CN109700998B - Compound skin injury regeneration repairing agent and preparation method thereof - Google Patents

Abstract

The invention provides a compound skin injury regeneration repairing agent and a preparation method thereof, and is characterized in that the repairing agent is prepared by using a plasma high-molecular protein extract, a tea tree root extract and ceramide as main materials and using borneol, polyethylene glycol (4000-8000), glycerol, epsilon-polylysine, carbomer and the like as auxiliary materials. The invention relates to a compound skin injury regeneration repairing agent which is prepared by scientifically proportioning a plurality of active ingredients such as macromolecule active protein extract, tea tree root extract, ceramide and the like in animal plasma. The wound healing cream has the effects of improving skin immunity, improving wound microcirculation, promoting angiogenesis and granulation tissue growth, remarkably accelerating wound healing speed and realizing perfect wound healing without scars. The invention belongs to a pure natural biological product, is easy to be absorbed by human body, has the advantages of quick curative effect, low price, convenient use, no toxic or side effect and the like, and has good medical development value and market application prospect.

Description

Compound skin injury regeneration repairing agent and preparation method thereof

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to a compound skin injury regeneration repairing agent and a preparation method thereof.

Background

Acute and chronic wounds, particularly chronic difficult-to-heal ulcers, have been the focus of research in the medical field. In recent years, although research on wound repair is more and is rapidly carried out, under the existing treatment level, more and more acute and chronic intractable ulcers are difficult for doctors and patients to deal with, and with the continuous improvement of living standard of people and the development of regenerative medicine, development of regenerative repair products and technologies capable of promoting perfect scar-free repair on the structure and function of a damaged part becomes a new research hotspot and direction in the field of wound repair.

Chronic hard-to-heal wounds on the body surface are caused by a series of wounds and diseases. Mainly comprises traumatic ulcer, lower limb venous ulcer, pressure ulcer, diabetic ulcer and the like. Although the wound surface does not cause death as rapidly as cancer, the wound surface has the characteristics of long disease course, more complications, large appearance influence and the like and causes great harm to the body, mind, life and working quality of patients due to the fact that the wound surface occurs on the body surface. Therefore, how to accelerate the regenerative repair of skin injury and realize perfect non-scar repair of the wound surface from the structure and the function becomes one of the problems to be solved urgently in the field of tissue repair.

The pathogenesis of chronic ulcer difficult to heal is not fully elucidated so far, and the current western medicine has the following arguments: 1. the down regulation of the gene expression of the wound extracellular matrix component cytoskeleton leads to the reduction of the generation of the wound extracellular matrix component cytoskeleton, and consequently, the movable scaffold of tissue repair cells is damaged, thereby delaying the healing of the wound. 2. In addition to the cellular environment, rapid apoptosis of mainly repairing cells, especially fibroblasts, further affects the production of extracellular matrix, especially collagen. 3. The influence of the concentration change of the local wound growth factor reduces the activity of the growth factor and changes the structure and the function of a target cell receptor, so that the normal combination of the factor and the receptor is obstructed, and the phenomenon of so-called uncoupling is generated. 4. The network of wound healing regulation is hindered. The traditional Chinese medicine considers that the chronic skin ulcer is caused by abnormal qi and blood circulation due to the fact that a human body is directly injured by trauma, crush injury, skin trauma and the like and then is infected by toxic factors, and the chronic skin ulcer cannot be cured after long-term treatment and seriously harms the health of a patient because the skin cannot be protected or is separated from an irritation environment.

At present, antibiotics are commonly used in western medicine in clinical treatment methods, the treatment effect is poor, the antibiotics are used for a long time, the dependence of a human body can be generated, the microbial balance of the human body can be damaged, and the liver and kidney functions can be damaged. In recent years, gene medicines, namely various cell growth factors, are clinically and significantly enhanced in skin repair and regeneration functions and accelerated in wound and sore healing effects, but a plurality of problems also occur in the application process. For example, EGF (epidermal growth factor), also called human oligopeptide-1, can directly promote the growth of epidermal cells, and with the development of scientific technology, detection and research find that EGF unidirectionally stimulates cell proliferation, even disorderly stimulates cell proliferation, and the stimulation may cause the problem that skin cells are difficult to predict. According to the relevant literature, the abuse of EGF also causes side effects, possibly causing excessive or unlimited growth of epidermal cells, thereby thickening the stratum corneum or inducing other canceration. Many cases of 'disfigurement' due to the abuse of EGF have appeared in plastic hospitals and cosmetics industries at present.

(reference 1, experimental study on epidermal growth factor promoting pancreatic cancer cell invasion and metastasis in No. 3, volume 3, month 15, 2008 of China general Foundation and clinical journal, reference 2, issue of EGF as a report of cosmetic raw material in No. 10, 2019, month 01, by State drug administration)

The traditional Chinese medicine mostly adopts Chinese herbal medicines such as violet herb oil, ruyi jinhuang powder, slough-removing and granulation-promoting powder and the like for treating the chronic skin ulcer, has small side effect, but has slow healing, long treatment period and poor curative effect.

According to the survey of China, Japan, United states and other countries, the following: the incidence rate of chronic difficult-to-heal wound patients in the comprehensive diseases is 7-23%; the incidence rate of cardiovascular diseases of patients is about 41 percent, the incidence rate of patients with neurological disorders is 27 percent, and the incidence rate of patients in plastic surgery is 15 percent. British survey showed that the incidence of diabetic ulcers has been increasing in recent years, with the incidence of ulcers accounting for 5.3% of patients with type ii diabetes; diabetic patients had 20% hospitalized with lower limb ulcers, with amputees about 0.6%. The investigation result of the Chinese part shows that: the main causes of ulceration are wound infection (67.5%), pressure ulcers (9.2%), venous ulcers (6.5%), diabetic ulcers (4.9%), other factors (11.9%).

The cost of treatment of chronic, refractory wounds on the body surface is quite expensive and has been reported: the U.S. annual treatment costs for venous ulcers are $ 1.65-10.78 million, with annual treatment costs for each patient being $ 2400, with the elderly being higher and tending to increase year by year. Therefore, the incidence rate and social hazards of chronic difficult-to-heal wounds on the body surface become important problems which must be faced by modern society. Therefore, the search for a skin regeneration and repair product with quick curative effect, small side effect and low cost is an urgent need of surgical clinic and is the desire and the need of solving the pain of patients.

Disclosure of Invention

The invention aims to provide a compound skin injury regeneration repairing agent which is specially researched and developed based on the situation and a preparation method thereof. The invention relates to a compound skin injury regeneration repairing agent which is prepared by scientifically proportioning a plurality of active ingredients such as macromolecule active protein extract, tea tree root extract, ceramide and the like in animal plasma. The wound healing cream has the effects of improving skin immunity, improving wound microcirculation, promoting angiogenesis and granulation tissue growth, remarkably accelerating wound healing speed and realizing perfect wound healing without scars.

The present invention belongs to a pure natural biological product, is easy to be absorbed by human body, has no toxic side effect, and can be extensively used for curing wound, burn, scald and various acute and chronic ulcers, etc. caused by various reasons. Compared with the existing products in the market, the product of the invention has the advantages of quick curative effect, low price, convenient use, no toxic or side effect and the like. The wound healing powder has obvious curative effect on the repair and regeneration of skin injury, particularly chronic difficult-to-heal wound surfaces, and has good medical development value and market application prospect.

The purpose of the invention is realized by the following technical scheme:

a compound skin injury regeneration repairing agent is prepared from plasma high molecular protein extract, tea root extract and ceramide as main materials, and one or more of Borneolum Syntheticum, polyethylene glycol (4000-; wherein the ratio of plasma high molecular protein extract is 1.5-10%, the ratio of tea tree root extract is 10-30%, and the ratio of ceramide is 0.05-0.2%.

The product of the invention can be prepared into dosage forms of aqua, ointment, freeze-dried powder and the like according to the requirements of patients and wound surfaces.

The water aqua product comprises the following components in percentage by mass:

1.5-10% of plasma high-molecular protein extract

10-30% of tea tree root extract

Ceramide 0.05-0.2%

10-20% of glycerin

0.01 to 0.5 percent of epsilon-polylysine

The balance of physiological saline, pH7.5-9.0.

