Application of turbellarian worm extract in preparation of product for promoting wound healing

Abstract

The invention provides application of a turbellarian worm extract in preparation of a product for promoting wound healing, a biological agent for promoting the proliferation activity and / or migration ability of fibroblast NIH3T3, a biological agent for improving the wound healing rate and the decrustation time and a biological agent for improving the collagen fiber deposition type in the wound healing process. The biological preparation is used for promoting regeneration of skin appendage organs in the wound healing process. Wherein the extraction of the turbellarian worm extract comprises the steps of turbellarian worm cleaning, tissue crushing, alcohol extraction, centrifugal separation and concentration drying. The turbellarian worm extract has good capacity of promoting wound healing, and has the effects of effectively improving skin wound healing efficiency, shortening decrustation time, inhibiting scar formation in the wound healing process, promoting regeneration of skin appendage organs in the wound healing process and the like.

Description

Technical Field

[0001] The present invention relates to the application of a planarian extract in the preparation of a product for promoting wound healing. Background Art

[0002] The skin is a complex structure composed of the epidermis, dermis, and skin appendages. It is the largest organ of the human body and serves as the first line of defense against external injuries, protecting the underlying organs from mechanical damage, microbial infection, ultraviolet radiation, and extreme temperatures. However, long-term exposure to the environment can cause damage to the skin. After skin trauma occurs in adults, it often cannot regenerate and repair, resulting in the formation of scars. The presence of scars seriously affects aesthetics and, in severe cases, can even lead to disfigurement, growth restriction, and permanent loss of organ function.

[0003] Skin repair requires different types of cells to work together. The repair processes of different cell types are often continuous, complex, and synchronous. In uninjured skin, the epidermis is the outer impermeable layer that resists the harsh external environment and includes accessory tissue organs such as sebaceous glands, sweat glands, and hair follicles. The dermis is rich in the extracellular matrix (ECM), vascular system, and mechanoreceptors, providing strength, nutrition, and immunity to the skin. The subcutaneous adipose tissue is located beneath the dermis and plays a role in energy storage. It is also a source of dermal growth factors. In addition to these cell types, each layer contains resident immune cells that constantly check for skin damage. When the skin is injured, multiple cell types in these three layers need to be coordinated at precise stages to achieve healing. The process of wound healing can be mainly divided into four periods, including the hemostasis period (within several hours after injury), the inflammation period (1 - 3 days after injury), the proliferation period (4 - 21 days after injury), and the remodeling period (21 days - 1 year after injury). These stages of hemostasis, inflammation, angiogenesis, growth, re-epithelialization, and remodeling occur in chronological order, but there is also overlap in the process. Skin repair can be regarded as one of the most complex processes in the human body. Therefore, exploring new mechanisms to promote the speed of skin wound healing and regenerative healing is one of the urgent problems to be solved in the field of clinical medicine.

[0004] It is known that the drug Kangfuxin Liquid, which uses the American cockroach with strong regenerative ability as the raw material, has the effects of promoting blood circulation and nourishing yin to generate flesh, and is often externally used in clinical practice to treat wounds such as trauma, ulcers, fistulas, burns, scalds, and pressure sores. Freshwater planarians are animals of the class Turbellaria in the phylum Platyhelminthes. They have a simple morphology but a complex anatomical structure, including a brain, eyespots, muscle tissue, intestines, protonephridia, and epidermis. Planarians have extremely powerful tissue regeneration and organ reconstruction abilities. After being damaged, they can regenerate any missing part of the body. Even if they are cut into 279 segments, each part can still grow into a complete individual. This powerful regenerative ability of planarians has become a research hotspot in regenerative medicine research. However, the application of planarian extracts in wound healing is currently unknown. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide an application of planarian extracts in the preparation of products for promoting wound healing.

[0006] Among them, for the planarian extract described in the present invention, its extraction process includes the following steps: a. Wash the planarians with PBS solution; b. Place the planarians in PBS solution and grind the tissues until there are no obvious tissue blocks, obtaining a planarian grinding solution; c. Place the planarian grinding solution obtained in step b in an alcohol solution so that the components in the planarian grinding solution are fully dissolved in the alcohol solution; d. Centrifuge and separate the mixture in step c, collect the supernatant, and filter the supernatant; and e. Concentrate and dry the filtered supernatant to obtain the planarian extract.

