Production test method of piceatannol-containing burn and scald dressing taking vaseline as carrier

Abstract

The invention belongs to the technical field of biological medicines, and discloses a piceatannol-containing burn and scald dressing production test method using vaseline as a carrier, and the method comprises the following specific steps: S1, dressing preparation raw material preparation: selecting pharmaceutical-grade high-quality vaseline as a basic carrier; firstly, pharmaceutical-grade high-quality vaseline is selected as a basic carrier to ensure the purity, stability and safety to skin of the dressing, secondly, the pharmaceutical dressing containing piceatannol is scientifically and reasonably prepared and is subjected to a contrast experiment with resveratrol, and the pharmaceutical dressing containing piceatannol is obtained. The potential of piceatannol in promoting cell proliferation, migration and scald wound healing in an inflammatory state is systematically evaluated, it is proved that piceatannol has a better drug effect at low concentration, and a new thought and a potential treatment means are provided for treatment of burns and scalds. Meanwhile, the curative effect of piceatannol is comprehensively verified through a cell viability test, a cell scratch experiment and an animal experiment.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a production and testing method for a burn and scald dressing containing piceatannol and taking vaseline as a carrier. Background Art

[0002] As the organ with the largest distribution area, the skin has the functions of resisting external pathogens, maintaining body temperature, protecting the body and stabilizing the internal environment. Burns and scalds are common health problems. After a burn, the skin loses its main functions, such as protecting itself from the external environment and pathogens, evaporation and heat dissipation. Burns and scalds not only affect the integrity of the body's tissue structure, but also form scars in the later healing process. The appearance and adhesion of scars also affect the patient's mental health and quality of life. Therefore, the treatment of burn wounds faces severe challenges, and the research and development of specific drugs for the treatment of burns and scalds is imminent.

[0003] In recent years, natural stilbene compounds have attracted great attention in the field of biomedicine. Piceatannol is a resveratrol metabolite, mainly found in grapes. Piceatannol has very strong antioxidant activity, and studies have shown that its antioxidant activity is better than its analogue resveratrol. Studies using grape seed extracts rich in resveratrol and piceatannol to test human immortalized keratinocytes in vitro have shown that the extract has wound healing activity. In addition to the lack of phytochemical research on piceatannol, there are also few studies on the application of this compound in the skin. Vaseline, as a commonly used moisturizer, has functional benefits to the skin, such as making the skin smooth and soft, increasing the moisture of the skin, and improving the optical properties of the skin; however, vaseline can also be used as a carrier to deliver specific ingredients to the skin. Therefore, piceatannol and vaseline can be used as key entry points to further explore and verify their value as burn and scald drugs around burn and scald coatings, and then develop targeted and systematic treatment plans to solve burn and scald diseases. Summary of the invention

[0004] The purpose of the present invention is to provide a production and testing method for burn and scald dressings containing piceatannol using vaseline as a carrier, so as to solve the problems raised in the above background technology.

