CN100441199C - Spleen extracts, its preparation method and use - Google Patents
Spleen extracts, its preparation method and use Download PDFInfo
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- CN100441199C CN100441199C CNB2005101027731A CN200510102773A CN100441199C CN 100441199 C CN100441199 C CN 100441199C CN B2005101027731 A CNB2005101027731 A CN B2005101027731A CN 200510102773 A CN200510102773 A CN 200510102773A CN 100441199 C CN100441199 C CN 100441199C
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Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The present invention discloses a spleen extract extracted and prepared from the spleen tissue of mammals except for people. The spleen tissue of mammals is treated by the steps of degreasing, homogenate, freeze thawing pulverization, precipitation, ultrafiltration, etc. for preparing the spleen extract; the spleen extract comprises the following components: polypeptide, free amino acid, nucleic acid and total sugar; the molecular weight of each component is smaller than 10000 daltons. The spleen extract is applied to the preparation of medicine for treating diseases, such as constitutional cellular immunity deficiency, subsequent cellular immunity deficiency that (such as eczema, thrombocytopenia, recurrent infection syndromes, etc.), respiratory tract infection, pulmonary infection, regular common cold, chronic hepatitis b, epidemic parotitis, recurrent aphtha, etc., auxiliary medicine for treating leukopenia, leukemia, reproduced disorder anemia, lymphomas and other malignant tumors due to radiotherapy and chemotherapy for improving the malignant deterioration and the virus infection of tumor patients and improving the weak health of postoperative patients or serious patients, etc.
Description
[technical field]
The present invention relates to a kind of spleen extract, Its Preparation Method And Use, specifically, relate to a kind of by from the mammal spleen tissue except that the people through spleen extract, the Preparation Method And The Use of extraction separation preparation, belong to field of pharmaceutical preparations.
[background technology]
As everyone knows, the various nutritional labelings and the active component 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 muscular 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 nutritional labeling of abundant cell and cytokine, particularly promote the active component of cellular metabolism such as polypeptides matter, free amino acid, nucleic acid, various saccharide, thereby be subjected to the extensive concern of researcher.Abroad to the research work of spleen extract early, the existing at present commodity that are used for cosmetics are as the health product of crydermine and raise immunity, as spleenP.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, liver, spleen; Select the sheep about being two months pregnant for use, get its embryo and Placenta Hominis, adopt modern biotechnology, carry out the science compatibility from wherein extracting active ingredients such as Cerebrolysin Vial, thymosin, 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; Invent described composite bio-active capsule, can reconcile human physiological functions, improve immunity, slow down aging, improving memory, resisting fatigue, the generation of liver,spleen,kidney and cardiovascular disease can be treated and prevent to looks improving and the skin nourishing again.
" Study on Preparation of Lien Sus domestica polypeptide " of " meat industry " 2002 2 phases discloses the preparation technology of pig spleen, and technological process is a spleen--＞degrease--and＞homogenate--＞defat--＞enzymolysis--＞cessation 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 n-hexane or the airtight at normal temperatures extracting 2h of ether defat in 4: 1 ratios, separate the spleen homogenate that obtains defat; 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 a period of 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% activated carbon adsorption decolouring, sucking 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 enzymatic isolation method, obtain polypeptide by protein in the enzymolysis spleen, the gained polypeptide becomes to be grouped into variable moving, and is 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 body immunity; To improve and to improve body's immunity, activate and the enhancing body non-specific immunity, 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 extract, described spleen extract is by extracting preparation in the mammal spleen tissue except that the people.
Another object of the present invention is to provide the preparation method of above-mentioned spleen extract.
A further object of the present invention provides above-mentioned spleen extract and is preparing treatment constitutional and Secondary cases cellular immunity deficiency (as eczema, thrombocytopenia, repeatedly infect syndrome etc.), respiratory tract and pulmonary infection, habitual common cold, chronic hepatitis B, mumps, the purposes of disease therapeuticing medicine aspects such as recurrent aphtha, and preparing the leukopenia that the treatment chemicotherapy causes, leukemia, reproducibility obstacle anemia, lymphoma and other malignant tumor, improve the tumor patient matter that cancerates, viral infection, the auxiliary purposes of using the medicine aspect when improving postoperative or patient with severe symptoms's physical weakness.
Spleen extract of the present invention, form for from the mammal spleen tissue except that the people, extracting, the component of following weight part ratio: 10～100 parts of polypeptide, 20～80 parts of free amino acids, 1～30 part of nucleic acid, 0～4 part of total sugar, all components molecular weight is all less than 10000 Dao Er; This extract and sulfosalicylic acid reaction, muddiness does not take place in mixing; With biuret reaction, show bluish violet to aubergine; With ninhydrin reaction, show bluish violet; There is absorption at the place at 260～270nm wavelength; The phenolsulfuric acid reagent reacting is positive.
Spleen extract of the present invention preferably contains the component of following weight part ratio: 20～80 parts of polypeptide, 30～60 parts of free amino acids, 5～20 parts of nucleic acid, 1～4 part of total sugar, and all components molecular weight is all less than 8000 dalton;
Most preferably contain: 32～48 parts of polypeptide, 40～60 parts of free amino acids, 8～12 parts of nucleic acid, 1～3 part of total sugar, all components molecular weight is all less than 6000 dalton.
Free amino acid has following aminoacid after measured in the spleen extract of the present invention: Aspartic Acid, glutamic acid, serine, histidine, glycine, threonine, alanine, arginine, valine, methionine, isoleucine, phenylalanine, leucine, lysine, proline, tryptophan, tyrosine etc.
Mammal of the present invention can be pig, Canis familiaris L., cattle, sheep, deer, horse or rabbit etc.
From fresh and healthy spleen tissues such as pig, Canis familiaris L., cattle, sheep, deer, horse or rabbit, all can extract spleen extract of the present invention.
