CN117797172B - Preparation method of anti-inflammatory, hemostatic and healing-promoting combination - Google Patents
Preparation method of anti-inflammatory, hemostatic and healing-promoting combination Download PDFInfo
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Abstract
The invention discloses a preparation method of an anti-inflammatory, hemostatic and healing-promoting combination, which comprises the steps of preparing a biological tissue membrane from fresh animal tissue, vacuum freeze-drying and grinding the biological tissue membrane into powder, and mixing the powder with chitosan and sodium alginate powder to prepare combination powder; adding acetic acid into the combination powder for dissolution, and stirring to prepare combination gel, wherein the combination powder or gel contains active ingredients such as natural collagen, glycosaminoglycan, cytokines and the like and various chemical ingredients with functions of anti-inflammatory repair and the like, and relates to the field of biomedicine. The product and the raw materials prepared by the invention have biodegradability, biocompatibility and functional stability, can effectively stop bleeding, resist inflammation, promote skin repair, prevent or treat scar generation, and have wide application prospects in the fields of medical treatment of various skin anomalies such as large-area burn wounds, various skin injuries, leucoderma and the like, medical treatment and acne removal, scar repair and cosmetics.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a preparation method of an anti-inflammatory, hemostatic and healing-promoting combination.
Background
The cosmetics are used as daily necessities of people nowadays, and the market demand is huge. The traditional anti-inflammatory and anti-acne cosmetics have unobvious treatment effect, and have the problems of high cost, allergy, endocrine disturbance, skin inflammation, pigmentation and the like caused by even hormone-containing products. Developing special effect cosmetics becomes a hot spot and a difficult point in the current market.
The natural biological tissue raw material contains active ingredients such as collagen, elastin, glycosaminoglycan, cytokines and the like, belongs to the structures in organisms, has the characteristics of active ingredients with various physiological functions and the like, can be used as a skin wound, anti-inflammatory and repairing treatment material in the medical and medical fields, and can also be used as a product and a raw material for special cosmetics. The chitosan is a product of removing partial acetyl of natural polysaccharide chitin, has multiple physiological functions of biodegradability, biocompatibility, nontoxicity, bacteriostasis, immunity enhancement and the like, and is widely applied to various fields such as food, beauty, cosmetics, antibacterial agents, medical dressing, artificial tissue materials, biomedical fields and the like. The chitosan has no toxicity, no smell, and antibacterial effect, and can be added into cosmetic to improve film forming property of product, and has antibacterial and moisturizing effects without causing any allergic irritation reaction. Sodium alginate is a natural high molecular polysaccharide polymer, and has the functions of moisturizing, promoting healing, resisting bacteria, diminishing inflammation, promoting tissue repair and the like. The modified polyurethane has unique thickening property, hydrophilicity, stability, gelation property and other characteristics, and is widely applied.
The skin is the largest organ of human body, is an important barrier of human body, and participates in important functions such as body temperature, metabolism, immunity and the like. It is susceptible to damage due to various external factors such as burn, wound, inflammation, etc., and metabolic abnormality such as vitiligo, etc. When defects and inflammation of the skin, or metabolic abnormalities reach a certain area and are difficult to heal to form scars, it is difficult to repair itself, and wound repair surgery or medicines are required to promote healing. It has long been a research hotspot to find an ideal dressing or gel-like substance that promotes wound healing faster. Most of the existing wound therapeutic dressings or gels have single components and do not heal fast. Therefore, how to select effective methods and medicines to promote the healing and repair of skin wounds to the maximum extent, and stopping bleeding, diminishing inflammation and resisting bacteria is an important subject in the current scientific research field.
