CN104740598A - Method for preparing earthworm maggot protein and application in preparation of medicine for preventing osteosarcoma - Google Patents
Method for preparing earthworm maggot protein and application in preparation of medicine for preventing osteosarcoma Download PDFInfo
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Abstract
The invention discloses a method for preparing an earthworm maggot protein and an application in preparation of a medicine for preventing osteosarcoma. The preparation method comprises the following steps: puffing and smashing an earthworm maggot in a normal temperature puffing device; adding into a puffing solvent system extracting device for extracting the earthworm maggot protein crude product; collecting a solution containing the earthworm maggot protein crude product and filtering out the impurities by a microporus filter; performing ultrafiltration by an ultrafilter and collecting the earthworm maggot protein ultrafiltration solution; dissolving the ultrafiltration solution in the acetonitrile, separating by a solid phase extracting column and collecting an elution component; dissolving the elution component in a solvent system and performing chromatography separation and purification by an ultrasound standing wave solution; preparing freeze dried protein powder by a vacuum freeze dryer, wherein freeze dried protein powder is used for preparing the anti-osteosarcoma medicine. The earthworm maggot protein preparation prepared by the invention is stable in quality, clear in structure, definite in pharmacology, obvious in effect, measurable in content, diverse in dosage forms, low in cost and suitable for industrial production.
Description
Technical field
The present invention relates to a kind of preparation method and novelty teabag thereof of medicine, especially the preparation method of Pheretima maggot protein and the application in the anti-osteosarcoma medicine of preparation thereof.
Background technology
Osteosarcoma is made up of the osteoid tissue of sarcomatous osteoblast and generation thereof and bone trabecula, is one of common bone malignant tumour.
That generally acknowledges at present should adopt Comprehensive Treatment to osteosarcoma, the general need that early stage patient can be performed the operation do staging tomography or radiotherapy, later operation amputation or full resected bone add artificial bone and plant and change, and postoperatively need combined chemotherapy several course for the treatment of with consolidate curative effect, eliminate may be remaining minimal disease.The inoperable patient of transporting patient, generally first does chemotherapy, is optionally performed the operation or radiotherapy later.
Patient condition, according to the situation of disease, is divided into that cloudy plug stagnates, erosion bone resistance network type by the traditional Chinese medical science, scorchingly hotly to pent up, qi stagnation and blood stasis type, and deficiency of kidney-essence, malicious stasis of blood expectorant junction type, suit the remedy to the case, Classification treatment.
CN102532261A discloses bone remoulding activator-cheese dried meat peptide and pharmaceutical composition thereof and purposes.The class that this invention finds the structure of modification of osteogenic growth PEPC end pentapeptide has the cheese dried meat peptide compounds activating bone remoulding activity, is expected to develop into a kind of medicine improving bone mass.
CN103304635A discloses application of a kind of cyclic peptide compound antitumor and preparation method thereof, and the preparation method that this invention relates to cyclic peptide compound generates product to ferment and carries out separation and obtains the activated cyclic peptide compound of tool.
CN103272221A discloses the application of WSB-1 albumen in preparation treatment osteosarcoma medicine.This invention describes WSB-1 albumen to treat osteosarcomatous medicine in preparation or diagnose the application in the product of osteosarcoma and the transfer of prediction osteosarcoma, this medicine comprises the product used RT-PCR, real-time quantitative PCR, immune detection, in situ hybridization or gene chip diagnosis osteosarcoma and predict osteosarcoma transfer.
CN103041366A discloses a kind of bone peptide composition and preparation method thereof, and said composition is with compositions such as bone peptide compound, aminoacid, nucleic acid, free fatties.
CN102053150A discloses a kind of human osteosarcoma cell stem cell related antigen label and application thereof.This invention describes a kind of human osteosarcoma cell stem cell related antigen label SSEA-4 and the application in osteogenic sarcoma targeted therapy thereof.
CN102792927A discloses the technique of artificial culture Pheretima maggot, this invention describes with Chinese traditional medicine sleeper etc. as raw material, grow generate Pheretima maggot through pulverizing, gelatinizing, sterilizing, training, the Pheretima maggot generated by a kind of gentle plain mode conversion eliminates the side effect that Pheretima easily causes, and improves the medical value of Pheretima simultaneously.
