Silkworm excrement extract as well as extraction method and application thereof

Abstract

The invention discloses a silkworm excrement extract as well as an extraction method and application thereof, and belongs to the technical field of silkworm excrement extracts. The extraction method comprises the following steps: crushing silkworm excrement, dissolving the crushed silkworm excrement in ethanol, and carrying out heating reflux extraction to obtain an extracting solution; concentrating the extracting solution to obtain a concentrated solution; extracting the concentrated solution with n-hexane, diethyl ether and ethyl acetate in sequence to obtain an ethyl acetate extract; and concentrating and drying the extract to obtain the silkworm excrement extract. Active substances are extracted from silkworm excrement, active ingredients capable of inhibiting prostaglandin D synthetase and 15-hydroxyprostaglandin dehydrogenase at the same time are screened out, the silkworm excrement extract can inhibit prostaglandin D synthetase and inhibit generation of prostaglandin D2 in the body, and the good hair loss preventing and hair growing effects are achieved; meanwhile, the effect exertion of 15-hydroxyprostaglandin dehydrogenase can be inhibited, release of extracellular prostaglandin E2 is promoted, the good effect of promoting wound healing is achieved, and the application range and value of silkworm excrement resources are widened.

Description

Technical Field

[0001] The invention belongs to the technical field of silkworm excrement extracts, and in particular relates to a silkworm excrement extract and an extraction method and application thereof. Background Art

[0002] With the development of biological science and technology, many molecular-level drug targets have been gradually discovered, and a batch of receptor antagonists, enzyme regulators, and even drugs acting at the gene level have been developed. silkworm excrement ) is the excrement of silkworms after eating mulberry leaves, which is a traditional Chinese medicinal material. At present, the most widely used silkworm excrement resources are directly used as fertilizers or to extract chlorophyll from them. A large amount of silkworm excrement produced in sericulture production is discarded, resulting in a serious waste of resources. As a Chinese medicinal material, silkworm excrement contains many useful active substances, but there are not many studies on the active substances in silkworm excrement, especially on the physiological activity of active substances in silkworm excrement. Therefore, it is particularly important to separate and extract active ingredients with new mechanisms of action from silkworm excrement, expand the application range of silkworm excrement resources, and avoid the waste of silkworm excrement resources. Summary of the invention

[0003] In view of this, the object of the present invention is to provide a method for extracting silkworm excrement extract, wherein the silkworm excrement extract obtained can simultaneously inhibit the production of prostaglandin D2 and promote the release of prostaglandin E2, and has good effects in preventing hair loss, promoting hair growth, and promoting wound healing.

[0004] Another object of the present invention is to provide a silkworm excrement extract obtained by extracting the silkworm excrement extract.

[0005] Another object of the present invention is to provide a use of the silkworm excrement extract in the preparation of a product for promoting hair growth, promoting hair regeneration or preventing and / or treating hair loss.

[0006] Another object of the present invention is to provide a 15-hydroxyprostaglandin dehydrogenase inhibitor.

[0007] Another object of the present invention is to provide a use of the silkworm excrement extract or the inhibitor in preparing a product for promoting wound healing.

[0008] Another object of the present invention is to provide a preparation.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a method for extracting silkworm excrement extract, comprising the following steps: Silkworm excrement is crushed, dissolved in ethanol, and extracted by heating under reflux to obtain an extract; the extract is concentrated to obtain a concentrated solution; the concentrated solution is extracted with n-hexane, ether and ethyl acetate in sequence to obtain an ethyl acetate extract; the extract is concentrated and dried to obtain a silkworm excrement extract.

[0010] Preferably, the ethanol is an ethanol solution with a volume fraction of 60% to 80%.

[0011] Preferably, the mass volume ratio of the silkworm excrement to the ethanol is 3g:5~15mL.

[0012] Preferably, the conditions for the heating reflux extraction are: heating reflux at 60-70° C. for 1-5 h.

[0013] Preferably, the volume ratio of the concentrated solution to the n-hexane is 1:0.5-2; the volume ratio of the concentrated solution to the ether is 1:0.5-2; the volume ratio of the concentrated solution to the ethyl acetate is 1:0.5-2.

[0014] The invention also provides a silkworm excrement extract obtained by the extraction method.

[0015] The present invention also provides an application of the silkworm excrement extract in a product for promoting hair growth, promoting hair regeneration or preventing and / or treating hair loss.

[0016] Preferably, the silkworm excrement extract promotes hair growth, hair regeneration or prevents and / or treats hair loss by inhibiting the production of prostaglandin D2.

[0017] Preferably, the hair loss comprises androgenic alopecia.

[0018] The present invention also provides a 15-hydroxyprostaglandin dehydrogenase (15-PGDH) inhibitor, which contains the silkworm excrement extract.

[0019] The present invention also provides an application of the silkworm excrement extract or the inhibitor in preparing a product for promoting wound healing.

[0020] Preferably, the silkworm excrement extract or the inhibitor promotes wound healing by promoting the release of prostaglandin E2 (PGE2).

[0021] The invention also provides a preparation, which contains the silkworm excrement extract.

[0022] Preferably, the dosage form of the preparation includes solution, ointment, plaster, cream or patch.

[0023] Preferably, the preparation is used for promoting hair growth and / or promoting wound healing.

[0024] Beneficial effects of the present invention: The present invention uses ethanol to extract silkworm excrement, and uses n-hexane, ether and ethyl acetate to extract in sequence to obtain ethyl acetate extract, and concentrates and dries to obtain silkworm excrement extract. The silkworm excrement extract extracted by the present invention has low cytotoxicity and can promote the proliferation of human hair papilla cells (HHDPCs). At the same time, prostaglandin D2 (PGD2) is generated by prostaglandin D synthase (PGDS) catalyzing the isomerization of unstable intermediate prostaglandin H2 (PGH2). The silkworm excrement extract of the present invention can inhibit the expression of prostaglandin synthase LPGDS and G protein coupled receptor 44 (GPR44) genes, reduce the release of PGD2 in HHDPCs cells, thereby playing an important role in hair growth and hair regeneration, and has a good effect on preventing and treating hair loss. The silkworm excrement extract extracted by the present invention has low cytotoxicity, can inhibit the performance of 15-PGDH, and promotes the release of extracellular PGE2 by inhibiting the expression of 15-PGDH gene and promoting the expression of intracellular MRP4 gene, and plays the effect of promoting wound healing, and has a good effect on promoting wound healing.

