CN110859834B - Composition for accelerating wound healing and preparation method thereof - Google Patents
Composition for accelerating wound healing and preparation method thereof Download PDFInfo
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- CN110859834B CN110859834B CN201911191125.6A CN201911191125A CN110859834B CN 110859834 B CN110859834 B CN 110859834B CN 201911191125 A CN201911191125 A CN 201911191125A CN 110859834 B CN110859834 B CN 110859834B
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- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
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Abstract
The invention relates to a composition for accelerating wound healing and a preparation method thereof, belonging to the technical field of wound healing. The invention provides a composition for accelerating wound healing, which comprises EGCG, sodium acetate, ascorbic acid, gelatin and chitosan. The preparation method of the composition for accelerating wound healing comprises the following steps: preparing gelatin buffer solution and chitosan buffer solution by using sodium acetate buffer solution, and performing membrane filtration; dissolving EGCG and ascorbic acid in chitosan buffer solution, dropwise adding into gelatin buffer solution, stirring, centrifuging, collecting centrifuged solid part, and freeze drying to obtain the composition for accelerating wound healing. Compared with the EGCG aqueous solution, the composition for promoting wound healing can obviously promote the healing of the wound of the diabetic mouse, is greatly improved, and has great potential application value for wound healing.
Description
Technical Field
The invention belongs to the technical field of compositions for wound healing, and relates to a composition for accelerating wound healing and a preparation method thereof.
Background
Wound healing is a complex biological process involving a variety of cellular and tissue components, including fibroblast proliferation, collagen secretion, neovascularization, epidermal cell proliferation, and re-epithelialization of the wound. Compared with non-diabetic patients, the inflammatory reaction cells at the wound part of the diabetic patient infiltrate relatively late, have long infiltration time and abnormal functions, so that the local part of the wound is in a chronic inflammatory reaction state and is difficult to transit to the later period of wound healing. Diabetic foot is one of the serious chronic complications of diabetes. Wounds to the diabetic foot are difficult to heal, which can lead to amputation and even death of the diabetic patient. Thus, the ideal wound healing composition would accelerate wound healing, relieve the diabetic patient from pain and reduce the cost of treatment.
EGCG (epigallocatechin gallate), is the main component of green tea polyphenol, is catechin monomer separated from tea, and has antibacterial, antiviral, antioxidant, arteriosclerosis resisting, thrombosis resisting, vascular proliferation resisting, antiinflammatory and antitumor effects. However, the EGCG is easily influenced by external environment and is unstable under strong acid, strong alkali, illumination and high heat conditions, and the factors limit the application of the EGCG.
At present, although EGCG has been proved to have an effect of accelerating diabetic wound healing, an aqueous solution of EGCG has low efficiency of accelerating diabetic wound healing, and the efficiency thereof is yet to be improved.
Disclosure of Invention
In order to solve the problem that the EGCG has low wound healing promoting efficiency, the invention provides a composition for promoting wound healing, and the components of the composition comprise EGCG, sodium acetate, ascorbic acid, gelatin and chitosan.
The preparation method of the composition for accelerating wound healing comprises the following steps:
firstly, preparing a sodium acetate solution with the concentration of 0.1-0.3 mol/L, and adjusting the pH value of the sodium acetate solution to 5.0-5.4 to obtain a sodium acetate buffer solution.
Secondly, preparing 0.1-0.3% gelatin buffer solution and 0.1-0.3% chitosan buffer solution by using the sodium acetate buffer solution, and removing insoluble impurities in the two solutions by using an organic filter membrane with the diameter of 0.22-0.45 mu m.
Dissolving EGCG and ascorbic acid in a chitosan buffer solution according to the final concentration of 3-4.5 mg/mL of EGCG and 0.02-0.03 mg/mL of ascorbic acid to obtain a mixed solution, dropwise adding the mixed solution into the 0.1-0.3% gelatin buffer solution according to the volume ratio of 1:1 after complete dissolution, uniformly mixing the two solutions, stirring, and completely mixing to obtain a suspension.