Summary of the invention
The object of this invention is to provide a kind of preparation method of Pheretima maggot protein and the application in the anti-osteosarcoma medicine of preparation thereof, adopt suitable method extraction and isolation Pheretima maggot protein in present invention process process, and be aided with corresponding material to the medicament being prepared into different dosage form according to the characteristic of Pheretima maggot protein.
The object of the invention is to be achieved through the following technical solutions:
(1) by the expanded pulverizing under normal temperature condition in room temperature expanding apparatus (200520108339.X) of 1000g Pheretima maggot.
In this step, described bulking pressure is 0.1 ~ 5MPa, and the time is 10 ~ 60min.
(2) material after expanded pulverizing joins in expanded solvents system extraction element (201210179076.6), adds expanded solvents and is forced into 2 ~ 10MPa, forms expanded solvents system, extracts Pheretima maggot protein crude product.
In this step, described expanded solvents is by 1 ~ 10Kg liquid carbon dioxide, 100 ~ 500mL methanol and 100 ~ 300gNH
3h
2o forms or is made up of 1 ~ 10Kg liquid carbon dioxide, 100 ~ 500mL ethanol and 100 ~ 300g ammonium chloride.
In this step, described Extracting temperature is 10 ~ 20 DEG C, and the time is 10 ~ 30min.
(3) collect the solution containing Pheretima maggot protein crude product and cross with microfilter and filter to remove impurity.
In this step, the aperture of described microfilter is 0.5 ~ 50 μm.
(4) use ultrafilter hyperfiltration treatment, collect the Pheretima maggot protein ultrafiltration solution containing corresponding molecular weight.
In this step, the aperture of described ultrafilter is 50 ~ 100nm.
(5) ultrafiltrate is dissolved in suitable solvent and is separated with solid-phase extraction column, collects eluting composition.
In this step, described solvent is acetonitrile.
(6) by eluting component dissolves in suitable dicyandiamide solution, with ultrasonic standing wave preparative liquid chromatogram (200920274485.8) separation and purification.
In this step, described dicyandiamide solution is made up of n-butyl alcohol-ethyl acetate-water (volume ratio 1.25:3.75:5) or 4.5% Polyethylene Glycol-7% Dextran T 500 (weight ratio) forms.
(7) the Pheretima maggot protein vacuum freeze drier after purification is prepared into lyophilized protein powder.
In this step, described vacuum lyophilization temperature is-30 ~ 50 DEG C, the time is 18 ~ 26h.
(8) give different medicinal auxiliary material, be prepared into injection, oral agents, suppository etc. respectively, can be used for preparing anti-osteosarcoma medicine.
Advantage of the present invention and beneficial effect as follows: recording relative molecular weight by the Pheretima maggot protein SDS-polyacrylamide gel electrophoresis of separation and purification of the present invention and sephadex G-75 gel filtration is 11.8 ~ 12.6KPa; Measuring its isoelectric point, IP with isoelectric focussing is pH9.6 ~ 10.7; Conventional chemistry analysis result shows it not containing saccharide and phosphate group; The Pheretima maggot protein content that ultraviolet spectrophotometry analysis measures purification at 260nm and 280nm is 98.2%; Molecular structure belongs to atypia, non-single insect protein, and do not contain disulfide bond with interchain in chain, its tertiary structure conformation unit is based on random coil, and the content ratio of α spiral and β-pleated sheet is substantially suitable.The Pheretima maggot protein quality of the pharmaceutical preparations prepared by the present invention is stablized, and structure is clear, and pharmacology is distinct, and curative effect is obvious, and content can be examined and can survey, and dosage form is various, and cost is lower, is applicable to suitability for industrialized production.
