CN104311632A - Silkworm polypeptide as well as preparation method and application thereof - Google Patents
Silkworm polypeptide as well as preparation method and application thereof Download PDFInfo
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- CN104311632A CN104311632A CN201410508548.7A CN201410508548A CN104311632A CN 104311632 A CN104311632 A CN 104311632A CN 201410508548 A CN201410508548 A CN 201410508548A CN 104311632 A CN104311632 A CN 104311632A
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Abstract
The invention relates to the field of medicine and in particular relates to silkworm polypeptide 1 with capability of preventing or treating diabetes mellitus. The sequence of the silkworm polypeptide 1 is RLGRRLSEDPATPADQ. A preparation method of the silkworm polypeptide 1 comprises the following steps: (1) decocting; (2) enzymatic hydrolysis; (3) protein separation; (4) semi-preparative RP-HPLC separation and purification; and (5) freeze-drying, wherein papain and alkaline protease are adopted to carry out enzymatic hydrolysis; macroporous resin and sephadex are adopted to carry out protein separation. The invention also relates to application of the silkworm polypeptide 1 in treatment of the diabetes mellitus and complications of the diabetes mellitus. The complications of the diabetes mellitus include diabetic nephropathy, diabetic hypertension, diabetic eye disease and diabetic neurologic disorder. Since polypeptide is extracted from silkworm, the silkworm polypeptide 1 disclosed by the invention is good in safety and is capable of effectively preventing or treating the 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 type 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.
Silkworm chrysalis, as insects food unique in " bread and cheese management new resource for food list ", has very high pharmaceutical use, and pharmacological action is also very extensive, except can growth promoting effects, improve immunizing power, also there is hypoglycemic effect.But silkworm chrysalis is the principal by product of silk, China can produce fresh silkworm chrysalis more than 500,000 tons every year.But because technology shortcoming, in the quite a long period, silkworm chrysalis is all used as word material substantially, result in certain wasting of resources.Therefore, if the compound that can extract treatment diabetes from silkworm chrysalis becomes the focus of drug development.However, the compound that extracts from silkworm chrysalis 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 silkworm chrysalis polypeptide 1, can safety, effectively treat diabetes.
Technical scheme
A kind of silkworm chrysalis polypeptide 1, its aminoacid sequence is RLGRRLSEDPATPADQ.The preparation method of described polypeptide 1, is characterized in that: step comprises (1) decocts, (2) enzymolysis, (3) protein separation, and RP-HPLC separation and purification is prepared, (5) freeze-drying in (4) half.Described enzymolysis adopts papoid and Sumizyme MP enzymolysis.Described protein separation adopts macroporous resin, dextrane gel.The application of described polypeptide 1 in treatment diabetes and diabetic complication.Described diabetic complication is diabetic nephropathy, diabetic hypertension, diabetes eye illness, diabetic neuropathic pathology.
Beneficial outcomes:
Silkworm chrysalis polypeptide 1 in the present invention, be extract polypeptide from silkworm chrysalis, security is good, and can effectively prevent or treat diabetes.Be more preferably the purposes in treatment diabetes and/or diabetes complicated disease drug.Described diabetes comprise type i diabetes and type ii diabetes; Described diabetic complication includes but not limited to: diabetic nephropathy, diabetic hypertension, diabetes eye illness, diabetic neuropathic pathology etc.
Embodiment
Embodiment 1
The preparation method of polypeptide
After silkworm chrysalis boiling 2h, be cooled to room temperature; Add 1% papoid and 0.5% Sumizyme MP enzymolysis 4h; Adopt macroporous resin according to molecular weight isolated protein, then with half preparation RP-HPLC separation and purification, after the product freeze-drying obtained, be silkworm chrysalis polypeptide 1.The silkworm chrysalis polypeptide 1 that application aforesaid method extracts, through Mass Spectrometric Identification, sequence is RLGRRLSEDPATPADQ, and this sequence is brand-new sequence.Also the raw work synthesis in Shanghai can be entrusted.
Embodiment 2
The cell in vitro determination of activity of silkworm chrysalis polypeptide 1
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 silkworm chrysalis polypeptide 1 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 15.04nmol/L.
Embodiment 3
Hypoglycemic experiment in the body of silkworm chrysalis polypeptide 1
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): 20 μ g/kg subcutaneous administrations (being dissolved in 0.1mol/L PBS damping fluid), one day twice, continuous 15d; Sample sets (silkworm chrysalis polypeptide 1): 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 1 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 1 is suitable with the effect of control group Bay g 5421.