The traditional high-pressure steam sterilization is generally carried out under the pressure of 0.105MPa, the temperature of 121 ℃ is 15-30 min, and the method is suitable for high-temperature resistant articles, instruments, culture mediums and the like, but is not suitable for powder and gel sterilization. Plasma sterilization uses the principle of ionization reaction of gas to sterilize thermolabile and moisture-proof surgical instruments and the like, and is not suitable for sterilization of liquid and powder. The ethylene oxide sterilization mainly comprises the processes of vacuum and humidification, and residual gas replacement after constant-temperature dosing, and the biological enzyme activity is inhibited by utilizing the alkylation principle of the ethylene oxide sterilization, so that the fumigating sterilization is carried out on articles which cannot be sterilized at high temperature, and the ethylene oxide sterilization is a low-temperature sterilization mode which is broad-spectrum, efficient, extremely strong in penetrating power and slight in damage to the articles. Conventional ethylene oxide sterilization is generally not used for liquids and powders.
Disclosure of Invention
The invention aims to provide a preparation method of an anti-inflammatory, hemostatic and healing-promoting combination body, which aims to solve the problems in the background technology.
In order to achieve the above purpose, the present invention provides the following technical solutions:
A method for preparing an anti-inflammatory, hemostatic and healing-promoting combination, which comprises the following steps:
S1: fresh small intestines or bladders of healthy animals are obtained, washed clean by sterile water, cut to the required length and prepared into tissue films;
s2: placing the prepared tissue membrane in a vacuum freeze dryer, and freeze-drying for 8-14 hours at the temperature below-50 ℃;
S3: taking out the tissue membrane, and preparing the tissue membrane into tissue powder I by grinding equipment;
S4: taking 1-3 parts of powder I, 6-12 parts of powder II and 6-12 parts of powder III to prepare composite powder, sterilizing by ethylene oxide, and preserving at a temperature of less than 10 ℃;
Powder two and powder three are auxiliary materials, which can promote the healing of skin;
wherein the ethylene oxide sterilization steps are as follows: weighing a proper amount of prepared powder, placing into a culture dish, wrapping the culture dish with medical sterilizing paper as a semipermeable membrane, sealing gaps between the culture dish and the medical sterilizing paper with a sterile adhesive tape, placing on a carrier of an ethylene oxide sterilizing box, sterilizing the sterilized articles with 80% space of the sterilizing chamber, placing a sterilizing indicator card, and setting vacuum negative pressure of 30-50KPa for sterilization.
In a preferred embodiment of the invention, the preparation of the tissue membrane comprises the steps of:
P1: placing fresh healthy animal small intestine or bladder in a disposable culture dish under aseptic condition, washing with sterile water, manually separating and removing each layer of tissue, and retaining submucosal tissue; continuously flushing submucosa tissues with sterile water, trimming to a required length, and soaking in a solution containing 80-120 mmol/L ethylenediamine tetraacetic acid (EDTA) and 8-12 mmol/L sodium hydroxide (NaOH);
P2: immersing the submucosa tissue in a solution containing 0.5-1.5 mmol/L hydrochloric acid (HCl) and 0.5-1.2 mmol/L sodium chloride (NaCl) after rinsing again;
P3: immersing the submucosa tissue in a Phosphate Buffer (PBS) solution containing 0.7-1.5 mmol/L sodium chloride (NaCl) after re-washing, wherein the submucosa tissue is prepared into extracellular matrix;
P4: taking out submucosa tissue, and soaking in PBS; taking out, washing, sterilizing, and storing.
In a preferred embodiment of the present invention, the second powder is chitosan powder and the third powder is sodium alginate powder.
In a preferred embodiment of the invention, 30-80ml of 1% -3% acetic acid solution is added into the combination powder, and the combination powder is stirred and dissolved to prepare combination gel, and the combination gel is refrigerated and stored after centrifugal defoaming.
In a preferred embodiment of the invention, the ethylene oxide sterilization step is also applicable to the combination gel.