Detailed description of the invention
Below in conjunction with embodiment, technical scheme of the present invention is further described; but do not limit to so; everyly technical solution of the present invention modified or equivalent to replace, and not departing from the spirit and scope of technical solution of the present invention, all should be encompassed in protection scope of the present invention.
embodiment 1:
(1) 1000g Pheretima maggot dry product is loaded in room temperature expanding apparatus (200520108339.X), to being filled with carbon dioxide in expanding apparatus and being forced into 0.1 ~ 5MPa, time 10 ~ 60min, then release pressure is to normal pressure rapidly, and Pheretima maggot is by expanded one-tenth powder body;
(2) the Pheretima maggot powder body after expanded pulverizing joins in expanded solvents system extraction element (201210179076.6), adds 1 ~ 10Kg liquid carbon dioxide, 100 ~ 500mL methanol and 100 ~ 300gNH
3h
2o, is forced into 2 ~ 10MPa, forms expanded solvents system, and keep temperature 10 ~ 20 DEG C, time 10 ~ 30min, the proteolytic in Pheretima maggot powder body, is then depressurized to normal pressure, generates Pheretima maggot protein crude product and methanol-NH
3h
2the mixed liquor of O;
(3) collect the solution containing Pheretima maggot protein crude product, filter place to go solid impurity with the microfilter in 0.5 ~ 50 μm, aperture;
(4) the solution ultrafilter hyperfiltration treatment of aperture 50 ~ 100nm, pressure 0.3 ~ 0.8MPa, temperature is room temperature, collects the Pheretima maggot protein ultrafiltration solution containing corresponding molecular weight;
(5) ultrafiltration solution 20% acetonitrile dissolves, C
18solid-phase extraction column 50 ~ 100% acetonitrile activating pretreatments, add 0.02 ~ 0.1% trifluoroacetic acid-water balance, the ultrafiltration solution upper prop dissolved with 10% acetonitrile solution, wash with the acetonitrile gradient of 10 ~ 80% is de-under room temperature, flow velocity 1 ~ 10mL/min, collects the eluting composition of associated gradients;
(6) by eluting component dissolves in 4.5% Polyethylene Glycol-7% Dextran T 500 (weight ratio) dicyandiamide solution, with ultrasonic standing wave preparative liquid chromatogram (200920274485.8) separation and purification, ultrasonic power 0.2 ~ 3.0KW, frequency 500 ~ 1000KHz, flow velocity 50 ~ 500mL/min, UV-detector wavelength 260 ~ 280nm;
(7) the Pheretima maggot protein vacuum freeze drier after purification is prepared into lyophilized protein powder in temperature-30 ~ 50 DEG C, time 18 ~ 26h drying.
embodiment 2:
(1) 1000g Pheretima maggot dry product is loaded in room temperature expanding apparatus (200520108339.X), to being filled with carbon dioxide in expanding apparatus and being forced into 0.1 ~ 5MPa, time 10 ~ 60min, then release pressure is to normal pressure rapidly, and Pheretima maggot is by expanded one-tenth powder body;
(2) the Pheretima maggot powder body after expanded pulverizing joins in expanded solvents system extraction element (201210179076.6), adds 5Kg liquid carbon dioxide, 300mL methanol and 200gNH
3h
2o, is forced into 5MPa, forms expanded solvents system, and keep temperature 15 DEG C, time 20min, the proteolytic in Pheretima maggot powder body, is then depressurized to normal pressure, generates Pheretima maggot protein crude product and methanol-NH
3h
2the mixed liquor of O;
(3) collect the solution containing Pheretima maggot protein crude product, filter place to go solid impurity with the microfilter in 30 μm, aperture;
(4) the solution ultrafilter hyperfiltration treatment of aperture 100nm, pressure 0.5MPa, temperature is room temperature, collects the Pheretima maggot protein ultrafiltration solution containing corresponding molecular weight;
(5) ultrafiltration solution 20% acetonitrile dissolves, C
18solid-phase extraction column 80% acetonitrile activating pretreatment, adds 0.05% trifluoroacetic acid-water balance, the ultrafiltration solution upper prop dissolved with 10% acetonitrile solution, and wash with the acetonitrile gradient of 10 ~ 80% is de-under room temperature, flow velocity 5mL/min, collects the eluting composition of associated gradients;
(6) by eluting component dissolves in 4.5% Polyethylene Glycol-7% Dextran T 500 (weight ratio) dicyandiamide solution, with ultrasonic standing wave preparative liquid chromatogram (200920274485.8) separation and purification, ultrasonic power 1.0KW, frequency 500KHz, flow velocity 200mL/min, UV-detector wavelength 260nm;
(7) the Pheretima maggot protein vacuum freeze drier after purification is prepared into lyophilized protein powder in temperature-30 ~ 40 DEG C, time 20h drying.
