CN104887661A - Medicinal composition for promoting wound healing and application thereof - Google Patents

Abstract

The present invention discloses a pharmaceutical composition for promoting wound healing and its use, which uses the following compound of formula I as its active ingredient: Thus, a pharmaceutical composition for promoting wound healing can be developed, especially using the above-mentioned compound of formula I as its active ingredient. The pharmaceutical composition further includes a pharmaceutically acceptable diluent or carrier, and can be prepared into various dosage forms such as liquid, paste, gel, dressing or spray, to promote the healing of open wounds caused by various external factors.

Description

Pharmaceutical compositions for promoting wound healing and uses thereof

technical field

The present invention relates to a compound isolated from Antrodia camphorata, in particular to a pharmaceutical composition for promoting wound healing and its use.

Background technique

The skin of the human body occupies an average total surface area of about 1.5-2.0 square meters. Functionally, it can maintain the temperature and moisture of the human body and avoid damage from bacteria and the external environment. Skin can be divided into epidermis, dermis and subcutaneous tissue according to its structure, each with different functions. The epidermis is the outermost layer of the skin that covers the entire body and has a protective function. The epidermis can be divided into stratum corneum, transparent layer, granular layer, echinoderm layer and basal layer from outside to inside. The thickness of the dermis is about 0.3-3 millimeters (mm), about seven times thicker than the epidermis, and contains a lot of water, which accounts for 18-40% of the total body water. It can be divided into papillary layer and reticular layer. The subcutaneous tissue is located in the lowermost layer of the skin and is mainly composed of fat. Its thickness varies with age, gender and health status. Its main function is heat preservation and shock resistance.

Wounds occur when the skin is injured, and the process from wound occurrence to healing can be divided into three stages, namely, the inflammatory stage, the proliferative stage, and the mature stage. After the skin is injured, the wound will become inflamed. At this time, white blood cells that swallow bacteria will gather, eat the necrotic tissue, stop bleeding, and secrete secretions that promote healing on the wound. At this time, the wound will become red, swollen, and hot. The inflammatory period usually lasts for three to four days, and in the elderly it lasts for up to a week. Subsequently, granulation tissue begins to grow, thin epidermal cells will grow into the wound, the activity of white blood cells increases, the synthesis of collagen tissue begins to take effect, new blood vessels grow, and the wound begins to shrink. The proliferative period usually lasts for ten days to two weeks. Finally, the wound will be scarred, the outside of the wound will be covered and shrunk, the collagen fibers will arrange themselves regularly, and the redundant new blood vessels will begin to degenerate and shrink. The mature period usually ranges from two weeks to six months. It takes two years.

Regardless of whether the wound is caused by trauma, burns, surgery, acute or chronic wounds, etc., wound healing has always been the focus of academia and the biomedical industry for a long time. Since the type and size of the wound, as well as the patient's nutritional status, age, other systemic diseases, and the drugs used, many factors will affect the process and time of wound healing. Therefore, how to develop a drug that can effectively promote wound healing, Even applying it to artificial skin, biomedical materials, and dressings is an urgent issue to be overcome.

Contents of the invention

Accordingly, an object of the present invention is to provide a pharmaceutical composition for promoting wound healing, which uses the compound of the following formula I as its active ingredient:

Another object of the present invention is to provide a pharmaceutical composition for the preparation of a medicine for promoting wound healing. The pharmaceutical composition uses the compound of the following formula I as its active ingredient:

Preferably, in the above-mentioned pharmaceutical composition and its use, the pharmaceutical composition further includes a pharmaceutically acceptable diluent or carrier, and the pharmaceutical composition is liquid, paste, gel, dressing or spray, The wound is an open wound.

Through the technical means disclosed in the present invention, a pharmaceutical composition for promoting wound healing can be developed, especially the compound of formula I above is used as its active ingredient, and it can be prepared into liquid, paste, gel Various dosage forms, such as dressings or sprays, aim at promoting the healing of open wounds caused by various external factors.

Description of drawings

Figures 1-2 show the results of animal wound healing tests and the wound healing conditions.

Figure 3 shows the analysis of wound tissue structure and recovery by slices.