Consider that from factors such as production cost, production technology, constant product quality, clinical efficacies the preferred calf spleen tissue of above-mentioned mammal cerebral tissue is as the raw materials for production of spleen extract.
Spleen extract of the present invention is for to make spleen extract from the mammal spleen tissue except that the people by steps such as degrease, homogenate, freeze thawing fragmentation, precipitation, ultrafiltration, and its molecular weight can be that the ultrafilter membrane of 10000 or 10000 following PSPPs is controlled by selecting molecular cut off for use.
The preparation method of spleen extract of the present invention comprises the steps:
A) the pretreated mammal spleen of learning from else's experience adds water or normal saline homogenate, regulates pH value, multigelation after 1-3 time centrifugal, get supernatant liquid filtering and get clear liquid I;
B) clear liquid I is regulated pH value; Perhaps clear liquid I is regulated the pH value post-heating; Carry out centrifugally then, get supernatant liquid filtering, clear liquid I I;
C) refilter after the freezing thawing of clear liquid I I, get stillness of night ultrafiltration and get spleen extract.
Wherein, described pretreatment 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 cell breakage by multigelation, and intracellular reactive polypeptide, free amino acid, nucleic acid, total sugar etc. are fully discharged.
Adopt freezing-thawing method in the preparation method of the present invention, extract polypeptide, aminoacid, nucleic acid quickly and easily from cerebral tissue, this method has the yield height, and the operating time is short, the characteristics that cost is low.
Centrifugal condition is 3000～4000 rev/mins, and centrifugation time is 15～30 minutes.
After the centrifugal filtration, adjust pH is 6.0～7.5.
In order to reach better centrifugal effect, after a centrifugal filtration, also can once heat cooling, centrifugal filtration again.
Specifically, the preparation method of spleen extract of the present invention comprises the steps:
A) get the fresh and healthy spleen, reject impurity and fatty tissue, the water for injection or the normal saline that add 0.5～2.5 times of spleen weight, pH value 3.0～5.0 is regulated in homogenate, multigelation 1～9 time, homogenate behind last the thawing is centrifugal, and centrifugal condition is 3000～4000 rev/mins, and centrifugation time is 15～30 minutes, get supernatant liquid filtering, get clear liquid I;
B) it is centrifugal that clear liquid I is regulated pH value 6.0～7.5 backs; Perhaps regulate pH value 6.0～7.5 and heating, be cooled to behind the room temperature centrifugally then, centrifugal condition is 3000～4000 rev/mins, and centrifugation time is 15～30 minutes, gets supernatant liquid filtering, clear liquid I I;
C) melt the freezing back of clear liquid I I, filter, and clear liquid ultrafilter membrane ultrafiltration 1～2 time, ultrafiltrate is a spleen extract.
Described freeze thawing is: freezing below-15 ℃, freeze real back and melt down at 10～40 ℃.
Above-mentioned B) in the step, after the centrifugal filtration, be heated to 50 ℃～90 ℃, be 15～30 minutes heat time heating time; Be cooled to 10～40 ℃.
More preferred, the preparation method of spleen 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 or normal saline, pH value 3.8～4.2 is regulated in homogenate ,-15 ℃～-25 ℃ freezing, freeze real back and melt down at 10～40 ℃, repeat freeze thawing 2～4 times; Homogenate behind last the thawing is centrifugal, and centrifugal condition is 3500～4000 rev/mins, and centrifugation time is 15～20 minutes, gets supernatant liquid filtering, gets clear liquid I;
B) clear liquid I is regulated pH value 6.5～7.0 backs centrifugal or adjusting pH value 6.0～7.5 and heating, heats 60 ℃～80 ℃, and be 15～20 minutes heat time heating time, be cooled to behind the room temperature centrifugally, centrifugal condition is 3500～4000 rev/mins, and centrifugation time is 15～20 minutes, get supernatant liquid filtering, get clear liquid I I;
C) clear liquid I I-15 ℃～-25 ℃ freezing, freezes real back and melt down at 10～40 ℃, filters, and clear liquid gets ultrafiltrate with molecular cut off 10000 following ultrafilter membrane ultrafiltration 1～2 time, is spleen extract.
Temperature during centrifugal in the above-mentioned steps can be set at room temperature, also can be to carry out under 0～4 ℃;
Describedly be filtered into paper pulp filtering general in the prior art or the clarification plate filters;
The said process adjust pH uses hydrochloric acid and/or sodium hydroxide;
When clear liquid was carried out ultrafiltration, can adopt molecular cut off was 6000～10000 daltonian ultrafilter membranes or the following ultrafilter membrane ultrafiltration of 6000 dalton, is the controlled step of the active polypeptide molecular weight in the spleen polypeptide extract.Such as, use molecular cut off 1000,3000,5000,6000,8000 or 10000 daltonian ultrafilter membranes, preferably using molecular cut off is 6000 daltonian ultrafilter membrane ultrafiltration.The ultrafilter 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 volume of production, the ultrafilter size selected for use.
Certainly, in order further to obtain the spleen extract of desired molecule amount section, can intercept with different ultrafilter membranes, get ultrafiltrate or trapped fluid, can obtain molecular weight like this is 1000-10000,1000-8000,1000-6000, the required spleen extract of 1000-5000 or 3000-8000 dalton or the like.
For making production technology 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 remove impurity.
Spleen extract of the present invention can be through post processing, 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 dosage form can be carried out according to the conventional method of acceptable forms on the preparation pharmaceutics.
The present invention most preferably specification of small-volume injection is 2ml; 5ml; It is that 3.2～4.8mg, free amino acid are that 4.0～6.0mg, nucleic acid are 0.8～1.2mg, total sugar 100～300 μ g that 10ml, every ml contain polypeptide.。
Intramuscular injection: 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.