embodiment 3:
(1) 1000g Pheretima maggot dry product is loaded in room temperature expanding apparatus (200520108339.X), to being filled with carbon dioxide in expanding apparatus and being forced into 0.1 ~ 5MPa, time 10 ~ 60min, then release pressure is to normal pressure rapidly, and Pheretima maggot is by expanded one-tenth powder body;
(2) the Pheretima maggot powder body after expanded pulverizing joins in expanded solvents system extraction element (201210179076.6), add 1 ~ 10Kg liquid carbon dioxide, 100 ~ 500mL ethanol and 100 ~ 300g ammonium chloride, be forced into 2 ~ 10MPa, form expanded solvents system, keep temperature 10 ~ 20 DEG C, time 10 ~ 30min, the proteolytic in Pheretima maggot powder body, then be depressurized to normal pressure, generate the mixed liquor of Pheretima maggot protein crude product and ethanol-ammonium chloride;
(3) collect the solution containing Pheretima maggot protein crude product, filter place to go solid impurity with the microfilter in 0.5 ~ 50 μm, aperture;
(4) the solution ultrafilter hyperfiltration treatment of aperture 50 ~ 100nm, pressure 0.3 ~ 0.8MPa, temperature is room temperature, collects the Pheretima maggot protein ultrafiltration solution containing corresponding molecular weight;
(5) ultrafiltration solution 20% acetonitrile dissolves, C
18solid-phase extraction column 50 ~ 100% acetonitrile activating pretreatments, add 0.02 ~ 0.1% trifluoroacetic acid-water balance, the ultrafiltration solution upper prop dissolved with 10% acetonitrile solution, wash with the acetonitrile gradient of 10 ~ 80% is de-under room temperature, flow velocity 1 ~ 10mL/min, collects the eluting composition of associated gradients;
(6) by eluting component dissolves in n-butyl alcohol-ethyl acetate-water (volume ratio 1.25:3.75:5) dicyandiamide solution, with ultrasonic standing wave preparative liquid chromatogram (200920274485.8) separation and purification, ultrasonic power 0.2 ~ 3.0KW, frequency 500 ~ 1000KHz, flow velocity 50 ~ 500mL/min, UV-detector wavelength 260 ~ 280nm;
(7) the Pheretima maggot protein vacuum freeze drier after purification is prepared into lyophilized protein powder in temperature-30 ~ 50 DEG C, time 18 ~ 26h drying.
embodiment 4:
(1) 1000g Pheretima maggot dry product is loaded in room temperature expanding apparatus (200520108339.X), to being filled with carbon dioxide in expanding apparatus and being forced into 2MPa, time 40min, then release pressure is to normal pressure rapidly, and Pheretima maggot is by expanded one-tenth powder body;
(2) the Pheretima maggot powder body after expanded pulverizing joins in expanded solvents system extraction element (201210179076.6), add 6Kg liquid carbon dioxide, 200mL ethanol and 150g ammonium chloride, be forced into 6MPa, form expanded solvents system, keep temperature 20 DEG C, time 10min, the proteolytic in Pheretima maggot powder body, then be depressurized to normal pressure, generate the mixed liquor of Pheretima maggot protein crude product and ethanol-ammonium chloride;
(3) collect the solution containing Pheretima maggot protein crude product, filter place to go solid impurity with the microfilter in 50 μm, aperture;
(4) the solution ultrafilter hyperfiltration treatment of aperture 100nm, pressure 0.5MPa, temperature is room temperature, collects the Pheretima maggot protein ultrafiltration solution containing corresponding molecular weight;
(5) ultrafiltration solution 20% acetonitrile dissolves, C
18solid-phase extraction column 60% acetonitrile activating pretreatment, adds 0.08% trifluoroacetic acid-water balance, the ultrafiltration solution upper prop dissolved with 10% acetonitrile solution, and wash with the acetonitrile gradient of 10 ~ 80% is de-under room temperature, flow velocity 8mL/min, collects the eluting composition of associated gradients;
(6) by eluting component dissolves in n-butyl alcohol-ethyl acetate-water (volume ratio 1.25:3.75:5) dicyandiamide solution, with ultrasonic standing wave preparative liquid chromatogram (200920274485.8) separation and purification, ultrasonic power 2.0KW, frequency 1000KHz, flow velocity 300mL/min, UV-detector wavelength 280nm;
(7) the Pheretima maggot protein vacuum freeze drier after purification is prepared into lyophilized protein powder in temperature-30 ~ 50 DEG C, time 18h drying.