[0025] The present invention extracts active substances from silkworm excrement and screens out active ingredients that can simultaneously inhibit PGDS and 15-PGDH. These natural active ingredients can inhibit prostaglandin D synthetase and inhibit the production of prostaglandin D2 in the body, and have good anti-hair loss and hair growth effects; at the same time, they can inhibit the efficacy of 15-PGDH and promote the release of extracellular PGE2, and have a good effect of promoting wound healing, thereby expanding the application scope and value of silkworm excrement resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The effect of silkworm excrement extract on cell activity in Experimental Example 1; Figure 2 The effect of silkworm excrement extract on the release of PGD2 in HHDPCs cells in Experimental Example 1; Figure 3 The effect of silkworm excrement extract SE3 on the gene expression of P27, Cyclin D1, LPGDS and GPR44 in HHDPCs cells in Experimental Example 1; Figure 4 The effect of silkworm excrement extract SE3 on HHDPCs cell growth in Experimental Example 1; Figure 5 The scratch healing efficiency of HHDPCs cells after treatment with silkworm excrement extract SE3 in Experimental Example 1; Figure 6The results and quantification of the expression of LPGDS and GPR44 proteins in HHDPCs cells after treatment with silkworm excrement extract SE3 in Experimental Example 1 are shown in Figure 1; A is the Western blot result of the expression of LPGDS and GPR44 proteins, and B is the quantitative result of the expression of LPGDS and GPR44 proteins; Figure 7 The hair growth effect of silkworm excrement extract SE3 on mice in Experimental Example 1; Figure 8 The cross section of the mouse skin in Experiment 1 and the hair growth effect of the mouse under a stereo microscope; a and b are the cross section of the mouse skin, and c and d are the hair growth effect of the mouse under a stereo microscope; Fig. 9 This is a verification diagram for the construction of the 15-PGDH expression vector in Experimental Example 2; Fig.10 This is the SDS-PAGE image of the separation and purification of 15-PGDH in Experimental Example 2; Fig.11 The effect of silkworm excrement extract on the release of PGE2 in HaCaT cells after treatment in Experimental Example 2; wherein A is the content of PGE2 released in HaCaT cells after treatment with SE1 to SE5 at a treatment concentration of 10 µg / mL, and B is the content of PGE2 released in HaCaT cells after treatment with SE3 and SE4; Fig.12 The effect of silkworm excrement extract SE3 on the gene expression of 15-PGDH, mPGES-1, PGT and MRP4 in HaCaT cells in Experimental Example 2; Fig.13 The photos of HaCaT cell scratch test after treatment with silkworm excrement extract SE3 in Experimental Example 2 at 0h and 48h; Fig.14 The wound healing efficiency in HaCaT cells after treatment with silkworm excrement extract SE3 in Experimental Example 2; Fig.15 The wound healing effect of silkworm excrement extract SE3 on SD rats in Experimental Example 2; Fig.16 This is the HE staining result of the wound healing tissue after treatment with silkworm excrement extract SE3 in Experimental Example 2. DETAILED DESCRIPTION

[0027] The present invention provides a method for extracting silkworm excrement extract, comprising the following steps: Silkworm excrement is crushed, dissolved in ethanol, and extracted by heating under reflux to obtain an extract; the extract is concentrated to obtain a concentrated solution; the concentrated solution is extracted with n-hexane, ether and ethyl acetate in sequence to obtain an ethyl acetate extract; the extract is concentrated and dried to obtain a silkworm excrement extract.

[0028] In the present invention, silkworm excrement is crushed, dissolved in ethanol, and heated under reflux extraction to obtain an extract. The present invention does not specifically limit the source of the silkworm excrement, and it can be conventionally selected according to actual needs. Before the silkworm excrement is crushed, it can be dried; the drying method is a conventional method in the field. As an implementable method, natural drying can be selected for drying. The method of crushing the silkworm excrement and the crushed particle size are not specifically limited, and they can be conventionally selected according to actual needs. The ethanol is preferably an ethanol solution with a volume fraction of 60% to 80%, and more preferably an ethanol solution with a volume fraction of 70% or 65% to 70% or 70% to 75%. The mass volume ratio of the silkworm excrement and the ethanol is preferably 3g:5~15mL, and more preferably 3g:7mL or 3g:6~7mL or 3g:7~10mL. In the present invention, as an embodiment, before the heating reflux extraction, cold soaking treatment is also included; the temperature of the cold soaking treatment is preferably 15~25℃, more preferably 20℃ or 18~20℃ or 20~22℃, and the time of the cold soaking treatment is preferably 12~24h, more preferably 12~16h or 16~18h; the temperature of the heating reflux extraction is preferably 60~70℃, more preferably 65℃ or 63~65℃ or 65~67℃; the time of the heating reflux extraction is preferably 1~5h, more preferably 3h or 2~3h or 3~4h. As an embodiment, after the heating reflux extraction, the extract is filtered under reduced pressure to obtain an extract. In the present invention, in order to extract the effective ingredients in the silkworm feces to the maximum extent, the above operation can be repeated for multiple extractions according to actual needs, and the number of repetitions is preferably 2~4 times, more preferably 3 times, and the extracts obtained each time are combined.

[0029] In the present invention, the extract is concentrated to obtain a concentrated solution. The concentration method can be a conventional concentration method. As an optional embodiment, the extract is concentrated under reduced pressure to obtain a concentrated solution. The concentrated volume is preferably 1 / 5 to 1 / 2 of the volume of the extract, and more preferably 1 / 3.

[0030] In the present invention, the concentrate is extracted with n-hexane, ether and ethyl acetate in sequence to obtain an ethyl acetate extract; the extract is concentrated and dried to obtain a silkworm excrement extract. The volume ratio of the concentrate to the n-hexane is preferably 1:0.5~2, more preferably 1:1 or 1:0.8~1 or 1:1~1.5; the volume ratio of the concentrate to the ether is preferably 1:0.5~2, more preferably 1:1 or 1:0.8~1 or 1:1~1.5; the volume ratio of the concentrate to the ethyl acetate is preferably 1:0.5~2, more preferably 1:1 or 1:0.8~1 or 1:1~1.5. As an implementable method, n-hexane is added to the concentrate for extraction, and after removing the n-hexane layer, ether is added to the remaining water layer for extraction, and after removing the ether layer, ethyl acetate is added to the remaining water layer for extraction to obtain an ethyl acetate extract. The concentration and drying method can be conventionally selected according to actual needs.

[0031] The invention also provides a silkworm excrement extract obtained by the extraction method.

[0032] The present invention extracts by heating ethanol under reflux, and extracts with n-hexane and ether in sequence, removes the components soluble in n-hexane and ether, and then extracts with ethyl acetate, and the obtained extract is concentrated and dried to obtain a silkworm excrement extract. The silkworm excrement extract extracted by the extraction method of the present invention has low cytotoxicity and can promote the proliferation of HHDPCs cells and HaCaT cells; at the same time, it can inhibit the expression of prostaglandin synthase LPGDS and GPR44 genes, reduce the generation of PGD2 in HHDPCs cells, and thus play an important role in hair growth and hair regeneration; the silkworm excrement extract of the present invention can inhibit the performance of 15-PGDH, and promote the release of extracellular PGE2 by inhibiting the expression of the 15-PGDH gene and promoting the expression of the intracellular MRP4 gene, and play the effect of promoting wound healing. The silkworm excrement extract extracted by the extraction method of the present invention has the effects of promoting hair growth and wound healing at the same time, and expands the application range of silkworm excrement resources.

[0033] The present invention also provides an application of the silkworm excrement extract in preparing a product for promoting hair growth, promoting hair regeneration or preventing and / or treating hair loss.

[0034] Studies have shown that prostaglandins (PGs) can regulate hair growth in mice; in contrast, prostaglandin D2 (PGD2) inhibits hair growth in humans and mice. PGD2 is elevated in the scalp of patients with androgenic alopecia and functionally inhibits hair elongation through its receptor GPR44. In addition, exogenous PGD2 inhibits hair growth in mouse and human hair follicle explants through the PGD2-GPR44 signaling pathway. These results suggest that there is a regulatory network in hair follicles and that PGD2 can inhibit hair follicle function. PGD2 is produced by the isomerization of the unstable intermediate prostaglandin H2 (PGH2) catalyzed by prostaglandin D synthase (PGDS). In other words, inhibiting prostaglandin D synthase can inhibit the production of PGD2, which can prevent hair loss and promote hair growth by inhibiting the production of prostaglandin D2. Brain-type PGDS (LPGDS), also known as glutathione-independent PGDS, is a small molecular weight secretory protein that is widely present in the extracellular fluid of the body. The silkworm excrement extract of the present invention can inhibit the expression of prostaglandin synthase LPGDS and GPR44 genes, thereby reducing the generation of PGD2, thereby achieving the effect of promoting hair growth and hair regeneration, and has a good effect on preventing and / or treating hair loss. The silkworm excrement extract of the present invention plays the effect of promoting hair growth, hair regeneration or preventing and / or treating hair loss by inhibiting the generation of prostaglandin D2. In the present invention, the hair loss preferably includes androgenic alopecia.

[0035] In the present invention, there is no special limitation on the type of product, including but not limited to medicines, cosmetics, etc.; the product can be in any form, including solutions, oils, essences, creams, ointments, gels, lotions, shampoos, conditioners, cleansers, moisturizers, sprays, etc.