[0005] In order to achieve the above object, the present invention provides the following technical solution: a method for producing and testing a burn and scald dressing containing piceatannol using vaseline as a carrier, the specific steps are as follows: S1: Dressing preparation Raw material preparation: Choose high-quality pharmaceutical-grade vaseline as the base carrier to ensure its purity, stability and safety to the skin; prepare piceatannol (PIC) powder as the active ingredient; prepare experimental equipment, clean beakers, pipettes, and sub-bottles for mixing and storing dressings; Dressing preparation: Heat and maintain a water bath at 60°C to ensure that vaseline can be melted evenly and safely. Put vaseline in a clean beaker and stir it in a water bath to melt it into liquid. Use a pipette to take liquid vaseline and put it in a sub-bottle. Add piceatannol to prepare a dressing with a concentration of 30 μM. Continue to place the sub-bottle in a water bath and stir to ensure that piceatannol powder can be completely dissolved in the vaseline to form a uniform drug dressing. Preparation of positive control group dressing: Use a pipette to take the same volume of liquid vaseline and place it in a sub-bottle, add resveratrol to prepare a dressing with a concentration of 30 μM, and continue to place the sub-bottle in a water bath for stirring to ensure that the resveratrol powder can be completely dissolved in the vaseline to form a uniform positive control group drug dressing; Storage of dressings: Store the two drugs in bottles in a refrigerator at 2-8°C to ensure the stability of the dressing and extend its shelf life; S2: Experimental cell plating preparation Cell culture: Human immortalized keratinocytes (Hacat) were selected for culture; Cell passaging: Closely observe the cell growth in the cell culture dish under a microscope. When the cell confluence density reaches 80% to 90%, cell passaging can be performed. Add an appropriate amount of trypsin to the culture dish to digest the connection between cells. Observe continuously during the digestion process. When the cells begin to shrink and become round, it means that the digestion is appropriate. At this time, culture medium containing serum should be added immediately to terminate the digestion. Use a pipette to gently blow the cell suspension to evenly disperse it. Then, divide the cell suspension into a new culture dish at a ratio of 1:3 and continue to culture. Experimental cell plating: Select human immortalized keratinocytes in the logarithmic growth phase for digestion to obtain a single cell suspension, count the cell suspension using a cell counter, and adjust the cell concentration. Add the adjusted cell suspension to the corresponding well plate according to the specifications of different well plates, slowly place the well plate with cells in an incubator at 37°C and 5% CO2 for culture, and after the cells are completely attached to the wall, perform subsequent processing according to the specific requirements of the experiment; S3: Cell viability test The drug effects of piceatannol and resveratrol were detected using a cell activity assay (CCK-8), and the 96-well plate prepared in step 2 was selected; Negative control group: normal cells without drug addition, used as baseline reference; Model group: 10 μg / ml lipopolysaccharide (LPS) was added to induce cell inflammation model; Experimental group 1: 10, 30, and 50 μM piceatannol were added to the cells in addition to 10 μg / ml lipopolysaccharide. Experimental group 2: In addition to the addition of 10 μg / ml lipopolysaccharide, 10, 30, and 50 μM resveratrol were added as a comparison; Experimental process: CCK-8 detection kit was used to detect cell viability. Hacat cells were inoculated in a 96-well plate at a density of 1×10^5 cells / ml. 100 μL of cell suspension was added to each well, and 5 replicates were set to ensure the reliability of the results. At the same time, a zeroing well was set and 100 μL of cell-free complete medium was added. 24 hours after cell inoculation, corresponding drugs and lipopolysaccharide were added according to the experimental groups. The culture dish was placed in a 5% CO2 incubator and incubated at 37°C for 24 hours. After the incubation, 10 μL of CCK-8 solution was added to each well, and the incubation continued in the incubator for 1 hour, and then the absorbance value of each well was detected using an enzyme marker. Experimental results: Through the human immortalized keratinocyte activity assay, piceatannol at a concentration of 10 μM has a promoting effect on the proliferation of human immortalized keratinocytes induced by lipopolysaccharide, and its promoting effect is better than that of resveratrol at the same concentration; this experiment can further illustrate that at low concentrations, piceatannol at an appropriate concentration has the potential to promote the proliferation of human immortalized keratinocytes under inflammatory conditions, and its effect is better than resveratrol, indicating that piceatannol has a better drug effect at low concentrations; S4: Cell scratch assay: The cell scratch assay was used to observe and evaluate the effect of piceatannol on the migration ability of human immortalized keratinocytes stimulated by lipopolysaccharide, further verifying its potential to promote wound healing; Negative control group: baseline group without drug treatment, used to evaluate normal cell migration ability; Model group: 10 μg / ml lipopolysaccharide was added to establish a cellular inflammation model to simulate the inflammatory environment of wounds in vivo; Positive control group: 10 μg / ml lipopolysaccharide was added, and 10 and 30 μM resveratrol was added respectively; Piceatannol experimental group: 10 μg / ml LPS was added with 10 and 30 μM piceatannol respectively; Experimental process: Take the immortalized human keratinocytes in the logarithmic growth phase in step 2, collect 6 groups of cells, and seed them in a 6-well cell culture plate according to the number of cells that can fill the 6-well cell culture plate on the second day. Prepare to scratch after the cells fill the culture plate. Use sterile tools to make uniform scratches in the cell culture plate to simulate wound formation. Then, replace it with serum-free culture medium, and add different concentrations of drugs for treatment. At 0 hours and 24 hours after the scratch, use a microscope to observe the migration of cells and perform 10x objective lens imaging; Experimental results: The cell scratch test found that compared with the model group stimulated by lipopolysaccharide, the cell migration distance in the positive control group and the piceatannol experimental group increased, further indicating that piceatannol can effectively offset the inhibitory effect of lipopolysaccharide on the proliferation of human immortalized cells; S5: Animal Experimentation: Select 6-8 week old C57 mice, which can enter formal experiments after one week of adaptive training; C57 mice were divided into four groups, with 6 mice in each group. The experimental groups were as follows: Negative control group: only scald modeling was performed without any drug treatment, serving as a baseline control; Vehicle control group: After burn modeling, non-drug vaseline was applied daily to evaluate the effect of vaseline itself on wound healing; Resveratrol positive control group: After burn modeling, apply the positive control group dressing in step 1 every day to evaluate the promoting effect of resveratrol on wound healing; Piceatannol experimental group: After burn modeling, piceatannol dressings in step 1 were applied daily to evaluate the promoting effect of piceatannol on wound healing; Experimental process: After the adaptive training was completed, the back of the mice was depilated. The next day, the temperature of the scalding instrument was set to 100 °C, the scalding time was set to 5s, and after the instrument was preheated, a uniformly scalded area was scalded on the back of the mice. The size of the scalding area on the back of each mouse was recorded with a camera. Different groups of mice were treated with different drugs every day after the injury, and the wounds were photographed and recorded to evaluate the wound healing. The weight of the mice in different groups was weighed and recorded on the 0th, 7th and 14th days to monitor the effect of the drugs on the growth of the mice. Experimental results: The recorded image results were processed uniformly, and the scratch area was measured using Image J software. Statistical analysis showed that the changes in the area of ​​scald wounds at different time points showed that the wound area of ​​the piceatannol experimental group was reduced faster and the healing effect was better than that of the resveratrol positive control group. At the same time, there were significant differences between these two groups and the negative control group and the vehicle control group. In the comparison of healing rates at different time points, the piceatannol experimental group also showed a better healing effect than the resveratrol positive control group. This result further proves the advantage of piceatannol in promoting the healing of scald wounds. After treatment with different drugs, there was no significant difference in the weight changes of mice in each group, indicating that the drugs had no obvious effect on the growth of mice.

