CN112957374A - Dog serum deproteinized eye gel for dogs and preparation method thereof - Google Patents
Dog serum deproteinized eye gel for dogs and preparation method thereof Download PDFInfo
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- CN112957374A CN112957374A CN202110455633.1A CN202110455633A CN112957374A CN 112957374 A CN112957374 A CN 112957374A CN 202110455633 A CN202110455633 A CN 202110455633A CN 112957374 A CN112957374 A CN 112957374A
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- extract
- dog serum
- gel
- deproteinized
- dog
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- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 229940010747 sodium hyaluronate Drugs 0.000 description 1
- JAJWGJBVLPIOOH-IZYKLYLVSA-M sodium taurocholate Chemical compound [Na+].C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCS([O-])(=O)=O)C)[C@@]2(C)[C@@H](O)C1 JAJWGJBVLPIOOH-IZYKLYLVSA-M 0.000 description 1
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- 238000013112 stability test Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
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- 238000012353 t test Methods 0.000 description 1
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Abstract
The invention provides a dog serum deproteinized eye gel for dogs and a preparation method thereof, the extraction method of the dog serum deproteinized extract is simple and effective, can overcome the defect that a plurality of bovine-derived proteins, polysaccharides, glycopeptides and other components exist in bovine serum extract, and does not need to add any exogenous chemical reagent or other components, so that the dog serum deproteinized eye gel does not contain exogenous proteins or impurities, can effectively remove the influence of endotoxin on pet suffering from diseases, improves the acceptability of pet suffering from diseases, and enhances the treatment effect. The ophthalmic gel for dogs is specially used for the sick dogs, and the main component of the ophthalmic gel is a dog serum deproteinized extract, so that rejection or other anaphylactic reactions are not generated, and the treatment effect is improved.
Description
Technical Field
The invention belongs to the technical field of biological medicine, and particularly relates to dog serum deproteinized eye gel for dogs and a preparation method thereof.
Background
DECB ophthalmic gel was first developed and manufactured by Solco pharmaceutical, Switzerland under the trade name Sulcosyl, and was first introduced into the market in Europe. The eye ointment is a kind of gel eye drops, is transparent and suitable for xerophthalmia, conjunctival ulcer, corneal ulcer or injury. The eye drop gel consists of calf blood extract, gel matrix and preservative. The calf blood extract contains small molecular peptides, amino acids and inorganic salts. The eye medicine can promote healing of eye wound, and has moistening, lubricating and nourishing effects for patients with mild and moderate xerophthalmia.
The high-speed eye gel for treating diseases enters China from 80 years and is widely applied clinically. The ophthalmic diseases of pets are increased rapidly in recent years, the sales of clinical ophthalmic preparations always occupy several places before the administration of pets, the demand is very large, no pet manufacturer in China produces the eye ointments, and the eye ointments are influenced by European mad cow disease, so that the products are difficult to import and have high price. The main raw material for producing the common ophthalmic gel at present is deproteinized calf blood extract. The deproteinized calf blood extract is a micromolecular bioactive substance separated and extracted from calf blood, has the functions of repairing and proliferating tissue cells, and can be widely used for healing skin, corneal ulcer, bedsore, burn, skin transplantation and wound erosion caused by various reasons. However, as the bovine serum extract contains a plurality of bovine-derived proteins, polysaccharides, glycopeptides and other components, when the bovine serum extract is applied to specific pets, allergic reaction of the pets, shock or death can be caused seriously, and the application of the medicine in pet treatment is seriously influenced. Moreover, the cattle may carry various viruses such as mad cow disease, foot and mouth disease and the like, so that the potential safety hazard of clinical application is brought to the subsequent treatment of pets.
In addition, although many methods for preparing deproteinized extract from blood of cow or fetal calf exist in the prior art, the methods are not suitable for pet treatment, and the existing methods for preparing deproteinized extract from calf blood are basically chemical extraction methods, so that foreign proteins or impurities are introduced to a greater or lesser extent during the extraction process, which further aggravates the rejection and conflict in pet treatment. In addition, the subsequent purification steps mainly comprise centrifugation, ethanol protein removal, acidolysis enzymatic protein removal, ultrafiltration macromolecular substance removal and other processes. However, in the prior art, it is difficult to remove protein and retain active substances to the maximum extent, which causes unstable product quality, large difference between batches, influences the curative effect of the product and is difficult to form a standardized treatment standard.
