CN105111293A - Amorphophallus konjac peptide and application thereof - Google Patents
Amorphophallus konjac peptide and application thereof Download PDFInfo
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- CN105111293A CN105111293A CN201510563848.XA CN201510563848A CN105111293A CN 105111293 A CN105111293 A CN 105111293A CN 201510563848 A CN201510563848 A CN 201510563848A CN 105111293 A CN105111293 A CN 105111293A
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- diabetes mellitus
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Gastroenterology & Hepatology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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Abstract
The invention relates to the field of medicines and in particular relates to an amorphophallus konjac peptide capable of preventing or treating diabetes mellitus. The sequence of the peptide is QLLSEKVSDHLIPHKTFSGNRPSL. A preparation method of the peptide comprises the following steps: (1) decoction, (2) enzymolysis, (3) protein separation, (4) separation and purification through semipreparative RP-HPLC (reverse phase high-performance liquid chromatography) and (5) freeze-drying, wherein neutral protease and compound protease are adopted for enzymolysis; and macroporous resin and sephadex are adopted for protein separation. The invention provides an application of the peptide to treatment of diabetes mellitus and complications of diabetes mellitus, wherein the complications of diabetes mellitus include diabetic nephropathy, diabetic hypertension, diabetic eye diseases and diabetic neuropathy. The amorphophallus konjac peptide is extracted from amorphophallus konjac, has good safety and can be used for effectively preventing or treating diabetes mellitus.
Description
Technical field:
The present invention relates to pharmaceutical field, be specifically related to the polypeptide extract being used for the treatment of diabetes.
Background technology
The metabolic disease of diabetes to be one group with hyperglycemia be feature is a kind of syndromes of glucose, protein and the lipid metabolism disorders caused by the cellular metabolism effect defect of insufficient insulin or Regular Insulin.In recent years, the number of diabetic subject increases gradually, is divided into I diabetes and type ii diabetes.Wherein the number of type ii diabetes patient accounts for the 85-90% of diabetes total number of persons, and the health and lives of patient in serious threat.If diabetes can not get good control, a lot of complication may be caused, such as: diabetic nephropathy, retinopathy, hypertension etc.
At present, Most patients helps blood sugar in control volume by insulin injection or orally-taken blood sugar reducing medicine, or regulates the method treatment diabetes such as insulin secretion.But these drug side effects are comparatively large, clinically can not life-time service, the clinical needs of diabetic subject can not be met.If the medicine of the safe and effective treatment diabetes can extracted from natural product, will bring glad tidings for numerous diabetic subjects.
Amorphophalus rivieri (formal name used at school Amorphophalluskonjac), is commonly called as konjaku, does again mill taro, and Araeceae mill taro belongs to per nnial herb, and ancient Chinese is also known as bewitching taro.Konjaku just has the title of " removing intestines sand " since ancient times.Konjaku is cold in nature, pungent, poisonous; Cure mainly the diseases such as hypotensive, hypoglycemic, scrofula subcutaneous nodule, the damage stasis of blood are swollen, constipation stomachache, swelling and pain in the throat, swelling and aching of gum.The absorption of konjaku energy delay glucose, reduces postprandial blood sugar effectively, thus alleviates the burden of pancreas, makes the carbohydrate metabolism of diabetic subject be in benign cycle, can not as some ofhypoglycemic medicine, blood sugar be declined suddenly and occur hypoglycemic effect.Especially the ultimate food of diabetic subject and obese person.Due to effect of konjaku hypoglycemic, fat-reducing, and have no side effect, therefore, Rhizoma amorphophalli extract can be developed to hypoglycemic drug.However, the compound that extracts from konjaku of unripe exploitation comes out, for prevention or treatment diabetes.
Summary of the invention:
The present invention seeks to the feature for existing hypoglycemic medicine, design a kind of konjaku polypeptide, can safety, effectively treat diabetes.
Technical scheme
A kind of konjaku polypeptide, its sequence is QLLSEKVSDHLIPHKTFSGNRPSL.The preparation method of described polypeptide, is characterized in that: step comprises (1) decocts, and (2) add amylase, 37 DEG C, stir 60-120 minute, (3) are centrifugal, 5000-10000rpm, 30 minutes, gets precipitation, (4) precipitation is redissolved in the damping fluid of pH9-13, and (5) are centrifugal, 5000-10000rpm, 30 minutes, get supernatant, (6) are by supernatant half preparation RP-HPLC separation and purification, (7) freeze-drying, to obtain final product.Described amylase can be α-amylase, beta-amylase, diastatic enzyme.Polypeptide is in the application for the treatment of diabetes.
Beneficial outcomes:
Konjaku polypeptide in the present invention, be extract polypeptide from konjaku, security is good, and can effectively prevent or treat diabetes.
Embodiment
Embodiment 1
The preparation method of polypeptide
After konjaku boiling 1.5h, add 1% diastatic enzyme, 37 DEG C, stir 60-120 minute, centrifugal, 5000-10000rpm, 30 minutes, get precipitation, precipitation is redissolved in the damping fluid of pH9-13, centrifugal, 5000-10000rpm, 30 minutes, get supernatant, by supernatant half preparation RP-HPLC separation and purification, after the product freeze-drying obtained, be konjaku polypeptide.The konjaku polypeptide that application aforesaid method extracts, through Mass Spectrometric Identification, sequence is QLLSEKVSDHLIPHKTFSGNRPSL, and this sequence is brand-new sequence.Also can be synthesized by the raw work in Shanghai.
