CN1079902A - The preparation method of soluble earthworm cellvibrio - Google Patents
The preparation method of soluble earthworm cellvibrio Download PDFInfo
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- CN1079902A CN1079902A CN93105054A CN93105054A CN1079902A CN 1079902 A CN1079902 A CN 1079902A CN 93105054 A CN93105054 A CN 93105054A CN 93105054 A CN93105054 A CN 93105054A CN 1079902 A CN1079902 A CN 1079902A
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- cellosolve
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Abstract
The present invention relates to a kind of preparation method of soluble earthworm cellvibrio.This method is mixed broken back adding distil water with Eisenia foetida through washing and is extracted, the centrifugal extracting solution of collecting, to extracting solution dialyse, ultrafiltration or the concentrated concentrated solution that obtains sterilizing of chemical dehydration, obtained by freeze drying Cellosolve crude product again.This crude product activity reaches 8500mm
2/ mg can directly be used as medicine.
Description
The present invention relates to a kind of method for preparing the solution fibrin medicine with earth-worm extractive as raw material.The Cellosolve of this method preparation is thrombus effectively, and tangible anticoagulation is arranged.
Known thrombolytic drug as streptokinase and urokinase, generally by intravenously administrable, and has the hemolytic danger of the whole body of generation, and its preparation method is also comparatively complicated, and it is raw material that the preparation of urokinase needs a large amount of male's freshly voided urines, collects comparatively difficulty.The medicine that some blood circulation promoting and blood stasis dispelling are also arranged in the Chinese medicine, as Flos Carthami, Radix Salviae Miltiorrhizae, Rhizoma Chuanxiong etc., but its thrombolytic DeGrain.Commercially available snake venom thrombolytics can not be oral, and tangible toxic reaction is arranged.
In recent years, many bibliographical informations about the earth-worm extractive as raw material thrombolytic effect being arranged both at home and abroad, mainly is the pharmacological research about the extraction of Lumbricus fibrinolytic enzyme, thrombolytic effect, few to the Application and Development report of such medicine.
Japan and South Korea are developed cooperatively with Lumbricus and are extracted molten fine medicine, are named " the imperial heart ", and are used for clinical.The preparation method of this medicine and part pharmacology, curative effect are disclosed in GB2116565A.This method mainly is that work Lumbricus (or certain portion of tissue) solubilizer is broken into suspension, under the temperature about 37 ℃, be incubated 72-100 hour, filter then or separate and obtain extracting solution, become concentrated solution through ultrafiltration or vacuum evaporation, carry out drying (vacuum or freezing) again and make rough molten fine powder, this crude product activity only is 142mm
2/ mg-350mm
2/ mg, can't be used as medicine, improve purity and activity through process for refining again with this crude product, subtractive process need add polar organic solvent, obtains having the pharmaceutical preparation of greater activity through precipitation → dissolving → dialysis → ion exchange → gel filtration (or affinity chromatography) → processing steps such as lyophilization then.The crude product activity that this method makes is lower, can not be used as medicine, and is necessary through process for refining, so complex manufacturing, and the production cycle is long, industrial-scale production cost height.In addition, the suspension insulation under 37 ℃ that makes with this method reaches 72-100 hour, and is perishable.Though the medicine of this method preparation has carried out molten fine experiment, real data is not seen in experiment to thrombolytic.
The flat 2-215724 of Japan Patent discloses the method that a kind of boulton process prepares earthworm dry powder, this method is put into Lumbricus and is contained the little acidity solution of organic or inorganic and put in a suitable place to breed, after treating to have in the inner chamber muck of bacterium fully to discharge, pulverizing obtains suspension, again through lyophilization, vacuum drying or negative pressure dehydration, vacuum rotary drying, obtain the earthworm dry powder crude product, this crude product is ground into fine powder under pulverizer.