[0036] The present invention also provides a 15-hydroxyprostaglandin dehydrogenase (15-PGDH) inhibitor, which contains the silkworm excrement extract.

[0037] 15-Hydroxyprostaglandin dehydrogenase (15-PGDH) is a key enzyme that can cause the degradation of prostaglandins. It is produced by the expression and translation of the HPGD gene on human chromosome 4. It causes the 15-hydroxyl group of prostaglandin E2 (PGE2) to undergo oxidation and synthesize 15-keto-prostaglandins, thereby inactivating prostaglandins in tissue cells. Therefore, this enzyme plays an important role in the inactivation of prostaglandins and is widely present in mammalian tissues. The 15-PGDH inhibitory efficacy of the silkworm excrement extract of the present invention is IC 50 It reached 1.34µg / mL, which had a good inhibitory ability on 15-PGDH, thereby inhibiting the degradation of PGE2.

[0038] In the present invention, the inhibitor may contain an active ingredient of the silkworm excrement extract that can inhibit 15-hydroxyprostaglandin dehydrogenase, or other active ingredients. The inhibitor may also contain an adjuvant acceptable to the dosage form, and there is no particular limitation on the type of adjuvant.

[0039] The present invention also provides an application of the silkworm excrement extract or the inhibitor in preparing a product for promoting wound healing.

[0040] In the present invention, the silkworm excrement extract or the inhibitor inhibits 15-PGDH and promotes the expression of intracellular MRP4 gene, thereby promoting the release of extracellular PGE2 and promoting wound healing.

[0041] In the present invention, there is no special limitation on the type of the product, including but not limited to medicines, cosmetics, etc.; the product can be in any form, including solutions, oils, essences, creams, ointments, gels, lotions, etc.

[0042] The invention also provides a preparation, which contains the silkworm excrement extract.

[0043] In the present invention, the preparation may contain only one active ingredient of the silkworm excrement extract, or may include other active ingredients. The present invention does not specifically limit the dosage form of the preparation, and may be conventionally selected according to actual needs, including but not limited to solutions, ointments, plasters, creams or patches. The preparation may also include various carriers added according to the dosage form requirements of the preparation; the carriers include but are not limited to diluents, lubricants, adhesives, disintegrants, colorants, antioxidants, preservatives, solubilizers, solvents, suspending agents, surfactants, penetration enhancers or combinations thereof. In the present invention, the preparation is preferably used to promote hair growth and / or promote wound healing.

[0044] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0045] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0046] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0047] Example 1 A method for extracting silkworm excrement extract: After the silkworm excrement is dried naturally, it is crushed and dissolved in 70% ethanol. The mass volume ratio of silkworm excrement to ethanol is 3g:7mL. It is cold soaked at 20℃ for 12h, heated to 65℃ for reflux extraction for 3h, and the extract is filtered under reduced pressure. Repeat the above operation 3 times, mix the extracts, and concentrate under reduced pressure to obtain a concentrated solution, which is concentrated to 1 / 3 of the volume of the extract. The concentrated solution is extracted with equal volumes of n-hexane, ether and ethyl acetate in turn to obtain an ethyl acetate extract. The extract is concentrated and dried to obtain a silkworm excrement extract.

[0048] Example 2 A method for extracting silkworm excrement extract: Silkworm excrement is naturally dried and crushed, dissolved in 60% ethanol, the mass volume ratio of silkworm excrement to ethanol is 3g:15mL, cold soaked at 25℃ for 12h, heated under reflux extraction at 60℃ for 5h, and the extract is filtered under reduced pressure. Repeat the above operation twice, mix the extracts, and concentrate under reduced pressure to obtain a concentrated solution, which is concentrated to 1 / 5 of the volume of the extract. The concentrated solution is extracted with 2 times the volume of n-hexane, ether and ethyl acetate in turn to obtain an ethyl acetate extract. The extract is concentrated and dried to obtain a silkworm excrement extract.

[0049] Example 3 A method for extracting silkworm excrement extract: After the silkworm excrement is dried naturally, it is crushed and dissolved in 80% ethanol. The mass volume ratio of silkworm excrement to ethanol is 3g:5mL. It is cold soaked at 15℃ for 24h, heated to reflux extraction at 70℃ for 1h, and the extract is filtered under reduced pressure. Repeat the above operation 4 times, mix the extracts, and concentrate under reduced pressure to obtain a concentrated solution, which is concentrated to 1 / 2 of the volume of the extract. The concentrated solution is extracted with 0.5 times the volume of n-hexane, ether and ethyl acetate in turn to obtain an ethyl acetate extract. The extract is concentrated and dried to obtain a silkworm excrement extract.

[0050] Example 4 A method for extracting silkworm excrement extract: Silkworm excrement is naturally dried and crushed, dissolved in 65% ethanol, the mass volume ratio of silkworm excrement to ethanol is 3g:10mL, cold soaked at 22℃ for 16h, heated under reflux at 63℃ for 4h, and the extract is filtered under reduced pressure. Repeat the above operation twice, mix the extracts, and concentrate under reduced pressure to obtain a concentrated solution, which is concentrated to 1 / 4 of the volume of the extract. The concentrated solution is extracted with 1.5 times the volume of n-hexane, ether and ethyl acetate in turn to obtain an ethyl acetate extract. The extract is concentrated and dried to obtain a silkworm excrement extract.

[0051] Example 5 A method for extracting silkworm excrement extract: Silkworm excrement is naturally dried and crushed, dissolved in 75% ethanol, the mass volume ratio of silkworm excrement to ethanol is 3g:6mL, cold soaked at 18℃ for 18h, heated under reflux at 67℃ for 2h, and the extract is filtered under reduced pressure. Repeat the above operation 3 times, mix the extracts, and concentrate under reduced pressure to obtain a concentrated solution, which is concentrated to 1 / 3 of the volume of the extract. The concentrated solution is extracted with 0.8 times the volume of n-hexane, ether and ethyl acetate in turn to obtain an ethyl acetate extract. The extract is concentrated and dried to obtain a silkworm excrement extract.

[0052] Test Example 1 1. Separation and extraction of silkworm excrement extract After the silkworm excrement is naturally dried in a ventilated place, 300g is taken, crushed into powder by a pulverizer, dissolved in 70% ethanol 700mL, cold soaked for 12h at 20℃, heated to reflux for 3h at 65℃, and the extract is filtered under reduced pressure with a Buchner funnel. Repeat the above operation three times, mix the extracts, and concentrate under reduced pressure to 1 / 3 of the volume of the extract. Extract with equal volumes of n-hexane, ether, ethyl acetate, and water-saturated n-butanol in turn, and concentrate to dryness. The remaining water layer after extraction is also concentrated and dried. Five silkworm excrement extracts are obtained in turn, recorded as SE1, SE2, SE3, SE4 and SE5, of which SE1 is the n-hexane layer, SE2 is the ether layer, SE3 is the ethyl acetate layer, SE4 is the water-saturated n-butanol layer, and SE5 is the water layer.

[0053] 2. Experiment on cytotoxicity and cell activity of five silkworm excrement extracts in HHDPCs In a 96-well plate, 5 × 10 4 / mL seeding density, 90μL of human hair follicle-derived dermal papilla cells (HHDPCs) were seeded in each well, and the culture plate was placed in a 37℃ incubator with 5% CO2 for pre-culture for 18 hours. The above five silkworm excrement extracts were dissolved in DMSO organic solvent at a concentration of 500mg / mL, and diluted with cell culture medium in a gradient to obtain five silkworm excrement extracts of different concentrations. 10μL of five silkworm excrement extracts of different concentrations were added to the culture plate, respectively, so that the final concentration gradient of each silkworm excrement extract was 500μg / mL, 100μg / mL, 10μg / mL, 1μg / mL, and 0.1μg / mL. The culture plate was placed in a 37℃ incubator with 5% CO2 and incubated for 24 hours. 10μL of CCK-8 solution was added to each well, and the culture plate was placed in an incubator for 3h. The absorbance at 450nm was measured with an enzyme marker. No silkworm excrement extract was added, and only 10μL of cell culture medium was added as a blank group. IC , the inhibitory concentration of cell growth, was obtained by statistical analysis. 50 and cell proliferation rate. The results are shown in Table 1 and Figure 1 shown.