[0007] Among them: in step a and step b, the concentration of the PBS solution is 0.01 M, and in step b, the dosage ratio of planarians to PBS solution is 1 g of planarians: (1 - 10) ml of PBS solution; Preferably: in step c, the alcohol solution is a methanol or ethanol solution, and its volume concentration is 30 - 100%.

[0008] Preferably: in step c, the volume ratio of the planarian crushing solution to the alcohol solution is 1: (2 - 50).

[0009] Preferably: the concentration and drying are vacuum concentration and drying.

[0010] The planarian extract obtained by the extraction of the present invention can be used in the preparation of products for promoting wound healing.

[0011] The planarian extract as described above in the present invention can also be used in the preparation of biological agents for promoting the proliferation activity and / or migration ability of fibroblast NIH3T3.

[0012] In addition, the planarian extract of the present invention can also be used to prepare a biological agent for improving the wound healing rate and eschar detachment time.

[0013] The planarian extract of the present invention can also be used to prepare a biological agent for improving the type of collagen fiber deposition during wound healing or a biological agent for promoting the regeneration of skin appendages during wound healing.

[0014] The planarian extract obtained by the extraction of the present invention has good ability to promote wound healing. It can effectively improve the skin wound healing efficiency, shorten the eschar detachment time, improve the proliferation activity of fibroblasts ((NIH3T3)) and effectively promote the migration of fibroblasts (NIH3T3), achieving the effects of rapidly repairing wounds, inhibiting scar formation and promoting the regeneration of skin appendages at the wound site, providing a new repair pathway for damaged skin. Description of the Drawings

[0015] Figure 1 Shows the analysis of the effect of planarian extract on the proliferation activity of fibroblasts; Figure 2 Shows the analysis of the effect of planarian extract on the migration of fibroblasts; Figure 3 Shows the analysis of the effect of planarian extract on the healing rate of full-thickness skin wounds in mice; Figure 4 Shows the analysis of the effect of planarian extract on the eschar detachment time of full-thickness skin wounds in mice; Figure 5 Shows the analysis of the effect of planarian extract on the type of collagen fiber precipitation in the wounds of full-thickness skin wounds in mice; and Figure 6 Shows the analysis of the effect of planarian extract on the regeneration of skin appendages in the wounds of full-thickness skin wounds in mice. Detailed Embodiments

[0016] First, for the planarian extract of the present invention, its extraction steps include: a. Wash the planarians with 0.01M PBS solution, wherein the planarians are Dugesia japonica Dugesia japonica ), which can be obtained by direct market purchase or self-cultivation in the laboratory; b. Place the washed planarians in step a in 0.01M PBS solution according to the ratio of 1 g of planarians: (1-10) ml of solution, and use a tissue grinder to break the tissue at room temperature until there are no obvious tissue blocks, obtaining a planarian grinding solution; c. Place the planarian grinding solution obtained in step b in an alcohol solution at a volume ratio of 1:(2 - 50), and incubate it by inversion at 4°C for 48 hours using a rotary mixer to fully dissolve the components in the planarian grinding solution in the alcohol solution; in the present invention, the alcohol solution can be an ethanol solution or a methanol solution, and its volume concentration is 30 - 100%; d. Place the incubated mixture in step c in a centrifuge and centrifuge it at a speed of 4000 rpm for 20 minutes, then collect the supernatant, and filter the supernatant using a needle-type sterilizing filter with a filtration accuracy of 0.22 μm - 0.45 μm; e. Concentrate and dry the filtered supernatant in step d using, for example, a vacuum centrifugal concentrator to obtain the solid planarian extract of the present invention.

