Preparation and application of galangin hydrogel

Abstract

The galangin hydrogel is constructed by adopting an inulin and chitosan quaternary ammonium salt cross-linking system and a galangin solution, the highest galangin release amount can reach 60%, and the OI / HACC hydrogel capable of releasing the drug galangin in an inflammatory environment is obtained. The skin psoriasis symptom of the mouse treated by the galangin hydrogel is obviously reduced; the spleen of the mice in the treatment group returns to normal and the spleen index is reduced; the PASI score Ki67 index and PCNA index of the skin tissue in the skin tissue of the mice in the treatment group are obviously reduced, the KRT14 immunofluorescence intensity is recovered to be close to normal, the abnormal proliferation of keratinocytes is inhibited, and the differentiation is improved; the number of F4 / 80 and CD11b positive cells is remarkably reduced, and inflammation is relieved. The expression levels of the SS100a9 gene, the S100a8 gene, the Krt6 gene and the Krt16 gene are obviously reduced, the epidermis abnormal proliferation is inhibited, and the anti-inflammatory effect is realized. The composite hydrogel constructed by the invention can effectively regulate the cuticle of the psoriasis, inhibit inflammatory response and improve the pathological state of the psoriasis, and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of skin external administration and traditional Chinese medicine effective ingredient preparations, and particularly relates to a galangin water gel composition, a preparation method thereof and an application of the galangin water gel composition in preparing medicines for preventing or treating psoriasis and other inflammatory skin diseases. BACKGROUND

[0002] Galangin is a natural flavonol compound extracted from galangal, also known as galangin, 3,5,7-trihydroxyflavone, and its CAS number is 548-83-4. A large number of studies have shown that galangin has multiple biological activities, including anti-mutation, anti-oxidation, anti-inflammatory, vasodilation and anti-tumor effects. A large number of studies have shown that galangin has multiple biological activities, and it has been found that it has multiple anti-tumor mechanisms, including anti-proliferation, pro-apoptosis, pro-autophagy, and inhibition of tumor cell metastasis.

[0003] Galangin is a hydrophobic flavonoid, and oral or conventional external preparations have problems such as low solubility, limited skin permeability and insufficient local sustained release, which limit its clinical application. Psoriasis is a chronic recurrent skin disease characterized by keratinization abnormalities and immune inflammation, and local administration is one of the first-line strategies because it "directly reaches the disease site and reduces systemic exposure". The existing traditional dosage forms of traditional Chinese medicine for external use (such as powders, ointments, lotions, etc.) still need to be improved in terms of modern quality control, skin permeability and patient compliance.

[0004] Therefore, it is urgent to develop a new galangin external dosage form with simultaneous solubilization, skin permeability promotion and sustained release functions to improve local effective exposure and reduce the risk of adverse reactions. SUMMARY

[0005] In view of the technical problem that galangin is a hydrophobic flavonoid, and oral or conventional external preparations have problems such as low solubility, limited skin permeability and insufficient local sustained release, which limit its clinical application, the present application aims to provide a galangin water gel, which realizes high drug loading, stability and controlled release of galangin through the synergistic effect of the three-dimensional network of inulin / chitosan quaternary ammonium salt mixture, and provides a preparation method thereof and an application thereof in preparing medicines for treating psoriasis and other inflammatory skin diseases.

[0006] In one aspect, the present application provides a galangin water gel, wherein the galangin water gel is composed of a polymer OI / HACC mixed with a galangin solution; the polymer OI / HACC is formed by blending reaction of OI and chitosan quaternary ammonium salt (HACC); and the galangin solution is prepared by dissolving galangin in Tween 80.

[0007] Further, the present application provides a preparation method of the galangin water gel, which is prepared by the following steps: S1: Inulin is oxidized by sodium periodate to produce OI; S2: OI and chitosan quaternary ammonium salt (HACC) are blended and reacted to form polymer OI / HACC; S3: Galangin is dissolved in Tween 80 to produce a galangin solution; S4: The polymer OI / HACC prepared in S2 is blended with the galangin solution prepared in S3, heated and stirred to form a gel.

[0008] Preferably, in S1, 400 mg of inulin is dissolved in 10 ml of deionized water. 200 mg of sodium periodate is dissolved in 2 ml of deionized water. The inulin solution is added to the sodium periodate solution in a dark environment, stirred and reacted for 24 hours. Appropriate amount of ethylene glycol is added to inactivate the excess sodium periodate, and then the solution is dialyzed in an 8 kDa dialysis bag for 3 days, with the dialysis solution being replaced every 12 hours. Finally, the solution is freeze-dried for 24 hours to obtain the inulin oxidation product (OI).

