CN101607990A - A kind of earthworm active protein and extracting method thereof and application - Google Patents
A kind of earthworm active protein and extracting method thereof and application Download PDFInfo
- Publication number
- CN101607990A CN101607990A CN 200910016682 CN200910016682A CN101607990A CN 101607990 A CN101607990 A CN 101607990A CN 200910016682 CN200910016682 CN 200910016682 CN 200910016682 A CN200910016682 A CN 200910016682A CN 101607990 A CN101607990 A CN 101607990A
- Authority
- CN
- China
- Prior art keywords
- earthworm
- active protein
- earthworm active
- desalination
- extracting method
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of earthworm active protein, extract by the following method: 1) get fresh and alive earthworm, clean, spend the night under the natural condition and tell mud; 2) distilled water cleans, and removes moisture, weighs; 3) add distilled water, high-speed homogenization under the condition of ice bath; 4) homogenate is left standstill 4 hours centrifugal collection supernatant; 5) under the condition of ice bath, adopt salting-out process or other method protein precipitation; 6) centrifugal collection albumen, heavy molten with the 0.1M phosphate buffered saline buffer; 7) will weigh solution after molten with filtering with microporous membrane after, adopt gel-filtration chromatography to carry out albumen sepn; 8) collect elution peak, desalination; 9) with the elutriant drying after the desalination, promptly get earthworm active protein.The present invention uses modern biochemical isolation technique and extract the albumen with pharmacologically active from fresh and alive earthworm, improved its pharmacologically active on the one hand, can effectively remove intravital other the various impurity of earthworm on the other hand, the untoward reaction of having avoided these impurity to cause.
Description
Technical field
The present invention relates to a kind of earthworm active protein and extracting method thereof and application.
Background technology
Earthworm also claims earthworm, earthworm, Annelita Oligochaeta terrestrial animal, and body contains abundant fatty acid, nucleic acid and derivative thereof, total free aminoacids, various trace element and multivitamin, and wherein protein content is particularly abundant.Fresh earthworm contains protein more than 20% (dry products is up to 70%), contains 18 seed amino acids of needed by human.Earthworm is a traditional Chinese medicine, its flavor saltiness is cold, effect with heat-clearing, flat liver, arresting convulsion, Zhichuan, vein relaxing, diuresis can be used to treat chronic bronchitis and bronchial asthma, stroke hemiplegia, hypertension, psychotic disorder, cancer, digestive tract ulcer, the ulcer of lower limb, mumps, banded lump rash, eczema, urticaria, empyrosis, wing skin calculus, the diseases such as smooth and cancer of urinating.
Utilize modern biotechnology, from earthworm, extract have pharmacologically active bioprotein or macromolecular substance, be one of current hot topic.
Summary of the invention
At above-mentioned prior art, the invention provides a kind of earthworm active protein, with and extracting method and the application in treatment is scalded.
The present invention is achieved by the following technical solutions:
A kind of earthworm active protein extracts by the following method, may further comprise the steps:
1) get fresh and alive earthworm, clean its surperficial earth after, spend the night under field conditions (factors) and tell mud;
2) clean the earthworm tell behind the mud with distilled water, remove moisture, weigh;
3) in 1: 1 ratio adding distilled water, use tissue refiner's high-speed homogenization under condition of ice bath;
4) homogenate is left standstill 4 hours, collect supernatant behind the 10000rpm/min high speed centrifugation;
5) under the condition of ice bath, adopt salting-out process or organic solvent precipitation method or polycation precipitator method protein precipitation;
6) albumen of the above-mentioned post precipitation of centrifugal collection, heavy molten with 2 times of volume 0.1M phosphate buffered saline buffers;
7) with behind the heavy filtering with microporous membrane of solution with 0.45 μ m after molten, adopt gel-filtration chromatography to carry out albumen sepn, flow velocity is 0.5ml/min, and the ultraviolet detection wavelength is 280nm;
8) collect elution peak, desalination;
9) with the elutriant drying after the desalination, promptly get earthworm active protein.
Described step 5) is specially: under the condition of ice bath, add ammonium sulfate and saltout in supernatant liquor.
In the described step 7), adopt Superdex 75 gel columns to carry out chromatographic separation, also can adopt the chromatography column of other gel filler to carry out chromatographic separation.
In the described step 8), adopt Sephadex G-25 gel column to carry out desalination, also can adopt modes such as other desalination chromatography columns and dialysis to carry out desalination.
