CN110215449A - Alkannic acid is inhibiting the application in insulin amyloid polypeptide aggregation - Google Patents
Alkannic acid is inhibiting the application in insulin amyloid polypeptide aggregation Download PDFInfo
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- CN110215449A CN110215449A CN201910396106.0A CN201910396106A CN110215449A CN 110215449 A CN110215449 A CN 110215449A CN 201910396106 A CN201910396106 A CN 201910396106A CN 110215449 A CN110215449 A CN 110215449A
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
- A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
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- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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Abstract
The present invention provides a kind of alkannic acids to inhibit the application in insulin amyloid polypeptide aggregation, specifically, it provides a kind of alkannic acid and is preparing the application in hIAPP agglutination inhibitor, by research shows that, alkannic acid provided by the invention can inhibit the aggregation of actrapid monotard's amyloid polypeptide from multiple angles, and then reduce and damaged caused by beta Cell of islet, achieve the purpose that prevention and/or treatment diabetes.
Description
Technical field
The present invention relates to field of medicaments more particularly to a kind of alkannic acid to inhibit answering in insulin amyloid polypeptide aggregation
With.
Background technique
Diabetes are a kind of using blood glucose rise as the multi-pathogenesis metabolic disorder syndrome of main feature, disease incidence year by year on
It rises, chronic complicating diseases spread each important organ of whole body, lead to dysfunction and failure, it is considered to be harm is only second to heart and brain blood
The Chronic Non-Communicable Diseases of pipe disease and malignant tumour.Diabetes were divided into four types in 1999 by the World Health Organization (WHO)
Type, i.e. type 1 diabetes, diabetes B, secondary and gestational diabetes mellitus, wherein diabetes B is most common type, accounts for about trouble
The 90% of person's sum.The current pathogenesis in relation to diabetes B not yet illustrates, 95% or more type 2 diabetic patient part
Have insulin amyloid polypeptide aggregation generate amyloid beta deposition, it is considered to be the important pathogenesis of diabetes B it
One.
Actrapid monotard's amyloid polypeptide (human islet amyloid polypeptide, hIAPP) is by 37 amino
The polypeptide of acid composition, synthesizes secretion by beta Cell of islet together with insulin, insulin etc. can be cooperateed under normal physiological condition
Blood glucose-control hormone more accurately adjusts blood sugar for human body, but simultaneously hIAPP be also be currently known aggregation tendency it is strongest
One of polypeptide.The aggregation of hIAPP is generally divided into: period of delay, rise period and plateau.Aggregation early stage be formed by oligomer and
Fiber precursor has stronger cytotoxicity.The interaction of toxicity and cell membrane that hIAPP aggregation generates is closely related, can lead to
Cross destruction beta Cell of islet membrane structure, inducing cell internal oxidition stress, the modes such as er stress and injury of mitochondria induce β
Cell death destroys pancreas islet normal physiological function, final to cause diabetes B lesion.
Therefore, the inhibitor and its inhibiting mechanism of research hIAPP aggregation reduces for developing potential drug
HIAPP assembles the toxic effect to beta Cell of islet, is of great significance to diabetes B is prevented and treated.
Summary of the invention
In view of this, inhibiting insulin amyloid technical problem to be solved by the present invention lies in a kind of alkannic acid is provided
Application in polypeptide aggregation, the present invention is by and then can be realized to 2- using alkannic acid as the inhibitor for inhibiting hIAPP aggregation
The prevention and/or treatment of patients with type Ⅰ DM.
The present invention provides a kind of alkannic acid answering in the inhibitor that preparation inhibits the aggregation of actrapid monotard's amyloid polypeptide
With.
The present invention also provides a kind of alkannic acids to prepare the application in the drug for preventing and/or treating diabetes.
Preferably, the diabetes are diabetes B.
Compared with prior art, the present invention provides a kind of alkannic acids to prepare the application in hIAPP agglutination inhibitor, leads to
Cross the experimental results showed that, alkannic acid provided by the invention can from multiple angles inhibit actrapid monotard's amyloid polypeptide aggregation,
And then reduce and damaged caused by beta Cell of islet, achieve the purpose that prevention and/or treatment diabetes.
