CN1634987A - Spleen polypeptide extract, its preparing process and use - Google Patents
Spleen polypeptide extract, its preparing process and use Download PDFInfo
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- CN1634987A CN1634987A CN 200410088895 CN200410088895A CN1634987A CN 1634987 A CN1634987 A CN 1634987A CN 200410088895 CN200410088895 CN 200410088895 CN 200410088895 A CN200410088895 A CN 200410088895A CN 1634987 A CN1634987 A CN 1634987A
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Abstract
The present invention discloses a spleen polypeptide extract which is extracted from splenic organ tissues of mammalians except human and prepared through steps of fats removing, homogenation, acid adjusting, freeze thawing and pulverizing, pH values regulation for precipitation, heating, centrifugal filtration, hyperfiltration and the like of mammalian tissues. Said drug, as a drug for immunomodulation, is capable of improving the immune function of bolt body; can be used for treating diseases such as primary and secondary cell immunoincompetence diseases (for example, eczema, reduction of blood platelet, recurrent infection syndromes, etc.), infection contamination of respiratory tract and lung, common cold, chronic hepatitis B, epidemic parotitis, relapsed aphtae and the like; can find auxiliary applications in curing leucocytopenia caused by chemotherapy, leukaemia, reproducibility disorder anaemia, lymphoid tumor and other malignant tumor, improving tumour patients bad deterioration, and improving general debility of postoperative patients.
Description
Technical field
The present invention relates to a kind of spleen polypeptide extract, Its Preparation Method And Use, specifically, relate to a kind of by the spleen polypeptide extract, the Preparation Method And The Use that from the Mammals spleen tissue except that the people, prepare through extraction separation.
Background technology
As everyone knows, the various nutritive ingredients and the activeconstituents of many needed by human body are arranged in the mammalian body, and then have much at the report that from mammiferous heart, liver and muscle tissue, extracts active polypeptide.
And spleen is mammiferous peripheral immune organ, it also is one of the most vigorous organ of animal body intracellular metabolite, owing to wherein contain basic nutritive ingredient of abundant cell and cytokine, particularly promote activeconstituents such as polypeptides matter and the nucleic acid and the immune-regulating factor-oligonucleoside peptide etc. of cellular metabolism, thereby be subjected to investigator's extensive concern.Abroad to the research work of spleen extract early, the existing at present commodity that are used for makeup are as the healthcare products of crydermine and raise immunity, as spleen P.M.G, IM-Encap, Gold-speen 500 etc.
Application number 03133572.1, denomination of invention is composite bio-active capsule and preparation method thereof, discloses a kind of composite bio-active capsule, selecting new born bovine for use is calf within nascent 14 hours, gets its brain, thymus gland, liver, spleen; Select the sheep about being two months pregnant for use, get its embryo and placenta, adopt modern biotechnology, carry out the science compatibility from wherein extracting effective ingredients such as Cerebrolysin Vial, Zadaxin, transfer factor, placenta pepton, hepatocyte generation element.The biological activity that this invention adopts plurality of raw materials to extract respectively can be combined into capsule or tablet by different proportion scales according to different objects.Composite bio-active capsule of the present invention can be reconciled human physiological functions, improves immunizing power, delay senility, and memory, antifatigue, the generation of liver,spleen,kidney and cardiovascular disease can be treated and prevent to beautifying face and moistering lotion again.
" Study on Preparation of pig spleen polypeptide " of " meat industry " 2002 2 phases discloses the preparation technology of pig spleen, and technical process is a spleen--＞degrease--and＞homogenate--＞degreasing--＞enzymolysis--＞termination reaction--＞filter--＞adsorption bleaching--＞gel chromatography--＞vacuum-drying--＞finished product.Concrete technology is: get a certain amount of fresh pig spleen, remove appearance fat, epidermis and the subcutaneous fascia of bulk, add 10 ℃ of aquae destillatas of 1: 1, use tissue mashing machine's homogenate, add normal hexane or the airtight at normal temperatures extracting 2h of ether degreasing in 4: 1 ratios, separate the spleen homogenate that obtains degreasing; With 400ml spleen homogenate, place thermostat water bath, adjust the temperature to (50 ± 2) ℃, with Ca (OH)
2The emulsion regulator solution adds the 30g enzyme to the enzyme optimum pH, after reaction for some time, with 1: 1HCl transfers pH to neutral, be warming up to 80 ℃ simultaneously and keep 15min, make the enzymatic inactivation stopped reaction, husky cloth filters, filtrate adds 5% charcoal absorption decolouring, suction filtration is crossed G-15 dextran gel column, the solution of capture range, 50 ℃ of vacuum concentration dryings get the spleen polypeptide finished product.This process using be enzymolysis process, obtain polypeptide by protein in the enzymolysis spleen, the gained polypeptide becomes to be grouped into variable moving and be not original naturally occurring polypeptide.