In the Chinese patent CN200910246717.3, sodium citrate, pepsin for enzymolysis, subtilisin, ethanol, xylitol and epsilon-polylysine are required to be added in the preparation process of the injection solution of the deproteinized calf blood extract composition. Such a process is not only complicated, but also requires the addition of large amounts of chemicals, thereby destroying the purity of the deproteinised calf blood extract.
In the Chinese patent CN200410013643, amino acids and small peptides in the deproteinized calf blood extract solution can be polymerized into peptides with large molecular weight and high molecular peptides, so that the medicine generates antigenicity, the biological activity of the medicine is reduced, and the clinical anaphylactic reaction is increased. The product prepared by the method cannot be used in the field of pet treatment aiming at reducing anaphylactic reaction, and although the technical difficulty is small and the production is easy, impurities, particularly protein, are difficult to completely remove, so that the purity of the product is influenced, and the health and even the life of a patient are harmed.
Another deproteinized calf blood extracting technique is disclosed in Chinese patent CN200610080520, which comprises the steps of decompression by a vacuum rotary evaporator to remove ethanol, enzyme hydrolysis, centrifugation, gel chromatographic desalting by glucan G15 and DEAE-Sephadex-50 gel chromatographic separation, finally membrane filtration and ultraviolet sterilization. The conditions for removing protein by enzyme hydrolysis are severe, more active substances are destroyed, the biological activity of the product is reduced, and a better treatment effect is difficult to realize.
Therefore, there is a need in the art for a simple and convenient method for preparing a deproteinized pet animal serum extract from pet animal blood to replace expensive bovine serum containing foreign impurities, so as to obtain a pet ophthalmic gel suitable for the Chinese situation, which is widely used in the treatment of pets.
Disclosure of Invention
The invention aims to provide a method for extracting a dog serum deproteinized extract.
The method specifically comprises the following steps:
firstly, raw material treatment: the dog serum is frozen and thawed for 2 times at the temperature of between 20 ℃ below zero and 40 ℃, and then is primarily filtered by gauze.
Centrifugation: the initially filtered canine serum was centrifuged at 8000rpm for 20min and the supernatant was collected.
③ pre-filtering: filtering the centrifuged supernatant with 500KD ultrafiltration column under pressure to obtain clear filtrate.
Affinity filtering: endotoxin was removed by purification using a ConA affinity column.
Ultra-filtering and subpackaging: and (3) ultrafiltering the filtrate by using a hollow fiber column with the molecular weight cutoff of 10KD to collect active polypeptide refined solution, and sampling to determine the content and the respiratory activity.
Sixthly, after the test is qualified, the dog serum deproteinized extract for injection is obtained by preparing the dog serum deproteinized extract with water for injection to the required concentration, sterilizing, filtering and subpackaging.
Further, the present invention provides a gel for canine eyes, the gel comprising by weight: 15-30% of dog serum deproteinized extract, 0.5-1% of sodium carboxymethylcellulose, 0.1-7% of hyaluronic acid, 0.1-8% of glycerol, 0.1-3% of methyl hydroxybenzoate, 0.1-3% of ethylparaben, 0.1-1% of gellan gum, 0.1-1% of xanthan gum, water for injection and the pH value of the water for injection is adjusted to 6.4-7.4.
Further, the invention provides a preparation method of the eye gel for dogs, which comprises the following specific preparation steps:
1. adding glycerol and sodium carboxymethylcellulose into a 1L container, and stirring and dispersing uniformly;
2. heating water for injection to dissolve methyl hydroxybenzoate, ethyl hydroxybenzoate, hyaluronic acid, gellan gum and xanthan gum;
3. mixing and stirring the step II and the step I, filtering insoluble substances after the sodium carboxymethylcellulose is completely dissolved, placing the insoluble substances in a sealed container, sterilizing by circulating steam at 100 ℃ for 30min, and then placing the mixture to room temperature for later use;
4. and (3) taking the deproteinized dog serum extract, sterilizing and filtering by using a 0.22-micron microporous filter, mixing with the solution obtained in the step (iii) under an aseptic condition, homogenizing and stirring uniformly, and carrying out aseptic subpackaging to obtain the dog serum deproteinized extract.
