CN114767786A

CN114767786A - External skin-generating oil for preventing and treating liquid nitrogen ultralow-temperature frostbite and preparation method and application thereof

Info

Publication number: CN114767786A
Application number: CN202210524959.XA
Authority: CN
Inventors: 唐和斌; 张朝; 李玉桑; 蒙莎莎; 唐晟杰
Original assignee: South Central University for Nationalities
Current assignee: South Central Minzu University
Priority date: 2022-05-13
Filing date: 2022-05-13
Publication date: 2022-07-22
Anticipated expiration: 2042-05-13
Also published as: CN114767786B

Abstract

The invention relates to the technical field of new drug indications, and particularly discloses an external skin-generating oil for preventing and treating liquid nitrogen ultralow-temperature frostbite, and a preparation method and application thereof. The external skin-generating oil is prepared by mixing the following raw materials in parts by weight in a batch heating mode, a flash extraction mode and the like: 10-50 parts of scutellaria baicalensis, 10-50 parts of coptis chinensis, 10-50 parts of phellodendron bark, 10-50 parts of rheum officinale, 10-30 parts of astragalus membranaceus, 10-30 parts of angelica sinensis, 10-30 parts of sanguisorba officinalis, 10-30 parts of frankincense, 10-30 parts of myrrh, 10-30 parts of rehmannia, 10-30 parts of bletilla striata, 10-30 parts of angelica dahurica, 10-30 parts of cynanchum atratum, 3-8 parts of borneol and 1500 parts of edible vegetable oil by weight. Scientific experiments prove that the external skin-generating oil preparation has better effects of purging fire, detoxifying, activating blood, removing stasis, removing slough and promoting granulation, can promote quick rewarming of rats in a short time after liquid nitrogen ultralow-temperature frostbite, and can accelerate early healing of the rat wound surface of the liquid nitrogen ultralow-temperature frostbite and traceless repair of the skin tissue structure of the frostbite wound surface.

Description

External skin-generating oil for preventing and treating liquid nitrogen ultralow-temperature frostbite and preparation method and application thereof

Technical Field

The invention relates to the technical field of new drug indications, in particular to an external skin-generating oil for preventing and treating liquid nitrogen ultralow-temperature frostbite, and a preparation method and application thereof.

Background

Frostbite is a localized inflammatory dermatosis at the tip part and even the injury of the whole body caused by cold (such as using liquid ammonia, dry ice and other substances in the work of medical treatment, scientific research and the like or extreme cold weather) under certain conditions, is a common disease in winter, and is characterized by congestive edema erythema at the part with poor peripheral blood circulation and the exposed part and skin pruritus at high temperature, and the phenomena of skin erosion, ulcer and the like at the affected part can occur in severe people. According to statistics of a certain cold region army in China on a frostbite group and vulnerable parts thereof in recent years, it is found that up to 9.35 percent of officers and soldiers are damaged by low-temperature frostbite, and the vulnerable parts comprise 52.8 percent of feet, 36.5 percent of hands and 11.7 percent of face.

After the local skin is stimulated by a cold environment, blood vessels shrink strongly to cause tissue ischemia, and if the temperature is further reduced, the tissue freezes. Rapidly freezing to form intracellular ice crystals, and slowly freezing to form intercellular ice crystals; the formation of ice crystals causes mechanical action on tissue cells, so that intercellular bridges are broken or cell membranes are ruptured, cells are dehydrated, cell contents are overflowed, and the concentrations of intracellular electrolyte enzymes, sugars and the like are increased, which is also an important cause of cell death. Once the tissue is free from freezing, in the process of rewarming, blood expands, and blood can quickly stagnate after entering expanded micro-blood vessels, so that exudate is increased, edema is formed, plasma extravasates, and blood is concentrated, so that thrombosis and microcirculation disturbance are caused, and the tissue is more ischemic and then necrotized. Meanwhile, the tissue is dehydrated (the dehydration degree can reach 85-90%) due to frostbite, protein denaturation and enzyme activity are reduced, cells are shrunk, and energy metabolites in the cells are exhausted and lost, so that the respiration rate of cell mitochondria is reduced, and a large amount of intermediate products are accumulated. This is the major cause of death of frozen tissue.

The professional treatment goal of frostbite is to rapidly rewet, prevent further cold exposure and restore blood circulation. Early treatments for frostbite included covering the frozen area or other body surface with clothing or warm hands to maintain adequate blood supply. At the same time, rapid water bath (38-42 ℃) is needed for rewarming, ice is prohibited to wipe the frozen limbs, and the body is dry and hot or slowly rewarming because the ice can further damage tissues. In addition, supportive care should be taken, such as bed rest, high protein/high calorie diet, wound protection and trauma avoidance. In hypothermic patients with frostbite, it is most important to complete fluid resuscitation and restore core body temperature before limb rewarming to prevent sudden hypotension and shock. Anticoagulants are suggested to prevent thrombosis and gangrene, antibacterial agents to prevent infection, and timely immunization with tetanus antitoxin. If there are local lesions such as blisters and necrotic tissues, surgical operation should be performed in time.

Traditional Chinese medicine believes that the frostbite is caused by lingering cold evil, skin and flesh, blood vessels and qi and blood stagnation, so that the limbs lose warm nourishment and become frostbite. If the skin is slightly rotten, the skin is infected with the pathogenic toxin again, the cold pathogen is transformed into heat, and the meat is rotten due to excessive heat, so that the fat is suppurated; if the disease is serious, the limbs are rotten, and the bones are disconnected with tendons to form gangrene. Especially severe cold entering zang-organs, internal closure of yin-qi, external collapse of yang-qi, accumulation of astringency in ying-wei, no circulation again, and cold stiffness; if the patient is treated early, blood, temperature and qi will be ventilated, otherwise, the patient will die due to vomiting. The principle of warming meridians and expelling cold, removing stasis and dredging collaterals is usually adopted by many doctors to treat the disease, and the medicines for tonifying qi and nourishing blood, and harmonizing ying and dredging collaterals are usually selected.