[0006] Preferably, the addition of lipopolysaccharide in step S3 can stimulate cells. Inflammation is an important factor affecting wound healing. In in vitro cell experiments, common toxoid lipopolysaccharide can be added to stimulate cells, so that they synthesize and release a variety of cytokines and inflammatory mediators through cell signal transduction to simulate the inflammatory response. Then, through drug treatment, the activity of cells after the action of the drug can be understood from the absorbance value after adding CCK-8 detected by an enzyme marker.

[0007] Preferably, after imaging in step S4, the scratch area is measured by taking 4 field sizes and applying Image J software to evaluate the migration distance and speed of the cells.

[0008] Preferably, when recording with a camera in step S5, it is necessary to ensure that the camera shooting height is consistent.

[0009] Preferably, the photographic record in step S5 requires photographing and recording the wound on days 0, 2, 4, 6, 8, 10, 12, and 14 to evaluate the wound healing condition.

[0010] Preferably, the reference concentration of the cell suspension for different well plate specifications in the experimental cell plating in step S2 is: 0.1 ml of cell suspension with a cell concentration of 1×104 cells / ml is added to each well of a 96-well plate; 1 ml of cell suspension with a cell concentration of 6×105 cells / ml is added to each well of a 12-well plate; and 2 ml of cell suspension with a cell concentration of 1×106 cells / ml is added to each well of a 6-well plate.

[0011] Preferably, the hair removal treatment in step S5 is performed by using an electric shaver and a hair removal cream in combination, so that the back skin of the mouse is fully exposed.

[0012] Preferably, the cell culture in step S2 needs to use DMEM culture medium as a base, and add 10% serum thereto, and preheat the prepared culture medium in an incubator at 37° C. and 5% CO 2 .

[0013] Preferably, the microplate reader in step S3 needs to detect the absorbance value of each well at a wavelength of 450 nm. Before using the microplate reader, the instrument should be calibrated to ensure the accuracy of the measurement results.

[0014] The beneficial effects of the present invention are as follows: First, by selecting high-quality pharmaceutical-grade vaseline as the basic carrier, the purity, stability and safety of the dressing to the skin were ensured. Secondly, by scientifically and rationally preparing medicated dressings containing piceatannol and conducting comparative experiments with resveratrol, the potential of piceatannol in promoting cell proliferation, migration and healing of burn wounds under inflammatory conditions was systematically evaluated, proving that piceatannol has better drug effects at low concentrations, providing new ideas and potential treatments for burns and scalds. At the same time, through cell activity tests, cell scratch experiments and animal experiments, the efficacy of piceatannol was fully verified, enhancing its scientificity and credibility, and it had no obvious effect on the growth of mice, indicating that it has good safety and practicality, providing new ideas and potential treatments for burns and scalds. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a comparison diagram of the experiment in S3 of the present invention; Figure 2 This is a comparison diagram of the experiment in S4 of the present invention; Figure 3 Schematic diagram of cell migration rate under different treatment conditions in S4 of the present invention; Figure 4 This is an example diagram of the back wound of the mouse scald model of the present invention; Figure 5 This is an example diagram of the back wound area of ​​scalded mice in different groups according to the present invention; Figure 6 This is a schematic diagram of the size of the wound edge area on the back of scalded mice in different groups according to the present invention; Figure 7 The histogram of the change of the back wound area of ​​scalded mice treated with different groups over time; Figure 8 This is an example diagram of the changes in the wound margin area on the back of mice in different drug groups of the present invention; Fig. 9 This is a schematic diagram of the changes in the wound margin area on the back of mice in different drug groups of the present invention; Fig.10 This is a histogram of burn healing rates of different drug groups of the present invention; Fig.11 It is a bar graph of weight change of mice of the present invention. DETAILED DESCRIPTION