The gel obtained by the invention has low organic solvent residue (the ethanol residue is less than or equal to 30ppm or lower through detection), and the gel strength is more than or equal to 1000g/cm2. The ion sensitive in-situ gel is prepared, and the preparation is administrated in a solution form and has phase change under the physiological condition of eyes. In aqueous solution, the carbonyl group in its molecule can react with cation (Na)+、K+、Ca2+、Mg2+Etc.) are combined, the random coil structure is changed into a double-spiral structure, then the double-spiral structure is reversely gathered, so that a three-dimensional reticular gel structure is formed, and the solution is changed into semisolid gel, so that the retention time of the eye part of the medicine is prolonged, the administration frequency is reduced, and the treatment effect is improved; meanwhile, the dosage absorbed into a circulatory system is reduced, and the adverse drug reaction is reduced.
In the gel, the surface activity of the sodium carboxymethyl cellulose is high, the prepared gel forms a layer of protective film after being dripped into eyes, and the surface viscosity can be increased by combining the gel with hyaluronic acid, so that the deproteinized extract of the contained restoration component canine serum can be better attached to eyeballs, and wounds and ulcer surfaces can be restored. And the hydroxypropyl methyl cellulose is dissolved in cold water to form a solution with a certain viscosity, the property of the solution is close to that of a viscoelastic substance (mucin) of the tear, and the solution can be used as an artificial tear. The hydroxypropyl methyl cellulose is used in eye drops, can reduce the surface tension of the eye drops, increase the permeability to the cornea, increase the absorption of the medicine and improve the bioavailability.
The glycerol has effects of lubricating and moistening, relieving pain due to friction of eyelid, and relieving dryness due to ocular inflammation, thereby preventing secondary injury of eye.
Sodium hyaluronate can promote the regeneration of injured skin and help repair corneal problems by promoting the proliferation and differentiation of epidermal cells and scavenging oxygen free radicals.
Methyl paraben and ethyl paraben are used as bacteriostatic agents, wherein the methyl paraben is effective on various moulds, yeasts and bacteria; the ethylparaben has strong fungal inhibition effect, and can be used for treating fungal corneal ulcer after being used in a small amount; the two are mixed for use, and have good synergy and addition.
The ophthalmic gel can be durably stable within 6 months after being accelerated, no new impurities are generated, and the ophthalmic gel is safe, effective and controllable in quality.
Further, the invention provides application of the gel in pet treatment, wherein the application is corneal and conjunctival wounds, inflammations and ulcers caused by various reasons of dog eyes.
Advantageous effects
The extraction method of the dog serum deproteinized extract provided by the invention is simple and effective, can overcome the defect that bovine serum extract contains a plurality of bovine-derived proteins, polysaccharides, glycopeptides and other components, does not need to add any exogenous chemical reagent or other components, does not contain exogenous proteins or impurities, can effectively remove the influence of endotoxin on patients and pets, improves the receptivity of the patients and enhances the treatment effect.
The ophthalmic gel for dogs is specially used for the sick dogs, and the main component of the ophthalmic gel is a dog serum deproteinized extract, so that rejection or other anaphylactic reactions are not generated, and the treatment effect is improved. The curative effect of the ophthalmic gel is similar to that of imported products, the ophthalmic gel can replace import, can relieve suffering from pet pain, relieve pet owner's economic pressure, and simultaneously improve development ability and company image, and has profound influence.
Drawings
FIG. 1 clinical pictures of dogs with dog serum before deproteinized gel
FIG. 2 clinical pictures of dogs with dog serum deproteinized gel
Detailed Description
Example 1 preparation of deproteinized extract of canine serum
The method specifically comprises the following steps:
firstly, raw material treatment: collecting venous blood of clean-grade Beagle dogs of 3-5 months ages under aseptic condition, stirring with a stirrer at a stirring speed of 15-100 r/min for 1-5 min to eliminate the influence of fibrinogen on blood coagulation and subsequent operation as much as possible, and putting the Beagle dogs in a refrigerator at 4 ℃ overnight to naturally separate serum I; the remaining part was centrifuged at 1000rpm at 4 ℃ for 10-30 minutes under sterile conditions to isolate serum II. Mixing the serum I and the serum II, repeatedly freezing and thawing at-20-40 ℃ for 2 times, roughly filtering by using gauze, and collecting filtrate;
centrifugation: centrifuging the preliminarily filtered dog serum at 8000rpm for 20min, collecting supernatant, and preliminarily removing impurities such as residual lipid, immunoglobulin, endotoxin, hemoglobin, etc. in the serum.
