CN105769926A - Skin mucus extracts of andrias davidianus Blanchard for preparing anti-breast cancer drugs and application thereof - Google Patents
Skin mucus extracts of andrias davidianus Blanchard for preparing anti-breast cancer drugs and application thereof Download PDFInfo
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- CN105769926A CN105769926A CN201610136184.3A CN201610136184A CN105769926A CN 105769926 A CN105769926 A CN 105769926A CN 201610136184 A CN201610136184 A CN 201610136184A CN 105769926 A CN105769926 A CN 105769926A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/56—Materials from animals other than mammals
- A61K35/65—Amphibians, e.g. toads, frogs, salamanders or newts
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Abstract
The invention discloses application of skin mucus extracts of andrias davidianus Blanchard to preparation of anti-breast cancer drugs. The application is characterized in that skin mucus is extracted through alkali liquor, and subjected to deproteinization and ion exchange and purified by gel chromatography to obtain vacuum dried extracts. In terms of mass fraction, the polysaccharide content is 38-41%, the protein content is 62-59%, polysaccharide proteins contain 6 monosaccharides of MaN, G1cUA, GaIUA, G1cN,G1c and Ga1, and the molecular weights of the proteins are 28KD. An inhibition test of human breast cancer cells MCF-7 is performed by adoption of an MTT (3-4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) method, and after the human breast cancer cells MCF-7 are treated for 48 hours by the skin mucus extracts of andrias davidianus Blanchard of which the concentrations are more than 0.5 mg/ml, the inhibition rate of the extracts is more than 90%. Injections prepared from the skin mucus extracts of the andrias davidianus Blanchard are used for treating breast cancers, without side effects.
Description
Technical field
The invention belongs to biomedicine field, be specifically related to the application in preparing anti-breast cancer medicines of the giant salamander skin mucus extract.
Background technology
Breast cancer is the primary tumor of harm WomanHealth, 5 years survival rates about 60% of patient with breast cancer, relapse and metastasis after nearly 50% patient therapy, advanced breast cancer patient's mean survival time only 18～30 months.The treatment means of breast cancer include traditional treatment means such as operative treatment, radiotherapy, chemotherapy, endocrine therapy.Operative treatment risk is high, and big to human body wound, it is adaptable to early stage patient, for centering, patients with terminal, art effect is the most undesirable, it is difficult to radical cure.The recurrence of breast cancer and transfer characteristic, radiotherapy also has lethal effect to the normal structure of irradiated region, causes iatrogenic injury, such as radiation myelitis, radiation pneumonitis, cerebral radiation necrosis etc..Chemotherapeutics is while suppression growth of tumour cell or killing cancer cell, the various types of cells of breeding normal in body the most also there is toxic action in various degree, the infringement that especially myeloid element and gastric epithelial cell are subject to is the heaviest, the serious toxicity of chemotherapeutics.Endocrinotherapy for breast cancer has become the critical treatment means in breast cancer each stage, but also can produce certain side effect.
Formed from animal evolutionary process in sp act composition and excavate anti-breast cancer new drug, be the new way of research and development anti-breast cancer medicines.Giant salamander (Andrias davidianus Blanchard) belongs to large-scale Amphibia, Caudata, Cryptobranchidae, there is the title of " living fossil ", for defence pathogenic microorganism and the infringement of natural enemy during natural evolution, giant salamander skin mucus cell release mucus, covers and defines one natural protective barrier on skin.Giant salamander skin mucus thing has special bioactive molecule containing abundant, has that broad-spectrum is antibacterial, antitumor action.
Summary of the invention
The mesh of the present invention is to be to provide one to have anti-breast cancer activity composition, derives from giant salamander skin extract slimy, and applies in preparing anti-breast cancer medicines.
Realize the technical scheme of above-mentioned purpose: the giant salamander more than healthy 3-5 age propagated artificially, after sterile water wash, mechanical stimulus scraping mucus, by mass fraction, add in mucus with the 0.3molNaoH aqueous solution in 20: 1 ratios, under the conditions of 40 DEG C, supersonic oscillations extract 1h, it is 1/5th that filtered fluid concentrates as original volume, taking off albumen three to four times by SAVEG method, in centrifugate, addition edible alcohol is to concentration 75% alcohol precipitation, stands 20-24h 0-4 DEG C of condition, it is centrifuged and removes supernatant, vacuum freeze drying giant salamander skin mucus extract.Mucus extract is through DEAE-52 ion-exchange chromatography, then purifies through G-100 sephadex chromatography, with 280nm for detection wavelength, collects out peak sample and concentrates, vacuum freeze drying.Mucus extract passes through SDS-polyacrylamide gel electrophoresis, has obvious protein band at 28KD molecular weight.Detect through efficient liquid phase and amino-acid analyzer, extraction purification thing contains 6 kinds of monose MaN, GlcUA, GalUA, GlcN, Glc, Gal, its molecule mole ratio is 63: 152: 40: 57: 37: 77, total sugar content is 38-41%, protein content is 62-59%, and giant salamander skin mucus extract is glycoprotein.