Fourthly, centrifuging the suspension for 20 to 60 minutes by a low-temperature high-speed centrifuge at the temperature of 4 +/-2 ℃ under the condition of 13000 +/-2000 r/min, and taking the centrifuged solid part.
Fifthly, freeze-drying the solid part after centrifugation to obtain the composition for accelerating wound healing.
Wherein the freeze drying condition is normal pressure freeze drying or vacuum freeze drying under the condition of-70 to-10 ℃.
Wherein the stirring treatment in the third step is specifically stirring for 20-50 min under the condition that the rotating speed is 300 +/-50 r/min.
Wherein the mixed solution is dropwise added into the 0.1-0.3% gelatin buffer solution according to the ratio of 1: 1.
Wherein the speed of dropwise adding the mixed solution is 0.5-1 mL/min.
Wherein, the EGCG and the ascorbic acid are dissolved in the chitosan buffer solution specifically by adopting ultrasonic-assisted dissolution.
Has the advantages that:
the positively charged EV nanoparticles are prepared through ionic crosslinking, EGCG is packaged in EV NPS, and the composition for promoting wound healing is obtained, wherein the packaging rate of the EGCG is 68.39 +/-2.60%, ascorbic acid is not added, the packaging rate of the EGCG is only less than 30% by adopting the same method, and the effect of promoting wound healing is almost the same as that of pure EGCG or EGCG aqueous solution.
The wound healing promoting composition prepared by the invention has uniform particle size, uniform distribution and no obvious aggregation. The composition contains a very small amount of ascorbic acid, and the addition of the ascorbic acid obviously improves the encapsulation rate of EGCG in the composition for promoting wound healing, and also improves the effect of promoting wound healing. Animal experiments show that the composition for accelerating wound healing prepared by the embodiment can obviously accelerate the healing speed of the wound of the diabetic mouse when the encapsulation rate of the encapsulated EGCG is high, and the lower the encapsulation rate is, the poorer the acceleration effect is. Whereas ascorbic acid alone has no effect on diabetic wound healing.
The mechanism of the invention for accelerating wound healing was analyzed, which may be to promote angiogenesis and reduce inflammatory cell infiltration of wounds in diabetic mice by increasing collagen accumulation. The effect of the composition for promoting wound healing on the wound healing of the diabetic mice is obviously better than that of the unloaded group and the EGCG group. The composition for promoting wound healing can obviously promote the healing of the wound of the diabetic mouse, and has great potential application value of wound healing. The composition has slow release effect, so that the acting time of EGCG is prolonged, and the EGCG solution is easily influenced and changed by external environment.
Description of the drawings:
figure 1 images of wound healing on different days for different groups of mice.
Figure 2 bar graph of wound healing rate of different groups of mice at different times.
Figure 3 representative images of HE staining of various groups of skin wounds on day five and day ten.
Figure 4 representative images of Masson skin trichrome staining of each group on day five and day ten.
FIG. 5 immunohistochemical stain images of the groups on day five and day ten.
Detailed Description
The present invention will be described in detail with reference to the following embodiments, wherein EGCG is purchased from Jiangsu DE and Biotechnology Co., Ltd, and has a purity of more than 98%, and other reagents are analytically pure.
Example 1
This example provides a composition for accelerating wound healing, which comprises sodium acetate, ascorbic acid, gelatin and chitosan as main ingredients, and is prepared by the following steps:
1. anhydrous sodium acetate is dissolved in ultrapure water to prepare a sodium acetate solution of 0.2mol/L, and the pH of the sodium acetate solution is adjusted to 5.4 with hydrochloric acid or sodium hydroxide to obtain a sodium acetate buffer solution.
2. Dissolving gelatin and chitosan in sodium buffer solution respectively to obtain 0.1% gelatin buffer solution and 0.1% chitosan buffer solution, and removing insoluble impurities from the two solutions with 0.45 μm organic filter membrane.