Figure 4 shows the analysis of collagen synthesis in wounds by slices.

Figure 5-6 shows the situation of angiogenesis and blood vessel distribution on the back of the wound.

Detailed ways

Extraction of Antrodia Antrodia

Antrodia camphorata (Antrodia camphorata) mycelia, fruiting bodies or a mixture of the two are extracted with water or organic solvents using conventional extraction methods to obtain water extracts or organic solvent extracts of Antrodia camphorata. Among them, organic solvents may include alcohols (such as methanol, ethanol or propanol), esters (such as ethyl acetate), alkanes (such as hexane) or haloalkanes (such as methyl chloride, ethyl chloride), but not This is the limit. Wherein the preferred one is alcohols, and the more preferred one is ethanol. The extracted Antrodia camphorata water extract or organic solvent extract can be further separated and purified by high performance liquid chromatography, and then the liquid separation is purified and used for subsequent experiments. In the present invention, it is mainly aimed at 4, 7 -Dimethoxy-5-methyl-1,3-benzodioxole (4,7-dimethoxy-5-methyl-1,3-benzodioxole) is discussed, hereinafter referred to as SY-1, as shown in formula I :

Regarding the extraction of 4,7-dimethoxy-5-methyl-1,3-benzodioxole (4,7-dimethoxy-5-methyl-l, 3-benzodioxole), please refer to Tu SH., J Agric Food Chem.2012Apr11;60(14):3612-8, no more details here. In previous related studies, SY-1 mainly focused on the study of its anti-tumor effect, and no research related to wound healing was proposed. However, the present invention uses the compound (hereinafter referred to as SY-1) to carry out related experiments on wound healing, so it can be regarded as a very novel use.

Animal wound healing test (non-infected wound model)

The experimental animals were purchased from the National Experimental Animal Center of C57BL/6JNarl strain mice, with a body weight of 40-45 g and an age of 8 weeks. They were raised in the Animal Center of the National Defense Medical College. The temperature of the animal room was maintained at 18-26 degrees, the relative humidity was 30%-70%, the photoperiod was 12 hours of light and 12 hours of darkness, and sufficient feed and drinking water were provided. Before the operation, the mice were given 0.6g/kg Avertin by intraperitoneal injection. After the mice were anesthetized, their back hair was shaved. Fix the paper card on the back of the mouse to determine the location of the wound, and draw an area of 1.2x1.2cm2 with a marker. Cut away the skin in this area using surgical scissors to create a wound. After taking an appropriate amount of medicine with a cotton swab, spread the medicine evenly on the wound, and use crude extract of Antrodia camphorata, Sy-1 at a concentration of 10 μg/ml and 50 μg/ml as the experimental group. Cover the wound with a breathable film of tegaderm, fix the tegaderm with a breathable tape, to prevent the tergaderm from falling off and wound infection, clean the wound every two days and replace the medicine, and take pictures of the wounds of the mice. In addition, neomycin, polymyxin B polymyxin B, Bacillus subtilis titanium ointment and other antibiotics containing different antibiotics were used as positive control group (positive control). The wound area of different days was quantified by computer software, and the Student’s-test statistical experiment results were tested by t distribution.

The results are shown in Figures 1 and 2. Compared with the Vaseline Vasel group, in the drug-treated group, the wound tended to shrink significantly with the increase of days, and the appearance of the wound was relatively complete. Hair grows, and the wound heals about 4-8 days ahead of schedule under the condition of giving drug treatment. Compared with the blank control of the blank culture group and the vasel control of the Vaseline culture group, the group with a concentration of 50 μg/ml had a significant effect on the initial wound healing (especially on the 0-8th day), while the group with a concentration of 10 μg/ml had a significant effect on wound healing. The late stage has a better effect (especially on the 8th to 18th day). The relevant mechanism of this different concentration that causes early and late wound healing still needs to be explored in follow-up studies, but its obvious effect has indeed been seen in this experiment. Wound healing effect. Looking at the timeliness of overall wound healing alone, it seems that the group given a concentration of 10 μg/ml has a better timeliness of wound healing. In addition to rapid healing at the initial stage, nearly 90% of the wounds can be healed on the 8th day healing rate.