③ pre-filtering: filtering the centrifuged supernatant with 500KD ultrafiltration column under pressure to obtain clear filtrate. The membrane was dialyzed thoroughly against a CLW-003 hollow fiber ultrafiltration membrane module, manufactured by Beijing Asahi Bangkong Membrane Equipment Limited company, and against an ultrafiltration column with a molecular weight cut-off of 500kd, manufactured by Spectrum, USA. The serum has high viscosity, and the filtering area of the ultrafiltration column should not be less than 50m2The pressure is less than or equal to 0.1 mpa.
Affinity filtering: endotoxin was removed by purification using a ConA affinity column. ConA filler ConA Sepharose 4B (GE Healthcare) was used. And (3) an equilibrium buffer: 20mM Tris-HCl, 0.5M NaCl, pH 7.4. Elution buffer: boric acid-borax buffer, 0.1M, pH 6.5. The specific method comprises the following steps: and (4) loading the sample obtained in the third step on a balanced ConA packed column, collecting flow-through liquid at the sample flow rate of 1 ml/min. The buffer was washed and eluted as above, and the ConA filler bound the polysaccharide and endotoxin, which were eluted and discarded. Cleaning and rehabilitation (CIP) of the ConA filler was carried out according to the manufacturer's instructions.
Ultra-filtering and subpackaging: and (3) ultrafiltering the filtrate with hollow fiber column of 005W-type hollow fiber ultrafiltration membrane component with cut-off molecular weight of 10KD, and collecting active polypeptide refined solution, and sampling to determine content and respiratory activity.
Sixthly, after the test is qualified, the dog serum deproteinized extract for injection is obtained by preparing the dog serum deproteinized extract with water for injection to the required concentration, sterilizing, filtering and subpackaging.
Example 2 quality testing of dog serum deproteinized extracts
(1) Comparison of the Components of the dog serum deproteinized extract with the bovine serum deproteinized extract
Since the prior art does not provide for the preparation of a dog serum deproteinized extract, it is desirable to compare the active ingredients in a dog serum deproteinized extract with those in a bovine serum deproteinized extract as is common in the art. Animal serum biochemical analysis is detected by a full-automatic blood biochemical analyzer (AU27200 type). Bovine serum FBS was purchased from hangzhou ilex bioengineering materials ltd, lot No.: 060912. the assay index includes urea nitrogen (BUN), Creatinine (CRE), Uric Acid (UA), Glucose (GLU), Total Protein (TP), Albumin (ALB), alanine Aminotransferase (ALT), alkaline phosphatase (ALP), glutamate transpeptidase (GGT), Cholinesterase (CHE), creatine phosphokinase (CK), creatine kinase isozyme (CK-MB), Lactate Dehydrogenase (LDH), hydroxybutyrate dehydrogenase (HBDH), Total Bilirubin (TBIL), Direct Bilirubin (DBIL), Cholesterol (CHO), Triglyceride (TG), potassium (K)+) Sodium, sodium (Na)+) Chloride (Cl)-) Calcium (Ca)2+) Phosphorus (Pi)3-)。
Statistical analysis was performed on the experimental results using SPSS10.0 statistical software. Data are presented as means ± standard deviation, and analysis of data between groups was performed using t-test. P<A difference of 0.05 is statistically significant. GGT, CK-MB, LDH and HBDH levels in FBS are obviously higher than those of Beagle-S (P)<0.05); the CHE level is obviously lower than that of the canine serum, and the comparison between the CHE level and the canine serum has obvious statistical significance. BUN, CRE, GLU, TP, ALB, ALT, ALP, TBIL, DBIL, TG, K in serum+、Na+、Cl-、Ca2+、Pi3-Levels were not significantly different between dog serum and fetal bovine serum.
