TWI527583B - Medical compositions for promoting wound healing and their use - Google Patents

Description

Medicinal composition for promoting wound healing and use thereof

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

The skin of the human body occupies an average surface area of about 1.5 to 2.0 square meters. It functionally maintains the temperature and moisture of the human body and is protected from bacteria and the external environment. The skin can be distinguished by the structure as epidermis, dermis and subcutaneous tissue, each having different functions. The epidermis is the outermost layer of the skin, covering the whole body and having a protective effect. The epidermis can be further divided into the stratum corneum, the transparent layer, the granular layer, the echinoderma layer and the basal layer from the outside to the inside. The thickness of the dermis is about 0.3~3 mm (mm), which is about seven times thicker than the epidermis. It contains a lot of water, and the water content accounts for 18-40% of the total body water. It can be divided into the nipple layer and the reticular layer. The subcutaneous tissue is located in the lowermost layer of the skin and is mainly composed of fat. Its thickness varies depending on age, gender and health. The main function is heat preservation and shockproof.

When the skin is damaged, a wound is produced, and the process from wound to healing can be divided into three stages, namely, an inflamed phase, a proliferative phase, and a mature phase. After the skin is injured, the wound will become inflamed. At this time, the white blood cells that devour the bacteria will gather, eat the necrotic tissue, stop bleeding, and secrete secretions that promote healing on the wound. At this time, the wound will produce redness, swelling and heat, and inflammation. The period usually lasts three to four days and the elders will last for one week. Subsequently, the granulation tissue begins to grow, the thin epidermal cells grow into the wound, the activity of white blood cells increases, the synthesis of collagen tissue begins to function, the new blood vessels grow, the wound begins to shrink, and the proliferative phase usually lasts for ten days to two. week. Finally, the wound will be crusted, the outside of the wound will be covered and shrunk, the collagen fibers will be regularly arranged, and the excess new blood vessels will begin to degenerate and shrink. The maturity usually lasts from two weeks to six months, and the wound must be completely healed. It takes two years.

Whether the wound is caused by trauma, burns, surgery, acute or chronic wounds, wound healing has always been the focus of academic and biomedical industries. Because of the type and size of the wound, as well as the patient's nutritional status, age, other systemic diseases, drugs used, etc., it will affect the process and time of wound healing. Therefore, how to develop a drug that can effectively promote wound healing, or even Applying it to artificial skin, biomedical materials, and dressings is an issue that needs to be overcome.

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

Another object of the present invention is to provide a pharmaceutical composition for the preparation of a medicament for promoting wound healing, which comprises the following compound of formula I as its active ingredient:

Preferably, in the above pharmaceutical composition and use thereof, the pharmaceutical composition is a liquid, a paste, a gel, a dressing or a spray, and 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, in particular, the compound of the above formula I is used as an active ingredient thereof, and can be prepared into a liquid, a paste, a gel, Various dosage forms such as dressings or sprays can promote wound healing for various open wounds caused by external factors.

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

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

Figure 4 shows the analysis of collagen synthesis at the wound by sectioning.

Figures 5-6 show the angiogenesis and vascular distribution on the back of the wound.

Extraction of burdock

The Antrodia camphorata mycelium, the fruit body or a mixture of the two is taken, and extracted by water or an organic solvent by a conventional extraction method to obtain an aqueous extract of Antrodia camphorata or an organic solvent extract. Wherein, the organic solvent may include alcohols (such as methanol, ethanol or propanol), esters (such as ethyl acetate), alkanes (such as hexane) or halogens (such as methyl chloride, ethyl chloride), but not This is limited to this. Among them, preferred are alcohols, and more preferably ethanol. The extracted aqueous extract of Antrodia camphorata or the organic solvent extract can be further separated and purified by high performance liquid chromatography, and then the fraction is purified and subjected to subsequent experiments, and in the present invention, mainly for 4, 7-Dimethoxy-5-methyl-1,3-benzodioxole (4,7-dimethoxy-5-methyl-1,3-benzodioxole), hereinafter referred to as SY-1, as in Formula I Show:

For the extraction of 4,7-dimethoxy-5-methyl-1,3-benzodioxole, refer to Tu SH., J Agric Food Chem. 2012 Apr 11; 60(14): 3612-8, and will not be repeated here. In previous related studies, SY-1 was mainly focused on the study of its anti-tumor effect, and no studies related to wound healing were proposed. However, the present invention utilizes the compound (hereinafter referred to as SY-1) for the related experiment of wound healing, and thus can be regarded as a very novel use.