Compared with blank control and vasel control, the group treated with Antrodia camphorata culture solution does not seem to have a better wound healing effect, but this is because the Antrodia camphorata culture solution is in the form of an aqueous solution, which leads to wound ulceration. Although the treatment was changed to an ointment formulation, the wound healing has been delayed. Therefore, the data of this group are for reference only, and follow-up related experiments are still needed to confirm its true effect.

wound tissue section

1. Tissue freezing and embedding

(1) Add an appropriate amount of non-prescription drug OCT into the aluminum foil tissue embedding box, put the freshly removed skin tissue into the embedding box (the incision of the tissue is parallel to the embedding box), then add OCT to cover the whole tissue, and put it in liquid nitrogen Fast freezing (tissues can be picked up after freezing to three-quarters to avoid tissue cracking), and stored at -70°C.

2. Tissue Cryosection

(1) Add an appropriate amount of OCT to the aluminum alloy tissue specimen table, place the pretreated tissue block on the specimen table, place the specimen table on the microtome freezing table to freeze, put the specimen table on the microtome and lock it. (2) First, flatten the tissue block with the coarse cutting mode (20 μm), and then start sectioning with the fine cutting mode (5-7 μm). (3) Flatten the curly slices from top to bottom with a watercolor pen. (4) Attach the glass slide to the tissue slice, soak the slide in acetone, and place at -20°C for 30 minutes. (5) Store the slides at -70°C.

3. Hematoxylin & Eosin Hematoxylin & Eosin staining

(1) Soak tissue slides in PBS for about 3 to 4 minutes. (2) Stain slides with Hematoxylin for 10 minutes. (3) After washing the slides with QH ₂ O three times, soak them in Q-H2O for 5, 5, 5, and 10 minutes each. (4) Stain the slide with Eosin for 15 seconds. (5) Soak slides in 95%, 95%, 100%, 100% alcohol for 1 minute each. Then soak in xylene for 1 minute. (6) Wipe the back of the slide with 70% alcohol and wait for the sample to dry. (7) Mount the slide with a cover slip (mounting medium: xylene = 2:1).

The results are shown in Figure 3, which respectively show the normal (normal), blank control, vasel control, Antrodia camphorata culture medium (crude extract), Sy-1 with a concentration of 10 μg/ml and 50 μg/ml, positive control and other groups of wounds Organizational structure and recovery. The results were used to further analyze the changes in tissue type. On the 21st day after the wound, the skin tissue around the wound was collected, and the changes in tissue type were observed by H&E staining. The experimental results showed that in the Blank and Vasel groups, the epidermis of the wound tissue was obviously not fully restored, the hair follicles were not fully developed, and there was no hair growth. In contrast, in the drug treatment group, the epidermis of the wound tissue has been completely restored, the structure of the dermis is similar to that of normal mice, and hair follicles have developed. Among them, in the SY-1 group, the skin structure was the most complete, the tissue layers were distinct, the hair follicles were obviously developed, and there was hair growth. The above results showed that under the administration of drug treatment (groups treated with Sy-1 at a concentration of 10 μg/ml and 50 μg/ml), the wound healing of mice was significantly promoted.

collagen synthesis

After sacrificing the mice, tissue sections and staining were performed in the above-mentioned manner to analyze the collagen synthesis in the wound. The results are shown in Figure 4. On the 21st day of trauma in mice, the skin tissue around the wound was taken, and the collagen synthesis in the skin tissue was observed by Masson's trichrome staining method. The experimental results showed that the collagen was stained blue. Compared with normal mice, in the Blank and Vasel groups, the amount of collagen synthesis in the tissue was small and the structure was loose; while in the treatment drug group, the collagen in the tissue The number of synthesis and the compactness of the structure are similar to those of normal mice. The above results show that under the administration of drug treatment, it has the effect of promoting collagen synthesis.