[0016] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0017] like Figures 1 to 11 As shown, the embodiment of the present invention provides a method for producing and testing a burn and scald dressing containing piceatannol using vaseline as a carrier, and the specific steps are as follows: S1: Dressing preparation Raw material preparation: Choose high-quality pharmaceutical-grade vaseline as the base carrier to ensure its purity, stability and safety to the skin; prepare piceatannol (PIC) powder as the active ingredient; prepare experimental equipment, clean beakers, pipettes, and sub-bottles for mixing and storing dressings; Dressing preparation: Heat and maintain a water bath at 60°C to ensure that vaseline can be melted evenly and safely. Put vaseline in a clean beaker and stir it in a water bath to melt it into liquid. Use a pipette to take liquid vaseline and put it in a sub-bottle. Add piceatannol to prepare a dressing with a concentration of 30 μM. Continue to place the sub-bottle in a water bath and stir to ensure that piceatannol powder can be completely dissolved in the vaseline to form a uniform drug dressing. Preparation of positive control group dressing: Use a pipette to take the same volume of liquid vaseline and place it in a sub-bottle, add resveratrol to prepare a dressing with a concentration of 30 μM, and continue to place the sub-bottle in a water bath for stirring to ensure that the resveratrol powder can be completely dissolved in the vaseline to form a uniform positive control group drug dressing; Storage of dressings: Store the two drugs in bottles in a refrigerator at 2-8°C to ensure the stability of the dressing and extend its shelf life; S2: Experimental cell plating preparation Cell culture: Human immortalized keratinocytes (Hacat) were selected for culture; Cell passaging: Closely observe the cell growth in the cell culture dish under a microscope. When the cell confluence density reaches 80% to 90%, cell passaging can be performed. Add an appropriate amount of trypsin to the culture dish to digest the connection between cells. Observe continuously during the digestion process. When the cells begin to shrink and become round, it means that the digestion is appropriate. At this time, culture medium containing serum should be added immediately to terminate the digestion. Use a pipette to gently blow the cell suspension to evenly disperse it. Then, divide the cell suspension into a new culture dish at a ratio of 1:3 and continue to culture. Experimental cell plating: Select human immortalized keratinocytes in the logarithmic growth phase for digestion to obtain a single cell suspension, count the cell suspension using a cell counter, and adjust the cell concentration. Add the adjusted cell suspension to the corresponding well plate according to the specifications of different well plates, slowly place the well plate with cells in an incubator at 37°C and 5% CO2 for culture, and after the cells are completely attached to the wall, perform subsequent processing according to the specific requirements of the experiment; S3: Cell viability test The drug effects of piceatannol and resveratrol were detected using a cell activity assay (CCK-8), and the 96-well plate prepared in step 2 was selected; Negative control group: normal cells without drug addition, used as baseline reference; Model group: 10 μg / ml lipopolysaccharide (LPS) was added to induce cell inflammation model; Experimental group 1: 10, 30, and 50 μM piceatannol were added to the cells in addition to 10 μg / ml lipopolysaccharide. Experimental group 2: In addition to the addition of 10 μg / ml lipopolysaccharide, 10, 30, and 50 μM resveratrol were added as a comparison; Experimental process: CCK-8 detection kit was used to detect cell viability. Hacat cells were inoculated in a 96-well plate at a density of 1×10^5 cells / ml. 100 μL of cell suspension was added to each well, and 5 replicates were set to ensure the reliability of the results. At the same time, a zeroing well was set and 100 μL of cell-free complete medium was added. 