The paste product comprises the following formula components in percentage by mass:

1.5-10% of plasma high-molecular protein extract

10-30% of tea tree root extract

Ceramide 0.05-0.2%

1 to 5 percent of borneol

0.01 to 0.5 percent of epsilon-polylysine

5-15% of glycerin

Carbomer 0.1-2%

The balance of physiological saline, pH7.5-9.0.

The freeze-dried powder comprises the following components in percentage by mass:

1.5-10% of plasma high-molecular protein extract

10-30% of tea tree root extract

Ceramide 0.05-0.2%

1 to 5 percent of borneol

5-10% of polyethylene glycol (4000-

0.01 to 0.5 percent of epsilon-polylysine

The balance of physiological saline, pH7.5-9.0.

A preparation method of a compound skin injury regeneration repairing agent comprises the following steps:

preparing plasma high molecular protein extract

(1) Collecting fresh animal (pig, cattle, sheep, etc.) anticoagulated plasma qualified by sanitary inspection, adding 20-25% saturation ammonium sulfate according to volume, stirring to completely dissolve the ammonium sulfate, standing for about 2 hours, filtering (300-. The filtrate or centrifugate can be used as animal protein byproduct by heat desalting.

(2) Virus inactivation: adding 3 times volume of pure water into the crude product of the macromolecular protein extract to dilute the crude product into porridge, putting the porridge into a reaction kettle, heating the porridge to 60 ℃, preserving the heat for 10 hours, and inactivating viruses.

(3) And (3) cyclic ultrafiltration desalination: and (3) performing virus inactivation on the crude product of the macromolecular protein extract according to the weight ratio of 1: 4-1: 5, diluting the solution with pure water, centrifuging (3000 + 4000r/min, 15-20 min) to remove insoluble precipitates, collecting the centrifugate, performing ultrafiltration treatment on the centrifugate by an ultrafiltration device with the molecular weight cut-off of 100KD, adding pure water with the volume of 4-5 times that of the cut-off into the cut-off when the cut-off is concentrated to 1/4-1/5 of the original volume, continuously performing ultrafiltration and desalination to 1/4-1/5 of the original volume, repeating the steps for 2-3 times until sulfate ions in the permeate are completely removed, and collecting the cut-off A. The permeate liquid containing ammonium sulfate in the cyclic desalting process can be reused as the dissolving liquid for dissolving the crude product of the macromolecular protein extract next time.

(4) And (3) ultrafiltration refining: and (4) carrying out ultrafiltration concentration on the trapped fluid A finally obtained in the step (3) by an ultrafiltration device with the molecular weight cut-off of 300KD until the volume of the trapped fluid is 1/3-1/5 of the original volume, and collecting the trapped fluid, namely the refined plasma high molecular protein extract (the total protein content is more than or equal to 20 mg/ml).

(II) preparing tea tree root extract

(1) Superfine grinding: collecting qualified tea tree root raw material, pulverizing with superfine pulverizer, placing into reaction kettle, adding water 8-10 times of the total weight of the raw material, adjusting pH to 6.0-7.0, adding 10-20% sodium chloride, and soaking for more than 24 hr. (2) Heat and ultrasonic treatment: the reaction kettle is heated to 85-90 ℃ under stirring and is thermally insulated for 30-60 minutes, and then is cooled to room temperature, and ultrasonic cracking (400-.

(3) Alkaline enzymolysis: adjusting pH of lysate to 11.0-12.0 with 10% sodium hydroxide, adding 1-5% mixed enzyme (pancreatin: cellulase =1: 2) and 0.5-2% active carbon, mixing well, heating to 45-50 deg.C under continuous stirring (rotation speed 130-.

(4) Filtering and clarifying: filtering the enzymolysis solution with 100-mesh and 300-mesh filter, clarifying the filtrate with a decanter centrifuge, and fine-filtering the crude tea tree root solution with 0.45-0.6 μm inorganic membrane. The filter residue is treated as a feed byproduct.

(5) And (3) ultrafiltration refining: and (3) passing the crude product liquid through an ultrafiltration device with the molecular weight cutoff of 1 ten thousand, wherein the permeate liquid is the refined extract of the tea tree root (the content of the total flavone is more than or equal to 30 percent, and the content of the tea polysaccharide is more than or equal to 10 percent). The collected trapped fluid can be added into the next batch of enzymolysis step to be reused by being matched with alkali liquor.

(III) the production process of the finished product:

1. an aqueous agent production process;

adding 1.5-10% of high molecular protein refined solution (total protein content is more than or equal to 20 mg/ml), 10-30% of tea root extract (total flavone content is more than or equal to 30%, tea polysaccharide content is more than or equal to 10%), 0.05-0.2% of ceramide, 0.01-0.5% of epsilon-polylysine and the balance of normal saline according to the formula proportion into active material liquid, performing fine filtration and microfiltration membrane sterilization on the active material liquid by using 1.0 mu m, 0.65 mu m and 0.22 mu m filter membranes respectively, adding 10-20% of autoclaved glycerol according to the proportion, mixing uniformly, adjusting the pH value to 7.5-9.0 by using 40% of sodium hydroxide, performing sterile subpackaging, and freezing and storing.

2. The production process of the finished paste product comprises the following steps:

adding 1.5-10% of high molecular protein refined solution (total protein content is more than or equal to 20 mg/ml), 10-30% of tea root extract (total flavone content is more than or equal to 30%, tea polysaccharide content is more than or equal to 10%), 0.05-0.2% of ceramide and 0.01-0.5% of epsilon-polylysine in sequence according to a paste formula, preparing active material liquid by using the balance of normal saline according to the formula proportion, performing fine filtration and microfiltration on the active material liquid by using filter membranes of 1.0 mu m, 0.65 mu m and 0.22 mu m respectively, adding 10-20% of autoclaved glycerol, 1-5% of borneol and 0.1-2% of carbomer according to the proportion, mixing uniformly, adjusting the pH value to 7.5-9.0, performing aseptic subpackaging and storing.

3. The production process of the freeze-dried powder comprises the following steps:

sequentially adding 1.5-10% of high molecular protein refined liquid (the total protein content is more than or equal to 20 mg/ml), 10-30% of tea root extract (the total flavone content is more than or equal to 30%, the tea polysaccharide content is more than or equal to 10%), 0.05-0.2% of ceramide and 0.01-0.5% of epsilon-polylysine according to a powder formula, preparing active material liquid by using the balance of normal saline according to the formula proportion, carrying out fine filtration and microfiltration membrane sterilization on the active material liquid by using filter membranes with the sizes of 1.0 mu m, 0.65 mu m and 0.22 mu m respectively, adding 1-5% of borneol and 5-10% of sterilized polyethylene glycol (4000 + 8000) according to the proportion, uniformly mixing, adjusting the PH value to be 7.5-9.0, putting into a freeze dryer for freeze-drying, carrying out aseptic subpackage, and preserving for cold storage.

The product formula mechanism is as follows:

the plasma high molecular protein extract in the components mainly contains high molecular active proteins such as fibrinogen (molecular weight is 340 KD), fibronectin (molecular weight is 450 KD), laminin (molecular weight is 804 KD), blood coagulation factors (such as blood coagulation factor VIII (molecular weight is more than 200 KD) and blood coagulation factor XIII (molecular weight is 15-30 KD), and the proteins have different structures, properties and physiological functions, but play a key role in the whole process of damaged tissue repair. They have multiple cellular biological activities, and can stimulate the production of cells such as epithelial cells, fibroblasts, vascular endothelial cells, macrophages and the like, and mediate the interaction between the cells and various proteins. These high molecular proteins can participate in complex inflammatory stress reaction of organisms, enhance the protection effect on extracellular or intercellular substance, and form good coordination effect with various enzymes in human bodies through the combination with corresponding receptors thereof, thereby playing roles in rapidly stopping bleeding, regulating the permeability of vascular walls, regulating the diffusion and the operation of protein, water and electrolyte, and the like. At the same time, they are multifunctional cell growth factors, can stimulate the growth of cells derived from mesoderm and neuroectoderm, such as fibroblasts, vascular endothelial cells, epithelial cells, nerve cells, muscle cells, bone cells and the like, and have wide biological activity.

