Turbellarian worm extract as well as extraction method and application thereof

Abstract

The invention discloses a turbellarian worm extract and a corresponding extraction method thereof. The extraction method comprises the steps of turbellarian worm cleaning, tissue crushing, alcohol extraction, centrifugal separation, concentration and drying and the like. The turbellarian worm extract can effectively activate the proliferation of hair papilla cells, inhibit the adverse effect of male hormone on hair follicles, increase the density of the hair follicles, effectively prolong the growth period of the hair follicles, promote the growth of new hair and improve the density and thickness of the hair.

Description

Technical Field

[0001] The invention relates to the field of planarian extraction, and in particular to a planarian extract and an extraction method thereof and application thereof in products for treating androgenic alopecia. Background Art

[0002] Androgenic alopecia (AGA) is a common type of hair loss that affects a large number of men and some women worldwide. It is mainly manifested by a receding hairline and sparse hair on the top of the head, which may eventually develop into permanent hair loss. The occurrence of AGA is mainly affected by genetic factors and androgen levels. Its core mechanism involves the effect of dihydrotestosterone (DHT) on hair follicles. DHT binds to androgen receptors in hair follicles, causing the hair follicles to gradually atrophy, resulting in a shortened hair growth period, and eventually causing the hair follicles to enter a resting phase and gradually lose their ability to grow hair. Although AGA does not directly affect physical health, it has a significant impact on patients' mental health, social life, and self-confidence. Therefore, seeking effective treatments has important clinical and market value.

[0003] At present, the treatment methods for androgenic alopecia mainly include drug therapy, hair transplant surgery and physical therapy. Among them, in terms of drug therapy, commonly used drugs such as minoxidil promote hair growth by dilating blood vessels, but hair loss is easy to recur after discontinuation; finasteride can inhibit DHT production and delay hair follicle atrophy, but it may cause side effects such as sexual dysfunction. In addition, some plant extracts, such as ginger and caffeine, have certain auxiliary effects, but their efficacy has not been widely recognized. Although hair transplant surgery can restore hair density, it is expensive, donor hair follicles are limited, and there are problems such as postoperative recovery. While physical therapy methods such as low-energy laser (LLLT), microneedles and PRP (platelet-rich plasma) can improve the scalp environment, they cannot fundamentally reverse hair loss. In general, the existing treatment methods for androgenic alopecia have more or less limited efficacy, obvious side effects, high prices or easy recurrence of hair loss, and new safe and effective treatment options are urgently needed.

[0004] Planarians are a type of lower invertebrate with extremely strong regenerative abilities. They are rich in stem cells that can rapidly proliferate, differentiate, and repair tissues after damage. However, there are not many studies on planarian extracts, especially on the treatment of androgenic alopecia. Therefore, it is particularly important to expand the application scope of planarian extracts and avoid the waste of planarian resources. Summary of the invention

[0005] The object of the present invention is to overcome the deficiencies of the prior art and provide a method for extracting planarian extract and the planarian extract obtained by this extraction. Another object of the present invention is to provide an application of the planarian extract in products for treating androgenetic alopecia.

[0006] To achieve the above object, the present invention adopts the following technical solutions: On the one hand, the present invention provides a method for extracting planarian extract, which includes the following extraction steps: a. Wash the planarians with PBS solution; b. Place the planarians in PBS solution and grind the tissue until there are no obvious tissue blocks left to obtain 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] Preferably: in steps a and 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.

[0008] Preferably: in step c, the alcohol solution is a methanol or ethanol solution, and its volume concentration is 30 - 100%.

[0009] Further: in step c, the volume ratio of the planarian crushing solution to the alcohol solution is 1: (2 - 50).

[0010] Further: the concentration and drying is vacuum concentration and drying.

[0011] The present invention also provides a planarian extract obtained by the aforementioned extraction method.

[0012] The planarian extract as described above in the present invention can be applied to the preparation of products for preventing and treating hair loss caused by androgens such as testosterone and dihydrotestosterone.

[0013] The planarian extract as described above in the present invention can also be applied to the preparation of products for repairing follicular atrophy, shortening the follicular growth period, and improving the follicular survival rate after hair transplantation, etc.

[0014] The planarian extract as described above in the present invention can also be applied in products for promoting the proliferation of dermal papilla cells, follicular regeneration medicine, and tissue engineering.

