CN114432430B - Pharmaceutical composition containing scutellarin, baicalin and/or breviscapine and application thereof - Google Patents
Pharmaceutical composition containing scutellarin, baicalin and/or breviscapine and application thereof Download PDFInfo
- Publication number
- CN114432430B CN114432430B CN202011191253.3A CN202011191253A CN114432430B CN 114432430 B CN114432430 B CN 114432430B CN 202011191253 A CN202011191253 A CN 202011191253A CN 114432430 B CN114432430 B CN 114432430B
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- growth factor
- scutellarin
- platelet
- vascular endothelial
- pharmaceutical composition
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Classifications
-
- 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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Abstract
The invention provides a medicinal composition containing scutellarin and analogues thereof and application thereof. The pharmaceutical composition comprises vascular endothelial growth factor and scutellarin and/or analogues thereof, wherein the analogues are selected from one or more of scutellarin and scutellarin. The invention provides a new application of scutellarin and analogues thereof in promoting wound healing and repair, which can cooperate with and increase the effects of Vascular Endothelial Growth Factor (VEGF) and platelet growth factor group (SGC) in the aspect, so that the scutellarin and analogues thereof can be used as a medicine or a cosmetic for treating and/or relieving skin wounds and/or scars, and have good clinical application prospect and market prospect.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a pharmaceutical composition containing scutellarin and analogues thereof and application thereof.
Background
Skin is the primary barrier between the human body and the environment. It is known to have many protective functions, including preventing body from losing water, preventing bacteria and toxic substances from outside the environment, etc., and therefore, it is very important to maintain the integrity and continuity of the skin.
Skin injuries can stimulate a wound healing response. In the wound healing reaction, the stages of blood clot formation, inflammation, re-epithelialization and the like are coordinated with each other, so that the skin is restored to a normal state as soon as possible. Re-epithelialization is critical at these various stages, with keratinocyte migration serving a central role. Keratinocyte migration is a complex process involving the action of multiple biomolecules.
When the skin is injured, platelets act as a first line defender and immediately secrete various growth factors to promote wound healing, such as Epidermal Growth Factor (EGF), vascular Endothelial Growth Factor (VEGF), fibroblast Growth Factor (FGF), platelet-derived growth factor (PDGF), etc. These growth factors play an important role in regulating the contraction of transitional keratinocytes in the wound bed.
It would be advantageous to develop an effective skin wound healing agent to promote wound healing.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a pharmaceutical composition containing scutellarin and analogues thereof and application thereof.
Specifically, the present invention provides:
(1) A pharmaceutical composition comprising vascular endothelial growth factor and scutellarin and/or an analogue thereof, wherein the analogue is selected from one or more of scutellarin and scutellarin.
(2) The pharmaceutical composition according to (1), wherein the molar ratio of the vascular endothelial growth factor to the scutellarin or analogue thereof is 1 (4000-12000).
(3) The pharmaceutical composition of (1), wherein the pharmaceutical composition comprises a population of platelet growth factors.
(4) The pharmaceutical composition according to (3), wherein the molar ratio of vascular endothelial growth factor in the platelet growth factor group to the scutellarin or analogue thereof is 1 (30000-150000).
(5) Use of scutellarin and analogues thereof in the manufacture of a medicament or skin care product for promoting the action of vascular endothelial growth factor and/or platelet growth factor group, wherein the analogues are selected from one or more of scutellarin and breviscapine.
(5) The use according to (5), wherein the effect comprises promoting keratinocyte migration, promoting wound repair and/or healing, promoting expression of matrix metalloproteinases, activating vascular endothelial growth factor specific receptor 2 and its mediated MAPK signaling pathway.
(7) The use according to (5), wherein the medicament is a medicament for treating and/or alleviating skin wounds and/or scars.
(8) The use according to (5), wherein the skin care product is a skin care product for treating and/or alleviating skin wounds and/or scars.
