CN100531723C - Calf serum protein-removing extract for injection and its preparation method - Google Patents
Calf serum protein-removing extract for injection and its preparation method Download PDFInfo
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Abstract
The present invention discloses a free protein serum of cow dry powder injection and the preparation method. The free protein serum of cow dry powder injection comprises components with the following weight and portion ratio, which comprises one portion of the free protein serum of cow dry powder, nine portions to eleven portions of mannitol, 4.5 portions to 5.5 portions of low molecular dextran. In the present invention, the free protein serum of cow dry powder injection not only overcomes defects of instable needle, infusion quality and prolongs period of production validity, but also extends clinical application and reduces rate of adverse effect.
Description
Technical field
The present invention relates to a kind of calf serum protein-removing extract for injection and preparation method thereof, particularly a kind of calf serum protein-removing extract freezing-dried powder injection and preparation method thereof.
Background technology
Calf serum protein-removing extract (claiming calf blood protein-removed extraction again) is mainly used in the treatment cerebrovascular disease.At present the extraction process of calf blood protein-removed extraction generally is to be that raw material adopts and adds evanescence of heat albumen acquisition calf blood protein-removed extraction (seeing Chinese patent ZL200410013643.6) with calf serum.The patent No. is that the patent documentation of ZL96120777.9 discloses a kind of method of extracting calf blood protein-removed extraction, adopts ethanol to remove albumen; The molecular weight of calf blood protein-removed extraction is less than 5000 dalton, because molecular weight is little, no antigen does not cause anaphylaxis.
To be the disclosed calf blood protein-removed extraction of the patent documentation of ZL96120777.9 exist with the state of lyophilized powder the patent No., and just for the ease of the preservation of calf blood protein-removed extraction, this simple lyophilized powder can not be as the clinical practice preparation.And this lyophilized powder has a strong impact on the stability of product, and deliquescence and degeneration just took place more than half a year.
Calf serum protein-removing extract class injection has only liquid drugs injection at present.Liquid drugs injection instability, room temperature are placed with regard to easy generation polymerization, photodissociation etc. and are reacted and the change composition, even precipitate, and product is guaranteed the quality shorter.
Summary of the invention
The purpose of this invention is to provide a kind of calf serum protein-removing extract for injection and preparation method thereof, i.e. calf serum protein-removing extract freezing-dried powder injection and preparation method thereof.
Calf serum protein-removing extract freezing-dried powder injection provided by the present invention, contain following components by weight portion:
1 part of calf serum protein-removing extract dry;
9-11 parts in mannitol;
4.5-5.5 parts of micromolecule dextrans.
Described calf serum protein-removing extract is that calf serum is removed the extracting solution that obtains behind the albumen.
Described calf serum protein-removing extract is that the quality percentage composition that will be in the calf serum adds two volumes is 96% ethanol, makes the albuminous degeneration precipitation, and centrifugal removal precipitation is removed the extracting solution that ethanol obtains.
The molecular weight of described calf serum protein-removing extract is below 5000 dalton.
The preparation method of calf serum protein-removing extract freezing-dried powder injection provided by the present invention, be mannitol and micromolecule dextran to be joined in the calf serum protein-removing extract according to above-mentioned ratio of weight and number, after 30-50 ℃ of dissolving, obtain mixed liquor, lyophilizing obtains the calf serum protein-removing extract freezing-dried powder injection.
The dry biomass percentage composition of described calf serum protein-removing extract is preferably 18-22mg/ml.
In the described method, described calf serum protein-removing extract is that the quality percentage composition that will be in the calf serum adds two volumes is 96% ethanol, makes the albuminous degeneration precipitation, and centrifugal removal precipitation is removed the extracting solution that ethanol obtains.
In the described method, comprise that also the extracting solution that described removal ethanol is obtained is 30-50 ℃ of evaporation and concentration.
In the described method, described freeze dried step is earlier-40 ℃ of pre-freezes 1 hour with described mixed liquor, then 20 ℃ of thawings, and then-40 ℃ of pre-freezes after 2 hours, under vacuum state, kept 8-10 hour at-20 ℃, kept 4 hours at-15 ℃, rise to-10 ℃ then, raise 10 ℃ every 1 hour, lyophilizing finishes during to 40 ℃.
In the described method, also being included in step of freeze drying preceding is that 5000 daltonian Hollow Fiber Ultrafiltration posts filter with the mixed liquor molecular cut off.