Unfortunately, no matter in western medicine or in clinical practice, a specific medicine with obvious and quick curative effect, convenient use and no toxic or side effect, especially an external medicine, is still lacked. Therefore, the active search for effective drugs for treating frostbite, especially frostbite caused by extreme cold conditions (such as ultra-low temperature frostbite) is the focus of attention of many medical researchers, and is also the main object of the present invention.

Disclosure of Invention

Aiming at the problem that the prior art is lack of specific medicines for preventing and treating the freezing injury of the liquid nitrogen at the ultralow temperature:

the first purpose of the invention is to provide the external skin-generating oil for effectively preventing and treating the frostbite caused by liquid nitrogen ultralow temperature.

The second purpose of the invention is to provide a preparation method of the external skin-growing oil for preventing and treating the cold injury at ultralow temperature by liquid nitrogen.

The third purpose of the invention is to provide the medical application of the external skin-generating oil for preventing and treating the cold injury caused by ultralow temperature and ultralow temperature of liquid nitrogen.

The invention concept of the invention is as follows:

the frostbite is the damage caused by the freezing and thawing process of the tissue, which is characterized in that the tissue cells are frozen, because the local or whole body of the human body is contacted with a severe cold environment or a medium (a refrigerant or liquid gas) to cause that the local tissue of the body is lower than the freezing temperature of the tissue (-3.6 to-2.5 ℃, also called as the biological freezing point). The result is that the limbs are often necrotized, amputated and other serious injuries, even life threatening. It is also a common disease in cold regions and plateau regions in China, has long course of disease, complex treatment and high disability rate, and particularly, up to 9.35 percent of officers and soldiers in troops in the region are easily threatened by frostbite.

The traditional treatment method for severe frostbite mainly comprises early-stage rapid rewarming and late-stage amputation treatment, and the frostbite guideline recommends rewarming (rewarming time is 15-60min) by using circulating antibacterial-drug-containing liquid (water temperature is 37-39 ℃) until the skin becomes red/purple or the extremity becomes flexible. Of course, the rapid rewarming can only solve the damage in the pre-freezing stage and the freezing stage so as to inhibit the formation of ice crystals by intracellular and extracellular fluids in the stage and reduce the tissue necrosis amputation rate. However, microthrombus formed by cryofreezing is a major factor in causing tissue ischemia and necrosis. The problem is often relieved by treatment such as vasodilatation (vasodilators such as prostaglandin E1, nitroglycerin, nifedipine and the like), thrombolysis (thrombolytics such as streptokinase and the like) and even surgical excision (amputation), thrombus prevention, circulation improvement, infection prevention and the like.

The traditional Chinese medicine considers that the cold injury is mainly caused by the insufficiency of body yang, or the extreme cold of muscles, the stagnation of qi and blood, the loss of smooth blood vessels and the blockage of channels and collaterals. Purple spots are formed at the beginning, become black after a long time, and become pus after rotten; it should warm yang and dispel cold, harmonize yingfen and weifen. At present, the medicines for treating frostbite in clinical traditional Chinese medicine can be divided into 3 types: group 1 are drugs for promoting blood circulation, promoting blood circulation and removing blood stasis, such as Carthami flos, radix Angelicae sinensis, radix Paeoniae, rhizoma Ligustici Chuanxiong, Saviae Miltiorrhizae radix, Notoginseng radix, caulis Spatholobi, etc.; class 2 is a heat-induced medicine for warming and dredging channels and collaterals, dispelling cold and relieving pain, such as ramulus Cinnamomi, herba asari, cortex Cinnamomi, radix Aconiti lateralis Preparata, Zingiberis rhizoma, Camphora, etc.; group 3 are herbs for tonifying healthy qi, tonifying blood and promoting qi circulation, such as Dang Shen, Huang Qi and shan Yao. Therefore, the medicines are usually selected by a plurality of Chinese medicine families to carry out compatibility, and the effects of promoting blood circulation to remove blood stasis, warming and activating meridians, and tonifying vital qi are exerted, so that the effect of improving the local frostbite is achieved.

The inventor of the technology is based on clinical experience of decades and scientific research accumulation of Chinese medicine pharmacology, is not completely considered to be the principle of the prescription medication aiming at the frostbite treatment of a plurality of Chinese medicine families (the essence of the principle is more suitable for the treatment of chilblain), and considers that the frostbite organism is attacked by extremely cold evil to cause stagnation of qi and blood and block channels and collaterals; and the pathogenic cold congeals the channel, causing rapid cold stasis and transformation of fire, which leads to pus formation and ulceration and sore formation. Therefore, the inventor of the technology puts forward a unique control view aiming at the pathogenesis of the formation of the frostbite, namely, the product for clearing heat and detoxicating, promoting blood circulation and removing blood stasis, astringing yin and promoting granulation is adopted to play the roles of relieving swelling and pain, removing slough and treating sore, and quickly repairing the cold frostbite. Just before, the inventors of the present technology have revealed that the topical skin-growing oil for treating skin burns and scalds meets the above-mentioned application principles and has the presumed effects. Therefore, the applicant speculates that the skin-regenerating oil for external use can also effectively cure the frostbite caused by the ultra-low temperature of liquid nitrogen.

Therefore, the applicant has conducted a great amount of scientific experimental examinations on the prevention and treatment of diseases caused by ultralow temperature of the skin oil, and researches the treatment effect of the prepared skin oil on diseases through external local administration by respectively establishing ultralow temperature frostbite animal models caused by liquid nitrogen. Research results show that when the skin-regenerating oil prepared by the method is used, the liquid nitrogen-induced cold injury of rats can be quickly restored to the original temperature (quickly evaluated within 5 minutes), the healing of the wound surfaces of the liquid nitrogen-induced cold injury rats can be accelerated (long-term monitoring within 20 days), and the skin tissue structures of the cold injury wound surfaces can be perfectly repaired; the prevention and treatment effect of the compound zinc oxide ointment is obviously superior to that of a contrast medicine composition (a combination of antibiotics and anti-inflammatory agents, namely the combination of mupirocin for external use combined with dexamethasone, and a combination of wound healing promoter combined with a microcirculation improving agent, namely the combination of compound zinc oxide ointment for external use and calcium dobesilate for internal use) which is commonly used in western medicine clinical practice.