24 hours after cell inoculation, corresponding drugs and lipopolysaccharide were added according to the experimental groups. The culture dish was placed in a 5% CO2 incubator and incubated at 37°C for 24 hours. After the incubation, 10 μL of CCK-8 solution was added to each well, and the incubation continued in the incubator for 1 hour, and then the absorbance value of each well was detected using an enzyme marker. Experimental results: Through the human immortalized keratinocyte activity assay, piceatannol at a concentration of 10 μM has a promoting effect on the proliferation of human immortalized keratinocytes induced by lipopolysaccharide, and its promoting effect is better than that of resveratrol at the same concentration; this experiment can further illustrate that at low concentrations, piceatannol at an appropriate concentration has the potential to promote the proliferation of human immortalized keratinocytes under inflammatory conditions, and its effect is better than resveratrol, indicating that piceatannol has a better drug effect at low concentrations; S4: Cell scratch assay: The cell scratch assay was used to observe and evaluate the effect of piceatannol on the migration ability of human immortalized keratinocytes stimulated by lipopolysaccharide, further verifying its potential to promote wound healing; Negative control group: baseline group without drug treatment, used to evaluate normal cell migration ability; Model group: 10 μg / ml lipopolysaccharide was added to establish a cellular inflammation model to simulate the inflammatory environment of wounds in vivo; Positive control group: 10 μg / ml lipopolysaccharide was added, and 10 and 30 μM resveratrol was added respectively; Piceatannol experimental group: 10 μg / ml LPS was added with 10 and 30 μM piceatannol respectively; Experimental process: Take the immortalized human keratinocytes in the logarithmic growth phase in step 2, collect 6 groups of cells, and seed them in a 6-well cell culture plate according to the number of cells that can fill the 6-well cell culture plate on the second day. Prepare to scratch after the cells fill the culture plate. Use sterile tools to make uniform scratches in the cell culture plate to simulate wound formation. Then, replace it with serum-free culture medium, and add different concentrations of drugs for treatment. At 0 hours and 24 hours after the scratch, use a microscope to observe the migration of cells and perform 10x objective lens imaging; Experimental results: The cell scratch test found that compared with the model group stimulated by lipopolysaccharide, the cell migration distance in the positive control group and the piceatannol experimental group increased, further indicating that piceatannol can effectively offset the inhibitory effect of lipopolysaccharide on the proliferation of human immortalized cells; S5: Animal Experimentation: Select 6-8 week old C57 mice, which can enter formal experiments after one week of adaptive training; C57 mice were divided into four groups, with 6 mice in each group. The experimental groups were as follows: Negative control group: only scald modeling was performed without any drug treatment, serving as a baseline control; Vehicle control group: After burn modeling, non-drug vaseline was applied daily to evaluate the effect of vaseline itself on wound healing; Resveratrol positive control group: After burn modeling, apply the positive control group dressing in step 1 every day to evaluate the promoting effect of resveratrol on wound healing; Piceatannol experimental group: After burn modeling, piceatannol dressings in step 1 were applied daily to evaluate the promoting effect of piceatannol on wound healing; Experimental process: After the adaptive training was completed, the back of the mice was depilated. The next day, the temperature of the scalding instrument was set to 100 °C, the scalding time was set to 5s, and after the instrument was preheated, a uniformly scalded area was scalded on the back of the mice. The size of the scalding area on the back of each mouse was recorded with a camera. Different groups of mice were treated with different drugs every day after the injury, and the wounds were photographed and recorded to evaluate the wound healing. The weight of the mice in different groups was weighed and recorded on the 0th, 7th and 14th days to monitor the effect of the drugs on the growth of the mice. Experimental results: The recorded image results were processed uniformly, and the scratch area was measured using Image J software. Statistical analysis showed that the changes in the area of ​​scald wounds at different time points showed that the wound area of ​​the piceatannol experimental group was reduced faster and the healing effect was better than that of the resveratrol positive control group. At the same time, there were significant differences between these two groups and the negative control group and the vehicle control group. In the comparison of healing rates at different time points, the piceatannol experimental group also showed a better healing effect than the resveratrol positive control group. This result further proves the advantage of piceatannol in promoting the healing of scald wounds. After treatment with different drugs, there was no significant difference in the weight changes of mice in each group, indicating that the drugs had no obvious effect on the growth of mice.