In the described method, also comprise before the described step of freeze drying mixed liquor is carried out filtration sterilization.
Calf serum protein-removing extract freezing-dried powder injection outward appearance exquisiteness of the present invention, constant product quality.Can be used to treat following disease: brain blood circulation and Dystrophy (ischemic lesions, craniocerebral trauma); Peripheral circulation disorders and the artery vessel disease that causes thereof, leg ulcer; Skin grafting; Skin burn, scald, erosion; Healing of wound (wound, decubital ulcer); Radio-induced skin, mucosa injury.Calf serum protein-removing extract freezing-dried powder injection of the present invention has overcome liquid drugs injection and the unsettled defective of injection quality, prolongs keeping life, and the expansion clinical practice reduces the untoward reaction odds.Carried out a step freeze thawing in the freeze-drying process in the method for preparing the calf serum protein-removing extract freezing-dried powder injection of the present invention, this method is the moisture in the release products thoroughly, makes the lyophilizing better effects if.
The specific embodiment
Method among the following embodiment if no special instructions, is conventional method.
Percentage composition among the following embodiment if no special instructions, is the quality percentage composition.
The constituent content screening of embodiment 1, calf serum protein-removing extract for injection
One, the preparation of calf serum protein-removing extract for injection (calf serum protein-removing extract freezing-dried powder injection)
The aseptic young cattle venous blood of 1-6 monthly age of adopting, isolate serum with reference to the method described in patent of invention (patent No. the is ZL96120777.9) embodiment 1, detect pH value, total serum protein, the serum albumin levels of calf serum, select the calf serum that meets the following conditions:
1) pH value: the pH value of calf serum should be 6.5-7.5.
2) total serum protein: 500 times of calf serum dilutions, measure The Total albumen content according to " Chinese biological goods rules " 35 pages of method, should be greater than 4.0g/100ml.
3) serum albumin levels: get calf serum 0.02ml,, measure serum albumin levels according to 644 pages of methods of " Chinese medical check pandect " (version in 1996), should be greater than 2.3g/100ml.
1, the preparation of calf serum protein-removing extract
Prepare the Deporteinnized calf serum extracting solution with reference to embodiment 1 described method in the patent of invention (patent No. is ZL96120777.9), concrete grammar is: (pH value is 7.2 to select above-mentioned isolating calf serum for use, The Total albumen content is 4.4g/100ml, serum albumin levels is 2.5g/100ml), add the quality percentage composition double the calf serum volume and be 96% ethanol, constantly stirred 30 minutes, static 60 minutes, make the albuminous degeneration precipitation, centrifugal 30 minutes through 10000rpm then at 4 ℃, obtain removing the albumen supernatant, supernatant is utilized the rotary evaporation in vacuo instrument, and at 37 ℃, 1 atmospheric pressure reduces pressure down and removes ethanol, in remaining liquid, add compound protease (purchase ooze in Ningxia flourish bio tech ltd) hydrolysis 24 hours, at 10000rpm, 4 ℃, centrifugal 30 minutes, stay supernatant, supernatant with 5000 dalton's hollow fiber column ultrafilter, is collected the filtrate that obtains and is the Deporteinnized calf serum extracting solution.Detect solid content (dry) content of this Deporteinnized calf serum extracting solution with the 1 described method of embodiment among the patent ZL96120777.9, the result shows that the solid quality percentage composition is 10mg/ml.
2, calf serum protein-removing extract freezing-dried powder injection preparation method
1) concentrate: the Deporteinnized calf serum extracting solution of getting four groups of 5000ml step 1 preparations respectively arrives 2500ml with Rotary Evaporators evaporation and concentration under 40 ℃ of conditions, show that with the 1 described method detection of embodiment among the patent ZL 96120777.9 this calf serum protein-removing extract amount of dry matter percentage composition is 20mg/ml.
2) add excipient: respectively according to the requirement of sample shown in the table 12, sample 3, sample 4, sample 5 constituent contents, calculate and accurately weighing mannitol and Dextran 10 to join the amount of dry matter percentage composition that step 1) obtains be the calf serum protein-removing extract of 20mg/ml, at 40 ℃ of thermosols, obtain rare dosing respectively.
3) ultrafiltration: with step 2) the rare dosing that obtains uses molecular weight cut off 5000 daltonian Hollow Fiber Ultrafiltration posts (purchase in the Shanghai group and step the laboratory equlpment company limited) the rare dosing of ultrafiltration to obtain ultrafiltrate respectively, collect ultrafiltrate respectively, it is standby to be settled to 3000ml respectively with distilled water.
