CN101697977B - Deproteinized calf blood extractive injection and preparation method thereof - Google Patents
Deproteinized calf blood extractive injection and preparation method thereof Download PDFInfo
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Landscapes
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention relates to a deproteinized calf blood extractive injection, every milliliter of which contains 160-200 mg of total solid, 100-150 mg of xylitol and 60-100 mg of epsilon-polylysine, wherein the total solid comprises the following components in parts by weight: 10-20 parts of amino acid, 25-35 parts of polypeptide and 15-25 parts of inorganic salt. The preparation method comprises thefollowing steps: taking calf blood and sodium citrate, adding water and stirring to rise the temperature, quickly cooling to -10-0 DEG C while stirring, centrifugating, regulating the pH value of thesupernatant, adding pepsin for enzymolysis, filtering, regulating the pH value, adding subtilisin for enzymolysis, and filtering; adding ethanol water solution to the filtrate, stirring, centrifugating, letting the supernatant flow through active carbon columns, combining the eluent and the cross-flow solution, concentrating until no ethanol smell is emitted, adding the xylitol and the epsilon-polylysine to the concentrated solution, adding water to dilute, and ultrafiltering; and packaging and filling the ultrafiltrate, sterilizing with ultraviolet light, and packing to obtain the injection.
Description
Technical field
The present invention relates to field of medicaments, specifically, relate to a kind of deproteinized calf blood extractive injection and preparation method thereof.
Background technology
Calf blood protein-removed extraction injection be fresh calf blood or serum through Deproteinization, concentrate, the aseptic aqueous solution that contains organic/inorganic substance and small organic molecule that technologies such as ultrafiltration or dialysis make, be flaxen clear liquid.This injection can strengthen histiocyte to oxygen and glucose uptake and utilization, improves the cell hypoxia state.Be applicable to the not congruent brain cell dysbolismus of cerebral ischemia, brain dementia, cerebral trauma and brain function treatment of diseases.
Disclosed at present preparation method has, such as patent 96120777 discloses and adopt young cattle venous blood, isolates serum, uses the ethanol Deproteinization, ethanol is removed in decompression, adds the compound protein enzyme hydrolysis, and supernatant is stayed in centrifugalize, ethanol in the supernatant is concentrated degerming, lyophilizing.In the resulting product, the inorganic ions ratio is 60%, and nucleotide, aminoacid, small-molecular peptides, keto acid equal proportion content are 40%.
Patent application 200410013509.6 discloses calf blood or calf serum, acid adjustment pH value to 3.0～5.5, transfer alkaline pH value to 8.0～10.5 again, after transferring neutrality, be heated to 30～80 ℃, go precipitation, use the ultrafilter membrane ultrafiltration then, molecular cut off is 5000～10000 dalton, collects filtrate and is calf blood or calf serum protein-removing extract solution.Prepared calf blood protein-removed extraction solid content contains that the total nitrogen weight portion is 0.10～0.90, free amino acid 0.01～0.40, and pH value should be 3.0～9.0, and molecular weight is 5000～10000, and ninhydrin reaction is positive.
Patent application 200410062657.7 discloses the collection bovine blood, add water after centrifugal, freeze molten, and then it is centrifugal, the supernatant pasteurization, with the daltonian ultrafilter membrane ultrafiltration in molecular cut off 5～100,000, reuse molecular cut off 0.5～1.5 ten thousand dalton's ultrafilter membrane ultrafiltration, filtrate is used and the monovalent cation rejection is reached 80～90% aromatic polyamide membrane desalination, re-use and the monovalent cation rejection reached 94～99% aromatic polyamide membrane reverse osmosis concentration, at last with after the decolouring of medicinal active carbon promptly.The calf blood protein-removed extraction breathing vigor of gained is 6.30 μ lO
2/ mgh, stimulation index is 5.40.
Patent application 200410013643.6 discloses gets the calf blood adjust pH to alkalescence, centrifugal, supernatant adjust pH to 7.5～8, centrifugal, supernatant adjust pH to 6.5～7.5, in 70～90 ℃ of heat treatments, centrifugal, 60～80 ℃ of concentrating under reduced pressure, the concentrated solution ultrafiltration, molecular cut off is 5000～10000 dalton, gets calf blood protein-removed extraction solution, gets osmotic pressure regulator, add the activated carbon heating after being dissolved in water, filter osmotic pressure regulator solution, more above-mentioned calf blood protein-removed extraction solution and osmotic pressure regulator solution are mixed, add the injection water and stir evenly promptly.Contain parts by weight aminoacid 20.0～30.0 in the prepared calf blood protein-removed extraction transfusion, total nitrogen 12.5～18.75, osmotic pressure regulator 50～100, pH value should be 5.5～8.0.
Patent application 200510076914.7 discloses stirs calf blood or serum, add sodium citrate, stir evenly the back and preserve 1～30h for 0～4 ℃, skim the top layer oil film, collect blood plasma, blood plasma is crossed pearl chitosan chromatographic column, collects the blood plasma behind the post, add ethanol, stir centrifugally, 75～85 ℃ of supernatant reclaim ethanol down, add protease hydrolyzed 10～38h, add 0.4～0.6 times of distilled water to enzymolysis solution, be heated to 75～85 ℃, cross chitin modified carbon column and filter, filtrate concentrates the back after carbon column, cross microporous filter membrane, fill is sterilized promptly.The calf blood protein-removed extraction injection of gained contains solid content 41～60mg for every milliliter, and wherein 55～65% weight are inorganic matter in the solid content, and 35～45% weight are organic active substance.
Patent application 200510084246.2 discloses centrifugalize under calf blood or the serum low temperature, getting supernatant adds sodium citrate and stirs evenly the back in 0～4 ℃ of stored refrigerated 1～30 hour, skim oil film, serum is crossed pearl chitosan chromatographic column, collected post serum, add ethanol, stir, leave standstill behind 20min～2h centrifugally, supernatant concentration is removed ethanol, add protease hydrolyzed 10～38h, enzymolysis solution adds distilled water, and the chitin modified carbon column good with activation filters, and filtrate concentrates the back filtering with microporous membrane, filtrate micropore ultrafilter molecular cut off is less than the component below 6000 dalton, fill sterilization back lyophilizing.Every milliliter of total solids content 0.035～0.055g of prepared injection, free aminoacid content 0.6～1.5mg, content of peptides 0.4～3.0mg, injection pH value are 6.0～8.0.
Patent application 200610018709.X is warming up to 50～95 ℃ after disclosing and having got aseptic fresh young cattle whole blood and purified water mixing, stir insulation 5～1000min, be cooled to room temperature, add 95% ethanol, stir 0.5～10h, clarification 1～24h gets extracting solution, extracting solution concentrates with film type evaporator, concentrated solution is poured Fei Erte clarifying filter clarification filtration while hot into, filtrate adds activated carbon and is heated to 60～95 ℃ of insulation 30min, use the 0.8um filtering with microporous membrane, filtrate concentrates with film type evaporator again and obtains calf blood protein-removed extraction with the filter membrane ultrafiltration of 5000 dalton Fuji.
Patent application 200610080520.3 discloses young cattle venous blood, isolate serum, use the ethanol Deproteinization, decompression removes ethanol, add the compound protein enzyme hydrolysis, centrifugal after the hydrolysis, the supernatant ultrafiltration, ultrafiltrate is earlier with the desalination of glucosan G-15 gel chromatography, there is the eluting part of special absorption at collection 280nm place, separates with the DEAE-Sephadex-50 gel chromatography again, collects the eluting part that there is special absorption at second 280nm place, reuse micro-pore-film filtration and ultraviolet radiation carry out inactivation of virus, obtain calf serum protein-removing extract.
The calf blood protein-removed extraction of method for preparing all exists active component content low, the defective that the holding time is short, in view of this, special proposition the present invention.
Summary of the invention
The present invention's first goal of the invention is to provide a kind of deproteinized calf blood extractive injection, and deproteinized calf blood extractive injection stability is higher in this injection provided by the present invention.
The present invention's second goal of the invention is to provide a kind of method that is used to prepare above-mentioned deproteinized calf blood extractive injection, and described method can further improve the content of active component in the calf blood protein-removed extraction.
In order to realize the foregoing invention purpose, the present invention takes following technical scheme:
A kind of deproteinized calf blood extractive injection, described injection contains following material for every milliliter: total solid 160～200mg, wherein wt umber are 0～20 part of amino acid/11,25～35 parts of polypeptide, 15～25 parts of inorganic salts; Xylitol 100～150mg; Epsilon-polylysine 60～100mg.
According to foregoing deproteinized calf blood extractive injection, described injection contains following material for every milliliter: total solid 180mg, wherein wt umber are 5 parts of amino acid/11s, 30 parts of polypeptide, 20 parts of inorganic salts; Xylitol 120mg; Epsilon-polylysine 80mg.
A kind of preparation method of deproteinized calf blood extractive injection noted earlier, described calf blood protein-removed extraction prepares according to following steps:
(1) get calf blood and add sodium citrate, add 3～5 times in water, be warming up to 50～60 ℃ under stirring, insulated and stirred 40～60min stirs down and is cooled to-10～0 ℃ rapidly, and temperature fall time is 2～5min, and is centrifugal at-10～0 ℃ of insulated and stirred 2～4h, gets supernatant;
(2) will going up the supernatant that makes of step, to regulate pH value be 3～4, adds the pepsin enzymolysis of supernatant weight 4%～5%, filters, and regulating pH value is 7.0～8.5, adds the subtilisin enzymolysis of supernatant weight 1%～1.5%, filters to get filtrate;
(3) will go up the filtrate adding ethanol water stirring that the step obtains, centrifugal, get supernatant;
(4) supernatant is crossed carbon column, merge eluent and percolation liquid, be concentrated into no ethanol flavor then, concentrated solution adds xylitol, and thin up contains xylitol 100～150mg, epsilon-polylysine 60～100mg, preferred xylitol 120mg to every ml soln, epsilon-polylysine 80mg, the solution microporous filter membrane ultrafiltration after the dilution gets ultrafiltrate;
(5) ultraviolet sterilization after the ultrafiltrate packing fill, packing promptly.