(9) Use of the pharmaceutical composition according to any one of (1) to (4) for the preparation of a medicament or skin care product for promoting the action of vascular endothelial growth factor and/or platelet growth factor group.
(10) The use according to (9), wherein the effect comprises promoting keratinocyte migration, promoting wound repair and/or healing, promoting expression of matrix metalloproteinases, activating vascular endothelial growth factor specific receptor 2 and its mediated MAPK signaling pathway.
(11) The use according to (9), wherein the medicament is a medicament for treating and/or alleviating skin wounds and/or scars.
(12) The use according to (9), wherein the skin care product is a skin care product for treating and/or alleviating skin wounds and/or scars.
(13) A pharmaceutical composition for promoting the action of vascular endothelial growth factor and/or platelet growth factor group, comprising the pharmaceutical composition of any one of (1) to (4).
(14) The medicament of (13), wherein the medicament further comprises pharmaceutically acceptable excipients.
(15) The medicament according to (13), wherein the medicament is in the form of a liniment or an injection.
(16) A skin care product for promoting the action of vascular endothelial growth factor and/or platelet growth factor group, comprising the pharmaceutical composition of any one of (1) to (4).
(17) The skin care product of (16), wherein the skin care product further comprises one or more formulation or additives.
(18) The skin care product according to (16), wherein the skin care product is in the form of a gel, an emulsion, an oil-in-water or water-in-oil two-phase emulsion, a mask, a lotion, a concentrate, a serum, a nanocapsule, a liposome, or a lipstick.
Compared with the prior art, the invention has the following advantages and positive effects:
the invention discovers that scutellarin and analogues thereof can be combined with vascular endothelial growth factor to increase migration of skin keratinocytes induced by vascular endothelial growth factor and platelet growth factor group, thereby promoting wound healing. The invention discovers that the mechanism of the action is that scutellarin and analogues thereof can promote the phosphorylation of the vascular endothelial growth factor receptor 2 (VEGFR 2) of the epithelial cells by combining with the vascular endothelial growth factor, so that the scutellarin can be activated, the expression and the phosphorylation of ErK in a downstream signal path MAPK of the receptor can be increased, and meanwhile, the expression of keratinocyte migration related gene matrix metalloproteinase (MMP 9) can be increased.
Based on the findings, the invention provides a novel application of scutellarin and analogues thereof in promoting wound healing and repair, which can cooperate with and increase the effects of Vascular Endothelial Growth Factor (VEGF) and platelet growth factor group (SGC) in the aspect, thereby being used as a medicament or cosmetic for treating and/or relieving skin wounds and/or scars, and having good clinical application prospect and market prospect.
In addition, the invention also discovers that the scutellarin has no cytotoxicity and is safe and reliable.
Drawings
FIG. 1 shows the results of cytotoxicity analysis of scutellarin.
Fig. 2 shows photographs (a) of scutellarin promoting vascular endothelial growth factor-induced keratinocyte (HaCaT) scratch healing and wound healing rate quantification results (B).
Fig. 3 shows a photograph (a) of scutellarin promoting healing of keratinocyte (HaCaT) scratch induced by platelet growth factor group and a result of quantification of wound healing rate (B).
FIG. 4 shows graphs of RT-PCR quantification results of the levels of transcription of scutellarin-promoted VEGF (A) or SGC (B) induced keratinocyte migration related gene matrix metalloproteinase-9 (MMP 9);
fig. 5 shows western blot photographs (a and C) of Erk phosphorylation in vascular endothelial growth factor specific receptor 2 (VEGFR 2) and its downstream signaling pathway in combination with scutellarin and corresponding quantification of band brightness plots (B and D), where the ordinate of plots B and D is a fold relative to baseline, with band brightness of the blank as baseline.
FIG. 6 shows a computer simulation of binding of scutellarin (A), baicalin (B) and breviscapine (C) to vascular endothelial growth factor molecules.
Detailed Description
The invention is further described below by means of the description of specific embodiments and with reference to the accompanying drawings, which are not intended to be limiting, but a person skilled in the art can make various modifications or improvements according to the basic idea of the invention, all without departing from the scope of the invention.