According to the result analysis, due to the difference of the components in the fetal bovine serum and the canine serum, the preparation method of the canine serum extract and the preparation method of the bovine serum extract cannot be simply replaced with each other, and a specific preparation method corresponding to the preparation method is needed, particularly, the specific parameters such as specific pore diameter and pressure in the filtering and ultrafiltration steps are involved, so that the component change of the prepared canine serum extract is influenced.
(2) Detection of endotoxin content
Dissolving limulus reagent with 100 μ L of endotoxin-free distilled water, adding 100 μ L of diluted 40 times of raw serum sample, mixing with limulus reagent, and incubating at 37 deg.C for 1 hr to determine endotoxin content range in specimen by observing agglutination condition.
Through three parallel tests, the serum extract samples added with the step (IV) endotoxin removal operation are not agglutinated, which shows that the bacterial endotoxin in the serum is lower than 0.1EU/mL (the detection limit of the kit is 0.1EU/mL), the requirement on the endotoxin content in the 2010 edition of Chinese pharmacopoeia is met and is less than or equal to 10EU/mL, and the concentration of the endotoxin in the samples which are not treated by the step (IV) is 55-69EU/mL (see Table 1).
TABLE 1 detection of endotoxin content
| Endotoxin (EU/mL) | Adding step IV | Omitting the step (iv) | Negative control |
| Repetition of 1 | - | 55 | - |
| Repetition 2 | - | 58 | - |
| Repetition of 3 | - | 69 | - |
EXAMPLE 3 preparation of ophthalmic gel for dogs
Firstly, raw materials are prepared according to specific requirements, and the method comprises the following steps: 20% of dog serum deproteinized extract, 0.5% of sodium carboxymethylcellulose, 5% of hyaluronic acid, 5% of glycerol, 1% of methyl hydroxybenzoate for injection, 1% of ethylparaben, 0.5% of gellan gum and 0.5% of xanthan gum.
Adding glycerol and sodium carboxymethylcellulose into a 1L container, and stirring and dispersing uniformly;
heating water for injection to 40-60 deg.C, and dissolving methyl hydroxybenzoate, ethylparaben, hyaluronic acid, gellan gum, and xanthan gum;
mixing the two liquids, stirring to dissolve the two solutions, filtering insoluble substances, placing in a sealed container, sterilizing with flowing steam at 100 deg.C for 30min, and cooling to room temperature;
and then taking the dog serum deproteinized extract, sterilizing and filtering by using a 0.22um microporous filter, mixing with the solution under the aseptic condition, homogenizing and stirring uniformly, and carrying out aseptic subpackaging to obtain the dog serum deproteinized extract.
Example 4 detection of bacteriostatic effect of ophthalmic gel for dogs:
selecting strains: escherichia coli, Staphylococcus aureus, Candida albicans, Pseudomonas aeruginosa
Respectively inoculating Escherichia coli and Staphylococcus aureus into broth culture solution, and culturing at 37 deg.C for 18 h; inoculating Candida albicans and Pseudomonas aeruginosa to Sabouraud's glucose liquid culture medium, and adjusting Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa to 106CFU/ml, adjusted to 10 with Candida albicans5CFU/ml。
0.1ml of each prepared bacterial suspension is respectively added into 4 parts of gel without bacteriostatic agent, each part is 10g, the mixture is uniformly mixed, plates are coated for 6h, 24h, 72h and 7d, and the counting is carried out. The results are shown in Table 2.
TABLE 2 growth of microorganisms in gels without bacteriostat (10)3CFU)
| Time | Escherichia coli | Staphylococcus aureus | Candida albicans | Pseudomonas aeruginosa |
| 6h | 723 | 243 | 76 | 625 |
| 24h | 8825 | 824 | 227 | 6627 |
| 72h | 11023 | 2634 | 624 | 12053 |
| 7d | 56231 | 4503 | 2008 | 30245 |
0.1ml of each prepared bacterial suspension is respectively added into 4 parts of sterile gel, 10g of each part of sterile gel is only added with methyl hydroxybenzoate, the mixture is uniformly mixed, plates are coated for 6h, 24h, 72h and 7d, and counting is carried out. The results are shown in Table 3.