Research worker of the present invention finds that through a large amount of experiments and pharmacodynamic study spleen extract medicine of the present invention is immunomodulator and tumor aid treatment medicine.This product has dual regulation to human body immune function, can the disorder of rectifier body immunity function, effect with activation and enhancing body non-specific immunity, 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 resistance of enhancing body infecting; Also but inducement interferon directly stops the synthetic of virus protein and duplicates, and can strengthen the cell surface antigen 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 leukocyte, and hemopoietic function is improved; This product can improve body to the virus and the immunocompetence of tumor, and propagation that can anticancer has certain antitumor action; This product can promote impaired hepatocellular reparation and leukocytic generation.
The stable preparation process maturation of spleen extract of the present invention, extract in the spleen natural existence and have active polypeptide, free amino acid, nucleic acid, the multiple sugared isoreactivity composition that special ratios is formed by means such as freeze thawing, centrifugal, ultrafiltration, molecular weight can be controlled below 10000 dalton, with at present main immunoregulation medicament relatively, the present invention has determined curative effect, can predict the little or nothing of toxic and side effects, advantage with low cost.Spleen extract of the present invention can be used for constitutional and Secondary cases cellular immunity deficiency (as eczema, thrombocytopenia, repeatedly infect syndrome etc.), respiratory tract and pulmonary infection, habitual common cold, chronic hepatitis B, mumps, the purposes of disease therapeuticing medicine aspects such as recurrent aphtha, and in the leukopenia that causes as the treatment chemicotherapy, leukemia, reproducibility obstacle anemia, lymphoma and other malignant tumor, improve the tumor patient matter that cancerates, viral infection, the auxiliary purposes of using the medicine aspect when improving postoperative or patient with severe symptoms's physical weakness.
[specific embodiment]
Following embodiment further describes the present invention, but described embodiment only is used to illustrate the present invention rather than restriction the present invention.
Embodiment 1
It is that 8.0mg, free amino acid are that 10.2mg, nucleic acid are that the molecular weight of 2.2mg, total sugar 260 μ g is less than 6000 daltonian spleen extracts that present embodiment provides every ml to contain polypeptide.Its preparation process is as follows:
A) get fresh and healthy calf spleen 10kg, remove impurity such as fascia and fatty tissue, wash with water for injection, add 10L water for injection, homogenate, it is freezing to regulate 3.8 ,-20 ℃ of pH value, freezing the taking-up of real back melts in 15 ℃ of water-baths, repeat freeze thawing 2 times, the homogenate behind last the thawing is centrifugal, and centrifugal condition is 4000 rev/mins, centrifugation time is 15 minutes, gets supernatant liquid filtering and obtains clear liquid I.
B) with behind the clear liquid I adjust pH 6.5, heat 80 ℃, be 15 minutes heat time heating time, centrifugal after being cooled to 35 ℃, centrifugal condition is 4000 rev/mins, centrifugation time is 20 minutes, gets supernatant liquid filtering, clear liquid I I;
C) clear liquid I I is freezing at-20 ℃, freezing the taking-up of real back melts in 12 ℃ of water-baths, filter the clear liquid molecular cut off be 6000 ultrafilter membrane (U.S. millipore company, the standard ultrafiltration system) ultrafiltration once, ultrafiltrate is spleen extract, molecular weight is less than 6000 dalton, and it is that 8.0mg, free amino acid are that 10.2mg, nucleic acid are 2.2mg, contain total sugar 260 μ g that every after measured ml contains polypeptide.
The injection with small volume that contains above-mentioned spleen extract is provided simultaneously:
Get above-mentioned spleen extract 1000ml, add the injection water to 2L, fill after the aseptic filtration, make the small-volume injection that 2ml/ props up, sterilized 15 minutes for 100 ℃, the packing warehouse-in, it is that 4.0mg, free amino acid are that 5.1mg, nucleic acid are 1.1mg, total sugar 130 μ g that every ml contains polypeptide.
Embodiment 2
It is that 5.0mg, free amino acid are that 7.0mg, nucleic acid are that the molecular weight of 1.6mg, total sugar 180 μ g is less than 8000 daltonian spleen extracts that present embodiment provides every ml to contain polypeptide.Its preparation process is as follows:
A) get fresh and healthy pig spleen 10kg, remove impurity such as fascia and fatty tissue, wash with water for injection, add 20L water for injection, homogenate, it is freezing to regulate 4.2 ,-18 ℃ of pH value, freezing the taking-up of real back melts in 10 ℃ of water-baths, repeat freeze thawing once, the homogenate behind last the thawing is centrifugal, and centrifugal condition is 3000 rev/mins, centrifugation time is 30 minutes, gets supernatant liquid filtering and obtains clear liquid I;
B) with behind the clear liquid I filtrate adjust pH 7.0, centrifugal, centrifugal condition is 4000 rev/mins, and centrifugation time is 30 minutes, gets supernatant liquid filtering, gets clear liquid I I;
C) clear liquid I I is freezing at-18 ℃, freezing the taking-up of real back melts in 25 ℃ of water-baths, filter, supernatant is 10000 a daltonian ultrafilter membranes (U.S. millipore companies with molecular cut off earlier, the standard ultrafiltration system) ultrafiltration once, get ultrafiltrate, the reuse molecular cut off is 8000 a daltonian ultrafilter membranes (U.S. millipore companies, the standard ultrafiltration system) ultrafiltration once, get ultrafiltrate, ultrafiltrate is spleen extract, and molecular weight is less than 8000 dalton, and it is that 5.0mg, free amino acid are that 7.0mg, nucleic acid are 1.6mg, total sugar 200 μ g that every after measured ml contains polypeptide.