This method technology is simple, and is high but vacuum dryer and process conditions require, in order to make aseptic crude product, its lyophilization temperature is at-10 ℃--and 60 ℃, vacuum drying temperature-60 ℃-+80 ℃ below the vacuum 10-100mmHg, reaches 100 hours most in this following operating time of process conditions.Even adopt the method for vacuum rotary drying, also under 10mmHg, be incubated operation 50 hours, just can obtain dry powder, vacuum rotary drying equipment requirements is also higher.
Purpose of the present invention: adjust common extraction process, the soluble earthworm cellvibrio crude product that makes preparation is nontoxic, aseptic, the active high characteristic of tool, can directly be used as medicine, thereby provide a kind of technology, equipment simple, with short production cycle, be suitable for the preparation method of the soluble earthworm cellvibrio of industrial-scale production.
Main points of the present invention:
It is raw material that this method is selected Eisenia foetida for use, remove mud with the clear water washing, reuse water recently distilled washing 3-5 time is made homogenate with tissue mashing machine, the adding distil water dilution is extracted, PH6.8-7.0 puts 4 ℃ of-6 ℃ of temperature and transferred 8-12 hour, collects supernatant with centrifugal method, sediment extracts 3-4 time with the same manner again, merge supernatant, be divided in the bag filter, dialysed 24-72 hour.Can remove inorganic ions and micromolecule pigment like this, improve purity, reduce toxicity, with ultrafiltration or the dehydration of chemical dehydration method, obtain concentrated solution then.Wherein suitably choosing ultrafilter membrane can sterilize, and as mesopore fiber or 5000-10000 ultrafilter membrane etc., also can dewater with polyethylene glycol 6000.Concentrated solution after the degerming is through lyophilization, temperature-20 ℃-60 ℃, and time 8-16 hour, promptly get the soluble earthworm cellvibrio crude product, this crude product is little yellow, and activity reaches 8500mm
2/ mg can grind the back direct drug injection, and also method such as available gel filtration is further refining purifies again.
Effect of the present invention: this method is aligned common process, when preparing, crude product adds technologies such as dialysis, ultrafiltration, make the activity and the purity of crude product improve 2-5 doubly, can directly be used as medicine, thereby provide a kind of process equipment simple, process conditions are easy to grasp, be suitable for the preparation method of the soluble earthworm cellvibrio of large-scale production.Various medicaments such as that this Cellosolve crude product can prepare is oral, injection, quiet, safe and reliable, the thrombolytic effect is remarkable.
1, proves its security reliability from following serial experiment.
(1) Cellosolve animal pharmacology experiment:
To the domestic cat group experiment, observe the influence of Cellosolve to cardiovascular system and respiratory system with the Cellosolve of the present invention preparation, test index is electrocardiogram, blood pressure, heart rate, respiratory frequency and respiratory depth.The result shows: more all do not have significant difference (P＞0.05) with matched group and see table 1, table 2 for details before and after administration.
Cellosolve with the present invention's preparation is tested the neural influence of white mice, the free movable number of test mice reaches the influence to pentobarbital subliminal hypnosis dosage, the result shows with matched group does not have significant difference (P＞0.05), promptly central nervous system of mice is not had bright work development and rings.See table 3, table 4 for details.
(2) Cellosolve long term toxicity test
Cellosolve with the present invention's preparation carries out long term toxicity test to Canis familiaris L., continuous oral three months, and observation index is: animal behavior, peripheral blood phase, liver, renal function and electrocardiogram etc.The result shows: various indexs are no abnormal, and animal receives food normally, vivaciously during whole administration, and weight average increases 3.5Kg, and the oral safety of this medicine, non-evident effect are described.See table 5, table 6 for details.
(3) acute, the sub-acute toxicity test of Cellosolve.
Cellosolve with the present invention's preparation carries out acute subacute toxicity test to white mice.
A, to white mice vein LD
50Mensuration: obtain Cellosolve fatality rate data (seeing Table 7).Obtaining 50% death point by table 7 data draws vertical line and gets LD
50=172mg/Kg.