The scutellarin is also called red calendula extract, and its plant source is stem, leaf, whole plant of Scutellaria baicalensis Georgi of Labiatae, etc., and its structural formula is shown in formula I below.
The clinical research results show that the scutellarin has the functions of reducing the cerebral vascular resistance, improving the cerebral blood circulation, increasing the cerebral blood flow and resisting the platelet aggregation, and is clinically used for treating paralysis after cerebral vascular diseases. However, the influence of scutellarin on skin wound healing and the action mechanism of scutellarin on skin keratinocytes are not reported.
The invention discovers that scutellarin and analogues thereof can be combined with vascular endothelial growth factor, effectively increases migration of skin keratinocytes induced by vascular endothelial growth factor and platelet growth factor group, and thereby promotes wound healing.
The invention also discovers that the mechanism of the action is that scutellarin and analogues thereof can effectively promote the phosphorylation of the vascular endothelial growth factor receptor 2 (VEGFR 2) of the epithelial cells through combining with the vascular endothelial growth factor, so that the scutellarin can activate the vascular endothelial growth factor receptor 2, and the expression and the phosphorylation of ErK in the MAPK of the downstream signal path of the receptor are increased, so that the MAPK signal path mediated by the VEGFR2 can be activated, and the effect of promoting the migration of the keratinocytes is exerted. In keratinocytes, VEGFR2 and its mediated signaling pathways are important for regulating cell survival, proliferation and migration. Meanwhile, scutellarin and analogues thereof can effectively increase the expression of keratinocyte migration related gene matrix metalloproteinase (MMP 9) promoted by VEGF or SGC.
Thus, the present invention has found that scutellarin and its analogues can enhance the biological functions of vascular endothelial growth factor and platelet growth factor group induced wound healing, and can be used as a wound healing agent.
Based on the above findings, the present invention provides the use of scutellarin and analogues thereof in the manufacture of a medicament or skin care product for promoting the action of vascular endothelial growth factor and/or platelet growth factor group, wherein the analogues of scutellarin are selected from one or more of scutellarin and breviscapine.
The baicalin has a structural formula shown in the following formula II, and the scutellarin has a structural formula shown in the following formula III.
The above-described actions of promoting vascular endothelial growth factor and/or platelet growth factor population include promoting keratinocyte migration, promoting wound repair and/or healing, promoting matrix metalloproteinase expression and activating VEGFR2 and its mediated MAPK signaling pathway.
Thus, scutellarin and analogs thereof can promote keratinocyte migration, promote wound repair and/or healing, promote expression of matrix metalloproteinases, activate VEGFR 2-mediated MAPK signaling pathways.
Thus, the present invention also provides the use of scutellarin and its analogues in the manufacture of a medicament or skin care product for promoting keratinocyte migration, promoting wound repair and/or healing, promoting expression of matrix metalloproteinases, activating VEGFR2, and/or activating VEGFR 2-mediated MAPK signaling pathway.
In the present invention, the term "effect of promoting vascular endothelial growth factor and/or platelet growth factor group" means that the scutellarin and its analogues increase the function as compared to the function exerted by vascular endothelial growth factor and platelet growth factor group alone, and the vascular endothelial growth factor and platelet growth factor group includes vascular endothelial growth factor and platelet growth factor group of the body itself, and vascular endothelial growth factor and platelet growth factor group used exogenously.
In the present invention, the term "platelet growth factor group" refers to platelet rich plasma extracted from whole blood, including vascular endothelial growth factor VEGF, epidermal growth factor EGF, platelet-derived growth factor PDGF, platelet 4-th factor PF4 and beta-platelet globulin, which are dispersed in platelet rich plasma. The platelet growth factor group can be obtained by the method described in example 2.
In some embodiments, the present invention provides the use of the scutellarin and its analogues in the manufacture of a medicament for the treatment and/or alleviation of skin wounds and/or scars.