The injection with small volume that contains above-mentioned spleen extract is provided simultaneously:
Get above-mentioned spleen extract 1000ml, add the injection water to 2L, fill after the aseptic filtration, make the small-volume injection that 5ml/ props up, sterilized 15 minutes for 100 ℃, the packing warehouse-in, it is that 2.5mg, free amino acid are that 3.5mg, nucleic acid are 0.8mg, total sugar 100 μ g that every ml contains polypeptide.
Embodiment 3
It is that 7.0mg, free amino acid are that 8.0mg, nucleic acid are that the molecular weight of 3.0mg, total sugar 360 μ g is less than 5000 daltonian spleen extracts that present embodiment provides every ml to contain polypeptide.Its preparation process is as follows:
A) get the dirty 10kg of fresh and healthy Lien Caprae seu ovis, remove impurity such as fascia and fatty tissue, wash with water for injection, add 15L water for injection, homogenate, it is freezing to regulate 3.0 ,-15 ℃ of pH value, freezing the taking-up of real back melts in 28 ℃ of water-baths, repeat freeze thawing 4 times, the homogenate behind last the thawing is centrifugal, and centrifugal condition is 3500 rev/mins, centrifugation time is 25 minutes, gets supernatant liquid filtering and obtains clear liquid I;
B) with behind the clear liquid I filtrate adjust pH 6.0, heat 75 ℃, be 15 minutes heat time heating time, centrifugal after being cooled to 25 ℃, centrifugal condition is 3500 rev/mins, centrifugation time is 30 minutes, gets supernatant liquid filtering, clear liquid I I;
C) clear liquid I I is freezing at-15 ℃, freezing the taking-up of real back melts in 15 ℃ of water-baths, filter, supernatant is 8000 a daltonian ultrafilter membranes (U.S. millipore companies with molecular cut off earlier, the standard ultrafiltration system) ultrafiltration once, get ultrafiltrate, the reuse molecular cut off is 5000 a daltonian ultrafilter membranes (U.S. millipore companies, the standard ultrafiltration system) ultrafiltration once, get ultrafiltrate, ultrafiltrate is spleen extract, and molecular weight is less than 5000 dalton, and it is that 7.0mg, free amino acid are that 8.0mg, nucleic acid are 3.0mg, total sugar 360 μ g that every after measured ml contains polypeptide.
The injection with small volume that contains above-mentioned spleen extract is provided simultaneously:
Get above-mentioned spleen extract 1000ml, add the injection water to 2L, fill after the aseptic filtration, make the small-volume injection that 10ml/ props up, sterilized 15 minutes for 100 ℃, the packing warehouse-in, it is that 3.5mg, free amino acid are that 4.0mg, nucleic acid are 1.5mg, total sugar 180 μ g that every ml contains polypeptide.
Embodiment 4
It is that 5.0mg, free amino acid are that 5.3mg, nucleic acid are that the molecular weight of 1.5mg, total sugar 160 μ g is less than the daltonian spleen extract of 1000-10000 that present embodiment provides every ml to contain polypeptide.Its preparation process is as follows:
A) get fresh and healthy pig spleen 10kg, remove impurity such as fascia and fatty tissue, wash with normal saline, add the 25L normal saline, homogenate, it is freezing to regulate 5.0 ,-25 ℃ of pH value, freezing the taking-up of real back melts in 40 ℃ of water-baths, repeat freeze thawing once, the homogenate behind last the thawing is centrifugal, and centrifugal condition is 3000 rev/mins, centrifugation time is 30 minutes, gets supernatant liquid filtering and obtains clear liquid I;
B) with behind the clear liquid I filtrate adjust pH 7.5, heat 65 ℃, be 20 minutes heat time heating time, centrifugal after being cooled to 20 ℃, centrifugal condition is 4000 rev/mins, centrifugation time is 30 minutes, gets supernatant liquid filtering, clear liquid I I;
C) clear liquid I I is freezing at-18 ℃, freezing the taking-up of real back melts in 35 ℃ of water-baths, filter, supernatant is 10000 a daltonian ultrafilter membranes (U.S. millipore companies with molecular cut off earlier, the standard ultrafiltration system) ultrafiltration once, get ultrafiltrate, the reuse molecular cut off is 1000 a daltonian ultrafilter membranes (U.S. millipore companies, the standard ultrafiltration system) ultrafiltration once, get trapped fluid, trapped fluid is spleen extract, and molecular weight is 1000-10000 dalton, and it is 5.0mg that every after measured ml contains polypeptide, free amino acid is 5.3mg, nucleic acid is 1.5mg, total sugar 160 μ g.
The injection with small volume that contains above-mentioned spleen extract is provided simultaneously:
Get above-mentioned spleen extract 1000ml, add the injection water to 2L, fill after the aseptic filtration, make the small-volume injection that 5ml/ props up, sterilized 15 minutes for 100 ℃, the packing warehouse-in, every ml contains polypeptide 2.5mg, it is 2.65mg that every ml contains free amino acid, and it is 0.75mg that every ml contains nucleic acid, and every ml contains total sugar 80 μ g.
Embodiment 5
It is that 8.0mg, free amino acid are that 10.0mg, nucleic acid are that the molecular weight of 2.0mg, total sugar 300 μ g is less than 6000 daltonian spleen extracts that present embodiment provides every ml to contain polypeptide.Its preparation process is as follows:
A) get fresh and healthy calf spleen 10kg, remove impurity such as fascia and fatty tissue, wash with water for injection, add 10L water for injection, pH value 4.0 is regulated in homogenate,-20 ℃ freezing, freeze the taking-up of real back and melt in 20 ℃ of water-baths, repeat freeze thawing 2 times, the homogenate behind last the thawing is centrifugal, centrifugal condition is 4000 rev/mins, centrifuging temperature is set at 4 ℃, and centrifugation time is 15 minutes, gets supernatant liquid filtering and obtains clear liquid I.