Giant salamander mucus extraction purification thing PBS is dissolved into 0.25, 0.50, 1.0mg/ml, positive control is made with 1mg/ml taxol, add in logarithmic phase human breast cancer cell MCF 1 cell that 5%CO2 cultivates, cultivate 48h, add 20ulMTT, continue to cultivate 4h, discard nutrient solution, add 150ul dmso solution, 37 DEG C of low speed concussion 10min, with ultraviolet specrophotometer, its absorbance is detected at 570nm, logarithmic phase human breast cancer cell MCF 1 inhibiting rate is respectively 85.20%, 90.62%, 91.42% and 59.6%, giant salamander mucus extract to the inhibiting rate of human breast cancer cell MCF 1 apparently higher than positive controls, and extract concentrations is less than positive drug.Giant salamander mucus extract-treated human breast cancer cell MCF 1 is after 48h, by observation by light microscope form, outer form shrinkage, change garden, comes off.Giant salamander mucus extract carries out anxious toxicity safety evaluation to small white mouse per os gavage, and giant salamander mucus extract LD50 value is about 4.64g/kg, and giant salamander mucus extract nontoxicity is safe, has good anti-breast cancer medicines DEVELOPMENT PROSPECT.
Detailed description of the invention
The present invention is further illustrated with the following Examples, the extracting method of giant salamander skin mucus extract of the present invention and suppression breast cancer cell test.
1) the giant salamander more than healthy 3-5 age that will propagate artificially, after sterile water wash, white mucus scraping mucus is produced with mechanical stimulus giant salamander, add in mucus with the 0.3molNaoH aqueous solution in 20: 1 ratios, under the conditions of 40 DEG C, supersonic oscillations extract 1h, it is 1/5th that filtered fluid concentrates as original volume, method takes off albumen three to four times, centrifugate adds edible alcohol to concentration 75% alcohol precipitation, stand 20-24h 0-4 DEG C of condition, be centrifuged and remove supernatant, vacuum freeze drying giant salamander skin mucus extract.
2) again through DEAE-52 ion-exchange chromatography, use 0.2mol/NacL eluant solution, be detection wavelength with 280nm, collect out peak sample, more than 7000D bag filter desalination, concentrate vacuum freeze drying.
3) through SePhadexG-100 gel chromatography, with distilled water for flowing phase, with 280nm for detection wavelength, collecting out peak sample and concentrate, vacuum freeze drying becomes powder.
4) giant salamander skin mucus extract, in terms of mass fraction, total sugar content is 38-41%, and protein content is 62-59%, and containing MaN, GlcUA, GalUA, GlcN, Glc, Gal6 kind monose in extract, molecular weight of albumen is 28.0KD.
5) mtt assay is used to have studied the giant salamander skin mucus extract inhibitory action to human breast cancer cell line Bcap-37, respectively with 0.25mg/mL, 0.5mg/mL, 1mg/mL giant salamander skin mucus extract processes 48h to breast cancer cell MCF-7, breast cancer cell inhibiting rate is respectively 85.20%, 90.62%, 91.42%, display giant salamander skin mucus extract has extremely strong inhibitory action to human breast cancer cell line Bcap-37.
6) by light microscope to extract-treated human breast cancer cell line Bcap-37 after 48h, observe form, result breast cancer cell volume diminishes, cell colony quantity is sparse, attached cell quantity reduces, shrinkage occurs, becomes round, show that giant salamander skin mucus extract has good suppression breast cancer cell effect.
7) giant salamander mucus extract carries out anxious poison test to small white mouse per os gavage, and giant salamander mucus extract LD50 value is about 5.35g/kg, and mucus extract is non-toxic, is safe for anti-breast cancer, has good anti-breast cancer medicines DEVELOPMENT PROSPECT.
8) giant salamander mucus extract is directly injected by animal abdominal cavity, emergency reaction does not occurs, and breast cancer affected area is directly administered treatment breast cancer.