4) degerming packing: behind the membrane filtration of the ultrafiltrate that step 3) is obtained with 0.2 micron pore size, sterile working's packing is filled to the cillin bottle splendid attire that the 10ml specification has been cleaned and dried with filtrate according to 3ml/ bottle sterile working branch respectively respectively.
5) the cillin bottle false add plug of above-mentioned degerming rear filtrate will be housed after, lyophilizing; Freeze dried step is as described below:
1. pre-freeze: the product after the packing is packed in the freeze dryer (model LYO-0.2 purchases in Dongfulong Sci-Tech Co., Ltd., Shanghai), before regulating the case shelf temperature to-40 ℃, pre-freeze 1 hour.
2. melt: close refrigeration, case shelf temperature to 20 ℃ before regulating heated 1-2 hour, and the ice that causes in the product bottle melts fully.
3. pre-freeze once more: before regulating the case shelf temperature to-40 ℃, pre-freeze 2 hours.
4. vacuum drying: before regulating the oven temperature, degree to-20 ℃, simultaneously before case begin evacuation, rear cabinet begins refrigeration; Vacuum drying 8-10 hour.
5. plait point insulation: keep vacuum state, the oven temperature, degree is incubated 4 hours to-15 ℃ before regulating.
6. parsing-desiccation: keep vacuum state, the case shelf temperature is to-10 ℃ before regulating, the rising shelf temperature was 10 ℃ in per 1 hour later on, to 40 ℃, be incubated to pressurize experiment qualified (case vacuum valve before closing, preceding case pressure raise preceding 10 seconds be that pressurize is qualified), compress plug less than 3Pa, lyophilizing finishes, and obtains the calf serum protein-removing extract lyophilized injectable powder.
Above-mentioned freeze-drying process does not have lay special stress on, all carries out according to freeze dried routine operation.
6) Zha Gai: from freeze dryer, take out goods, prick rapidly and go up aluminium lid, promptly get the calf serum protein-removing extract for injection (sample 2 shown in the table 1, sample 3, sample 4 or sample 5) that meets the demands.
Above-mentioned Deporteinnized calf serum extracting solution 5000ml is carried out degerming packing lyophilizing according to embodiment among the patent ZL 96120777.9 1 obtain sample 1 in the table 1.
The screening of table 1. Ox blood serum deprotein extract freezing-dried powder injection constituent content
| Sample | Constituent content | Calf serum protein-removing extract (the mg dry /) | Mannitol (mg/ props up) | Micromolecule stone revolves sugared acid anhydride (mg/ props up) |
| 1 | According to the patent No. is the lyophilized powder of the patent documentation production of ZL96120777.9 | 50 | ||
| 2 | Calf serum protein-removing extract dry matter weight part is 1, and the mannitol weight portion is 15 | 50 | 750 | |
| 3 | Calf serum protein-removing extract dry matter weight part is 1, and the mannitol weight portion is 10, and the Dextran 10 weight portion is 5 | 50 | 500 | 250 |
| 4 | Calf serum protein-removing extract dry matter weight part is 1, and the mannitol weight portion is 5, and the Dextran 10 weight portion is 10 | 50 | 250 | 500 |
| 5 | Calf serum protein-removing extract dry matter weight part is 1, and the Dextran 10 weight portion is 15 | 50 | 750 |
Observed character, moisture content, activity, the clarity of above-mentioned 5 groups of samples respectively at 0th month, 6 months, 12 months, 18 months, 24 months, with the reasonability of 5 groups of sample prescriptions relatively.
Wherein active (SI) method that detects is carried out according to document (in great scholar, plain 2000 the 2nd phases of the bioactive experimentation of injection " Chinese Journal of New Drugs " of the short cellular metabolism of Xiao Jianying, 96-98); The characteristic index perusal; Moisture content and clarity are according to " method that Chinese pharmacopoeia is 2005 editions 2 ones detects.