Described step 9) is specially: with the elutriant lyophilize after the desalination, get lyophilized powder, be earthworm active protein; Also can adopt other drying meanss that can keep protein-active to carry out drying.
Described earthworm active protein has higher pharmaceutical use, that the pharmacologically active that is had comprises is anticancer, thrombolysis, antibiotic, treatment burn and scald etc., therefore, it can be mixed separately or with other drug, be prepared into various formulations, be applied to the treatment of burn and scald, also can be applied to the inflammation treatment of wound healing or open wound, or be applied to the treatment or the assisting therapy of other various diseases reactions.
The present invention uses modern biochemical isolation technique and extract the albumen with pharmacologically active from fresh and alive earthworm, improved its pharmacologically active on the one hand, can effectively remove intravital other the various impurity of earthworm on the other hand, the untoward reaction of having avoided these impurity to cause.
Embodiment
The present invention is further illustrated below in conjunction with embodiment:
Embodiment 1: extract activated protein from fresh and alive earthworm, step is as follows:
1) get fresh and alive earthworm, clean its surperficial earth after, spend the night under field conditions (factors) and tell mud;
2) clean the earthworm tell behind the mud with distilled water, remove moisture, weigh;
3) in 1: 1 ratio adding distilled water, use tissue refiner's high-speed homogenization under condition of ice bath;
4), collect supernatant behind the 10000rpm/min high speed centrifugation with the homogenate standing over night;
5) under the condition of ice bath, the ammonium sulfate of porphyrize is added above-mentioned supernatant liquor saltout;
6) the centrifugal collection postprecipitation of saltouing, heavy molten with 2 times of volume Tris-HCl damping fluids;
7) with behind the heavy filtering with microporous membrane of solution with 0.45 μ m after molten, last Superdex 75 gel columns carry out chromatography, and flow velocity is 0.5ml/min, and the ultraviolet detection wavelength is 280nm;
8) collect elution peak, with the desalination of Sephadex G-25 gel column;
9) elutriant after the desalination is carried out lyophilize, the obtained freeze-drying powder promptly obtains earthworm active protein.
Embodiment 2: from fresh and alive earthworm, extract activated protein, step 1) to step 6) with embodiment 1, adopt the chromatography column of dextrane gel, polyacrylamide gel or sepharose filler to carry out chromatography in the step 7); Step 8) to step 9) with embodiment 1.
Embodiment 3: from fresh and alive earthworm, extract activated protein, step 1) to step 7) with embodiment 1, adopt dextrane gel, polyacrylamide gel or sepharose chromatography column and dialysis mode desalination in the step 8); Step 9) is with embodiment 1.
Embodiment 4: from fresh and alive earthworm, extract activated protein, step 1) to step 8) with embodiment 1, step 9) adopts spraying drying or vacuum-drying to carry out drying.
Experimental example 1: earthworm active protein is to the treatment of burn and scald
1.1 test materials
1.1.1 reagent and consumptive material
Be subjected to test product: according to the earthworm active protein of technical solution of the present invention preparation;
Reference substance: MEIBAO SHIRUN SHAOSHANG GAO (Shantou Mebo Pharmaceuticals Co., Ltd.);
Vetanarcol (west, Shanghai biotechnology company of Tang);
Diameter 3cm copper rod (1kg).
1.1.2 animal
(6～8 weeks, were provided conformity certification numbering: SCXK (Shandong)-20030004 to male Wistar rat by 210～280g) 40 by Shandong University's Experimental Animal Center.
1.2 experimental technique
Rat is divided into model group, positive drug group, high dose group, middle dosage group and low dose group at random, every group of 8 rats.With 2.5% vetanarcol rat is carried out intraperitoneal injection anesthesia, lost hair or feathers in the rat back both sides then.With 2.5% vetanarcol rat is anaesthetized, copper rod is put in boils 5min in the boiling water, then copper rod be put in pulled out the hair after the rat back both sides scalded respectively 4 seconds.The basic, normal, high dosage group of earthworm active protein respectively with 5mg/ml, 20mg/ml, 100mg/ml every day 3 times to the scald wound administration.Model group is with the physiological saline administration, and control group is with the control drug administration.
1.3 result and discussion
Scalding healing time and scald are repaired the back pathological section and are shown that earthworm active protein has unusual effect to the treatment of burn and scald.Wherein, the result of treatment of earthworm active protein high dose group surpasses the positive control medicine.