In other embodiments, the present invention provides the use of said scutellarin and analogues thereof for the preparation of a skin care product for the treatment and/or alleviation of skin wounds and/or scars.
Based on the application of the scutellarin and the analogues thereof, the scutellarin and/or the analogues thereof can be independently prepared into medicines or made into skin care products, and after the scutellarin and/or the analogues thereof are used, the effects of VEGF and SGC of the scutellarin and the analogues thereof in the body can be enhanced; or used together with other VEGF and/or SGC drugs or products to enhance the effect of the VEGF and/or SGC drugs or products. Scutellarin and/or analogues thereof may also be used in combination with VEGF and/or SGC in the form of a medicament or cosmetic.
Accordingly, the present invention also provides a pharmaceutical composition characterized by comprising vascular endothelial growth factor and scutellarin and/or an analogue thereof, wherein the analogue of scutellarin is selected from one or more of scutellarin and scutellarin.
Preferably, the molar ratio of the vascular endothelial growth factor to the scutellarin or analogue thereof is 1 (4000-12000). In the case where the pharmaceutical composition comprises scutellarin and analogues thereof, preferably the molar ratio of vascular endothelial growth factor to the sum of both is 1 (4000-12000).
In some embodiments, the pharmaceutical composition comprises a platelet growth factor group comprising vascular endothelial growth factor, in which case the molar ratio of vascular endothelial growth factor to said scutellarin or analog thereof in the platelet growth factor group may be 1 (30000-150000). In the case where the pharmaceutical composition comprises scutellarin and analogues thereof, the molar ratio of vascular endothelial growth factor in the platelet growth factor group to the sum of both may be 1 (30000-150000).
The invention also provides application of the pharmaceutical composition in preparing medicines or skin care products for promoting the action of vascular endothelial growth factors and/or platelet growth factor groups.
In some embodiments, the effect comprises promoting keratinocyte migration, promoting wound repair and/or healing, promoting expression of matrix metalloproteinases, activating vascular endothelial growth factor specific receptor 2 and its mediated MAPK signaling pathway.
In some embodiments, the medicament is a medicament for treating and/or alleviating skin wounds and/or scars.
In other embodiments, the skin care product is a skin care product for treating and/or alleviating skin wounds and/or scars.
The invention also provides a medicament for promoting the action of vascular endothelial growth factor and/or platelet growth factor group, which comprises the pharmaceutical composition of the invention.
In some embodiments, the medicament is for treating and/or alleviating skin wounds and/or scars.
A single dose of the medicament may comprise a daily dose of the active ingredient.
When the medicament comprises either scutellarin or an analogue thereof, the daily dose of scutellarin may be 10-200mg; the daily dose of the analogue of scutellarin may be 10-200mg. Where the medicament comprises both scutellarin and analogues thereof, the daily dose of the combination of scutellarin and analogues thereof may be in the range of 10-200mg.
The daily dose of vascular endothelial growth factor may be 10-30. Mu.g. When the drug comprises a population of platelet growth factors, the daily dose of the population of platelet growth factors may be 10-100 μg.
According to practical applications, the above-mentioned pharmaceutical composition of the present invention may contain scutellarin and/or its analogues, and vascular endothelial growth factor or platelet growth factor group in amounts for formulating a single dose of the drug, and may also contain these active ingredients in amounts for formulating multiple doses of the drug.
The medicament may be in the form of a liniment or an injection.
The medicament may also comprise pharmaceutically acceptable auxiliary materials.
The auxiliary materials can be selected according to the required dosage form. For example, when the dosage form is a liniment, the adjuvant may be one or more selected from glycerol, allantoin, xanthan gum, squalane and phenoxyethanol; when the dosage form is an intravenous injection, the auxiliary material can be one or more selected from mannitol, lactose, dextran xylitol, sorbitol glucose and sodium chloride.
The content of the pharmaceutically acceptable auxiliary materials in the single-dose medicament can be adjusted in the common range in the field according to actual needs.