Animal wound healing test (non-infectious trauma mode)

The experimental animals were purchased from C57BL/6JNarl strain mice of the National Experimental Animal Center, weighing 40-45 g, and aged for 8 weeks. Raised in the Animal Center of the National Defense Medical College, the animal room temperature is maintained at 18 to 26 degrees, the relative humidity is 30% to 70%, the light cycle is 12 hours light, 12 hours and night, and sufficient feed and water are provided. Mice were given 0.6 g/kg Avertin by intraperitoneal injection before surgery. After the mice were anesthetized, their back hair was shaved. The paper card was fixed on the back of the mouse to determine the wound position, and the area of 1.2 x 1.2 cm ^{2 was} drawn with a stylus. The skin in this area is cut off using a surgical scissors to form a wound. After a proper amount of the drug was applied using a cotton swab, the drug was evenly spread on the wound, and a crude extract, a concentration of 10 μg/ml, and 50 μg/ml of Sy-1 was used as an experimental group. Cover the wound with tegaderm, fix tegaderm with breathable tape, avoid tergaderm shedding and wound infection, clean the wound every two days to change the drug, and take a picture of the wound in the mouse. In addition, neomycin, polymyxin B, bacitracin ointment and the like contain different antibiotics as a positive control. The wound area of different days was quantified by computer software, and the results were statistically analyzed using Student's-test.

The results are shown in Fig. 1 and Fig. 2. Compared with the Vasel group, in the group treated with drugs, the wound has a significant shrinkage trend with the increase of the number of days, and the appearance of the wound is relatively complete, and the skin on the back of the mouse has been The hair grows, and in the case of drug treatment, the wound heals about 4-8 days earlier. Compared with blank control and vasel control, the concentration of 50μg/ml had a significant effect in the early stage of wound healing (particularly on days 0-8), while the group with concentration of 10μg/ml had a better effect in the late stage of wound healing. (Specially significant on days 8-18), the mechanism of early and late wound healing caused by such different concentrations remains to be explored in subsequent studies, but its obvious wound healing effect has indeed been seen in this experiment. If the overall time of wound healing is taken alone, it seems that a group with a concentration of 10 μg/ml has better wound healing aging, and in addition to rapid healing at the beginning, nearly 90% of wounds can be reached on the 8th day. Healing rate.

However, the group treated with the burdock culture solution did not seem to have a better wound healing effect compared with the blank control and the vasel control, but this was because the burdock culture solution was in the form of an aqueous solution, which caused the ulcer to be ulcerated. Although it is treated with an ointment, the wound has healed. Delay, so the data of this group is for reference only, and it is still necessary to carry out relevant experiments to confirm its real effect.

Wound tissue section

Tissue freezing and embedding

(1) Add appropriate amount of OCT into the aluminum foil tissue embedding box, put the freshly removed skin tissue into the embedding box (the tissue cutting surface is parallel to the embedding box), then add OCT to cover the whole tissue and place it on liquid nitrogen. Quickly freeze (tissue frozen to three-quarters to pick up, avoid tissue cracking), and store at -70 °C.

2. Tissue cryosection

(1) Add appropriate amount of OCT on the aluminum alloy tissue sample table, place the pre-treated tissue block on the specimen table, place the specimen table on the slicer freezer, and place the specimen table on the slicer and lock it. (2) First, the tissue block was formed into a flat surface in a rough cut mode (20 μm), and then sectioning was started using a fine cut mode (5 to 7 μm). (3) The curled sheet after cutting out is flattened from top to bottom with a watercolor pen. (4) The slide was attached to the tissue sheet, the slide was immersed in acetone, and placed at -20 ° C for 30 minutes. (5) Store the slide at -70 °C.

3. Hematoxylin & Eosin staining

(1) Soak the tissue slide in PBS for about 3 to 4 minutes. (2) The slide was stained with Hematoxylin for 10 minutes. (3) The slides were washed three times with QH ₂ O and then immersed in QH ₂ O for 5, 5, 5, and 10 minutes each. (4) The slide was stained with Eosin for 15 seconds. (5) Soak the slides in 95%, 95%, 100%, 100% alcohol for 1 minute each. Soak in xylene for 1 minute. (6) Wipe the back of the slide with 70% alcohol and wait for the sample to dry. (7) Cover with a cover glass (munting medium: xylene = 2:1).

