CN102657848B - Application of toad maggot protein in preparing medicine used for treating colon cancer - Google Patents
Application of toad maggot protein in preparing medicine used for treating colon cancer Download PDFInfo
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- CN102657848B CN102657848B CN201210179129.4A CN201210179129A CN102657848B CN 102657848 B CN102657848 B CN 102657848B CN 201210179129 A CN201210179129 A CN 201210179129A CN 102657848 B CN102657848 B CN 102657848B
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Abstract
The invention discloses the application of toad maggot protein in preparing a medicine used for treating colon cancer and belongs to the technical field of medicines. Aiming at the problems of the existing medicine used for treating colon cancer, the invention combines the toad maggot protein and appropriate pharmaceutical necessities for use and provides a novel application of the toad maggot protein in a medicine used for treating colon cancer. The invention is designed to aim at the special symptom of colon cancer; the content of toad maggot protein which is extracted and purified is 96.5-98.1%; and the toad maggot protein with the purity is used for preparing a medicine in various forms. The medicine has definite ingredients, good consistency, controllable quality, good curative effect for colon cancer and convenience for use.
Description
Technical field
The invention belongs to medical technical field, relate to the application of a kind of Bufo siccus maggot protein in preparation treatment colon cancer medicine.
Background technology
Colon cancer refers to the malignant change that epithelium of intestinal mucosa occurs under the multiple carcinogenic factor effects such as environment or heredity.Pathogenic factor has certain relation with heredity, adenoma of colon, polyposis, chronic inflammation characteristic of disease pathological changes, few fiber, high fat diet custom.
CN101024085 discloses a kind of colon cancer to treatment prodrug and preparation method, is mainly a kind of precursor medicine for target treatment of colon cancer and the preparation method synthetic with the polysaccharide containing galactose about anticancer chemotherapeutic agent 5-fluorouracil (5-FU).
CN101810624A discloses a kind of medicine for the treatment of colon cancer and preparation method thereof, changes medicine and is from charred Radix Sanguisorbae through extracting to separate and obtain the medicament that 3 beta-hydroxies-28-disappearance-12,17 (18)-diene ursanes and pharmaceutic adjuvant are made.
CN101676301 discloses a kind of antibody that suppresses Growth of Human Colon Cancer and, for the preparation of the application of medicine and test kit, this invention provides a kind of monoclonal antibody, and antibody is the IgM immunoglobulin like protein, the sugar chain structure that identification antigen is mucin surface sialic acid.
CN101537165 discloses a kind of Chinese medicine preparation for the treatment of colon cancer, by Radix Codonopsis, Radix Notoginseng, the Radix Astragali, spend in vain, the Chinese medicine such as Herba Hedyotidis Diffusae, the Rhizoma Atractylodis Macrocephalae, Poria, Radix Polygalae, Radix Glycyrrhizae, Arillus Longan, Semen Ziziphi Spinosae, Rhizoma Zingiberis Recens, Rhizoma Chuanxiong, the Radix Aucklandiae, Rhizoma Coptidis, Fructus Crataegi forms.
CN101278196 discloses colon cancer related gene TOM 34, and this invention has provided the method for diagnosis and detection colon cancer.
CN102309485A discloses inhibitor against colon carcinoma cells effect and the pharmaceutical preparation thereof of piperine, and this disclosure of the invention is purposes and the formulation method at inhibitor against colon carcinoma cells with piperine.
[0009] aforesaid several preparation may be used to treat the preparation of colon cancer medicine in principle, but, because aforesaid several prescription Chinese medicines are various, every kind of Chinese herbal medicine, due to the difference of planting region, weather, kind, collecting time, the process of preparing Chinese medicine and processing method, is difficult to guarantee concordance and the quality control of product in suitability for industrialized production.
Summary of the invention
The problem existed for existing treatment colon cancer medicine, the present invention is combined with Bufo siccus maggot protein with suitable pharmaceutical necessities, the new purposes of a kind of Bufo siccus maggot protein in preparation treatment colon cancer medicine is provided.
