CN112237570B - Novel carbamide peroxide preparation for preparing medicine for repairing pulmonary vascular injury and preparation method thereof - Google Patents
Novel carbamide peroxide preparation for preparing medicine for repairing pulmonary vascular injury and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a preparation method of a novel carbamide peroxide preparation for preparing a medicament for repairing pulmonary vascular injury, which comprises the following steps: the ratio of the amount of the substances is (1-2): 1, putting the hydrogen peroxide and the urea into a reactor, stirring at room temperature to dissolve the hydrogen peroxide and the urea, then adding 0.05-0.2 g of sorbic acid, and stirring at room temperature for 30-200 min to obtain a reaction solution; and after the reaction is finished, cooling and crystallizing the reaction solution at the temperature of 0-5 ℃ for 12-36 h, taking out and filtering, and freeze-drying the filter cake in a freeze-dryer for 5-20 h to obtain the product. The preparation method of the novel carbamide peroxide preparation has the advantages of mild reaction conditions, high yield, simple components, no need of heating and low energy consumption. The novel carbamide peroxide can obviously promote the proliferation activity of HPAECs and inhibit the apoptosis activity of HPAECs, has more obvious effect especially under the condition of hypoxia, can be used for promoting angiogenesis and enhancing the cell proliferation capacity, and can be applied to preparing medicaments for repairing pulmonary vascular injury.
Description
Technical Field
The invention relates to the field of pulmonary vascular injury repair preparations, in particular to a novel carbamide peroxide preparation for preparing a medicament for repairing pulmonary vascular injury and a preparation method thereof.
Background
Endothelial Cells (ECs) are a layer of well-differentiated monolayer of flattened cells located on the inner wall of blood vessels and lymphatic vessels, and the vascular endothelium formed by endothelial cells is located between the blood circulation in the lumen and the vessel wall and is the main barrier for maintaining vascular permeability. Endothelial cells are mainly distributed in organs and tissues such as brain, lung, liver, etc., and are smaller than mesothelial cells and are arranged closely. The main role of Human Pulmonary Artery Endothelial Cells (HPAECs) is to maintain vascular homeostasis. The cells synthesize and secrete activating and inhibitory factors in the coagulation and fibrinolytic systems, as well as certain mediators that influence platelet adhesion and aggregation. Endothelial cells can also release molecules that function to regulate cell proliferation and vessel wall tension. Activation of adenosine receptors on human pulmonary artery endothelial cells by lipopolysaccharide results in the release of IL-6, which is involved in the pathophysiological processes of acute lung injury.
Cell proliferation and apoptosis are the fundamental vital activities of a multicellular organism during development. Cell proliferation is the proliferation by cell division to generate new cells to replenish senescent or dead cells in vivo. Apoptosis is the active suicide process of cells in a specific environment. Apoptosis is a major feature of pathological changes in many diseases in clinic. Loss or functional impairment of endothelial cells may cause a number of diseases, such as pulmonary hypertension.
The carbamide peroxide preparation (CP) has the molecular formula of CO (NH) 2 ) 2 ·H 2 O 2 The molecular weight is 94.07, and the appearance is white transparent flaky crystals or white powdery solids. It is easily soluble in water, absorbs heat when dissolved in water, and has a melting point of 75-85 deg.C. The urea can be decomposed into hydrogen peroxide and urea when dissolved in water, has the properties of the urea and the hydrogen peroxide, has nearly neutral pH of an aqueous solution, is non-toxic and odorless, has good stability and long action effect time, can be used for sterilization, disinfection, bleaching and the like, and can be widely applied to different fields of medicine, daily chemical industry, agriculture, food and the like. The existing production method has better reaction conditionsThe method has the advantages of high yield, low yield and complex components, most of carbamide peroxide preparations need to be heated, which causes the problems of serious energy consumption and the like, and simultaneously, the stabilizer is added, and other elements except C, H, O, N are introduced, so that the addition of the proper stabilizer and the reduction of the energy consumption are one of the problems to be solved next.
Disclosure of Invention
The invention aims to provide a novel carbamide peroxide preparation for preparing a medicament for repairing pulmonary vascular injury and a preparation method thereof aiming at the defects in the prior art.
