CN104645307A - Application of gecko and maggot protein in preparation of anti-ovarian cancer medicines - Google Patents
Application of gecko and maggot protein in preparation of anti-ovarian cancer medicines Download PDFInfo
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- CN104645307A CN104645307A CN201510092375.XA CN201510092375A CN104645307A CN 104645307 A CN104645307 A CN 104645307A CN 201510092375 A CN201510092375 A CN 201510092375A CN 104645307 A CN104645307 A CN 104645307A
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Abstract
The invention discloses an application of gecko and maggot protein in preparation of anti-ovarian cancer medicines. A preparation method comprises the following steps: puffing and crushing geckos and maggots in a room-temperature puffing device, adding to a swelling solvent system extraction device, and extracting out a crude product of the gecko and maggot protein; collecting a solution containing the crude product of the gecko and maggot protein, filtering and removing impurities by virtue of a micro-pore filter; carrying out ultra-filtration treatment by virtue of an ultrafilter, collecting an ultra-filtration solution of the gecko and maggot protein; dissolving the ultra-filtration solution in acetonitrile, separating by virtue of a solid-phase extraction column, collecting an eluting component; dissolving the eluting component into a solvent system, and carrying out liquid-phase preparation and chromatographic separation and purification with an ultrasonic standing wave; and preparing freeze-dried protein powder by virtue of a vacuum freeze drier to prepare the anti-ovarian cancer medicines. The gecko and maggot protein preparation prepared by the method is stable in quality, clear in structure, clear in pharmacology, obvious in curative effect, datable in content, diverse in dosage form, relatively low in cost and suitable for industrialized production.
Description
Technical field
The present invention relates to a kind of novelty teabag of Gekko Swinhonis maggot protein, especially Gekko Swinhonis maggot protein is preparing the application in ovarian cancer resistance medicine.
Background technology
Ovarian cancer is the malignant tumor betiding ovary tissue, and he is one of common malignant tumor of important threat WomanHealth, and in gynecologic malignant tumor, sickness rate accounts for the 3rd, is only second to cervical cancer and carcinoma of endometrium at China's sickness rate.
Ovarian cancer classification is following three kinds: (1) epithelial tumor: comprise myxedema, serosity adenoma, endometrioid adenoma, clear cell tumor, suddenly strangle tumor and mixed epithelium tumor; (2) sex cord-stromal tumor: comprise granule Leydig's cell tumor, embryoma, polyembryoma, choriocarcinoma, teratoma etc.; (4) lipoid glucagonoma, the Non-specific soft tissue neoplasms of non-ovary, gonadoblastoma, metastatic tumo(u)r, tumor-like lesion etc.
Generally acknowledge the cause of disease and pathogenesis all relevant with ovarian tumors with environmental factors, endocrine factors, inherited genetic factors, dietary habit, viral infection, chemistry, lonizing radiation, Nervous and Mental Factors etc.
Therapeutic Method adopts operative surgery treatment, radiotherapy, chemotherapy, and the symptom of ovarian cancer to be then divided in yang deficiency of spleen and stomache, liver-kidney yin deficiency, deficiency of qi and blood type, blood stasis junction type in junction type and irritability by the traditional Chinese medical science, suits the remedy to the case, classifying type treatment.
CN103146703A discloses the SiRNA and recombinant vector thereof and application that suppress epithelial ovarian tumor growth.This invention screens a kind of recombinant vector suppressing the interference sequence of epithelial ovarian oncogene expression and construct containing this interference sequence.
CN102670944A discloses a kind of compound Chinese medicinal preparation for the treatment of ovarian cancer and preparation method thereof.This compound Chinese medicinal preparation makes solid or liquid dosage form by the extract of 11 kinds of Chinese herbal medicine such as the Radix Astragali, Radix Codonopsis, the Radix Rehmanniae, Herba Scutellariae Barbatae, the Rhizoma Atractylodis Macrocephalae, Fructus Lycii, the Radix Aucklandiae, Radix Asparagi, the Radix Paeoniae Alba, shows to have comparatively obvious curative effects through zoopery.
CN102876770A discloses a kind of neoplasm metastasis marker gene and application thereof, the invention provides a kind of neoplasm metastasis marker gene and the application for the exploitation of anticancer novel drugs thereof, additionally provide neoplasm metastasis marker gene and preparing the application of diagnostic agent of cancer diagnosis.
CN102908321A discloses a kind of albumin bound type effect of nano-paclitaxel lyophilized formulations and preparation method thereof.The preparation of this invention is prepared into lyophilized formulations by paclitaxel, human serum albumin, surfactant, oil phase, the agent of lyophilizing skeleton, stabilizing agent, pH adjusting agent.
CN102783461A discloses the technique of artificial culture Gekko Swinhonis maggot, the present invention grows culture medium with raw material trainings such as Gekko Swinhonis, make Gekko Swinhonis body macromole be converted to micromolecule composition by a kind of temperate condition, cultivating process eliminates toxin and the anaphylactogen of Gekko Swinhonis, expands the medicinal scope of Gekko Swinhonis.
Summary of the invention
The object of this invention is to provide a kind of Gekko Swinhonis maggot protein and prepare the application in ovarian cancer resistance medicine, adopt suitable method extraction and isolation Gekko Swinhonis 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 Gekko Swinhonis 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 Gekko Swinhonis 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 Gekko Swinhonis 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 Gekko Swinhonis 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 Gekko Swinhonis 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 Gekko Swinhonis 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 ovarian cancer resistance medicine.