B, oral Cellosolve toxicity: oral back was observed 7 days, and the result does not have animal dead, and autonomic activities does not have change, and continuous 15 days, leukocytes red to peripheral blood and platelet and liver,spleen,kidney function and control animals relatively do not have obvious influence (seeing Table 8).
C, big and small mouse upgrowth curve show that growth promoter and matched group relatively do not have obvious influence.
Accompanying drawing 1 is a rat growth curve contrast table.As shown in Figure 1: 1 is the rat growth curve of oral Cellosolve 500mg/kg, and 2 is the matched group growth curve.
Accompanying drawing 2 is a mice growth curve contrast table.As shown in Figure 2: 3 is oral Cellosolve 100mg/Kg mice growth curve, and 4 is oral 200mg/Kg mice growth curve, and 5 is mice matched group growth curve.
Except that above-mentioned experimental data, the Cellosolve of the present invention's preparation obtains the licence (seeing Appendix) of Beijing's health medicine inspection department.
(2) medicine of this method preparation is used for clinically, and effect is obvious, not only the body inner fibrin is had dissolution, and thrombolytic effect is more obvious, particularly the old thrombosis is also demonstrated beyond thought curative effect.Case is as follows:
A, patient women are diagnosed as left ilium femoral vein thrombosis, and be invalid with urokinase 5 days (totally 100 ten thousand units), and drug withdrawal was withdrawn the previous remark and obeyed the capsule of the present invention's preparation after 15 days, and 1g/ time, 3 times/day, oral 14 people, healing is left hospital.
B, patient are the male, 61 years old, because of being installed, pacemaker causes left subclavian vein thrombosis, invalid through urine hormone treatment 4 days, drug withdrawal one day, the Cellosolve capsule that the clothes the present invention that withdraws the previous remark prepares, gram divided and took for three times every days 8, use gram every days 4 after 10 days instead, divide and to take for 4 times, with (totally 21 days) after 11 days, thromboembolism is left hospital.Before and after the last two routine patients radiography basis is arranged all.
C, two routine ophthalmology patients are the male, and the age was respectively 62 years old, 73 years old, be diagnosed as the quiet bolt thrombosis in right eye optical fundus retinal vein thrombosis and retina of left eye below, the seasonal disease eye vision of being admitted to hospital is respectively 0.04 and 0.02, wherein 1 routine oral Cellosolve 1 gram, restrain every days 2, medication 12 days, and it is invalid that another example is dripped with ligustrazine and low molecular dextran vein earlier, drug withdrawal 5 days, withdraw the previous remark and obey Cellosolve capsule, 1g/ time, 2 times/day, obeyed altogether 21 days, vision reaches 0.2 and leaves hospital after the two routine patient.
Through practical application, the Cellosolve of the present invention's preparation has multiple route of administration such as oral, quiet notes, intramuscular injection.Can be used for treating various plug diseases such as cerebral thrombosis, cerebral embolism, myocardial infarction, limbs misfortune arteries and veins, vein, the dynamic and static plug in optical fundus.
Below in conjunction with embodiment to the detailed description of the invention.
Accompanying drawing 3 is process charts of the present invention.
As shown in Figure 3: with bright Lumbricus (Eisenia foetida) 200 grams, remove mud with the clear water washing, reuse water recently distilled washing 3-5 time adds 100 milliliters of mixing of water recently distilled (PH6.8-7.0), be broken into homogenate with tissue mashing machine, the adding distil water dilution is extracted, and puts 4 ℃ of refrigerator 8-12 hours, with 3000rpm * 15 ' centrifugal collection supernatant, sediment extracts 3-4 time with the same manner, merge supernatant, cumulative volume is 500ml, and residue is abandoned it.Extracting solution is divided in the bag filter, dialyses after 2-3 days, with mesopore cellulosic ultrafiltration (or polyethylene glycol 6000) dehydration, about 200 milliliters of concentrated extracting solution, cool-drying, temperature-20 ℃--60 ℃.Get the soluble earthworm cellvibrio crude product, take out a small amount of molten fine its activity of surveying, remainder or fine ground direct pharmacy, or further refining purification.