TABLE 3 growth of microorganisms on gels containing methylparaben (10)3CFU)
| Time | Escherichia coli | Staphylococcus aureus | Candida albicans | Pseudomonas aeruginosa |
| 6h | 235 | 45 | 24 | 312 |
| 24h | 7 | 2 | 14 | 217 |
| 72h | 0 | 0 | 10 | 183 |
| 7d | 0 | 0 | 12 | 100 |
0.1ml of each prepared bacterial suspension is respectively added into 4 parts of sterile gel, 10g of sterile gel is added with only ethylparaben, the mixture is uniformly mixed, plates are coated for 6 hours, 24 hours, 72 hours and 7 days, and counting is carried out. The results are shown in Table 4.
TABLE 4 growth of microorganisms in gels containing methylparaben (10)3CFU)
| Time | Escherichia coli | Staphylococcus aureus | Candida albicans | Pseudomonas aeruginosa |
| 6h | 302 | 57 | 13 | 305 |
| 24h | 0 | 0 | 0 | 272 |
| 72h | 0 | 0 | 0 | 154 |
| 7d | 0 | 0 | 0 | 64 |
0.1ml of each prepared bacterial suspension is respectively added into 4 parts of sterile gel, 10g of each part of sterile gel is added with methyl hydroxybenzoate and ethyl hydroxybenzoate, the mixture is uniformly mixed, plates are coated for 6h, 24h, 72h and 7d, and counting is carried out. The results are shown in Table 5.
TABLE 5 growth of microorganisms on gels containing ethylparaben (10)3CFU)
| Time | Escherichia coli | Staphylococcus aureus | Candida albicans | Pseudomonas aeruginosa |
| 6h | 127 | 46 | 19 | 152 |
| 24h | 0 | 0 | 3 | 76 |
| 72h | 0 | 0 | 0 | 18 |
| 7d | 0 | 0 | 0 | 0 |
From the above data, it can be seen that methyl paraben and ethyl paraben are used as bacteriostatic agents, wherein methyl paraben is effective on various molds, yeasts and bacteria; the ethylparaben has strong fungal inhibition effect, and can be used for treating fungal corneal ulcer after being used in a small amount; the two are mixed for use, have good synergy and addition, have the effect that 1+1 is far more than 2, and are not enough in bacteriostatic effect.
Example 5 quality testing of ophthalmic gel for dogs
(1) And (3) biological activity determination:
an effective method for determining the protein-depleted extract is to calculate its respiratory activity (QO)2) And irritation index (SI), which is obtained by calculating oxygen consumption of two products by using SKW-3 micro-respiration pressure detector, calculating respiratory activity and irritation index, and comparing biological activity and quality of the two products, and the results are shown in Table 6 below.
5.1 instruments
SKW-3 microscopical breath pressure tester (Shanghai university); an electronic balance; a sample injector; a stopwatch; surgical instrument 5.2 reagent
Sodium hydroxide solution (10%), Soerensen buffer (pH 7.40.0667mol. L-1), weighing disodium hydrogen phosphate (Na)2HPO4.2H2O)9.72g and monopotassium phosphate (KH)2PO4)1.65g, dissolved in 1000mL of deionized water. Reference is made to the standard (austria) and the test article (austria). Brodie pressure measuring liquid is prepared by dissolving 23g of sodium chloride and 5g of sodium taurocholate in 100mg of Yiwenlan in water to obtain 500m L.
5.3 liver homogenate preparation
One male guinea pig with the weight of 250-: 9(w/v) phosphate buffer was added and triturated in an ice bath to make a liver homogenate.
5.4 test preparation
Measuring the respiratory activity of the homogenate of the liver of the guinea pig with a Wasser microaspiration pressure detector, measuring the oxygen consumption, and calculating the respiratory activity QO2(μLO2Mg h) and stimulation index SI. The specific operation steps are as follows:
firstly, filling pressure measuring liquid into a rubber pipe, then inserting the pressure measuring pipe into the rubber pipe to ensure that no fine bubbles exist, finally fixing the pressure measuring pipe on a frame, and adjusting the liquid level of the pressure measuring pipe to 150 mm.
② adding 0.2mL of 10 percent potassium hydroxide solution into a reaction bottle small cup (in order to increase the absorption area of sodium hydroxide to carbon dioxide, a small piece of filter paper is inserted into the potassium hydroxide solution).