B) with behind the clear liquid I adjust pH 6.5, heat 80 ℃, be 20 minutes heat time heating time, centrifugal after being cooled to 25 ℃, centrifugal condition is 4000 rev/mins, centrifuging temperature is set at 4 ℃, centrifugation time is 20 minutes, gets supernatant liquid filtering, clear liquid I I;
C) clear liquid I I is freezing at-20 ℃, freezing the taking-up of real back melts in 15 ℃ of water-baths, filter, the clear liquid molecular cut off is 6000 a daltonian ultrafilter membranes (U.S. millipore companies, the standard ultrafiltration system) ultrafiltration once, ultrafiltrate is spleen extract, and molecular weight is less than 6000, and it is that 8.0mg, free amino acid are that 10.0mg, nucleic acid are 2.0mg, contain total sugar 300 μ g that every after measured ml contains polypeptide.
The injection with small volume that contains above-mentioned spleen extract is provided simultaneously:
Get above-mentioned spleen extract 1000ml, add the injection water to 2L, fill after the aseptic filtration, make the small-volume injection that 2ml/ props up, sterilized 15 minutes for 100 ℃, the packing warehouse-in, it is that 4.0mg, free amino acid are that 5.0mg, nucleic acid are 1.0mg, total sugar 150 μ g that every ml contains polypeptide.
Embodiment 6
With reference to embodiment 1～3, after the spleen extract preparation, by known method, after earlier extract suitably being concentrated, make soft material after adding an amount of dry starch mixing, cross 16 mesh sieves and make granule, add the lubricant mixing after the drying, with the compacting of 10mm punch die in flakes, promptly get medicinal tablet.
Embodiment 7
With reference to embodiment 4～5, after the spleen extract preparation, add suitable excipient (as Dextran 40, mannitol etc.), carry out the degerming fill, by known method, the freeze-dried powder product is made in lyophilization.
Embodiment 8
With reference to embodiment 1～3, spleen extract is put in the dilute preparing tank; Drop into the sodium chloride of 0.9% final preparation cumulative volume in dense preparing tank, add 0.4 ‰ active carbons 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 9
With reference to embodiment 4～5, after the spleen extract preparation,, after earlier extract suitably being concentrated, make soft material after adding an amount of dry starch mixing by known method, cross 16 mesh sieves and make granule, behind the concentrate drying, granulate, pack, granule.
Embodiment 10
With reference to embodiment 1～3, after the spleen extract preparation,, spleen 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, cold preservation is filtered.Embedding with 100 ℃ of flowing steam sterilizations 30 minutes, promptly gets oral liquid in the 10ml oral liquid bottle.
Embodiment 11
With reference to embodiment 4～5, after the spleen extract preparation,, after earlier extract suitably being concentrated, make soft material after adding an amount of dry starch mixing by known method, cross 16 mesh sieves and make granule, granulate behind 60 ℃ of bake dryings, encapsulated, capsule.
Experimental example 1
This experimental example is that to contain polypeptide be that 8.0mg, free amino acid are that 10.2mg, nucleic acid are the detection of the molecular weight of 2.2mg, total sugar 260 μ g less than protein, high molecular weight material and other project of 6000 daltonian spleen extracts to the embodiment of the invention 1 described every ml.
Character: this product is faint yellow clear liquid.
PH value: 6.5.
Protein: get this product 2ml, add 20% sulfosalicylic acid solution 2ml, muddiness does not take place in mixing.
Get this product 1ml, add ninhydrin solution number droplet, heating shows bluish violet.
Get this product 2ml, add biuret solution and (get copper sulfate 1.50g and sodium potassium tartrate tetrahydrate 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 shows bluish violet to aubergine.
Get this product, add water and make the solution that contains polypeptide 50 μ g among every ml, measure, absorption maximum is arranged at the wavelength place of 265 ± 2nm according to spectrophotography (Chinese Pharmacopoeia appendix IV in 2000 A).
High molecular weight material: measure according to high performance liquid chromatography (two appendix V of Chinese Pharmacopoeia version in 2000 D).
Chromatographic condition and system suitability test gel chromatographic columns (as TSK GEL 2000SW 7.8mm * 300mm, 5 μ m); Mobile 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 insulin (molecular weight 5800) is got in the preparation of reference substance solution, makes the solution that contains 1mg among every 1ml with mobile phase, in contrast product solution.
This product is got in the preparation of need testing solution, adds mobile phase and is diluted to the solution that contains polypeptide 1mg among every 1ml, as need testing solution.
Algoscopy is got reference substance solution and each 20 μ l of need testing solution, injects chromatograph of liquid respectively, the record chromatogram.Calculate by area normalization method, must not cross 4.0% less than the peak area sum of insulin spikes retention time.
Experimental example 2
This experimental example is the detection and the qualitative analysis of injection inspection item of appearance character, pH value, protein, high molecular weight material and other regulation of the embodiment of the invention 1 described specification 2ml injection (molecular weight is less than 6000 dalton, and it is that 4.0mg, free amino acid are that 5.1mg, nucleic acid are 1.1mg, total sugar 130 μ g that every ml contains polypeptide).
Character: 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: get this product 2ml, add 20% sulfosalicylic acid solution 2ml, muddiness does not take place in mixing.
Get this product 1ml, add ninhydrin solution number droplet, heating shows bluish violet.
Get this product 2ml, add biuret solution and (get copper sulfate 1.50g and sodium potassium tartrate tetrahydrate 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 shows bluish violet to aubergine.
Get this product, add water and make the solution that contains polypeptide 50 μ g among every ml, measure, absorption maximum is arranged at the wavelength place of 265 ± 2nm according to spectrophotography (Chinese Pharmacopoeia appendix IV in 2000 A).
High molecular weight material: measure according to high performance liquid chromatography (two appendix V of Chinese Pharmacopoeia version in 2000 D).