3. Respectively weighing 30mg of EGCG and 0.2mg of ascorbic acid, ultrasonically dissolving the EGCG and the ascorbic acid in 10mL of 0.1% chitosan buffer solution, slowly dripping the EGCG and the ascorbic acid into 10mL of 0.1% gelatin buffer solution after the EGCG and the ascorbic acid are completely dissolved, wherein the dripping speed is 0.5-1 mL/min. After the two are uniformly mixed, the magnetic stirring is continued for 30min at the rotating speed of 300r/min, and the suspension is obtained after the complete mixing. Wherein the ultrasonic power is within the range of 20-40 kHz.
4. And centrifuging the suspension for 30 minutes at 13000 r/min by using a low-temperature high-speed centrifuge at the temperature of 4 +/-2 ℃, and taking a centrifuged solid part.
5. Freeze-drying the centrifuged solid part at-18 deg.C to obtain the composition for accelerating wound healing.
The inventor conducts animal experiments on the composition, and the specific method is as follows.
1) Establishment of diabetes model
30 male ICR mice, 28-30 g in weight, eight weeks old, were provided by Zhejiang Weitongli Hua Limited liability company. The method is characterized by being arranged in an SPF animal house, the environment temperature is 25 +/-2 ℃, the relative humidity is 70-80%, the illumination is carried out for 12 hours, and the adaptive breeding is carried out for one week under the dark condition of 12 hours. Fasting was performed for 12 hours in the evening before the experiment. Body weight and fasting plasma glucose were measured and a single intraperitoneal injection of STZ solution (120 mg/kg) was made. The blank control group was injected with citric acid-sodium citrate buffer by body weight. It was determined that the diabetic model fasted overnight for 12 hours without water deprivation. Mice with blood glucose values above 11.1mmol/L were considered successful in the diabetes-causing model. And randomly grouping and stabilizing the successfully molded mice for two days, and then molding the skin wounds.
2) Establishing skin wound model
Mice were divided into five groups, each: blank Control group (Control), Model group (Model), No-load NPS group (No-load NPS), EGCG group (EGCG), composition group of this example (EV NPS).
Blank control group: the mice used were non-diabetic normal mice, and the wounds were swabbed with 0.9% physiological saline.
Model group: the mice used were those with diabetes, and the wounds were wiped with 0.9% physiological saline.
And (3) no-load group: the mice used were diabetic and the wounds were swabbed with equal amounts of gelatin, chitosan suspension prepared according to the method of this example without the addition of EGCG.
Group EGCG: the mice used were those with diabetes, and the wounds were swabbed with an equivalent amount of 10mg/ml EGCG solution.
Composition set of the present example: the mice used were diabetic mice and the wounds were swabbed with an equal amount of a solution of the same wound healing accelerating composition as the EGCG group prepared in this example.
After all mice were anesthetized by intraperitoneal injection with 10% chloral hydrate (0.004 ml/g), the back hairs were removed with depilatory cream, two circularly symmetric 6mm wounds were made with a punch, and the mice were housed in a single cage. The initial wound area was photographed and recorded as 0D with a digital camera. The administration was once daily for ten days. Photographs were taken at 2D, 4D, 6D, 8D, 10D, the rate of healing was calculated and the healing was recorded. Three were dissected at 5D and 10D each, and the back wound skin was cut, placed in 4% formalin, fixed for more than 24 hours, embedded with paraffin and sectioned.
In fig. 2, data are presented as mean ± SEM (n = 6). (# 0.05) is significantly different from the control group. Significant differences from the model group (P < 0.05). The No-load NPS group was significantly different (P < 0.05). ● was significantly different from the EGCG group (P < 0.05).
The wound was monitored for 10 consecutive days as shown in fig. 1 and 2. The results show that the wound area is significantly reduced for the composition group of this example compared to the other groups. The percentage of wound healing is shown in figure 1. From 2D to 8D, the composition group of this example had significantly higher wound healing rates than the model, empty and EGCG groups (all P < 0.001). From 2D to 8D, the wound healing rates were significantly different in the model group compared to the control group (P <0.05 or P < 0.001). This indicates that diabetes can indeed delay the healing of the wound. We know that EGCG can accelerate the healing of diabetic wounds, as confirmed by our studies. However, the effect of the composition group of this example on wound healing was significantly higher than that of the EGCG group. In short, the wound healing effect of the composition group of this example was very good.