③ 1.1mL of phosphate buffer solution and 1.0mL of liver homogenate are added into the reaction bottle, and then 0.2mL of the sample is added into the reaction bottle.
Fourthly, 1.1mL of phosphate buffer solution and 1.0mL of liver homogenate are firstly added into a blank bottle, and then 0.2mL of phosphate buffer solution is added into a reaction bottle. (since liver homogenates themselves have an oxygen-consuming effect).
Fifthly, adding 1.1mL of phosphate buffer solution and 1.0mL of phosphate buffer solution into the correction bottle, and then adding 0.2mL of phosphate buffer solution into the reaction bottle. The test tube was stabilized to observe the change in pressure (Δ C) under exactly the same conditions as the test tube to eliminate errors due to the effects of temperature and atmospheric pressure.
Sixthly, the reaction bottles are correspondingly connected with the pressure detecting tubes one by one according to the marks (the reaction bottles are sealed without air leakage) and are placed in an oscillator of a constant temperature water bath at 37 ℃.
And (c) starting an oscillator to shake for 10min (120 times/minute) to ensure that the internal temperature and the external temperature of the reaction bottle are consistent, ensuring that a three-way piston of the pressure detecting pipe is in an open state during shaking, adjusting the right side liquid level of the pressure detecting pipe to 150mm after 10min, recording a left side liquid level reading (A), closing the three-way piston, adjusting the right side liquid level of the pressure measuring meter to 150mm after timing reaction for 30min, and recording a left side liquid level reading (B). Repeat twice, note readings (C) and (D).
Accurately measuring 1.0mL liver homogenate, placing the liver homogenate in a constant-weight weighing bottle filled with sea sand, drying the liver homogenate at 105 ℃ to constant weight, and calculating the weight difference delta W (mg) of the two times
5.5 calculation method:
QO2(μLO2(A-B + C-D) x K1- Δ C x K2/(G.T) wherein K1 is the reaction flask constant of the test sample or the control; k2 is the constant of the reaction bottle of the correction tube group; delta C is the sum of the pressure changes of the correction pipe group; g is the dry weight per ml of liver homogenate; g ═ Δ W-9.3 (mg); g is the dry weight per ml of liver homogenate; g ═ Δ W-9.3 (mg); 9.3 Dry weight (mg) of phosphate buffer in 1mL liver homogenate; t is reaction time (h); stimulation Index (SI) ═ quality of test article QO2Blank QO2。
TABLE 6 comparison of the bioactivity and quality of the product with the Gaojiejie eye gel
(2) Stability detection
Taking the prepared dog eye gel, and placing for 6 months under the conditions that the temperature is 35 +/-2 ℃ and the humidity is 75% +/-5%. Samples were taken at 0 month, 1 month, 2 months, 3 months and 6 months for measurement. The results are shown in Table 7. The results demonstrate that ophthalmic gels prepared according to the present application are stable for 6 months with an accelerated rate.
TABLE 7 accelerated stability test of ophthalmic gels
(3) Clinical efficacy testing
The experiment selects 15 healthy dogs with the age of 2-3 months and the weight of 0.8-1.2 kg. The group was divided into 3 groups, control group, Sugaojie treatment group, and dog blood gel group for eye use. On the day before the experiment, 0.5% procaine was locally anesthetized for both eyes, corneal epithelium was uniformly scraped with a scalpel, and neomycin sulfate eye drops were added dropwise. And (3) dropwise adding 2% fluorescein sodium for coloring, observing the damaged area, and starting administration after the damage reaches 100%. The medicine is taken three times a day for 5 days continuously, and the neomycin sulfate eye drops are dripped into two eyes to prevent bacterial infection. The cornea was observed for healing by daily staining with 2% fluorescein sodium.