The invention also provides a skin care product for promoting the action of vascular endothelial growth factor and/or platelet growth factor group, which comprises the pharmaceutical composition.
In some embodiments, the skin care product is used to treat and/or alleviate skin wounds and/or scars.
The skin care product in a single dose may comprise a daily dose of active ingredient.
When the skin care product comprises one of scutellarin and its analogues, the daily dose of scutellarin may be 10-200mg; the daily dose of the analogue of scutellarin may be 10-200mg. Where the skin care product comprises both scutellarin and its analogues, the daily dosage of the combination of scutellarin and its analogues may be 10-200mg.
The daily dose of vascular endothelial growth factor may be 10-30. Mu.g. When the skin care product comprises a platelet growth factor group, the daily dose of platelet growth factor group may be 10-100 μg.
The skin care product may be in the form of a gel, emulsion, oil-in-water or water-in-oil two-phase emulsion, mask, lotion, concentrate, serum, nanocapsule, liposome or lipstick.
The skin care product may also comprise one or more formulation or additives. In some embodiments, the formulation or additive is selected from penetrants, thickeners, surfactants, demulcents, polydimethylsiloxanes, cyclomethicones, emulsifiers, preservatives, oils, UV-Sup>A and UV-B filters, pigments, dyes, film formers, minerals and fragrances.
The content of the formulation or additives in the single-dose skin care product can be adjusted according to actual needs within the range commonly used in the art.
The following further illustrates or describes the present invention by way of examples, which should not be construed as limiting the scope of the invention.
Examples of the invention
Unless otherwise indicated, the experimental procedures, materials, and conditions used in the examples below were all conducted using conventional experimental procedures, materials, and conditions in the art.
Example 1: cytotoxicity assays
To assess whether scutellarin is cytotoxic in keratinocytes and would stimulate skin cell proliferation, cell viability was analyzed using the MTT assay.
Human HaCaT keratinocytes (purchased from AddexBio corporation, san diego) were cultured in DMEM medium supplemented with 10% fbs, 1% penicillin/streptomycin for 2 days to 80% growth density. The adherent cells at the bottom of the dish were digested with 0.25% trypsin solution to prepare a cell suspension, which was counted and then prepared to a cell concentration of 1X 10 4 The suspensions were inoculated in 96-well plates at a volume of 1000 cells per well, placed in CO 2 Incubator (5% CO) 2 Culturing at 37℃for 24 hours. Then adding scutellarin with different final concentrations shown in figure 2 for treatment, continuously culturing for 24 hours, sucking the culture medium in a 96-well plate, adding an MTT solution with the concentration of 0.5mg/mL, and culturing in an incubator for 4 hours. After the incubation, DMSO solution was added and shaken on a shaker for 15 minutes, absorbance at 570nm was measured to analyze viability of the cells.
As shown in fig. 1, haCaT keratinocytes showed no significant decrease in cell number at concentrations of 0.5-100 μm scutellarin. The results demonstrate that scutellarin does not adversely affect skin cells and that its safety is reliable.
Example 2: keratinocyte scratch test
Inoculation of 5X 10 5 Cells were plated in 12-well plates at 5X 10 cells per well 5 And (3) when the cell fusion monolayer reaches 100%, making scratches with the same width on the cell fusion monolayer in each hole by using a 200 mu L pipette tip to be perpendicular to the hole wall, and taking the time point as the 0 th hour. The cells were washed three times with 1 XPBS, scraped off suspended cell debris was aspirated, serum-free DMEM medium was added, and different drug treatments were then added: cells treated with 10 μm vascular endothelial growth factor VEGF alone served as positive controls, and this concentration of VEGF was able to promote the closure of keratinocyte lesions; to use VEGF (10. Mu.M) and VEGF inhibitor Avastin (2)00 μg/mL) treated cells served as negative control; cells without any drug treatment (with equal volume of DMEM) were used as blank; experiment 1 was performed on cells treated with 3. Mu.M scutellarin alone, and experiment 2 was performed on cells treated with 10. Mu.M VEGF and 3. Mu.M scutellarin. Placing cells into CO 2 Incubator (5% CO) 2 Culturing At 37 ℃) and sampling At0 and 20 hours, photographing, analyzing and quantifying cell coverage by using TScratch software (CSE Lab), and calculating the wound healing rate At20 hours, wherein the calculation method of the wound healing rate is as follows: wound healing rate (%) = (At 0-At 20)/At 20×100%.