In fig. 3, (a) representative image of HE staining of skin wounds on day 5. (B) Representative images of HE staining of skin wounds on day 10. The magnification was 40 times and 100 times, respectively.
The wounds of the control group (non-diabetic group) showed partial healing at 5D. From a histological point of view, a filling of granulation tissue is observed. In the new tissue formation stage, fibroblasts migrate to the wound and are converted into myofibroblasts, leading to contraction and collagen deposition, forming granulation tissue. In addition, the keratinocytes at the wound margins are highly proliferative, forming a new epidermis over the wound, which is the re-epithelialization of the wound. Compared with the control group, the wound surface of the model group has less granulation tissue, and only infiltrates a small amount of epithelial cells and a large amount of inflammatory cells. In the unloaded NPS group, some new blood vessels and epidermis were formed in the wound, and inflammatory cells and fibroblasts were less in the wound. In the EGCG group, a small number of inflammatory cells were present in the wound and the dermis began to grow towards the center of the wound. The composition group of this example had more abundant neovascularization, thicker granulation tissue, fewer inflammatory cells, and easier wound healing. On day ten, the control group wounds substantially healed. New blood vessels are abundant, fibroblasts are regularly arranged, but inflammatory cell infiltration is still present. The model group still had a large inflammatory cell infiltration and the granulation tissue and epidermis were not fully formed. There were few new vessels compared to the empty group. At the same time, the wound in the unloaded group had healed. However, the number of inflammatory cells is large, the fibroblasts are well-aligned, and the epidermis is already healed. In the EGCG group, the epidermis was thin and some new blood vessels were formed. There is infiltration of inflammatory cells and hair follicles and other skin attachments are partially formed. The epithelial cells of the composition group of this example were completely regenerated, and the epithelial cells were thick. More new blood vessels, hair follicles and other skin appendages are formed. In short, the wound healing effect of the composition group of this example was superior to that of the other groups.
In fig. 4, (a): representative images of Masson skin trichrome staining on day 5. (B) The method comprises the following steps Representative images of trichrome staining of Masson wound skin on day 10. The magnification was 40 times and 100 times, respectively. (C) The method comprises the following steps The average area percentage of collagen deposition in Masson trichrome stained sections was quantified. (# 0.05) is significantly different from the control group. Significant differences from the model group (P < 0.05). It is clearly different from the unloaded group (P < 0.05). ● was significantly different from the EGCG group (P < 0.05).
FIG. 4 shows collagen deposition observed by Masson staining. In the wound healing process, fibroblasts align collagen fibers by depositing and degrading collagen molecules, thereby reconstructing the patched dermal structure. Studies have shown that the healing of diabetic wounds is affected by many physiological factors, such as reduced collagen deposition, reduced number of new granulation tissue, and reduced fibroblast migration and proliferation. On day 5, the collagen deposition was significantly higher in the control, non-loaded NPS and EGCG groups than in the model group (P < 0.001). The collagen deposition amount of the composition group of the present example was much higher than that of the model group, the non-load NPS group and the EGCG group (P <0.001 or P < 0.05). The EGCG group tended to be ordered, but there was no significant difference between the two (P > 0.05). The composition group of this example had good wound healing, collagen fibers arranged in a dark blue color, and the difference in collagen deposition between the unloaded group and the EGCG group was significant (both P < 0.001). In short, the wound healing accelerating compositions prepared in the examples can increase collagen deposition in wounds.