Table 2. detected the result in 0th month
| Sample | Character | Moisture content | Active | Clarity |
| 1 | Pale yellow powder | 9％ | SI＝3.3 | Up to specification |
| 2 | Faint yellow cellular agglomerate | 1％ | SI＝3.2 | Up to specification |
| 3 | Pale yellow powder | 1.5％ | SI＝3.2 | Up to specification |
| 4 | Pale yellow powder | 6％ | SI＝3.2 | Up to specification |
| 5 | Pale yellow powder | 8％ | SI＝3.2 | Up to specification |
Table 3. detected the result in 6th month
| Sample | Character | Moisture content | Active | Clarity |
| 1 | Pale yellow powder | 9％ | SI＝3.3 | Flocculent deposit appears |
| 2 | Faint yellow cellular agglomerate | 1％ | SI＝3.1 | Up to specification |
| 3 | Pale yellow powder | 1.5％ | SI＝3.2 | Up to specification |
| 4 | Pale yellow powder | 6％ | SI＝3.0 | Up to specification |
| 5 | Pale yellow powder | 8％ | SI＝2.8 | Up to specification |
Table 4. detected the result in 12nd month:
| Sample | Character | Moisture content | Active | Clarity |
| 1 | Pale yellow powder | 10％ | SI＝3.3 | Flocculent deposit appears |
| 2 | Faint yellow cellular agglomerate | 1％ | SI＝3.1 | Up to specification |
| 3 | Pale yellow powder | 1％ | SI＝3.1 | Up to specification |
| 4 | Pale yellow powder | 6％ | SI＝2.6 | Up to specification |
| 5 | Pale yellow powder | 8％ | SI＝2.6 | Up to specification |
Table 5. detected the result in 18th month
| Sample | Character | Moisture content | Active | Clarity |
| 1 | Pale yellow powder | 10％ | SI＝3.0 | Flocculent deposit appears |
| 2 | Faint yellow cellular agglomerate | 1％ | SI＝3.1 | Up to specification |
| 3 | Pale yellow powder | 1％ | SI＝3.1 | Up to specification |
| 4 | Pale yellow powder | 6％ | SI＝2.2 | Up to specification |
| 5 | Pale yellow powder | 8％ | SI＝2.0 | Up to specification |
Table 6. detected the result in 24th month
| Sample | Character | Moisture content | Active | Clarity |
| 1 | Pale yellow powder | 10％ | SI＝2.4 | Flocculent deposit appears |
| 2 | Faint yellow cellular agglomerate | 1％ | SI＝3.0 | Up to specification |
| 3 | Pale yellow powder | 1％ | SI＝3.1 | Up to specification |
| 4 | Pale yellow powder | 6％ | SI＝1.6 | Up to specification |
| 5 | Pale yellow powder | 8％ | SI＝1.2 | Up to specification |
Above data show, except that sample 3 every technical specifications in 24 months the no change, gliding all appearred after 6 months in indivedual indexs of other samples, therefore, the prescription of sample 3 is more reasonable.
The preparation of embodiment 2, calf serum protein-removing extract freezing-dried powder injection and Detection of Stability thereof
One, the preparation of calf serum protein-removing extract freezing-dried powder injection and injection
The aseptic young cattle venous blood of 1-6 monthly age of adopting, isolate serum with reference to the method among patent of invention (patent No. the is ZL 96120777.9) embodiment 1, detect pH value, total serum protein, the serum albumin levels of calf serum, select the calf serum that meets the following conditions:
1) pH value: the pH value of calf serum should be 6.5-7.5.
2) total serum protein: 500 times of calf serum dilutions, measure The Total albumen content according to " Chinese biological goods rules " 35 pages of method, should be greater than 4.0g/100ml.
3) serum albumin levels: get calf serum 0.02ml,, measure serum albumin levels according to 644 pages of methods of " Chinese medical check pandect " (version in 1996), should be greater than 2.3g/100ml.
1, the preparation of calf serum protein-removing extract
Prepare the Deporteinnized calf serum extracting solution with reference to embodiment 1 described method in the patent of invention (patent No. is ZL 96120777.9), concrete grammar is: (pH value is 7.3 to select above-mentioned isolating calf serum for use, The Total albumen content is 4.2g/100ml, serum albumin levels is 2.6g/100ml), add the quality percentage composition double the calf serum volume and be 96% ethanol, constantly stirred 30 minutes, static 60 minutes, make the albuminous degeneration precipitation, centrifugal 30 minutes through 10000rpm then at 4 ℃, obtain removing the albumen supernatant, supernatant is utilized the rotary evaporation in vacuo instrument, and at 37 ℃, 1 atmospheric pressure reduces pressure down and removes ethanol, in remaining liquid, add compound protease (purchase ooze in Ningxia flourish bio tech ltd) hydrolysis 24 hours, at 10000rpm, 4 ℃, centrifugal 30 minutes, stay supernatant, supernatant with 5000 dalton's hollow fiber column ultrafilter, is collected the filtrate that obtains and is the Deporteinnized calf serum extracting solution.Detect solid content (dry) content of this Deporteinnized calf serum extracting solution with the 1 described method of embodiment among the patent ZL 96120777.9, the result shows that the solid quality percentage composition is 10mg/ml.