Angiogenesis

After sacrificing the mice, the distribution of blood vessels on the back of the wound was analyzed by photographing and recording, and the results of angiogenesis were observed. The results are shown in Figures 5-6. On the 21st day after the trauma of the mouse, the skin tissue around the wound was taken to analyze the proliferation of blood vessels on the back of the skin. The experimental results show that from the appearance of the skin, it can be found that compared with the Blank and Vasel groups, the area, length and branch development of the blood vessels were significantly promoted in the drug treatment group. According to the quantitative data, in terms of vascular distribution area, length, vascular branch joint joint and path pointer, the treatment drug group significantly surpassed the Blank and Vasel groups, showing that SY-1 has a significant role in promoting angiogenesis, Angiogenesis is an essential process for wound healing.

in conclusion

The above results show that SY-1 can indeed promote the effect of wound healing in C57BL6/J mice, and a high concentration of SY-1 (50ug/mL) in the initial wound healing, a low concentration of SY-1 (10ug/mL) It has a significant effect in the end stage of wound healing. The results of tissue section staining showed that the tissue integrity of the SY-1-treated group was good, and SY-1 could significantly promote collagen deposition and wound angiogenesis, indicating that it was better at promoting healing.

The inventor conducted another experiment on the infection group, but the results showed that SY-1 has no bactericidal effect, so the relevant experimental data are not provided here. However, according to the results of the infection group and the non-infection group, SY-1 can significantly promote wound healing in C57BL6/J mice. Regardless of the infection or non-infection group, high concentration of SY-1 (50ug/mL) has a significant effect on the initial wound healing, and low concentration of SY-1 (10ug/mL) has a significant effect on the end of wound healing. In the non-infected group, SY-1 can obviously proliferate wound blood vessels. In the infection group, the cinnabar culture solution had obvious angiogenesis effect because of the incomplete wound healing. Cinnamomum camphora culture solution was initially used as an aqueous solution, but it caused wound ulceration. Later, it was changed to an ointment formulation, but the wound healing has been delayed, so the values are listed here for reference only. In the infection group, SY-1 can also significantly promote collagen deposition, showing that it promotes healing better. The hematoxylin & eosin staining method HEstaining is used to test the integrity of the tissue and is also a reference. SY-1 has no bactericidal effect, and its effect of promoting wound healing may be mainly related to its anti-inflammatory effect, and its mechanism of action still needs to be further explored in follow-up experiments.

Through the above-mentioned results, a kind of pharmaceutical composition for promoting wound healing can be provided, and a kind of purposes of this pharmaceutical composition is used in the preparation of medicine for promoting wound healing, and this pharmaceutical composition is the compound of following formula I (namely SY- 1) As its active ingredient:

Preferably, in the above-mentioned pharmaceutical composition or its use, the pharmaceutical composition further includes a pharmaceutically acceptable diluent or carrier, and the pharmaceutical composition is liquid, paste, gel, dressing or spray, The wound is an open wound.

Through the above results, a pharmaceutical composition for promoting wound healing can be developed, which uses SY-1 as its active ingredient, and the pharmaceutical composition further includes a pharmaceutically acceptable diluent or carrier, which can be It is prepared in various dosage forms such as liquid, paste, gel, dressing or spray, etc., to promote wound healing for various open wounds caused by external factors.

The pharmaceutical composition and its use for promoting wound healing provided by the present invention are indeed of industrial utility value, but the above description is only a description of the preferred embodiments of the present invention, and those who are proficient in this skill can rely on Above-mentioned explanation and do other various improvements, but these changes still belong to the spirit of the present invention and in the scope of the patent defined below.

Claims (8)

1. for promoting a medical component for wound healing, it is characterized in that, is using compounds of Formula I as its active component:

2. medical component according to claim 1, is characterized in that, this medical component is liquid, body of paste, gel, dressing or spraying.

3. medical component according to claim 1, is characterized in that, this medical component comprises a pharmaceutically acceptable diluent or carrier further.

4. medical component according to claim 1, is characterized in that, this wound is open wound.

5. medical component is for the preparation of the purposes promoting wound healing medicine, and it is characterized in that, this medical component is using compounds of Formula I as its active component:

6. purposes according to claim 5, is characterized in that, this medical component is liquid, body of paste, gel, dressing or spraying.

7. purposes according to claim 5, is characterized in that, this medical component comprises a pharmaceutically acceptable diluent or carrier further.

8. purposes according to claim 5, is characterized in that, this wound is open wound.