The results are shown in Table 8, with scores based on the following criteria for the extent of corneal damage:
no damage: 0
Damage is within 25%: 1
Between 26-50% damage: 2
Lesions ranged between 51-75%: 3
Damage is greater than 75%: 4
TABLE 8 clinical effectiveness test grading table
The result shows that the damage area of the cornea of the control group is more than 75 percent on the first day of administration, and the damage area of the vegetarian high-speed treatment group and the gel group for dog blood eyes is more than 51 to 75 percent; the second day of administration, the injured area of cornea of control group is 51-75%, and the injured area of vegetarian agility group and gel group for dog blood eye is 26-50% more; on the third day of administration, the injured area of cornea of the control group is 51-75%, the injured area of the prime high agility treatment group and the gel group for dog blood eyes is half 26-50%, and half is within 25%; on the fourth day of administration, the injured area of cornea of control group is 26-50%, the injured area of the prime high-speed treatment group is mostly within 25%, the injured area of the prime high-speed treatment group is healed individually, and the injured area of the gel group for dog blood eye is mostly healed within 25%; on the fifth day of administration, the corneal injury area of the control group is mostly between 26 and 50 percent, the minority is recovered to be within 25 percent, the corneal injury of the prime mover treatment group is mostly healed, the minority is within 25 percent, and the corneal injury of the canine blood ophthalmic gel group is basically healed.
While the invention has been described in detail with reference to the preferred embodiments thereof, it will be understood by those skilled in the art that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Those of ordinary skill in the art will understand that: the discussion of any embodiment above is meant to be exemplary only, and is not intended to intimate that the scope of the disclosure, including the claims, is limited to these examples; within the idea of the invention, also features in the above embodiments or in different embodiments may be combined, steps may be implemented in any order, and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity.
Claims (6)
1. A method for extracting a dog serum deproteinized extract is characterized by comprising the following steps:
firstly, raw material treatment: the dog serum is frozen and thawed for 2 times at the temperature of between 20 ℃ below zero and 40 ℃, and then is primarily filtered by gauze.
Centrifugation: the initially filtered canine serum was centrifuged at 8000rpm for 20min and the supernatant was collected.
③ pre-filtering: filtering the centrifuged supernatant with 500KD ultrafiltration column under pressure to obtain clear filtrate.
Affinity filtering: endotoxin was removed by purification using a ConA affinity column.
Ultra-filtering and subpackaging: and (3) ultrafiltering the filtrate by using a hollow fiber column with the molecular weight cutoff of 10KD to collect active polypeptide refined solution, and sampling to determine the content and the respiratory activity.
Sixthly, after the test is qualified, the dog serum deproteinized extract for injection is obtained by preparing the dog serum deproteinized extract with water for injection to the required concentration, sterilizing, filtering and subpackaging.
2. The dog serum deproteinized extract prepared by the method for extracting dog serum deproteinized extract according to claim 1.
3. An ophthalmic gel for dogs comprising an effective amount of the dog serum deproteinized extract of claim 2.
4. A gel for the eyes of a dog as claimed in claim 3 wherein said gel comprises by weight:
15-30% of dog serum deproteinized extract, 0.5-1% of sodium carboxymethylcellulose, 0.1-7% of hyaluronic acid, 0.1-8% of glycerol, 0.1-3% of methyl hydroxybenzoate, 0.1-3% of ethylparaben, 0.1-1% of gellan gum, 0.1-1% of xanthan gum, water for injection and the pH value of the water for injection is adjusted to 6.4-7.4.
5. A method for preparing the eye gel for dogs as claimed in claim 3 or 4, which comprises the following steps:
firstly, taking a 1L container, adding glycerol and sodium carboxymethylcellulose, and uniformly stirring and dispersing;
heating water for injection to dissolve methyl hydroxybenzoate, ethyl hydroxybenzoate, hyaluronic acid, gellan gum and xanthan gum;
mixing and stirring the step II and the step I, filtering insoluble substances after the sodium carboxymethyl cellulose is completely dissolved, placing the insoluble substances in a sealed container, sterilizing the insoluble substances by circulating steam at 100 ℃ for 30min, and then placing the insoluble substances to room temperature for later use;
and fourthly, taking the deproteinized dog serum extract, sterilizing and filtering the deproteinized dog serum extract by using a 0.22um microporous filter, mixing the deproteinized dog serum extract with the solution obtained in the third step under an aseptic condition, homogenizing and stirring the mixture uniformly, and performing aseptic subpackaging on the mixture to obtain the dog serum albumin.
6. The use of an eye gel for dogs according to claim 3 or 4, wherein said use is the use of said gel in pet animal treatment, and further wherein said use is corneal and conjunctival trauma, inflammation and ulceration due to various causes of canine eye.
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