[0054] Table 1 Cytotoxicity of silkworm excrement extracts

[0055] From the results in Table 1, it can be seen that the inhibitory cell growth concentrations of the five silkworm excrement extracts SE1, SE2, SE3, SE4, and SE5 are IC 50 The concentrations were 89.35µg / mL, 66.04µg / mL, 266.13µg / mL, 315.00µg / mL, and >500µg / mL, respectively. Among them, silkworm excrement extracts SE3, SE4, and SE5 showed less cytotoxicity.

[0056] Figure 1 It can be seen that compared with the blank group, silkworm excrement extracts SE3 and SE4 have cell proliferation effects at concentrations of 0.1, 1, and 10 μg / mL, and the cell proliferation effect is best at 0.1 μg / mL.

[0057] 3. Effects of five silkworm excrement extracts on the release of PGD2 in HHDPCs cells In a 6-well plate, 2.5 × 10 5 / mL inoculation density, 2mL of HHDPCs cells were inoculated in each well, and incubated in a 37℃ incubator with 5% CO2 for 18 hours. Five silkworm excrement extracts were added to make the final concentration 10µg / mL. No silkworm excrement extract was added as a blank control (Control). After incubation in a 37℃ incubator with 5% CO2 for 12 hours, the supernatant was collected. The PGD2 release amount was detected using a PGD2 kit. The results are shown in Figure 2 shown.

[0058] Depend on Figure 2 It can be seen that after treatment with SE1, SE2, SE4, and SE5, the content of PGD2 released in HHDPCs cells was higher than that in the blank group, while after treatment with SE3, the content of PGD2 released in HHDPCs cells was significantly lower than that in the blank control, indicating that silkworm feces extract SE3 has an inhibitory effect on the release of PGD2 from HHDPCs cells.

[0059] 4. Effect of SE3 on the expression of LPGDS, CyclinD1, P27 and GPR44 genes in HHDPCs cells LPGDS, cyclinD1, P27 and GPR44 genes are genes related to hair growth. Fluorescence quantitative PCR was used to evaluate the effect of silkworm excrement extract SE3 on the expression of these four genes.

[0060] (1) Cell culture HHDPCs cells were cultured in DMEM containing 10% fetal bovine serum and 1% double antibody (penicillin-streptomycin mixture) in a 5% CO2 incubator at 37°C. The cells were observed to be adherent cells under a microscope. 3×105 / mL of HHDPCs cells were incubated in a 37℃ incubator with 5% CO2 for 18 hours. Silkworm excrement extract SE3 was added to a final concentration of 1µg / mL, and SE3 was not added as a blank control (Control). After incubation in a 37℃ incubator with 5% CO2 for 12 hours, the cells were collected.

[0061] (2) RNA extraction 1) Add 1 mL of TRIzol reagent to the cells and remove them by centrifugation at 12000 g for 10 minutes at 4°C.

[0062] 2) Place the homogenate at room temperature for 5 minutes to allow nucleic acids and proteins to fully dissociate. Add 0.2 mL of chloroform to every 1 mL of TRIzol reagent, cover the tube tightly, shake vigorously by hand for 15 seconds, and then place at room temperature for 2-3 minutes.

[0063] 3) Centrifuge at 10,000 g for 10 min at 4°C.

[0064] 4) Carefully pipette the upper aqueous phase (colorless) into a new test tube, and calculate the volume of the aqueous phase you pipette. Do not pipette too much to prevent the inhalation of DNA and protein impurities (middle layer, white).

[0065] 5) Add an equal volume of pre-cooled isopropanol to the absorbed water, cap the tube tightly, and shake gently.

[0066] 6) Let stand at room temperature for 10 minutes to allow the RNA to fully precipitate.

[0067] 7) Centrifuge at 10,000 g for 10 min at 4°C.

[0068] 8) Discard the supernatant (be careful not to discard the precipitate), add 1 mL of 75% ethanol to each tube for rinsing, cover the tube tightly, and gently shake the tube to remove residual isopropanol and salt. Centrifuge at 7500g at 4℃ for 5 minutes.

[0069] 9) Discard the supernatant (be careful not to discard the precipitate), open the tube cap, and dry the RNA precipitate (evaporate at room temperature or vacuum dry). Note that the RNA precipitate cannot be completely dried, otherwise it will be difficult to dissolve.

[0070] 10) Dissolve the RNA precipitate in an appropriate amount (20-50 μL) of RNase-free water. If the RNA precipitate is not completely dissolved, the OD260 / OD280 will be ≤1.6. This RNA solution can be used for further experiments or stored at -70°C.

[0071] (3) Fluorescence quantitative PCR Randomly take samples for preliminary experiments, and then determine the reaction conditions as follows: 1) Preparation of reverse transcription reaction system, as shown in Table 2: Table 2 Reverse transcription reaction system (total volume 20 μL)

[0072] 2) Reverse transcription reaction conditions: 25°C for 5 minutes, 42°C for 50 minutes, and 85°C for 5 minutes; 3) Preparation of fluorescent quantitative PCR reaction system, as shown in Table 3: Table 3 Fluorescence quantitative PCR reaction system (total volume 50 μL)

[0073] 4) Fluorescence quantitative PCR amplification conditions were set as follows: 94°C for 4 minutes; 94°C for 20 seconds, 60°C for 25 seconds, 72°C for 30 seconds, 35 cycles; 72°C for signal detection. Figure 3 shown.

[0074] Depend on Figure 3 It can be seen that after SE3 treatment, the expression of P27 gene decreased compared with the blank control, the expression of CyclinD1 gene was not significantly different from the blank control, the expression of prostaglandin synthase LPGDS gene decreased compared with the blank control, and the expression of GPR44 gene decreased compared with the blank control. This indicates that the reduction of PGD2 content in HHPDCs cells is because silkworm excrement extract SE3 inhibits the expression of LPGDS prostaglandin synthase and receptor gene GPR44, thereby reducing the extracellular PGD2 content.

[0075] 5. Scratch effect of SE3 on HHDPCs cells (1) Cell culture HHDPCs cells were cultured in DMEM containing 10% fetal bovine serum and 1% double antibody (penicillin-streptomycin mixture) in an incubator at 37°C and 5% CO2. The cells were observed to be adherent cells under a microscope.

[0076] (2) Cell scratching In order to study the effect of silkworm excrement extract SE3 on the growth of HHDPCs cells, scratches were made in HHDPCs cells, silkworm excrement extract SE3 was used as the experimental group, TGF-β1 was used as the control group, and photos were taken at 0 and 24 hours after the scratch. The specific process is as follows: 1) Digest and count the cells and take 8×10 5 35mm 2 Incubate overnight in a Petri dish; 2) Draw a line on the bottom of the dish with a marker pen as a mark, aspirate the culture medium, and use a 10 μL pipette tip to scratch the cells in the dish perpendicular to the marker mark; 3) Wash with PBS to remove the scratched cells and perform experimental treatment. Each group has 2 replicates. The experimental groups are as follows: Blank group (Control): add normal serum-free culture medium; Control group (TGF-β1): serum-free culture medium containing 1 ng / mL TGF-β1 was added; Experimental group (SE3): serum-free culture medium containing 10 μg / mL SE3 was added; 4) Place in a 37°C incubator for culture; 5) Take photos at each time point at 0h / 24h after scratching. When taking photos, select the intersection of the line drawn by the marker and the cell scratch as the observation point for fixed-point observation. Figure 4 The scratch healing efficiency of HHDPCs cells in each group was calculated according to the following formula. The results are shown in Figure 5 shown.