In order to realize the purpose, the invention adopts the technical scheme that:
the first aspect of the invention provides a preparation method of a novel carbamide peroxide preparation for preparing a medicine for repairing pulmonary vascular injury, which comprises the following steps:
s1, mixing the following substances in a ratio of (1-2): 1, putting the hydrogen peroxide and the urea into a reactor, stirring at room temperature to dissolve the hydrogen peroxide and the urea, then adding 0.05-0.2 g of sorbic acid, and stirring at room temperature for 30-200 min to obtain a reaction solution;
and S2, after the reaction is finished, cooling and crystallizing the reaction solution at the temperature of 0-5 ℃ for 12-36 h, taking out and filtering, and freeze-drying the filter cake in a freeze-drying machine for 5-20 h to obtain the novel carbamide peroxide preparation.
Preferably, the lyophilization time is 20h.
Preferably, the crystallization time is 24h.
Preferably, the crystallization temperature is 5 ℃.
Preferably, the stirring time is 30min.
Preferably, the sorbic acid is added in an amount of 0.1g.
Preferably, the mass ratio between the hydrogen peroxide and urea is 1.6.
Preferably, the mass fraction of hydrogen peroxide is 30%.
The second aspect of the invention provides a novel carbamide peroxide preparation obtained by the preparation method for repairing pulmonary vascular injury.
By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:
the preparation method of the novel carbamide peroxide preparation has the advantages of mild reaction conditions, high yield, simple components, no need of heating and low energy consumption.
The novel carbamide peroxide preparation can obviously promote the proliferation activity of HPAECs and inhibit the apoptosis activity of HPAECs, has more obvious effect particularly under the condition of hypoxia, can be used for promoting angiogenesis and enhancing the cell proliferation capability, and can be applied to preparing medicaments for repairing pulmonary vascular injury.
Drawings
FIG. 1 is a graph showing the effect of the novel carbamide peroxide formulation of the present invention on the proliferation of HPAECs;
FIG. 2 is a graph showing the effect of the novel carbamide peroxide formulation of the present invention on apoptosis of HPAECs under normoxic conditions;
FIG. 3 is a graph showing the effect of the novel carbamide peroxide formulation of the present invention on apoptosis of HPAECs under hypoxia;
FIG. 4 is a graph showing the effect of the novel carbamide peroxide formulation of the present invention on migration of HPAECs;
figure 5 is a graph showing the effect of the novel carbamide peroxide formulation of the invention on migration of HPAECs.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.
The invention is further described with reference to the following drawings and specific examples, which are not intended to be limiting.
Example 1
The embodiment provides a novel carbamide peroxide preparation for preparing a medicine for repairing pulmonary vascular injury, which comprises urea, hydrogen peroxide and sorbic acid; the preparation method comprises the following steps:
s1, filling 30% hydrogen peroxide and urea with the mass ratio of 1.6 into a reactor, stirring at room temperature to dissolve the hydrogen peroxide and urea, then adding 0.1g of sorbic acid, and stirring at room temperature for 30min to obtain a reaction solution;
and S2, after the reaction is finished, cooling and crystallizing the reaction solution at the temperature of 5 ℃ for 24 hours, taking out and filtering, and freeze-drying a filter cake in a freeze-drying machine for 20 hours to obtain the novel carbamide peroxide preparation.
In conclusion, the preparation method of the novel carbamide peroxide preparation has the advantages of mild reaction conditions, high yield, simple components, no need of heating and low energy consumption.
The novel carbamide peroxide preparation is prepared into 10 -6 ～10 -12 M solution with different concentrations is ready for use.
Detection example 1
The activity of promoting the proliferation of endothelial cells (HPAECs) in vitro was tested: the effect of the novel carbamide peroxide formulation of example 1 on the proliferation activity of HPAECs was examined.