The results are shown in Fig. 3, which show normal, blank control, vase control, crude extract, concentration of 10 μg/ml, 50 μg/ml of Sy-1, and positive control. Wound tissue structure and recovery. The tissue type change was further analyzed from the results. The skin tissue around the wound was taken 21 days after the wound in the mouse, and the tissue type change was observed by H&E staining. The experimental results show that in the treatment of Blank and Vasel groups, the epidermal layer of the wound tissue is not completely restored, the hair follicle is not fully developed and no hair growth. In contrast, in the treatment group, the epidermal layer of the wound tissue was completely restored, and the dermis structure was similar to that of normal mice, and hair follicles developed. Among them, in the treatment of SY-1 group, the skin structure is the most complete, the tissue layer is distinct, the hair follicles are obviously developed and hair growth is possible. From the above results, it was revealed that the wound healing was significantly promoted in the mice under the administration of the drug (the group treated with Sy-1 at a concentration of 10 μg/ml and 50 μg/ml).

Collagen synthesis

After the mice were sacrificed, tissue sections and staining were performed in the above manner to analyze the collagen synthesis at the wound. As shown in Fig. 4, on the 21st day of the mouse wound, the skin tissue around the wound was taken, and the collagen synthesis in the skin tissue was observed by Masson's trichrom staining. The experimental results show that collagen is stained blue. Compared with normal mice, in the Blank and Vasel groups, the amount of collagen synthesis inside the tissue is small and the structure is loose; in the treatment drug group, the tissue internal collagen The amount of synthesis and the tightness of the structure are similar to those of normal mice. From the above results, it has been shown to promote the synthesis of collagen under the administration of a drug.

Angiogenesis

After the mice were sacrificed, the distribution of blood vessels on the back of the wound was analyzed by photographing and the results of angiogenesis were observed. As shown in Fig. 5-6, on the 21st day of the mouse wound, the skin tissue around the wound was taken, and the vascular proliferation on the back side of the skin was analyzed. Experimental results show that the appearance from the skin can be It was found that compared to the Blank and Vasel groups, there was a significant promotion of vascular area, length and branch development in the treated drug group. From the quantitative data, in the vascular distribution area, length, vascular branch joint and path indicators, the treatment drug group significantly exceeded the Blank and Vasel groups, showing that SY1 has a significant role in promoting angiogenesis, and angiogenesis is wound healing. The necessary process.

in conclusion

From the above results, it was shown that SY-1 can indeed promote the wound healing effect of C57BL6/J mice, and high concentration of SY-1 (50ug/mL) in the early stage of wound healing, low concentration of SY-1 (10ug/mL) It has a significant effect at the end 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 promoted healing better.

The inventors also conducted an experiment in the infection group, but the results showed that SY-1 had no bactericidal efficacy, and thus no relevant experimental data was provided herein. However, in terms of the results of the infected group and the non-infected group, SY-1 can significantly promote wound healing in C57BL6/J mice. High concentrations of SY-1 (50 ug/mL) in the infected or non-infected group, at the early stage of wound healing, low concentrations of SY-1 (10 ug/mL) had a significant effect at the end of wound healing. In the non-infected group, SY-1 can significantly affect wound angiogenesis. In the infected group, the burdock culture solution was not completed due to wound healing, and the angiogenesis effect was obvious. The burdock culture solution was initially treated with an aqueous solution, which caused the wound to fester, and later changed to an ointment dosage form, but the wound healing has been delayed, and its value is only for reference. In the infected group, SY-1 also significantly promoted collagen deposition, indicating that it promoted healing better. HE staining is a reference for testing tissue integrity. SY-1 has no bactericidal effect, and its effect on promoting wound healing may be mainly related to its anti-inflammatory effect, and its action is still to be further explored in subsequent experiments.

From the above results, there can be provided a pharmaceutical composition for promoting wound healing, and a use of the pharmaceutical composition for the preparation of a medicament for promoting wound healing, which is a compound of the following formula I (ie, SY-1) ) as its active ingredient:

Preferably, in the above pharmaceutical composition or use thereof, the pharmaceutical composition is a liquid, a paste, a gel, a dressing or a spray, and the wound is an open wound.

From the above results, a pharmaceutical composition for promoting wound healing can be developed, which uses SY-1 as its active ingredient, and can be prepared into a liquid, a paste, a gel, a dressing or a spray. The dosage form is suitable for various open wounds caused by external factors to promote wound healing.

The pharmaceutical composition for promoting wound healing and the use thereof provided by the present invention have industrial use value, but the above description is only a description of the preferred embodiment of the present invention, and those skilled in the art can Other modifications are possible in the above description, but such modifications are still within the spirit of the invention and the scope of the invention as defined below.

Claims (3)

A pharmaceutical composition for the preparation of a medicament for promoting wound healing, the pharmaceutical composition comprising the following compound of formula I as its active ingredient: The use of claim 1, wherein the pharmaceutical composition is a liquid, a paste, a gel, a dressing or a spray. The use of claim 1, wherein the wound is an open wound.