embodiment 5:
Get Pheretima maggot lyophilized protein powder 100 ~ 150g, lecithin 1 ~ 15g, liquid paraffin 200 ~ 350g, three is stirred, pasty state is ground well into 30 ~ 60r/min with grinder, separately get oleum sapii 100 ~ 200g heat in water-bath 40 ~ 60 DEG C fusing after, be cooled to 25 ~ 38 DEG C, pour in the Pheretima maggot protein-lecithin-liquid paraffin mixture ground well and stir rapidly, be cast in while hot in suppository moulds, condense to 10 ~ 20 DEG C, from suppository moulds, take out the Pheretima maggot protein suppository being molding, arrange smooth rear packed products.
embodiment 6:
Get Pheretima maggot lyophilized protein powder 120g, lecithin 10g, liquid paraffin 250g, three is stirred, pasty state is ground well into 60r/min with grinder, separately get oleum sapii 150g heat in water-bath 60 DEG C fusing after, be cooled to 30 DEG C, pour in the Pheretima maggot protein-lecithin-liquid paraffin mixture ground well and stir rapidly, be cast in while hot in suppository moulds, condense to 15 DEG C, from suppository moulds, take out the Pheretima maggot protein suppository being molding, arrange smooth rear packed products.
embodiment 7:
Prepared by lipidosome injection:
(1) after the 1mol Phosphatidylserine glass planar method of forming makes thin film, add 1000 ~ 3000L normal saline hydration-treated, then use ultrasound wave at frequency 500 ~ 2000KHz, power 0.01 ~ 5KW process 1 ~ 5min.
(2) 0.1 ~ 1000mLCaCl that above-mentioned solution 1 ~ 100mL joins content 0.001 ~ 0.01mol/L is got
2in solution, under rotating speed 30 ~ 75r/min condition, stir 10 ~ 30min, then under 25 ~ 50 DEG C of conditions, leave standstill 30 ~ 120min.
(3) product is placed in centrifuge, at rotating speed 1500 ~ 3000r/min centrifugalize 5 ~ 30min, after being taken out from centrifuge by centrifugal thing, add 2 ~ 50g Pheretima maggot protein mix homogeneously, under 10 ~ 25 DEG C of conditions, leave standstill 10 ~ 120min.
(4) above-mentioned object adds in the normal saline solution 0.1 ~ 10L of the ethylenediaminetetraacetic acid containing 0.01 ~ 10mol/L, and mix homogeneously leaves standstill 30 ~ 180min, obtains unilamellar liposome.
(5) get 1 ~ 10mg unilamellar liposome to mix with 10 ~ 200mL normal saline solution, preparation becomes injection.
embodiment 8:
Prepared by lipidosome injection:
(1), after the 1mol Phosphatidylserine glass planar method of forming makes thin film, 2000L normal saline hydration-treated is added, then with ultrasound wave at frequency 1000KHz, power 2KW process 5min.
(2) 1000mLCaCl that above-mentioned solution 50mL joins content 0.005mol/L is got
2in solution, under rotating speed 45r/min condition, stir 30min, then under 30 DEG C of conditions, leave standstill 60min.