Detailed description of the invention
Fig. 1 is that the result of electron spray ionic mobility mass spectral analysis alkannic acid and hIAPP interaction is utilized in embodiment 1
Figure；
Fig. 2-A is that thioflavin T fluorometric detects the result figure that alkannic acid assembles inhibiting effect to hIAPP in embodiment 2；
Fig. 2-B is the inhibiting rate result figure that various concentration alkannic acid assembles hIAPP；
Fig. 3 is the result figure of hydrophobicity variation in Nile red fluorescence detection hIAPP accumulation process in embodiment 3；
Fig. 4 is the transmission electron microscope picture of hIAPP self-controlled group；
Fig. 5 is the transmission electron microscope picture that hIAPP and alkannic acid are incubated for jointly with molar ratio 1: 1；
Fig. 6 is the transmission electron microscope picture that hIAPP and alkannic acid are incubated for jointly with molar ratio 1: 5；
Fig. 7 is the transmission electron microscope picture that hIAPP and alkannic acid are incubated for jointly with molar ratio 1: 10；
Fig. 8 is hIAPP and INS-1 cell survival rate result after various concentration alkannic acid is added；
Fig. 9 is hIAPP and the measurement result of INS-1 cell release LDH content after various concentration alkannic acid is added.
Specific embodiment
The present invention provides application of the alkannic acid in the inhibitor that preparation inhibits the aggregation of actrapid monotard's amyloid polypeptide.
The present invention also provides a kind of alkannic acids to prepare the middle application for preventing and/or treating the drug of diabetes；
Wherein, the diabetes are preferably diabetes B.
Alkannic acid provided by the invention inhibits the inhibitor of actrapid monotard's amyloid polypeptide aggregation as preparation, and existing
Inhibit the inhibitor of actrapid monotard's amyloid polypeptide aggregation to compare, has good compatibility with hIAPP, can effectively inhibit hIAPP
Aggregation, and be easy to be absorbed by organisms, there is various biological activity can inhibit hIAPP aggregation, and then reduction pair from multiple angles
It is damaged caused by beta Cell of islet, achievees the purpose that prevention and/or treatment diabetes, in treatment diabetes B and/or diabetes
The Field of Drug Discovery of complication has good application prospect.
It is clearly and completely described below in conjunction with the technical solution of the embodiment of the present invention, it is clear that described implementation
Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common
Technical staff's every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
Embodiment 1
Electron spray-influence of ionic mobility mass spectrum (ESI-IM-MS) the analysis alkannic acid to hIAPP monomer conformation
Concrete operations: hIAPP is dissolved in hexafluoroisopropanol (HFIP), and 150 μM of concentration, ultrasound after 4 DEG C of placement 4h
30min.Being lyophilized after the packing of polypeptide stock solution, -20 DEG C are saved.Alkannic acid and the hIAPP of freeze-drying are dissolved in 10mM ammonium acetate solution
In, hIAPP concentration is 20 μM, and hIAPP carries out electron spray-ionic mobility mass spectrum (ESI- after mixing with alkannic acid with molar ratio 1: 5
IM-MS it) analyzes.
As a result as shown in FIG. 1, FIG. 1 is electron spray ionic mobility mass spectral analysis alkannic acid and hIAPP phase are utilized in embodiment 1
The result figure of interaction；It can be seen from the figure that as indicated at a, alkannic acid and hIAPP have good compatibility, it is able to observe that
The spectral peak of the compound for 1: 1,1: 2 or 1: 3 (hIAPP: alkannic acid) that hIAPP monomer is formed in conjunction with alkannic acid；For another example 1-B institute
Show, hIAPP band+3 or+4 charges monomer, containing there are two types of tripe systems as.A kind of conformation that its drift time is relatively short is
Stable conformation；And another drift time longer conformation for opposite stretching, extension, it has been also considered as stronger aggregation tendency
Conformation, aggregation form fiber approach in play an important role.And alkannic acid forms compound in conjunction with hIAPP monomer
Afterwards, thus it is possible to vary the relative intensity of two kinds of conformations makes hIAPP monomer more be in metastable conformational state, to send out
Wave the effect for inhibiting aggregation.
Embodiment 2
The inhibiting effect that thioflavine T (ThT) fluorescence detection alkannic acid assembles hIAPP
Concrete operations: dissolving a sample in 25mM PBS (25mM NaCl, pH 7.4) buffer, and 96 hole of black is added
In plate, making hIAPP and ThT final concentration in detection architecture is 20 μM, and the molar ratio of hIAPP and alkannic acid is respectively 1: 0.5,1:
1,1: 5 and 1: 10.At 25 DEG C, continuous fluorescence monitoring, reflection kinetics of aggregation variation, excitation and transmitting are carried out in microplate reader
Wavelength is respectively 440nm and 485nm.