According to foregoing preparation method, the middle sodium citrate consumption of step (1) is 0.1～0.2 times of calf blood weight, preferred 0.15 times; Described centrifugal be rotating speed 2～3krpm, time 30～40min.
According to foregoing preparation method, it is at 55～60 ℃ of following enzymolysis 10～20min that step (2) adds the pepsin enzymolysis, is preferably at 58 ℃ of following enzymolysis 15min; Adding the subtilisin enzymolysis is at 30～40 ℃ of following enzymolysis 15～20min, is preferably at 35 ℃ of following enzymolysis 17min.
According to foregoing preparation method, the described ethanol water of step (3) is 80% ethanol water of 3～5 times of filtrate weight, and described stirring is 40～50 ℃ and stirred 10～20 minutes down.
According to foregoing preparation method, the described carbon column of step (4) is for getting 1～2 times of described supernatant weight, preferred 1.5 times medicinal active carbon, and the post draw ratio is 4: 1～5: 1, preferred 4.5: 1.
According to foregoing preparation method, the described carbon column of crossing of step (4) is poured in the carbon column for the supernatant that step (3) is obtained, equal to the supernatant liquid level with the activated carbon upper surface, standing adsorption 10～20min, preferred 15min, emit percolation liquid, put into the residue supernatant again, according to the above-mentioned steps repeatable operation to putting into whole supernatant, use 80% ethanol water eluting then, weight consumption is 2 times of supernatant weight, and reuse 70% ethanol water eluting, weight consumption are 1 times of supernatant weight.
According to foregoing preparation method, 80% ethanol water eluting described in the step (4) is elution time 20～30min, preferred 25min; Described 70% ethanol water eluting is elution time 40～50min, preferred 45min.
According to foregoing preparation method, the described ultraviolet sterilization of step (5) is the irradiation under ultraviolet ray 25min of 240nm at 54 ℃ with the wavelength preferably for being irradiation under ultraviolet ray 20～30min of 230～260nm at 50～58 ℃ with wavelength.
Below technical solution of the present invention is elaborated:
A kind of deproteinized calf blood extractive injection, described injection contains following material for every milliliter: total solid 160～200mg, wherein wt umber are 0～20 part of amino acid/11,25～35 parts of polypeptide, 15～25 parts of inorganic salts; Xylitol 100～150mg; Epsilon-polylysine 60～100mg.
The present invention has added xylitol in described deproteinized calf blood extractive injection, can be used to regulate osmotic pressure, makes calf blood protein-removed extraction injection of the present invention can be directly used in injection.
Further, the applicant finds that also the deproteinized calf blood extractive injection stability of having added xylitol increases greatly, and this shows that xylitol has special Stabilization for this extract.
In addition, the present invention has also further added epsilon-polylysine with its holding time of better raising.
When every milliliter of described injection contains total solid 160～200mg, during xylitol 100～150mg, especially can improve the stability of this injection.
When the further preferred injection of deproteinized calf blood extractive injection of the present invention contains following material for every milliliter: total solid 180mg, during xylitol 120mg, stability reaches optimum.
And can the preferred weight umber be 5 parts of amino acid/11s, 30 parts of polypeptide, 20 parts of inorganic salts in the described total solid.
The content of aminoacid and polypeptide can better play a role greater than inorganic salt content in the deproteinized calf blood extractive injection solid content provided by the present invention.
Above-mentioned realization with more stable deproteinized calf blood extractive injection, the calf blood protein-removed extraction that can adopt any method of prior art to extract, for example can the reference background technology in those cited preparation methoies.But the present invention is in order further to improve the content of active component in the calf blood protein-removed extraction, preferred following preparation method:
A kind of preparation method of deproteinized calf blood extractive injection noted earlier, described calf blood protein-removed extraction prepares according to following steps:
(1) get calf blood and add sodium citrate, add 3～5 times in water, be warming up to 50～60 ℃ under stirring, insulated and stirred 40～60min stirs down and is cooled to-10～0 ℃ rapidly, and temperature fall time is 2～5min, and is centrifugal at-10～0 ℃ of insulated and stirred 2～4h, gets supernatant;
The present invention takes refrigerated mode, can destroy blood inner cell structure, discharges intracellular material, and the extract activity that this mode prepares will be higher than conventional method and only extract the extract that obtains from serum.But common is freezing, and cooling rate is slow, the slow crystallization of extracellular hydrone, and intracellular matter can all not discharge.And the present invention adopts rapid cooling freezing, can discharge intracellular matter so that the hydrone crystallization in the blood cell destroys cell membrane more completely from cell interior, makes that active constituent content increases greatly in the extracting solution.
Wherein the sodium citrate consumption can be with reference to the consumption of sodium citrate during similar extracting method in the prior art, and the present invention taked preferred for calf blood weight 0.1～0.2 times, more preferably 0.15 times.
Described centrifugal also can be with reference to the centrifugally operated of the similar extracting method of prior art, what the present invention preferably adopted is according to the centrifugal 30～40min of rotating speed 2～3krpm.
(2) will going up the supernatant that makes of step, to regulate pH value be 3～4, adds the pepsin enzymolysis of supernatant weight 4%～5%, filters, and regulating pH value is 7.0～8.5, adds the subtilisin enzymolysis of supernatant weight 1%～1.5%, filters to get filtrate;
The present invention adopts two kinds of different protease hydrolyzeds, is to obtain by a large amount of experiment sievings in numerous proteolytic enzymes.Specificity according to Proteolytic enzyme enzyme hydrolysis target, the present invention at first adopts the stronger pepsin hydrolysis of a large amount of specificitys, and then adopt a spot of site-specific nature poor, and the subtilisin hydrolysis more responsive to the aminoacid Acidity of Aikalinity, can further optimize aminoacid kind in the product, make this extract activity have more significantly and improve.
Hydrolysis temperature of the present invention and time can be with reference to the hydrolysis of this protease of prior art operations, are the pepsin enzymolysis at 55～60 ℃ of following enzymolysis 10～20min, more preferably at 58 ℃ of following enzymolysis 15min and the present invention preferably adopts; The subtilisin enzymolysis is at 30～40 ℃ of following enzymolysis 15～20min, is preferably at 35 ℃ of following enzymolysis 17min.
(3) will go up the filtrate adding ethanol water stirring that the step obtains, centrifugal, get supernatant;
Adding ethanol water of the present invention stirs and can for example add 95% ethanol and stir 5 minutes for 0.5 times with reference to similar operation in the prior art; And the present invention is preferably 80% ethanol water that adds 3～5 times of filtrate weight, stirs 10～20 minutes down at 40～50 ℃.
Described centrifugal this area operation commonly used that is, those skilled in the art need not to pay more creative work for this reason, and that the present invention preferably adopts is centrifugal for the centrifugal 5～10min of 2～3krpm.
(4) supernatant is crossed carbon column, merge eluent and percolation liquid, be concentrated into no ethanol flavor then, concentrated solution adds xylitol, and thin up contains xylitol 100～150mg, epsilon-polylysine 60～100mg, preferred xylitol 120mg to every ml soln, epsilon-polylysine 80mg, the solution microporous filter membrane ultrafiltration after the dilution gets ultrafiltrate;
In order further to improve the purity of effective ingredient in the product, remove other impurity in the blood, the present invention has attempted multiple column chromatography or other means of purification, find at last to use carbon column to carry out column chromatography, not only can reduce the content of other impurity, and can farthest avoid absorption for effective ingredient.
Carbon column chromatography of the present invention can be with reference to the operation of similar column chromatography in the prior art, and the present invention preferably adopts be, described activated carbon consumption is 1～2 times of a described supernatant weight, more preferably 1.5 times, the preferred medicinal active carbon that adopts, the post draw ratio is preferably 4: 1～and 5: 1, more preferably 4.5: 1.The carbon column of this specification can further improve column efficiency.
The present invention can also pour in the carbon column for the supernatant that step (3) is obtained by the further preferred more described carbon column of crossing, equal to the supernatant liquid level with the activated carbon upper surface, standing adsorption 10～20min, preferred 15min, emit percolation liquid, put into the residue supernatant again, according to the above-mentioned steps repeatable operation to putting into whole supernatant, use 80% ethanol water eluting then, weight consumption is 0.5 times of activated carbon weight, reuse 70% ethanol water eluting, weight consumption are 1 times of activated carbon weight.
Wherein can also be again for preferably, described 80% ethanol water eluting is elution time 20～30min, more preferably 25min; Described 70% ethanol water eluting is preferably elution time 40～50min, more preferably 45min.
The microporous filter membrane that described microporous filter membrane can be adopted when other calf blood protein-removed extraction prepares in the prior art, those skilled in the art know the effective ingredient that the filter membrane that uses which kind of aperture can keep suitable molecular weight usually, and remove other impurity of macromolecule.
And microporous filter membrane molecular cut off 4000～7000 dalton that the present invention preferably adopts are preferably 5000～6000 dalton.
(5) ultraviolet sterilization after the ultrafiltrate packing fill, packing promptly.