[0077] Scratch efficiency = (scratch distance of sample at 0 hours - scratch distance of sample at 24 hours) / (scratch distance of blank at 0 hours - scratch distance of blank at 24 hours) Depend on Figure 4 It can be seen that the cell growth effect of the control group and the experimental group is significantly better than that of the blank group. The cell growth effect of the experimental group is the best compared with the blank group and the control group. Figure 5 It can be seen that after 24 hours, compared with the blank group, the cell scratch healing efficiency of the control group reached 171%, and the cell scratch healing efficiency of the experimental group reached 205%, indicating that silkworm excrement extract SE3 can promote the growth of HHDPCs cells.

[0078] 6. Western blot experiment (1) Cell culture HHDPCs cells were cultured in DMEM containing 10% fetal bovine serum and 1% double antibody (penicillin-streptomycin mixture) in an incubator at 37°C and 5% CO2. The cells were observed to be adherent cells under a microscope. 2.5×10 5 / mL HHDPCs cells 2mL, 5% CO2, 37℃ incubated for 18 hours. Silkworm excrement extract SE3 was added at a final concentration of 1µg / mL as the experimental group (SE3), silkworm excrement extract SE3 was not added as the blank control (Control), and minoxidil was added at a final concentration of 10μM as the positive control (minoxidil), and the cells were collected after incubation at 37℃ with 5% CO2 for 24 hours.

[0079] (2) Western blotting After the cells were collected, they were washed twice with 0.01M PBS. 100μL RIPA lysis buffer (1mMol / L PMSF, broad-spectrum protease inhibitors and phosphatase inhibitors were added to the cell pellet) was added to each group of cells. The centrifuge tube was placed on ice and sonicated at 150W for three times, each for 5 seconds, with a 30-second pause in between. Then, the cells were lysed on ice for 2 hours. Note that no bubbles should be generated during the sonication process. The cells were centrifuged at 12000g at 4℃, and the supernatant was aspirated for protein quantification. The proteins were separated on a 12% SDS-polyacrylamide gel and then transferred to a PVDF membrane (Biosharp, Beijing, China). The blot was blocked in a TBST solution (containing 1×TBS and 0.1% Tween20) dissolved with 5% skim milk powder at 37℃ for 1 hour, washed with TBST, and incubated with the primary antibody (Table 4) at 4℃ overnight. After washing, the membrane was reacted with the diluted secondary antibody (1:5000) at room temperature for 1 hour. The immunostained bands were visualized by enhanced chemiluminescence. Figure 6 shown.

[0080] Table 4 Western blot antibodies and dilution factors

[0081] Depend on Figure 6 It can be seen that compared with the blank control, the expression of LPGDS and GPR44 proteins related to hair growth decreased after treatment with silkworm excrement extract SE3. Compared with the blank control, the expression of LPGDS protein in the positive control group decreased compared with the blank control, but the expression of GPR44 protein increased compared with the blank control. Silkworm excrement extract SE3 has an inhibitory effect on the production of prostaglandin D2, and the mechanism of hair growth is also greatly related to the production of PGD2 factors.

[0082] 7. Hair growth animal model experiment To evaluate the hair growth effect of silkworm excrement extract SE3, a hair growth model was established using C57BL / 6 mice.

[0083] (1) Animal grouping: Sixteen C57BL / 6 mice (7-week-old, male mice) were randomly divided into two groups, 8 mice in each group, after acclimating to the environment for 3 days, namely the blank group (Control) and the sample group (SE3).

[0084] (2) Drug preparation for the sample group: Dissolve silkworm excrement extract SE3 in propylene glycol to a concentration of 30 μg / mL; (3) Animal administration: After 3 days of adaptation, all animals were shaved with an electric shaver and depilatory cream. Starting from the next day, 0.5 mL of the drug was taken with a pipette and applied to the back skin of C57BL / 6 mice twice a day. The second application was applied after the first application was completely dry. After the administration was completed, the drug was covered with vaseline to help drug absorption. The drug was administered once in the morning and evening for 14 days. The blank group was administered with 0.5 mL of propylene glycol twice, and then vaseline was applied once in the morning and evening for 14 days.

[0085] (4) Detection indicators: All mice were photographed on the day of shaving, and photographed on the 7th, 9th, and 11th days of drug administration. On the 11th day of drug administration, 2 mice in each group were killed and the skin surface was photographed under a stereomicroscope. At the same time, the back skin was removed of excess tissue such as fat, embedded in paraffin, sliced, and the cross-section of the skin was photographed. The results are as follows: Figure 7-Figure 8 shown.

[0086] Depend on Figure 7 It can be seen that there was no obvious effect in the blank group and the sample group on the 7th day, but on the 11th day, the hair growth of the mice in the SE3 sample group was significantly stronger than that in the blank group. Figure 8 As can be seen from c and d, the hair of the SE3 sample group grows faster and more luxuriantly than that of the blank group. Figure 8 As can be seen from a and b, the hair growth in the SE3 sample group was significantly stronger than that in the blank group.

[0087] It can be seen from the above test results that the silkworm excrement extract obtained by the present invention has low cytotoxicity, can promote the proliferation of HHDPCs cells, and can inhibit the expression of LPGDS prostaglandin synthase and receptor gene GPR44, reduce the release of PGD2 by HHDPCs cells, and thus play an important role in promoting hair growth and hair regeneration. It shows that the silkworm excrement extract obtained by the extraction method of the present invention can promote hair growth and hair regeneration, and has a good effect on preventing and treating hair loss.

[0088] Test Example 2 1. Separation and extraction of silkworm excrement extract The same method as in Experimental Example 1 was used to obtain five silkworm excrement extracts, SE1, SE2, SE3, SE4 and SE5.

[0089] 2. Preparation of 15-PGDH protein (1) Construction of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) expression system 1) 15-PGDH gene synthesis and target gene amplification The nucleotide sequence of 15-PGDH was searched in the NCBI gene library, and the sequence Genebank number was: NM_000860.6; the nucleotide sequence of the target 15-PGDH gene was known, and the nucleotide synthesis was completed by Flash Biotechnology Company; primers were designed according to the nucleotide sequence of the 15-PGDH gene included in NCBI; PCR amplification was performed using the designed primers and the synthesized 15-PGDH gene as a template.

[0090] The PCR reaction system is as follows: 5×Green GoTaq Flexi buffer 10µL; MgCl2 solution 4µL; dNTP (10mM) 4µL; forward and reverse primers Primer F1 / R1 2.5µL; Taq polymerase 0.5µL; Template 1µL; H2O28µL.

[0091] The PCR reaction program was set as follows: 94°C for 2 min; 95°C for 1 min, 55°C for 45 s, 72°C for 1 min, 30 cycles; 72°C for 5 min; after the reaction was completed, stored at 4°C; The PCR products were detected by 1% agarose gel electrophoresis (120 V, 30 min).

[0092] 2) PCR product purification The PCR products were electrophoresed on 1% agarose gel and then recovered using the PCR recovery kit produced by Tiangen.

[0093] 3) Restriction enzyme digestion Use restriction endonucleases (BamHI / XhoI) to digest the 15-PGDH PCR product and plasmid vector PGEX-4T-1. The digestion reaction system is as follows: 10× digestion buffer 3μL; PCR product or plasmid vector 10μL; BamHI 2.5μL; XhoI 2.5μL; H2O 12μL. Place the reaction solution at 37℃ for 2 hours and inactivate at 65℃. Then use the ordinary agarose gel DNA recovery kit produced by Tiangen to recover the PCR product.

[0094] 4) Ligation of PCR products and vector The ligation reaction solution is as follows: 1 μL of 10× ligation buffer; 5 μL of ligation sample (PCR product and plasmid vector after enzyme digestion); 1 μL of ligase; 3 μL of H2O.