Pharmacological research shows that various plasma high molecular proteins have promoting effects of different degrees in three stages in the wound repair process, namely a local inflammatory reaction stage, a cell proliferation and differentiation stage, a granulation tissue formation stage and a tissue reconstruction stage. In the inflammation period, the medicine has obvious chemotactic activity on wound cells and can stimulate fibroblasts, vascular endothelial cells and the like to move to the wound part. When cells migrate to the site of injury, the phase of proliferation and repair begins, one of the most critical steps being the formation of granulation tissue. The nature of granulation tissue is a large number of capillaries and abundant fibroblasts. The high molecular proteins such as fibrinogen, fibronectin, laminin, blood coagulation factors and the like are efficient growth promoters of fibroblasts, vascular endothelial cells and vascular smooth muscle cells, can strongly promote the formation of new capillaries, obviously increase the number of the capillaries of granulation tissues and blood flow, improve wound microcirculation and provide necessary oxygen and rich nutrient substances for tissue repair; meanwhile, the composition also has a remarkable promoting effect on the regeneration of nerve fibers at the damaged tissue part, and can accelerate the recovery of the function of the damaged tissue. A large number of studies and clinical practices prove that: in the whole process of wound healing, the biological activities of the macromolecular proteins can supplement each other, promote each other and restrict each other in the body to form a good synergistic effect, so that various cells and fibers of the body are orderly arranged and grow, and the formation of scar tissues is avoided. If any one of the components is purified and used independently, not only the production cost is increased, but also the effect of wound tissue repair and healing is greatly reduced. Therefore, the invention jointly extracts and comprehensively applies various high molecular proteins in the plasma, fully exerts the team effect of the high molecular proteins such as fibrinogen, fibronectin, laminin, blood coagulation factors and the like, realizes the complementary advantages of the various high molecular proteins, comprehensively participates and mediates three processes of wound repair, and promotes the rapid scar-free repair of the wound.

(reference: fibrin-based biomedical materials, vol.2, 2 nd 2 of 2008, journal of Chinese repair and reconstruction surgery;

reference documents: journal of Sichuan physiology and sciences 2002; 24 (2) human fibrinogen structure and function studies;

reference documents: clinical application of plasma fibronectin to skin wound repair, Xuyanxushuang, etc., in the 14 th volume, 4 th phase of the journal of Chinese biologies 2001;

reference documents: 1998,18(1) Advance in laminin research in foreign medical science, physiology, pathology, and clinical handbook;

reference documents: the physician's journal of advanced repair at 1983, 6 th section "coagulation factors and coagulation principles";

reference documents: new progress of clinical application of cryoprecipitate (Leikanhong, Yedong, etc.), Nanjing military area Nanjing general Hospital transfusion department, Jiangsu Nanjing 210002 medical research institute, 17 st volume, 2004 year 1 month 1. )

The tea tree root serving as a formula component is a common Chinese herbal medicine, is bitter in taste and mild in nature, and has the effects of tonifying heart, promoting urination, reducing blood fat and blood sugar, resisting tumors, resisting bacteria, diminishing inflammation, resisting gout and the like. It is used clinically in treating heart disease, edema, hepatitis, sore, ulcer, aphtha, burns, herpes zoster, psoriasis, etc. The tea tree root extract mainly contains total flavonoids, total saponins, procyanidine, tea polysaccharide, polyphenol compounds and other components; has antioxidant effect, and can scavenge free radicals such as superoxide ion; can obviously improve the blood viscosity; preventing cancer and resisting mutation; tea polysaccharide is another important substance with physiological activity in tea, which is discovered after tea polyphenol, is acid glycoprotein and is combined with a large amount of mineral elements. The protein part mainly comprises about 20 common amino acids, the sugar part mainly comprises arabinose, xylose, fucose, glucose, galactose and the like, and the mineral elements mainly comprise calcium, magnesium, iron, manganese and the like and a small amount of trace elements such as rare earth elements and the like. Pharmacological research shows that the tea polysaccharide has the functions of reducing blood sugar, reducing blood fat, reducing blood pressure, resisting cancer, resisting blood coagulation, resisting thrombus, resisting atherosclerosis, resisting anoxia, preventing radiation, preventing and treating cardiovascular diseases, increasing coronary blood flow, enhancing immunity of organisms and the like. In conclusion, the tea tree root extract contains various active ingredients such as total flavonoids, tea polysaccharides and the like, has multiple functions of resisting inflammation, easing pain, repairing body surface ulcers, enhancing body immunity, improving local blood circulation and the like, and can effectively assist the regeneration and repair of chronic healing-difficult wounds with complex pathogenesis.

(reference 1: research on anti-inflammatory and analgesic effects of tea Tree root at No. 4 of 26 th volume of 2012 in modern Chinese medicine research and practice; (reference 2: adjustment of lipid in rat plasma by tea Tree root at No. 8 of 1992 in Shanghai J.Med.); (reference 3: action of extract of tea Tree root on solid tumor at No. 2 of 12 nd volume of 1999 in foreign medical and Chinese medicine Subdivision))

Ceramide is the main component of skin stratum corneum lipid, has effects of keeping moisture, resisting allergy, resisting aging, and can also assist whitening and antioxidant. Ceramide can form a network structure in the horny layer of epidermis to associate water molecules, so that the ceramide has strong moisturizing effect on the skin; in the process of skin aging, lipid synthesis is reduced, the content of ceramide in the horny layer is reduced, and the skin is dry, desquamation, rough and lustrous; the ceramide can increase ceramide content in skin stratum corneum, and can improve symptoms of xerosis cutis, desquamation, pachylosis, etc.; meanwhile, ceramide can increase the thickness of the horny layer of the skin, improve the water holding capacity of the skin, reduce wrinkles, increase the elasticity of the skin, delay skin aging and ensure that skin cells are well recovered and regenerated. (reference 4: research progress of plant ceramide in 11 th 2013 th 11 th month of volume 36 of Chinese medicinal herbs; reference 5: physiological action of ceramide on skin and application thereof in cosmetics in 2007 first phase of food and fat)

Other components in the formula: borneol, polyethylene glycol (4000-. The Borneolum Syntheticum can be easily absorbed by mucosa and subcutaneous tissue, and has effects of clearing away heat and toxic materials, removing necrotic tissue, promoting granulation, and relieving swelling and pain. Polyethylene glycol (4000-. Polyethylene glycol can also absorb water in solution, improve the biocompatibility of medical polymer materials contacting with blood, is commonly used for promoting cell fusion or protoplast fusion, is beneficial to the organism to absorb DNA in the transformation process, has strong universality, and can be used for various cells of animals, plants and microorganisms. Carbomer has an inherent unique cross-linking structure, and has good viscosity and hydrophilic gel property, so that carbomer has good regulation and control and slow release effects on the release of drugs. The carbomer has good biological adhesion performance, hydrogel and no toxicity or irritation to organisms, and the formed hydrogel is uniform and transparent, has good stability, is comfortable after use, and is easy to coat and clean. The auxiliary materials are all collected and carried in Chinese pharmacopoeia.

Epsilon-polylysine is a natural preservative, has a wide antibacterial spectrum, and has certain antibacterial effects on gram-positive bacteria, gram-negative bacteria, yeast and mould. The epsilon-polylysine has good bacteriostatic effect on gram-negative escherichia coli and salmonella which are difficult to inhibit by other natural preservatives, and has inhibitory effect on thermotolerant bacillus and some viruses.

The product of the invention is prepared by scientific proportioning according to the action mechanism of the functional components and the performance of various auxiliary materials, and the same documents and patent reports are not found so far.

Compared with the prior art, the innovation points of the invention are mainly as follows:

1. rich raw materials and can realize high value-added utilization of resources

The product of the invention utilizes animal blood plasma and plant tea tree roots with rich resources as main raw materials, enlarges the comprehensive utilization of the resources and improves the added value of the resources. The natural plant feed additive is pure natural, free of irritation, free of chemical residue, safe, effective, low in price, wide in market application prospect, and good in economic benefit and social benefit.