In order to achieve the first object, the invention adopts the following technical measures:

an external skin-generating oil for preventing and treating liquid nitrogen ultralow-temperature frostbite is prepared from the following raw materials in parts by weight: 10-50 parts of scutellaria baicalensis, 10-50 parts of coptis chinensis, 10-50 parts of phellodendron, 10-50 parts of rheum officinale, 10-30 parts of astragalus membranaceus, 10-30 parts of angelica sinensis, 10-30 parts of sanguisorba officinalis, 10-30 parts of frankincense, 10-30 parts of myrrh, 10-30 parts of rehmannia, 10-30 parts of bletilla striata, 10-30 parts of angelica dahurica, 10-30 parts of cynanchum atratum, 3-8 parts of borneol and 1500 parts of edible vegetable oil by weight;

the edible vegetable oil is selected from at least one of sesame oil, coconut oil, peanut oil, soybean oil, linseed oil, castor oil and olive oil, preferably at least one of sesame oil and coconut oil.

In order to achieve the second purpose, the invention adopts the following technical measures:

a preparation method of the external skin-growing oil for preventing and treating the cold injury at the ultralow temperature by using the liquid nitrogen comprises the following steps: putting edible vegetable oil into a container, adding dry powder of Scutellariae radix, radix et rhizoma Rhei, Coptidis rhizoma, cortex Phellodendri, radix astragali, radix Angelicae sinensis, radix Sangusorbae, rehmanniae radix, rhizoma Bletillae, radix Angelicae Dahuricae, and radix Cynanchi Atrati, and stirring; standing and soaking for 3-10 days, stirring and heating, keeping at 50-90 deg.C for 1-2 hr under stirring, stopping heating, filtering to remove residue, adding Olibanum and Myrrha powder, stirring at 50-90 deg.C for 5-30 min, stopping heating, high-speed shearing at 20000rpm for 3-5 times (5 min each) with a flash extraction device, sieving with 5000 mesh sieve under negative pressure to remove residue, collecting oil, cooling to 30-40 deg.C, adding Borneolum Syntheticum, stirring, and cooling to obtain topical skin-growth oil for preventing and treating cold injury due to liquid nitrogen.

Furthermore, the dry powder of 13 traditional Chinese medicinal materials such as scutellaria baicalensis, rheum officinale, coptis chinensis, golden cypress, astragalus membranaceus, angelica sinensis, garden burnet, rehmannia glutinosa, angelica dahurica, cynanchum atratum, bletilla striata, frankincense, myrrh and the like is sieved by a 120-mesh sieve.

In order to achieve the third object of the present invention, the present invention further adopts the following technical measures:

the invention also provides application of the external skin-generating oil in preparation of medicines for preventing and treating frostbite caused by liquid nitrogen ultralow temperature.

The formula principle of each traditional Chinese medicine raw material of the invention is as follows: the scutellaria baicalensis, the coptis chinensis, the golden cypress and the rhubarb are used for clearing heat and drying dampness, purging intense heat and removing toxicity, and are monarch drugs; the astragalus, the angelica, the garden burnet, the frankincense and the myrrh are used as ministers for promoting blood circulation, removing blood stasis, relieving swelling and pain; rehmannia root, bletilla striata, angelica dahurica and cynanchum atratum are used as adjuvant drugs for astringing yin and promoting granulation, removing putrefaction and treating sore, and reducing swelling and expelling pus; the borneol and the vegetable oil coordinate with the other drugs to achieve the effects of detoxification and tissue regeneration.

The liquid nitrogen ultralow temperature rat model establishment and index evaluation are as follows: a standard weight (cylinder bottom) of 50g stainless steel soaked in liquid nitrogen for 15min is tightly attached to the skin of the back of the unhaired rat for 10s, and a liquid nitrogen frostbite model of the skin of the back of the rat is established. And then monitoring the temperature change and the wound area before and after the treatment of the frostbite model mouse by adopting an infrared thermometer and a Sony single-reflex micro-range camera, finally, killing the experimental animal according to the ethical requirement after the experiment is finished, and taking the skin of the tested part of the experimental animal to perform HE staining and histopathological analysis.

Compared with the prior art, the invention has the following advantages and effects: research results show that the skin-growing oil prepared by the invention can effectively play the roles of purging fire, detoxifying, activating blood circulation, removing stasis, removing putrefaction and promoting tissue regeneration after being used preventively and curatively, obviously and quickly rewarming the skin temperature of the liquid nitrogen ultralow-temperature frostbite part, reducing the inflammatory reaction of the frostbite part, achieving the effect of preventing or rehabilitating the ultralow-temperature frostbite, and obviously superior to the treatment effect of the clinical treatment drug combination of western medicines (the combination of antibiotics and anti-inflammatory agents, the combination of mupirocin for external use and dexamethasone, the combination of wound healing promoter and microcirculation improver, the combination of compound zinc oxide ointment for external use and calcium dobesilate for internal use).

The skin-growing oil disclosed by the invention has the following advantages for preventing and treating ultralow-temperature frostbite: the cost of the medicine is low; secondly, the external use is convenient; and the liquid nitrogen ultralow temperature frostbite part has good rehabilitation effect.

Drawings

FIG. 1 is a graph showing the effect of topical skin oil (within five minutes) prepared in example 1 on the rapid rewarming of rats with liquid nitrogen ultra-low temperature (skin infrared thermometry). In the figures, and, respectively, indicate that the p-value is less than 0.01, 0.001 compared to the model group.