[0015] In addition, the planarian extract of the present invention as described above is also applicable to the preparation of pet hair care products.

[0016] The planarian extract obtained by the extraction method of the present invention can activate the proliferation of dermal papilla cells, and can effectively slow down hair loss caused by male hormones such as testosterone and dihydrotestosterone. The planarian extract of the present invention can also effectively inhibit the adverse effects of male hormones on hair follicles, delay hair follicle atrophy and increase the number of hair follicles, and inhibit hair follicles from entering the regression period. In addition, the planarian extract of the present invention can also effectively promote hair growth, improve hair coverage and thickness, and promote the vitality and proliferation ability of dermal papilla cells. The planarian extract of the present invention can be made into external application ointments, serums, shampoos or oral preparations, etc., and has broad market application prospects, providing a new and efficient option for the treatment of male pattern hair loss. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Shows the effect of planarian extract on the appearance change of the male pattern hair loss model in mice; Figure 2 Shows the results of HE staining analysis of the effect of planarian extract on hair follicle tissue; Figure 3 Shows the effect of planarian extract on the thickness of the male pattern hair loss model in mice; Figure 4 Shows the results of CCK-8 assay analysis of the effect of planarian extract on the viability of dermal papilla cells; and Figure 5 Shows the EdU detection results of the effect of planarian extract on the proliferation of dermal papilla cells. DETAILED DESCRIPTION OF THE INVENTION

[0018] According to the first aspect of the present invention, the present invention provides a method for extracting a planarian extract, which comprises the following extraction steps: a. Wash 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, to obtain a planarian grinding solution; c. Place the planarian grinding solution obtained in step b in an alcohol solution according to a volume ratio of 1: (2-50), and incubate it with a rotary mixer at 4°C for 48 hours 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 at 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 a vacuum centrifugal concentrator, for example, to obtain the solid planarian extract of the present invention.

[0019] According to another aspect of the present invention, there is provided a study on the use of the above-mentioned extracted planarian extract in the treatment of androgenetic alopecia, mainly including the following research contents: (1) Establish a mouse androgenetic alopecia model and conduct drug intervention: Specifically, male C57BL / 6 mice aged 6 - 8 weeks are raised according to animal health care and handling procedures (all experimental mice of the present invention are 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 experiment complies with the "Regulations on the Administration of Animal Experiments"). During the experiment, a testosterone-induced mouse androgenetic alopecia model is used to simulate the androgen-mediated follicular atrophy process. This method can effectively simulate androgenetic alopecia and provide a reliable experimental basis for studying the role of planarian extract in AGA treatment; (2) Observe the hair growth situation in terms of morphology: The specific process is to record the hair growth on the back of the mice through a camera, and take pictures to record the hair growth on the back of the mice on the day (day 0), day 3, day 7, day 10, day 14, day 18, and day 26 when the mice are intervened with the planarian extract of the present invention; (3) Study at the histological level whether the follicular atrophy and reduction caused by testosterone are effectively inhibited: Specifically, use the HE staining method (hematoxylin-eosin staining method) to observe the follicular structure of the mice, and measure the follicle number, diameter, and the proportion of growth-phase cysts, etc.; (4) The effect of planarian extract on the thinning of mouse hair caused by testosterone treatment: Use an environmental scanning electron microscope to observe and calculate the hair shaft diameter of the mouse hair in the above content (2) to evaluate the change in hair thickness; (5)Detection and analysis of the promotion of dermal papilla cell viability and proliferation ability by planarian extract: The CCK-8 detection method (Cell Counting Kit-8) and EdU staining method were used to detect the dermal papilla cells in the dihydrotestosterone (DHT) interference group (final concentration of DHT was 150 μM) and the planarian extract intervention treatment group; among them, the CCK-8 method was to add CCK-8 solution to the cell culture environment and measure the absorbance (OD value) at specific time points to evaluate the proliferation viability of cells. The EdU staining method was to label the proliferating cells (EdU-positive cells) and observe and quantitatively analyze the proliferation of dermal papilla cells under a fluorescence microscope.

[0020] It should be noted that the planarian extract solution involved in the above research process refers to the solution obtained by dissolving the planarian extract in sterile deionized water to the required use concentration and filtering and sterilizing it with a filter with a filtration accuracy of 0.22 μm - 0.45 μm, such as a needle-type sterilizing filter, for later use.