Chromatographic condition and system suitability test gel chromatographic columns (as TSK GEL 2000SW 7.8mm * 300mm, 5 μ m); Mobile 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 insulin (molecular weight 5800) is got in the preparation of reference substance solution, makes the solution that contains 1mg among every 1ml with mobile phase, in contrast product solution.
This product is got in the preparation of need testing solution, adds mobile phase and is diluted to the solution that contains polypeptide 1mg among every 1ml, as need testing solution.
Algoscopy is got reference substance solution and each 20 μ l of need testing solution, injects chromatograph of liquid respectively, the record chromatogram.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 I B of Chinese Pharmacopoeia) under the injection item.
Experimental example 3
It is that 7.0mg, free amino acid are that 8.0mg, nucleic acid are that the molecular weight of 3.0mg, total sugar 360 μ g is measured less than polypeptide quantitative assay in the 5000 daltonian spleen extracts that every ml that this experimental example provides for the embodiment of the invention 3 contains polypeptide.
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 algoscopy.
Reagent alkaline copper solution is got sodium hydroxide 10g, and sodium carbonate 50g adds water 400ml and makes dissolving, as first liquid; Get Soluble tartar. 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 maneuver 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 standard 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 psychrolusia 10 minutes, and measured trap at the wavelength place of 650nm according to spectrophotography (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 algoscopy 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.
Experimental example 4
It is that 5.0mg, free amino acid are that 5.3mg, nucleic acid are that the molecular weight of 1.5mg, total sugar 160 μ g is less than the contained free amino acid quantitative assay of 5000 daltonian spleen extract injection with small volume that every ml that this experimental example provides for embodiment 4 contains polypeptide.
This product is got in the free amine group acidity test and standard amino acid is an amount of, carries out amino acid analysis with amino-acid analyzer or HPLC, and its free aminoacid content should be among every 1ml and contains 4.0～6.0mg.
It is 5.3mg/ml that every 1ml contains free amino acid, wherein:
Aspartic Acid 0.28mg/ml glutamic acid 0.63mg/ml serine 0.19mg/ml
The sweet sour 0.29mg/ml threonine 0.25mg/ml of histidine 0.24mg/ml
Alanine 0.34mg/ml arginine 0.32mg/ml valine 0.29mg/ml
Methionine 0.16mg/ml isoleucine 0.23mg/ml phenylalanine 0.34mg/ml
Leucine 0.85mg/ml lysine 0.52mg/ml proline 0.18mg/ml
Tryptophan 0.09mg/ml tyrosine 0.10mg/ml
Experimental example 5
This experimental example is that to contain polypeptide be that 8.0mg, free amino acid are that 10.0mg, nucleic acid are that the molecular weight of 2.0mg, total sugar 300 μ g is measured less than the contained nucleic acid quantification of 6000 daltonian spleen extracts to the embodiment of the invention 5 described every ml.
Nucleic acid determination: get this product 1.0ml, thin up is to 50ml, as need testing solution.Measure absorption value down according to spectrophotography (two appendix IV of Chinese Pharmacopoeia version in 2000 A) fourth 260nm wavelength, press E
1cm 1%200 calculate nucleic acid content.
It is 2.0mg that embodiment 5 described spleen extracts contain nucleic acid.
Experimental example 6
This experimental example is that to contain polypeptide be that 8.0mg, free amino acid are that 10.0mg, nucleic acid are that the molecular weight of 2.0mg, total sugar 300 μ g is less than the contained total sugar quantitative assay of 6000 daltonian spleen extracts to the embodiment of the invention 5 described every ml.
Total sugar determination: the preparation of reference substance solution: take by weighing the about 50mg of the glucose that is dried to constant weight, put in the 50ml measuring bottle, the adding distil water dissolving also is settled to scale, mixing.Get this solution 5.0ml, add water and be settled to 50ml, mixing, promptly.
The preparation of need testing solution: get this product 10ml, add water and be settled to 50ml, as need testing solution.
The preparation of standard curve: precision is measured reference substance solution 0.0,0.2,0.4,0.6,0.8,1.0ml puts respectively in the tool plug test tube, respectively add entry to 1.0ml, each adds 5% phenol solution 1.0ml, shake up, add 5ml sulphuric acid, put in 37 ℃ of water-baths and accurately heated 10 minutes, be cooled to room temperature rapidly, with No. 0 pipe is blank, according to spectrophotography (two appendix IV of Chinese Pharmacopoeia version in 2000 A), measure trap at the wavelength place of 487nm, be abscissa with concentration, trap is that the vertical coordinate drawing standard curve line linearity of going forward side by side returns, and its correlation coefficient should be greater than 0.99.
Algoscopy: precision is measured need testing solution 1ml, and the preparation method of sighting target directrix curve from " respectively adding 5% phenol solution 1.0ml ", is measured in accordance with the law.From regression equation, obtain total sugar content.
The contained total sugar of embodiment 5 described spleen extracts is 300 μ g.
Experimental example 7
This experimental example is the toxicity test of the embodiment of the invention 5 described spleen extract injection with small volume (it is that 4.0mg, free amino acid are that 5.0mg, nucleic acid are 1.0mg, total sugar 150 μ g that every ml contains polypeptide):
√ 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 Cavia porcellus, 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.
Inspection technique is got 6 of above-mentioned Cavia porcelluss, every other day lumbar 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 anaphylactic reaction must not occur in 15 minutes, as perpendicular hair, and the two or more persons in 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 antibacterial 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 requirement, does not have any toxic action, uses human body safety.
Experimental example 8
This experimental example is that the embodiment of the invention 5 described spleen extract injection with small volume (it is that 4.0mg, free amino acid are that 5.0mg, nucleic acid are 1.0mg, total sugar 150 μ g that every ml contains polypeptide) are being treated the recurrent respiratory tract infection clinical effectiveness, and the good efficacy of injection of the present invention is described.