(3) product is placed in centrifuge, at rotating speed 2000r/min centrifugalize 20min, after being taken out from centrifuge by centrifugal thing, add 20g Pheretima maggot protein mix homogeneously, under 15 DEG C of conditions, leave standstill 60min.
(4) above-mentioned object adds in the normal saline solution 5L of the ethylenediaminetetraacetic acid containing 1mol/L, and mix homogeneously leaves standstill 80min, obtains unilamellar liposome.
(5) get 5mg unilamellar liposome to mix with 100mL normal saline solution, preparation becomes injection.
embodiment 9:
The preparation of enteric coated capsule:
Get 10 ~ 100g Pheretima maggot protein, 500 ~ 2000g pregelatinized starch and 5-50g lactose fully to mix, being packed into preparation in hydroxypropyl emthylcellulose enteric coated capsule becomes Pheretima maggot protein enteric coated capsule.
embodiment 10:
The preparation of enteric coated capsule:
Get 50g Pheretima maggot protein, 1000g pregelatinized starch and 30g lactose fully to mix, being packed into preparation in hydroxypropyl emthylcellulose enteric coated capsule becomes Pheretima maggot protein enteric coated capsule.
embodiment 11:
The preparation of externally-applied soft ointment:
Get 10 ~ 20g Pheretima maggot protein and be placed in sterilizing mortar, add and use 120 ~ 150 DEG C of dry heat sterilization 30 ~ 60min in advance and 10 ~ 20mL the liquid paraffin let cool, grind to form pasty state, then add and use 150 ~ 160 DEG C of dry heat sterilization 60 ~ 90min in advance and 2 ~ 5g the vaseline let cool grinds well, obtain ointment.
embodiment 12:
The preparation of externally-applied soft ointment:
Get 150g Pheretima maggot protein and be placed in sterilizing mortar, add in advance with 130 DEG C of dry heat sterilization 40min and the 15mL liquid paraffin let cool, grind to form pasty state, then add in advance with 155 DEG C of dry heat sterilization 80min and the 4g vaseline let cool grind well, obtain ointment
embodiment 13:
Osteosarcoma staging:
0 phase: cancer is confined to mucous layer, without lymphatic metastasis;
phase: within tumor is confined to muscularis propria, without lymphatic metastasis;
phase: tumor-infiltratedly exceed muscularis propria, but without lymphatic metastasis;
phase: lymphatic metastasis;
phase: metastasis (brain, liver, lung etc.) or peritoneum transfer.
Therapeutic Method:
Patient need strictly give up cigarette, wine, maror, patient by body weight 70Kg, every day injecting lipid body medicament 1 time, each 300 μ g; Or every day take enteric coated capsule 2 times, each 3; Or suppository in treatment, every day 1 time, each 1; Or externally-applied soft ointment, every day 3 times, each 0.2g.
Therapeutic outcome grade scale:
, short term effect (40 days end with treat before compare)
(1) complete incidence graph: focus disappears completely, occurs without new focus;
(2) partial rcsponse: focus narrows down to 50% or less before treatment, occurs without new focus, during multifocal pathological changes, neither one focus increases;
(3) stable phase:
minor responses: focus reduces area 30% or area increased 25%, subjective symptoms is improved, and physical basal conditions rises;
basicly stable: focus reduces and is no more than more than 25% less than 25% or area increased, subjective symptoms is improved, and physical basal conditions rises.
(4) expand: single area or multiple focus gross area are than increase more than 25% before treatment or occur new pathological changes, and clinical symptoms increases the weight of, and physical basal conditions declines.
, mid-term effects (7 weeks ~ 1 year)
(1) complete incidence graph: survive without tumor;
(2) partial rcsponse:
area more than 50% before corpus carcinosus returns and changes to treatment, or there is new focus;
corpus carcinosus is without increase or reduce, and disease is improved or disappeared;
muscle power basal conditions rises.
(3) transfer or expansion: cause of disease stove expands development, and occur new metastasis, clinical symptoms increases the weight of, and physical situation declines.
, late result (1 ~ 5 year)
(1) survive without tumor;
(2) tumor existence is with;
(3) dead.