2, calf serum protein-removing extract injection production method
Behind the above-mentioned membrane filtration of Deporteinnized calf serum extracting solution through 0.2 micron pore size, sterile working's branch is filled in the ampoule that cleans up that specification is 5ml, fills nitrogen and seals, and promptly gets the calf serum protein-removing extract injection.
3, the production of calf serum protein-removing extract for injection (freezing-dried powder injection)
1) concentrate: the Deporteinnized calf serum extracting solution of getting three parts of 5000ml step 1 preparations respectively arrives 2500ml with Rotary Evaporators evaporation and concentration under 40 ℃ of conditions, show that with the 1 described method detection of embodiment among the patent ZL 96120777.9 this Deporteinnized calf serum extracting solution concentrated solution amount of dry matter percentage composition is 20mg/ml.
2) add excipient: according to the requirement of 2,3,4 constituent contents of sample shown in the table 7, calculate and accurately weighing mannitol and Dextran 10 to join the amount of dry matter percentage composition that step 1) obtains be the calf serum protein-removing extract of 20mg/ml, at 40 ℃ of thermosols, obtain rare dosing respectively.
3) ultrafiltration: with step 2) the rare dosing that obtains obtains ultrafiltrate with molecular weight cut off 5000 daltonian Hollow Fiber Ultrafiltration posts (purchase in the Shanghai group and step the laboratory equlpment company limited) the rare dosing of ultrafiltration, collect ultrafiltrate respectively, it is standby to be settled to 3000ml respectively with distilled water.
4) degerming packing: respectively with behind the membrane filtration of ultrafiltrate with 0.2 micron pore size, sterile working's packing is filled to the cillin bottle splendid attire that the 10ml specification has been cleaned and dried with filtrate according to 3ml/ bottle sterile working branch respectively.
5) the cillin bottle false add plug of above-mentioned degerming rear filtrate will be housed after, lyophilizing; Freeze dried step is as described below:
1. pre-freeze: the product after the packing is packed in the freeze dryer (model LYO-0.2 purchases in Dongfulong Sci-Tech Co., Ltd., Shanghai), before regulating the case shelf temperature to-40 ℃, pre-freeze 1 hour.
2. melt: close refrigeration, case shelf temperature to 20 ℃ before regulating heated 1-2 hour, and the ice that causes in the product bottle melts fully.
3. pre-freeze once more: before regulating the case shelf temperature to-40 ℃, pre-freeze 2 hours.
4. vacuum drying: before regulating the oven temperature, degree to-20 ℃, simultaneously before case begin evacuation, rear cabinet begins refrigeration; Vacuum drying 8-10 hour.
5. plait point insulation: keep vacuum state, the oven temperature, degree is incubated 4 hours to-15 ℃ before regulating.
6. parsing-desiccation: keep vacuum state, the case shelf temperature is to-10 ℃ before regulating, the rising shelf temperature was 10 ℃ in per 1 hour later on, to 40 ℃, be incubated to pressurize experiment qualified (case vacuum valve before closing, preceding case pressure raise preceding 10 seconds be that pressurize is qualified), compress plug less than 3Pa, lyophilizing finishes, and obtains the calf serum protein-removing extract lyophilized injectable powder.
Above-mentioned freeze-drying process does not have lay special stress on, all carries out according to freeze dried routine operation.
6) Zha Gai: from freeze dryer, take out goods, prick rapidly and go up aluminium lid, promptly get the calf serum protein-removing extract for injection (sample 2 shown in the table 7, sample 3, sample 4) that meets the demands.
Observe four indexs of character, activity, pH value, clarity of observing calf serum protein-removing extract freeze-dried powder and liquid drugs injection at 0th month, 6 months, 12 months, 18 months, 24 months respectively under the room temperature condition, relatively the stability of liquid drugs injection and freeze-dried powder.