2. The process has strong operability and is convenient for large-scale production

We have completed the research from laboratory trial production to medium and small scale production and the observation of product stability, and have proved that the production process has strong operability, low cost and stable quality, and can realize large scale production. The invention innovatively improves the extraction processes of the main functional components of the plasma high-molecular protein extract and the tea tree root extract. At present, similar products and production reports are not seen at home and abroad, so the technology has obvious technical advantages.

The conventional extraction process of the high molecular protein usually adopts a freeze-thaw method, a cold precipitation method, an affinity chromatography method and the like, has high production cost and is inconvenient for large-scale production. The process adopts a method combining salting out with circulating ultrafiltration desalination and ultrafiltration refinement with different molecular weight cut-off, has high production efficiency, low cost, convenient operation and large-scale production.

The active pharmacological components in the tea tree root are mainly total flavonoids, polysaccharides, sapogenins and the like, and the prior art uses a large amount of organic solvents such as alcohol and the like, so that the pollution to personnel and environment is caused, and the large-scale production, development and application of the product are limited. The method adopts the combined process of ultrasound, enzyme and alkaline hydrolysis to effectively avoid the defects of high energy consumption and heavy pollution of the conventional organic solvent extraction method, is simple and easy to control, has high target component yield, can realize large-scale production, and is favorable for high-valued utilization of tea tree root resources.

3. The product has innovative formula, complementary component advantages and obvious curative effect

Through research of recent years, the method has the following findings: the combined application of the plasma high molecular protein extract, the tea tree root extract and the ceramide has better effect than the single application. The three effective components are combined for application, the efficacy advantages of the traditional Chinese herbal medicine extract and the animal tissue extract are combined, the effects of complementation shortage, complementation, mutual promotion and stable curative effect can be achieved, the selected auxiliary materials are non-toxic and non-irritant, the selected auxiliary materials are good lubricants, slow-release agents and stabilizers, and the bioavailability and the stability of the effective components can be effectively improved. Meanwhile, the application effect of the product is subjected to multiple animal experiments and tracking investigation of voluntary test population, the curative effect is obvious, and the favorable comment of patients is obtained. So far, the formula is not reported in the same documents and patents.

4. The whole production process is green and environment-friendly

According to the production process, leftovers produced in each extraction step can be comprehensively utilized, other byproducts can be produced or can be recycled, waste water and waste residues are not discharged basically, and the environment is protected in the true sense.

Detailed Description

The following examples are intended to further illustrate the invention and are not to be construed as limiting the invention in any way.

Example 1

The product of the invention can be prepared into aqua according to the requirements of patients and wound surfaces. The product comprises the following specific formula in parts by weight: 10% of plasma high molecular protein extract, 30% of tea tree root extract, 0.2% of ceramide, 20% of glycerol, 0.5% of epsilon-polylysine and the balance of normal saline.

The preparation method comprises the following steps:

preparing plasma high molecular protein extract

(1) Collecting fresh animal sheep anticoagulant plasma qualified by health inspection, adding 25% saturation ammonium sulfate according to volume, stirring to completely dissolve the ammonium sulfate, standing for about 2 hours, filtering with 500 meshes, and collecting precipitate to obtain a crude product of the macromolecular protein extract. The filtrate can be used as animal protein by-product after heating and desalting.

(2) Virus inactivation: adding 3 times volume of pure water into the crude product of the macromolecular protein extract to dilute the crude product into porridge, putting the porridge into a reaction kettle, heating the porridge to 60 ℃, preserving the heat for 10 hours, and inactivating viruses.

(3) And (3) cyclic ultrafiltration desalination: and (3) performing virus inactivation on the crude product of the macromolecular protein extract according to the weight ratio of 1: 5, diluting the solution with pure water, centrifuging (4000 r/min, 20 min) to remove insoluble precipitates, collecting the centrifugate, performing ultrafiltration treatment on the centrifugate by an ultrafiltration device with the molecular weight cutoff of 100KD, adding pure water with the volume 5 times that of the retentate into the retentate when the retentate is concentrated to 1/5 of the original volume, continuously performing ultrafiltration desalination to 1/5 of the original volume, repeating the step for 3 times until sulfate ions in the permeate are completely removed, and collecting the retentate. The permeate liquid containing ammonium sulfate in the cyclic desalting process can be reused as the dissolving liquid for dissolving the crude product of the macromolecular protein extract next time.

(4) And (3) ultrafiltration refining: and (3) carrying out ultrafiltration concentration on the trapped fluid in the step (2) by using ultrafiltration equipment with the molecular weight cutoff of 300KD, carrying out ultrafiltration until the volume of the trapped fluid is 1/5 of the original volume, and collecting the trapped fluid, namely the refined liquid of the plasma high molecular protein extract (the total protein content is more than or equal to 20 mg/ml).

(II) preparing tea tree root extract

(1) Superfine grinding: collecting qualified tea tree root raw material, pulverizing with an ultrafine pulverizer, placing into a reaction kettle, adding water 10 times of the total weight of the raw material, adjusting pH to 7.0, adding 20% sodium chloride, and soaking for more than 24 hr. (2) Heat and ultrasonic treatment: the autoclave was heated with stirring to 90 ℃ for 60 minutes, allowed to cool to room temperature, and subjected to ultrasonic lysis (500W) for 60 minutes.

(3) Alkaline enzymolysis: adjusting pH of lysate to 12.0 with 10% sodium hydroxide, adding 5% mixed enzyme (pancreatin and cellulase) and 2% active carbon, mixing, heating to 50 deg.C under continuous stirring (rotation speed 150 r/min), and hydrolyzing for 10 hr.

(4) Filtering and clarifying: filtering the enzymolysis solution with 300 mesh, clarifying the filtrate with a decanter centrifuge, and fine filtering with 0.6 μm inorganic membrane to obtain crude tea tree root solution. The filter residue is treated as a feed byproduct.

(5) And (3) ultrafiltration refining: and (3) passing the crude product liquid through an ultrafiltration device with the molecular weight cutoff of 1 ten thousand, wherein the permeate liquid is the refined extract of the tea tree root (the content of the total flavone is more than or equal to 30 percent, and the content of the tea polysaccharide is more than or equal to 10 percent). The collected trapped fluid can be added into the next batch of enzymolysis step to be reused by being matched with alkali liquor.

(III) preparing the finished product

Adding 10% of high molecular protein refined solution, 30% of tea tree root extract, 0.2% of ceramide and 0.5% of epsilon-polylysine in sequence according to a formula, preparing active material liquid by using the balance of normal saline according to the formula proportion, carrying out fine filtration and microporous membrane sterilization on the active material liquid by using 1.0 mu m, 0.65 mu m and 0.22 mu m filter membranes respectively, adding 20% of autoclaved glycerol according to the proportion, mixing uniformly, adjusting the pH value to 9.0, carrying out sterile subpackage, and carrying out freezing storage.

Example 2

The product of the invention can be prepared into aqua according to the requirements of patients and wound surfaces. The product comprises the following specific formula in parts by weight: 2% of plasma high molecular protein extract, 10% of tea tree root extract, 0.05% of ceramide, 10% of glycerol, 0.02% of epsilon-polylysine and the balance of physiological saline.

The preparation method comprises the following steps:

preparing plasma high molecular protein extract

(1) Collecting fresh animal cattle anticoagulated plasma qualified by sanitary inspection, adding 20 saturation ammonium sulfate according to volume, stirring to completely dissolve the ammonium sulfate, standing for about 2 hours, centrifuging (4000 r/min, 20 min), and collecting precipitate as crude product of high molecular protein extract. The centrifugate can be used as animal protein by-product after heating and desalting.

(2) Virus inactivation: adding 3 times volume of pure water into the crude product of the macromolecular protein extract to dilute the crude product into porridge, putting the porridge into a reaction kettle, heating the porridge to 60 ℃, preserving the heat for 10 hours, and inactivating viruses.