At present, mostly the disease of puzzlement human health is because human body immune function descends and to cause, so should constantly research and develop the medicine that some can improve the human immunological competence; To improve and to improve body's immunity, activate and the enhancing body non-specific immune function, improve the anti-disease ability of human body.
Summary of the invention
The object of the present invention is to provide a kind of spleen polypeptide extract, this product is to contain the spleen polypeptide extract that extracts preparation from the Mammals spleen tissue except that the people.
Another object of the present invention is to provide the preparation method of spleen polypeptide extract.
A further object of the present invention provides the purposes of spleen polypeptide extract medicine aspect disease therapeuticing medicines such as preparation treatment primary and Secondary cases cellular immunity deficiency (as eczema, thrombopenia, repeatedly infect syndromes etc.), respiratory tract and pulmonary infection, customary cold, chronic hepatitis B, mumps, recurrent aphtha.
A further object of the present invention provides the leukopenia that the spleen polypeptide extract medicine causes at preparation treatment chemicotherapy, leukemia, reproducibility obstacle anaemia, lymphoma and other malignant tumours, improve tumour patient cancerate matter, improve postoperative or patient with severe symptoms auxiliary use when in poor health.
Spleen polypeptide extract of the present invention, for extract the active small molecular polypeptide that forms from the Mammals spleen tissue except that the people, molecular weight is less than 10000 dalton.
Wherein, preferred molecular weight is less than 8000 dalton, and most preferred molecular weight is less than 6000 dalton.
Mammals of the present invention can be pig, dog, ox, sheep, deer, horse or rabbit etc.From fresh and healthy spleen tissues such as pig, dog, ox, sheep, deer, horse or rabbit, all can extract active polypeptide of the present invention.
Consider that from factors such as production cost, production technique, constant product quality, clinical efficacies the preferred calf spleen tissue of above-mentioned Mammals cerebral tissue is as the raw materials for production of spleen polypeptide extract.
Spleen polypeptide extract of the present invention by degrease, homogenate, acid adjustment, freeze thawing fragmentation, regulate steps such as pH value precipitation, heating, centrifuging, ultrafiltration and make spleen polypeptide extract, its molecular weight can be that the ultra-filtration membrane of 10000 or 10000 following PSPPs is controlled by selecting molecular weight cut-off for use.
The preparation method of spleen polypeptide extract of the present invention comprises the steps:
A) the pretreated Mammals spleen of learning from else's experience adds water homogenate, regulates the pH value, multigelation;
B) through 1-3 time centrifugal, filter, regulate pH value, must clear liquid;
C) refilter after the freezing thawing, get spleen polypeptide extract.
Wherein, described pre-treatment is that impurity, fat, epidermis and the subcutaneous fascia of fresh healthy spleen are anticipated.
Adjust pH to 3.0 after the homogenate～5.0, number of freezing and thawing is 1～9 time, makes cytoclasis by multigelation, and the intracellular reactive polypeptide is fully discharged.
Centrifugal condition is 3000～4000 rev/mins, and centrifugation time is 15～30 minutes, but the filtration means that filtration paper pulp filtering or those skilled in the art use always, to play pre-filtering, pretreated effect.After the centrifuging, adjust pH is 6.0～7.5.
In order to reach better centrifugal effect, after a centrifuging, also can once heat cooling, centrifuging again.
Specifically, the preparation method of spleen polypeptide extract of the present invention comprises the steps:
A) get the fresh and healthy spleen, reject impurity and fatty tissue, add the water for injection of 0.5～2.5 times of spleen weight, homogenate is regulated pH value 3.0～5.0, multigelation 1～9 time;
B) homogenate behind last the thawing is centrifugal, and centrifugal condition is 3000～4000 rev/mins, and centrifugation time is 15～30 minutes, filters, supernatant liquor is regulated pH value 6.0～7.5, and heating cooling back is centrifugal, and centrifugal condition is 3000～4000 rev/mins, centrifugation time is 15～30 minutes, filters, and gets clear liquid;
C) melt freezing back, filter, and clear liquid ultra-filtration membrane ultrafiltration 1～2 time, ultrafiltrated is a spleen polypeptide extract.
Described freeze thawing is: freezing below-15 ℃, freeze real back and melt down at 10～40 ℃.
B) in the step, after the centrifuging, be heated to 50 ℃～90 ℃, be 15～30 minutes heat-up time; Be cooled to 10～40 ℃.