FIG. 2 is a graph showing the effect of topical crude oil prepared in example 2 (within twenty days) on the improvement of body temperature of rats frozen at ultra-low temperature with liquid nitrogen (skin infrared thermometry). Graphs indicate p values of less than 0.05, 0.01, 0.001 compared to the model group, respectively.

FIG. 3 is a graph showing the effect of topical skin oil (within twenty days) prepared in example 2 on wound repair in rats with liquid nitrogen ultralow temperature frostbite (general skin view). In the figures,. indicates p values of less than 0.05, 0.01, 0.001, respectively, compared to the model group.

FIG. 4 is a graph simulating the effect of clinical combination of antibiotics and anti-inflammatory agents on wound repair (gross skin view), body temperature improvement (infrared skin temperature measurement) and wound repair pathology (HE staining). In the figures, each indicates that the p-value is less than 0.01 compared to the model group.

FIG. 5 is a pathological graph (HE staining graph) of wound repair of rats frostbitten by liquid nitrogen (within twenty days) with skin oil for external use prepared in example 2. In the figures, each indicates a p-value of less than 0.001 compared to the control group; #, # #, and # # # in the figure identify p values less than 0.05, 0.01, 0.001, respectively, as compared to the model group.

Detailed Description

The applicant shall now make further details of the technical solution of the present invention with reference to specific examples in order to make the present application more clearly understood and appreciated by those skilled in the art.

The dry powder of 13 traditional Chinese medicinal materials such as scutellaria baicalensis, rheum officinale, coptis chinensis, golden cypress, astragalus membranaceus, angelica sinensis, garden burnet, rehmannia glutinosa, angelica dahurica, cynanchum atratum, bletilla striata, frankincense, myrrh and the like used in the following examples is sieved by a 120-mesh sieve.

Example 1: a preparation method of external skin-growing oil for preventing and treating liquid nitrogen ultralow-temperature frostbite comprises the following steps:

putting 1L of sesame oil into a container, then adding 30g of dry powder of each of radix Scutellariae, radix et rhizoma Rhei, rhizoma Coptidis and cortex Phellodendri, and 10g of dry powder of each of radix astragali, radix Angelicae sinensis, radix Sangusorbae, radix rehmanniae, radix Angelicae Dahuricae, radix Cynanchi Atrati and rhizoma Bletillae, and stirring; standing and soaking for 10 days, stirring and heating, keeping at 50 deg.C under stirring for 2 hr, stopping heating, filtering to remove residue, adding dry powder of Olibanum and Myrrha 10g each, heating to 50 deg.C, and stirring for 30 min; stopping heating, and shearing at 20000rpm for 5 times (5 min each time) by flash extraction device; sieving with 5000 mesh sieve under negative pressure, and collecting filtered oil; when the oil temperature is reduced to 40 ℃, adding 3g of borneol into the filtered oil, fully stirring, cooling to room temperature to obtain the external skin-generating oil 1, and storing in a 100ml sterilization bottle in a sealed manner.

Example 2: a preparation method of external skin-growing oil for preventing and treating liquid nitrogen ultralow-temperature frostbite comprises the following steps:

putting 1.5L of sesame oil into a container, then adding 50g of dry powder of scutellaria baicalensis, rheum officinale, coptis chinensis and golden cypress and 30g of dry powder of astragalus membranaceus, angelica sinensis, garden burnet, rehmannia glutinosa, angelica dahurica, cynanchum atratum and bletilla striata, and uniformly stirring; standing and soaking for 3 days, stirring and heating, keeping at 90 deg.C under stirring for 1 hr, stopping heating, filtering to remove residue, adding dry powder of Olibanum and Myrrha 10g each, and stirring for 10 min; shearing at 20000rpm for 3 times (5 min each time) with flash extraction device; sieving with 5000 mesh sieve under negative pressure, and collecting the filtered oil; when the temperature of the oil is reduced to 30 ℃, 8g of borneol is added into the filtered oil, the mixture is fully stirred, the mixture is cooled to the room temperature to obtain the external skin-generating oil 2, and the external skin-generating oil is sealed and stored after being filled into a 100ml sterilization bottle.

Example 3 Effect of skin oil 1 prepared in example 1 on wound surface skin temperature recovery in rats with liquid nitrogen frostbite model

The animals tested were 30 SPF grade SD (6 weeks old) rats (provided by longevous biotechnology, liaison limited). The water diet is freely drunk, the temperature is kept at 25 +/-1 ℃ during the breeding period, and the light and shade period is 12 hours.

1) Group modeling and drug delivery

30 rats were divided into 6 groups: a control group (Normal), a Model group (Model), a raw skin oil prevention group (SFO-BF), a raw skin oil treatment group (SFO-AF), a compound zinc oxide and calcium dobesilate combined group (ZnO + CD) and sesame oil group (SO). After adaptive feeding for 7 days, roughly shearing off the mouse hair at the experimental positions on both sides of the back of each rat by tightly adhering the skin to a razor, unhairing the circular area with the diameter of about 4cm in the exposed area of the skin position, and completely washing the rat with warm water immediately after unhairing to expose the skin of the experimental position for later use. A50 g stainless steel standard weight was completely immersed in liquid nitrogen for 15min and allowed to cool sufficiently to a temperature equivalent to that of liquid nitrogen (-196 ℃ C.). The rats with the prepared skin are placed beside a liquid nitrogen tank so as to reduce experimental errors; then, except that the control group is not subjected to any damage treatment, all rats of the model group are subjected to ether inhalation anesthesia and are placed on a fixing frame; then, 1 standard weight of 50g stainless steel is taken out, the bottom of the cylinder body is attached to the experimental part on one side of the back of a rat immediately, the experimental part is maintained for 10s, and local ultralow-temperature frostbite of the skin (1 frostbite wound surface is formed on each of the two sides of the back) caused by liquid nitrogen is simulated.