The beneficial effects of the invention are as follows:
1. Compared with the existing powder or gel substances, the product is used as a novel composite of natural biological tissue chemical raw materials, the prepared composite powder or gel product is used for dressing, can accelerate blood coagulation, platelet adhesion and thrombin generation, has high hydration characteristic, can be closely connected with cells and tissues, is used for medical beauty, can be prepared into various repairing products in the cosmetic field, and has the advantages of promoting skin repair, promoting inflammation resolution, preventing or fading scar generation, along with low cost, better economic and social benefits;
2. The product can be used for repairing skin of patients in the medical field such as burn, wound vitiligo, refractory scar, etc., promoting various injuries and healing of skin, removing acne and reducing speckle in the medical field, and can be used as product and raw material in the cosmetic field;
3. The invention adopts a special packaging mode, because the pore diameter of the medical sterilizing paper is larger than that of the ethylene oxide gas and smaller than that of gel or powder to be sterilized, the ethylene oxide gas can penetrate the sterilizing paper to enter a culture dish under the use condition of 30-50KPa pressure, and the gel or powder is fully sterilized. Meanwhile, gel or powder in the culture dish cannot overflow from medical sterilizing paper, and the properties and the efficacy of the powder and the gel are maintained and are not changed during sterilization.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 shows the actual effect of the combination gel on days 3-12 after subject number 1;
FIG. 2 shows the actual effect of the combination gel on days 3-20 after subject number 2;
Fig. 3 is a graph of a control group of nude mice with wound not being healed and a control group of nude mice with wound using gel, showing wound healing and scar growth after the experimental operation and the third to nineteenth days after the operation, respectively;
FIG. 4 is a powder end view of the combination;
fig. 5 is a gel end view of the combination.
Detailed Description
Reference will now be made in detail to the present embodiment combinations of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to like or similar elements or elements having like or similar functionality throughout. The embodiments described below by referring to the drawings are illustrative only and are not to be construed as limiting the invention.
A method for preparing an anti-inflammatory, hemostatic and healing-promoting combination, which comprises the following steps:
S1: fresh small intestines or bladders of healthy animals are obtained, after the small intestines or bladders are washed clean by sterile water, the small intestines or the bladders are cut to the required length of 5cm to 15cm, and are prepared into tissue films, wherein the tissue films are prepared as follows: placing fresh healthy animal small intestine or bladder in a disposable culture dish under aseptic condition, washing with sterile water for several times, separating and removing each layer of tissue, and retaining submucosal tissue; continuously flushing submucosa tissues with sterile water for a plurality of times, trimming to 2-6 cm, and soaking in a solution containing 80-120 mmol/L ethylenediamine tetraacetic acid (EDTA) and 8-12 mmol/L sodium hydroxide (NaOH) for 10-18 hours;
immersing the submucosa tissue in a solution containing 0.5-1.5 mmol/L hydrochloric acid (HCl) and 0.5-1.2 mmol/L sodium chloride (NaCl) for 5-10 hours after rinsing again;
rinsing the submucosa tissue again, and then soaking the submucosa tissue in a Phosphate Buffer (PBS) solution containing 0.7-1.5 mmol/L sodium chloride (NaCl) for 12-18 hours, wherein the submucosa tissue is prepared into extracellular matrix;
Taking out submucosa tissue, and soaking in PBS for 1-3 hours; taking out, washing, sterilizing, and storing;
s2: placing the prepared tissue membrane in a vacuum freeze dryer, and freeze-drying for 8-14 hours at the temperature below-50 ℃;
s3: taking out the freeze-dried membrane tissue, and preparing tissue powder I by grinding equipment;
s4: taking 1-3 parts of powder I, 6-12 parts of powder II and 6-12 parts of powder III to prepare composite powder, wherein the powder II and the powder III are auxiliary materials which can promote the healing of skin, for example, the powder II is chitosan powder and the powder III is sodium alginate powder;
Placing the powder of the combination of the first powder, the second powder and the third powder in a beaker, adding 30-80ml of 1% -3% acetic acid solution, stirring and dissolving to prepare gel, centrifuging and defoaming, refrigerating and preserving, and setting parameters of a centrifuge to 10000rpm for 5min;
Specifically, 0.1-0.3 g of powder I, biological tissue powder, 0.6-1.2 g of powder II, chitosan powder, 0.6-1.2 g of powder III and sodium alginate powder are weighed to prepare composite powder, and the composite powder is placed in a beaker; adding 30-80 ml of 1% -3% acetic acid solution, stirring and dissolving with a stirrer, preparing gel after about 0.5-2 hours, sterilizing with ethylene oxide, and sealing and packaging;
preparing the composite powder into gel, sterilizing the composite powder and the gel by ethylene oxide, wherein the sterilization steps are as follows: weighing a proper amount of prepared powder or gel, placing the powder or gel into a culture dish, sealing the culture dish by a semipermeable membrane, placing the culture dish on a carrier of an ethylene oxide sterilization box, placing a sterilization indicator card, and setting vacuum negative pressure of 30KPa for sterilization; the semipermeable membrane is medical sterilizing paper, which can allow ethylene oxide gas to enter the culture dish for sterilization, and simultaneously prevent the powder of the combination from falling out of the culture dish under the suction of negative pressure.