More preferred, the preparation method of spleen polypeptide extract of the present invention comprises the steps:
A) get the fresh and healthy spleen, reject impurity and fatty tissue, add 1～2 times of spleen weight water for injection, pH value 3.8～4.2 is regulated in homogenate, freezing below-15 ℃, freeze real back and melts down at 10～40 ℃, repeats freeze thawing 2～4 times;
B) homogenate behind last the thawing is centrifugal, centrifugal condition is 3500～4000 rev/mins, and centrifugation time is 15～20 minutes, filters, supernatant liquor is regulated pH value 6.5～7.0, heat 65 ℃～80 ℃, be 15～20 minutes heat-up time, and the cooling back is centrifugal, centrifugal condition is 3500～4000 rev/mins, centrifugation time is 15～20 minutes, filters, and gets clear liquid;
℃ C)-15 freezing below, freeze real back and melt down at 10～40 ℃, filter, clear liquid gets ultrafiltrated with molecular weight cut-off 10000 following ultra-filtration membrane ultrafiltration 1～2 time, is spleen polypeptide extract.
When clear liquid was carried out ultrafiltration, can adopt molecular weight cut-off was 5000～10000 ultra-filtration membrane or 5000 following ultra-filtration membrane ultrafiltration, is the controlled step of the active polypeptide molecular weight in the spleen polypeptide extract.Such as, use the ultra-filtration membrane of molecular weight cut-off 1000,3000,5000,6000,8000 or 10000, preferably using molecular weight cut-off is 5000 ultra-filtration membrane ultrafiltration.The ultra-filtration membrane that uses is the commercially available prod, and its manufacturer should be the production unit of industry approval, and the filter membrane model of use and size can be determined according to concrete turnout, the ultra-fine filter size selected for use.
Certainly, in order further to obtain the spleen polypeptide extract of desired molecule amount section, can intercept with different ultra-filtration membranes, get ultrafiltrated or trapped fluid, can obtain molecular weight like this is 1000-10000,1000-8000,1000-6000, the spleen polypeptide extract that 1000-5000 or 3000-8000 or the like are required.
For making production technique of the present invention, constant product quality, take to regulate pH value 3.8～4.2 after the homogenate after, freeze thawing again, preparation process B) in for the first time centrifugal back re-adjustment pH value 6.5～7.0, and heat centrifugal freezing again removal of impurities.
Spleen polypeptide extract of the present invention can be through aftertreatment, make acceptable different dosage form on the pharmaceutics, such as tablet, capsule, oral liquid, small-volume injection, bulk capacity injection or lyophilized injectable powder etc., be preferably small-volume injection, lyophilized injectable powder, most preferably be small-volume injection.Described formulation can be carried out according to the ordinary method of acceptable forms on the preparation pharmaceutics.
The present invention most preferably specification of small-volume injection is the 2ml:8mg polypeptide; The 5ml:20mg polypeptide; The 10ml:40mg polypeptide.
Intramuscularly: a 2～8ml, 1 time on the one or follow the doctor's advice.Intravenous drip: a 10ml is dissolved in 0.9% sodium chloride injection or 5%～10% glucose injection of 500ml for 1 time on the one or follow the doctor's advice.
Researchist of the present invention finds through a large amount of experiments and pharmacodynamic study, spleen polypeptide extract medicine of the present invention is an immunomodulator, human body immune function there is dual regulation, can the disorder of rectifier body immunity function, effect with activation and enhancing body non-specific immune function, can promote that the T lymphocyte is ripe and can make not primed lymphocyte activation becoming primed lymphocyte, thereby improved lymphocyte immunologic function, triggered and the resistibility of enhancing body infecting; Also but inducement interferon directly stops the synthetic of virus protein and duplicates, and can strengthen the cell-surface antigens expression, promotes the cytotoxic activity of NK cell, regulates lymphocyte and macrophage function, can obviously improve the body cell immunologic function; This product can stimulate proliferation of bone marrow cells, produces a large amount of white corpuscles, and hemopoietic function is improved.
The stable preparation process maturation of spleen polypeptide extract of the present invention, extract in the spleen natural existence and have the active polypeptide that specified proportion is formed by means such as freeze thawing, centrifugal, ultrafiltration, active polypeptide component molecules amount can be controlled in 1000～10000 scope, with at present main immunoregulation druge relatively, the present invention has determined curative effect, can predict the little or nothing of toxic side effect, advantage with low cost.Spleen polypeptide extract medicine of the present invention can be used for the purposes of disease therapeuticing medicine aspects such as primary and Secondary cases cellular immunity deficiency (as eczema, thrombopenia, repeatedly infect syndromes etc.), respiratory tract and pulmonary infection, customary cold, chronic hepatitis B, mumps, recurrent aphtha, and as the leukopenia that causes of treatment chemicotherapy, leukemia, reproducibility obstacle anaemia, lymphoma and other malignant tumours, improve tumour patient cancerate matter, improve postoperative or patient with severe symptoms auxiliary use when in poor health.
Embodiment
Further describe the present invention with embodiment below, help understanding, but described embodiment only is used to illustrate the present invention rather than restriction the present invention the present invention and advantage thereof, better effects if.