In a modeling experiment, in addition to the skin oil prevention group which previously applied skin oil 1 (10 minutes before injury treatment; 0.25ml each) to the site to be frosted, the model group, the skin oil treatment group, the compound zinc oxide and calcium dobesilate group, and the sesame oil group were immediately applied with 0.25ml of physiological saline, 0.25ml of the skin oil 1 prepared in example 1, 0.25ml of compound zinc oxide (boric acid zinc oxide ointment, beijing shuangji pharmaceutical co., ltd.) and 0.25ml of sesame oil to each rat site to be frosted, respectively, and the compound zinc oxide and calcium dobesilate group was simultaneously applied with calcium dobesilate (calcium dobesilate capsule, shanghai chui pharmaceutical ltd.; first administration was completed 10 minutes before frostbite) daily to perform gastric lavage (the capsule contents were dissolved with physiological saline to prepare 4g/L calcium dobesilate solution) according to 13.2 mg/kg.

2) Skin temperature measurement of the test rats before and after the frostbite: 5 time periods of 1 minute, 3 minutes, 5 minutes and the like immediately before and after the frostbite are selected, and the temperature of the wound surface of the rat is recorded and photographed by an infrared camera. The resulting temperature change data (fig. 1) was then analyzed using GraphPad Prism (v5.01) scientific statistical mapping software and expressed as mean ± SEM, with two-way anova between and within groups, and finally with a difference of p <0.05 as statistically significant.

3) The experimental results are as follows:

as shown in fig. 1, at 1 minute after the frostbite, compared with the model group rats (1.9 ± 0.1 ℃), the wound temperatures of the two groups of rats were quickly adjusted back to 17.8 ± 1.4 ℃ or higher, and there was a significant difference (p <0.001) regardless of whether the skin oil was applied in advance or immediately after the frostbite. And the rewarming effect of the two groups of the skin-generating oil for preventing and treating is much better than that of the compound zinc oxide and calcium dobesilate group (3.3 +/-1.0 ℃ and p < 0.01). After 3 minutes of frostbite, compared with a model group rat (28.4 +/-4.1 ℃), the skin oil prevention group (36.3 +/-0.2 ℃), p <0.01) and the skin oil treatment group (32.8 +/-3.5 ℃), p <0.01) still have good rewarming effect and are close to normal skin temperature. The rewarming effect of the compound zinc oxide and calcium dobesilate and sesame oil treatment on the wound skin of the rat with the frostbite is similar to that of a model group which is not subjected to prevention or treatment. The skin temperature of rats in each test group was close to normal 5 minutes after the frostbite.

It should be noted that: in FIG. 1A, the infrared thermometry data (temperature at each time) are displayed by selecting the specific temperature of the skin of one experimental animal of representative interest in each group, while the results depicted in FIG. 1B are the average of the temperatures determined for each group of experimental animals and are therefore expressed in the form of X + -Y deg.C text. From the results, no matter the skin generating oil is used for preventing and treating before being subjected to frostbite or the skin generating oil is used for treating immediately after being subjected to frostbite, the skin generating oil has good rewarming effect on the wound surface skin of the rat with ultralow temperature caused by liquid nitrogen. In contrast, the effect of the combination of wound healing promoter and microcirculation improver and the sesame oil group simulating clinical treatment of frostbite is not ideal.

Example 4 Effect of skin oil 2 prepared in example 2 on wound skin temperature recovery in rats with liquid nitrogen frostbite model

The test animals were 40 SPF-grade SD (6 weeks old) rats (provided by Liaoning Biotechnology GmbH, Inc.). The water diet was freely drunk, the temperature was kept at 25 + -1 deg.C during the feeding period, and the light and dark cycle was 12 hours.

1) Group modeling and drug delivery

40 rats were divided into 7 groups: control group (Normal, 5), Model group (Model, 10), raw skin oil prevention group (SFO-BF, 5), raw skin oil treatment group (SFO-AF, 5), compound zinc oxide combined calcium dobesilate group (ZnO + CD, 5), sesame oil group (SO, 5) and mupirocin combined dexamethasone group (DXMS + MUP, 5). After adaptive feeding for 7 days, roughly shearing off the mouse hair at the experimental positions on both sides of the back of each rat by tightly adhering the skin to a razor, unhairing the circular area with the diameter of about 4cm in the exposed area of the skin position, and completely washing the rat with warm water immediately after unhairing to expose the skin of the experimental position for later use. A50 g stainless steel standard weight was fully immersed in liquid nitrogen for 15min and allowed to cool sufficiently to a temperature equivalent to that of liquid nitrogen (-196 ℃ C.). The rats with prepared skin are placed beside a liquid nitrogen tank so as to reduce experimental errors; then, carrying out ether inhalation anesthesia on all rats of the model building group except for the control group without any damage treatment, and placing the rats on a fixing frame; then, 1 standard weight of 50g stainless steel is taken out, the bottom of the cylinder body is attached to the experimental part on one side of the back of a rat immediately, the experimental part is maintained for 10s, and the local ultralow temperature frostbite of the skin (1 frostbite wound surface is formed on each of the two sides of the back) caused by liquid nitrogen is simulated.

In a modeling experiment, except that the skin oil prevention group is previously coated with skin oil (10 minutes before injury treatment; 0.25ml each) on a part to be frozen, the model group, the skin oil treatment group, the compound zinc oxide and calcium dobesilate group, the sesame oil group and the mupirocin and dexamethasone group are respectively and immediately coated with 0.25ml of normal saline, 0.25ml of the skin oil 2 prepared in example 2, 0.25ml of the compound zinc oxide, 0.25ml of sesame oil and 0.25ml of mupirocin (mupirocin ointment, mezzanine acetate, medetocin) and dexamethasone (compound dexamethasone acetate, sanjiu hua medicine, ltd) on the part to be frozen of each rat (when in use, the mupirocin ointment and the dexamethasone ointment are firstly mixed uniformly according to the equal volume). And the compound zinc oxide and calcium dobesilate group is simultaneously administrated with 4g/L calcium dobesilate normal saline solution per day and is intragastrically infused according to 13.2 mg/kg.