Claims (3)
1. giant salamander skin mucus extract application in preparing anti-breast cancer medicines.It is characterized in that giant salamander skin
Mucus is extracted by alkali lye, de-albumen, the vacuum drying extract after ion exchange and gel chromatography,
In terms of mass fraction, polyoses content is 38-41%, and protein content is 62-59%, in polysaccharide containing MaN,
GlcUA, GalUA, GlcN, Glc, Gal6 kind monose, molecular weight of albumen is 28KD, giant salamander skin mucus
Extract has inhibitory action to MCF-7 MCF～7 test.
2. according to the answering in preparing anti-breast cancer medicines of the giant salamander skin mucus extract described in claim 1
With, it is characterised in that: giant salamander skin mucus extract is dissolved in PBS, in terms of mass fraction, and 0.5mg/ml
Above extract concentrations, after MCF-7 MCF～7 is processed 48h, its inhibiting rate reaches more than 90%.
3. according to the answering in preparing anti-breast cancer medicines of the giant salamander skin mucus extract described in claim 1
With, it is characterised in that: giant salamander skin mucus extract makes injection injection for curing breast cancer.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610136184.3A CN105769926A (en) | 2016-02-28 | 2016-02-28 | Skin mucus extracts of andrias davidianus Blanchard for preparing anti-breast cancer drugs and application thereof |
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| CN201610136184.3A CN105769926A (en) | 2016-02-28 | 2016-02-28 | Skin mucus extracts of andrias davidianus Blanchard for preparing anti-breast cancer drugs and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106361776A (en) * | 2016-11-16 | 2017-02-01 | 张家界金姑大鲵开发有限公司 | Effect of giant salamander mucus for hemostasis |
| CN106511399A (en) * | 2016-11-16 | 2017-03-22 | 太鲵生物科技（天津）有限公司 | A use of mucus of andrias davidianus Blanchard |
| CN107648276A (en) * | 2017-09-28 | 2018-02-02 | 凯瑞欧（天津）生物技术有限公司 | A kind of process of low temperature ultrasonic cracking extraction giant salamander mucus active ingredient |
| CN108421080A (en) * | 2018-05-07 | 2018-08-21 | 魏泓 | Giant salamander secretion hydrogel and its preparation method and application |
| CN110818773A (en) * | 2018-08-10 | 2020-02-21 | 浙江楚沅生物科技有限公司 | Method for purifying active substance in amniotic fluid of non-human animal |
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| CN103621869A (en) * | 2013-11-15 | 2014-03-12 | 贵阳学院 | Giant salamander oligosaccharide peptide health-care product |
| CN104840489A (en) * | 2015-05-23 | 2015-08-19 | 汉中领维生物科技发展有限公司 | Application of andrias davidianus Blanchard skin mucus extract to preparation of medicines against lung cancers |
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2016
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Patent Citations (3)
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| CN1451393A (en) * | 2002-04-19 | 2003-10-29 | 清华大学 | Clam extract, and preparing process and use thereof |
| CN103621869A (en) * | 2013-11-15 | 2014-03-12 | 贵阳学院 | Giant salamander oligosaccharide peptide health-care product |
| CN104840489A (en) * | 2015-05-23 | 2015-08-19 | 汉中领维生物科技发展有限公司 | Application of andrias davidianus Blanchard skin mucus extract to preparation of medicines against lung cancers |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106361776A (en) * | 2016-11-16 | 2017-02-01 | 张家界金姑大鲵开发有限公司 | Effect of giant salamander mucus for hemostasis |
| CN106511399A (en) * | 2016-11-16 | 2017-03-22 | 太鲵生物科技（天津）有限公司 | A use of mucus of andrias davidianus Blanchard |
| CN106511399B (en) * | 2016-11-16 | 2023-12-01 | 太鲵生物科技（天津）有限公司 | Application of giant salamander mucus |
| CN106361776B (en) * | 2016-11-16 | 2024-03-19 | 张家界金姑大鲵开发有限公司 | Giant salamander mucus used for hemostasis |
| CN107648276A (en) * | 2017-09-28 | 2018-02-02 | 凯瑞欧（天津）生物技术有限公司 | A kind of process of low temperature ultrasonic cracking extraction giant salamander mucus active ingredient |
| CN108421080A (en) * | 2018-05-07 | 2018-08-21 | 魏泓 | Giant salamander secretion hydrogel and its preparation method and application |
| CN108421080B (en) * | 2018-05-07 | 2021-01-26 | 魏泓 | Giant salamander secretion hydrogel and preparation method and application thereof |
| CN110818773A (en) * | 2018-08-10 | 2020-02-21 | 浙江楚沅生物科技有限公司 | Method for purifying active substance in amniotic fluid of non-human animal |
| CN110818773B (en) * | 2018-08-10 | 2023-04-11 | 浙江楚沅生物科技有限公司 | Method for purifying active substance in amniotic fluid of non-human animal |
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