Clinical settings:
Recurrent respiratory tract infection patient's 50 examples, wherein the male 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. CD3 and CD4 all≤2s; 3. CD3≤2s and CD3/CD8＜1.5 meet 1 in above-mentioned 3 at least.
Therapeutic Method:
The equal intramuscular injection of patient: the 4ml embodiment of the invention 5 described small-volume injections, the last fortnight 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 immune indexes no change or lower.
Therapeutic outcome:
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 situation of change before and after the treatment: IgA (g/L), CD3 (%), CD4 (%) all significantly improve, all P＜0.01; IgG (g/L), IgM (g/L), CD8 (%), BC all make moderate progress but are not obvious, all 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 immunity, 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 resistance of enhancing body infecting.Recurrent respiratory tract infection patient IgG, IgA, CD3, CD4/CD8 obviously reduce, and show that its cellular immunization is low, immunologic function disorder, and 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 symptoms and sign all have and alleviate.Illustrate that small-volume injection of the present invention has recovery and potentiation to patient's body's immunity.
Experimental example 9
This experimental example is the embodiment of the invention 5 described spleen extract injection with small volume (it is that 4.0mg, free amino acid are that 5.0mg, nucleic acid are 1.0mg, total sugar 150 μ g that every ml contains polypeptide) at treatment chronic hepatitis B clinical efficacy, illustrates that injection of the present invention has good efficacy.
1, clinical settings: chronic hepatitis B patient 60 examples, be divided into treatment group and matched group, each male 15 example, women 15 examples, 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, Therapeutic Method: the treatment group is used injection of the present invention and the treatment of conventional hepatic, intramuscular injection injection 5ml of the present invention, and inferior on every Wendesdays, one month is a course of treatment, matched 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, therapeutic effect:
The treatment group: cure 15 examples, 10 examples that take a turn for the better, total effective rate is 83.3%;
Matched group: cure 8 examples, cure 1 example substantially, 3 examples that take a turn for the better, total effective rate 4%.
Experimental example 10
Leukopenia is one of modal untoward reaction in tumor patient radiotherapy, the chemotherapy.This example has illustrated research worker of the present invention in 3 years, Application Example 1 product, and treatment is put, chemotherapy leukopenia 124 examples, the curative effect ideal, other embodiment products also have corresponding effects in application.Particular content is as follows:
Object: cancer patient's 124 examples, nasopharyngeal carcinoma 15 examples wherein, pulmonary carcinoma 18 examples, the esophageal carcinoma 24 examples, mastocarcinoma 32 examples, gastric cancer 24 examples, colorectal cancer 8 examples, oral cancer 3 examples.
Man's 66 examples, women 58 examples.
Age: 30-40 year is 24 examples, and 41-50 year is 46 examples, and 51-60 year is 35 examples, is 19 examples more than 61 years old.Radiotherapy 68 examples, chemotherapy 56 examples.
Leukocyte count is lower than 1.0*10
9/ L is 2 examples, and (1.1-2.0) * 10
9/ L is 25 examples, and (2.1-3.0) * 10
9/ L is 48 examples, and (3.1-4.0) * 10
9/ L is 49 examples.
Therapeutic Method: intravenous drip 10mL/ time, is dissolved in the 500mL0.9% sodium chloride injection 1 time/day.Two weeks were a course of treatment.
Criterion of therapeutical effect: through 1 course of treatment, leukocyte count reaches 4.0*10
9The above person of/L is a produce effects; Do not reach normal value for effective than raising before the treatment 50% or more; Do not raise 10% for invalid.
Therapeutic outcome: produce effects 72 examples (accounting for 58%); Effective 39 examples (accounting for 31%); Invalid 13 examples (accounting for 11%).Total effective rate is more than 89%.
Claims (12)
1, a kind of spleen extract is characterized in that, described spleen extract forms for extracting from the mammal spleen tissue except that the people, and described spleen extract preparation method comprises the steps:
A) the pretreated mammal spleen of learning from else's experience adds water or normal saline homogenate, regulates pH value, multigelation after 1-3 time centrifugal, get supernatant liquid filtering and get clear liquid I;
B) it is centrifugal that clear liquid I is regulated pH value, gets supernatant liquid filtering and get clear liquid I I; Perhaps clear liquid I is regulated the pH value post-heating, and is centrifugal, gets supernatant liquid filtering and gets clear liquid I I;
C) refilter after the freezing thawing of clear liquid I I, get the clear liquid ultrafiltration and get spleen extract;
Described spleen extract contains the component of following weight part ratio: 10～100 parts of polypeptide, 20～80 parts of free amino acids, 1～30 part of nucleic acid, 0～4 part of total sugar, all components molecular weight is all less than 10000 dalton.
2, spleen extract according to claim 1, it is characterized in that, described spleen extract contains the component of following weight part ratio: 20～80 parts of polypeptide, 30～60 parts of free amino acids, 5～20 parts of nucleic acid, 1～4 part of total sugar, all components molecular weight is all less than 8000 dalton.
3, spleen extract according to claim 1 and 2, it is characterized in that, described spleen extract contains the component of following weight part ratio: 32～48 parts of polypeptide, 40～60 parts of free amino acids, 8～12 parts of nucleic acid, 1～3 part of total sugar, all components molecular weight is all less than 6000 dalton.
4, spleen extract according to claim 1 is characterized in that, described spleen extract is for extracting and form by degrease, homogenate, freeze thawing fragmentation, precipitation, ultrafiltration step from the mammal spleen tissue except that the people.
5, spleen extract according to claim 1 is characterized in that, described mammal be in pig, Canis familiaris L., cattle, sheep, deer, horse or the rabbit any one.