In fig. 5, (a): immunohistochemical staining images of 5D and 10D, macrophages in skin wounds (F4/80) were brown and nuclei were stained with hematoxylin. The magnification is 200 times. (B) The method comprises the following steps Quantification of the average area% of the F4/80 immunostained area. (# 0.05) is significantly different from the control group. Significant differences from the model group (P < 0.05). It is significantly different from the No-load NPS group (P < 0.05). ● was significantly different from the EGCG group (P < 0.05).
As can be seen in fig. 5, inflammatory cells gradually decreased over time. At 5D and 10D, the number of inflammatory cells was significantly higher in the model group than in the control group (P <0.05 or P < 0.01). The inflammatory cells in the unloaded group were significantly lower than in the model group (P < 0.001). However, at 10 days, inflammatory cells were significantly more in the EGCG group than in the unloaded NPS group (P < 0.05). On days 5 and 10, the inflammatory cell infiltration was significantly lower in the composition group of this example than in the model, unloaded and EGCG groups (P <0.001, P < 0.01). The results show that the wound healing accelerating composition prepared in the examples can significantly reduce the number of inflammatory cells in the wound of diabetic mice.
Example 2
This example provides a composition for accelerating wound healing, which comprises sodium acetate, ascorbic acid, gelatin and chitosan as main ingredients, and is prepared by the following steps:
1. anhydrous sodium acetate is dissolved in ultrapure water to prepare a sodium acetate solution of 0.3mol/L, and the pH of the sodium acetate solution is adjusted to 5.0 by hydrochloric acid or sodium hydroxide to obtain a sodium acetate buffer solution.
2. Dissolving gelatin and chitosan in sodium buffer solution respectively to obtain 0.3% gelatin buffer solution and 0.3% chitosan buffer solution, and removing insoluble impurities from the two solutions with 0.22 μm organic filter membrane.
3. Respectively weighing 30mg of EGCG and 0.2mg of ascorbic acid, ultrasonically dissolving the EGCG and the ascorbic acid in 10mL of 0.3% chitosan buffer solution, slowly dripping the EGCG and the ascorbic acid into 10mL of 0.3% gelatin buffer solution after the EGCG and the ascorbic acid are completely dissolved, wherein the dripping speed is 0.5-1 mL/min. After the two are uniformly mixed, the mixture is continuously stirred for 50min by magnetic force, the rotating speed is 350r/min, and suspension is obtained after complete mixing. Wherein the ultrasonic power is within the range of 20-40 kHz.
4. The suspension is centrifuged for 20 minutes at 15000 r/min by a low-temperature high-speed centrifuge at 4 +/-2 ℃, and the centrifuged solid part is taken.
5. Freeze-drying the centrifuged solid part at-10 deg.C to obtain the composition for accelerating wound healing.
Example 3
This example provides a composition for accelerating wound healing, which comprises sodium acetate, ascorbic acid, gelatin and chitosan as main ingredients, and is prepared by the following steps:
1. anhydrous sodium acetate is dissolved in ultrapure water to prepare a sodium acetate solution of 0.1mol/L, and the pH of the sodium acetate solution is adjusted to 5.2 by hydrochloric acid or sodium hydroxide to obtain a sodium acetate buffer solution.
2. Dissolving gelatin and chitosan in sodium buffer solution respectively to obtain 0.2% gelatin buffer solution and 0.2% chitosan buffer solution, and removing insoluble impurities from the two solutions with 0.45 μm organic filter membrane.
3. Respectively weighing 45mg of EGCG and 0.3mg of ascorbic acid, ultrasonically dissolving the EGCG and the ascorbic acid in 10mL of 0.2% chitosan buffer solution, slowly dripping the EGCG and the ascorbic acid into 10mL of 0.2% gelatin buffer solution after the EGCG and the ascorbic acid are completely dissolved, wherein the dripping speed is 0.5-1 mL/min. After the two are uniformly mixed, the mixture is continuously stirred for 20min by magnetic force, the rotating speed is 250r/min, and suspension is obtained after the mixture is completely mixed. Wherein the ultrasonic power is within the range of 20-40 kHz.