[0095] Mix the sample and centrifuge briefly on ice to allow all samples to settle to the bottom of the tube; place the reaction solution at 16°C overnight.

[0096] 5) Ligation product transformation Take 100μL of calcified bacteria DH5a stored at -70℃, melt in ice bath; add appropriate amount of ligation product, mix gently, and ice bath for 30min; heat shock at 42℃ for 90s, quickly transfer to ice bath, and continue ice bath for 2min; add LB liquid culture medium 200μL, incubate at 37℃ with slow shaking for 45min; apply appropriate amount of culture on 1.5% agar LB plate, and when there is no liquid flowing on the surface of the gel, invert and culture in 37℃ incubator for 12-16hr. Obtain the transformed plasmid bacteria PGEX-4T-1-15-PGDH / BL21(DE3).

[0097] 6) Confirmation of transformation products The transformed plasmid bacteria PGEX-4T-1-15-PGDH / BL21 (DE3) were purified using a plasmid DNA purification kit to recover the PCR product. The specific DNA recovery procedure is as follows: a. Pick a single colony growing on LB solid medium, inoculate it into 2.0 mL LB liquid medium (containing corresponding antibiotics), and culture it at 37°C and 200 rpm with shaking overnight (about 12-14 hours).

[0098] b. Take 1.5 mL of the culture into a microcentrifuge tube, centrifuge at room temperature at 8000 g for 1 min, discard the supernatant, invert the centrifuge tube to allow the liquid to drain as much as possible, and store the remaining culture at 4°C.

[0099] c. Resuspend the bacterial precipitate in 100 μL of pre-cooled solution I and shake vigorously to disperse and mix the bacteria. Make sure that the bacterial precipitate is completely dispersed in solution I. The bottoms of two microcentrifuge tubes can be touched and shaken to quickly disperse the precipitate.

[0100] d. Add 200 μL of freshly prepared Solution II, invert several times to mix (do not shake violently), and place the centrifuge tube on ice for 2-3 minutes to lyse the cell membrane (Solution II is the lysis solution, so the bacterial solution in the centrifuge tube gradually becomes clear).

[0101] e. Add 150 μL of pre-cooled solution III, gently invert the tube several times to mix, and white flocculent precipitate will appear, which can be placed on ice for 3-5 minutes. Solution III is a neutralizing solution, at which time plasmid DNA is renatured, chromosomes and proteins are irreversibly denatured, forming an insoluble complex, and K+ precipitates the SDS-protein complex.

[0102] f. Add 450 μL of phenol / chloroform / isoamyl alcohol, vortex to mix, and centrifuge at 12,000 g for 10 min at 4°C.

[0103] g. Carefully remove the supernatant and place it in a new microcentrifuge tube. Add 2.5 times the volume of pre-cooled anhydrous ethanol, mix well, leave at room temperature for 2-5 minutes, and centrifuge at 12000g for 15 minutes at 4°C.

[0104] h. Wash the precipitate 1-2 times with 1 mL of pre-cooled 70% ethanol, centrifuge at 8000 g for 7 min at 4°C, discard the supernatant, and dry the precipitate at room temperature.

[0105] i. Dissolve the precipitate in 20 μL TE (containing 20 μg / mL RNaseA), incubate in a 37°C water bath for 30 min to degrade RNA molecules, and store at -20°C for later use.

[0106] j. The purified plasmid DNA was double digested with restriction enzymes BamHI and XhoI, and then detected by 1% agarose gel electrophoresis (120 V, 30 min).

[0107] (2) 15-PGDH protein purification 1) Fermentation with engineered bacteria a. Pick a small amount of PGEX-4T-1-15-PGDH / BL21(DE3) bacteria and inoculate them into 500 mL LB liquid medium containing Amp (final concentration 100 μg / mL), and culture them at 37°C overnight.

[0108] b. Inoculate the primary culture at a 1% inoculation rate into 500 mL LB liquid medium containing Amp (final concentration 100 μg / mL). After culturing at a constant temperature for 2 h (OD600 is about 0.5), add the inducer IPTG to a final concentration of 1 mM and continue culturing at 37°C.

[0109] c. After 12 hours of induction, the cells were collected and centrifuged at 6000 rpm for 10 min at 4°C. The supernatant was discarded and the cell pellet was stored at -20°C for later use.

[0110] 2) Cell disruption a. Place the culture medium (500 mL) of the recombinant bacteria in a centrifuge cup and centrifuge at 4000 r / min for 20 min at room temperature. Collect the bacteria, place them in plastic centrifuge tubes, and store them at -20°C.

[0111] b. Add 20 mL of 0.01 mol / L pH 8.0 Tris-HCl buffer to the recombinant bacteria and stir to mix.

[0112] c. Place the bacterial suspension in a 10 mL beaker and disrupt the cells in an ultrasonicator under ice bath conditions. The ultrasonicator settings are: power 400 W, work 5 s, interval 5 s, and work 60 times.

[0113] d. Place the cell wall disruption solution in a plastic centrifuge tube and centrifuge in a low-temperature high-speed centrifuge at 4°C and 12,000 r / min for 20 min.

[0114] 3) Isolation and purification of recombinant proteins a. Dissolve the bacterial pellet in an appropriate amount of Lysis buffer 2 (Lysis buffer under denaturing conditions), stir and blow the pellet at room temperature to avoid foaming.

[0115] b. Centrifuge at 10,000 g, 4°C for 30 min and collect the supernatant.

[0116] c. Fill the column with glutathione agarose resin and connect it to the Pharmarcia low-pressure liquid chromatography system. Equilibrate the glutathione agarose resin with 5 column volumes of lysis buffer 2 and adjust the A280 value to zero.

[0117] d. Load an appropriate amount of supernatant onto a glutathione agarose resin column and wash with lysis buffer until the A280 value is less than 0.01.

[0118] e. Wash the column with 5 to 10 column volumes of Wash buffer 1 and Wash buffer 2 respectively until the A280 value is less than 0.01.

[0119] f. Elute the recombinant protein with elution buffer and collect all the eluates containing the recombinant protein after the peak appears under the monitoring of A280 value.

[0120] The 15-PGDH gene sequence was obtained from the NCBI gene library. After the obtained gene was synthesized, primers were designed for PCR gene amplification. The amplified gene was restriction enzyme digested and subcloned into the PGEX-4T-1 expression vector. The results are as follows Fig. 9 As shown, double enzyme digestion identification confirmed that the expression vector of 15-PGDH protein was successfully constructed.

[0121] The recombinant 15-PGDH vector was transfected into BL21 (DE3) Escherichia coli, and the induced bacteria were broken and the protein was separated and purified using glutathione agarose resin. Fig.10 As shown, it is shown that the separated and purified 15-PGDH protein is obtained.

[0122] 3. Cell subculture HaCaT cells were cultured in DMEM containing 10% fetal bovine serum and 1% double antibody (penicillin-streptomycin mixture) in an incubator at 37°C and 5% CO2. The cells were observed to be adherent cells under a microscope.

[0123] 4. Cytotoxicity assay Add 5 × 10 4 / mL of the above-mentioned cell subcultured HaCaT cells 90μL, 5% CO2, 37℃ incubated for 18 hours. The above 5 kinds of silkworm excrement extracts were dissolved in DMSO organic solvent at a concentration of 500mg / mL, and diluted with cell culture medium in a gradient to obtain 5 kinds of silkworm excrement extracts of different concentrations. 10μL of 5 kinds of silkworm excrement extracts of different concentrations were added to the culture plate, so that the final concentration gradient of each silkworm excrement extract was 0.1μg / mL, 1μg / mL, 10μg / mL, 100μg / mL, 500μg / mL, 5% CO2, 37℃ incubated for 48 hours, and then 10μL MTT was added and cultured for 4 hours. After 4 hours, 150μL DMSO was added again, and the absorbance was detected at OD540nm using an enzyme reader after shaking for 10 minutes. The results are shown in Table 5.