2.1 test materials
2.1.1 reagent and consumptive material
Be subjected to test product: according to the earthworm active protein of technical solution of the present invention preparation;
DMEM (U.S. JRH);
Foetal calf serum (U.S. GIBCO);
Trypsin U.S. GIBCO):
MTT (U.S. Sigma).
2.1.2 cell
Rat fibroblast (NIH3T3): Chinese typical culture collection center (Wuhan).
2.2 experimental technique
The NIH3T3 cell is connected in the cell culture fluid that contains 10% tire calf serum, cultivates 24h, add earthworm active protein solution, after continuing to cultivate 24h, carry out MTT and measure.
Other takes the logarithm vegetative period NIH3T3 cell through 0.25% trysinization, make single cell suspension, be inoculated in 96 orifice plates, cultivate after 48 hours with putting down mouthful micropipet at the standardized even vestige of central vertical, the foetal calf serum DMEM nutrient solution that more renews, add earthworm active protein solution, detect 12h, the cell cut spacing of 24h and three time points of 48h.
2.3 result and discussion
Above-mentioned cell proliferation experiment and cell cut wound healing result of experiment show, the short significantly cell-proliferation activity of earthworm active protein tool, and therefore, this albumen is answered the tool significant curative effect to the treatment of wound.
Experimental example 3: earthworm active protein is to the treatment of inflammation
3.1 test materials
Rat interleukin-6 ELISA test kit (west, Shanghai Tang bio tech ltd);
Rat tumor necrosin ELISA test kit (west, Shanghai Tang bio tech ltd).
3.2 experimental technique
Get the scalded skin of experimental example 1 each rat, use rat interleukin-6 ELISA test kit and rat tumor necrosin ELISA its TNF-a of kit measurement and IL-6 content after the homogenate respectively.
3.3 result and discussion
Inflammatory factor assay result shows that earthworm active protein has good inhibition effect to inflammatory factor, and therefore, this albumen is answered the tool unusual effect for the treatment of inflammation.
Therefore the prepared earthworm active protein of the present invention has remarkable pharmacological activity, can be used for the treatment of burn and scald, and can develop fully becomes newtype drug.
Claims (9)
1. the extracting method of an earthworm active protein may further comprise the steps:
1) get fresh and alive earthworm, clean its surperficial earth after, spend the night under field conditions (factors) and tell mud;
2) clean the earthworm tell behind the mud with distilled water, remove moisture, weigh;
3) in 1: 1 ratio adding distilled water, use tissue refiner's high-speed homogenization under condition of ice bath;
4) homogenate is left standstill 4 hours, collect supernatant behind the 10000rpm/min high speed centrifugation;
5) under the condition of ice bath, adopt salting-out process or organic solvent precipitation method or polycation precipitator method protein precipitation;
6) albumen of the above-mentioned post precipitation of centrifugal collection, heavy molten with 2 times of volume 0.1M phosphate buffered saline buffers;
7) with behind the heavy filtering with microporous membrane of solution with 0.45 μ m after molten, adopt gel-filtration chromatography to carry out albumen sepn, flow velocity is 0.5ml/min, and the ultraviolet detection wavelength is 280nm;
8) collect elution peak, desalination;
9) with the elutriant drying after the desalination, promptly get earthworm active protein.
2. the extracting method of a kind of earthworm active protein according to claim 1, it is characterized in that: described step 5) is specially: under the condition of ice bath, add ammonium sulfate and saltout in supernatant liquor.
3. the extracting method of a kind of earthworm active protein according to claim 1 is characterized in that: in the described step 7), adopt Superdex 75 gel columns to carry out chromatographic separation.
4. the extracting method of a kind of earthworm active protein according to claim 1 is characterized in that: in the described step 8), adopt Sephadex G-25 gel column to carry out desalination.
5. the extracting method of a kind of earthworm active protein according to claim 1, it is characterized in that: described step 9) is specially: with the elutriant lyophilize after the desalination, lyophilized powder, be earthworm active protein.
6. use the earthworm active protein of the extracting method preparation of the described a kind of earthworm active protein of claim 1.
7. the application of the described earthworm active protein of claim 6 in preparation treatment scald medicament.
8. the application of the described earthworm active protein of claim 6 in preparation promotion wound healing medicine.