As shown in fig. 2A and B, 3 μm scutellarin alone did not promote healing of keratinocyte scratches, whereas scratches of experimental group 2 healed significantly at20 hours and healed significantly faster than other groups, indicating that 3 μm scutellarin enhanced the effect of VEGF on keratinocyte migration.
Since VEGF is highly enriched in the platelet growth factor group (SGC), the scored HaCaT cell monolayer was drug treated in the same manner with the platelet growth factor group: cells treated with SGC alone at a high concentration of 0.5% (v/v) were used as positive controls; cells treated with 0.5% (v/v) SGC and avastin (200. Mu.g/mL) were used as negative controls; cells without any drug treatment (with equal volume of DMEM) were used as blank; experiment 1 was a cell treated with 3. Mu.M scutellarin and a low concentration of 0.1% (v/v) SGC, experiment 2 was a cell treated with 3. Mu.M scutellarin alone, and experiment 3 was a cell treated with a low concentration of 0.1% (v/v) SGC alone.
As shown in fig. 3, the composition of scutellarin and the platelet growth factor group significantly promoted the migration of HaCaT cells in the scratch gap compared to the control group, indicating that scutellarin can promote the effect of the growth factor or the platelet growth factor group on the keratinocyte migration.
The preparation method of the platelet growth factor group comprises the following steps: after taking human whole blood with anticoagulant, the whole blood sample was centrifuged at 3800rpm for 20 minutes at 4 ℃. Whole blood plasma was separated after the first centrifugation. The plasma is then brought to 1250rp at 4℃m centrifugation for 10 min. 2/3 of the supernatant was discarded as platelet poor plasma, and the remaining 1/3 was resuspended, called Platelet Rich Plasma (PRP). The platelet growth factor group was obtained by adding 10mM EDTA/EGTA protease inhibitor to PRP, removing ferrous ions with a magnetic stent (available from Merck Millipore, berlington, U.S.A.), and stripping the platelets through one freeze-thawing cycle (-80 ℃ for 10 hours and 37 ℃ for 10 minutes). After about 30 minutes of ultraviolet sterilization, the plasma sample was subjected to LabconcoThe cells were lyophilized in a freeze drying system (Labconco, mitsubii) under vacuum for 16 hours and stored at 4℃for use.
Example 3: scutellarin in combination with VEGF or SGC promotes expression of keratinocyte migration related gene matrix metalloproteinase-9 (MMP 9)
To further confirm the effect of scutellarin in combination with VEGF or SGC on keratinocyte migration, RT-PCR was used to measure the transcript levels of MMP9 genes. First, haCaT cells were prepared to have a cell concentration of 3X 10 5 The individual/mL suspensions were inoculated onto 12-well plates, each well being inoculated with 3X 10 5 Individual cells. Put into CO 2 Incubator (5% CO) 2 Incubation for 24 hours at 37 ℃) followed by addition of different concentrations of drug: the dosing setup was essentially the same as in example 2, except that this example increased the group of cells treated with 1. Mu.M scutellarin and low concentration of 0.1% (v/v) SGC (experimental group 4), and the group of cells treated with 3. Mu.M scutellarin and medium concentration of 0.3% (v/v) SGC (experimental group 5). Culturing was continued for 24 hours after dosing, mRNA was extracted from HaCaT cells, and RT-PCR was performed using primers for MMP 9. The primer sequences were as follows: MMP 9F-primer 5'-GGAGCGAGATCCCTCCAAAAT-3' (SEQ ID NO. 1) and MMP 9R-primer 5'-GGCTGTTGTCATACTTCTCATGG-3' (SEQ ID NO. 2).