6, the preparation method of one of any described spleen extract of claim 1～5 is characterized in that described method comprises the steps:
A) the pretreated mammal spleen of learning from else's experience adds water or normal saline homogenate, regulates pH value, multigelation after 1-3 time centrifugal, get supernatant liquid filtering and get clear liquid I;
B) it is centrifugal that clear liquid I is regulated pH value, gets supernatant liquid filtering and get clear liquid I I; Perhaps clear liquid I is regulated the pH value post-heating, and is centrifugal, gets supernatant liquid filtering and gets clear liquid I I;
C) refilter after the freezing thawing of clear liquid I I, get the clear liquid ultrafiltration and get spleen extract.
7, preparation method according to claim 6 is characterized in that, adjust pH to 3.0～5.0 after the homogenate of A step in the described method, and number of freezing and thawing is 1～9 time, and centrifugal condition is 3000～4000 rev/mins, and centrifugation time is 15～30 minutes; The adjust pH of B step is 6.0～7.5, and centrifugal condition is 3000～4000 rev/mins, and centrifugation time is 15～30 minutes; Use the ultrafilter membrane ultrafiltration of molecular cut off below 10000 in the C step.
8, according to claim 6 or 7 described preparation methoies, it is characterized in that described method comprises the steps:
A) get the fresh and healthy spleen, reject impurity and fatty tissue, add the water for injection or the normal saline of 0.5～2.5 times of spleen weight, homogenate is regulated pH value 3.0～5.0, multigelation 1～9 time; Homogenate behind last the thawing is centrifugal, and centrifugal condition is 3000～4000 rev/mins, and centrifugation time is 15～30 minutes, gets supernatant liquid filtering, gets clear liquid I;
B) it is centrifugal that clear liquid I is regulated pH value 6.0～7.5 backs; Perhaps clear liquid I is regulated pH value 6.0～7.5 and heating, be cooled to then carry out behind the room temperature centrifugal; Centrifugal condition is 3000～4000 rev/mins, and centrifugation time is 15～30 minutes, gets supernatant liquid filtering after centrifugal, supernatant II;
C) melt the freezing back of clear liquid I I, filter, and the clear liquid following ultrafilter membrane ultrafiltration of molecular cut off 10000 dalton 1～2 time, ultrafiltrate is a spleen extract.
9, preparation method according to claim 8 is characterized in that, described method 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 or normal saline, homogenate, it is freezing to regulate 3.8～4.2 ,-15 ℃～-25 ℃ of pH value, melts down at 10～40 ℃, repeats freeze thawing 2～5 times; Homogenate behind last the thawing is centrifugal, and centrifugal condition is 3500～4000 rev/mins, and centrifugation time is 15～20 minutes, gets supernatant liquid filtering, gets clear liquid I;
B) it is centrifugal or regulate pH value 6.0～7.5 that clear liquid I is regulated pH value 6.5～7.0 backs, heats 60 ℃～80 ℃, and be 15～20 minutes heat time heating time, is cooled to behind the room temperature centrifugal; Centrifugal condition is 3500～4000 rev/mins, and centrifugation time is 15～20 minutes, gets supernatant liquid filtering, gets clear liquid I I;
C) clear liquid I I-15 ℃～-25 ℃ freezing, freeze real back and melt down at 10～40 ℃, filter, clear liquid is with the following ultrafilter membrane ultrafiltration of molecular cut off 8000 dalton 1～2 time, ultrafiltrate, be spleen extract.
10, preparation method according to claim 8, it is characterized in that, described spleen extract is made acceptable different dosage form on the pharmaceutics through post processing, and it is oral liquid, small-volume injection, bulk capacity injection, tablet, capsule or lyophilized injectable powder.
11, preparation method according to claim 9, it is characterized in that, described spleen extract is made acceptable different dosage form on the pharmaceutics through post processing, and it is oral liquid, small-volume injection, bulk capacity injection, tablet, capsule or lyophilized injectable powder.
12, one of any described spleen extract of claim 1～5 is at preparation treatment constitutional and Secondary cases cellular immunity deficiency, and it is eczema, thrombocytopenia, repeatedly infects syndrome, respiratory tract and pulmonary infection, habitual common cold, chronic hepatitis B, mumps, recurrent aphtha disease medicament; Leukopenia, leukemia, reproducibility obstacle anemia, lymphoma and other malignant tumor that causes at preparation treatment chemicotherapy, improve tumor patient cancerate matter, viral infection, the purposes when improving postoperative or patient with severe symptoms's physical weakness aspect the medicine.
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| CN107960653A (en) * | 2017-12-11 | 2018-04-27 | 长春三真实业有限公司 | A kind of spleen extract frozen dried food that can improve the elderly's body's immunity |
| CN108095073A (en) * | 2017-12-11 | 2018-06-01 | 长春三真实业有限公司 | A kind of kidney extract frozen dried food that can improve old human organism's renal function |
| CN111375048A (en) * | 2018-12-27 | 2020-07-07 | 北京第一生物化学药业有限公司 | Application of spleen aminopeptide in preparation of medicines for treating leukopenia |
| CN114306269A (en) * | 2021-12-27 | 2022-04-12 | 西华大学 | Preparation method of lemon polypeptide effervescent tablets |
| CN115624187B (en) * | 2022-12-08 | 2023-04-04 | 北京第一生物科技开发有限公司 | Application of bovine spleen peptide powder in improving sleep |
Citations (1)
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| CN1089852A (en) * | 1993-01-21 | 1994-07-27 | 陈玉春 | The natural biological oral liquid that contains the bioactie agent nucleotide polypeptide type |
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| CN1089852A (en) * | 1993-01-21 | 1994-07-27 | 陈玉春 | The natural biological oral liquid that contains the bioactie agent nucleotide polypeptide type |
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