9. the described earthworm active protein of claim 6 has application in the anti-inflammatory effect medicine in preparation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200910016682 CN101607990A (en) | 2009-07-03 | 2009-07-03 | A kind of earthworm active protein and extracting method thereof and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200910016682 CN101607990A (en) | 2009-07-03 | 2009-07-03 | A kind of earthworm active protein and extracting method thereof and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101607990A true CN101607990A (en) | 2009-12-23 |
Family
ID=41481908
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200910016682 Pending CN101607990A (en) | 2009-07-03 | 2009-07-03 | A kind of earthworm active protein and extracting method thereof and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101607990A (en) |
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102525866A (en) * | 2012-01-11 | 2012-07-04 | 山东大学威海分校 | Cosmetic additive, and preparation method and application thereof |
| CN103755801A (en) * | 2014-01-22 | 2014-04-30 | 山东大学（威海） | Application of combination of earthworm fibrinolytic-activity protein and paclitaxel in preparing drugs for inhibiting cancer cells |
| CN104887541A (en) * | 2015-02-27 | 2015-09-09 | 济宁华能制药厂有限公司 | Cosmetic containing earthworm active protein |
| CN105168258A (en) * | 2015-08-07 | 2015-12-23 | 湖南农业大学 | Anti-injury preparation with earthworm and method for preparing anti-injury preparation |
| CN106074612A (en) * | 2016-07-22 | 2016-11-09 | 杨凌三度蚯蚓科技有限公司 | A kind of Lumbricus lyophilized powder regulating cardiovascular function and its production and use |
| CN106723024A (en) * | 2016-11-30 | 2017-05-31 | 洛阳华以生物工程有限公司 | The preparation method of person in middle and old age's cardiovascular and cerebrovascular disease health products |
| CN107897498A (en) * | 2017-12-29 | 2018-04-13 | 宁夏万辉生物环保科技有限公司 | The preparation method of earthworm protein powder rich in anthocyanidin |
| CN108142639A (en) * | 2017-12-29 | 2018-06-12 | 宁夏万辉生物环保科技有限公司 | The preparation method of selenium-enriched earthworm albumen powder |
| CN108837140A (en) * | 2018-06-26 | 2018-11-20 | 陕西中医药大学 | A kind of preparation method and application of pheretima protein microsphere nanometer wound compound |
| CN109260231A (en) * | 2017-07-18 | 2019-01-25 | 首都儿科研究所 | The preparation method of the anti-inflammatory antimicrobial extract of cough-relieving apophlegmatic in a kind of earthworm |
| CN109758574A (en) * | 2019-03-25 | 2019-05-17 | 山东大学 | A kind of Lumbrokinase hollow suppository and its preparation method and application |
| CN110170047A (en) * | 2019-05-30 | 2019-08-27 | 重庆多拉吧生物科技有限公司 | A kind of antibacterial ointment of glusulase |
| CN115025126A (en) * | 2022-06-23 | 2022-09-09 | 广元市中医医院 | Application of earthworm in preparing medicament for preventing and/or treating fatty liver |
-
2009
- 2009-07-03 CN CN 200910016682 patent/CN101607990A/en active Pending
Cited By (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102525866B (en) * | 2012-01-11 | 2013-07-10 | 山东大学威海分校 | Cosmetic additive, and preparation method and application thereof |
| CN102525866A (en) * | 2012-01-11 | 2012-07-04 | 山东大学威海分校 | Cosmetic additive, and preparation method and application thereof |
| CN103755801A (en) * | 2014-01-22 | 2014-04-30 | 山东大学（威海） | Application of combination of earthworm fibrinolytic-activity protein and paclitaxel in preparing drugs for inhibiting cancer cells |
| CN103755801B (en) * | 2014-01-22 | 2016-03-16 | 山东大学（威海） | The application of earthworm fibrinolytic protein associating taxol in the medicine preparing anticancer |
| CN104887541A (en) * | 2015-02-27 | 2015-09-09 | 济宁华能制药厂有限公司 | Cosmetic containing earthworm active protein |
| CN105168258A (en) * | 2015-08-07 | 2015-12-23 | 湖南农业大学 | Anti-injury preparation with earthworm and method for preparing anti-injury preparation |
| CN106074612A (en) * | 2016-07-22 | 2016-11-09 | 杨凌三度蚯蚓科技有限公司 | A kind of Lumbricus lyophilized powder regulating cardiovascular function and its production and use |
| CN106723024A (en) * | 2016-11-30 | 2017-05-31 | 洛阳华以生物工程有限公司 | The preparation method of person in middle and old age's cardiovascular and cerebrovascular disease health products |
