low 5KD donkey-hide gelatin water of (freeze-drying) group low | 10 10 10 10 10 10 10 10 10 | - 0.167g/ml 0.08g/ml 0.02g/ml 0.01g/ml 0.005g/ml 0.02g/ml 0.01g/ml 0.005g/ml | ip ig ip ip ip ip ip ip ip | 0.2ml/10g 0.4ml/10g 0.2ml/10g 0.2ml/10g 0.2ml/10g 0.2ml/10g 0.2ml/10g 0.2ml/10g 0.2ml/10g |
Three, result
Result of the test sees Table 12.
The influence of Fn in table 12 pair serum and the granulation tissue (x ± s)
| Group | Dosage | Organ index (mg/g) | |
| Thymus | Spleen | ||
| Normal group e-jiao hematinic ointment group Amino Acid Compound Injection group 10KD donkey-hide gelatin enzyme is high, and to separate in the parenteral solution high (freeze-drying) group of separating in the parenteral solution of the low 5KD donkey-hide gelatin enzyme of (freeze-drying) group low | - 0.167g/ml 0.08g/ml 0.02g/ml 0.01g/ml 0.005g/ml 0.02g/ml 0.01g/ml 0.005g/ml | 2.10±0.13 3.62±0.22 ** 3.34±0.17 * 3.72±0.25 ** 3.67±0.18 ** 3.57±0.29 ** 3.77±0.18 ** 3.69±0.27 ** 3.57±0.17 ** | 4.32±0.11 5.62±0.23 ** 5.12±0.13 * 5.73±0.14 ** 5.67±0.27 ** 5.43±0.13 ** 5.77±0.29 ** 5.61±0.32 ** 5.50±0.11 ** |
Annotate: compare with the normal control group
*P＜0.05,
*P＜0.01
Conclusion: 10KD, 5KD Colla Corii Asini hydrolysis injection (lyophilizing) have obvious effect of gain to the mouse immune organ, relatively have utmost point significant difference with the normal control group, and with the increase of dosage enhancing trend are arranged.
Specification: 100ml: 5g (the total protein quality is not less than 1.5g, and total nitrogen is not less than 0.25g)
Prescription:
Colla Corii Asini protolysate 50g
Sodium chloride for injection 9g
10% sodium hydroxide solution is an amount of
Add the injection water to 1000ml
Make 10 bottles
Preparation method: the Colla Corii Asini protolysate that obtains behind the enzymolysis of learning from else's experience with the water for injection dissolving, adds sodium chloride for injection again, stirring and dissolving, transferring pH value with 10% sodium hydroxide solution is 6.0～8.0,0.45 ц m filtering with microporous membrane, filtrate is divided the 100ml infusion bottle of packing into, gland, sterilization, promptly.
Specification: 100ml: 5g (the total protein quality is not less than 1.5g, and total nitrogen is not less than 0.25g)
Prescription:
Colla Corii Asini protolysate 50g
Glucose for injection 5g
0.1mol hydrochloric acid solution is an amount of
Add the injection water to 1000ml
Make 10 bottles
Preparation method: the Colla Corii Asini protolysate that obtains behind the enzymolysis of learning from else's experience with the water for injection dissolving, adds glucose for injection again, stirring and dissolving, transferring pH value with the 0.1mol hydrochloric acid solution is 5.0～6.0,0.45 ц m filtering with microporous membrane, filtrate is divided the 100ml infusion bottle of packing into, gland, sterilization, promptly.
Embodiment 3 Colla Corii Asini protein hydrolysate injections
Specification: 10ml: 0.25g 10ml: 0.5g 2ml: 50mg 5ml: 0.1g
Prescription (2ml: 50mg)
Colla Corii Asini protolysate 25g
EDTA-Na 2g
Sodium pyrosulfite 5g
0.1mol hydrochloric acid solution is an amount of
Add the injection water to 1000ml
Make 500
Preparation method: EDTA-Na, the sodium pyrosulfite of recipe quantity are dissolved in water for injection, the Colla Corii Asini protolysate of learning from else's experience again and obtaining behind the enzymolysis, add in the above solution, transferring pH value with the 0.1mol hydrochloric acid solution is 6.0～8.0,0.45 ц m filtering with microporous membrane, filtrate is divided pack into ampule sealing, sterilization, promptly.
Embodiment 4 injection Colla Corii Asini protolysates ()
Specification: 50mg
Prescription (50mg)
Colla Corii Asini protolysate 25g
0.1mol hydrochloric acid solution is an amount of
Add the injection water to 1000ml
Make 500 bottles
Preparation method: the Colla Corii Asini protolysate that obtains behind the enzymolysis of learning from else's experience, with the water for injection dissolving, reuse 0.1mol hydrochloric acid solution adjust pH is 6.0～8.0,0.22 ц m filtering with microporous membrane, the cillin bottle of packing into filtrate branch, lyophilizing, gland, promptly.
Specification: 1g, 2g, 5g
Preparation method: get Colla Corii Asini protolysate solution, pallet is gone in packing behind the 0.22 ц m filtering with microporous membrane, lyophilization, and lyophilized products is pulverized the back and is divided the cillin bottle of packing into, and gland is promptly.
Claims (4)
1, a kind of Colla Corii Asini injection, it is characterized in that containing the Colla Corii Asini protolysate, wherein said Colla Corii Asini protolysate is that Colla Corii Asini is earlier with subtilisin hydrolysis reuse papain hydrolysis, small peptide and the aminoacid of molecular weight below 10KD that obtains through ultrafiltration then.
2, Colla Corii Asini injection as claimed in claim 1 comprises vein infusion solution, lyophilized injectable powder, sterile powder injection, aqueous injection.
3, the preparation method of Colla Corii Asini injection as claimed in claim 1, its feature requires the Colla Corii Asini protolysate described in 1 to dissolve with water for injection in the weighting profit, add sodium chloride for injection again, stirring and dissolving, transferring pH value with 10% sodium hydroxide solution is 6.0～8.0,0.45 μ m filtering with microporous membrane, the 100ml infusion bottle of packing into filtrate branch, gland, sterilization, promptly.
4, the preparation method of Colla Corii Asini injection as claimed in claim 1, it is characterized in that the weighting profit requires the Colla Corii Asini protolysate described in 1 to dissolve with water for injection, add glucose for injection again, stirring and dissolving, transferring the ph value with the 0.1mol hydrochloric acid solution is 5.0～6.0,0.4.5 μ m filtering with microporous membrane, the 100ml infusion bottle of packing into filtrate branch, gland, sterilization, promptly.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100227435A CN1290574C (en) | 2004-06-07 | 2004-06-07 | Injection of donkey hide gelatin and its preparation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100227435A CN1290574C (en) | 2004-06-07 | 2004-06-07 | Injection of donkey hide gelatin and its preparation |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1583162A CN1583162A (en) | 2005-02-23 |
| CN1290574C true CN1290574C (en) | 2006-12-20 |
Family
ID=34600701
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2004100227435A Expired - Lifetime CN1290574C (en) | 2004-06-07 | 2004-06-07 | Injection of donkey hide gelatin and its preparation |
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| Country | Link |
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| CN (1) | CN1290574C (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100582214C (en) * | 2007-01-05 | 2010-01-20 | 浙江省医学科学院 | Donkey-hide gelatin polypeptide wine and production process thereof |
-
2004
- 2004-06-07 CN CNB2004100227435A patent/CN1290574C/en not_active Expired - Lifetime
Also Published As
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|---|---|
| CN1583162A (en) | 2005-02-23 |
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