Embodiment 1
Step (1):
A) get fresh and healthy calf spleen 10kg, remove impurity such as manadesma and fatty tissue,, add 10L water for injection with the water for injection washing, homogenate, 3.8 ,-20 ℃ of adjusting pH values are freezing, freeze the taking-up of real back and melt in 15 ℃ of water-baths, repeat freeze thawing 2 times.
B) homogenate behind last the thawing is centrifugal, centrifugal condition is 4000 rev/mins, and centrifugation time is 15 minutes, and supernatant liquor is through paper pulp filtering, behind the filtrate adjust pH 6.5, heat 80 ℃, be 15 minutes heat-up time, centrifugal after being cooled to 35 ℃, centrifugal condition is 4000 rev/mins, centrifugation time is 20 minutes, through paper pulp filtering, gets clear liquid;
C)-20 ℃ freezing, freeze real back and take out and in 12 ℃ of water-baths, melt, behind the paper pulp filtering, the clear liquid molecular weight cut-off be 6000 ultra-filtration membrane (U.S. millipore company, standard ultrafiltration system) ultrafiltration once, ultrafiltrated is spleen polypeptide extract, molecular weight is less than 6000, and content of peptides is 8mg/ml.
Step (2):
With above-mentioned spleen polypeptide extract 1000ml, add the injection water to 2L, the small-volume injection that 2ml/ props up is made in can after the sterile filtration, sterilized 15 minutes for 100 ℃, the packing warehouse-in, every ml contains polypeptide 4mg.
Embodiment 2
Step (1):
A) get fresh and healthy pig spleen 10kg, remove impurity such as manadesma and fatty tissue,, add 20L water for injection with the water for injection washing, homogenate, 4.2 ,-18 ℃ of adjusting pH values are freezing, freeze the taking-up of real back and melt in 10 ℃ of water-baths, repeat freeze thawing once.
B) homogenate behind last the thawing is centrifugal, and centrifugal condition is 3000 rev/mins, and centrifugation time is 30 minutes, supernatant liquor is through paper pulp filtering, behind the filtrate adjust pH 7.0, heat 70 ℃, be 20 minutes heat-up time, centrifugal after being cooled to 20 ℃, centrifugal condition is 4000 rev/mins, and centrifugation time is 30 minutes, through paper pulp filtering, get clear liquid ,-18 ℃ freezing;
C) freezing the taking-up of real back melts in 25 ℃ of water-baths, behind the paper pulp filtering, supernatant liquor is that 10000 ultra-filtration membrane (U.S. millipore company, standard ultrafiltration system) ultrafiltration is once got ultrafiltrated with molecular weight cut-off earlier, be 8000 ultra-filtration membrane (U.S. millipore company again with molecular weight cut-off, the standard ultrafiltration system) ultrafiltration is once got ultrafiltrated, and ultrafiltrated is spleen polypeptide extract, molecular weight is less than 8000, and content of peptides is 5mg/ml.
Step (2):
With above-mentioned spleen polypeptide extract 1000ml, add the injection water to 2L, the small-volume injection that 5ml/ props up is made in can after the sterile filtration, sterilized 15 minutes for 100 ℃, the packing warehouse-in, every ml contains polypeptide 2.5mg.
Embodiment 3
Step (1):
A) get fresh and healthy sheep spleen 10kg, remove impurity such as manadesma and fatty tissue,, add 15L water for injection with the water for injection washing, homogenate, 3.0 ,-15 ℃ of adjusting pH values are freezing, freeze the taking-up of real back and melt in 28 ℃ of water-baths, repeat freeze thawing 4 times.
B) homogenate behind last the thawing is centrifugal, and centrifugal condition is 3500 rev/mins, and centrifugation time is 25 minutes, supernatant liquor is through paper pulp filtering, behind the filtrate adjust pH 6.0, heat 75 ℃, be 15 minutes heat-up time, centrifugal after being cooled to 25 ℃, centrifugal condition is 3500 rev/mins, and centrifugation time is 30 minutes, through paper pulp filtering, get clear liquid ,-15 ℃ freezing.
C) freezing the taking-up of real back melts in 15 ℃ of water-baths, behind the paper pulp filtering, supernatant liquor is that 8000 ultra-filtration membrane (U.S. millipore company, standard ultrafiltration system) ultrafiltration is once got ultrafiltrated with molecular weight cut-off earlier, be 5000 ultra-filtration membrane (U.S. millipore company again with molecular weight cut-off, the standard ultrafiltration system) ultrafiltration is once got ultrafiltrated, and ultrafiltrated is spleen polypeptide extract, molecular weight is less than 5000, and content of peptides is 7mg/ml.