(1) Taking frozen HPAECs, thawing and recovering in a water bath at 37 ℃, adding 1mL of ECM culture medium, transferring to a 15mL centrifuge tube, centrifuging in a low-speed centrifuge at 1000rpm for 5min, sucking supernatant, and then adding 1mL of ECM culture medium for resuspension;
(2) Transferring the resuspended cells to a new culture flask, adding appropriate amount of ECM culture medium, and placing in CO 2 Incubator (5% 2 Concentration, 37 ℃) culture;
(3) When the cells grow to 80%, digesting and blowing out the cells, counting, and adjusting the number of the cells to be about 1 × 10 5 one/mL, added to a 96-well plate at 100. Mu.L per well;
(4) After the cells adhere to the wall, 10 mu L of novel carbamide peroxide preparations with different concentrations are added to ensure that the cells are concentrated finallyDegree of 10 respectively -6 M、10 -7 M、10 -8 M、10 -9 M、10 -10 M、10 -11 M and 10 -12 M; set 6 secondary wells to reduce errors and use water as a negative control;
(5) Plating in CO 2 Incubator (5% CO) 2 Concentration) or low oxygen incubator (5%O) 2 Concentration) for 24h; respectively adding 10 mu L of CCK-8 solution;
(6) After another incubation for 4h, the absorbance at 450nm was measured using a microplate reader.
The results of the experiment are shown in FIG. 1, and the administration 10 is carried out under the condition of normal oxygen -9 At M, HPAECs increased to 126.68% (p) of control after 24h<0.05). Under hypoxic conditions, administration of 10 -9 At M, HPAECs increased to 134.39% (p = 0.001) of the control after 24h; that is, the novel carbamide peroxide preparation of example 1 has the effect of promoting the proliferation of HPAECs.
Detection example 2
Assaying the Activity to inhibit endothelial cell (HPAECs) apoptosis in vitro: the novel carbamide peroxide formulation of example 1 was tested for its effect on apoptotic activity of HPAECs.
(1) Thawing frozen HPAECs in a water bath at 37 ℃ for resuscitation, adding 1mL of ECM culture medium, transferring to a 15mL centrifuge tube, centrifuging in a low-speed centrifuge at 1000rpm for 5min, sucking supernatant, and then adding 1mL of ECM culture medium for resuspension;
(2) Transferring the resuspended cells to a new culture flask, adding appropriate amount of ECM culture medium, and placing in CO 2 Incubator (5% CO) 2 Concentration, 37 ℃) culture;
(3) When the cells grow to 80%, digesting and blowing out the cells, counting, and adjusting the number of the cells to be about 1 × 10 5 Adding the mixture into a 24-well plate at a concentration of 200 mu L per well;
(4) After the cells are attached to the wall, 10 mu L of novel carbamide peroxide preparations with different concentrations are added into the normoxic group, so that the final concentration is 10 -8 M、10 -9 M and 10 -10 M; 2 secondary wells were set to reduce errors and water was used as a negative control; the hypoxia group is added with 10 μ L of novel peroxygen with different concentrationsThe carbamide preparation is treated to make the final concentration be 10 respectively -8 M、10 -9 M and 10 -10 M; 2 secondary wells were set to reduce errors and water was used as a negative control;
(5) Plating the plates in CO 2 Incubator (5% CO) 2 Concentration) or low oxygen incubator (5%O) 2 Concentration) for 24h;
(6) The culture solution in the culture plate is sucked off, washed for 2 times by PBS, digested, and cells are resuspended in a 1.5mL centrifuge tube, the suspension is centrifuged for 5min at 1000rpm, and the supernatant is discarded; washing with PBS, and repeating the steps twice;
(7) Adding the prepared 1X Annexin V Binding Solution to prepare the final concentration of 1X 10 6 Cell suspension per mL; taking 100 mu L of the suspension, and adding the suspension into a new EP tube;
(8) Adding 5 mu L of Annexin V and a FITC conjugate into the cell suspension, and then adding 5 mu L of PI Solution; culturing at room temperature in dark for 15min; finally, 400 mu L of Annexin V Binding Solution is added;
(9) The apoptosis rate of HPAECs was measured using flow cytometry.
As shown in fig. 2 and 3, a large amount of cell production was observed under both normoxic and hypoxic conditions, and the increased rate of apoptosis was probably due to cell death caused by insufficient oxygen supply; namely, the novel carbamide peroxide formulation of example 1 had the effect of inhibiting apoptosis in HPAECs.
Detection example 3
Testing the capacity of promoting the migration of endothelial cells (HPAECs) in vitro: the effect of the novel carbamide peroxide formulation of example 1 on the migration ability of HPAECs was examined.