Embodiment 2
The cell in vitro determination of activity of konjaku polypeptide
Adopt MTT colorimetry.By the insulinoma cell (INS-1 cell) of logarithmic growth, add in 96 well culture plates with 1.0 × 105, cultivate 24h, experimental port, positive drug control hole add the Experimental agents konjaku polypeptide of different concns respectively; Blank group adds the solvent of same volume.Five multiple holes are established in every hole, and cultivate 48h, every hole adds MTT, after effect 4h, add DMSO, hatch 30min, absorbance A value is measured, by formula growth and proliferation of cell rate=(experimental group light absorption value/control group light absorption value-1) × 100% at microplate reader 570nm place.The EC50 calculating Experimental agents is 39.76 μ g/L.
Embodiment 3
Hypoglycemic experiment in the body of konjaku polypeptide
Kunming mice, the high sugar of high fat is fed to body weight 18 ~ 22g, male and female half and half, and before experiment, water 24h is can't help in fasting, abdominal cavity disposable injection streptozotocin (STZ) 50mg/kg (citrate buffer of 1% dissolves preparation).After 1 week, blood sugar (BS) is surveyed in mouse tail vein blood sampling, and BS is modeling success higher than 16mmol/L.After modeling, mouse is divided into 3 groups, often organizes 10, diabetic model group (DM group): the PBS damping fluid subcutaneous injection giving equivalent; Control group (Bay g 5421): 2.0mg/kg subcutaneous administrations (being dissolved in 0.1mol/LPBS damping fluid), one day twice, continuous 15d; Sample sets (konjaku polypeptide): same to control group; Separately get 10 normal mouses as blank group.After medication, 15d is in tail venous blood sampling Quick Measurement blood sugar.After 15d, in drug withdrawal after 1 week, fasting be can't help water and is carried out glucose tolerance test in 16 hours.By 25% glucose solution (2g/kg) gavage, after gavage, 0,120 minute tail venous blood sampling detects blood sugar.
Result: glucose transporter protein polypeptide has the effect (P<0.05, table 1) reducing diabetic mice blood sugar; Meanwhile, can the sugar tolerance (table 2) of diabetes-alleviating model mice.The effect of its polypeptide is suitable with the effect of control group Bay g 5421.
Table 1 konjaku polypeptide is to mouse hypoglycemic activity
* p<0.05, * * p<0.01 is compared with model group
Table 2 konjaku polypeptide is to glucose tolerance in mice experimental result
* p<0.05, * * p<0.01 is compared with model group
Conclusion: konjaku polypeptide has therapeutic action to diabetic mice.
SEQUENCELISTING
Pu Luoda bio tech ltd, <110> Suzhou
<120> konjaku polypeptide and application thereof
<130>
<160>1
<170>PatentInversion3.5
<210>1
<211>24
<212>PRT
<213> artificial sequence
<400>1
GlnLeuLeuSerGluLysValSerAspHisLeuIleProHisLysThr
151015
PheSerGlyAsnArgProSerLeu
20
Claims (4)
1. a konjaku polypeptide, is characterized in that: its sequence is QLLSEKVSDHLIPHKTFSGNRPSL.
2. a preparation method for polypeptide described in claim 1, is characterized in that: step comprises (1) decocts, and (2) add amylase, 37 DEG C, stir 60-120 minute, (3) are centrifugal, 5000-10000rpm, 30 minutes, gets precipitation, (4) precipitation is redissolved in the damping fluid of pH9-13, and (5) are centrifugal, 5000-10000rpm, 30 minutes, get supernatant, (6) are by supernatant half preparation RP-HPLC separation and purification, (7) freeze-drying, to obtain final product.
3. the preparation method of polypeptide according to claim 2, is characterized in that: described amylase can be α-amylase, beta-amylase, diastatic enzyme.
4. the konjaku polypeptide of claim 1 is in the application for the treatment of diabetes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510563848.XA CN105111293A (en) | 2015-09-08 | 2015-09-08 | Amorphophallus konjac peptide and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201510563848.XA CN105111293A (en) | 2015-09-08 | 2015-09-08 | Amorphophallus konjac peptide and application thereof |
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| CN105111293A true CN105111293A (en) | 2015-12-02 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN201510563848.XA Pending CN105111293A (en) | 2015-09-08 | 2015-09-08 | Amorphophallus konjac peptide and application thereof |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1384196A (en) * | 2002-06-04 | 2002-12-11 | 上海复旦迪恩生物技术有限公司 | Coding sequence and application of konjaku agglutinic protein |
-
2015
- 2015-09-08 CN CN201510563848.XA patent/CN105111293A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1384196A (en) * | 2002-06-04 | 2002-12-11 | 上海复旦迪恩生物技术有限公司 | Coding sequence and application of konjaku agglutinic protein |
Non-Patent Citations (3)
| Title |
|---|
| 吕婧等: "发酵法制备魔芋多肽工艺研究", 《食品科技》 * |
| 毛跟年等: "魔芋多肽酶解法制备工艺研究", 《粮食与油脂》 * |
| 邵静可等: "魔芋多肽制备工艺研究", 《食品科技》 * |
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Application publication date: 20151202 |