[0017] The present invention also provides a study on the promotion of wound healing by the planarian extract obtained by the above method, which mainly includes the following research contents: (1) Establish a full-thickness skin injury model in mice: Male C57BL / 6 mice aged 6 - 8 weeks are raised according to the animal health care and handling procedures (the experimental mice in the present invention are all purchased from Liaoning Changsheng Biotechnology Co., Ltd., animal production license number: SCXK(Liao)2020 - 0001, experimental unit use license number: SYXK(Yu)2024 - 0010, and the whole process of the experimental operation complies with the "Regulations on the Administration of Animal Experiments"). According to the body weight of the mice, anesthetize the mice by intraperitoneal injection of 4% chloral hydrate solution at a dose of 100 μL / 10 g. After depilating and disinfecting the back skin of the mice, create two symmetrical circular wounds with a full cortex resection of 6 mm on both sides along the midline of the back, approximately 6 mm below the ear and 4 mm outside the midline. Then fix a silicone ring with a diameter of 12 mm around the wound to avoid the interference of the healing method of rodent skin contraction and wound healing on the subsequent experiment. The two wounds are used as the experimental group and the control group respectively to avoid the error of the healing result caused by individual animal differences. After modeling, the animals are raised individually in cages.

[0018] (2) Administer the drug to the mice with the trauma model: After dissolving the planarian extract of the present invention in sterile normal saline to an appropriate concentration (generally 1 - 50 mg / mL), apply it to the wounds of the mice after modeling in experiment (1) according to the standard of 50 μg - 100 μg of planarian extract per wound to ensure that the wound position is fully covered by the drug, and apply the drug once a day for 7 consecutive days. At the same time, the control group is applied with sterile normal saline.

[0019] (3)Evaluate the efficacy of the planarian extract on wound repair: The efficacy evaluation includes determination of wound healing rate, eschar separation time, analysis of collagen production, and detection of the regeneration of skin appendages; among them, the methods for determining the wound healing rate and eschar separation time are as follows: After topical administration in the above experiment (2), starting from day 0, place a scale at the wound level every 2 days, take pictures of the wound surface with the same photographing tool, calculate the wound healing rate with software, and record the eschar separation time; the analysis of collagen production adopts microscopic examination analysis after Masson staining: Take the skin wound tissues of the experimental group and the control group of mice on the 24th day after modeling in the above experiment (1), fix, dehydrate, impregnate with wax, embed, solidify, trim the block, section, perform Masson staining, seal with neutral gum, and use a microscope for microscopic examination and image acquisition to analyze the type of collagen fibers in the regenerated skin at the wound position; the detection of the regeneration of skin appendages adopts microscopic examination analysis after HE staining: Take the skin wound tissues of the experimental group and the control group of mice on the 24th day after modeling in the above experiment (1), fix, dehydrate, impregnate with wax, embed, solidify, trim the block, section, perform HE staining, seal with neutral gum, and use a microscope for microscopic examination and image acquisition to analyze the regeneration of appendages such as sebaceous glands and hair follicles in the regenerated skin at the wound position.

[0020] (4)Evaluate the effects of the planarian extract on the proliferation activity and migration ability of mouse fibroblasts (NIH3T3): Use the Cell Counting Kit-8 (CCK8) method to detect the effect of the planarian extract on cell proliferation activity at different time points during cultivation; the detection of the migration ability of fibroblasts NIH3T3 adopts the scratch test method. Take pictures at the 0th, 12th, and 24th hours respectively to determine the effect of the planarian extract on cell migration ability, and use ImageJ software for analysis.

[0021] It should be noted that the planarian extract solutions involved in the present invention are all prepared by dissolving the planarian extract in sterile deionized water to the required use concentration, and filtering and sterilizing with a filter with a filtration accuracy of 0.22 μm - 0.45 μm, such as a needle-type sterilizing filter, and then waiting for use.

[0022] Example 1 An extraction method of a planarian extract, the extraction steps of which include: a. Wash the planarians twice with a PBS solution with a concentration of 0.01 M; b. Place the planarians washed in step a in a PBS solution with a concentration of 0.01 M according to the ratio of 1 g of planarians: 1 ml of solution, and use a tissue grinder to break the tissue at room temperature until there are no obvious tissue blocks, obtaining a planarian grinding solution; c. Place the planarian grinding solution obtained in step b into an ethanol solution with a volume concentration of 40% at a volume ratio of 1:50, and incubate it by inversion at 4 °C for 48 hours using a rotary mixer to fully dissolve the components in the planarian grinding solution in the ethanol solution; d. Place the incubated mixture in step c into a centrifuge and centrifuge it at a speed of 4000 rpm for 20 minutes, then collect the supernatant and filter the supernatant with a 0.22 μm needle-type sterile filter; e. Use a vacuum centrifugal concentrator to concentrate and dry the filtered supernatant in step d at room temperature and a vacuum degree of 20 hPa to obtain a solid planarian extract.