2) Skin temperature monitoring and wound area determination of the tested rats before and after frostbite: 6 time periods of 1 day, 5 days, 10 days, 15 days, 20 days and the like before and after the frostbite are selected, and the wound surface temperature and the wound surface of the rat are recorded by an infrared camera and a Sony single-reflex microspur camera respectively. Then, the obtained temperature change (figure 2) and wound area (figure 3; a ruler is put on the skin of the wound margin of the tested animal when a microspur picture is taken so as to calculate the size of each wound by software at the later stage) data are analyzed by GraphPad Prism (v5.01) scientific statistical plotting software, mean + -SEM is used for representing, two-way variance analysis is adopted between groups and in groups, and finally, the difference with p <0.05 is taken as statistical significance.

3) HE staining and pathological analysis of the wounded tissue of the test rats:

after 20 days of administration (i.e., day of experiment end), all animals were sacrificed under anesthesia, and the skin tissue at the wound site was removed, wrapped with gauze, and fixed in formalin for 24 hours. The skin tissues were then sequentially subjected to running water washing, dehydration, paraffin embedding, followed by tissue sectioning, HE routine staining, and observation under an optical microscope, and the skin tissues were pathologically scored (fig. 5).

4) The experimental results are as follows:

(1) skin temperature monitoring of ultralow temperature frostbite rats

The skin temperature of the wound surface of the rat with ultralow temperature frostbite is monitored in a segmented mode in 5 time periods (1 day, 5 days, 10 days, 15 days and 20 days after frostbite) to be recorded in a planned implementation mode, and the result is shown in figure 2. The skin temperature of the rats in the model group started to decrease (35.3 + -0.4 ℃ C.; p <0.01) at the wound site by 1 day after the cold injury, whereas the skin temperature of the affected part was maintained at a high level (36.9 + -0.0 ℃ C.) in all the treatment groups, whereas the skin temperature of the rats in the model group was significantly different between the skin oil prevention group (37.2 + -0.3 ℃ C.; the control model group, p <0.01) and the skin oil treatment group (36.9 + -0.0 ℃ C.; the control model group, p < 0.05). The skin temperature of the wound surface of the rat in the model group suddenly rose (36.5 + -0.1 ℃) 10 days after the frostbite and fell back (34.9 + -0.4 ℃) 15 days after the frostbite (p <0.05 in the control group), and the temperature was maintained for 20 days (35.4 + -0.3 ℃) basically. Compared with the abnormal change of the wound skin temperature of rats in the model group, the temperature of rats in other groups is basically maintained near normal, and particularly, the wound skin temperature of rats in the skin oil generation prevention group is always in the optimal state (37.5 +/-0.1 ℃ at 10 days; 37.1 +/-0.2 ℃ at 15 days; p is less than 0.001 in the comparison model group; 36.9 +/-0.3 ℃ at 20 days; p is less than 0.05 in the comparison model group). Although the treatment effect of the skin oil-treated group was slightly lower than that of the preventive group, they had almost the same ability to recover the wound skin temperature 20 days after the frostbite (37.1. + -. 0.5 ℃ C.; comparative model group, p < 0.01). Completely different from the prevention and treatment effect of skin oil, the skin temperature of the wound surface of rats in the compound zinc oxide and calcium dobesilate combined group is always in an ascending state within 5 to 20 days after frostbite, and is higher than the normal skin temperature (37.6 +/-0.4 ℃ C.; a comparative model group, p is less than 0.001) within 20 days after frostbite. The sesame oil group used as a negative treatment control shows that the skin temperature of the wound surface of a rat always fluctuates between the model group and the skin oil prevention and treatment group (35.8 +/-0.3-36.9 +/-0.1 ℃), which indicates that the sesame oil group has a limited rewarming effect on frostbite.

It should be noted that: in FIG. 2A, the infrared thermometry data (temperature at each time) are displayed by selecting the specific temperature of the skin of one experimental animal of representative interest in each group, while the results depicted in FIG. 2B are the average of the temperatures determined for each group of experimental animals and are therefore expressed in the form of X + -Y deg.C text. From the results, the skin-generating oil has good long-term effect on the skin rewarming of the wound surface of a rat with ultralow temperature frostbite caused by liquid nitrogen no matter the skin-generating oil is used for preventing and administering before frostbite or is used for treating the skin-generating oil immediately after frostbite. In contrast, the drug combination of the wound healing promoter and the microcirculation improver which simulates clinical treatment of the frostbite is effective for rewarming, but the long-term use effect is not ideal.

(2) Wound surface area determination of ultralow temperature frostbite rat

As shown in FIG. 3, it was observed that the skin of the wound surface of the rats in the model group was whitish and edematous first (day of frostbite), then turned deep red (1 day after frostbite), then turned purple (5 days after frostbite), and finally formed a light purple crust (20 days after frostbite) from 1 day to 20 days after the liquid nitrogen ultralow temperature frostbite. The surface area of the whole frostbite part is also reduced from large to small (2.87 +/-0.01 cm)²～0.65±0.04cm²) (ii) a Compared with the model group, the indexes of wound swelling, scab, wound area and the like of rats in each treatment group are improved, especially the prevention group of skin oil (the skin of the affected part is light red on the day of frostbite, edema is not obvious, the skin of the wound is light purple on 1 day after frostbite, most of the scab peels off 10 days after frostbite, the skin color is close to normal, and the area is 2.91 +/-0.07 cm²～0.10±0.01cm²) And treatment group (the affected skin was red and edema was not evident on the day after frostbite; the skin of the wound surface is purple after 1 day of frostbite, most of crust skin falls off after 15 days of frostbite, and the skin color is close to normal; the area is 2.90 +/-0.03 cm²～0.19±0.04cm²) There is a significant improvement. Of course, compared with the treatment effect of prevention and treatment of raw skin oil, the clinical compound zinc oxide and calcium dobesilate combination (the skin of the affected part is dark red and edema is obvious on the day of frostbite, the skin of the wound surface is purple on the 5 days after frostbite, a small part of crust skin falls off on the 10 days after frostbite, the skin color is dark red after decrustation, most of crust skin falls off on the 20 days after frostbite, the skin color is light red after decrustation, and the area is 2.88 +/-0.06 cm²～0.35±0.04cm²) There is little difference. Interestingly, the sesame oil group used as a negative treatment control also showed a certain protective effect in the repair of liquid nitrogen frostbitten skin (the affected skin was deep red and edema was obvious on the day after frostbite, the wound skin was purple on 5 days after frostbite, a small part of crust skin fell off 10 days after frostbite, the skin color was red after decrustation, most of crust skin fell off 15 days after frostbite, the skin color was close to normal after decrustation, and the area was 2.85 + -0.02 cm²～0.22±0.02cm²) The result is even better than that of the compound zinc oxide and calcium dobesilate combination.