Wherein determination of activity: get the 10ml crude product, be added in and record every 10ul in the 2.5ml normal saline and contain the 0.04mg activity, the area size is 324-361mm
2, promptly about 8500mm
2/ mg activity.
Embodiment 2: the Cellosolve 200-500mg that use-case 1 makes grinds into fine powder, incapsulates, and obtains oral capsule.
Embodiment 3: the Cellosolve 10-12mg that use-case 1 makes adds injection water 5ml, directly intramuscular injection.
Embodiment 4: the Cellosolve that use-case 1 makes adds quiet of normal saline by 12-18mg/Kg.
Table 3 Cellosolve is to the impact of mouse autonomic activities
| Group | Dosage | Number of animals (only) | Mouse autonomic activities number of times X ± SD |
| Control group | 25ml/kg | 10 | 137±56 |
| Cellosolve | 120mg/kg | 10 | 139±32 |
| Cellosolve | 240mg/kg | 10 | 128±70 |
Table 4 Cellosolve is to the impact of pentobarbital sodium sub-threshold lull dosage
| Group | Dosage | Number of animals (only) | The number of animals (only) of sleep takes place |
| Control group | 25ml/kg | 10 | 6 |
| Cellosolve | 120mg/kg | 10 | 5 |
| Control group | 25ml/kg | 10 | 8 |
| Cellosolve | 240mg/kg | 10 | 9 |
Table 5 hemochrome, red blood cell, leucocyte measurement result
| Number of animals | Cellosolve dosage mg(ten thousand U)/kg | Hemochrome (g) | Leucocyte (1 * 10/mm) | Red blood cell (1 * 10/mm) | |||
| Before the administration | After the administration | Before the administration | After the administration | Before the administration | After the administration | ||
| 1 2 | Contrast | 12.3 13.0 | 11.0 13.0 | 1.9 1.3 | 1.4 1.4 | 756 806 | 739 724 |
| 3 4 | 500 (15.63) | 11.0 12.0 | 12.0 13.0 | 1.4 1.1 | 1.3 1.1 | 665 754 | 733 630 |
| 5 6 | 800 (25.00) | 12.0 12.0 | 12.0 11.0 | 1.2 1.3 | 1.1 1.2 | 883 748 | 769 694 |
SGPT, BUN measurement result before and after table 6 administration
| Numbering | Precious dosage mg(ten thousand U of dragon)/kg | SGPT U/L | BUN MG% | ||
| Before the administration | After the administration | Before the administration | After the administration | ||
| 1 2 | Contrast | 31.0 44.0 | 42.7 34.8 | 13.2 14.0 | 12.8 13.7 |
| 3 4 | 500 (15.63) | 33.3 32.9 | 40.5 35.7 | 11.4 14.5 | 10.8 13.7 |
| 5 6 | 800 (25.00) | 35.7 31.0 | 40.5 32.8 | 15.3 11.4 | 13.8 12.0 |
Table 7 soluble earthworm cellvibrio lethal dose data
| Group | Dosage mg(ten thousand U)/kg | The death rate | Probability |
| 1 2 3 4 5 6 | 124.6(3.89) 150.0(4.69) 180.6(5.64) 217.4(6.79) 315.2(9.85) 379.5(11.86) | 2/8(25%) 4/8(50%) 5/8(62.5%) 6/9(66.7%) 7/9(77.8%) 9/9(100%) | 4.32 5.00 5.30 5.43 5.78 |
Claims (3)
1, a kind of preparation method of soluble earthworm cellvibrio, comprise technologies such as the washing of work Lumbricus, fragmentation, separation, dryings, it is characterized in that: Eisenia foetida is mixed fragmentation after washing, obtain homogenate, add distilled water extraction, pH value is 6.8-7.0, place 4 ℃-6 ℃ down insulations 8-12 hour, centrifugal collection supernatant is after supernatant dialysed 24-72 hour, carry out ultrafiltration or chemical dehydration and concentrate sterilization, the concentrated solution lyophilization that obtains made Cellosolve in 8-16 hour.