Step (2):
With above-mentioned spleen polypeptide extract 1000ml, add the injection water to 2L, the small-volume injection that 10ml/ props up is made in can after the sterile filtration, sterilized 15 minutes for 100 ℃, the packing warehouse-in, every ml contains polypeptide 3.5mg.
Embodiment 4
Step (1):
A) get fresh and healthy pig spleen 10kg, remove impurity such as manadesma and fatty tissue,, add 25L water for injection with the water for injection washing, homogenate, 5.0 ,-25 ℃ of adjusting pH values are freezing, freeze the taking-up of real back and melt in 40 ℃ of water-baths, repeat freeze thawing once.
B) homogenate behind last the thawing is centrifugal, and centrifugal condition is 3000 rev/mins, and centrifugation time is 30 minutes, supernatant liquor is through paper pulp filtering, behind the filtrate adjust pH 7.5, heat 65 ℃, be 20 minutes heat-up time, centrifugal after being cooled to 20 ℃, centrifugal condition is 4000 rev/mins, and centrifugation time is 30 minutes, through paper pulp filtering, get clear liquid ,-18 ℃ freezing;
C) freezing the taking-up of real back melts in 35 ℃ of water-baths, behind the paper pulp filtering, supernatant liquor is that 10000 ultra-filtration membrane (U.S. millipore company, standard ultrafiltration system) ultrafiltration is once got ultrafiltrated with molecular weight cut-off earlier, be 1000 ultra-filtration membrane (U.S. millipore company again with molecular weight cut-off, the standard ultrafiltration system) ultrafiltration is once got trapped fluid, and trapped fluid is spleen polypeptide extract, molecular weight is 1000-10000, and content of peptides is 5mg/ml.
Step (2):
With above-mentioned spleen polypeptide extract 1000ml, add the injection water to 2L, the small-volume injection that 5ml/ props up is made in can after the sterile filtration, sterilized 15 minutes for 100 ℃, the packing warehouse-in, every ml contains polypeptide 2.5mg.
Embodiment 5
With reference to embodiment 1～3, after the spleen polypeptide extract preparation, by known method, after earlier extract suitably being concentrated, make softwood after adding an amount of dry starch mixing, cross 16 mesh sieves and make particle, add the lubricant mixing after the drying, with the compacting of 10mm punch die in flakes, promptly get medicinal tablet.
Embodiment 6
With reference to embodiment 1～3, after the spleen polypeptide extract preparation, add suitable excipient (as Dextran 40, N.F,USP MANNITOL etc.), carry out the degerming can, by known method, the freeze-dried powder product is made in lyophilize.
Embodiment 7
With reference to embodiment 1～3, spleen polypeptide extract is put in the dilute preparing tank; Drop into the sodium-chlor of 0.9% final preparation cumulative volume in dense preparing tank, add 0.4 ‰ gacs and boil decarbonization filtering, after the cooling, medical filtration to dilute preparing tank, is added water for injection to the dosing amount in dilute preparing tank, transferring PH is 6.8～7.0, filters.Embedding is in infusion bottle, and 100 ℃ of flowing steam sterilizations 30 minutes promptly get infusion products.
Embodiment 8
With reference to embodiment 1～3, after the spleen polypeptide extract preparation,, after earlier extract suitably being concentrated, make softwood after adding an amount of dry starch mixing by known method, cross 16 mesh sieves and make particle, behind the concentrate drying, whole grain, pack, granule.
Embodiment 9
With reference to embodiment 1～3, after the spleen polypeptide extract preparation,, spleen polypeptide extract is added an amount of water for injection by known method, filter, add simple syrup, Sodium Benzoate again, add water to 1000 milliliters again, refrigeration is filtered.Embedding with 100 ℃ of flowing steam sterilizations 30 minutes, promptly gets oral liquid in the 10ml oral liquid bottle.
Embodiment 10
With reference to embodiment 1～3, after the spleen polypeptide extract preparation,, after earlier extract suitably being concentrated, make softwood after adding an amount of dry starch mixing by known method, cross 16 mesh sieves and make particle, whole grain is encapsulated behind 60 ℃ of bake dryings, capsule.
Experimental example 1
This experimental example is the detection of injection inspection item of appearance character, pH value, protein, high molecular weight material and other regulation of the specification 2ml:8mg polypeptide (molecular weight is less than 6000 dalton) of most preferably small-volume injection of the present invention.
√ proterties this product is faint yellow clear liquid.
√ pH value should be 5.5～7.5 (two appendix VI of Chinese Pharmacopoeia version in 2000 H).
√ protein is got this product 2ml, adds 20% sulphosalicylic acid solution 2ml, and muddiness must not take place mixing.
The √ high molecular weight material is measured according to high performance liquid chromatography (two appendix VD of Chinese Pharmacopoeia version in 2000).