[0021] Example 1 An extraction method of planarian extract, the extraction steps of which include: a. Wash the planarians twice with PBS solution with a concentration of 0.01 M; b. Place the washed planarians in step a in 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 into no obvious tissue blocks at room temperature to obtain a planarian grinding solution; c. Place the planarian grinding solution obtained in step b in an ethanol solution with a volume concentration of 40% according to a volume ratio of 1:50, and incubate it by inversion at 4°C for 48 hours using a rotary mixer to make the components in the planarian grinding solution fully dissolve in the ethanol solution; 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 with 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 20 hPa to obtain a solid planarian extract.

[0022] Example 2 An extraction method of planarian extract, the extraction steps of which include: a. Wash the planarians twice with 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 planarians : 1 ml of solution, and use a tissue grinder to break the tissue at room temperature until there are no obvious tissue chunks left, obtaining 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 use a rotary mixer to incubate with inversion at 4°C for 48 hours 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 at a speed of 4000 rpm for 20 minutes, then collect the supernatant and filter the supernatant with 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 30 hPa to obtain a solid planarian extract.

[0023] 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 planarians : 2 ml of solution, and use a tissue grinder to break the tissue at room temperature until there are no obvious tissue chunks left, obtaining 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 use a rotary mixer to incubate with inversion at 4°C for 48 hours 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 at a speed of 4000 rpm for 20 minutes, then collect the supernatant and filter the supernatant with 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 planarian extract.

[0024] Experimental Example 1 Appearance effect of planarian extract on male hormone alopecia model in mice Prepare a 5 mg / mL testosterone solution (TES solution): Weigh 500 mg of testosterone, fully dissolve it with 50% ethanol, and make up the volume to 100 mL. After aliquoting, store it at -20°C.

[0025] SPF-grade male C57BL / 6 mice aged 6 - 8 weeks (purchased from Liaoning Changsheng Biotechnology Co., Ltd., animal production license number: SCXK (Liao) 2020 - 0001) were randomly grouped. After 1 week of adaptive feeding, the mice were anesthetized by intraperitoneal injection of 4% chloral hydrate solution at a dose of 100 μL / 10 g. A hair clipper was used to depilate an area of approximately 2 cm × 2 cm on the back of the mice, and then a depilatory cream was used to thoroughly remove the remaining hair in this area.

[0026] Drug intervention was carried out on the day when the hair removal of the mouse model was completed. The mice were divided into three groups: a control group, an AGA group, and an AGA treatment group. Among them, the control group was subcutaneously injected with normal saline in the morning and 100 μL of 50% ethanol solution in the afternoon; the AGA group was subcutaneously injected with normal saline in the morning and 100 μL of 5 mg / mL TES solution in the afternoon; the AGA treatment group was subcutaneously injected with 2 mg / mL of the planarian extract solution prepared in Example 1 of the present invention in the morning and 100 μL of 5 mg / mL TES solution in the afternoon. During the experiment, the minimum interval between morning and afternoon subcutaneous injections was 6 hours. The hair growth on the back of the mice was observed, and the hair growth conditions of the mice were recorded and photographed on the day of the experiment (day 0), day 3, day 7, day 10, day 14, day 18, and day 26. The photos taken each time were analyzed to evaluate indicators such as the hair growth rate, hair coverage area, and hair density of each group, and the entire experimental cycle ended on day 26.

[0027] Figure 1 The results of the observed changes in the appearance of the back hair of the mice photographed during the entire experimental cycle are shown. From Figure 1 It can be seen that compared with the control group, the mice in the AGA group showed significantly inhibited hair growth on their backs under the treatment of testosterone, while the hair on the backs of the mice in the AGA treatment group intervened with the planarian extract was significantly more than that in the AGA group, and the hair coverage area increased. This shows that the use of the planarian extract can effectively repair the inhibitory effect of testosterone on hair growth and restore the ability of normal hair growth on the back.

[0028] Experimental Example 2 Analysis of the effect of planarian extract on hair follicle tissue by HE staining At the end of the experiment in Experimental Example 1 (day 26), the mice were sacrificed and the skin tissue of the depilated area on the back was removed. The skin tissue was fixed with 4% paraformaldehyde solution for 24 hours, and then the fixed skin tissue was paraffin-embedded. The paraffin-embedded skin tissue was cut into thin slices of 5 - 7 μm using a microtome, collected using glass slides, and air-dried for staining.