2, method according to claim 1 is characterized in that, ultrafilter membrane adopts the ultrafilter membrane of mesopore fiber or 5000-10000.
3, method according to claim 1 is characterized in that, chemical dehydration is dewatered with polyethylene glycol 6000
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN93105054A CN1044560C (en) | 1993-05-18 | 1993-05-18 | Method of preparation of soluble earthworm cellvibrio |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN93105054A CN1044560C (en) | 1993-05-18 | 1993-05-18 | Method of preparation of soluble earthworm cellvibrio |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1079902A true CN1079902A (en) | 1993-12-29 |
| CN1044560C CN1044560C (en) | 1999-08-11 |
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ID=4985512
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN93105054A Expired - Fee Related CN1044560C (en) | 1993-05-18 | 1993-05-18 | Method of preparation of soluble earthworm cellvibrio |
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| Country | Link |
|---|---|
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1051229C (en) * | 1994-11-30 | 2000-04-12 | 南京金宁信息技术研究所 | Preparation method of active dried earthworm powder |
| CN1062140C (en) * | 1997-05-29 | 2001-02-21 | 湖北省荆门卫生学校 | Method for extracting earthworm element from Jpan Dula earthworm |
| CN1089369C (en) * | 1999-04-01 | 2002-08-21 | 孙启良 | Earthworm kinase prepn. tech. |
| CN101857634A (en) * | 2010-05-17 | 2010-10-13 | 成都瑞盛高科技有限责任公司 | Activator protein for fibrinolytic system |
| CN101890052B (en) * | 2009-05-21 | 2011-11-23 | 龚之森 | Earthworm crystal powder, preparation process and application thereof |
| CN103656630A (en) * | 2012-09-11 | 2014-03-26 | 江苏仁寿药业有限公司 | Method for purifying plasmin in animal medicine and preparing traditional Chinese medicine composition |
-
1993
- 1993-05-18 CN CN93105054A patent/CN1044560C/en not_active Expired - Fee Related
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1051229C (en) * | 1994-11-30 | 2000-04-12 | 南京金宁信息技术研究所 | Preparation method of active dried earthworm powder |
| CN1062140C (en) * | 1997-05-29 | 2001-02-21 | 湖北省荆门卫生学校 | Method for extracting earthworm element from Jpan Dula earthworm |
| CN1089369C (en) * | 1999-04-01 | 2002-08-21 | 孙启良 | Earthworm kinase prepn. tech. |
| CN101890052B (en) * | 2009-05-21 | 2011-11-23 | 龚之森 | Earthworm crystal powder, preparation process and application thereof |
| CN101857634A (en) * | 2010-05-17 | 2010-10-13 | 成都瑞盛高科技有限责任公司 | Activator protein for fibrinolytic system |
| CN101857634B (en) * | 2010-05-17 | 2012-10-03 | 成都瑞益科技有限责任公司 | Activator protein for fibrinolytic system |
| CN103656630A (en) * | 2012-09-11 | 2014-03-26 | 江苏仁寿药业有限公司 | Method for purifying plasmin in animal medicine and preparing traditional Chinese medicine composition |
| CN103656630B (en) * | 2012-09-11 | 2015-07-08 | 江苏仁寿药业有限公司 | Method for purifying plasmin in animal medicine and preparing traditional Chinese medicine composition |
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| Publication number | Publication date |
|---|---|
| CN1044560C (en) | 1999-08-11 |
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