Example 1:
Referring to fig. 1, after daily basic cleaning, the prepared gel is uniformly smeared on the lower jaw of the left and right sides of a first subject, and the facial skin condition is observed on the 3 rd day, the 8 th day and the 12 th day respectively, as shown in fig. 1, before the gel is smeared, the acne on the face of the subject is more obvious, and a certain swelling and redness occur, and more acne marks exist. The red and swelling degree is improved when the acne cream is used on the 3 rd day, the acne marks start to be reduced, the red and swelling starts to be removed when the acne cream is used on the 5 th day, the acne marks are obviously reduced, the skin condition is obviously improved, the red and swelling completely removes when the acne cream is used on the 7 th day, the acne marks basically remove, the facial skin basically recovers to be smooth, and the color is relatively uniform.
Example 2:
Referring to the figure, after 2 days of basic cleaning, the prepared gel is uniformly smeared on the forehead of a second subject, and the forehead skin condition is observed on the 3 rd day, the 7 th day and the 20 th day respectively, as shown in figure 2, before the gel is smeared, acne at the forehead of the subject is serious, acne marks are obvious, the redness and swelling degree of the acne is improved when the gel is smeared on the 3 rd day, the number of the acne marks is reduced, and the acne marks are basically resolved when the gel is smeared on the 7 th day; the forehead skin has no red swelling on the 20 th day of gel application, the acne marks are basically resolved, the skin flatness is obviously improved, and the skin color is restored uniformly.
Example 3:
Referring to fig. 3, nude mice were divided into two groups, anesthetized with 10% chloral hydrate (0.35 mL/100 g), and after iodophor sterilization, the back central skin was held with forceps, circular skin with a diameter of 1cm was cut off with tissue, subcutaneous tissue was exposed, the two groups of nude mice were divided into a non-wound treatment group-control group, and a gel treatment group-experimental group for wound, the wound on the back of the control group nude mice was directly debrided and wrapped with an elastic bandage for wrapping with sterile gauze, and the wound was periodically sterilized and dressing was replaced; the back wound of the nude mice of the experimental group is smeared with the combined body gel, the wound is wrapped by an elastic bandage after being wrapped by sterile gauze, and the wound is disinfected and dressing is replaced regularly; it can be observed that the back wound of the nude mice in the control group is at the 3 rd day after operation, scar is found; wound scar increase at day 7 post-operation; on the 11 th day after operation, the wound scar is increased and is fully distributed on the outer edge of the wound, the recovery of the skin defect is poor, and the healing is slow; the back skin wound of the nude mice of the experimental group is obviously healed on the 10 th day after operation, and no scar exists; on day 14, the wound was substantially healed; by day 19, the wound had healed substantially and no scar was found.