[0023] Example 2 A method for extracting a planarian extract, the extraction steps of which include: a. Wash the planarians twice with a PBS solution with a concentration of 0.01 M; b. Place the planarians washed in step a into a PBS solution with a concentration of 0.01 M at a ratio of 1 g of planarian: 1 ml of solution, and use a tissue grinder to break the tissue at room temperature until there are no obvious tissue blocks left to obtain a planarian grinding solution; c. Place the planarian grinding solution obtained in step b into a methanol solution with a volume concentration of 75% at a volume ratio of 1:20, and incubate it by inversion at 4 °C for 48 hours using a rotary mixer to fully dissolve the components in the planarian grinding solution in the methanol solution; d. Place the incubated mixture in step c into a centrifuge and centrifuge it at a speed of 4000 rpm for 20 minutes, then collect the supernatant and filter the supernatant with a 0.22 μm needle-type sterile filter; e. Use a vacuum centrifugal concentrator to concentrate and dry the filtered supernatant in step d at room temperature and a vacuum degree of 30 hPa to obtain a solid planarian extract.

[0024] Example 3 A method for extracting a planarian extract, the extraction steps of which include: a. Wash the planarians twice with a PBS solution with a concentration of 0.01 M; b. Place the planarians washed in step a into a PBS solution with a concentration of 0.01 M at a ratio of 1 g of planarian: 2 ml of solution, and use a tissue grinder to break the tissue at room temperature until there are no obvious tissue blocks left to obtain a planarian grinding solution; c. Place the planarian grinding solution obtained in step b into an ethanol solution with a volume concentration of 90% at a volume ratio of 1:20, and incubate it by inversion at 4 °C for 48 hours using a rotary mixer to fully dissolve the components in the planarian grinding solution in the ethanol solution; d. Place the incubated mixture in step c in a centrifuge and centrifuge at 4000 rpm for 20 minutes. Then collect the supernatant and filter the supernatant through a 0.22 μm needle-type sterilizing filter; e. Use a vacuum centrifugal concentrator to concentrate and dry the filtered supernatant in step d at room temperature and a vacuum degree of 25 hPa to obtain a solid-state planarian extract.

[0025] Experimental Example 1 Effect of Planarian Extract on the Viability of Fibroblasts Place fibroblasts NIH3T3 at a density of 3×10 3 cells / well in a 96-well plate, add 100 μL of complete medium (DMEM medium (Thermo Fisher Scientific)+10% FBS (fetal bovine serum)), and place it in an incubator to culture overnight; after the culture is completed, add the planarian extract solution with a final concentration of 1 mg / mL of the planarian extract in Example 1 to each well of the experimental group, and add an equal volume of sterile normal saline to the control group. Place the cells in a CO2 incubator at 37 °C for culture.

[0026] Detect the cell proliferation viability at 0, 6, 12, and 24 h: Use a pipette to add 1 / 10 of the medium volume of CCK8 solution to each well of the culture plate, continue to culture the culture plate in the incubator for 1.5 hours, and measure the absorbance at 450 nm with an enzyme-linked immunosorbent assay reader. And calculate the cell proliferation viability through the following formula: Cell viability (%) = [A 加药 -A 空白 / [A 0加药 -A 空白 ×100% Among them, A 加药 : OD value of the well with cells, CCK-8 solution, and planarian extract solution; A 0加药 : OD value of the well with cells and CCK-8 solution but without planarian extract solution; A 空白 : OD value of the well without cells.

[0027] Figure 1 The analysis results are shown. It can be seen from the figure that by treating the cells with the planarian extract solution, the cell proliferation viability of fibroblasts NIH3T3 begins to increase significantly from 6 hours after treatment, indicating that the planarian extract has an obvious promoting effect on the proliferation viability of fibroblasts NIH3T3.