Comparing the results of FIG. 4, it was also difficult to find combinations of drugs (mopipid) that mimic clinical antibacterial and anti-inflammatory effectsLuoxing in combination with dexamethasone) seems not to have promotion effect on wound healing of rats with ultralow temperature frostbite caused by liquid nitrogen, but presents an obstructive result. As shown in fig. 4A and 4B, from the general view of the skin: the mupirocin and dexamethasone are combined to cause local whitening and edema on the same day after the frostbite; mauve when 1 day after frostbite; when the skin is frozen for 15 days, a small part of crust peels off, and the skin color is whitish after decrustation; the area is 2.82 +/-0.18 cm²～1.55±0.08cm². From the examination of skin temperature at 5 time periods (1 day, 5 days, 10 days, 15 days, 20 days after the frostbite) (as shown in fig. 4C and 4D), the trend of the temperature trend of rats is consistent with that of rats in the model group, the temperature starts to decrease at 5 days of the frostbite, the temperature rises again at 10 days, and the temperature decreases again at 15 days (and no significant difference exists in each time period compared with the model group). From the analysis of the pathological structure of the skin (as shown in fig. 4E and 4F): the pathological morphology of the mupirocin in combination with dexamethasone presented abnormalities of epidermal structure, inflammatory infiltration of the dermis, collagen disorders and disappearance of cutaneous appendages similar to those of the model group, as well as a more hypertrophic acanthosis, more severe keratosis and more severe disorders of the proportion of epidermal structure (with a pathological score of 2.16 ± 0.16; slightly lower than that of the model group). The reason is considered that the drug combination of antibiosis and anti-inflammation is only used for possibly blocking the pathway of arachidonic acid epoxidase, reducing the generation of prostaglandin and thromboxane, further shrinking blood vessels of a wound surface and aggravating skin ischemia, and further causing the shrinkage of arterioles and capillaries in the dermis of the wound surface due to the wrong use of drugs after frostbite to hinder wound repair.

It should be noted that: the infrared thermometry data (temperature at each time) in FIG. 4C are presented by selecting the specific temperature of the skin of one experimental animal of representative interest in each group, and the results depicted in FIG. 4D are the average of the temperatures determined for each group of experimental animals and are therefore expressed in X + -Y deg.C text. From the results, the skin oil has good promotion effect on wound healing of rats with ultralow temperature frostbite caused by liquid nitrogen no matter the skin oil is used for preventing and treating before frostbite or the skin oil is used for treating immediately after frostbite. In contrast, the combination of the wound healing promoter and the microcirculation improving agent which simulates clinical treatment aiming at the treatment of the frostbite has a certain improvement effect, but the effect is not more ideal than that of the negative treatment control sesame oil group. The mupirocin and dexamethasone group (antibacterial and anti-inflammatory drug combination) can prevent the frostbite wound surface from being repaired.

(3) Pathological analysis of wound skin of rat with ultralow temperature and frostbite