Chromatographic condition and system suitability test gel chromatographic columns (as TSK GEL 2000SW 7.8mm * 300mm, 5 μ m); Moving phase is trifluoracetic acid-acetonitrile-water (0.05: 10: 90): the detection wavelength is 214nm.Number of theoretical plate calculates by insulin spikes should be lower than 3000.
It is an amount of that Regular Insulin (molecular weight 5800) is got in the preparation of reference substance solution, makes the solution that contains 1mg among every 1ml with moving phase, in contrast product solution.
This product is got in the preparation of need testing solution, adds moving phase and is diluted to the solution that contains polypeptide 1mg among every 1ml, as need testing solution.
Assay method is got reference substance solution and each 20 μ l of need testing solution, injects liquid chromatograph respectively, the record color atlas.Calculate by area normalization method, must not cross 4.0% less than the peak area sum of insulin spikes retention time.
Other should meet relevant every regulation (2000 editions two appendix IB of Chinese Pharmacopoeia) under the injection item √.
Experimental example 2
This experimental example is the qualitative test of the specification 2ml:8mg polypeptide (molecular weight is less than 6000 dalton) of most preferably small-volume injection of the present invention.
√ gets this product 2ml, adds biuret solution and (gets copper sulfate 1.50g and Seignette salt 6.0g, add water 500ml and make dissolving, add 10% sodium hydroxide solution 300ml while stirring, be diluted with water to 1000ml, mixing) 4ml, mixing, room temperature was placed 15 minutes, and solution should show bluish voilet to red-purple.
√ gets this product 1ml, adds ninhydrin solution number droplet, and heating should show bluish voilet.
Experimental example 3
This experimental example is the contained polypeptide quantitative assay of specification 2ml:8mg polypeptide (molecular weight is less than 6000 dalton) of most preferably small-volume injection of the present invention.Contain polypeptide and should be 90.0%～110.0% of labelled amount
It is an amount of that determining content of peptides is got this product, adds water and make the solution that contains the polypeptide of 0.15mg among every 1ml approximately, and as need testing solution, precision is measured 1.0ml, measures according to forint phenol assay method.
Reagent alkaline copper solution is got sodium hydroxide 10g, and yellow soda ash 50g adds water 400ml and makes dissolving, as first liquid; Get soluble tartrate 0.5g, add water 50ml and make its dissolving, other gets copper sulfate 0.25g, adds water 30ml and makes its dissolving, two liquid is mixed, as second liquid.
Before facing usefulness, merge first, second two liquid, and add water to 500ml.
The bovine serum albumin reference substance is got in the preparation of methodology reference substance solution, adds water and makes the solution that contains 0.3mg among every 1ml.
Preparing of need testing solution according to the method preparation of regulation down of each kind item.
The preparation precision of typical curve is measured reference substance solution 0.0ml, 0.1ml, 0.3ml, 0.5ml, 0.7ml, 0.9ml, put respectively in the tool plug test tube, respectively add water to 1.0ml, add alkaline copper test solution 1.0ml more respectively, shake up, each adds forint phenol test solution (getting the stock solution 1 → 16 in the forint test solution) 4.0ml, mixing immediately, put in 55 ℃ of water-baths accurate response 5 minutes, put cooling bath 10 minutes, and measured optical density at the wavelength place of 650nm according to spectrophotometry (two appendix IV of Chinese Pharmacopoeia version in 2000 B); Manage as blank with No. 0.Calculate regression equation with reference substance solution concentration and respective absorption degree.
The assay method precision is measured need testing solution 1.0ml, the method under the preparation of sighting target directrix curve, and " from adding the alkaline copper test solution " measured in accordance with the law, from the content of regression equation calculation polypeptide, and multiply by extension rate, promptly.
By three batches of mensuration, every ml content results sees the following form:
| Lot number | Polypeptide (mg) |
| ????20031201 | ????4.12 |
| ????20031202 | ????3.96 |
| ????20031203 | ????4.24 |
Experimental example 4
This experimental example is specification 2ml:8mg polypeptide (molecular weight is less than the 6000 dalton) toxicity test of most preferably small-volume injection of the present invention:
√ is unusual, and disposition is got this product, checks in accordance with the law and (two appendix XI of Chinese Pharmacopoeia version in 2000 C) presses the intravenous injection administration, should be up to specification.
The √ hypersensitive test is got this product, checks (attached) in accordance with the law, should be up to specification.
For examination animal health cavy, male and female all can, body weight 250～350g before the test and in the observation period of test, all should raise by normal raising condition, does the animal of this test and must not reuse.
Test procedure is got 6 of above-mentioned cavys, every other day abdominal injection this product 0.5ml, and 3 times, be divided into two groups then, 3 every group, inject back the 14th day and intravenous injection 1.0ml on the 21st respectively for the first time,
The result judges after the intravenous injection all anaphylaxis must not occur in 15 minutes, as perpendicular hair, and the two or more persons in expiratory dyspnea, sneeze, retch or the phenomenon such as cough 3: or one of rale, tic, collapse or phenomena of mortality person should be judged to the positive.