[0028] Experimental Example 2 Effect of Planarian Extract on the Migration of Fibroblasts Before the experiment, use a marker pen to draw horizontal lines evenly on the back of a 6-well plate. The interval between adjacent horizontal lines is about 0.5 cm to 1 cm, crossing the wells, and there are at least 5 lines per well; add about 5×105 cells, and the specific number is judged according to the cell doubling time, and it is controlled to be fully covered overnight; the next day, use a pipette tip to draw a straight scratch along the horizontal line on the back; wash with PBS buffer solution with a concentration of 0.01M until all cell debris is washed away, add 1 mL of serum-free DMEM medium (Thermo Fisher Scientific), and at the same time add 50 μg of the planarian extract solution in Example 1 with a final concentration of 0.5 mg / mL to each well of the experimental group, and add an equal volume of sterile PBS solution of 0.01M to the control group, and place it in a CO2 incubator at 37°C for static culture; take pictures at 0 and 24 hours respectively, and use ImageJ software for analysis, and the results are as Figure 2 shown.

[0029] As can be seen from Figure 2 it, after the cells were treated with the planarian extract for 24 hours, compared with the control group, significantly more cells in the experimental group migrated from the surrounding area to the central scratch area, and the difference was statistically significant, indicating that the use of the planarian extract can effectively promote the migration ability of fibroblasts NIH3T3.

[0030] Experimental Example 3 Effect of Planarian Extract on the Wound Healing Rate and Eschar Separation Time of Full-Thickness Skin Lesions in Mice After applying the drug to the mice, starting from day 0, place the scale at the same wound level position of the mice every 2 days, take pictures with the same photographing tool to record the wound surface conditions, and use ImageJ software to calculate the wound healing rate and record the eschar separation time. The wound healing rate is calculated by the following formula:

[0031] where: n is the number of days after modeling.

[0032] Figure 3 The analysis results are shown. As can be seen from Figure 3 it, as the time after injury prolonged, on the 14th day after modeling, the wound healing rate of the skin of the mice in the experimental group was significantly higher than that of the mice in the control group, and the difference was statistically significant, indicating that the use of the planarian extract can effectively promote the healing of the wounds of the mice.

[0033] Example 4 Effect of Planarian Extract on the Collagen Deposition Type of Wound in Full-Thickness Skin Lesions of Mice On the 24th day after modeling, the skin wound tissues of the experimental group and the control group of mice were placed in 4% paraformaldehyde and fixed at 4°C for 24 hours, with the fixing solution changed once in the middle; after the samples were taken out, they were immersed in 0.01M PBS solution and rinsed 3 times for 3 minutes each. Gradient alcohol and xylene dehydration were carried out with turning, as follows: 75% ethanol for 4 hours, 85% ethanol for 2 hours, 90% ethanol for 2 hours, 95% ethanol for 1 hour, absolute ethanol twice for 30 minutes each, 25% xylene + 75% absolute ethanol for 10 minutes, 50% xylene + 50% absolute ethanol for 10 minutes, 75% xylene + 25% absolute ethanol for 10 minutes, and 100% xylene twice for 30 minutes each until the tissue became transparent. At 37°C, 50% ethanol + 50% paraffin for 1 hour, pure paraffin was added at 60°C, and the tissue was infiltrated with paraffin for 12 hours. Pure wax was changed and the process was repeated four times. Finally, the materials were embedded, solidified, trimmed, and sectioned. The sections were routinely dewaxed to water: Staining was carried out according to the instructions of the Masson staining kit (Beijing Solarbio Science & Technology Co., Ltd.). The process was as follows: Hematoxylin staining - differentiation - bluing - washing with water - Ponceau staining - washing with weak acid working solution - differentiation with phosphomolybdic acid - washing with weak acid working solution - Aniline blue staining - weak acid washing - rapid dehydration and clearing - mounting. Microscopic examination was carried out, and the types of collagen fibers in the regenerated skin at the wound position were analyzed by image acquisition. The results are as Figure 4 shown: It can be seen from Figure 4 that compared with the skin injury of the control group mice, the eschar shedding time of the skin injury of the experimental group mice was significantly advanced, and the difference was statistically significant, indicating that the planarian extract can effectively promote the rapid healing of mouse wounds.