As a result as shown in Fig. 2-A, Fig. 2-A is that thioflavin T fluorometric detects alkannic acid to hIAPP aggregation inhibition in embodiment 2
The result figure of effect；It can be seen from the figure that when hIAPP individualism, as the increase hIAPP of incubation time gradually occurs to gather
Collection, fluorescence intensity gradually increase.After the alkannic acid of various concentration is added, the speed that hIAPP assembles significantly slows down, and reaches
The fluorescence intensity of plateau is significantly reduced, and alkannic acid is in concentration dependent to the inhibitory effect of aggregation.
The dose relationship between inhibiting effect and concentration further to acquire aggregation of the alkannic acid to hIAPP, according to not
With the inhibiting rate that concentration alkannic acid assembles hIAPP, corresponding inhibiting rate-dose curve is drawn.As shown in fig. 2-b, Fig. 2-B is not
With the inhibiting rate result figure that concentration alkannic acid assembles hIAPP, related formula fitting shows between inhibiting rate and Asian puccoon acid concentration
Property it is good, can be obtained alkannic acid inhibit hIAPP aggregation IC50Value is 8.91 μM, i.e., when hIAPP is 20 μM, only needs 8.91 μM
Alkannic acid be can inhibit 50% hIAPP aggregation.Since the hIAPP concentration that design needed for testing uses is 20 μM, it is much higher than
The concentration (being typically maintained in pM) of hIAPP in actual human body, therefore under this inhibiting rate-dose relationship, alkannic acid can sufficiently be sent out
Wave its inhibiting effect to hIAPP aggregation.
Embodiment 3
Hydrophobicity changes in Nile red (Nile red) fluorescence detection hIAPP accumulation process
Concrete operations: 20 μM of hIAPP and alkannic acid press 1: 0.5,1: 1,1: 5 and 1: 10 molar ratio at room temperature respectively
It is incubated for, and Nile red is added to make 10 μM of its concentration.With the hydrophobic region of microplate reader detection Nile red and hIAPP aggregation exposure
Change in fluorescence in conjunction with after.Excitation wavelength 550nm is taken, fluorescence intensity of the launch wavelength range in 575-700nm is recorded.
As a result as shown in figure 3, Fig. 3 is hydrophobicity variation in Nile red fluorescence detection hIAPP accumulation process in embodiment 3
Result figure；It can be seen from the figure that the exposure of hydrophobic region can occur in accumulation process for hIAPP, when it is in conjunction with Nile red
After fluorescence intensity can be made to enhance, emission peak blue shift.In the case where the alkannic acid with various concentration coexists, hIAPP combination Buddhist nun sieve
Red fluorescence intensity is substantially reduced, and emission peak blue shift is gradually reduced, and function and effect are related to Asian puccoon acid concentration, the result and reality
2 result of example is consistent.
Embodiment 4
Electron microscope experiment observes the fibrosis that alkannic acid inhibits hIAPP
Concrete operations: 8 μ L are incubated for sample spot for 24 hours on copper mesh, and static 10min blots redundant sample with filter paper, then uses
1% acetic acid uranium carries out dyeing 5min, then blots surplus liquid, and room temperature is dried, and is observed using Electronic Speculum.
As a result as shown in Figure 4 to 7, Fig. 4 is the transmission electron microscope picture of hIAPP self-controlled group；Fig. 5 is hIAPP and Asian puccoon
The transmission electron microscope picture that acid is incubated for jointly with molar ratio 1: 1；It is saturating that Fig. 6 is that hIAPP and alkannic acid are incubated for jointly with molar ratio 1: 5
Penetrate electron microscope；Fig. 7 is the transmission electron microscope picture that hIAPP and alkannic acid are incubated for jointly with molar ratio 1: 10；It can be seen that from Fig. 4~7
HIAPP control group itself forms a large amount of typical slim line fiber, and with the increase that alkannic acid dosage is added,
HIAPP assembles the fiber shape to be formed and substantially reduces.Further prove that alkannic acid is populated with obvious inhibiting effect to hIAPP.