The main components of the product comprise active components such as collagen, elastin, glycosaminoglycan, cytokines and the like and natural chemical components with various functions of anti-inflammatory repair and the like, the product has biodegradability, biocompatibility and functional stability, can effectively stop bleeding, resist inflammation, promote skin repair, prevent or treat scar generation, has good application effect, has wide application prospect in the fields of medical treatment of large-area burn wounds and various skin injuries such as leucoderma and the like, medical treatment and beauty acne removal, repair scars and cosmetics, the important components of the anti-inflammatory, hemostatic and healing-promoting composite powder and gel product are derived from natural animal tissues, and can be used as anti-inflammatory and healing materials for treating all acute and chronic skin injuries such as vitiligo, difficult scar and the like by utilizing the characteristics of structural materials in organisms and various physiological functions, adding natural chemical raw materials, having biodegradability and biocompatibility, functional stability and film forming property, resisting inflammation, promoting metabolism, accelerating skin repair, preventing or fading scar generation, and being used as skin wound large-area burn wounds in the medical and medical field.
While embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that: many changes, modifications, substitutions and variations may be made to the embodiments without departing from the spirit and principles of the invention, the scope of which is defined by the claims and their equivalents.
Claims (5)
1. A method for preparing an anti-inflammatory, hemostatic and healing-promoting combination, which is characterized by comprising the following steps:
S1: fresh small intestines or bladders of healthy animals are obtained, washed clean by sterile water, cut to the required length and prepared into tissue films;
s2: placing the prepared tissue membrane in a vacuum freeze dryer, and freeze-drying for 8-14 hours at a temperature below-50 ℃;
S3: taking out the tissue membrane, and preparing the tissue membrane into tissue powder I by grinding equipment;
S4: taking 1-3 parts of powder I, 6-12 parts of powder II and 6-12 parts of powder III to prepare composite powder, sterilizing by ethylene oxide, and preserving at a temperature of less than 10 ℃;
Powder two and powder three are auxiliary materials, which can promote the healing of skin;
Wherein the ethylene oxide sterilization steps are as follows: weighing a proper amount of prepared powder, placing the powder into a culture dish, wrapping the culture dish with medical sterilizing paper as a semipermeable membrane, sealing gaps between the culture dish and the medical sterilizing paper by using a sterile adhesive tape, placing the culture dish and the medical sterilizing paper on a carrier of an ethylene oxide sterilizing box, placing sterilized articles accounting for 80% of the space of the sterilizing chamber, placing a sterilizing indicator card, and setting vacuum negative pressure of 30-50KPa for sterilization;
In S1, the preparation of the tissue membrane comprises the following steps:
P1: placing fresh healthy animal small intestine or bladder in a disposable culture dish under aseptic condition, washing with sterile water, manually separating and removing each layer of tissue, and retaining submucosal tissue; continuously flushing submucosa tissues with sterile water, trimming to a required length, and soaking in a solution containing 80-120 mmol/L ethylenediamine tetraacetic acid (EDTA) and 8-12 mmol/L sodium hydroxide (NaOH);
P2: immersing the submucosa tissue in a solution containing 0.5-1.5 mmol/L hydrochloric acid (HCl) and 0.5-1.2 mmol/L sodium chloride (NaCl) after rinsing again;
P3: immersing the submucosa tissue in a Phosphate Buffer (PBS) solution containing 0.7-1.5 mmol/L sodium chloride (NaCl) after re-washing, wherein the submucosa tissue is prepared into extracellular matrix;
P4: taking out submucosa tissue, and soaking in PBS; taking out, washing, sterilizing, and storing.
2. The method for preparing the anti-inflammatory, hemostatic and healing-promoting combination according to claim 1, wherein the method comprises the following steps: the second powder is chitosan powder, and the third powder is sodium alginate powder.
3. The method for preparing an anti-inflammatory, hemostatic and healing-promoting composition according to claim 1, wherein 30-80ml of 1% -3% acetic acid solution is added into the composition powder, and the mixture is stirred and dissolved to prepare a composition gel, and the composition gel is refrigerated and stored after centrifugal defoaming.
4. A method of preparing an anti-inflammatory, hemostatic, and healing-promoting composition according to claim 3, wherein the ethylene oxide sterilization step is also applicable to the composition gel.
5. An anti-inflammatory, hemostatic and healing-promoting combination, characterized in that: the combination prepared by the preparation method of any one of claims 1 to 4, which is in the form of powder or gel.
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