Described ultraviolet sterilization can be with reference to the ultraviolet disinfecting action of any injection, and just the concrete parameter of ultraviolet disinfecting action can not influence the realization of the object of the invention.But the present invention preferably adopts is to be irradiation under ultraviolet ray 20～30min of 230～260nm at 50～58 ℃ with wavelength, is the irradiation under ultraviolet ray 25min of 240nm at 54 ℃ with the wavelength more preferably.
Detect through experiment, calf blood protein-removed extraction provided by the present invention is breathed vigor can reach 6～8 μ 1O
2/ mgh is preferably 6.5～7.5 μ lO
2/ mgh, more preferably 6.8～7.2 μ lO
2/ mgh; Stimulation index is 5～7.5, is preferably 5.5～7, more preferably 6～6.5.
Technical scheme of the present invention has following advantage:
(1) deproteinized calf blood extractive injection provided by the present invention has higher stability, has further improved patient's drug safety.
(2) deproteinized calf blood extractive injection provided by the invention has higher activity, has further improved the effectiveness of product.
(3) preparation method provided by the present invention is simple and easy to do, and is with low cost.
(4) preparation method provided by the present invention can further improve content of effective in the product.
The specific embodiment
Below with embodiment technical scheme of the present invention is further described; to help advantage to technical scheme of the present invention; effect has further to be understood, and embodiment does not limit protection scope of the present invention, and protection scope of the present invention is decided by claim.
Embodiment 1
A kind of deproteinized calf blood extractive injection, injection contains following material for every milliliter: total solid 180mg, wherein wt umber are 5 parts of amino acid/11s, 30 parts of polypeptide, 20 parts of inorganic salts; Xylitol 120mg; Epsilon-polylysine 80mg.
Described deproteinized calf blood extractive injection breathing vigor is 7 μ lO
2/ mgh, stimulation index is 6.3.
Described calf blood protein-removed extraction prepares according to following steps: getting calf blood adding weight is 0.15 times sodium citrate of calf blood weight, add 4 times in water, be warming up to 55 ℃ under stirring, insulated and stirred 50min, stir down and be cooled to-5 ℃ rapidly, temperature fall time is 3min, insulated and stirred 3h, with the centrifugal 35min of 2.5krpm, get supernatant; It is 3～4 that supernatant is regulated pH value, and adding weight is the pepsin of supernatant weight 4.5%, at 58 ℃ of following enzymolysis 15min, filter, regulating pH value is 7.5, and adding weight is the subtilisin of supernatant weight 1%, at 35 ℃ of following enzymolysis 17min, filter to get filtrate; Filtrate is added 80% ethanol water that weight is 4 times of filtrate weight, stir,, get supernatant according to the centrifugal 7min of rotating speed 2.5kpm; Get the medicinal active carbon dress post of 1.5 times of supernatant weight, post draw ratio 4.5: 1, supernatant is put into carbon column, equal to the supernatant liquid level with the activated carbon upper surface, standing adsorption 15min, emit percolation liquid, put into the residue supernatant again, equal to liquid level with the activated carbon upper surface, standing adsorption 15min, emit percolation liquid, repeatable operation to whole supernatant pass through carbon column, are 80% ethanol water eluting of 2 times of supernatant with weight, and the control elution speed makes that elution time is 25min, reuse weight is 70% ethanol water eluting of 1 times of supernatant, and the control elution speed makes that elution time is 45min; Merge eluent and percolation liquid, be concentrated into no ethanol flavor then, concentrated solution adds xylitol, and thin up extremely contains xylitol 120mg in every ml soln, epsilon-polylysine 80mg, and the solution microporous filter membrane ultrafiltration after the dilution gets ultrafiltrate; Be the irradiation under ultraviolet ray 25min ultraviolet sterilization of 240nm with the wavelength at 54 ℃ after the ultrafiltrate packing fill, packing promptly.
Embodiment 2
A kind of deproteinized calf blood extractive injection, injection contains following material for every milliliter: total solid 180mg, wherein wt umber are 5 parts of amino acid/11s, 30 parts of polypeptide, 20 parts of inorganic salts; Xylitol 120mg; Epsilon-polylysine 80mg.
Described deproteinized calf blood extractive injection breathing vigor is 7 μ lO
2/ mgh, stimulation index is 6.3.
Described calf blood protein-removed extraction prepares according to following steps: getting calf blood adding weight is 0.15 times sodium citrate of calf blood weight, add 4 times in water, be warming up to 55 ℃ under stirring, insulated and stirred 50min, stir down and be cooled to-5 ℃ rapidly, temperature fall time is 3min, insulated and stirred 3h, with the centrifugal 35min of 2krpm, get supernatant; It is 3～4 that supernatant is regulated pH value, and adding weight is the pepsin of supernatant weight 4.5%, at 57 ℃ of following enzymolysis 17min, filter, regulating pH value is 8, and adding weight is the subtilisin of supernatant weight 1.5%, at 32 ℃ of following enzymolysis 17min, filter to get filtrate; Filtrate is added 80% ethanol water that weight is 4 times of filtrate weight, stir,, get supernatant according to the centrifugal 7min of rotating speed 2kpm; Get the medicinal active carbon dress post of 1.5 times of supernatant weight, post draw ratio 4.5: 1, supernatant is put into carbon column, equal to the supernatant liquid level with the activated carbon upper surface, standing adsorption 15min, emit percolation liquid, put into the residue supernatant again, equal to liquid level with the activated carbon upper surface, standing adsorption 15min, emit percolation liquid, repeatable operation to whole supernatant pass through carbon column, are 80% ethanol water eluting of 2 times of supernatant with weight, and the control elution speed makes that elution time is 25min, reuse weight is 70% ethanol water eluting of 1 times of supernatant, and the control elution speed makes that elution time is 42min; Merge eluent and percolation liquid, be concentrated into no ethanol flavor then, concentrated solution adds xylitol, and thin up extremely contains xylitol 120mg in every ml soln, epsilon-polylysine 80mg, and the solution microporous filter membrane ultrafiltration after the dilution gets ultrafiltrate; Be the irradiation under ultraviolet ray 25min ultraviolet sterilization of 240nm with the wavelength at 54 ℃ after the ultrafiltrate packing fill, packing promptly.
Embodiment 3
A kind of deproteinized calf blood extractive injection, injection contains following material for every milliliter: total solid 180mg, wherein wt umber are 5 parts of amino acid/11s, 30 parts of polypeptide, 20 parts of inorganic salts; Xylitol 120mg; Epsilon-polylysine 70mg.
Described deproteinized calf blood extractive injection breathing vigor is 7 μ lO
2/ mgh, stimulation index is 6.3.
Described calf blood protein-removed extraction prepares according to following steps: getting calf blood adding weight is 0.15 times sodium citrate of calf blood weight, add 3 times in water, be warming up to 50 ℃ under stirring, insulated and stirred 55min, stir down and be cooled to-5 ℃ rapidly, temperature fall time is 3min, insulated and stirred 3h, with the centrifugal 35min of 2.5krpm, get supernatant; It is 3～4 that supernatant is regulated pH value, and adding weight is the pepsin of supernatant weight 4.5%, at 55 ℃ of following enzymolysis 10min, filter, regulating pH value is 7.5, and adding weight is the subtilisin of supernatant weight 1.2%, at 35 ℃ of following enzymolysis 16min, filter to get filtrate; Filtrate is added 80% ethanol water that weight is 3 times of filtrate weight, stir,, get supernatant according to the centrifugal 9min of rotating speed 2.5kpm; Get the medicinal active carbon dress post of 1 times of supernatant weight, post draw ratio 4: 1, supernatant is put into carbon column, equal to the supernatant liquid level with the activated carbon upper surface, standing adsorption 12min, emit percolation liquid, put into the residue supernatant again, equal to liquid level with the activated carbon upper surface, standing adsorption 12min, emit percolation liquid, repeatable operation to whole supernatant pass through carbon column, are 80% ethanol water eluting of 2 times of supernatant with weight, and the control elution speed makes that elution time is 23min, reuse weight is 70% ethanol water eluting of 1 times of supernatant, and the control elution speed makes that elution time is 44min; Merge eluent and percolation liquid, be concentrated into no ethanol flavor then, concentrated solution adds xylitol, and thin up extremely contains xylitol 120mg in every ml soln, epsilon-polylysine 70mg, and the solution microporous filter membrane ultrafiltration after the dilution gets ultrafiltrate; Be the irradiation under ultraviolet ray 23min ultraviolet sterilization of 240nm with the wavelength at 52 ℃ after the ultrafiltrate packing fill, packing promptly.
Embodiment 4
A kind of deproteinized calf blood extractive injection, injection contains following material for every milliliter: total solid 180mg, wherein wt umber are 5 parts of amino acid/11s, 30 parts of polypeptide, 20 parts of inorganic salts; Xylitol 120mg; Epsilon-polylysine 80mg.
Described deproteinized calf blood extractive injection breathing vigor is 7 μ lO
2/ mgh, stimulation index is 6.2.