Embodiment 5
The cytotoxicity that alkannic acid induces hIAPP
Concrete operations: by INS-1 cell with 6 × 103The density kind in a/hole after culture for 24 hours, is removed old on 96 orifice plates
Culture medium, each group are added the culture medium containing 15 μM of hIAPP and various concentration alkannic acid, continue culture for 24 hours.Then, every hole is again
After the MTT solution incubation 4h of 20 μ L 5mg/ml is added, culture medium is removed, the DMSO that 150 μ L are added shakes 10min, uses enzyme mark
Instrument detects absorbance value under 570nm wavelength.
Lactic dehydrogenase (LDH) assay tests identical cultural method using with MTT, group of cells through hIAPP and
After alkannic acid effect, the supernatant of cell culture is collected.Sample is handled according to the method for kit specification introduction, uses enzyme mark
Instrument is discharged into the LDH content in culture solution after measurement cellular damage at 450nm wavelength.
As a result as shown in Fig. 8~Fig. 9, Fig. 8 is hIAPP and INS-1 cell survival rate after various concentration alkannic acid is added
As a result；INS-1 cell discharges the amount measurement result that LDH contains after Fig. 9 is hIAPP and various concentration alkannic acid is added.From figure
As can be seen that lactic dehydrogenase (LDH) content is tested and detects by MTT to evaluate alkannic acid to the thin of hIAPP aggregation inducing
The protective effect of cellular toxicity.15 μM of hIAPP have stronger cytotoxicity, and cell survival rate is made to drop to 65%, are added different dense
After the alkannic acid collective effect of degree, cellular damage caused by hIAPP can obviously reduce, improve cell survival rate (Fig. 8).Work as cell
Under oxidative stress status, LDH leakage rate can be obviously increased, and LDH level is higher to show that its cellular damage is more serious.With MTT
As a result consistent, the addition of various concentration alkannic acid can inhibit LDH leakage, play its protective effect (Fig. 9) to cell.
The above description of the embodiment is only used to help understand the method for the present invention and its core ideas.It should be pointed out that pair
For those skilled in the art, without departing from the principle of the present invention, the present invention can also be carried out
Some improvements and modifications, these improvements and modifications also fall within the scope of protection of the claims of the present invention.
Claims (3)
1. application of the alkannic acid in the inhibitor that preparation inhibits the aggregation of actrapid monotard's amyloid polypeptide.
2. alkannic acid is preparing the application in the drug for preventing and/or treating diabetes.
3. application according to claim 2, which is characterized in that the diabetes are diabetes B.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201910396106.0A CN110215449A (en) | 2019-05-13 | 2019-05-13 | Alkannic acid is inhibiting the application in insulin amyloid polypeptide aggregation |
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| CN201910396106.0A CN110215449A (en) | 2019-05-13 | 2019-05-13 | Alkannic acid is inhibiting the application in insulin amyloid polypeptide aggregation |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2024077977A1 (en) * | 2022-10-11 | 2024-04-18 | 上海市皮肤病医院 | Use of lithospermic acid in preparation of drugs used for treating diabetic ulcers |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994006809A2 (en) * | 1992-09-09 | 1994-03-31 | I.R.2.M. | Pharmaceutical compositions containing associations of metals and tannins or tannin mixtures |
| US6267992B1 (en) * | 1999-09-29 | 2001-07-31 | Shiva Biomedical Llc | Treatment of diabetic nephropathy and microalbuminuria |
-
2019
- 2019-05-13 CN CN201910396106.0A patent/CN110215449A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994006809A2 (en) * | 1992-09-09 | 1994-03-31 | I.R.2.M. | Pharmaceutical compositions containing associations of metals and tannins or tannin mixtures |
| US6267992B1 (en) * | 1999-09-29 | 2001-07-31 | Shiva Biomedical Llc | Treatment of diabetic nephropathy and microalbuminuria |
| CN1387439A (en) * | 1999-09-29 | 2002-12-25 | 希瓦生物医学股份有限公司 | Treatment of diabetic nephropathy and microalbuminuria |
Non-Patent Citations (1)
| Title |
|---|
| 张盼盼: "牛至叶提取物的体外降糖活性及其作用机制研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2024077977A1 (en) * | 2022-10-11 | 2024-04-18 | 上海市皮肤病医院 | Use of lithospermic acid in preparation of drugs used for treating diabetic ulcers |
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