Table 7. sample requirement
Two, the system Detection of Stability of calf serum protein-removing extract freeze-dried powder
Observed character, activity, pH value, the clarity of above-mentioned 4 groups of samples respectively at 0th month, 6 months, 12 months, 18 months, 24 months, with the stability of liquid drugs injection and powder pin relatively.Wherein active (SI) detection method is carried out according to document (in great scholar, plain 2000 the 2nd phases of the bioactive experimentation of injection " Chinese Journal of New Drugs " of the short cellular metabolism of Xiao Jianying); The characteristic index perusal; PH value and clarity are according to " method that Chinese pharmacopoeia is 2005 editions 2 ones detects.
Table 8. detected the result in 0th month
| Sample | Character | Active | PH value | Clarity |
| 1 | Light yellow transparent liquid | SI＝3.3 | 7.3 | Up to specification |
| 2 | Pale yellow powder | SI＝3.2 | 7.2 | Up to specification |
| 3 | Pale yellow powder | SI＝3.2 | 7.2 | Up to specification |
| 4 | Pale yellow powder | SI＝3.2 | 7.2 | Up to specification |
Table 9. detected the result in 6th month
| Sample | Character | Active | PH value | Clarity |
| 1 | Light yellow transparent liquid | SI＝3.1 | 7.6 | Up to specification |
| 2 | Pale yellow powder | SI＝3.1 | 7.2 | Up to specification |
| 3 | Pale yellow powder | SI＝3.2 | 7.1 | Up to specification |
| 4 | Pale yellow powder | SI＝3.2 | 7.2 | Up to specification |
Table 10. detected the result in 12nd month
| Sample | Character | Active | PH value | Clarity |
| 1 | Light yellow transparent liquid | SI＝2.8 | 7.6 | Flocculent deposit appears |
| 2 | Pale yellow powder | SI＝3.2 | 7.1 | Up to specification |
| 3 | Pale yellow powder | SI＝3.1 | 7.2 | Up to specification |
| 4 | Pale yellow powder | SI＝3.1 | 7.2 | Up to specification |
Table 11. detected the result in 18th month
| Sample | Character | Active | PH value | Clarity |
| 1 | Light yellow transparent liquid | SI＝2.6 | 8.4 | Flocculent deposit appears |
| 2 | Pale yellow powder | SI＝3.1 | 7.1 | Up to specification |
| 3 | Pale yellow powder | SI＝3.1 | 7.2 | Up to specification |
| 4 | Pale yellow powder | SI＝3.2 | 7.2 | Up to specification |
Table 12. detected the result in 24th month
| Sample | Character | Active | PH value | Clarity |
| 1 | Light yellow transparent liquid | SI＝2.3 | 8.8 | Flocculent deposit appears |
| 2 | Pale yellow powder | SI＝3.2 | 7.1 | Up to specification |
| 3 | Pale yellow powder | SI＝3.1 | 7.2 | Up to specification |
| 4 | Pale yellow powder | SI＝3.3 | 7.3 | Up to specification |
Above data show in 24 months of observation period, just occurs changing since 6th month liquid drugs injection, and its active continuing descends, and pH value continues to raise, and occurs flocculent deposit on the 12nd month; Sample 2,3,4 each index of freeze-dried powder dosage form do not have significant change.
Embodiment 3, calf serum protein-removing extract freezing-dried powder injection are to the influence of white mice normal pressure hypoxia-bearing capability
Select 80 of healthy Kunming kind one-level white mice (available from the SPF of Liaoning Medical University Experimental Animal Center), body weight 18-22g, male and female half and half, be divided into four groups, the sample of embodiment 2 preparations 2 groups of (calf serum protein-removing extract freezing-dried powder injection), 3 groups in samples of embodiment 2 preparations, 4 groups in sample, the normal saline matched group of embodiment 2 preparations.
The sample of embodiment 2 preparations 2 groups of (calf serum protein-removing extract freezing-dried powder injection), 3 groups in the sample of embodiment 2 preparations, samples of embodiment 2 preparations are injected the sample 2 of embodiment 2 preparations, the sample 3 of embodiment 2 preparations, the sample 4 of embodiment 2 preparations according to 1.5mg calf serum protein-removing extract dry/kg body weight respectively for 4 groups, once a day, continuous four days.Administering mode is intravenous injection (with the physiological saline solution dissolving, carrying out dissolved dilution according to every white mice injected dose for the 0.5ml calculating concentration).Matched group is injected the normal saline of equal volume ' (0.5m/ only) every day.