[0124] Table 5 Cytotoxicity of silkworm excrement extracts

[0125] From the results in Table 5, it can be seen that the toxicity of SE1~SE5 silkworm excrement extracts in HaCaT cells were 48.38µg / mL, 98.65µg / mL, 348.11µg / mL, 289.65µg / mL and greater than 500µg / mL, respectively, among which SE3, SE4 and SE5 had less toxicity to HaCaT cells.

[0126] 5. 15-PGDH activity detection Add 50mM Tris–HCl (pH 7.5), 0.1mM DTT, and 0.25mM NAD into the cuvette. + , 10 μg of isolated and purified 15-PGDH protein, 21 μM PGE2 and different concentrations of 15-PGDH inhibitors (silkworm excrement extracts SE1 to SE5), the content of NADH formed by the reaction was determined by a fluorescence spectrophotometer at an excitation wavelength of 340 nm and an emission wavelength of 468 nm, and the 15-PGDH inhibitory potency was determined using the drawn NADH standard curve. The results are shown in Table 6.

[0127] Table 6 15-PGDH inhibitory activity of silkworm excrement extracts

[0128] From the results in Table 6, it can be seen that the inhibitory efficacy of 15-PGDH of SE1 to SE5 is IC 50 They were 20.49µg / mL, 84.12µg / mL, 1.34µg / mL, 1.87µg / mL and 482.63µg / mL respectively, among which SE3 and SE4 had the best inhibitory efficacy.

[0129] 6. PGE2 content determination Add 2.5×10 5 / mL HaCaT cells 2mL, 5% CO2, 37℃ incubation for 18 hours. Five silkworm excrement extracts were added at a final concentration of 10µg / mL, and no silkworm excrement extract was added as a blank control (Control). After incubation at 5% CO2, 37℃ for 12 hours, the supernatant was collected. The PGE2 release was detected using a PGE2 kit. The results are shown in Fig.11 As shown in A.

[0130] Fig.11 Figure A is the content of PGE2 released in HaCaT cells after treatment with SE1 to SE5 at a treatment concentration of 10µg / mL. It can be seen that the release content of PGE2 in HaCaT cells after SE3 treatment is 2988µg / mL, and the release content of PGE2 in HaCaT cells after SE4 treatment is 1660µg / mL. After treatment with these two extracts, the levels increased by 24 times and 13 times respectively compared with the blank group, which is the highest release content among the five extracts.

[0131] Add 2.5×10 5 / mL HaCaT cells 2mL, 5% CO2, 37℃ incubation for 18 hours. Silkworm excrement extracts SE3 and SE4 were added at 6 different final concentrations (0µg / mL, 0.1µg / mL, 1µg / mL, 10µg / mL, 20µg / mL, 30µg / mL), 5% CO2, 37℃ incubation for 12 hours, and then the supernatant was collected. The PGE2 release was detected using a PGE2 kit. The results are shown in Fig.11 As shown in B.

[0132] Fig.11 Figure B is the content of PGE2 released in HaCaT cells after treatment with SE3 and SE4. It can be seen that the PGE2 content increased significantly with the increase of SE3 or SE4 concentration. When the concentration of SE3 and SE4 was 30µg / mL, the release of PGE2 reached 12880µg / mL and 9120µg / mL, respectively.

[0133] 7. Effect of SE3 on the expression of 15-PGDH, mPGES-1, PGT and MRP4 genes in HaCaT cells (1) Cell culture HaCaT cells were cultured in DMEM containing 10% fetal bovine serum and 1% double-antibody (penicillin-streptomycin mixture) at 37°C and 5% CO2 in an incubator. The cells were observed to be adherent cells under a microscope. 3×10 5 / mLHaCaT cells, 5% CO2, 37℃ for 18 hours. Silkworm excrement extract SE3 was added at a final concentration of 1µg / mL, and SE3 was not added as a blank control (Control), and the cells were collected after incubation at 5% CO2, 37℃ for 12 hours.

[0134] (2) Extract RNA from the recovered cells and synthesize cDNA using a reverse transcription method. The specific method is the same as that in Experimental Example 1.

[0135] (3) Fluorescence quantitative PCR 1) Fluorescence quantitative PCR reaction system: Add 25µL 2×HotstartFluo-PCR mix, 1µL forward primer, 1µL reverse primer, 0.5µL probe, 1µL template, and 21.5µL DEPC water into 50µL reaction system and perform fluorescence quantitative PCR amplification.

[0136] 2) Fluorescence quantitative PCR amplification conditions: 94℃ 4min; 94℃ 20s, 60℃ 25s, 72℃ 30s, 35 cycles, 72℃ detection signal. After 35 cycles of reaction, the Ct value will be read, and the final melting curve analysis will be carried out. Relevant data were analyzed by relative quantitative analysis to detect the expression differences at the transcriptional level. Each sample had 3 replicates to ensure the accuracy of the data. The results are as follows Fig.12 shown.

[0137] 15-PGDH, mPGES-1, PGT and MRP4 genes are PGE2 release-related genes, and the expression levels of these genes were detected by fluorescence quantitative PCR. Fig.12 The results show that the gene expression of 15-PGDH and mPGES-1 decreased after SE3 treatment compared with the blank control, indicating that the increase in the content of PGE2 in the cell is due to the inhibition of 15-PGDH. PGT protein is a protein that transports PGE2 into the cell. After SE3 treatment, the gene expression of PGT decreased slightly compared with the blank control, and the expression level was not high; MRP4 protein is a protein that transports PGE2 from the cell to the outside of the cell. After SE3 treatment, the gene expression of MRP4 increased significantly by many times compared with the blank control, indicating that most of the PGE2 is released outside the cell. It shows that SE3 increases the content of PGE2 released outside the cell by inhibiting the 15-PGDH gene and increasing the expression of the MRP4 gene in the cell.

[0138] 8. Cell scratching (1) Cell culture HaCaT cells were cultured in DMEM containing 10% fetal bovine serum and 1% double antibody (penicillin-streptomycin mixture) in an incubator at 37°C and 5% CO2. The cells were observed to be adherent cells under a microscope.

[0139] (2) Cell scratching 1) Digest and count the cells and take 8×10 5 35mm 2 Incubate overnight in a Petri dish; 2) Draw a line on the bottom of the dish with a marker pen as a mark, aspirate the culture medium, and use a 10 μL pipette tip to scratch the cells in the dish perpendicular to the marker mark; 3) Wash with PBS to remove the scratched cells and then treat them. Each group has 2 replicates. The experimental groups are as follows: Blank group (Control): add normal serum-free culture medium; Control group (TGF-β1): serum-free culture medium containing 1 ng / mL TGF-β1 was added Experimental group (SE3): serum-free culture medium with 10 μg / mL SE3 4) Place in a 37°C incubator for culture; 5) Take photos at each time point at 0h / 24h / 48h after scratching. When taking photos, select the intersection of the line drawn by the marker and the cell scratch as the observation point for fixed-point observation. Fig.13 The scratch healing efficiency of HaCaT cells in each group was calculated according to the following formula. The results are shown in Fig.14 shown.

[0140] Scratch efficiency = (scratch distance of sample at 0 hours - scratch distance of sample at 48 hours) / (scratch distance of blank at 0 hours - scratch distance of blank at 48 hours) Depend on Fig.13 The results show that the cell growth effects of the control group and the experimental group are significantly better than those of the blank group, and the cell growth effect of the SE3 group is stronger than that of the control group. Fig.14 The results showed that after 48 hours, compared with the blank group, the cell scratch healing efficiency of the control group reached 238%, and that of the experimental group reached 283%, indicating that silkworm excrement extract SE3 can promote the growth of HaCaT cells.