[0018] Burn dressings were prepared by using vaseline as a carrier and integrating piceatannol powder. Cell activity tests were performed, and the absorbance value was accurately measured using an enzyme marker to evaluate cell proliferation and survival. Cell scratch experiments and animal experiments were also performed to comprehensively and systematically evaluate the effects of piceatannol in promoting cell proliferation, cell migration and burn wound healing under inflammatory conditions. At the same time, piceatannol was compared with resveratrol. The results showed that piceatannol had better drug effects at low concentrations and had no significant effect on the growth of mice, providing new ideas and potential therapeutic methods for the treatment of burns and scalds.

[0019] Among them, the addition of lipopolysaccharide in step S3 can stimulate cells. Inflammation is an important factor affecting wound healing. In in vitro cell experiments, common toxin-like lipopolysaccharides can be added to stimulate cells, so that they synthesize and release a variety of cytokines and inflammatory mediators through cell signal transduction to simulate the inflammatory response. Then, through drug treatment, the absorbance value after adding CCK-8 can be detected by an enzyme marker to understand the activity of the cells after the action of the drug.

[0020] As the instruction manual Figure 1 As shown, piceatannol has a stronger effect on promoting the proliferation of human immortalized keratinocytes induced by lipopolysaccharide than resveratrol. LPS 10ug / ml was added to cause damage, and then different concentrations of drugs containing lipopolysaccharide were added for treatment. In the figure, * indicates a significant difference compared with the Control, *p <0.05, **p <0.01, ***p <0.001), and # indicates a significant difference compared with the lipopolysaccharide group, #p <0.05, ##p <0.01, ###p <0.001, ####p <0.0001.

[0021] After imaging in step S4, the scratch area is measured by using Image J software at 4 field sizes to evaluate the migration distance and speed of the cells.

[0022] As the instruction manual Figure 2 And the instruction manual Figure 3 As shown, piceatannol exhibited significant activity in promoting cell migration at a concentration of 10 μM, which was significantly better than 10 μM and 30 μM resveratrol.

[0023] When recording with a camera in step S5, it is necessary to ensure that the camera shooting height is consistent.

[0024] By controlling the height at which the camera takes pictures, errors in calculating the burn area of ​​mice caused by height errors can be avoided.

[0025] Among them, the photographic record in step S5 needs to take photographs of the wound on the 0th, 2nd, 4th, 6th, 8th, 10th, 12th and 14th days to evaluate the wound healing condition.

[0026] As the instruction manual Figure 7 As shown, the wound area of ​​the piceatannol experimental group was reduced faster than that of the resveratrol positive control group. In the figure, * indicates a significant difference compared with the Control, *p <0.05, **p <0.01, ***p <0.001, ****p <0.0001.

[0027] The reference concentration of the cell suspension for different well plates in the experimental cell plating in step S2 is: 0.1 ml of the system is added to each well of the 96-well plate, and the cell concentration is 1×10 4 1 ml of the system was added to each well of a 12-well plate, and the cell concentration was 6×10 5 1×10 cells / ml cell suspension; 2 ml of the system was added to each well of a 6-well plate, and the cell concentration was 1×10 6 / ml cell suspension.

[0028] By preparing cell suspensions of different well plate specifications, different types of experiments can be adapted to facilitate subsequent experiments.

[0029] In step S5, the hair removal treatment uses an electric shaver and a depilatory cream to fully expose the mouse back skin.

[0030] By completely removing the hair on the back of the mouse, the skin can be completely exposed, avoiding the presence of hair when the mouse back is scalded, thereby preventing the effect of the scald and the recovery of the wound from being affected.

[0031] In the step S2, the cell culture needs to use DMEM culture medium as the base, and 10% serum is added thereto, and the prepared culture medium is placed in an incubator at 37° C. and 5% CO 2 for preheating.

[0032] Serum is added to the culture dish to provide the nutrients and growth factors required for cell growth, and the culture dish is placed in an incubator under appropriate conditions to ensure that the cells grow in an optimal temperature and gas environment.

[0033] In step S3, the microplate reader needs to detect the absorbance value of each well at a wavelength of 450 nm. Before using the microplate reader, the instrument should be calibrated to ensure the accuracy of the measurement results.

[0034] The microplate reader can indirectly reflect the activity status of cells by measuring the absorbance value of each well after adding CCK-8 reagent. The higher the absorbance value, the more living cells there are and the better the cell activity.