[0029] The sliced tissues were successively placed into xylene, 100% ethanol, 95% ethanol, 85% ethanol, and 75% ethanol to remove paraffin and hydrate. The sections were stained with HE (hematoxylin-eosin) staining kit (Solarbio) for HE staining, dehydration, and mounting. The HE-stained sections were observed under an optical microscope to analyze the morphological changes of hair follicles, and the morphology and number of hair follicles were counted to analyze the hair follicle density. The results are as Figure 2 shown.

[0030] According to Figure 2 the optical microscope photos, compared with the control group, the mice in the AGA group showed significant inhibition of the hair follicles on the back skin from entering the growth phase under the treatment of testosterone, with relatively shorter hair follicle lengths and lower hair follicle densities; while the hair follicle lengths on the back skin of the mice in the AGA treatment group under the intervention of planarian extract were significantly longer than those in the AGA group, and the hair follicle density per unit area was significantly increased. It shows that planarian extract can effectively repair the inhibitory effect of testosterone on hair follicle growth.

[0031] Experimental Example 3 Effect of Planarian Extract on the Thickness of the Male Hormone Alopecia Model in Mice At the end of the experiment in Experimental Example 1, the back hair of the mice was taken, fixed, and processed into samples suitable for environmental scanning electron microscope detection. The hair was observed under an environmental scanning electron microscope to analyze the thickness and surface morphology of the hair. The results are as Figure 3 shown.

[0032] According to Figure 3 the environmental scanning electron microscope scanning results, compared with the control group, the hair diameter of the mice in the AGA group became significantly thinner under the treatment of testosterone, while the hair diameter of the mice in the AGA treatment group under the intervention of planarian extract increased significantly and the hair became thicker, indicating that after treatment with planarian extract, the damage caused by testosterone to hair follicle function was repaired, effectively promoting the healthy growth of hair.

[0033] Experimental Example 4 Analysis of the Effect of Planarian Extract on the Proliferation Ability of Dermal Papilla Cells Using the CCK-8 kit (Beyotime Biotechnology Research Institute), according to 0, 2.5×10 3 , 5×10 3 , 1×10 4 , 2.5×10 4 , 5×10 4At each density, dermal papilla cells were seeded in 96-well plates with 3 replicate wells per density. 100 μL of complete medium (Gibco™ Essential 8™ medium (Thermo Fisher Scientific)+14% FBS (fetal bovine serum)) and 10 μL of CCK8 solution were added to each well. After reacting for 2 hours, the absorbance was measured at 450 nm using a microplate reader, and a standard curve was plotted based on the cell density and absorbance values. Prepare a 150 μM dihydrotestosterone (DHT) solution. Pipette 15 μL of a 10 mM DHT solution and make up to 1 mL with the above complete medium (Gibco™ Essential 8™ medium + 14% FBS). Prepare fresh as needed. At a density of 5×10 3 dermal papilla cells per well were seeded in 96-well plates, and 100 µL of complete medium (Gibco™ Essential8 TM medium + 14% FBS) was added, and the plates were incubated overnight in an incubator. The cells after overnight culture were divided into three groups: a control group, an AGA group, and an AGA treatment group. Among them, in the AGA group, a DHT solution with a final concentration of 150 μM was added to each well, and in the AGA treatment group, a DHT solution with a final concentration of 150 μM and a planarian extract solution with a final concentration of 1 mg / mL prepared in Example 3 of the present invention were added to each well. In the control group, an equal volume of sterile 0.01 M PBS solution was added to each well, and then the cells were cultured in a CO2 incubator at 37 °C. At 0 hours and 24 hours of culture, 10 µL of CCK-8 solution was added to each well, and the culture plates were further incubated in the incubator for 2 hours. The absorbance was measured at 450 nm using a microplate reader. The proliferation fold of the cells in each group relative to 0 hours at each time point was calculated according to the standard curve, and the results are as Figure 4 shown.