According to following step, prepared by treatment colon cancer medicine of the present invention:
One, extract Bufo siccus maggot protein:
(1) the dry Bufo siccus maggot of 1000g is put into to the room temperature expanding apparatus, be filled with 1～10kg nitrogen, 0.1～0.5kg ammonia, be forced into 0.2～1.0Mpa, time 10～30min, gas in the abrupt release device then, the Bufo siccus maggot is by expanded pulverizing;
(2) the puffing powder broken material joins in expanded solvents system extraction element, is filled with 200～2000g carbon dioxide, 100～500g ethanol and 100～200g dimethyl formamide, is forced into 1～8Mpa, 10～25 ℃ of temperature, time 10～60min, then release pressure, to normal pressure, is collected mixed liquor;
(3) mixed liquor separates with 1500～5000r/min with seperator, collects liquid phase;
(4) collect liquid phase and filter with membrane aperture 0.5～50 μ m Microfilter, obtain filtrate;
(5) filtrate is injected in the solvent resistant column of saphacryls-100HR and filters under pH=3～12 conditions;
(6) filtrate is dissolved in the dicyandiamide solution be comprised of according to the volume ratio of 4:3:3:2 chloroform-ethanol-methanol-water, pump in ultrasonic standing wave preparative liquid chromatogram, ultrasonic standing wave power 0.2～5.0KW, frequency 500～1000kHz, flow velocity 50～500mL/min, UV-detector wavelength 280nm, separation and purification goes out Bufo siccus maggot protein;
(7) purification Bufo siccus maggot protein under vacuum 200～800Kpa, temperature-50～30 ℃, the condition of time 6～24h, is dried to the freeze-dried protein finished product of moisture content 5～8% with vacuum freeze drier;
Two, mix: mix according to proportioning, be processed into the treatment colon cancer medicine of various dosage forms.
Bufo siccus maggot protein is the biological activity protein of extraction separation and purification from the Bufo siccus maggot, molecular structure is clear, medicinal potency ratio is higher, high specificity, toxic reaction relatively a little less than, be difficult in vivo producing accumulating, there is the awareness of molecule with the affinity of body inner recipient, and almost there is no alienation biological metabolism toxicity.
The present invention is directed to the colon cancer characteristic symptom and design, a kind of Bufo siccus maggot protein content that extracts purification reaches 96.5～98.1%, become again the medicine of various dosage forms with the Bufo siccus maggot protein preparation of this purity, this drug ingredient is clear and definite, high conformity, quality controllable, to colon cancer disease good effect and easy to use.
The accompanying drawing explanation
The structural representation that Fig. 1 is expanded solvents system extraction element.
The specific embodiment
The specific embodiment one: present embodiment prepares the medicine for the treatment of colon cancer by the following method:
(1) the dry Bufo siccus maggot of 1000g is put into to room temperature expanding apparatus (ZL200520108339.X), be filled with 1～10kg nitrogen, 0.1～0.5kg ammonia, be forced into 0.2～1.0Mpa, time 10～30min, then gas in the abrupt release device, the Bufo siccus maggot is by expanded pulverizing;
(2) the puffing powder broken material joins in expanded solvents system extraction element, is filled with 200～2000g carbon dioxide, 100～500g ethanol and 100～200g dimethyl formamide, is forced into 1～8Mpa, 10～25 ℃ of temperature, time 10～60min, then release pressure, to normal pressure, is collected mixed liquor;
(3) mixed liquor separates with 1500～5000r/min with seperator, collects liquid phase;
(4) collect liquid phase and filter with membrane aperture 0.5～50 μ m Microfilter, obtain filtrate;
(5) filtrate is injected in the solvent resistant column of saphacryls-100HR and filters under pH=3～12 conditions;
(6) filtrate is dissolved in the dicyandiamide solution be comprised of according to the volume ratio of 4:3:3:2 chloroform-ethanol-methanol-water, pump in ultrasonic standing wave preparative liquid chromatogram (ZL200920274485.8), ultrasonic standing wave power 0.2～5.0KW, frequency 500～1000kHz, flow velocity 50～500mL/min, UV-detector wavelength 280nm, separation and purification goes out Bufo siccus maggot protein;
(7) purification Bufo siccus maggot protein under vacuum 200～800Kpa, temperature-50～30 ℃, the condition of time 6～24h, is dried to the freeze-dried protein finished product of moisture content 5～8% with vacuum freeze drier;
(8) get above-mentioned freeze-dried protein 10～15g, lecithin 0.1～1.5g, liquid paraffin 20～50g, stir, grind well into pasty state, separately get oleum sapii 100～200g is heated to be cooled to 35～38 ℃ after 45～55 ℃ of fusings in water-bath, pour in the protein that grinds well into pasty state and lecithin and liquid paraffin mixture and grind rapidly evenly, be cast in while hot in the suppository mould, condense to 10～20 ℃, take out molding Bufo siccus maggot protein suppository from the suppository mould, packed products.