Described calf blood protein-removed extraction prepares according to following steps: getting calf blood adding weight is 0.15 times sodium citrate of calf blood weight, add 4 times in water, be warming up to 60 ℃ under stirring, insulated and stirred 5min, stir down and be cooled to-5 ℃ rapidly, temperature fall time is 3min, insulated and stirred 3h, with the centrifugal 40min of 2.5krpm, get supernatant; It is 3～4 that supernatant is regulated pH value, and adding weight is the pepsin of supernatant weight 4, at 58 ℃ of following enzymolysis 14min, filter, regulating pH value is 8.5, and adding weight is the subtilisin of supernatant weight 1.4%, at 35 ℃ of following enzymolysis 17min, filter to get filtrate; Filtrate is added 80% ethanol water that weight is 3 times of filtrate weight, stir,, get supernatant according to the centrifugal 5min of rotating speed 2.3kpm; Get the medicinal active carbon dress post of 1.5 times of supernatant weight, post draw ratio 4.2: 1, supernatant is put into carbon column, equal to the supernatant liquid level with the activated carbon upper surface, standing adsorption 13min, emit percolation liquid, put into the residue supernatant again, equal to liquid level with the activated carbon upper surface, standing adsorption 13min, emit percolation liquid, repeatable operation to whole supernatant pass through carbon column, are 80% ethanol water eluting of 2 times of supernatant with weight, and the control elution speed makes that elution time is 26min, reuse weight is 70% ethanol water eluting of 1 times of supernatant, and the control elution speed makes that elution time is 48min; Merge eluent and percolation liquid, be concentrated into no ethanol flavor then, concentrated solution adds xylitol, and thin up extremely contains xylitol 120mg in every ml soln, epsilon-polylysine 80mg, and the solution microporous filter membrane ultrafiltration after the dilution gets ultrafiltrate; Be the irradiation under ultraviolet ray 24min ultraviolet sterilization of 230nm with the wavelength at 57 ℃ after the ultrafiltrate packing fill, packing promptly.
Embodiment 5
A kind of deproteinized calf blood extractive injection, injection contains following material for every milliliter: total solid 180mg, wherein wt umber are 5 parts of amino acid/11s, 30 parts of polypeptide, 20 parts of inorganic salts; Xylitol 120mg; Epsilon-polylysine 80mg.
Described deproteinized calf blood extractive injection breathing vigor is 7 μ lO
2/ mgh, stimulation index is 6.3.
Described calf blood protein-removed extraction prepares according to following steps: getting calf blood adding weight is 0.1 times sodium citrate of calf blood weight, add 3 times in water, be warming up to 55 ℃ under stirring, insulated and stirred 50min, stir down and be cooled to 0 ℃ rapidly, temperature fall time is 3min, insulated and stirred 3h, with the centrifugal 30min of 3krpm, get supernatant; It is 3～4 that supernatant is regulated pH value, and adding weight is the pepsin of supernatant weight 4.2%, at 59 ℃ of following enzymolysis 12min, filter, regulating pH value is 7, and adding weight is the subtilisin of supernatant weight 1.5%, at 35 ℃ of following enzymolysis 17min, filter to get filtrate; Filtrate is added 80% ethanol water that weight is 4 times of filtrate weight, stir,, get supernatant according to the centrifugal 8.5min of rotating speed 3kpm; Get the medicinal active carbon dress post of 1.5 times of supernatant weight, post draw ratio 4.7: 1, supernatant is put into carbon column, equal to the supernatant liquid level with the activated carbon upper surface, standing adsorption 17min, emit percolation liquid, put into the residue supernatant again, equal to liquid level with the activated carbon upper surface, standing adsorption 17min, emit percolation liquid, repeatable operation to whole supernatant pass through carbon column, are 80% ethanol water eluting of 2 times of supernatant with weight, and the control elution speed makes that elution time is 28min, reuse weight is 70% ethanol water eluting of 1 times of supernatant, and the control elution speed makes that elution time is 45min; Merge eluent and percolation liquid, be concentrated into no ethanol flavor then, concentrated solution adds xylitol, and thin up extremely contains xylitol 120mg in every ml soln, epsilon-polylysine 80mg, and the solution microporous filter membrane ultrafiltration after the dilution gets ultrafiltrate; Be the irradiation under ultraviolet ray 21min ultraviolet sterilization of 240nm with the wavelength at 54 ℃ after the ultrafiltrate packing fill, packing promptly.
Embodiment 6
A kind of deproteinized calf blood extractive injection, injection contains following material for every milliliter: total solid 180mg, wherein wt umber are 5 parts of amino acid/11s, 32 parts of polypeptide, 20 parts of inorganic salts; Xylitol 120mg; Epsilon-polylysine 90mg.
Described deproteinized calf blood extractive injection breathing vigor is 7 μ lO
2/ mgh, stimulation index is 6.4.
Described calf blood protein-removed extraction prepares according to following steps: getting calf blood adding weight is 0.15 times sodium citrate of calf blood weight, add 5 times in water, be warming up to 55 ℃ under stirring, insulated and stirred 40min, stir down and be cooled to-8 ℃ rapidly, temperature fall time is 4min, insulated and stirred 3h, with the centrifugal 35min of 3krpm, get supernatant; It is 3～4 that supernatant is regulated pH value, and adding weight is the pepsin of supernatant weight 4.8%, at 55 ℃ of following enzymolysis 18min, filter, regulating pH value is 8.5, and adding weight is the subtilisin of supernatant weight 1%, at 37 ℃ of following enzymolysis 15min, filter to get filtrate; Filtrate is added 80% ethanol water that weight is 4 times of filtrate weight, stir,, get supernatant according to the centrifugal 10min of rotating speed 2.5kpm; Get the medicinal active carbon dress post of 2 times of supernatant weight, post draw ratio 5: 1, supernatant is put into carbon column, equal to the supernatant liquid level with the activated carbon upper surface, standing adsorption 19min, emit percolation liquid, put into the residue supernatant again, equal to liquid level with the activated carbon upper surface, standing adsorption 19min, emit percolation liquid, repeatable operation to whole supernatant pass through carbon column, are 80% ethanol water eluting of 2 times of supernatant with weight, and the control elution speed makes that elution time is 22min, reuse weight is 70% ethanol water eluting of 1 times of supernatant, and the control elution speed makes that elution time is 47min; Merge eluent and percolation liquid, be concentrated into no ethanol flavor then, concentrated solution adds xylitol, and thin up extremely contains xylitol 120mg in every ml soln, epsilon-polylysine 90mg, and the solution microporous filter membrane ultrafiltration after the dilution gets ultrafiltrate; Be the irradiation under ultraviolet ray 27min ultraviolet sterilization of 250nm with the wavelength at 53 ℃ after the ultrafiltrate packing fill, packing promptly.
Embodiment 7
A kind of deproteinized calf blood extractive injection, injection contains following material for every milliliter: total solid 180mg, wherein wt umber are 4 parts of amino acid/11s, 28 parts of polypeptide, 19 parts of inorganic salts; Xylitol 115mg; Epsilon-polylysine 80mg.
Described deproteinized calf blood extractive injection breathing vigor is 6.8 μ lO
2/ mgh, stimulation index is 6.1.
Described calf blood protein-removed extraction prepares according to following steps: getting calf blood adding weight is 0.2 times sodium citrate of calf blood weight, add 4 times in water, be warming up to 60 ℃ under stirring, insulated and stirred 55min, stir down and be cooled to-3 ℃ rapidly, temperature fall time is 5min, insulated and stirred 2h, with the centrifugal 35min of 2krpm, get supernatant; It is 3～4 that supernatant is regulated pH value, and adding weight is the pepsin of supernatant weight 5%, at 58 ℃ of following enzymolysis 19min, filter, regulating pH value is 7, and adding weight is the subtilisin of supernatant weight 1%, at 40 ℃ of following enzymolysis 18min, filter to get filtrate; Filtrate is added 80% ethanol water that weight is 5 times of filtrate weight, stir,, get supernatant according to the centrifugal 6min of rotating speed 2.7kpm; Get the medicinal active carbon dress post of 1 times of supernatant weight, post draw ratio 4.8: 1, supernatant is put into carbon column, equal to the supernatant liquid level with the activated carbon upper surface, standing adsorption 15min, emit percolation liquid, put into the residue supernatant again, equal to liquid level with the activated carbon upper surface, standing adsorption 15min, emit percolation liquid, repeatable operation to whole supernatant pass through carbon column, are 80% ethanol water eluting of 2 times of supernatant with weight, and the control elution speed makes that elution time is 25min, reuse weight is 70% ethanol water eluting of 1 times of supernatant, and the control elution speed makes that elution time is 49min; Merge eluent and percolation liquid, be concentrated into no ethanol flavor then, concentrated solution adds xylitol, and thin up extremely contains xylitol 115mg in every ml soln, epsilon-polylysine 80mg, and the solution microporous filter membrane ultrafiltration after the dilution gets ultrafiltrate; Be the irradiation under ultraviolet ray 20min ultraviolet sterilization of 240nm with the wavelength at 51 ℃ after the ultrafiltrate packing fill, packing promptly.
Embodiment 8
A kind of deproteinized calf blood extractive injection, injection contains following material for every milliliter: total solid 180mg, wherein wt umber are 3 parts of amino acid/11s, 30 parts of polypeptide, 17 parts of inorganic salts; Xylitol 120mg; Epsilon-polylysine 90mg.
Described deproteinized calf blood extractive injection breathing vigor is 6.8 μ lO
2/ mgh, stimulation index is 6.0.