Therapeutic outcome:
Claims (10)
1. a preparation method for Pheretima maggot protein, is characterized in that described preparation method step is as follows:
(1) by the expanded pulverizing under normal temperature condition in room temperature expanding apparatus of 1000g Pheretima maggot;
(2) material after expanded pulverizing joins in expanded solvents system extraction element, adds by 1 ~ 10Kg liquid carbon dioxide, 100 ~ 500mL methanol and 100 ~ 300gNH
3h
2the expanded solvents that O forms or is made up of 1 ~ 10Kg liquid carbon dioxide, 100 ~ 500mL ethanol and 100 ~ 300g ammonium chloride is also forced into 2 ~ 10MPa, forms expanded solvents system, extracts Pheretima maggot protein crude product;
(3) collect the solution containing Pheretima maggot protein crude product and cross with microfilter and filter to remove impurity;
(4) use ultrafilter hyperfiltration treatment, collect Pheretima maggot protein ultrafiltration solution;
(5) ultrafiltrate is dissolved in acetonitrile and is separated with solid-phase extraction column, collects eluting composition;
(6) by eluting component dissolves by n-butyl alcohol-ethyl acetate-water dicyandiamide solution that 1.25:3.75:5 forms or is made up of by weight 4.5:7 Polyethylene Glycol-Dextran T 500 by volume, use ultrasonic standing wave preparative liquid chromatogram separation and purification;
(7) the Pheretima maggot protein vacuum freeze drier after purification is prepared into lyophilized protein powder.
2. the preparation method of Pheretima maggot protein according to claim 1, it is characterized in that described bulking pressure is 0.1 ~ 5MPa, the time is 10 ~ 60min.
3. the preparation method of Pheretima maggot protein according to claim 1, it is characterized in that described Extracting temperature is 10 ~ 20 DEG C, the time is 10 ~ 30min.
4. the preparation method of Pheretima maggot protein according to claim 1, is characterized in that the aperture of described microfilter is 0.5 ~ 50 μm, and the aperture of ultrafilter is 50 ~ 100nm.
5. the preparation method of Pheretima maggot protein according to claim 1, is characterized in that described vacuum lyophilization temperature is-30 ~ 50 DEG C, the time is 18 ~ 26h.
6. the Pheretima maggot protein that prepared by the arbitrary described method of claim 1-5 is preparing the application in anti-osteosarcoma medicine.
7. the application of Pheretima maggot protein according to claim 6 in the anti-osteosarcoma medicine of preparation, it is characterized in that the dosage form of described anti-osteosarcoma medicine is suppository, preparation method is as follows:
Get Pheretima maggot lyophilized protein powder 100 ~ 150g, lecithin 1 ~ 15g, liquid paraffin 200 ~ 350g, three is stirred, pasty state is ground well into 30 ~ 60r/min with grinder, separately get oleum sapii 100 ~ 200g heat in water-bath 40 ~ 60 DEG C fusing after, be cooled to 25 ~ 38 DEG C, pour in the Pheretima maggot protein-lecithin-liquid paraffin mixture ground well and stir rapidly, be cast in while hot in suppository moulds, condense to 10 ~ 20 DEG C, from suppository moulds, take out the Pheretima maggot protein suppository being molding.
8. the application of Pheretima maggot protein according to claim 6 in the anti-osteosarcoma medicine of preparation, it is characterized in that the dosage form of described anti-osteosarcoma medicine is lipidosome injection, preparation method is as follows:
(1) after the 1mol Phosphatidylserine glass planar method of forming makes thin film, add 1000 ~ 3000L normal saline hydration-treated, then use ultrasound wave at frequency 500 ~ 2000KHz, power 0.01 ~ 5KW process 1 ~ 5min;
(2) 0.1 ~ 1000mLCaCl that above-mentioned solution 1 ~ 100mL joins content 0.001 ~ 0.01mol/L is got
2in solution, under rotating speed 30 ~ 75r/min condition, stir 10 ~ 30min, then under 25 ~ 50 DEG C of conditions, leave standstill 30 ~ 120min;
(3) product is placed in centrifuge, at rotating speed 1500 ~ 3000r/min centrifugalize 5 ~ 30min, after being taken out from centrifuge by centrifugal thing, add 2 ~ 50g Pheretima maggot protein mix homogeneously, under 10 ~ 25 DEG C of conditions, leave standstill 10 ~ 120min;
(4) above-mentioned object adds in the normal saline solution 0.1 ~ 10L of the ethylenediaminetetraacetic acid containing 0.01 ~ 10mol/L, and mix homogeneously leaves standstill 30 ~ 180min, obtains unilamellar liposome;
(5) get 1 ~ 10mg unilamellar liposome to mix with 10 ~ 200mL normal saline solution, preparation becomes injection.