(3) And (3) cyclic ultrafiltration desalination: and (3) performing virus inactivation on the crude product of the macromolecular protein extract according to the weight ratio of 1: 4, diluting the solution with pure water, centrifuging (3000 r/min, 15 min) to remove insoluble precipitates, collecting centrifugate, performing ultrafiltration treatment on the centrifugate by an ultrafiltration device with the molecular weight cutoff of 100KD, adding pure water with the volume 4 times that of the retentate into the retentate when the retentate is concentrated to 1/4 of the original volume, continuously performing ultrafiltration desalination to 1/4 of the original volume, repeating the step for 2 times until sulfate ions in the permeate are completely removed, and collecting the retentate. The permeate liquid containing ammonium sulfate in the cyclic desalting process can be reused as the dissolving liquid for dissolving the crude product of the macromolecular protein extract next time.

(4) And (3) ultrafiltration refining: and (3) carrying out ultrafiltration concentration on the trapped fluid in the step (2) by using ultrafiltration equipment with the molecular weight cutoff of 300KD, carrying out ultrafiltration until the volume of the trapped fluid is 1/3 of the original volume, and collecting the trapped fluid, namely the refined liquid of the plasma high molecular protein extract (the total protein content is more than or equal to 20 mg/ml).

(II) preparing tea tree root extract

(1) Superfine grinding: collecting qualified tea tree root raw material, pulverizing with an ultrafine pulverizer, placing into a reaction kettle, adding water 8 times of the total weight of the raw material, adjusting pH to 6.0, adding 10 times of sodium chloride, and soaking for more than 24 hr. (2) Heat and ultrasonic treatment: the autoclave was heated with stirring to 85 ℃ for 40 minutes, cooled to room temperature, and subjected to ultrasonic lysis (400W) for 50 minutes.

(3) Alkaline enzymolysis: adjusting pH of lysate to 11.0 with 10% sodium hydroxide, adding 2% mixed enzyme (pancreatin and cellulase) and 0.5% active carbon, mixing, heating to 45-50 deg.C under continuous stirring (rotation speed 130 r/min), and hydrolyzing for 8-10 hr.

(4) Filtering and clarifying: filtering the enzymolysis solution with 100 mesh, clarifying the filtrate with a decanter centrifuge, and fine filtering with 0.45 μm inorganic membrane to obtain crude tea tree root solution. The filter residue is treated as a feed byproduct.

(5) And (3) ultrafiltration refining: and (3) passing the crude product liquid through an ultrafiltration device with the molecular weight cutoff of 1 ten thousand, wherein the permeate liquid is the refined extract of the tea tree root (the content of the total flavone is more than or equal to 30 percent, and the content of the tea polysaccharide is more than or equal to 10 percent). The collected trapped fluid can be added into the next batch of enzymolysis step to be reused by being matched with alkali liquor.

(III) preparing the finished product

Adding 2% of high molecular protein refined solution, 10% of tea tree root extract, 0.05% of ceramide and 0.02% of epsilon-polylysine in sequence according to a formula, preparing active material liquid by using the balance of normal saline according to the formula proportion, carrying out fine filtration and microporous membrane sterilization on the active material liquid by using 1.0 mu m, 0.65 mu m and 0.22 mu m filter membranes respectively, adding 10% of autoclaved glycerol according to the proportion, uniformly mixing, adjusting the pH value to 7.5, carrying out sterile subpackage, and carrying out frozen storage.

Example 3

The product of the invention can be prepared into ointment according to the requirements of patients and wound surfaces. The product comprises the following specific formula in parts by weight: 10% of plasma high molecular protein extract, 30% of tea tree root extract, 0.2% of ceramide, 5% of borneol, 0.5% of epsilon-polylysine, 15% of glycerol, 2% of carbomer and the balance of normal saline.

The preparation method comprises the following steps:

preparing plasma high molecular protein extract

(1) Collecting fresh animal pig anticoagulant plasma qualified by health inspection, adding 22% saturation ammonium sulfate according to volume, stirring to completely dissolve the ammonium sulfate, standing for about 2 hours, filtering (400 meshes), and collecting precipitate as a crude product of the macromolecular protein extract. The filtrate can be used as animal protein by-product after heating and desalting.

(2) Virus inactivation: adding 3 times volume of pure water into the crude product of the macromolecular protein extract to dilute the crude product into porridge, putting the porridge into a reaction kettle, heating the porridge to 60 ℃, preserving the heat for 10 hours, and inactivating viruses.

(3) And (3) cyclic ultrafiltration desalination: and (3) performing virus inactivation on the crude product of the macromolecular protein extract according to the weight ratio of 1: 5, diluting the solution with pure water, centrifuging (4000 r/min, 20 min) to remove insoluble precipitates, collecting the centrifugate, performing ultrafiltration treatment on the centrifugate by an ultrafiltration device with the molecular weight cutoff of 100KD, adding pure water with the volume 5 times that of the retentate into the retentate when the retentate is concentrated to 1/5 of the original volume, continuously performing ultrafiltration desalination to 1/5 of the original volume, repeating the step for 3 times until sulfate ions in the permeate are completely removed, and collecting the retentate. The permeate liquid containing ammonium sulfate in the cyclic desalting process can be reused as the dissolving liquid for dissolving the crude product of the macromolecular protein extract next time.

(4) And (3) ultrafiltration refining: and (3) carrying out ultrafiltration concentration on the trapped fluid in the step (2) by using ultrafiltration equipment with the molecular weight cutoff of 300KD, carrying out ultrafiltration until the volume of the trapped fluid is 1/5 of the original volume, and collecting the trapped fluid, namely the refined liquid of the plasma high molecular protein extract (the total protein content is more than or equal to 20 mg/ml).

(II) preparing tea tree root extract

(1) Superfine grinding: collecting qualified tea tree root raw material, pulverizing with an ultrafine pulverizer, placing into a reaction kettle, adding water 10 times of the total weight of the raw material, adjusting pH to 6.5, adding 15% sodium chloride, and soaking for more than 24 hr. (2) Heat and ultrasonic treatment: the autoclave was heated with stirring to 90 ℃ for 50 minutes, allowed to cool to room temperature, and subjected to ultrasonic lysis (450W) for 50 minutes.

(3) Alkaline enzymolysis: adjusting pH of lysate to 11.0 with 10% sodium hydroxide, adding 4% mixed enzyme (pancreatin and cellulase) and 2% active carbon, mixing, heating to 50 deg.C under continuous stirring (rotation speed 150 r/min), and hydrolyzing for 10 hr.

(4) Filtering and clarifying: filtering the enzymolysis solution with 200 mesh, clarifying the filtrate with a decanter centrifuge, and fine filtering with 0.6 μm inorganic membrane to obtain crude tea tree root solution. The filter residue is treated as a feed byproduct.

(5) And (3) ultrafiltration refining: and (3) passing the crude product liquid through an ultrafiltration device with the molecular weight cutoff of 1 ten thousand, wherein the permeate liquid is the refined extract of the tea tree root (the content of the total flavone is more than or equal to 30 percent, and the content of the tea polysaccharide is more than or equal to 10 percent). The collected trapped fluid can be added into the next batch of enzymolysis step to be reused by being matched with alkali liquor.

(III) preparing the finished product

Adding 10% of high molecular protein refined solution, 30% of tea tree root extract, 0.2% of ceramide and 0.5% of epsilon-polylysine in sequence according to a paste formula, preparing active material liquid by using the balance of normal saline according to the formula proportion, carrying out fine filtration and microporous membrane sterilization on the active material liquid by using 1.0 mu m, 0.65 mu m and 0.22 mu m filter membranes respectively, adding 15% of autoclaved glycerol, 5% of borneol and 2% of carbomer according to the proportion, mixing uniformly, adjusting the pH value to 9.0, carrying out sterile subpackage, and refrigerating for storage.

Example 4

The product of the invention can be prepared into ointment according to the requirements of patients and wound surfaces. The product comprises the following specific formula in parts by weight: 3% of plasma high-molecular protein extract, 20% of tea tree root extract, 0.06% of ceramide, 1% of borneol, 0.02% of epsilon-polylysine, 5% of glycerol, 0.5% of carbomer and the balance of physiological saline.