Advantage of the present invention and beneficial effect as follows: recording relative molecular weight by the Gekko Swinhonis 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 Gekko Swinhonis 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 Gekko Swinhonis 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 Gekko Swinhonis 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 Gekko Swinhonis maggot is by expanded one-tenth powder body;
(2) the Gekko Swinhonis 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 Gekko Swinhonis maggot powder body, is then depressurized to normal pressure, generates Gekko Swinhonis maggot protein crude product and methanol-NH
3h
2the mixed liquor of O;
(3) collect the solution containing Gekko Swinhonis 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 Gekko Swinhonis 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 Gekko Swinhonis 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 Gekko Swinhonis 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 Gekko Swinhonis maggot is by expanded one-tenth powder body;
(2) the Gekko Swinhonis 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 Gekko Swinhonis maggot powder body, is then depressurized to normal pressure, generates Gekko Swinhonis maggot protein crude product and methanol-NH
3h
2the mixed liquor of O;
(3) collect the solution containing Gekko Swinhonis 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 Gekko Swinhonis 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 Gekko Swinhonis 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 Gekko Swinhonis 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 Gekko Swinhonis maggot is by expanded one-tenth powder body;
(2) the Gekko Swinhonis 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 Gekko Swinhonis maggot powder body, then be depressurized to normal pressure, generate the mixed liquor of Gekko Swinhonis maggot protein crude product and ethanol-ammonium chloride;
(3) collect the solution containing Gekko Swinhonis 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 Gekko Swinhonis 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 Gekko Swinhonis 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 Gekko Swinhonis 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 Gekko Swinhonis maggot is by expanded one-tenth powder body;
(2) the Gekko Swinhonis 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 Gekko Swinhonis maggot powder body, then be depressurized to normal pressure, generate the mixed liquor of Gekko Swinhonis maggot protein crude product and ethanol-ammonium chloride;
(3) collect the solution containing Gekko Swinhonis 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 Gekko Swinhonis 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 Gekko Swinhonis 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 Gekko Swinhonis 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 Gekko Swinhonis 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 Gekko Swinhonis maggot protein suppository being molding, arrange smooth rear packed products.
embodiment 6:
Get Gekko Swinhonis 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 Gekko Swinhonis 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 Gekko Swinhonis 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 Gekko Swinhonis 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 Gekko Swinhonis 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 Gekko Swinhonis maggot protein, 500 ~ 2000g pregelatinized starch and 5-50g lactose fully to mix, being packed into preparation in hydroxypropyl emthylcellulose enteric coated capsule becomes Gekko Swinhonis maggot protein enteric coated capsule.
embodiment 10:
The preparation of enteric coated capsule:
Get 50g Gekko Swinhonis maggot protein, 1000g pregelatinized starch and 30g lactose fully to mix, being packed into preparation in hydroxypropyl emthylcellulose enteric coated capsule becomes Gekko Swinhonis maggot protein enteric coated capsule.
embodiment 11:
Ovarian cancer disease is by stages:
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.
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 (5)
1. a Gekko Swinhonis maggot protein is preparing the application in ovarian cancer resistance medicine.
2. Gekko Swinhonis maggot protein according to claim 1 is preparing the application in ovarian cancer resistance medicine, it is characterized in that the preparation process of described Gekko Swinhonis maggot protein is as follows:
(1) by the expanded pulverizing under normal temperature condition in room temperature expanding apparatus of 1000g Gekko Swinhonis 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 Gekko Swinhonis maggot protein crude product;
(3) collect the solution containing Gekko Swinhonis maggot protein crude product and cross with microfilter and filter to remove impurity;
(4) use ultrafilter hyperfiltration treatment, collect Gekko Swinhonis 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 Gekko Swinhonis maggot protein vacuum freeze drier after purification is prepared into lyophilized protein powder.
3. Gekko Swinhonis maggot protein according to claim 1 is preparing the application in ovarian cancer resistance medicine, and it is characterized in that the dosage form of described ovarian cancer resistance medicine is suppository, preparation method is as follows:
Get Gekko Swinhonis 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 Gekko Swinhonis 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 Gekko Swinhonis maggot protein suppository being molding.
4. Gekko Swinhonis maggot protein according to claim 1 is preparing the application in ovarian cancer resistance medicine, and it is characterized in that the dosage form of described ovarian cancer resistance 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 Gekko Swinhonis 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.
5. Gekko Swinhonis maggot protein according to claim 1 is preparing the application in ovarian cancer resistance medicine, and it is characterized in that the dosage form of described ovarian cancer resistance medicine is enteric coated capsule, preparation method is as follows:
Get 10 ~ 100g Gekko Swinhonis maggot protein, 500 ~ 2000g pregelatinized starch and 5-50g lactose fully to mix, be packed in hydroxypropyl emthylcellulose enteric coated capsule, preparation becomes Gekko Swinhonis maggot protein enteric coated capsule.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1535694A (en) * | 2003-04-07 | 2004-10-13 | 谢东泽 | Chinese medicine for curing tumor and its production method |
| 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 |
| CN102783461A (en) * | 2012-08-31 | 2012-11-21 | 哈尔滨工业大学 | Process for artificially breeding wall gecko maggots |
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2015
- 2015-03-02 CN CN201510092375.XA patent/CN104645307A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1535694A (en) * | 2003-04-07 | 2004-10-13 | 谢东泽 | Chinese medicine for curing tumor and its production method |
| 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 |
| CN102783461A (en) * | 2012-08-31 | 2012-11-21 | 哈尔滨工业大学 | Process for artificially breeding wall gecko maggots |
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