As shown in Figure 1, the system of expanded solvents described in present embodiment extraction element is by spherical expanded solvents system extractor 1, head tank 2, solid-liquid separator 3, gas-solid separator 4, finished pot 5, external source carbon dioxide cylinder 6, carbon dioxide storage tank 7, carbon dioxide circulating pressure pump 8 and solvent tank 9 form, spherical expanded solvents system extractor 1 by pipeline respectively with head tank 2, solid-liquid separator 3, carbon dioxide circulating pressure pump 8 and solvent tank 9 are connected, solid-liquid separator 3 is connected with gas-solid separator 4, gas-solid separator 4 is connected with finished pot 5 and carbon dioxide storage tank 7 respectively by pipeline, carbon dioxide storage tank 7 is connected with external source carbon dioxide cylinder 6 and carbon dioxide circulating pressure pump 8 respectively by pipeline.
The specific embodiment two: present embodiment prepares the medicine for the treatment of colon cancer by the following method:
(1) use beater at 500～1000r/min making beating, 10～30min the bright Bufo siccus maggot of 1000g, become slurry;
(2) add in slurry containing 0.01～0.5% aqueous sodium carbonate 5～20L, stir, place at normal temperatures 30～120min;
(3) with centrifuge centrifugal 10～30min under 1500～5000r/min condition, collect liquid phase;
(4) filter above-mentioned liquid phase with membrane aperture 0.2～5.0 μ m Microfilter, collect filtrate;
(5) filtrate is injected in the saphacryls-100HR solvent resistant column and filters to obtain filtrate in pH=3～12;
(6) filtrate is dissolved in the dicyandiamide solution be comprised of according to the volume ratio of 4::3:3:2 chloroform-ethanol-methanol-water, pump in ultrasonic standing wave preparative liquid chromatogram, ultrasonic standing wave power 0.2～5.0KW, frequency 500～1000kHz, flow 50～500mL/min, UV-detector wavelength 280nm, continuous purification separates Bufo siccus maggot protein;
(7) with vacuum freeze drier, at vacuum 50～500Kpa, temperature-30～30 ℃, be dried to moisture content 5～8% finished products to purification Bufo siccus maggot protein under time 6～24h condition;
(8) 100g lecithin is become to I liquid with joining in 500～1000ml ether after the mixing of 30～80g cholesterol, 10～50g Bufo siccus maggot protein joins stirring and dissolving in 3～10mmol/L phosphate buffered solution and makes II liquid, after being mixed with II liquid, I liquid use ultrasound wave at power 0.1～0.5KW, frequency 300～500KHz, sonic oscillation 10～30min under room temperature, then heat 50～80 ℃ in rotary evaporator, vacuum 100～500Kpa, rotating speed 30～120r/min is evaporated to and presents gel, after taking off the evaporation flask and being placed on gyrate shaker and making the gel phase inversion with 30～200r/min vibration, to evaporate flask puts into water-bath and heats 60～80 ℃ again, remove ether, again material is put into to supercentrifuge inherence 15000～50000r/min ultracentrifugation, 30～60min, separate and remove the Bufo siccus maggot protein do not coated, 1～2L deionized water wash 2～3 times for precipitate, centrifuge is sloughed water at 150～750r/min, obtain Bufo siccus maggot protein liposome, collecting packing and get final product.
Colon cancer cancer staging:
0 phase: cancer is confined to mucous layer, without lymphatic metastasis;
The I phase: tumor is confined in muscularis propria, without lymphatic metastasis;
The II phase: neoplasm invasiveness surpasses muscularis propria, but without lymphatic metastasis;
The III phase: lymphatic metastasis;
The IV phase: metastasis (liver, lung etc.) or peritoneum shift.
Therapeutic Method:
The patient needs strict restriction to drink.By body weight 70kg, supp anal administration every day, 3 of every days, each 1, or the liposome local injection, 2 doses of every days, every dose of 250～300 μ g, a course for the treatment of 2 Mondays.
The therapeutic outcome grade scale:
One, short term effect (4 weekends are relatively front with treatment)
Alleviate fully: focus disappears fully, without new focus, occurs;
Partial rcsponse: 50% before the treatment that focus is dwindled or less, without new focus, occur, when many focal diseases become, the neither one focus increases;
Stable phase: 1. slightly alleviate: focus is dwindled area 20% or area increased 25%, and subjective symptoms is improved, and physical basal conditions rises; 2. basicly stable: focus dwindle less than 25% or area increased be no more than more than 25%, subjective symptoms is improved, physical basal conditions rises.
Expansion: single area or a plurality of focus gross area increase more than 25% than treatment is front, or new pathological changes occurs, and clinical symptoms increases the weight of, and physical basal conditions descends.