The preparation method comprises the following steps:

preparing plasma high molecular protein extract

(1) Collecting fresh animal cattle anticoagulated plasma qualified by health inspection, adding 20 saturation ammonium sulfate according to volume, stirring to completely dissolve the ammonium sulfate, standing for about 2 hours, centrifuging (3000 min, 15 min), and collecting precipitate as crude product of high molecular protein extract. The centrifugate can be used as animal protein by-product after heating and desalting.

(2) Virus inactivation: adding 3 times volume of pure water into the crude product of the macromolecular protein extract to dilute the crude product into porridge, putting the porridge into a reaction kettle, heating the porridge to 60 ℃, preserving the heat for 10 hours, and inactivating viruses.

(3) And (3) cyclic ultrafiltration desalination: and (3) performing virus inactivation on the crude product of the macromolecular protein extract according to the weight ratio of 1: 4, diluting the solution with pure water, centrifuging (3000 r/min, 18 min) to remove insoluble precipitates, collecting centrifugate, performing ultrafiltration treatment on the centrifugate by an ultrafiltration device with the molecular weight cutoff of 100KD, adding pure water with the volume 4 times that of the retentate into the retentate when the retentate is concentrated to 1/4 of the original volume, continuously performing ultrafiltration desalination to 1/4 of the original volume, repeating the step for 2 times until sulfate ions in the permeate are completely removed, and collecting the retentate. The permeate liquid containing ammonium sulfate in the cyclic desalting process can be reused as the dissolving liquid for dissolving the crude product of the macromolecular protein extract next time.

(4) And (3) ultrafiltration refining: and (3) carrying out ultrafiltration concentration on the trapped fluid in the step (2) by using ultrafiltration equipment with the molecular weight cutoff of 300KD, carrying out ultrafiltration until the volume of the trapped fluid is 1/3 of the original volume, and collecting the trapped fluid, namely the refined liquid of the plasma high molecular protein extract (the total protein content is more than or equal to 20 mg/ml).

(II) preparing tea tree root extract

(1) Superfine grinding: collecting qualified tea tree root raw material, pulverizing with an ultrafine pulverizer, placing into a reaction kettle, adding water 8 times of the total weight of the raw material, adjusting pH to 6.0, adding 10% sodium chloride, and soaking for more than 24 hr. (2) Heat and ultrasonic treatment: the autoclave was heated with stirring to 85 ℃ for 30 minutes, allowed to cool to room temperature, and subjected to ultrasonic lysis (400W) for 30 minutes.

(3) Alkaline enzymolysis: adjusting pH of lysate to 11.0 with 10% sodium hydroxide, adding 1% mixed enzyme (pancreatin and cellulase) and 0.5% active carbon, mixing well, heating to 45 deg.C under continuous stirring (rotation speed 130 r/min), and hydrolyzing for 8 hr.

(4) Filtering and clarifying: filtering the enzymolysis solution with 100 mesh, clarifying the filtrate with a decanter centrifuge, and fine filtering with 0.45 μm inorganic membrane to obtain crude tea tree root solution. The filter residue is treated as a feed byproduct.

(5) And (3) ultrafiltration refining: and (3) passing the crude product liquid through an ultrafiltration device with the molecular weight cutoff of 1 ten thousand, wherein the permeate liquid is the refined extract of the tea tree root (the content of the total flavone is more than or equal to 30 percent, and the content of the tea polysaccharide is more than or equal to 10 percent). The collected trapped fluid can be added into the next batch of enzymolysis step to be reused by being matched with alkali liquor.

(III) preparing the finished product

Adding 3% of high molecular protein refined solution, 20% of tea tree root extract, 0.06% of ceramide and 0.02% of epsilon-polylysine in sequence according to a paste formula, preparing active material liquid by using the balance of normal saline according to the formula proportion, carrying out fine filtration and microporous membrane sterilization on the active material liquid by using 1.0 mu m, 0.65 mu m and 0.22 mu m filter membranes respectively, adding 5% of autoclaved glycerol, 1% of borneol and 0.5% of carbomer according to the proportion, mixing uniformly, adjusting the pH value to 8.5, carrying out sterile subpackage, and refrigerating and storing.

Example 5

The product of the invention can be prepared into freeze-dried powder according to the requirements of patients and wound surfaces. The product comprises the following specific formula in parts by weight: 9% of plasma high molecular protein extract, 25% of tea tree root extract, 0.2% of ceramide, 4% of borneol, 10% of polyethylene glycol (6000), 0.4% of epsilon-polylysine and the balance of normal saline.

The preparation method comprises the following steps:

preparing plasma high molecular protein extract

(1) Collecting fresh animal cattle anticoagulated plasma qualified by health inspection, adding 25% saturation ammonium sulfate according to volume, stirring to completely dissolve ammonium sulfate, standing for about 2 hr, centrifuging (3500 r/min, 20 min), and collecting precipitate as crude product of high molecular protein extract. The centrifugate can be used as animal protein by-product after heating and desalting.

(2) Virus inactivation: adding 3 times volume of pure water into the crude product of the macromolecular protein extract to dilute the crude product into porridge, putting the porridge into a reaction kettle, heating the porridge to 60 ℃, preserving the heat for 10 hours, and inactivating viruses.

(3) And (3) cyclic ultrafiltration desalination: and (3) performing virus inactivation on the crude product of the macromolecular protein extract according to the weight ratio of 1: 5, diluting the solution with pure water, centrifuging (4000 r/min, 20 min) to remove insoluble precipitates, collecting the centrifugate, performing ultrafiltration treatment on the centrifugate by an ultrafiltration device with the molecular weight cutoff of 100KD, adding pure water with the volume 5 times that of the retentate into the retentate when the retentate is concentrated to 1/5 of the original volume, continuously performing ultrafiltration desalination to 1/5 of the original volume, repeating the step for 3 times until sulfate ions in the permeate are completely removed, and collecting the retentate. The permeate liquid containing ammonium sulfate in the cyclic desalting process can be reused as the dissolving liquid for dissolving the crude product of the macromolecular protein extract next time.

(4) And (3) ultrafiltration refining: and (3) carrying out ultrafiltration concentration on the trapped fluid in the step (2) by using ultrafiltration equipment with the molecular weight cutoff of 300KD, carrying out ultrafiltration until the volume of the trapped fluid is 1/4 of the original volume, and collecting the trapped fluid, namely the refined liquid of the plasma high molecular protein extract (the total protein content is more than or equal to 20 mg/ml).

(II) preparing tea tree root extract

(1) Superfine grinding: collecting qualified tea tree root raw material, pulverizing with an ultrafine pulverizer, placing into a reaction kettle, adding water 9 times the total weight of the raw material, adjusting pH to 7.0, adding 15% sodium chloride, and soaking for more than 24 hr. (2) Heat and ultrasonic treatment: the autoclave was heated with stirring to 88 ℃ for 55 minutes, allowed to cool to room temperature, and subjected to ultrasonic lysis (500W) for 60 minutes.

(3) Alkaline enzymolysis: adjusting pH of lysate to 12.0 with 10% sodium hydroxide, adding 5% mixed enzyme (pancreatin and cellulase) and 2% active carbon, mixing, heating to 50 deg.C under continuous stirring (rotation speed 140 r/min), and hydrolyzing for 10 hr.

(4) Filtering and clarifying: filtering the enzymolysis solution with 250 mesh, clarifying the filtrate with a decanter centrifuge, and fine filtering with 0.6 μm inorganic membrane to obtain crude tea tree root solution. The filter residue is treated as a feed byproduct.

(5) And (3) ultrafiltration refining: and (3) passing the crude product liquid through an ultrafiltration device with the molecular weight cutoff of 1 ten thousand, wherein the permeate liquid is the refined extract of the tea tree root (the content of the total flavone is more than or equal to 30 percent, and the content of the tea polysaccharide is more than or equal to 10 percent). The collected trapped fluid can be added into the next batch of enzymolysis step to be reused by being matched with alkali liquor.

(III) preparing the finished product

Adding 9% of high molecular protein refined solution, 25% of tea tree root extract, 0.2% of ceramide and 0.4% of epsilon-polylysine in sequence according to a powder formula, preparing active material liquid by using the balance of normal saline according to the formula proportion, carrying out fine filtration and microporous membrane sterilization on the active material liquid by using 1.0 mu m, 0.65 mu m and 0.22 mu m filter membranes respectively, adding 4% of borneol and 10% of sterilized polyethylene glycol (6000) according to the proportion, mixing uniformly, adjusting the pH value to 9.0, putting into a freeze dryer for freeze-drying, carrying out sterile subpackaging, and refrigerating and storing.