| CN109260231A (en) * | 2017-07-18 | 2019-01-25 | 首都儿科研究所 | The preparation method of the anti-inflammatory antimicrobial extract of cough-relieving apophlegmatic in a kind of earthworm |
| CN109260231B (en) * | 2017-07-18 | 2021-09-03 | 首都儿科研究所 | Preparation method of earthworm extract with cough stopping, phlegm eliminating, anti-inflammatory and antimicrobial functions |
| CN107897498A (en) * | 2017-12-29 | 2018-04-13 | 宁夏万辉生物环保科技有限公司 | The preparation method of earthworm protein powder rich in anthocyanidin |
| CN108142639A (en) * | 2017-12-29 | 2018-06-12 | 宁夏万辉生物环保科技有限公司 | The preparation method of selenium-enriched earthworm albumen powder |
| CN108837140A (en) * | 2018-06-26 | 2018-11-20 | 陕西中医药大学 | A kind of preparation method and application of pheretima protein microsphere nanometer wound compound |
| CN108837140B (en) * | 2018-06-26 | 2021-12-07 | 陕西中医药大学 | Preparation method and application of earthworm protein microsphere nano wound compound |
| CN109758574A (en) * | 2019-03-25 | 2019-05-17 | 山东大学 | A kind of Lumbrokinase hollow suppository and its preparation method and application |
| CN110170047A (en) * | 2019-05-30 | 2019-08-27 | 重庆多拉吧生物科技有限公司 | A kind of antibacterial ointment of glusulase |
| CN115025126A (en) * | 2022-06-23 | 2022-09-09 | 广元市中医医院 | Application of earthworm in preparing medicament for preventing and/or treating fatty liver |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101607990A (en) | A kind of earthworm active protein and extracting method thereof and application | |
| CN104844723A (en) | Preparation method and application of dendrobium officinale extract | |
| CN101463061B (en) | Ginseng saponin Rg1 and Rb1 in pseudo-ginseng and preparation of total saponin thereof | |
| EP2829275B1 (en) | Total flavone extract of abelmoschus manihot and preparation method thereof | |
| TWI501772B (en) | Plectranthus amboinicus fraction having anti-arthritis activity | |
| KR100347880B1 (en) | Extracts of shark cartilage, process of producti0n and uses thereof | |
| KR102304498B1 (en) | Composition for anti-transmissible gastroenteritis virus (tgev) and pharmaceutical composition including the same | |
| CN105624252A (en) | Saussurea involucrate micro-molecular peptide extract as well as preparation method and application thereof | |
| CN110101731B (en) | Chrysanthemum stem and leaf active extract with function of preventing and treating eye diseases and preparation method and application thereof | |
| CN114949034B (en) | Application of perilla extract in preparing medicine for preventing and treating acute pneumonia | |
| CN114891067B (en) | Dandelion anti-inflammatory active peptide and preparation method and application thereof | |
| CN105601707A (en) | Dandelion seed antioxidant tetrapeptide and preparation method thereof | |
| US20100074975A1 (en) | Pharmaceutical composition for the prevention and treatment of liver disease comprising a lonicera caerulea L. Var. Edulis extract | |
| KR102272237B1 (en) | Composition for improved atopy skin and skin moisturizing comprising natural extract | |
| Hao et al. | Cynanchum komarovii extract for the treatment of rheumatoid arthritis by acting on synovial cells in vitro and in vivo | |
| CN101229271B (en) | Rheumatism ostealgia medicated wine and preparing method thereof | |
| KR20110095765A (en) | Anti-allergic composition containing scrophularia buergeriana extract | |
| TW201247212A (en) | Use of ginseng flower extract to prevent or cure diseases or symptoms caused by abnormally high blood uric acid concentration | |
| KR101093006B1 (en) | Method for producing functional health food containing bezoar bovis used microbes and the functional health food produced by the same | |
| JP6688585B2 (en) | Barrier function improver | |
| CN117683150B (en) | Sea buckthorn fruit peel polysaccharide and preparation method and application thereof | |
| KR20200035785A (en) | Composition for Enhancing Immunity comprising Agastachis Herba Extract as effective component | |
| KR102048565B1 (en) | Composition for Preventing or Treating Osteoarthritis comprising the complex extracts of allium hookeri and turmeric | |
| Coopoosamy | In-vitro studies on lectin derivatives of Aloe excelsa (Berger) | |
| CN112107645B (en) | Traditional Chinese medicine composition for improving reproductive function and improving quality of life |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20091223 |