[0035] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0036] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A production and testing method for burn and scald dressings containing piceatannol using vaseline as a carrier, characterized in that: The specific steps are as follows: S1: Dressing preparation Raw material preparation: Select high-quality pharmaceutical-grade vaseline as the base carrier to ensure its purity, stability and safety to the skin; prepare piceatannol (PIC) powder as the active ingredient; Prepare laboratory equipment, clean beakers, pipettes, and dispensing bottles for mixing and storing dressings; Dressing preparation: Heat and maintain a water bath at 60°C to ensure that vaseline can be melted evenly and safely. Put vaseline in a clean beaker and stir it in a water bath to melt it into liquid. Use a pipette to take liquid vaseline and put it in a sub-bottle. Add piceatannol to prepare a dressing with a concentration of 30 μM. Continue to place the sub-bottle in a water bath and stir to ensure that piceatannol powder can be completely dissolved in the vaseline to form a uniform drug dressing. Preparation of positive control group dressing: Use a pipette to take the same volume of liquid vaseline and place it in a sub-bottle, add resveratrol to prepare a dressing with a concentration of 30 μM, and continue to place the sub-bottle in a water bath for stirring to ensure that the resveratrol powder can be completely dissolved in the vaseline to form a uniform positive control group drug dressing; Storage of dressings: Store the two drugs in bottles in a refrigerator at 2-8°C to ensure the stability of the dressing and extend its shelf life; S2: Experimental cell plating preparation Cell culture: Human immortalized keratinocytes (Hacat) were selected for culture; Cell passaging: Closely observe the cell growth in the cell culture dish under a microscope. When the cell confluence density reaches 80% to 90%, cell passaging can be performed. Add an appropriate amount of trypsin to the culture dish to digest the connection between cells. Observe continuously during the digestion process. When the cells begin to shrink and become round, it means that the digestion is appropriate. At this time, culture medium containing serum should be added immediately to terminate the digestion. Use a pipette to gently blow the cell suspension to evenly disperse it. Then, divide the cell suspension into a new culture dish at a ratio of 1:3 and continue to culture. Experimental cell plating: Select human immortalized keratinocytes in the logarithmic growth phase for digestion to obtain a single cell suspension, count the cell suspension using a cell counter, and adjust the cell concentration. Add the adjusted cell suspension to the corresponding well plate according to the specifications of different well plates, slowly place the well plate with cells in an incubator at 37°C and 5% CO2 for culture, and after the cells are completely attached to the wall, perform subsequent processing according to the specific requirements of the experiment; S3: Cell viability test The drug effects of piceatannol and resveratrol were detected using a cell activity assay (CCK-8), and the 96-well plate prepared in step 2 was selected; Negative control group: normal cells without drug addition, used as baseline reference; Model group: 10 μg / ml lipopolysaccharide (LPS) was added to induce cell inflammation model; Experimental group 1: 10, 30, and 50 μM piceatannol were added to the cells in addition to 10 μg / ml lipopolysaccharide. Experimental group 2: In addition to the addition of 10 μg / ml lipopolysaccharide, 10, 30, and 50 μM resveratrol were added as a comparison; Experimental process: CCK-8 detection kit was used to detect cell viability. Hacat cells were inoculated in a 96-well plate at a density of 1×10^5 cells / ml. 100 μL of cell suspension was added to each well, and 5 replicates were set to ensure the reliability of the results. At the same time, a zeroing well was set and 100 μL of cell-free complete medium was added. 24 hours after cell inoculation, corresponding drugs and lipopolysaccharide were added according to the experimental groups. The culture dish was placed in a 5% CO2 incubator and incubated at 37°C for 24 hours. After the incubation, 10 μL of CCK-8 solution was added to each well, and the incubation continued in the incubator for 1 hour, and then the absorbance value of each well was detected using an enzyme marker. Experimental results: Through the human immortalized keratinocyte activity assay, piceatannol at a concentration of 10 μM has a promoting effect on the proliferation of human immortalized keratinocytes induced by lipopolysaccharide, and its promoting effect is better than that of resveratrol at the same concentration; this experiment can further illustrate that at low concentrations, piceatannol at an appropriate concentration has the potential to promote the proliferation of human immortalized keratinocytes under inflammatory conditions, and its effect is better than resveratrol, indicating that piceatannol has a better drug effect at low concentrations; S4: Cell scratch assay: The cell scratch assay was used to observe and evaluate the effect of piceatannol on the migration ability of human immortalized keratinocytes stimulated by lipopolysaccharide, further verifying its potential to promote wound healing; Negative control group: baseline group without drug treatment, used to evaluate normal cell migration ability; Model group: 10 μg / ml lipopolysaccharide was added to establish a cellular inflammation model to simulate the inflammatory environment of wounds in vivo; Positive control group: 10 μg / ml lipopolysaccharide was added, and 10 and 30 μM resveratrol was added respectively; Piceatannol experimental group: 10 μg / ml LPS was added with 10 and 30 μM piceatannol respectively; Experimental process: Take the immortalized human keratinocytes in the logarithmic growth phase in step 2, collect 6 groups of cells, and seed them in a 6-well cell culture plate according to the number of cells that can fill the 6-well cell culture plate on the second day. Prepare to scratch after the cells fill the culture plate. Use sterile tools to make uniform scratches in the cell culture plate to simulate wound formation. Then, replace it with serum-free culture medium, and add different concentrations of drugs for treatment. At 0 hours and 24 hours after the scratch, use a microscope to observe the migration of cells and perform 10x objective lens imaging; Experimental results: The cell scratch test found that compared with the model group stimulated by lipopolysaccharide, the cell migration distance in the positive control group and the piceatannol experimental group increased, further indicating that piceatannol can effectively offset the inhibitory effect of lipopolysaccharide on the proliferation of human immortalized cells; S5: Animal Experimentation: Select 6-8 week old C57 mice, which can enter formal experiments after one week of adaptive training; C57 mice were divided into four groups, with 6 mice in each group. The experimental groups were as follows: Negative control group: only scald modeling was performed without any drug treatment, serving as a baseline control; Vehicle control group: After burn modeling, non-drug vaseline was applied daily to evaluate the effect of vaseline itself on wound healing; Resveratrol positive control group: After burn modeling, apply the positive control group dressing in step 1 every day to evaluate the promoting effect of resveratrol on wound healing; Piceatannol experimental group: After burn modeling, piceatannol dressings in step 1 were applied daily to evaluate the promoting effect of piceatannol on wound healing; Experimental process: After the adaptive training was completed, the back of the mice was depilated. The next day, the temperature of the scalding instrument was set to 100 °C, the scalding time was set to 5s, and after the instrument was preheated, a uniformly scalded area was scalded on the back of the mice. The size of the scalding area on the back of each mouse was recorded with a camera. Different groups of mice were treated with different drugs every day after the injury, and the wounds were photographed and recorded to evaluate the wound healing. The weight of the mice in different groups was weighed and recorded on the 0th, 7th and 14th days to monitor the effect of the drugs on the growth of the mice. Experimental results: The recorded image results were processed uniformly, and the scratch area was measured using Image J software. Statistical analysis showed that the changes in the area of ​​scald wounds at different time points showed that the wound area of ​​the piceatannol experimental group was reduced faster and the healing effect was better than that of the resveratrol positive control group. At the same time, there were significant differences between these two groups and the negative control group and the vehicle control group. In the comparison of healing rates at different time points, the piceatannol experimental group also showed a better healing effect than the resveratrol positive control group. This result further proves the advantage of piceatannol in promoting the healing of scald wounds. After treatment with different drugs, there was no significant difference in the weight changes of mice in each group, indicating that the drugs had no obvious effect on the growth of mice.