Example 6

The product of the invention can be prepared into freeze-dried powder according to the requirements of patients and wound surfaces. The product comprises the following specific formula in parts by weight: 3% of plasma high molecular protein extract, 12% of tea tree root extract, 0.08% of ceramide, 2% of borneol, 6% of polyethylene glycol (8000), 0.05% of epsilon-polylysine and the balance of normal saline.

The preparation method comprises the following steps:

preparing plasma high molecular protein extract

(1) Collecting fresh animal sheep anticoagulant plasma qualified by health inspection, adding 20% saturation ammonium sulfate according to volume, stirring to completely dissolve the ammonium sulfate, standing for about 2 hours, filtering (300 meshes), and collecting precipitate as crude product of high molecular protein extract. The filtrate can be used as animal protein by-product after heating and desalting.

(2) Virus inactivation: adding 3 times volume of pure water into the crude product of the macromolecular protein extract to dilute the crude product into porridge, putting the porridge into a reaction kettle, heating the porridge to 60 ℃, preserving the heat for 10 hours, and inactivating viruses.

(3) And (3) cyclic ultrafiltration desalination: and (3) performing virus inactivation on the crude product of the macromolecular protein extract according to the weight ratio of 1: 4, diluting the solution with pure water, centrifuging (3000 r/min, 15 min) to remove insoluble precipitates, collecting centrifugate, performing ultrafiltration treatment on the centrifugate by an ultrafiltration device with the molecular weight cutoff of 100KD, adding pure water with the volume 4 times that of the retentate into the retentate when the retentate is concentrated to 1/4 of the original volume, continuously performing ultrafiltration desalination to 1/4 of the original volume, repeating the step for 2 times until sulfate ions in the permeate are completely removed, and collecting the retentate. The permeate liquid containing ammonium sulfate in the cyclic desalting process can be reused as the dissolving liquid for dissolving the crude product of the macromolecular protein extract next time.

(4) And (3) ultrafiltration refining: and (3) carrying out ultrafiltration concentration on the trapped fluid in the step (2) by using ultrafiltration equipment with the molecular weight cutoff of 300KD, carrying out ultrafiltration until the volume of the trapped fluid is 1/3 of the original volume, and collecting the trapped fluid, namely the refined liquid of the plasma high molecular protein extract (the total protein content is more than or equal to 20 mg/ml).

(II) preparing tea tree root extract

(1) Superfine grinding: collecting qualified tea tree root raw material, pulverizing with an ultrafine pulverizer, placing into a reaction kettle, adding water 8 times of the total weight of the raw material, adjusting pH to 6.0, adding 10% sodium chloride, and soaking for more than 24 hr. (2) Heat and ultrasonic treatment: the autoclave was heated with stirring to 85 ℃ for 35 minutes, allowed to cool to room temperature, and subjected to ultrasonic lysis (400W) for 35 minutes.

(3) Alkaline enzymolysis: adjusting pH of lysate to 11.0 with 10% sodium hydroxide, adding 2% mixed enzyme (pancreatin and cellulase) and 0.5% active carbon, mixing well, heating to 45 deg.C under continuous stirring (rotation speed 130 r/min), and hydrolyzing for 8 hr.

(4) Filtering and clarifying: filtering the enzymolysis solution with 100 mesh, clarifying the filtrate with a decanter centrifuge, and fine filtering with 0.45 μm inorganic membrane to obtain crude tea tree root solution. The filter residue is treated as a feed byproduct.

(5) And (3) ultrafiltration refining: and (3) passing the crude product liquid through an ultrafiltration device with the molecular weight cutoff of 1 ten thousand, wherein the permeate liquid is the refined extract of the tea tree root (the content of the total flavone is more than or equal to 30 percent, and the content of the tea polysaccharide is more than or equal to 10 percent). The collected trapped fluid can be added into the next batch of enzymolysis step to be reused by being matched with alkali liquor.

(III) preparing the finished product

Adding 3% of high molecular protein refined solution, 12% of tea tree root extract, 0.08% of ceramide and 0.05% of epsilon-polylysine in sequence according to a powder formula, preparing active material liquid by using the balance of normal saline according to the formula proportion, carrying out fine filtration and microporous membrane sterilization on the active material liquid by using 1.0 mu m, 0.65 mu m and 0.22 mu m filter membranes respectively, adding 2% of borneol and 6% of sterilized polyethylene glycol (8000) according to the proportion, mixing uniformly, adjusting the pH value to 8.5, putting into a freeze dryer for freeze-drying, carrying out sterile subpackaging, and refrigerating and storing.

Example 7 pharmacodynamic experiment: the research on the application effect of the compound skin injury regeneration repairing agent on the rabbit III-degree burn is provided.

Test animals: 60 common New Zealand white rabbits with a clean grade, 3-4 months old rabbits, half female rabbits and half male rabbits, 2.5 +/-0.2 kg of body mass, purchased from Zhengzhou university laboratory animal center, all the rabbits are mixed and artificially fed in cages at the environmental temperature of 20-22 ℃ and the relative humidity of 50%, and after the rabbits are artificially fed with the mixed feed for 1 week, a III-degree burn model is constructed by an ethanol combustion method.

Test grouping and administration: the 60 model rabbits were randomly divided into 5 groups, an experimental group, a control group 1, a control group 2, a control group 3, a blank control group, and 12 rabbits per group. The medicine is taken 24 hours after the wounds of each group, and is taken once every morning and evening, and the specific grouping conditions of the medicines are shown in table 1 based on the fact that the medicines completely cover the wounds:

TABLE 1 administration of the experimental group and each control group

Group of	Administration of drugs	Preparation method
			Experimental group	Compound skin injury regeneration repairing agent	Same formulation phase as prepared in example 1All in one
Control group 1	Fibronectin formulations	Zhengzhou Fubor Biotechnology Ltd
			Control group 2	Beifuji (a medicine for rehabilitation)	Purchased from Zhuhaisheng biopharmaceutical Limited
Control group 3	Powder for removing putrefaction and promoting tissue regeneration	Chinese medicine Hospital supply of Henan province
			Control blank group	Physiological saline	0.85% sterilized normal saline

Observation indexes are as follows:

healing time of the wound surface: i.e. the time required for complete epithelialization of the wound surface. The standard for wound healing is that the crust skin of the wound falls off and new epithelium is completely formed. The observation was continued with naked eyes for 35 days.

Degree of skin healing:

healing in stage I: the repaired part contains a small amount of fibrotic tissues, no scar or a small amount of scar hyperplasia, the structure of the dermal layer is clear and complete, the fibroblasts and the collagen fibers are arranged orderly, and the sebaceous membrane is complete.

Healing in the II stage: the wound surface is mainly repaired by fibrous tissues, scar contracture or hyperplasia subcutaneous fibers are arranged in disorder, and the muscle lipid membrane is incomplete.

TABLE 2 curative effect of the compound skin injury regeneration repairing agent of the present invention on rabbit III degree burn

Group of	Time of healing	Number of healings in phase I	Number of healings in phase II
				Experimental group	17±1.2	9	3
Control group 1	18±1.5	8	4
				Control group 2	22±1.7	2	10
Control group 3	24±0.7	3	9
				Control blank group	26±1.3	12	0

As can be seen from table 2, the compound skin injury regeneration repair agent, the fibronectin group, the Beifuji group and the slough-removing and granulation-promoting powder group of the invention can shorten the healing time of the wound surface and have the effect of promoting healing after injury compared with the control group, and simultaneously, the compound skin injury regeneration repair agent and the fibronectin group of the invention also have the effects of remarkably promoting healing of the III degree burn in the stage I and reducing the hyperplasia of scar tissues of the III degree burn. The compound skin injury regeneration repairing agent group of the invention has the effect of promoting the healing of the wound in the I stage better than that of the fibronectin group, and has obvious difference. Therefore, the compound skin injury regeneration repairing agent has the function of promoting wound surface regeneration repairing without scars, and can be clinically applied to treatment of various skin injuries.