(1) Thawing frozen HPAECs in a water bath at 37 ℃ for resuscitation, adding 1mL of ECM culture medium, transferring to a 15mL centrifuge tube, centrifuging in a low-speed centrifuge at 1000rpm for 5min, sucking supernatant, and then adding 1mL of ECM culture medium for resuspension;
(2) Transferring the resuspended cells to a new culture flask, adding appropriate amount of ECM culture medium, and placing in CO 2 Incubator (5% CO) 2 Concentration ofCulturing at 37 ℃;
(3) When the cells grow to 80%, digesting and blowing out the cells, counting, and adjusting the number of the cells to be about 1 × 10 5 one/mL, added to a Transwell in a 24-well plate at 100. Mu.L per well; 3 secondary wells were set to reduce errors and water was used as a negative control; adding 10 μ L of novel carbamide peroxide preparation with different concentrations into the normoxic group to make the final concentration be 10 -8 M、10 -9 M and 10 -10 M; 2 secondary wells were set to reduce errors and water was used as a negative control; the hypoxic group was supplemented with 10. Mu.L of novel carbamide peroxide formulations at different concentrations to a final concentration of 10 -8 M、10 -9 M and 10 -10 M; 2 secondary wells were set to reduce errors and water was used as a negative control;
(4) Pre-culturing for 24h, taking out each Transwell hole of the 24-hole plate, slightly wiping off intimal cells in the small holes by using a cotton swab, slightly sliding down a mulching film of the Transwell holes along the periphery by using a blade, reversely buckling the film on a glass slide, carrying out DAPI staining, and then taking a picture by inverting a microscope; the amount of migration capacity of the cells after administration can be determined by comparing the ratio of the number to the total number of DAPI on the membrane.
Experimental results As shown in FIGS. 4 and 5, the novel carbamide peroxide formulation enhanced the migration of HPAECs under normoxic conditions and was administered 10 -8 M, the transfer capacity is improved by 28.23 percent (p) compared with that of a control group<0.05). While the novel carbamide peroxide preparation can obviously enhance the migration capability of HPAECs under the low oxygen condition, the administration 10 -9 M, the transfer capacity of the composition is improved by 32.37 percent (p) compared with that of a control group<0.05 ); that is, the novel carbamide peroxide formulation of example 1 had the effect of enhancing migration ability of HPAECs.
In conclusion, the novel carbamide peroxide preparation can remarkably promote the proliferation activity of HPAECs, inhibit the apoptosis activity of HPAECs and improve the migration capacity of HPAECs, has more remarkable effect particularly under the condition of hypoxia, can be used for promoting angiogenesis and enhancing the cell proliferation capacity, and can be applied to preparing medicaments for repairing lung vascular injury.
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made without departing from the spirit and scope of the invention.
Claims (8)
1. The application of a carbamide peroxide preparation in preparing a medicine for repairing pulmonary vascular injury is characterized in that the preparation steps of the carbamide peroxide preparation comprise:
s1, mixing the following substances in a ratio of (1-2): 1, putting the hydrogen peroxide and the urea into a reactor, stirring at room temperature to dissolve the hydrogen peroxide and the urea, then adding 0.05-0.2 g of sorbic acid, and stirring at room temperature for 30-200 min to obtain a reaction solution;
and S2, after the reaction is finished, cooling and crystallizing the reaction solution at the temperature of 0-5 ℃ for 12-36 h, taking out and filtering, and freeze-drying the filter cake in a freeze-drying machine for 5-20 h to obtain the novel carbamide peroxide preparation.
2. Use according to claim 1, wherein the lyophilization time is 20h.
3. Use according to claim 1, characterized in that the crystallization time is 24h.
4. Use according to claim 1, wherein the crystallization temperature is 5 ℃.
5. Use according to claim 1, characterized in that the stirring time is 30min.
6. Use according to claim 1, characterized in that the sorbic acid is added in an amount of 0.1g.
7. Use according to claim 1, characterized in that the mass ratio between hydrogen peroxide and urea is 1.6.
8. Use according to claim 1, characterized in that the hydrogen peroxide is present in a mass fraction of 30%.
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| GB191120242A (en) * | 1911-03-31 | 1911-11-30 | Chem Fab Vormals Dr Heinrich B | Manufacture of a Stable Preparation from Hydrogen Peroxide and Urea. |
| CN101235008A (en) * | 2008-02-29 | 2008-08-06 | 利尔化学股份有限公司 | Method for preparing urea peroxide |
| CN110563613A (en) * | 2019-09-09 | 2019-12-13 | 上海理工大学 | synthesis method and application of urea peroxide |
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