The results of pathological analysis of all rats after anesthesia and death (20 days after frostbite) by HE staining of the affected skin are shown in FIG. 5. The epidermal and dermal cells of the blank rats are arranged normally without edema, a large number of hair follicles and other accessory organs can be seen, and no abnormal pathological morphological changes exist. Compared with a blank control group, the rat frostbite wound skin of the model group shows that: the epidermal layer structure is incomplete, the epidermal layer is obviously thickened, the intercellular edema is obvious, and the gaps are thickened; secondly, a large amount of neutrophil and monocyte infiltration, interstitial congestion and edema can be seen in the dermis, long scar tissues exist, collagen fiber cells are seriously disordered, and a large amount of red blood cells overflow in the dermis; the hair follicle and other skin appendages disappear; the pathological score of the skin tissue (2.6 +/-0.3; p <0.001) is obviously less than that of a blank group (10.0 +/-0.0). Compared with the model group, the skin oil generation prevention group rat frostbite wound skin presents: firstly, the epidermal layer is clear and complete, the arrangement of the keratinized epithelial cells is normal, the thickening of the epidermal layer is not obvious, and the intercellular edema is not obvious; the dermis layer has very little infiltration of neutrophils and mononuclear cells; collagen fiber cells are basically arranged in order, and a large number of hair follicles and hair follicles grow; and the pathological score of the skin tissue is obviously more than that of the model group (7.2 +/-0.3; p is less than 0.001). Of course, the skin oil-producing treatment group also exhibited a considerably good treatment effect: firstly, the epidermal layer is clear and complete, the arrangement of the keratinized epithelial cells is normal, the thickening of the epidermal layer is not obvious, and the intercellular edema is not obvious; secondly, a small amount of neutrophil granulocytes and mononuclear cells are seen in the dermis; collagen fiber cells are basically arranged in order, and a large number of hair follicles and hair follicles grow; and the pathological score of the skin tissue is obviously higher than that of a model group (6.4 +/-0.5; p is less than 0.001). However, compared with the model group and the prevention and treatment group of skin oil, the compound zinc oxide and calcium dobesilate group also shows a certain curative effect: the epidermal layer has clear and complete structure, obviously thickened epidermal layer, small amount of edema in intercellular substance and thickened interstitial space; the dermis can be infiltrated by neutrophilic granulocytes and mononuclear cells, interstitial congestion and edema and scar tissues are formed; collagen fiber cells are disordered, a large number of red blood cells overflow, and hair follicles and other skin accessory organs are not seen; and the pathological score of the skin tissue is obviously higher than that of a model group (5.2 +/-0.5; p is less than 0.01). The antibacterial and anti-inflammatory drug combination mupirocin and dexamethasone are not good in treatment on the liquid nitrogen ultralow temperature frostbite wound surface: the epidermis has incomplete structure and seriously maladjusted structure proportion, hyperkeratosis of the epidermis (thickening of a horny layer and a granular layer), pachytene, large amount of edema of intercellular substance and obvious thickening of gaps; secondly, severe neutrophil and monocyte infiltration can be seen in the dermis layer, collagen fibrosis is accompanied with fibroblast hyperplasia, blood vessels in connective tissues can be seen as edema and congestion, and a large amount of overflowing red blood cells can be seen; the hair follicle and other skin appendages disappear completely; the pathological score of the skin tissue (2.16 +/-0.16; p <0.001) is obviously less than that of a blank group (10.0 +/-0.0). The sesame oil group used as negative treatment control has limited protective effect on liquid nitrogen ultralow temperature frostbite wound surfaces: the epidermal layer has clear and complete structure, thickened epidermal layer, small amount of edema in intercellular substance and thickened interstitial space; secondly, a small amount of neutrophil granulocytes and mononuclear cells are infiltrated in the dermis layer, and edema exists in interstitial substances; ③ the collagen fiber cells are obviously disordered, a large amount of red blood cells overflow, and hair follicles and other skin accessory organs are not seen; and the pathological score of the skin tissue is obviously higher than that of a model group (4.2 +/-0.5; p is less than 0.05).

Claims

1. The application of the external skin-generating oil in preparing the medicine for preventing and treating the frostbite is characterized in that the external skin-generating oil is prepared from the following raw materials in parts by weight: 10-50 parts of scutellaria baicalensis, 10-50 parts of coptis chinensis, 10-50 parts of phellodendron, 10-50 parts of rheum officinale, 10-30 parts of astragalus membranaceus, 10-30 parts of angelica sinensis, 10-30 parts of sanguisorba officinalis, 10-30 parts of frankincense, 10-30 parts of myrrh, 10-30 parts of rehmannia, 10-30 parts of bletilla striata, 10-30 parts of angelica dahurica, 10-30 parts of cynanchum atratum, 3-8 parts of borneol and 1500 parts of edible vegetable oil by weight;

the preparation method of the external skin-generating oil comprises the following steps: putting edible vegetable oil into a container, adding dry powder of Scutellariae radix, radix et rhizoma Rhei, Coptidis rhizoma, cortex Phellodendri, radix astragali, radix Angelicae sinensis, radix Sangusorbae, rehmanniae radix, rhizoma Bletillae, radix Angelicae Dahuricae, and radix Cynanchi Atrati, and stirring; standing and soaking for 3-10 days, stirring and heating, keeping at 50-90 deg.C for 1-2 hr under stirring, stopping heating, filtering to remove residue, adding Olibanum and Myrrha dry powder, stirring at 50-90 deg.C for 5-30 min, filtering to remove residue, collecting oil, cooling to 30-40 deg.C, adding Borneolum, stirring, and cooling to obtain the topical skin-care oil.

2. The use of the skin-growth oil for external use according to claim 1 for the preparation of a medicament for preventing and treating frostbite, wherein the frostbite is caused by ultra-low temperature of liquid nitrogen.

3. The use of the external skin-generating oil according to claim 1 or 2 in the preparation of a medicament for preventing and treating frostbite, wherein the preparation method of the external skin-generating oil comprises the following steps: putting edible vegetable oil into a container, adding dry powder of Scutellariae radix, radix et rhizoma Rhei, Coptidis rhizoma, cortex Phellodendri, radix astragali, radix Angelicae sinensis, radix Sangusorbae, rehmanniae radix, rhizoma Bletillae, radix Angelicae Dahuricae, and radix Cynanchi Atrati, and stirring; standing and soaking for 3-10 days, stirring and heating, keeping at 50-90 deg.C for 1-2 hr under stirring, stopping heating, filtering to remove residue, adding Olibanum and Myrrha powder, stirring at 50-90 deg.C for 5-30 min, stopping heating, high-speed shearing at 20000rpm for 3-5 times with a flash extraction device, sieving with 5000 mesh sieve under negative pressure to remove residue, collecting oil filtrate, cooling to 30-40 deg.C, adding Borneolum Syntheticum, stirring, and cooling to obtain the topical skin-regenerating oil.

4. The use of the topical skin-generating oil in the preparation of a medicament for preventing and treating frostbite according to claim 1 or 2, wherein the dry powder of the traditional Chinese medicinal materials of scutellaria baicalensis, rheum officinale, coptis chinensis, phellodendron amurense, astragalus mongholicus, angelica sinensis, sanguisorba officinalis, rehmannia glutinosa, angelica dahurica, cynanchum atratum, bletilla striata, frankincense and myrrh is sieved by a 120-mesh sieve.

5. Use of the skin regenerating oil for external use according to claim 1 or 2, characterized in that the edible vegetable oil is at least one selected from sesame oil, coconut oil, peanut oil, soybean oil, linseed oil, castor oil and olive oil in the preparation of the medicine for preventing and treating frostbite.

6. Use of the skin-growth oil for external use according to claim 1 or 2, for the preparation of a medicament for preventing and treating frostbite, wherein the edible vegetable oil is selected from at least one of sesame oil and coconut oil.