Table 1 silkworm chrysalis polypeptide 1 pair of mouse hypoglycemic activity
* p<0.05, * * p<0.01 is compared with model group
Table 2 silkworm chrysalis polypeptide 1 pair of glucose tolerance in mice experimental result
* p<0.05, * * p<0.01 is compared with model group
Conclusion: silkworm chrysalis polypeptide 1 pair of diabetic mice has therapeutic action.
SEQUENCE LISTING
Pu Luoda bio tech ltd, <110> Suzhou
<120> silkworm chrysalis polypeptide and preparation method thereof, application
<130>
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 16
<212> PRT
<213> artificial sequence
<400> 1
Arg Leu Gly Arg Arg Leu Ser Glu Asp Pro Ala Thr Pro Ala Asp Gln
1 5 10 15
Claims (6)
1. a silkworm chrysalis polypeptide, is characterized in that: its aminoacid sequence is RLGRRLSEDPATPADQ.
2. a preparation method for polypeptide described in claim 1, is characterized in that: step comprises (1) decocts, (2) enzymolysis, (3) protein separation, and RP-HPLC separation and purification is prepared, (5) freeze-drying in (4) half.
3. the preparation method of polypeptide according to claim 2, is characterized in that: enzymolysis used in enzymolysis in step (2) adopts papoid and Sumizyme MP.
4. the preparation method of polypeptide according to claim 2, is characterized in that: the protein separation described in step (3) adopts macroporous resin, dextrane gel.
5. the application of polypeptide according to claim 1 in preparation treatment diabetes and diabetes complicated disease drug.
6. application according to claim 5, is characterized in that described diabetic complication is diabetic nephropathy, diabetic hypertension, diabetes eye illness or diabetic neuropathic pathology.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410508548.7A CN104311632A (en) | 2014-09-28 | 2014-09-28 | Silkworm polypeptide as well as preparation method and application thereof |
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| CN201410508548.7A CN104311632A (en) | 2014-09-28 | 2014-09-28 | Silkworm polypeptide as well as preparation method and application thereof |
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| CN104311632A true CN104311632A (en) | 2015-01-28 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105661554A (en) * | 2016-01-14 | 2016-06-15 | 安徽生物肽产业研究院有限公司 | Silkworm chrysalis small molecule peptide chelate for improving immunity and preparation method thereof |
| CN106943586A (en) * | 2017-03-31 | 2017-07-14 | 山东岐伯堂生物科技有限公司 | A kind of male health-care yang-invigorating formulation production method |
| CN108395467A (en) * | 2018-03-08 | 2018-08-14 | 浙江汇能生物股份有限公司 | The isolation and purification method of silkworm pupa protein polypeptide |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2011251952A (en) * | 2010-06-03 | 2011-12-15 | Genome Soyaku Kenkyusho:Kk | Hypoglycemic substance |
| CN104031963A (en) * | 2014-04-02 | 2014-09-10 | 浙江省农业科学院 | Method for preparing hpyerglycemic peptide by using silkworm chrysalis |
-
2014
- 2014-09-28 CN CN201410508548.7A patent/CN104311632A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2011251952A (en) * | 2010-06-03 | 2011-12-15 | Genome Soyaku Kenkyusho:Kk | Hypoglycemic substance |
| CN104031963A (en) * | 2014-04-02 | 2014-09-10 | 浙江省农业科学院 | Method for preparing hpyerglycemic peptide by using silkworm chrysalis |
Non-Patent Citations (2)
| Title |
|---|
| ZUOWEI SONG，ET AL.: "Reducing blood glucose levels in TIDM mice with an orally administered extract of sericin from hIGF-I-transgenic silkworm cocoons.", 《FOOD AND CHEMICAL TOXICOLOGY》 * |
| 张海娜: "蚕蛹活性多肽的制备及降血压、降血糖活性研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105661554A (en) * | 2016-01-14 | 2016-06-15 | 安徽生物肽产业研究院有限公司 | Silkworm chrysalis small molecule peptide chelate for improving immunity and preparation method thereof |
| CN106943586A (en) * | 2017-03-31 | 2017-07-14 | 山东岐伯堂生物科技有限公司 | A kind of male health-care yang-invigorating formulation production method |
| CN108395467A (en) * | 2018-03-08 | 2018-08-14 | 浙江汇能生物股份有限公司 | The isolation and purification method of silkworm pupa protein polypeptide |
| CN108395467B (en) * | 2018-03-08 | 2021-06-15 | 浙江汇能生物股份有限公司 | Separation and purification method of silkworm pupa protein polypeptide |
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