As shown in fig. 4, scutellarin significantly promoted VEGF or SGC induced transcript levels of MMP9 genes, with an enhancement of about 130% of the placebo. These results indicate that scutellarin in combination with VEGF or SGC has a significant effect on promoting wound healing by increasing the expression of MMP 9.
Example 4: scutellarin specifically activates vascular endothelial growth factor specific receptor 2 (VEGFR 2) and signal pathway mediated thereby, and plays a role in promoting wound healing
The effect of scutellarin on VEGF-dependent wound healing in a platelet growth factor group was evaluated using western blot experiments. HaCaT cells were seeded in 12-well plates (1.5X10) 5 Cell/well), placed in CO 2 Incubator (5% CO) 2 Culturing at 37℃for 24 hours. Serum-free DMEM was added for starvation for 3 hours, and 0.5% (v/v) SGC, 0.1% (v/v) SGC, 3. Mu.M scutellarin, and a combination of 0.1% (v/v) SGC and 3. Mu.M scutellarin were added, respectively. Samples were collected at0, 5, 10 min, lysed using low concentration lysates (100 mM Tris-Cl pH6.8,8% sodium dodecyl sulfate, 0.2% bromophenol blue, 20% glycerol, 400mM 2-mercaptoethanol), and cell lysates were collected and loaded on an 8% SDS-PAGE gel. Antibodies used for western blotting were: 1:1000 phosphorylated Phospho-VEGFR2 rabbit anti-monoclonal antibody (CST, 2478S), 1:1000 phosphorylated Phospho-p44/42MAPK (Erk 1/2) (CST, 9101S), 1:1000p44/42MAPK (Erk 1/2) (CST, 8544S).
The results are shown in FIGS. 5A-D. Figures 5A and B show that scutellarin in combination with low concentration SGC induced VEGFR2 phosphorylation, wherein the expression level of phosphorylated VEGFR2 in the placebo group (without drug but with equal volume of DMEM) and in the low concentration SGC treated group did not significantly change after 5 minutes, 10 minutes, the expression level of phosphorylated VEGFR2 in the high concentration SGC treated group was about 3-fold after 10 minutes, and the expression level of phosphorylated VEGFR2 in the 0.1% (v/v) SGC in combination with 3 μm scutellarin was about 2.5-fold compared to the positive control (0.5% (v/v) SGC alone). FIGS. 5C and D show that scutellarin in combination with low concentration SGC exhibited induction of Erk phosphorylation downstream of the receptor, wherein the phosphorylated Erk expression level of 0.1% (v/v) SGC in combination with 3. Mu.M scutellarin increased to about 1.5 fold after 10 minutes, as compared to the effect of 0.5% (v/v) SGC induction in the positive control.
Example 5: molecular fitting experiment
Molecular interactions between scutellarin and its analogs and vascular endothelial growth factor were examined using molecular binding software (Molecular Docking, vina, phyMOL). Molecular docking was simulated using PyMOL molecular image analysis software and SwissDock tool to find scutellarin and its analogues: erigeron breviscapus and baicalin can be combined with VEGF. Binding affinity data were obtained from the molecular docking tool SwissDock, detailed steps as follows:
1. the pdb structure file of the target protein VEGF was imported, and VEGF pdb used in the present invention was numbered 3QTK.
2. Preparing a small molecular structure mol2 file of small molecules (scutellarin, scutellarin and baicalin).
3. And uploading pdb and mol2 files on the SwissDock website respectively, and clicking and submitting to obtain the molecular docking score. The following table shows:
the structural interactions between scutellarin and vascular endothelial growth factor are shown in figure 6A. The binding affinity of scutellarin and VEGF protein is between-5.57 and-8.28, which shows that the binding affinity of scutellarin and VEGF protein is strong. The structural interactions of scutellarin analogues, scutellarin and VEGF are shown in figures 6B and C, and the binding affinities are-5.59 to-8.15 and-6.34 to-8.26 respectively, which shows that the binding affinities of scutellarin and scutellarin are also stronger.