[0141] 9. Animal model experiments (1) Model construction SD rats were purchased from the Sibor-Bikai Animal Experiment Center. After one week of adaptive feeding, the rats were anesthetized with ether, the hair on the back of the rats was shaved with a shaver, and the skin was wiped with alcohol. A 1 cm × 1 cm circular wound was cut on the back skin of the rats. In order to prevent the experimental results from being affected by skin contraction, the wound was sutured with a rubber ring to fix the surrounding skin. The constructed rat model was randomly divided into 2 groups, 7 in each group, namely the blank group (Control) and the experimental group (SE3).

[0142] (2) Method of administration Blank group: Normal saline (sterile 0.9% sodium chloride solution) was used. 0.2 mL of normal saline was taken with a pipette and dropped on the wound every morning and afternoon. Wound photos were taken every three days. The medication lasted for 15 days. Experimental group: Silkworm excrement extract SE3 was dissolved in DMSO at a concentration of 50 mg / mL, and diluted with saline to a concentration of 0.15 mg / mL for administration, 200 μL each time, i.e. 30 μg / mouse / time. 0.2 mL of the prepared drug was respectively drawn with a pipette and dropped on the wound in the morning and afternoon, and the wound was photographed every three days for 15 days.

[0143] In order to evaluate the wound healing effect of silkworm excrement extract SE3 in vivo, especially its effect on epithelial tissue regeneration, skin defects were created on the backs of SD rats using punch biopsy and photographs were taken every three days. The results are shown in Figure 2. Fig.15 As shown. It can be seen that compared with the blank group, the experimental group has a significant wound healing effect starting from the 3rd day, and the wounds of the experimental group have been basically healed by the 15th day. This shows that silkworm excrement extract SE3 has a significant effect on promoting wound healing.

[0144] (3) HE staining 1) Sampling: On the 4th and 7th day after surgery, one rat from each group was randomly selected for sacrifice by cervical dislocation, and the unhealed skin of the wound was fixed and embedded for subsequent HE staining. After the experiment, all rats were sacrificed by cervical dislocation, and the healed wound tissues on the body were fixed and embedded for subsequent HE staining.

[0145] 2) HE staining sample embedding and fixation Sampling and fixation: Use sharp knives and scissors when cutting tissue. When cutting tissue blocks, pull the knife backwards from the root of the knife to cut the tissue. The thickness of the tissue block is about 0.2~0.3cm, and the size is preferably 1.5cm×1.5cm×0.3cm. The tissue block is fixed in 10% formalin for 48 hours; Washing and dehydration: Rinse the fixed tissue with running water to remove residual fixative and impurities. Dehydrate in different concentrations of ethanol, 50%, 70%, 85%, 95% to pure alcohol (anhydrous ethanol), 2 hours per level. Dehydration must be done in a covered bottle to prevent high-concentration ethanol from absorbing moisture in the air, causing the concentration to decrease and making dehydration incomplete. Materials that need to be preserved can be dehydrated to 70% ethanol and remain in it. If long-term storage is required, an equal amount of glycerol can be added; Transparency: Place the tissue block in a mixture of equal volumes of pure ethanol and xylene for 2 hours, then in pure xylene for 2 hours, and then in pure xylene again for 2 hours; after the material is made transparent, it will show the effect of the previous dehydration step. If the dehydration is thorough, the tissue will appear transparent. If there is white cloudiness in the tissue, it means that the dehydration is not complete and rework is required, but the rework effect is often not good. When using xylene for transparency, avoid its evaporation and absorption of moisture in the air, and keep it in anhydrous state; Wax dipping: Wax dipping must be done in a constant temperature box. First, immerse the tissue material block in an equal amount of melted paraffin and xylene for 1 to 2 hours, then move it into two melted paraffin solutions and immerse it for about 3 hours each; Embedding: Embedding is to wrap the tissue block soaked in wax in paraffin. The specific method is: first prepare a paper box, pour the molten wax into the box, quickly use pre-warmed tweezers to pick up the tissue block and place it flat on the bottom of the paper box, with the cut surface facing down, then gently lift the paper box and place it flat in cold water. After the surface paraffin solidifies, immediately press the paper box into the water to quickly cool and solidify it, and take it out after 30 minutes; Slicing: Place the wax block in a -20℃ refrigerator for at least 30 minutes before slicing to increase hardness. Install the slicer on the blade holder of the blade machine and fix it tightly, fix the wax block base or wax block, adjust the wax block and the knife to the appropriate position, and the blade is at a 5-degree angle to the surface of the wax block. Adjust the slice thickness on the slicer to 4-7μm, and then slice.

[0146] 3) HE staining Place the slide in a 65°C constant temperature oven and bake for 30 minutes; soak in xylene I for 15 minutes, and then soak in xylene II for 15 minutes. Soak the dewaxed sections in 100% alcohol, 95% alcohol, 85% alcohol, and 75% alcohol for 5 minutes respectively, and rinse with tap water for 10 minutes. Place the sections that have been immersed in distilled water in a hematoxylin aqueous solution for staining for 5 minutes, and separate the colors in ammonia water for a few seconds. Rinse with running water for 15 minutes, and dehydrate in 70% and 90% alcohol for 10 minutes each. Stain in alcohol eosin staining solution for 1-2 minutes, and dehydrate the stained sections with pure alcohol. Place the slide in xylene to make it transparent for 3 minutes × 2 times, seal the slide with neutral gum, and place it in a 65°C oven for 15 minutes. Take pictures under a microscope, and collect and analyze the relevant parts of the sample. The results are as follows Fig.16 shown.

[0147] Depend on Fig.16 The results show that the HE staining results on the 4th and 7th days showed that the wounds in the experimental group were significantly smaller than those in the blank group; on the 15th day, the surrounding epidermal tissue in the blank group had completed epithelialization and formed thick granulation tissue, and red blood traces were also seen, and new capillaries were formed at the wound (red arrows), while the new epithelialized tissue at the wound in the experimental group was thinner than that in the blank group, and began to form epidermal ridges (black frame), complete new capillaries (red arrows), and began to form primary hair follicle tissue (indicated by the blue arrow). The wounds in the experimental group had more hair follicles and hair bulbs than the blank group, and the epidermis was thinner than that in the blank group. This shows that the SE3 drug group has a good wound healing effect.

[0148] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for extracting silkworm excrement extract, characterized in that: The extraction method comprises the following steps: Silkworm excrement is crushed, dissolved in ethanol, and extracted by heating under reflux to obtain an extract; the extract is concentrated to obtain a concentrated solution; the concentrated solution is extracted with n-hexane, ether and ethyl acetate in sequence to obtain an ethyl acetate extract; the extract is concentrated and dried to obtain a silkworm excrement extract.

2. The extraction method according to claim 1, characterized in that The ethanol is an ethanol solution with a volume fraction of 60% to 80%.

3. The extraction method according to claim 1, characterized in that The mass volume ratio of the silkworm excrement to the ethanol is 3g:5-15mL.

4. The extraction method according to claim 1, characterized in that The conditions for the heating reflux extraction are: heating reflux at 60-70° C. for 1-5 hours.

5. The extraction method according to claim 1, characterized in that The volume ratio of the concentrated liquid to the n-hexane is 1:0.5-2; the volume ratio of the concentrated liquid to the ether is 1:0.5-2; the volume ratio of the concentrated liquid to the ethyl acetate is 1:0.5-2.

6. The silkworm excrement extract obtained by the extraction method according to any one of claims 1 to 5.

7. Use of the silkworm excrement extract according to claim 6 in the preparation of products for promoting hair growth, promoting hair regeneration or preventing and / or treating hair loss.

8. A 15-hydroxyprostaglandin dehydrogenase inhibitor, characterized in that The inhibitor comprises the silkworm excrement extract according to claim 6.

9. Use of the silkworm excrement extract according to claim 6 or the inhibitor according to claim 8 in the preparation of a product that promotes wound healing.

10. A preparation, characterized in that The preparation comprises the silkworm excrement extract according to claim 6.

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