Described calf blood protein-removed extraction prepares according to following steps: getting calf blood adding weight is 0.15 times sodium citrate of calf blood weight, add 3 times in water, be warming up to 50 ℃ under stirring, insulated and stirred 60min, stir down and be cooled to-9 ℃ rapidly, temperature fall time is 5min, insulated and stirred 4h, with the centrifugal 40min of 3krpm, get supernatant; It is 3～4 that supernatant is regulated pH value, and adding weight is the pepsin of supernatant weight 4.5%, at 57 ℃ of following enzymolysis 20min, filter, regulating pH value is 7.5, and adding weight is the subtilisin of supernatant weight 1.3%, at 30 ℃ of following enzymolysis 19min, filter to get filtrate; Filtrate is added 80% ethanol water that weight is 5 times of filtrate weight, stir,, get supernatant according to the centrifugal 5min of rotating speed 2.5kpm; Get the medicinal active carbon dress post of 2 times of supernatant weight, post draw ratio 4.5: 1, supernatant is put into carbon column, equal to the supernatant liquid level with the activated carbon upper surface, standing adsorption 15min, emit percolation liquid, put into the residue supernatant again, equal to liquid level with the activated carbon upper surface, standing adsorption 15min, emit percolation liquid, repeatable operation to whole supernatant pass through carbon column, are 80% ethanol water eluting of 2 times of supernatant with weight, and the control elution speed makes that elution time is 22min, reuse weight is 70% ethanol water eluting of 1 times of supernatant, and the control elution speed makes that elution time is 45min; Merge eluent and percolation liquid, be concentrated into no ethanol flavor then, concentrated solution adds xylitol, and thin up extremely contains xylitol 120mg in every ml soln, epsilon-polylysine 90mg, and the solution microporous filter membrane ultrafiltration after the dilution gets ultrafiltrate; Be the irradiation under ultraviolet ray 25min ultraviolet sterilization of 250nm with the wavelength at 57 ℃ after the ultrafiltrate packing fill, packing promptly.
Embodiment 9
A kind of deproteinized calf blood extractive injection, injection contains following material for every milliliter: total solid 180mg, wherein wt umber are 5 parts of amino acid/11s, 34 parts of polypeptide, 22 parts of inorganic salts; Xylitol 120mg; Epsilon-polylysine 70mg.
Described deproteinized calf blood extractive injection breathing vigor is 7.2 μ lO
2/ mgh, stimulation index is 6.5.
Described calf blood protein-removed extraction prepares according to following steps: getting calf blood adding weight is 0.15 times sodium citrate of calf blood weight, add 3 times in water, be warming up to 55 ℃ under stirring, insulated and stirred 52min, stir down and be cooled to-10 ℃ rapidly, temperature fall time is 2min, insulated and stirred 2h, with the centrifugal 38min of 2.5krpm, get supernatant; It is 3～4 that supernatant is regulated pH value, and adding weight is the pepsin of supernatant weight 4.5%, at 57 ℃ of following enzymolysis 15min, filter, regulating pH value is 8, and adding weight is the subtilisin of supernatant weight 1.5%, at 32 ℃ of following enzymolysis 17min, filter to get filtrate; Filtrate is added 80% ethanol water that weight is 4 times of filtrate weight, stir,, get supernatant according to the centrifugal 7min of rotating speed 2.2kpm; Get the medicinal active carbon dress post of 1.5 times of supernatant weight, post draw ratio 5: 1, supernatant is put into carbon column, equal to the supernatant liquid level with the activated carbon upper surface, standing adsorption 20min, emit percolation liquid, put into the residue supernatant again, equal to liquid level with the activated carbon upper surface, standing adsorption 20min, emit percolation liquid, repeatable operation to whole supernatant pass through carbon column, are 80% ethanol water eluting of 2 times of supernatant with weight, and the control elution speed makes that elution time is 21min, reuse weight is 70% ethanol water eluting of 1 times of supernatant, and the control elution speed makes that elution time is 45min; Merge eluent and percolation liquid, be concentrated into no ethanol flavor then, concentrated solution adds xylitol, and thin up extremely contains xylitol 120mg in every ml soln, epsilon-polylysine 70mg, and the solution microporous filter membrane ultrafiltration after the dilution gets ultrafiltrate; Be the irradiation under ultraviolet ray 29min ultraviolet sterilization of 260nm with the wavelength at 54 ℃ after the ultrafiltrate packing fill, packing promptly.
Embodiment 10
A kind of deproteinized calf blood extractive injection, injection contains following material for every milliliter: total solid 180mg, wherein wt umber are 6 parts of amino acid/11s, 31 parts of polypeptide, 20 parts of inorganic salts; Xylitol 130mg; Epsilon-polylysine 80mg.
Described deproteinized calf blood extractive injection breathing vigor is 7.2 μ lO
2/ mgh, stimulation index is 6.4.
Described calf blood protein-removed extraction prepares according to following steps: getting calf blood adding weight is 0.1 times sodium citrate of calf blood weight, add 4 times in water, be warming up to 60 ℃ under stirring, insulated and stirred 57min, stir down and be cooled to-5 ℃ rapidly, temperature fall time is 4min, insulated and stirred 3h, with the centrifugal 32min of 2.5krpm, get supernatant; It is 3～4 that supernatant is regulated pH value, and adding weight is the pepsin of supernatant weight 4.9%, at 59 ℃ of following enzymolysis 15min, filter, regulating pH value is 7, and adding weight is the subtilisin of supernatant weight 1%, at 37 ℃ of following enzymolysis 18min, filter to get filtrate; Filtrate is added 80% ethanol water that weight is 4 times of filtrate weight, stir,, get supernatant according to the centrifugal 10min of rotating speed 2.5kpm; Get the medicinal active carbon dress post of 1.5 times of supernatant weight, post draw ratio 4.5: 1, supernatant is put into carbon column, equal to the supernatant liquid level with the activated carbon upper surface, standing adsorption 15min, emit percolation liquid, put into the residue supernatant again, equal to liquid level with the activated carbon upper surface, standing adsorption 15min, emit percolation liquid, repeatable operation to whole supernatant pass through carbon column, are 80% ethanol water eluting of 2 times of supernatant with weight, and the control elution speed makes that elution time is 28min, reuse weight is 70% ethanol water eluting of 1 times of supernatant, and the control elution speed makes that elution time is 43min; Merge eluent and percolation liquid, be concentrated into no ethanol flavor then, concentrated solution adds xylitol, and thin up extremely contains xylitol 130mg in every ml soln, epsilon-polylysine 80mg, and the solution microporous filter membrane ultrafiltration after the dilution gets ultrafiltrate; Be the irradiation under ultraviolet ray 25min ultraviolet sterilization of 240nm with the wavelength at 54 ℃ after the ultrafiltrate packing fill, packing promptly.
Embodiment 11
A kind of deproteinized calf blood extractive injection, injection contains following material for every milliliter: total solid 170mg, wherein wt umber are 7 parts of amino acid/11s, 29 parts of polypeptide, 21 parts of inorganic salts; Xylitol 120mg; Epsilon-polylysine 80mg.
Described deproteinized calf blood extractive injection breathing vigor is 6.3 μ lO
2/ mgh, stimulation index is 5.7.
Described calf blood protein-removed extraction prepares according to following steps: getting calf blood adding weight is 0.2 times sodium citrate of calf blood weight, add 5 times in water, be warming up to 60 ℃ under stirring, insulated and stirred 55min, stir down and be cooled to-5 ℃ rapidly, temperature fall time is 3min, insulated and stirred 3h, with the centrifugal 30min of 2.5krpm, get supernatant; It is 3～4 that supernatant is regulated pH value, and adding weight is the pepsin of supernatant weight 5%, at 60 ℃ of following enzymolysis 17min, filter, regulating pH value is 8.5, and adding weight is the subtilisin of supernatant weight 1%, at 35 ℃ of following enzymolysis 20min, filter to get filtrate; Filtrate is added 80% ethanol water that weight is 3 times of filtrate weight, stir,, get supernatant according to the centrifugal 7min of rotating speed 2.5kpm; Get the medicinal active carbon dress post of 2 times of supernatant weight, post draw ratio 4.5: 1, supernatant is put into carbon column, equal to the supernatant liquid level with the activated carbon upper surface, standing adsorption 14min, emit percolation liquid, put into the residue supernatant again, equal to liquid level with the activated carbon upper surface, standing adsorption 14min, emit percolation liquid, repeatable operation to whole supernatant pass through carbon column, are 80% ethanol water eluting of 2 times of supernatant with weight, and the control elution speed makes that elution time is 26min, reuse weight is 70% ethanol water eluting of 1 times of supernatant, and the control elution speed makes that elution time is 45min; Merge eluent and percolation liquid, be concentrated into no ethanol flavor then, concentrated solution adds xylitol, and thin up extremely contains xylitol 120mg in every ml soln, epsilon-polylysine 80mg, and the solution microporous filter membrane ultrafiltration after the dilution gets ultrafiltrate; Be the irradiation under ultraviolet ray 30min ultraviolet sterilization of 230nm with the wavelength at 53 ℃ after the ultrafiltrate packing fill, packing promptly.
Embodiment 12
A kind of deproteinized calf blood extractive injection, injection contains following material for every milliliter: total solid 160mg, wherein wt umber are 4 parts of amino acid/11s, 30 parts of polypeptide, 23 parts of inorganic salts; Xylitol 120mg; Epsilon-polylysine 80mg.
Described deproteinized calf blood extractive injection breathing vigor is 6.0 μ lO
2/ mgh, stimulation index is 5.0.