All white mice were put into the 250ml wide mouthed bottle that 10g soda lime is housed respectively in 30 minutes after the last administration, airtight normobaric hypoxia is observed the white mice time-to-live, measures the content of glucose and lactic acid in the cerebral tissue behind the mice dying; Brain lactic acid and glucose content are measured according to the described method of document (hair victory, Chen Jincao GM1 rolled up 6 phases, 449-452 in 2004 39 to the influence " Medical University Of Anhui's journal " of the lactic acid and the glucose content of cerebral ischemia) and are carried out.
Normobaric hypoxia white mice time-to-live measurement result is as shown in table 13, and the result shows that 3 groups in the sample of 2 groups in the sample of embodiment 2 preparations, embodiment 2 preparations, 4 groups of white mice time-to-live of sample of embodiment 2 preparations all obviously are longer than the normal saline matched group; There is not significant difference between 2,3,4 groups in the sample of embodiment 2 preparation.Calf serum protein-removing extract freezing-dried powder injection of the present invention has protective effect to the central nervous system under the normobaric hypoxia environment; And each set product curative effect indifference.
The table 13. normobaric hypoxia white mice time-to-live
| Group | The example number | Route of administration | Dosage (mg/kg body weight) | Time-to-live (min) |
| 2 groups in the sample of embodiment 2 preparations | 20 | Intravenous injection | 1.5 | 46.63±7.03 * |
| 3 groups in the sample of embodiment 2 preparations | 20 | Intravenous injection | 1.5 | 46.37±8.20 * |
| 4 groups in the sample of embodiment 2 preparations | 20 | Intravenous injection | 1.5 | 44.44±8.82 * |
| Matched group | 20 | Intravenous injection | Normal saline | 35.65±5.61 |
Shoulder motes * represents to compare P＜0.01 with matched group
Normobaric hypoxia white mice brain lactic acid content measurement result is as shown in table 14, the result shows, 4 groups of white mice brains of sample lactic acid content of 3 groups in the sample of 2 groups in the sample of embodiment 2 preparation, embodiment 2 preparations, embodiment 2 preparations is starkly lower than the normal saline matched group, indifference between 2,3,4 groups in the sample of embodiment 2 preparations.Calf serum protein-removing extract freezing-dried powder injection of the present invention can obviously promote aerobic metabolism; And each set product curative effect indifference.
Table 14. normobaric hypoxia white mice brain lactic acid content
| Group | The example number | Route of administration | Dosage (mg/kgB.W) | Brain lactic acid content (mmol/g.wet.wt) |
| 2 groups in the sample of embodiment 2 preparations | 20 | Intravenous injection | 1.5 | 9.68±0.61 * |
| 3 groups in the sample of embodiment 2 preparations | 20 | Intravenous injection | 1.5 | 9.71±0.56 * |
| 4 groups in the sample of embodiment 2 preparations | 20 | Intravenous injection | 1.5 | 9.63±0.70 * |
| Matched group | 20 | Intravenous injection | Normal saline | 12.36±0.85 |
Shoulder motes * represents to compare P＜0.01 with matched group
Normobaric hypoxia white mice brain glucose content measurement result is as shown in Table 15, the result shows, 4 groups of white mice brains of sample glucose content of 3 groups in the sample of 2 groups in the sample of embodiment 2 preparation, embodiment 2 preparations, embodiment 2 preparations is all apparently higher than matched group, indifference between 2,3,4 groups in the sample of embodiment 2 preparations; Show that calf serum protein-removing extract freezing-dried powder injection of the present invention can obviously promote the picked-up of central nervous system to glucose; And each set product curative effect indifference.
Table 15. normobaric hypoxia white mice brain glucose content
| Group | The example number | Route of administration | Dosage (mg/kgB.W) | Brain glucose content (mmol/g.wet.wt) |
| 2 groups in the sample of embodiment 2 preparations | 20 | Intravenous injection | 1.5 | 5.65±0.72 * |
| 3 groups in the sample of embodiment 2 preparations | 20 | Intravenous injection | 1.5 | 5.46±0.68 * |
| 4 groups in the sample of embodiment 2 preparations | 20 | Intravenous injection | 1.5 | 5.57±0.61 * |
| Matched group | 20 | Intravenous injection | Normal saline | 3.74±0.18 |
Shoulder motes * represents to compare P＜0.01 with matched group
Above-mentioned experiment shows that calf serum protein-removing extract freezing-dried powder injection of the present invention can promote normobaric hypoxia white mice brain cell to carry out aerobic metabolism; Can promote blood or other tissue glucose to transport to cerebral tissue; Can prolong the normobaric hypoxia white mice time-to-live.Show that calf serum protein-removing extract freezing-dried powder injection of the present invention contains several physiological active substances, can strengthen glucose and cell is gone in oxygen transport, increase the utilization of glucose.