Claims (7)

1. A compound skin injury regeneration repairing agent is characterized in that the agent is prepared by taking plasma high molecular protein extract, tea tree root extract and ceramide as main materials and taking one or more of borneol, polyethylene glycol (4000-; wherein the ratio of the plasma high molecular protein extract is 1.5-10%, the ratio of the tea tree root extract is 10-30%, and the ratio of the ceramide is 0.05-0.2%; the compound skin injury regeneration repairing agent is in the form of aqua, paste or freeze-dried powder;

the plasma high-molecular protein extract is prepared by the following steps:

(1) collecting fresh animals qualified by hygienic inspection: adding 20-25% saturated ammonium sulfate into anticoagulant plasma of pig, cattle and sheep, stirring to completely dissolve ammonium sulfate, standing for about 2 hr, filtering or centrifuging at 300-mesh sieve of 500 meshes, collecting precipitate as crude product of high molecular protein extract, and heating and desalting filtrate or centrifugate to obtain animal protein byproduct;

(2) virus inactivation: adding 3 times of pure water into the crude product of the macromolecular protein extract to dilute the crude product into a porridge shape, putting the porridge into a reaction kettle, heating the porridge to 60 ℃, preserving heat for 10 hours, and inactivating viruses;

(3) and (3) cyclic ultrafiltration desalination: and (3) performing virus inactivation on the crude product of the macromolecular protein extract according to the weight ratio of 1: 4-1: 5, diluting the filtrate into solution by pure water, centrifuging to remove insoluble precipitate, collecting centrifugate, performing ultrafiltration treatment on the centrifugate by ultrafiltration equipment with the cut-off molecular weight of 100KD, when the cut-off liquid is concentrated to 1/4-1/5 of the original volume, adding pure water with the volume 4-5 times that of the cut-off liquid into the cut-off liquid, continuing ultrafiltration and desalination to 1/4-1/5 of the original volume, repeating the steps for 2-3 times until sulfate ions in the permeate are completely removed, and collecting cut-off liquid A; the permeate liquid containing ammonium sulfate in the cyclic desalting process can be reused as a dissolving liquid for dissolving the crude product of the macromolecular protein extract next time;

(4) and (3) ultrafiltration refining: ultrafiltering and concentrating the trapped fluid A finally obtained in the step (3) by using ultrafiltration equipment with the molecular weight cut-off of 300KD until the volume of the trapped fluid is 1/3-1/5 of the original volume, and collecting the trapped fluid, namely the refined liquid of the plasma high-molecular protein extract, wherein the total protein content in the refined liquid of the extract is more than or equal to 20 mg/ml;

the tea tree root extract is prepared by the following steps:

(1) superfine grinding: collecting qualified tea tree root raw material, pulverizing with an ultrafine pulverizer, placing into a reaction kettle, adding water 8-10 times of the total weight of the raw material, adjusting pH to 6.0-7.0, adding 10-20% sodium chloride, and soaking for more than 24 hr;

(2) heat and ultrasonic treatment: heating the reaction kettle to 85-90 ℃ under stirring, carrying out heat preservation for 30-60 minutes, cooling to room temperature, and carrying out ultrasonic cracking for 30-60 minutes;

(3) alkaline enzymolysis: the lysate is adjusted to pH11.0-12.0 with 10% sodium hydroxide, 1-5% mixed enzyme is added: pancreatin, cellulase and 0.5-2% of active carbon are evenly mixed, and the mixture is heated to 45-50 ℃ under continuous stirring for hydrolysis for 8-10 hours;

(4) filtering and clarifying: filtering the enzymolysis solution with 100-mesh and 300-mesh filter, clarifying the filtrate with a decanter centrifuge, and fine-filtering the crude tea tree root solution with 0.45-0.6 μm inorganic membrane; the filter residue is treated as a feed byproduct;

(5) and (3) ultrafiltration refining: passing the crude product liquid through an ultrafiltration device with the molecular weight cutoff of 1 ten thousand, wherein the permeate liquid is the refined liquid of the tea root extract, the content of the total flavonoids in the refined liquid of the extract is more than or equal to 30 percent, and the content of the tea polysaccharide is more than or equal to 10 percent; the collected trapped fluid can be added into the next batch of enzymolysis step to be reused by being matched with alkali liquor.

2. The compound skin injury regeneration repairing agent as claimed in claim 1, wherein the aqueous formula comprises the following components by mass percent:

1.5-10% of plasma high-molecular protein extract

10-30% of tea tree root extract

Ceramide 0.05-0.2%

10-20% of glycerin

0.01 to 0.5 percent of epsilon-polylysine

The balance of physiological saline, pH7.5-9.0.

3. The compound skin injury regeneration repairing agent according to claim 1, wherein the ointment formula comprises the following components in percentage by mass:

1.5-10% of plasma high-molecular protein extract

10-30% of tea tree root extract

Ceramide 0.05-0.2%

1 to 5 percent of borneol

0.01 to 0.5 percent of epsilon-polylysine

5-15% of glycerin

Carbomer 0.1-2%

The balance of physiological saline, pH7.5-9.0.

4. The compound skin injury regeneration repairing agent as claimed in claim 1, wherein the freeze-dried powder formulation comprises the following components by mass percent:

1.5-10% of plasma high-molecular protein extract

10-30% of tea tree root extract

Ceramide 0.05-0.2%

1 to 5 percent of borneol

5-10% of polyethylene glycol (4000-

0.01 to 0.5 percent of epsilon-polylysine

The balance of physiological saline, pH7.5-9.0.

5. A method for preparing the compound skin injury regeneration and repair agent as claimed in claim 1 or 2, which is characterized in that: the preparation method of the repairing agent aqueous solution comprises the following steps:

adding 1.5-10% of plasma high molecular protein extract, 10-30% of tea tree root extract, 0.05-0.2% of ceramide and 0.01-0.5% of epsilon-polylysine according to a formula in turn, preparing an active material liquid by using the balance of normal saline according to the formula proportion, carrying out fine filtration and micropore membrane sterilization on the active material liquid by using filter membranes with the diameters of 1.0 mu m, 0.65 mu m and 0.22 mu m respectively, adding 10-20% of autoclaved glycerol according to the proportion, mixing uniformly, adjusting the pH value to 7.5-9.0, carrying out sterile subpackaging and freezing storage.

6. A method for preparing the compound skin injury regeneration and repair agent as claimed in claim 1 or 3, which is characterized in that: the preparation method of the repairing agent paste comprises the following steps:

adding 1.5-10% of plasma high molecular protein extract, 10-30% of tea tree root extract, 0.05-0.2% of ceramide and 0.01-0.5% of epsilon-polylysine according to a formula in sequence, preparing an active material liquid by using the balance of normal saline according to the formula proportion, performing fine filtration and microporous membrane sterilization on the active material liquid by using a filter membrane with the diameter of 1.0 mu m, the diameter of 0.65 mu m and the diameter of 0.22 mu m respectively, adding 10-20% of autoclaved glycerol, 1-5% of borneol and 0.1-2% of carbomer according to the proportion, mixing uniformly, adjusting the pH value to 7.5-9.0, performing sterile subpackaging, and refrigerating for storage.

7. A method for preparing the compound skin injury regeneration and repair agent as claimed in claim 1 or 4, which is characterized in that: the preparation method of the repairing agent freeze-dried powder comprises the following steps:

adding 1.5-10% of plasma high molecular protein extract, 10-30% of tea tree root extract, 0.05-0.2% of ceramide and 0.01-0.5% of epsilon-polylysine according to a powder formula in sequence, preparing an active material liquid by using the balance of normal saline according to the formula proportion, carrying out fine filtration and microporous membrane sterilization on the active material liquid by using filter membranes with the particle size of 1.0 mu m, 0.65 mu m and 0.22 mu m respectively, adding 1-5% of borneol and 5-10% of sterilized polyethylene glycol (4000-8000), uniformly mixing, adjusting the pH value to 7.5-9.0, freeze-drying in a freeze dryer, carrying out sterile subpackaging, and carrying out refrigeration storage.