[0034] From Figure 4 the absorbance results, it can be seen that compared with the control group, the treatment with DHT significantly reduced the proliferation fold of dermal papilla cells in the AGA group, while the proliferation fold of the AGA treatment group under the intervention of the planarian extract was significantly increased compared with that of the AGA group, indicating that the planarian extract can effectively relieve the stress of DHT on dermal papilla cells and restore the proliferation ability of dermal papilla cells.

[0035] Experimental Example 5 EdU detection analysis of the effect of planarian extract on the proliferation of dermal papilla cells Using an Edu detection kit (BeyoClick™ EdU-488 Cell Proliferation Detection Kit, Beyotime), dermal papilla cells were seeded at a density of 5×10 4Cells were seeded at a density of [cells per well] in 6-well plates, and 1 mL of complete medium (Gibco™ Essential 8™ medium (Thermo Fisher Scientific) + 14% FBS (fetal bovine serum)) was added to each well. The cells were divided into three groups: a control group, an AGA group, and an AGA treatment group. In the AGA group, a DHT solution with a final concentration of 150 μM was added to each well. In the AGA treatment group, a DHT solution with a final concentration of 150 μM and a solution of planarian extract prepared in Example 1 of the present invention with a final concentration of 1 mg / mL were added to each well. The control group was added with an equal volume of sterile 0.01 M PBS solution, and the cells were placed in a CO2 incubator at 37 °C and cultured for another 24 hours. Each group had 3 replicate wells. Subsequently, EdU-labeled proliferating cells were detected according to the instructions of the BeyoClick™ EdU-488 Cell Proliferation Detection Kit. The time for treating the cells with the EdU working solution was 2 hours. Imaging was performed using an inverted fluorescence microscope, with 6 fields of view per well. The number of EdU-positive cells and the total number of cells in each group were counted, and the proportion of EdU-positive cells was calculated for statistical analysis. The promotion effect of the planarian extract on the proliferation of dermal papilla cells was evaluated by the number of EdU-positive cells. Figure 5 The imaging results of the inverted fluorescence microscope are shown.

[0036] As can be seen from Figure 5 it, compared with the control group, DHT treatment significantly reduced the proportion of dermal papilla cells in the proliferation phase in the AGA group. Compared with the AGA group, the proportion of proliferating cells in the AGA treatment group with planarian extract intervention was significantly increased, indicating that the planarian extract can effectively promote the proliferation of dermal papilla cells.

[0037] Through the above experimental studies, it can be known that the planarian extract prepared in the present invention can effectively inhibit the negative effects of androgens on hair follicles, reduce hair follicle regression, alleviate the process of androgenetic alopecia, enhance the resistance of hair follicles to androgen damage, and restore the vitality of dermal papilla cells.

[0038] The present invention has been described in detail through 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 illustrating 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) are the same as those understood by those of ordinary skill in the art to which the present invention belongs. Any modification and improvement of this product, and the substitution and use of similar or closely related substances within the scope or category of the patent, all belong to the protection scope of the present invention patent.

Claims

1. A method for extracting a planarian extract, characterized in that: It includes the following extraction steps: a. Wash the planarians with PBS solution; b. Place the planarians in PBS solution and grind the tissue until there are no obvious tissue chunks left to obtain 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 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.

2. The extraction method of the planarian extract according to claim 1, characterized in that: 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.

3. The extraction method of the planarian extract according to claim 1, characterized in that: In step c, the alcohol solution is methanol or ethanol solution, and its volume concentration is 30 - 100%.

4. The extraction method of the planarian extract according to claim 3, wherein: In step c, the volume ratio of the planarian crushing solution to the alcohol solution is 1 : (2 - 50).

5. The extraction method of the planarian extract according to claim 1, characterized in that: The concentration and drying is vacuum concentration and drying.

6. A planarian extract, characterized in that: The planarian extract is obtained by the extraction method described in any one of claims 1 - 5.

7. Use of the planarian extract according to claim 6 in anti - hair loss products.

8. Use of the planarian extract according to claim 6 in products for repairing atrophied hair follicles, products for shortening the hair follicle growth period, and products for increasing the survival rate of hair follicles after hair transplantation.

9. Use of the planarian extract according to claim 6 in products for promoting the proliferation of dermal papilla cells, hair follicle regeneration medicine, and tissue engineering.

10. Use of the planarian extract according to claim 6 in the preparation of pet hair care products.

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