Claims (6)
1, a kind of calf serum protein-removing extract freezing-dried powder injection, contain following components by weight portion:
1 part of calf serum protein-removing extract dry below molecular weight 5000 dalton;
9-11 parts in mannitol;
4.5-5.5 parts of micromolecule dextrans;
Wherein, described calf serum protein-removing extract is that the quality percentage composition that will be in the calf serum adds two volumes is 96% ethanol, makes the albuminous degeneration precipitation, and centrifugal removal precipitation is removed the extracting solution that ethanol obtains.
2, the preparation method of the described calf serum protein-removing extract freezing-dried powder injection of claim 1, be mannitol and micromolecule dextran to be joined in the calf serum protein-removing extract according to the described ratio of weight and number of claim 1, after 30-50 ℃ of dissolving, obtain mixed liquor, lyophilizing obtains the calf serum protein-removing extract freezing-dried powder injection.
3, method according to claim 2 is characterized in that: the dry biomass content of described calf serum protein-removing extract is 18-22mg/ml.
4, according to claim 2 or 3 described methods, it is characterized in that: described freeze dried step is earlier-40 ℃ of pre-freezes 1 hour with described mixed liquor, then 20 ℃ of thawings, and then-40 ℃ of pre-freezes after 2 hours, under vacuum state, kept 8-10 hour at-20 ℃, kept 4 hours at-15 ℃, rise to-10 ℃ then, raise 10 ℃ every 1 hour, lyophilizing finishes during to 40 ℃.
5, method according to claim 4 is characterized in that: in the described method, also being included in step of freeze drying preceding is that 5000 daltonian Hollow Fiber Ultrafiltration posts filter with the mixed liquor molecular cut off.
6, method according to claim 5 is characterized in that: also comprise before the described step of freeze drying mixed liquor is carried out filtration sterilization.
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| CNB2007101191557A CN100531723C (en) | 2007-07-17 | 2007-07-17 | Calf serum protein-removing extract for injection and its preparation method |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11604026B2 (en) | 2019-03-14 | 2023-03-14 | Terumo Bct Biotechnologies, Llc | Lyophilization loading tray assembly and system |
| US11634257B2 (en) | 2017-10-09 | 2023-04-25 | Terumo Bct Biotechnologies, Llc | Lyophilization container and method of using same |
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2007
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11634257B2 (en) | 2017-10-09 | 2023-04-25 | Terumo Bct Biotechnologies, Llc | Lyophilization container and method of using same |
| US11604026B2 (en) | 2019-03-14 | 2023-03-14 | Terumo Bct Biotechnologies, Llc | Lyophilization loading tray assembly and system |
| US11609043B2 (en) | 2019-03-14 | 2023-03-21 | Terumo Bct Biotechnologies, Llc | Lyophilization container fill fixture, system and method of use |
| US11609042B2 (en) | 2019-03-14 | 2023-03-21 | Terumo Bct Biotechnologies, Llc | Multi-part lyophilization container and method of use |
| US11740019B2 (en) | 2019-03-14 | 2023-08-29 | Terumo Bct Biotechnologies, Llc | Lyophilization loading tray assembly and system |
| US11747082B2 (en) | 2019-03-14 | 2023-09-05 | Terumo Bct Biotechnologies, Llc | Multi-part lyophilization container and method of use |
| US11815311B2 (en) | 2019-03-14 | 2023-11-14 | Terumo Bct Biotechnologies, Llc | Lyophilization container fill fixture, system and method of use |
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| CN101120924A (en) | 2008-02-13 |
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Address after: 121013 Songshan street, Taihe District, Jinzhou, Liaoning Province, No. 55 Patentee after: Aohong Pharmaceutical Co., Ltd., Jinzhou Address before: 121013 No. 10 West exposed street, Taihe District, Liaoning, Jinzhou Patentee before: Aohong Pharmaceutical Co., Ltd., Jinzhou |