9. the application of Pheretima maggot protein according to claim 6 in the anti-osteosarcoma medicine of preparation, it is characterized in that the dosage form of described anti-osteosarcoma medicine is enteric coated capsule, preparation method is as follows:
Get 10 ~ 100g Pheretima maggot protein, 500 ~ 2000g pregelatinized starch and 5-50g lactose fully to mix, be packed in hydroxypropyl emthylcellulose enteric coated capsule, preparation becomes Pheretima maggot protein enteric coated capsule.
10. the application of Pheretima maggot protein according to claim 6 in the anti-osteosarcoma medicine of preparation, it is characterized in that the dosage form of described anti-osteosarcoma medicine is externally-applied soft ointment, preparation method is as follows:
Get 10 ~ 20g Pheretima maggot protein and be placed in sterilizing mortar, add and use 120 ~ 150 DEG C of dry heat sterilization 30 ~ 60min in advance and 10 ~ 20mL the liquid paraffin let cool, grind to form pasty state, then add and use 150 ~ 160 DEG C of dry heat sterilization 60 ~ 90min in advance and 2 ~ 5g the vaseline let cool grinds well, obtain ointment.
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| CN107389580A (en) * | 2017-07-12 | 2017-11-24 | 广西壮族自治区农业科学院植物保护研究所 | A kind of method for detecting brown paddy plant hopper protein total content |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1606929A (en) * | 2003-10-16 | 2005-04-20 | 吴永胜 | Highly active multifunctional products series of earthworm and its production method |
| CN101057886A (en) * | 2006-04-17 | 2007-10-24 | 孙辅 | Medicine for treating early bone tumor |
| CN102702308A (en) * | 2012-06-02 | 2012-10-03 | 哈尔滨工业大学 | Expansion solvent system extraction device and process for separating toad maggot proteins by using same |
| CN102792927A (en) * | 2012-08-31 | 2012-11-28 | 哈尔滨工业大学 | Process for artificially incubating earthworm maggots |
| CN104095116A (en) * | 2014-06-16 | 2014-10-15 | 胡安然 | Tea capable of alleviating bone cancer |
-
2015
- 2015-03-02 CN CN201510092399.5A patent/CN104740598A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1606929A (en) * | 2003-10-16 | 2005-04-20 | 吴永胜 | Highly active multifunctional products series of earthworm and its production method |
| CN101057886A (en) * | 2006-04-17 | 2007-10-24 | 孙辅 | Medicine for treating early bone tumor |
| CN102702308A (en) * | 2012-06-02 | 2012-10-03 | 哈尔滨工业大学 | Expansion solvent system extraction device and process for separating toad maggot proteins by using same |
| CN102792927A (en) * | 2012-08-31 | 2012-11-28 | 哈尔滨工业大学 | Process for artificially incubating earthworm maggots |
| CN104095116A (en) * | 2014-06-16 | 2014-10-15 | 胡安然 | Tea capable of alleviating bone cancer |
Non-Patent Citations (2)
| Title |
|---|
| 彭劲甫等: "地龙的药理作用与保健功能", 《中药材》 * |
| 程春旭等: "抗肿瘤药物作用机制的研究进展", 《吉林医学》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107389580A (en) * | 2017-07-12 | 2017-11-24 | 广西壮族自治区农业科学院植物保护研究所 | A kind of method for detecting brown paddy plant hopper protein total content |
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