Described calf blood protein-removed extraction prepares according to following steps: getting calf blood adding weight is 0.2 times sodium citrate of calf blood weight, add 5 times in water, be warming up to 50 ℃ under stirring, insulated and stirred 50min, stir down and be cooled to-2 ℃ rapidly, temperature fall time is 3min, insulated and stirred 4h, with the centrifugal 35min of 2krpm, get supernatant; It is 3～4 that supernatant is regulated pH value, and adding weight is the pepsin of supernatant weight 4.6%, at 56 ℃ of following enzymolysis 18min, filter, regulating pH value is 7.5, and adding weight is the subtilisin of supernatant weight 1.4%, at 39 ℃ of following enzymolysis 17min, filter to get filtrate; Filtrate is added 80% ethanol water that weight is 4 times of filtrate weight, stir,, get supernatant according to the centrifugal 7min of rotating speed 3kpm; Get the medicinal active carbon dress post of 1.5 times of supernatant weight, post draw ratio 4.5: 1, supernatant is put into carbon column, equal to the supernatant liquid level with the activated carbon upper surface, standing adsorption 18min, emit percolation liquid, put into the residue supernatant again, equal to liquid level with the activated carbon upper surface, standing adsorption 18min, emit percolation liquid, repeatable operation to whole supernatant pass through carbon column, are 80% ethanol water eluting of 2 times of supernatant with weight, and the control elution speed makes that elution time is 25min, reuse weight is 70% ethanol water eluting of 1 times of supernatant, and the control elution speed makes that elution time is 43min; Merge eluent and percolation liquid, be concentrated into no ethanol flavor then, concentrated solution adds xylitol, and thin up extremely contains xylitol 120mg in every ml soln, epsilon-polylysine 80mg, and the solution microporous filter membrane ultrafiltration after the dilution gets ultrafiltrate; Be the irradiation under ultraviolet ray 25min ultraviolet sterilization of 240nm with the wavelength at 55 ℃ after the ultrafiltrate packing fill, packing promptly.
Embodiment 13
A kind of deproteinized calf blood extractive injection, injection contains following material for every milliliter: total solid 190mg, wherein wt umber are 8 parts of amino acid/11s, 30 parts of polypeptide, 19 parts of inorganic salts; Xylitol 150mg; Epsilon-polylysine 90mg.
Described deproteinized calf blood extractive injection breathing vigor is 7.8 μ lO
2/ mgh, stimulation index is 7.2.
Described calf blood protein-removed extraction prepares according to following steps: getting calf blood adding weight is 0.1 times sodium citrate of calf blood weight, add 4 times in water, be warming up to 50 ℃ under stirring, insulated and stirred 52min, stir down and be cooled to-1 ℃ rapidly, temperature fall time is 4min, insulated and stirred 2h, with the centrifugal 35min of 3krpm, get supernatant; It is 3～4 that supernatant is regulated pH value, and adding weight is the pepsin of supernatant weight 4.4%, at 57 ℃ of following enzymolysis 15min, filter, regulating pH value is 8, and adding weight is the subtilisin of supernatant weight 1.1%, at 34 ℃ of following enzymolysis 19min, filter to get filtrate; Filtrate is added 80% ethanol water that weight is 4 times of filtrate weight, stir,, get supernatant according to the centrifugal 7min of rotating speed 2.7kpm; Get the medicinal active carbon dress post of 1 times of supernatant weight, post draw ratio 4.2: 1, supernatant is put into carbon column, equal to the supernatant liquid level with the activated carbon upper surface, standing adsorption 19min, emit percolation liquid, put into the residue supernatant again, equal to liquid level with the activated carbon upper surface, standing adsorption 19min, emit percolation liquid, repeatable operation to whole supernatant pass through carbon column, are 80% ethanol water eluting of 2 times of supernatant with weight, and the control elution speed makes that elution time is 25min, reuse weight is 70% ethanol water eluting of 1 times of supernatant, and the control elution speed makes that elution time is 48min; Merge eluent and percolation liquid, be concentrated into no ethanol flavor then, concentrated solution adds xylitol, and thin up extremely contains xylitol 150mg in every ml soln, epsilon-polylysine 90mg, and the solution microporous filter membrane ultrafiltration after the dilution gets ultrafiltrate; Be the irradiation under ultraviolet ray 25min ultraviolet sterilization of 240nm with the wavelength at 57 ℃ after the ultrafiltrate packing fill, packing promptly.
Embodiment 14
A kind of deproteinized calf blood extractive injection, injection contains following material for every milliliter: total solid 185mg, wherein wt umber are 5 parts of amino acid/11s, 27 parts of polypeptide, 18 parts of inorganic salts; Xylitol 130mg; Epsilon-polylysine 80mg.
Described deproteinized calf blood extractive injection breathing vigor is 7.5 μ lO
2/ mgh, stimulation index is 6.8.
Described calf blood protein-removed extraction prepares according to following steps: getting calf blood adding weight is 0.15 times sodium citrate of calf blood weight, add 3 times in water, be warming up to 60 ℃ under stirring, insulated and stirred 58min, stir down and be cooled to-5 ℃ rapidly, temperature fall time is 5min, insulated and stirred 4h, with the centrifugal 35min of 2.5krpm, get supernatant; It is 3～4 that supernatant is regulated pH value, and adding weight is the pepsin of supernatant weight 4.5%, at 58 ℃ of following enzymolysis 15min, filter, regulating pH value is 7, and adding weight is the subtilisin of supernatant weight 1.5%, at 35 ℃ of following enzymolysis 15min, filter to get filtrate; Filtrate is added 80% ethanol water that weight is 4 times of filtrate weight, stir,, get supernatant according to the centrifugal 8min of rotating speed 2.5kpm; Get the medicinal active carbon dress post of 1.5 times of supernatant weight, post draw ratio 4.5: 1, supernatant is put into carbon column, equal to the supernatant liquid level with the activated carbon upper surface, standing adsorption 15min, emit percolation liquid, put into the residue supernatant again, equal to liquid level with the activated carbon upper surface, standing adsorption 15min, emit percolation liquid, repeatable operation to whole supernatant pass through carbon column, are 80% ethanol water eluting of 2 times of supernatant with weight, and the control elution speed makes that elution time is 30min, reuse weight is 70% ethanol water eluting of 1 times of supernatant, and the control elution speed makes that elution time is 45min; Merge eluent and percolation liquid, be concentrated into no ethanol flavor then, concentrated solution adds xylitol, and thin up extremely contains xylitol 130mg in every ml soln, epsilon-polylysine 80mg, and the solution microporous filter membrane ultrafiltration after the dilution gets ultrafiltrate; Be the irradiation under ultraviolet ray 28min ultraviolet sterilization of 260nm with the wavelength at 54 ℃ after the ultrafiltrate packing fill, packing promptly.
Embodiment 15
A kind of deproteinized calf blood extractive injection, injection contains following material for every milliliter: total solid 165mg, wherein wt umber are 0 part of amino acid/11,32 parts of polypeptide, 16 parts of inorganic salts; Xylitol 120mg; Epsilon-polylysine 100mg.
Described deproteinized calf blood extractive injection breathing vigor is 6.1 μ lO
2/ mgh, stimulation index is 5.2.
Described calf blood protein-removed extraction prepares according to following steps: getting calf blood adding weight is 0.15 times sodium citrate of calf blood weight, add 5 times in water, be warming up to 55 ℃ under stirring, insulated and stirred 51min, stir down and be cooled to-7 ℃ rapidly, temperature fall time is 2min, insulated and stirred 3h, with the centrifugal 40min of 3krpm, get supernatant; It is 3～4 that supernatant is regulated pH value, and adding weight is the pepsin of supernatant weight 4.5%, at 57 ℃ of following enzymolysis 16min, filter, regulating pH value is 7, and adding weight is the subtilisin of supernatant weight 1%, at 37 ℃ of following enzymolysis 16min, filter to get filtrate; Filtrate is added 80% ethanol water that weight is 5 times of filtrate weight, stir,, get supernatant according to the centrifugal 5min of rotating speed 2kpm; Get the medicinal active carbon dress post of 2 times of supernatant weight, post draw ratio 5: 1, supernatant is put into carbon column, equal to the supernatant liquid level with the activated carbon upper surface, standing adsorption 15min, emit percolation liquid, put into the residue supernatant again, equal to liquid level with the activated carbon upper surface, standing adsorption 15min, emit percolation liquid, repeatable operation to whole supernatant pass through carbon column, are 80% ethanol water eluting of 2 times of supernatant with weight, and the control elution speed makes that elution time is 25min, reuse weight is 70% ethanol water eluting of 1 times of supernatant, and the control elution speed makes that elution time is 50min; Merge eluent and percolation liquid, be concentrated into no ethanol flavor then, concentrated solution adds xylitol, and thin up extremely contains xylitol 120mg in every ml soln, epsilon-polylysine 100mg, and the solution microporous filter membrane ultrafiltration after the dilution gets ultrafiltrate; Be the irradiation under ultraviolet ray 29min ultraviolet sterilization of 240nm with the wavelength at 54 ℃ after the ultrafiltrate packing fill, packing promptly.
Embodiment 16
A kind of deproteinized calf blood extractive injection, injection contains following material for every milliliter: total solid 175mg, wherein wt umber are 2 parts of amino acid/11s, 30 parts of polypeptide, 20 parts of inorganic salts; Xylitol 140mg; Epsilon-polylysine 80mg.
Described deproteinized calf blood extractive injection breathing vigor is 6.6 μ lO
2/ mgh, stimulation index is 5.4.
Described calf blood protein-removed extraction prepares according to following steps: getting calf blood adding weight is 0.2 times sodium citrate of calf blood weight, add 3 times in water, be warming up to 60 ℃ under stirring, insulated and stirred 48min, stir down and be cooled to-8 ℃ rapidly, temperature fall time is 3min, insulated and stirred 3h, with the centrifugal 40min of 3krpm, get supernatant; It is 3～4 that supernatant is regulated pH value, and adding weight is the pepsin of supernatant weight 5%, at 55 ℃ of following enzymolysis 20min, filter, regulating pH value is 8, and adding weight is the subtilisin of supernatant weight 1%, at 39 ℃ of following enzymolysis 17min, filter to get filtrate; Filtrate is added 80% ethanol water that weight is 4 times of filtrate weight, stir,, get supernatant according to the centrifugal 8min of rotating speed 3kpm; Get the medicinal active carbon dress post of 1.7 times of supernatant weight, post draw ratio 4: 1, supernatant is put into carbon column, equal to the supernatant liquid level with the activated carbon upper surface, standing adsorption 13min, emit percolation liquid, put into the residue supernatant again, equal to liquid level with the activated carbon upper surface, standing adsorption 13min, emit percolation liquid, repeatable operation to whole supernatant pass through carbon column, are 80% ethanol water eluting of 2 times of supernatant with weight, and the control elution speed makes that elution time is 25min, reuse weight is 70% ethanol water eluting of 1 times of supernatant, and the control elution speed makes that elution time is 42min; Merge eluent and percolation liquid, be concentrated into no ethanol flavor then, concentrated solution adds xylitol, and thin up extremely contains xylitol 140mg in every ml soln, epsilon-polylysine 80mg, and the solution microporous filter membrane ultrafiltration after the dilution gets ultrafiltrate; Be the irradiation under ultraviolet ray 25min ultraviolet sterilization of 240nm with the wavelength at 54 ℃ after the ultrafiltrate packing fill, packing promptly.