[0034] Experimental Example 5 Effect of Planarian Extract on the Regeneration of Skin Appendages in the Wounds of Full-thickness Skin Defects in Mice On the 24th day after modeling, the skin wound tissues of the experimental group and the control group of mice were fixed, dehydrated, infiltrated with paraffin, embedded, solidified, trimmed, and sectioned. The process was the same as that in Experimental Example 4 above; the sections were routinely dewaxed to water: The samples were placed in the prepared hematoxylin staining solution for nuclear staining for 5 - 20 minutes, and the dye and floating color on the slides were washed off with running water. Differentiation was carried out with hydrochloric acid alcohol differentiation solution for 30 seconds, followed by washing with water. The samples were placed in eosin staining solution for cytoplasmic staining for 1 - 3 minutes, and the surface staining solution was slightly rinsed off with distilled water. Routine dehydration, clearing, and mounting were carried out with neutral gum; Microscopic examination was carried out, and the regeneration of sebaceous glands, hair follicles and other appendages in the regenerated skin at the wound position was analyzed by image acquisition. The results are shown in Figure 5 and Figure 6 respectively.

[0035] The formation of scar tissue is accompanied by collagen fiber deposition. The typical feature of scar tissue is that the collagen fibers are dense and arranged in parallel bundles. From Figure 5It can be seen that on the 24th day after modeling, the collagen deposition type in the skin wound tissue of the experimental group mice was significantly closer to that of the uninjured skin, indicating that the use of planarian extract can effectively inhibit scar formation. According to Figure 6 (In the figure, the white dotted line marks the wound edge, the yellow arrow marks the hair follicle primordium at the wound site, the red arrow marks the hair follicles at the wound site, and the green arrow marks the sebaceous glands at the wound site), it can be seen that on the 24th day after modeling, the skin wound tissue of the experimental group mice showed the regeneration of skin appendages such as hair follicles and sebaceous glands, while the control group mice did not show obvious regeneration of skin appendages, indicating that the use of planarian extract can inhibit scar formation and promote the regenerative repair of wounds.

[0036] From the above research of the present invention, it can be known that the planarian extract has the functions of promoting wound healing and inhibiting scar formation, and its effects are closely related to promoting the proliferation and migration of fibroblasts and the deposition and remodeling of collagen fibers.

[0037] The present invention has been described in detail by way of preferred embodiments. However, through the study of the foregoing, changes and additions to each embodiment are also obvious to those of ordinary skill in the art. The applicant's intention is that all such changes and additions fall within the protection scope of the claims of the present invention. The terms used herein are only for explaining specific embodiments, and they are not intended to limit the present invention. Unless otherwise defined, all terms used herein (including technical terms and scientific terms) have the same meaning as understood by those of ordinary skill in the art to which the present invention pertains. Any modification and improvement of this product, and the substitution and use of similar or closely related substances within the scope of the patent, fall within the protection scope of this invention patent.

Claims

1. Application of planarian extract in preparing products for promoting wound healing.

2. Application of planarian extract in preparing biological agents for promoting the proliferation vitality and / or migration ability of fibroblast NIH3T3.

3. Application of planarian extract in preparing biological agents for increasing wound healing rate and eschar separation time.

4. Application of planarian extract in preparing biological agents for improving the type of collagen fiber deposition during wound healing.

5. Application of planarian extract in preparing biological agents for promoting the regeneration of skin appendages during wound healing.

6. The application according to any one of claims 1-5, characterized in that: The extraction steps of the planarian extract include: a. Washing planarians with PBS solution; b. Placing planarians in PBS solution and grinding the tissue until there are no obvious tissue blocks, obtaining a planarian grinding solution; c. Placing the planarian grinding solution obtained in step b in an alcohol solution to fully dissolve the components in the planarian grinding solution in the alcohol solution; d. Centrifuging and separating the mixture in step c, collecting the supernatant, and filtering the supernatant; and e. Concentrating and drying the filtered supernatant to obtain the planarian extract.

7. The application according to claim 6, wherein: In steps a and b, the concentration of the PBS solution is 0.01M, and in step b, the dosage ratio of planarians to PBS solution is 1 g of planarians: (1 - 10) ml of PBS solution.

8. The application according to claim 6, wherein: In step c, the alcohol solution is methanol or ethanol solution, and its volume concentration is 30 - 100%.

9. The application according to claim 6, wherein: In step c, the volume ratio of the planarian crushing solution to the alcohol solution is 1: (2 - 50).

10. The application according to claim 6, characterized in that: The concentration and drying is vacuum concentration and drying.

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