2. The method for producing and testing a burn and scald dressing containing piceatannol and using vaseline as a carrier according to claim 1, characterized in that: The addition of lipopolysaccharide described in step S3 can stimulate cells. Inflammation is an important factor affecting wound healing. In in vitro cell experiments, common toxin-like lipopolysaccharides can be added to stimulate cells, so that they synthesize and release a variety of cytokines and inflammatory mediators through cell signal transduction to simulate the inflammatory response. Then, through drug treatment, the absorbance value after adding CCK-8 can be detected by an enzyme marker to understand the activity of the cells after the action of the drug.

3. The method for producing and testing a burn and scald dressing containing piceatannol and using vaseline as a carrier according to claim 1, characterized in that: After imaging as described in step S4, the scratch area was measured using Image J software by taking four field sizes to assess the migration distance and speed of the cells.

4. The method for producing and testing a burn and scald dressing containing piceatannol and using vaseline as a carrier according to claim 1, characterized in that: When recording with a camera in step S5, it is necessary to ensure that the camera shooting height is consistent.

5. The method for producing and testing a burn and scald dressing containing piceatannol and using vaseline as a carrier according to claim 1, characterized in that: The photographic record in step S5 needs to take photographs of the wound on days 0, 2, 4, 6, 8, 10, 12, and 14 to evaluate the wound healing condition.

6. The method for producing and testing a burn and scald dressing containing piceatannol and using vaseline as a carrier according to claim 1, characterized in that: The reference concentrations of the cell suspensions for different well plate specifications in the experimental cell plating described in step S2 are: 0.1 ml of a cell suspension with a cell concentration of 1×104 cells / ml is added to each well of a 96-well plate; 1 ml of a cell suspension with a cell concentration of 6×105 cells / ml is added to each well of a 12-well plate; and 2 ml of a cell suspension with a cell concentration of 1×106 cells / ml is added to each well of a 6-well plate.

7. The method for producing and testing a burn and scald dressing containing piceatannol and using vaseline as a carrier according to claim 1, characterized in that: The hair removal treatment in step S5 uses an electric shaver and a depilatory cream to fully expose the mouse back skin.

8. The method for producing and testing a burn and scald dressing containing piceatannol and using vaseline as a carrier according to claim 1, characterized in that: The cell culture in step S2 needs to use DMEM culture medium as a base, and add 10% serum thereto, and preheat the prepared culture medium in an incubator at 37° C. and 5% CO 2 .

9. The method for producing and testing a burn and scald dressing containing piceatannol and using vaseline as a carrier according to claim 1, characterized in that: The microplate reader in step S3 needs to detect the absorbance value of each well at a wavelength of 450 nm. Before using the microplate reader, the instrument should be calibrated to ensure the accuracy of the measurement results.

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