Two, intermediate period treatment: (5 weeks～1 year)
Alleviate fully, survive without tumor.
Partial rcsponse: 1. corpus carcinosus is returned to area before treatment more than 50% or new focus occurs; 2. corpus carcinosus is without increasing or dwindling doing well,improving or disappearance; 3. physical basal conditions rises.
Shift and expansion: the cause of disease kitchen range enlarges development, new metastasis occurs, and clinical symptoms increases the weight of, and physical situation descends.
Three, late result: (1 year～5 years)
Without tumor, survive.
The existence of band tumor.
Dead.
Therapeutic outcome: in Table 1.
Table 1
| Short term effect | Alleviate fully | Partial rcsponse | Stable phase | Obvious effective rate % | Effective percentage % |
| Patient 17 people | 13 | 3 | 1 | 94 | 100 |
| Mid-term effects | Alleviate fully | Partial rcsponse | Shift and expansion | Obvious effective rate % | Effective percentage % |
| Patient 20 people | 15 | 3 | 2 | 90 | 90 |
| Late result | Without tumor, survive | The existence of band tumor | Dead | Obvious effective rate % | Effective percentage % |
| Patient 15 people | 8 | 3 | 4 | 73 | 73 |
Above embodiment is only for the present invention is further illustrated, and scope of the present invention is not subject to the limitation of lifted embodiment.
Claims (3)
1. the application of Bufo siccus maggot protein in preparation treatment colon cancer medicine is characterized in that described Bufo siccus maggot protein obtains by the following method:
(1) the dry Bufo siccus maggot of 1000g is put into to the room temperature expanding apparatus, be filled with 1～10kg nitrogen, 0.1～0.5kg ammonia, be forced into 0.2～1.0Mpa, time 10～30min, gas in the abrupt release device then, the Bufo siccus maggot is by expanded pulverizing;
(2) the puffing powder broken material joins in expanded solvents system extraction element, be filled with 200～2000g carbon dioxide, 100～500g ethanol and 100～200g dimethyl formamide, be forced into 1～8Mpa, 10～25 ℃ of temperature, time 10～60min, then release pressure is to normal pressure, collect mixed liquor, described expanded solvents system extraction element is by spherical expanded solvents system extractor (1), head tank (2), solid-liquid separator (3), gas-solid separator (4), finished pot (5), external source carbon dioxide cylinder (6), carbon dioxide storage tank (7), carbon dioxide circulating pressure pump (8) and solvent tank (9) form, spherical expanded solvents system extractor (1) by pipeline respectively with head tank (2), solid-liquid separator (3), carbon dioxide circulating pressure pump (8) and solvent tank (9) are connected, solid-liquid separator (3) is connected with gas-solid separator (4), gas-solid separator (4) is connected with finished pot (5) and carbon dioxide storage tank (7) respectively by pipeline, carbon dioxide storage tank (7) is connected with external source carbon dioxide cylinder (6) and carbon dioxide circulating pressure pump (8) respectively by pipeline,
(3) mixed liquor separates with 1500～5000r/min with seperator, collects liquid phase;
(4) collect liquid phase and filter with membrane aperture 0.5～50 μ m Microfilter, obtain filtrate;
(5) filtrate is injected in the solvent resistant column of saphacryls-100HR and filters under pH=3～12 conditions;
(6) filtrate is dissolved in dicyandiamide solution, pump in ultrasonic standing wave preparative liquid chromatogram, ultrasonic standing wave power 0.2～5.0KW, frequency 500～1000kHz, flow velocity 50～500mL/min, UV-detector wavelength 280nm, separation and purification goes out Bufo siccus maggot protein, and by chloroform-ethanol-methanol-water, the volume ratio according to 4:3:3:2 forms described dicyandiamide solution;
(7) purification Bufo siccus maggot protein under vacuum 200～800Kpa, temperature-50～30 ℃, the condition of time 6～24h, is dried to the freeze-dried protein finished product of moisture content 5～8% with vacuum freeze drier.
2. the application of Bufo siccus maggot protein according to claim 1 in preparation treatment colon cancer medicine, is characterized in that described treatment colon cancer medicine is the suppository of being made by Bufo siccus maggot protein 10～15g, lecithin 0.1～1.5g, liquid paraffin 20～50g, oleum sapii 100～200g.
3. the application of Bufo siccus maggot protein according to claim 1 in preparation treatment colon cancer medicine, is characterized in that described treatment colon cancer medicine is the liposome of being made by 100g lecithin, 30～80g cholesterol, 10～50g Bufo siccus maggot protein.
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