Embodiment 17
A kind of deproteinized calf blood extractive injection, injection contains following material for every milliliter: total solid 180mg, wherein wt umber are 4 parts of amino acid/11s, 30 parts of polypeptide, 22 parts of inorganic salts; Xylitol 110mg; Epsilon-polylysine 60mg.
Described deproteinized calf blood extractive injection breathing vigor is 7.1 μ lO
2/ mgh, stimulation index is 6.4.
Described calf blood protein-removed extraction prepares according to following steps: getting calf blood adding weight is 0.1 times sodium citrate of calf blood weight, add 3 times in water, be warming up to 60 ℃ under stirring, insulated and stirred 43min, stir down and be cooled to-10 ℃ rapidly, temperature fall time is 4min, insulated and stirred 2h, with the centrifugal 40min of 3krpm, get supernatant; It is 3～4 that supernatant is regulated pH value, and adding weight is the pepsin of supernatant weight 4%, at 55 ℃ of following enzymolysis 13min, filter, regulating pH value is 8.5, and adding weight is the subtilisin of supernatant weight 1.4%, at 35 ℃ of following enzymolysis 20min, filter to get filtrate; Filtrate is added 80% ethanol water that weight is 3 times of filtrate weight, stir,, get supernatant according to the centrifugal 6min of rotating speed 2.9kpm; Get the medicinal active carbon dress post of 1.8 times of supernatant weight, post draw ratio 4.2: 1, supernatant is put into carbon column, equal to the supernatant liquid level with the activated carbon upper surface, standing adsorption 17min, emit percolation liquid, put into the residue supernatant again, equal to liquid level with the activated carbon upper surface, standing adsorption 17min, emit percolation liquid, repeatable operation to whole supernatant pass through carbon column, are 80% ethanol water eluting of 2 times of supernatant with weight, and the control elution speed makes that elution time is 27min, reuse weight is 70% ethanol water eluting of 1 times of supernatant, and the control elution speed makes that elution time is 45min; Merge eluent and percolation liquid, be concentrated into no ethanol flavor then, concentrated solution adds xylitol, and thin up extremely contains xylitol 110mg in every ml soln, epsilon-polylysine 60mg, and the solution microporous filter membrane ultrafiltration after the dilution gets ultrafiltrate; Be the irradiation under ultraviolet ray 30min ultraviolet sterilization of 260nm with the wavelength at 58 ℃ after the ultrafiltrate packing fill, packing promptly.
Embodiment 18
A kind of deproteinized calf blood extractive injection, injection contains following material for every milliliter: total solid 175mg, wherein wt umber are 5 parts of amino acid/11s, 25 parts of polypeptide, 24 parts of inorganic salts; Xylitol 120mg; Epsilon-polylysine 80mg.
Described deproteinized calf blood extractive injection breathing vigor is 6.5 μ lO
2/ mgh, stimulation index is 5.4.
Described calf blood protein-removed extraction prepares according to following steps: getting calf blood adding weight is 0.15 times sodium citrate of calf blood weight, add 5 times in water, be warming up to 55 ℃ under stirring, insulated and stirred 45min, stir down and be cooled to-5 ℃ rapidly, temperature fall time is 5min, insulated and stirred 4h, with the centrifugal 30min of 2krpm, get supernatant; It is 3～4 that supernatant is regulated pH value, and adding weight is the pepsin of supernatant weight 4.5%, at 58 ℃ of following enzymolysis 20min, filter, regulating pH value is 8, and adding weight is the subtilisin of supernatant weight 1%, at 40 ℃ of following enzymolysis 17min, filter to get filtrate; Filtrate is added 80% ethanol water that weight is 4 times of filtrate weight, stir,, get supernatant according to the centrifugal 8min of rotating speed 2.5kpm; Get the medicinal active carbon dress post of 1.3 times of supernatant weight, post draw ratio 4.7: 1, supernatant is put into carbon column, equal to the supernatant liquid level with the activated carbon upper surface, standing adsorption 18min, emit percolation liquid, put into the residue supernatant again, equal to liquid level with the activated carbon upper surface, standing adsorption 18min, emit percolation liquid, repeatable operation to whole supernatant pass through carbon column, are 80% ethanol water eluting of 2 times of supernatant with weight, and the control elution speed makes that elution time is 24min, reuse weight is 70% ethanol water eluting of 1 times of supernatant, and the control elution speed makes that elution time is 47min; Merge eluent and percolation liquid, be concentrated into no ethanol flavor then, concentrated solution adds xylitol, and thin up extremely contains xylitol 120mg in every ml soln, epsilon-polylysine 80mg, and the solution microporous filter membrane ultrafiltration after the dilution gets ultrafiltrate; Be the irradiation under ultraviolet ray 25min ultraviolet sterilization of 240nm with the wavelength at 54 ℃ after the ultrafiltrate packing fill, packing promptly.
Embodiment 19
A kind of deproteinized calf blood extractive injection, injection contains following material for every milliliter: total solid 185mg, wherein wt umber are 5 parts of amino acid/11s, 34 parts of polypeptide, 21 parts of inorganic salts; Xylitol 120mg; Epsilon-polylysine 90mg.
Described deproteinized calf blood extractive injection breathing vigor is 7.6 μ lO
2/ mgh, stimulation index is 7.1.
Described calf blood protein-removed extraction prepares according to following steps: getting calf blood adding weight is 0.15 times sodium citrate of calf blood weight, add 4 times in water, be warming up to 55 ℃ under stirring, insulated and stirred 60min, stir down and be cooled to-2 ℃ rapidly, temperature fall time is 5min, insulated and stirred 2h, with the centrifugal 35min of 2krpm, get supernatant; It is 3～4 that supernatant is regulated pH value, and adding weight is the pepsin of supernatant weight 4.5%, at 60 ℃ of following enzymolysis 12min, filter, regulating pH value is 7, and adding weight is the subtilisin of supernatant weight 1%, at 35 ℃ of following enzymolysis 18min, filter to get filtrate; Filtrate is added 80% ethanol water that weight is 4 times of filtrate weight, stir,, get supernatant according to the centrifugal 7min of rotating speed 3kpm; Get the medicinal active carbon dress post of 1.5 times of supernatant weight, post draw ratio 4.5: 1, supernatant is put into carbon column, equal to the supernatant liquid level with the activated carbon upper surface, standing adsorption 20min, emit percolation liquid, put into the residue supernatant again, equal to liquid level with the activated carbon upper surface, standing adsorption 20min, emit percolation liquid, repeatable operation to whole supernatant pass through carbon column, are 80% ethanol water eluting of 2 times of supernatant with weight, and the control elution speed makes that elution time is 25min, reuse weight is 70% ethanol water eluting of 1 times of supernatant, and the control elution speed makes that elution time is 45min; Merge eluent and percolation liquid, be concentrated into no ethanol flavor then, concentrated solution adds xylitol, and thin up extremely contains xylitol 120mg in every ml soln, epsilon-polylysine 90mg, and the solution microporous filter membrane ultrafiltration after the dilution gets ultrafiltrate; Be the irradiation under ultraviolet ray 25min ultraviolet sterilization of 240nm with the wavelength at 54 ℃ after the ultrafiltrate packing fill, packing promptly.
Embodiment 20
A kind of deproteinized calf blood extractive injection, injection contains following material for every milliliter: total solid 200mg, wherein wt umber are 20 parts in aminoacid, 35 parts of polypeptide, 25 parts of inorganic salts; Xylitol 100mg; Epsilon-polylysine 70mg.
Described deproteinized calf blood extractive injection breathing vigor is 8.0 μ lO
2/ mgh, stimulation index is 7.5.
Described calf blood protein-removed extraction prepares according to following steps: getting calf blood adding weight is 0.15 times sodium citrate of calf blood weight, add 4 times in water, be warming up to 55 ℃ under stirring, insulated and stirred 50min, stir down and be cooled to-5 ℃ rapidly, temperature fall time is 4min, insulated and stirred 3h, with the centrifugal 45min of 3.5krpm, get supernatant; It is 3～4 that supernatant is regulated pH value, and adding weight is the pepsin of supernatant weight 4.5%, at 57 ℃ of following enzymolysis 15min, filter, regulating pH value is 7.5, and adding weight is the subtilisin of supernatant weight 1.2%, at 35 ℃ of following enzymolysis 17min, filter to get filtrate; Filtrate is added 80% ethanol water that weight is 5 times of filtrate weight, stir,, get supernatant according to the centrifugal 10min of rotating speed 2kpm; Get the medicinal active carbon dress post of 1 times of supernatant weight, post draw ratio 4: 1, supernatant is put into carbon column, equal to the supernatant liquid level with the activated carbon upper surface, standing adsorption 10min, emit percolation liquid, put into the residue supernatant again, equal to liquid level with the activated carbon upper surface, standing adsorption 10min, emit percolation liquid, repeatable operation to whole supernatant pass through carbon column, are 80% ethanol water eluting of 2 times of supernatant with weight, and the control elution speed makes that elution time is 25min, reuse weight is 70% ethanol water eluting of 1 times of supernatant, and the control elution speed makes that elution time is 45min; Merge eluent and percolation liquid, be concentrated into no ethanol flavor then, concentrated solution adds xylitol, and thin up extremely contains xylitol 100mg in every ml soln, epsilon-polylysine 70mg, and the solution microporous filter membrane ultrafiltration after the dilution gets ultrafiltrate; Be the irradiation under ultraviolet ray 26min ultraviolet sterilization of 260nm with the wavelength at 57 ℃ after the ultrafiltrate packing fill, packing promptly.