The √ bacterial endotoxin is got this product, check in accordance with the law contain rhzomorph in the bacterium among (two appendix XI of Chinese Pharmacopoeia version in 2000 E) every 1ml should be less than 2EU.
Conclusion: medicine composition injection of the present invention meets the injection liquid requirement, does not have any toxic action, uses human body safety.
Experimental example 5
This experimental example is that the specification 2ml:8mg polypeptide (molecular weight is less than 6000 dalton) of most preferably small-volume injection of the present invention is being treated the recurrent respiratory tract infection clinical effectiveness, and the good efficacy of injection liquid of the present invention is described.
Clinical settings:
Recurrent respiratory tract infection patient's 50 examples, wherein the male sex is 28 examples, the women is 22 examples.Age 8-15 year, average 11.3 years old.
All cases are recurrent respiratory tract infection patient symptoms such as (have in cough, heating, antiadoncus and the lung) rales, and the mensuration of patient's immune indexes meets following standard: 1. IgA≤2s; 2. CD
3And CD
4All≤2s; 3. CD
3≤ 2s and CD
3/ CD
8＜1.5, meet 1 in above-mentioned 3 at least.
Methods of treatment:
The equal intramuscularly of patient: a 4ml most preferably small-volume injection of the present invention, first two weeks 3 times weekly, 2 .3 were a course of treatment in individual month weekly later on.
Efficacy determination:
The mensuration of immune indexes: respectively check immune indexes 1 time before the medication and after finishing the course of treatment, carry out clinical observation and record before and after the treatment, followed up a case by regular visits to 1 time in every month.(1) produce effects: do not fall ill or only fell ill I time in 3 months, it is normal that immune indexes recovers substantially.(2) effective: the morbidity number of times reduces or does not reduce, but the state of an illness is more preceding light, and the course of disease shortens, and immune indexes transfers to normal or the same.(3) invalid: morbidity number of times, the course of disease all end change, and immune indexes is become a full member normal or lower.
Treatment result:
3 months treatment backs: 1, patient's curative effect: produce effects 31 examples, account for 62%, effective 14 examples account for 28%, and invalid 5 examples account for 10%, and total effective rate is 90%.2, immune indexes changing conditions before and after the treatment: IgA (g/L), CD
3(%), CD
4(%) all significantly improve, all P＜0.01; IgG (g/L), IgM (g/L), CD
8(%), BC all makes moderate progress but not obvious, equal P＞0.05.3, symptom and sign changes before and after the treatment: rale all obviously alleviates in treatment back cough, heating, antiadoncus and the lung, learns by statistics and handles. and difference has highly significant meaning (P is all＜0.01) before and after the treatment.
Conclusion:
Small-volume injection of the present invention has dual regulation to human body immune function, can the disorder of rectifier body immunity function, effect with activation and enhancing body non-specific immune function, can promote that the T lymphocyte is ripe and can make not primed lymphocyte activation becoming primed lymphocyte, thereby improved lymphocyte immunologic function, triggered and the resistibility of enhancing body infecting.Recurrent respiratory tract infection patient IgG, IgA, CD
3, CD
4/ CD
8Obviously reduce, show that its cellular immunization is low, immunologic function disorder, this is the major reason that recurrent respiratory tract infection produces.Using small-volume injection of the present invention to treat back 7 immune indexes all improves.Along with the raising of immune indexes, the morbidity number of times reduces, and has shortened the course of disease, and clinical symptom and sign all have and alleviate.Illustrate that small-volume injection of the present invention has recovery and enhancement to patient's body's immunity.
Experimental example 6
This experimental example be the specification 5ml:20mg polypeptide (molecular weight is less than 6000 dalton) of most preferably small-volume injection of the present invention at treatment chronic hepatitis B clinical efficacy, illustrate that injection liquid of the present invention has good efficacy.
1, clinical settings:
Chronic hepatitis B patient 60 examples are divided into treatment group and control group, each male 15 example, women 15 examples, and the age is 25-40 year, and two groups of courses of disease are 1-2, and average 1.5 years, all patients all met the viral hepatitis standard.
2, methods of treatment:
The treatment group is used injection liquid of the present invention and the treatment of conventional hepatic, intramuscular injection injection liquid 5ml of the present invention, and inferior on every Wendesdays, one month is a course of treatment, control group is only used conventional hepatic treatment.Medication before and after look liver function and hepatitis B virus five indices.
3, efficacy determination:
Cure: HBsAg.HBeAg turns out cloudy, and liver function recovery is normal; The basic healing: HBeAg changes sun, and liver function recovery is normal; Take a turn for the better; More than three have one to transfer to normal; Invalid: every index no change before and after the treatment.