SEQUENCE LISTING
<110> university of hong Kong science and technology
<120> pharmaceutical composition comprising scutellarin and analogues thereof and use thereof
<130> FI-205149-59:52/C
<160> 2
<170> PatentIn version 3.5
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Claims (10)
1. A pharmaceutical composition comprising vascular endothelial growth factor and scutellarin.
2. The pharmaceutical composition of claim 1, wherein the vascular endothelial growth factor is at a molar concentration of 10 μΜ and the scutellarin is at a molar concentration of 3 μΜ.
3. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition comprises a platelet growth factor group that is platelet rich plasma extracted from whole blood, including vascular endothelial growth factor, epidermal growth factor, platelet-derived growth factor, platelet factor 4, and beta-platelet globulin.
4. The pharmaceutical composition of claim 3, wherein the platelet growth factor group has a volume concentration of 0.1% and the scutellarin has a molar concentration of 1 μΜ or 3 μΜ; or the volume concentration of the platelet growth factor group is 0.3%, and the molar concentration of the scutellarin is 3 mu M.
5. Use of a pharmaceutical composition according to any one of claims 1-4 for the preparation of a medicament having the effect of promoting wound repair and/or healing.
6. The use according to claim 5, wherein the effect comprises an effect of promoting vascular endothelial growth factor and/or a group of platelet growth factors, promoting keratinocyte migration, promoting expression of matrix metalloproteinases, activating vascular endothelial growth factor specific receptor 2 and its mediated MAPK signaling pathway.
7. The use according to claim 5, wherein the medicament is a medicament for the treatment and/or alleviation of skin wounds and/or scars.
8. A medicament for the treatment and/or alleviation of skin wounds and/or scars comprising a pharmaceutical composition according to any one of claims 1-4.
9. The medicament of claim 8, wherein the medicament further comprises pharmaceutically acceptable excipients.
10. The medicament of claim 8, wherein the medicament is in the form of a liniment or an injection.
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| CN202011191253.3A CN114432430B (en) | 2020-10-30 | 2020-10-30 | Pharmaceutical composition containing scutellarin, baicalin and/or breviscapine and application thereof |
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| CN202011191253.3A CN114432430B (en) | 2020-10-30 | 2020-10-30 | Pharmaceutical composition containing scutellarin, baicalin and/or breviscapine and application thereof |
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| CN114432430A CN114432430A (en) | 2022-05-06 |
| CN114432430B true CN114432430B (en) | 2024-01-26 |
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| CN202011191253.3A Active CN114432430B (en) | 2020-10-30 | 2020-10-30 | Pharmaceutical composition containing scutellarin, baicalin and/or breviscapine and application thereof |
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| CN115887528B (en) * | 2022-11-23 | 2023-12-12 | 广东药科大学 | Biological rehabilitation preparation for promoting stem cell function and further repairing osteoarthritis |
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| CN103655547A (en) * | 2013-12-18 | 2014-03-26 | 成都中医药大学 | Novel application of scutellarein |
| CN104739913A (en) * | 2015-03-26 | 2015-07-01 | 鼎旺生物科技有限公司 | Preparation for accelerating wound healing, and preparation method and application thereof |
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2020
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| CN102247397A (en) * | 2011-05-30 | 2011-11-23 | 浙江大学 | Application of astragaloside to preparation of drug |
| CN102349925A (en) * | 2011-08-23 | 2012-02-15 | 上海中医药大学附属岳阳中西医结合医院 | Application of astragaloside in preparation of drugs for promoting epithelization in wound healing |
| CN103655547A (en) * | 2013-12-18 | 2014-03-26 | 成都中医药大学 | Novel application of scutellarein |
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