The present invention also provides following test example, so that further the present invention will be described:
Test example 1
Because 2005 editions the 3rd chronicity and study on the stability to biological preparation of Chinese Pharmacopoeia do not done regulation, so the experimental implementation that this experiment is investigated with reference to stability and the chronicity about pharmaceutical preparation of 2005 editions second appendix XIXC of Chinese Pharmacopoeia.
Table 1, calf blood protein-removed extraction stability long-term experiment are investigated
This test example has been investigated the stability of calf blood protein-removed extraction provided by the present invention.Wherein 1 is embodiment 1 product; 2 is the product of embodiment 11; 3 is the method preparation of embodiment 11, and according to the proportioning of embodiment 11, difference is to have removed epsilon-polylysine; 4 is the product of embodiment 20; Be 5 for according to the preparation of the method for patent application 200510084246.2, and add xylitol and epsilon-polylysine according to the proportioning of embodiment 1; 6 is the product according to the method preparation of patent application 200410062657.7 embodiment 1.
Wherein polypeptide is to be 100% with initial content of peptides in the product, detects thereafter the content of peptides percentage ratio of its initial content relatively then.
As can be seen, the present invention has added xylitol and epsilon-polylysine can increase the calf blood protein-removed extraction stability of formulation greatly from this table.For example number 1,2 product, the polypeptide composition changes very little.Do not add epsilon-polylysine and number 3 product, but still keep comparatively stable content of peptides, confirm that xylitol has certain stability action.Though numbering 4 product has more high-load polypeptide and higher stimulation index because active component keeps too much when extracting, thereby may be residual more impurity, and it is very fast to make that effective ingredient and stimulation index reduce.Numbering 5 is according to the art methods preparation, but has added xylitol and epsilon-polylysine, and its product can be realized better stability equally.Numbering 6 is complete product according to prior art for preparing, and its stability is relatively poor relatively.
Test example 2
This test example has been investigated the influence of preparation method for properties of product.
Table 2, preparation method influence
| Cool-down method | Enzymolysis | Aminoacid | Polypeptide | |
| 1 | 3min reduces to-5 ℃ | Elder generation's supernatant weight 4.5% pepsin, the subtilisin of supernatant weight 1% again | 41.5mg | 83.1mg |
| 2 | 4min reduces to-1 ℃ | The pepsin of elder generation's supernatant weight 4.4%, the subtilisin of supernatant weight 1.1% again | 51mg | 85.1mg |
| 3 | 4min reduces to-5 ℃ | Supernatant weight 4.5% pepsin | 30.2mg | 70.9mg |
| 4 | 10min reduces to-5 ℃ | Supernatant weight 4.5% pepsin | 35.7mg | 50.2mg |
| 5 | 4min reduces to-5 ℃ | The subtilisin of supernatant weight 1% | 32.8mg | 67.2mg |
| 6 | 4min reduces to-5 ℃ | The pepsin of elder generation's supernatant weight 10%, the subtilisin of supernatant weight 5% again | 44.8mg | 48.1mg |
| 7 | 2min reduces to-15 ℃ | Elder generation's supernatant weight 4.5% pepsin, the subtilisin of supernatant weight 1% again | 34.8mg | 75.2mg |
| 8 | 10min reduces to-5 ℃ | Elder generation's supernatant weight 4.5% pepsin, the subtilisin of supernatant weight 1% again | 34.4mg | 65.8mg |
| 9 | 10min reduces to-5 ℃ | Add supernatant weight 4.5% compound protein enzyme hydrolysis 24h | 28.4mg | 45.1mg |
Wherein number 1 for carrying out according to embodiment 1 operation fully, number 2 for carrying out according to embodiment 13 operations.Find by above-mentioned test, take twice different protease hydrolyzed of rapid frozen cooling of the present invention and difference, can increase content of effective greatly.
Through overtesting, find that the prepared product of other embodiments of the invention also all meets above-mentioned test example trend, have similar effect with embodiment 1,11,13.The present invention repeats no longer one by one.
Claims (17)
1. a deproteinized calf blood extractive injection is characterized in that, described injection contains following material for every milliliter: total solid 160～200mg, wherein wt umber are 0～20 part of amino acid/11,25～35 parts of polypeptide, 15～25 parts of inorganic salts; Xylitol 100～150mg; Epsilon-polylysine 60～100mg.
2. deproteinized calf blood extractive injection according to claim 1 is characterized in that, described injection contains following material for every milliliter: total solid 180mg, wherein wt umber are 5 parts of amino acid/11s, 30 parts of polypeptide, 20 parts of inorganic salts; Xylitol 120mg; Epsilon-polylysine 80mg.
3. the preparation method of claim 1 or 2 described deproteinized calf blood extractive injections is characterized in that described calf blood protein-removed extraction prepares according to following steps:
(1) get calf blood and add sodium citrate, add 3～5 times in water, be warming up to 50～60 ℃ under stirring, insulated and stirred 40～60min stirs down and is cooled to-10～0 ℃ rapidly, and temperature fall time is 2～5min, and is centrifugal at-10～0 ℃ of insulated and stirred 2～4h, gets supernatant;
(2) will going up the supernatant that makes of step, to regulate pH value be 3～4, adds the pepsin enzymolysis of supernatant weight 4%～5%, filters, and regulating pH value is 7.0～8.5, adds the subtilisin enzymolysis of supernatant weight 1%～1.5%, filters to get filtrate;
(3) will go up the filtrate adding ethanol water stirring that the step obtains, centrifugal, get supernatant;
(4) supernatant is crossed carbon column, merge eluent and percolation liquid, be concentrated into no ethanol flavor then, concentrated solution adds xylitol, and thin up contains xylitol 100～150mg to every ml soln, epsilon-polylysine 60～100mg, and the solution microporous filter membrane ultrafiltration after the dilution gets ultrafiltrate;
(5) ultraviolet sterilization after the ultrafiltrate packing fill, packing promptly.
4. preparation method according to claim 3 is characterized in that, the middle sodium citrate consumption of step (1) is 0.1～0.2 times of calf blood weight; Described centrifugal be rotating speed 2～3krpm, time 30～40min.
5. preparation method according to claim 4 is characterized in that, the middle sodium citrate consumption of step (1) is 0.15 times of calf blood weight.
6. preparation method according to claim 3 is characterized in that, it is at 55～60 ℃ of following enzymolysis 10～20min that step (2) adds the pepsin enzymolysis; Adding the subtilisin enzymolysis is at 30～40 ℃ of following enzymolysis 15～20min.
7. preparation method according to claim 6 is characterized in that, it is at 58 ℃ of following enzymolysis 15min that step (2) adds the pepsin enzymolysis; Adding the subtilisin enzymolysis is at 35 ℃ of following enzymolysis 17min.
8. preparation method according to claim 3 is characterized in that, the described ethanol water of step (3) is 80% ethanol water of 3～5 times of filtrate weight, and described stirring is 40～50 ℃ and stirred 10～20 minutes down.
9. preparation method according to claim 3 is characterized in that, thin up contains xylitol 120mg, epsilon-polylysine 80mg in the step (4) to every ml soln.
10. preparation method according to claim 3 is characterized in that, the described carbon column of step (4) is a medicinal active carbon of getting 1～2 times of described supernatant weight, and the post draw ratio is 4: 1～5: 1.
11. preparation method according to claim 10 is characterized in that, the described carbon column of step (4) is a medicinal active carbon of getting 1.5 times of described supernatant weight; The post draw ratio is 4.5: 1.
12. preparation method according to claim 3, it is characterized in that, the described carbon column of crossing of step (4) is poured in the carbon column for the supernatant that step (3) is obtained, equal to the supernatant liquid level with the activated carbon upper surface, standing adsorption 10～20min, emit percolation liquid, put into the residue supernatant again, according to the above-mentioned steps repeatable operation to putting into whole supernatant, use 80% ethanol water eluting then, weight consumption is 2 times of supernatant weight, and reuse 70% ethanol water eluting, weight consumption are 1 times of supernatant weight.
13. preparation method according to claim 12 is characterized in that, the described standing adsorption of step (4) is 15min.
14. preparation method according to claim 12 is characterized in that, 80% ethanol water eluting described in the step (4) is elution time 20～30min; Described 70% ethanol water eluting is elution time 40～50min.
15. preparation method according to claim 14 is characterized in that, 80% ethanol water eluting described in the step (4) is elution time 25min; Described 70% ethanol water eluting is elution time 45min.
16. preparation method according to claim 3 is characterized in that, the sterilization of the described ultraviolet of step (5) is for being irradiation under ultraviolet ray 20～30min of 230～260nm at 50～58 ℃ with wavelength.
17. preparation method according to claim 16 is characterized in that, the sterilization of the described ultraviolet of step (5) is for being the irradiation under ultraviolet ray 25min of 240nm at 54 ℃ with the wavelength.
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