4, result of treatment:
The treatment group: cure 15 examples, 10 examples that take a turn for the better, total effective rate is 83.3%; Control group: cure 8 examples, cure 1 example substantially, 3 examples that take a turn for the better, total effective rate 4%.
Although the present invention has been done detailed explanation and has quoted some specific exampless as proof, for a person skilled in the art, only otherwise leave that the spirit and scope of the present invention can be done various variations or correction is obvious.
Claims (10)
1. a spleen polypeptide extract is characterized in that, for extract the active small molecular polypeptide that forms from the Mammals spleen tissue except that the people, molecular weight is less than 10000 dalton.
2. a kind of spleen polypeptide extract according to claim 1 is characterized in that, the molecular weight of described active small molecular polypeptide is less than 8000 dalton, and preferred molecular weight is less than 6000 dalton.
3. a kind of spleen polypeptide extract according to claim 1 is characterized in that, described Mammals be in pig, dog, ox, sheep, deer, horse or the rabbit any one, be preferably ox.
4. a preparation method who prepares the described spleen polypeptide extract of claim 1 is characterized in that, comprises the steps:
A) the pretreated Mammals spleen of learning from else's experience adds water homogenate, regulates the pH value, multigelation;
B) through 1-3 time centrifugal, filter, regulate pH value, must clear liquid;
C) refilter after the freezing thawing, get spleen polypeptide extract.
5. the preparation method of spleen polypeptide extract according to claim 4 is characterized in that, adjust pH to 3.0 after the homogenate～5.0, and number of freezing and thawing is 1～9 time; Centrifugal condition is 3000～4000 rev/mins, and centrifugation time is 15～30 minutes, and after the centrifuging, adjust pH is 6.0～7.5.
6. according to claim 4 or 5 described spleen polypeptide extract process for preparing medicine, it is characterized in that, comprise the steps:
A) get the fresh and healthy spleen, reject impurity and fatty tissue, add the water for injection of 0.5～2.5 times of spleen weight, homogenate is regulated pH value 3.0～5.0, multigelation 1～9 time;
B) homogenate behind last the thawing is centrifugal, and centrifugal condition is 3000～4000 rev/mins, and centrifugation time is 15～30 minutes, filters, supernatant liquor is regulated pH value 6.0～7.5, and heating cooling back is centrifugal, and centrifugal condition is 3000～4000 rev/mins, centrifugation time is 15～30 minutes, filters, and gets clear liquid;
C) melt freezing back, filter, and clear liquid ultra-filtration membrane ultrafiltration 1～2 time, ultrafiltrated is a spleen polypeptide extract.
7. according to claim 4 or 5 described spleen polypeptide extract process for preparing medicine, it is characterized in that, comprise the steps: A) get the fresh and healthy spleen, reject impurity and fatty tissue, add 1～2 times of spleen weight water for injection, homogenate, regulate pH value 3.8～4.2, freezing below-15 ℃, melt down at 10～40 ℃, repeat freeze thawing 2～4 times;
B) homogenate behind last the thawing is centrifugal, centrifugal condition is 3500～4000 rev/mins, and centrifugation time is 15～20 minutes, filters, supernatant liquor is regulated pH value 6.5～7.0, heat 65 ℃～80 ℃, be 15～20 minutes heat-up time, and the cooling back is centrifugal, centrifugal condition is 3500～4000 rev/mins, centrifugation time is 15～20 minutes, filters, and gets clear liquid;
℃ C)-15 freezing below, freeze real back and melt down at 10～40 ℃, filter, clear liquid gets ultrafiltrated with the ultra-filtration membrane ultrafiltration of molecular weight cut-off 10000 below 1～2 time, is spleen polypeptide extract.
8. spleen polypeptide extract according to claim 1 and 2, it is characterized in that, described spleen polypeptide extract can be through aftertreatment, make acceptable different dosage form on the pharmaceutics, such as oral liquid, small-volume injection, bulk capacity injection, tablet, capsule or lyophilized injectable powder, be preferably small-volume injection, lyophilized injectable powder, most preferably be small-volume injection.
9. claim 1 or 2 described spleen polypeptide extracts are at preparation treatment primary and Secondary cases cellular immunity deficiency, as eczema, thrombopenia, repeatedly infect syndromes, the purposes of respiratory tract and pulmonary infection, customary cold, chronic hepatitis B, mumps, recurrent aphtha disease medicament aspect.
10. the leukopenia that causes at preparation treatment chemicotherapy of claim 1 or 2 described spleen polypeptide extracts, leukemia, reproducibility obstacle anaemia, lymphoma and other malignant tumours, improve tumour patient cancerate matter, improve the purposes of postoperative or patient with severe symptoms medicine aspect when in poor health.
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