4. And centrifuging the suspension for 60 minutes by using a low-temperature high-speed centrifuge at the temperature of 4 +/-2 ℃ at 11000 r/min, and taking a centrifuged solid part.
5. Freeze-drying the centrifuged solid part at-70 deg.C to obtain the composition for accelerating wound healing.
Claims (7)
1. A composition for accelerating wound healing, comprising: the components of the composition for accelerating wound healing comprise EGCG, sodium acetate, ascorbic acid, gelatin and chitosan;
the preparation method of the composition for accelerating wound healing comprises the following steps:
firstly, preparing a sodium acetate solution with the concentration of 0.1-0.3 mol/L, and adjusting the pH value of the sodium acetate solution to 5.0-5.4 to obtain a sodium acetate buffer solution;
secondly, preparing 0.1-0.3% gelatin buffer solution and 0.1-0.3% chitosan buffer solution by using the sodium acetate buffer solution, and removing insoluble impurities in the two solutions by using a 0.22-0.45 mu m organic filter membrane;
dissolving EGCG and ascorbic acid in a chitosan buffer solution according to the final concentration of 3-4.5 mg/mL of EGCG and 0.02-0.03 mg/mL of ascorbic acid to obtain a mixed solution, dropwise adding the mixed solution into the 0.1-0.3% gelatin buffer solution after complete dissolution, uniformly mixing the EGCG and the ascorbic acid, stirring, and completely mixing to obtain a suspension;
fourthly, centrifuging the suspension for 20 to 60 minutes at the temperature of 4 +/-2 ℃ under the condition of 13000 +/-2000 r/min, and taking a centrifuged solid part;
fifthly, freeze-drying the solid part after centrifugation to obtain the composition for accelerating wound healing.
2. A method of preparing a composition for enhancing wound healing, comprising: the preparation method of the composition for accelerating wound healing comprises the following steps:
firstly, preparing a sodium acetate solution with the concentration of 0.1-0.3 mol/L, and adjusting the pH value of the sodium acetate solution to 5.0-5.4 to obtain a sodium acetate buffer solution;
secondly, preparing 0.1-0.3% gelatin buffer solution and 0.1-0.3% chitosan buffer solution by using the sodium acetate buffer solution, and removing insoluble impurities in the two solutions by using a 0.22-0.45 mu m organic filter membrane;
dissolving EGCG and ascorbic acid in a chitosan buffer solution according to the final concentration of 3-4.5 mg/mL of EGCG and 0.02-0.03 mg/mL of ascorbic acid to obtain a mixed solution, dropwise adding the mixed solution into the 0.1-0.3% gelatin buffer solution after complete dissolution, uniformly mixing the EGCG and the ascorbic acid, stirring, and completely mixing to obtain a suspension;
fourthly, centrifuging the suspension for 20 to 60 minutes at the temperature of 4 +/-2 ℃ under the condition of 13000 +/-2000 r/min, and taking a centrifuged solid part;
fifthly, freeze-drying the solid part after centrifugation to obtain the composition for accelerating wound healing.
3. The method of preparing a composition for enhancing wound healing according to claim 2, wherein: the freeze drying condition is normal pressure freeze drying or vacuum freeze drying under the condition of-70 to-10 ℃.
4. The method of preparing a composition for enhancing wound healing according to claim 2, wherein: and the stirring treatment in the third step is specifically stirring for 20-50 min under the condition that the rotating speed is 300 +/-50 r/min.
5. The method of preparing a composition for enhancing wound healing according to claim 2, wherein: the mixed solution is dropwise added into the 0.1-0.3% gelatin buffer solution, and the specific mixing conditions are as follows: according to the volume ratio, the mixed solution: gelatin buffer =1: 1.
6. The method of preparing a composition for enhancing wound healing according to claim 2, wherein: the speed of dropwise adding the mixed solution is 0.5-1 mL/min.
7. The method of preparing a composition for enhancing wound healing according to claim 2, wherein: the EGCG and the ascorbic acid are dissolved in the chitosan buffer solution specifically by adopting ultrasonic-assisted dissolution.
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