[0009] Preferably, in S2, OI and chitosan quaternary ammonium salt (HACC) are blended in a mass ratio of (2%-10%):(10%-2%). 800 mg of chitosan quaternary ammonium salt (HACC) is dissolved in 7 ml of deionized water, and the OI oxidation product is dissolved in 1 ml of deionized water. The HACC is added to form an OI / HACC hydrogel matrix system.

[0010] Preferably, in S3, 80 mg of galangin reference substance is dissolved in 10 ml of Tween 80 to obtain a galangin solution.

[0011] Preferably, in S4, 2 ml of the galangin solution prepared in S3 is added dropwise to the OI / HACC hydrogel matrix system prepared in S2, and stirred for half an hour to obtain a galangin hydrogel.

[0012] Further, the application also provides the use of the above-mentioned galangin hydrogel in improving an IMQ-induced mouse psoriasis model.

[0013] The use is the use in reducing the Ki67 index and PCNA index in the skin tissue of IMQ-treated mice.

[0014] The use is the use in restoring the KRT14 immunofluorescence intensity in the skin tissue of IMQ-treated mice to near normal, inhibiting abnormal proliferation of keratinocytes, and improving differentiation.

[0015] The use is the use in significantly reducing the number of F4 / 80 and CD11b positive cells in the skin tissue of IMQ-treated mice, and reducing inflammation.

[0016] The application described is in IMQ-treated mouse skin tissue. S100a9, S100a8, Krt6, and Krt16 It significantly reduces gene expression levels and has applications in inhibiting abnormal epidermal proliferation and anti-inflammation.

[0017] The present invention also provides a drug for treating psoriasis, which is made with the above-mentioned galangin hydrogel as the active ingredient.

[0018] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages: (1) This invention is the first to prepare galangin hydrogel, which significantly improves the water solubility and stability of galangin and solves the problems of poor solubility and low absorption efficiency in traditional preparations. Secondly, a hydrogel matrix constructed using an inulin and chitosan quaternary ammonium salt crosslinking system provides a composite hydrogel for local application. The composite hydrogel constructed in this invention effectively regulates the stratum corneum of psoriasis, inhibits inflammatory responses, and improves the pathological state of psoriasis, showing great application potential.

[0019] (2) The galangin hydrogel system prepared in this application showed a small amount of galangin release from the OI / HACC@GA hydrogel in PBS (pH 7.4). In PBS (pH 5.5), galangin was released from the three-dimensional structure of the hydrogel after 12 h. After incubation for 24 h, the release amount reached up to 60%, indicating that the release efficiency of the galangin prepared in this application is improved under inflammatory conditions. The addition of galangin to the hydrogel matrix resulted in a unique encapsulation structure, indicating that the galangin hydrogel system was successfully prepared. The technical solution provided in this application synthesizes an OI / HACC hydrogel capable of releasing the drug galangin under inflammatory conditions.

[0020] (3) The galangin hydrogel prepared using the technical solution provided in this application was applied to IMQ-treated mice in a smear experiment. Compared with the control group, the IMQ-treated mice showed epidermal erythema, desquamation, and thickening on day 3. The galangin hydrogel-treated mice showed a significant reduction in psoriasis symptoms. Furthermore, the PASI scores of the galangin hydrogel-treated mice were significantly reduced. The spleen of the galangin hydrogel-treated mice returned to normal, and the spleen index decreased ( P< 0.001 ).

[0021] (4) H&E staining of IMQ-treated mouse skin tissue showed that galangin hydrogel significantly alleviated IMQ-induced psoriasis-like symptoms in mice. P<0.001 Immunohistochemical results for Ki67 showed that the Ki67 index was elevated in the skin tissue of IMQ-treated mice, indicating vigorous proliferation of keratinocytes and significant abnormal thickening of the epidermis. P<0.001). The Ki67 index of the mouse skin tissue treated by the galangin hydrogel decreased, indicating that the keratinocyte proliferation could be improved P<0.01 ). The PCNA immunohistochemistry results showed that the PCNA index of the mouse skin tissue treated by the IMQ increased, indicating that the proliferation activity of the keratinocyte increased P<0.001 ). The PCNA index of the mouse skin tissue treated by the galangin hydrogel decreased, indicating that the proliferation activity of the keratinocyte could be improved P<0.001 .

[0022] (5) The KRT14 immunofluorescence results of the mouse skin tissue treated by the IMQ showed that the KRT14 immunofluorescence intensity of the mouse skin tissue treated by the IMQ increased, and the epidermis thickness increased P<0.001 ). The KRT14 immunofluorescence intensity of the mouse skin tissue treated by the galangin hydrogel returned to normal, the abnormal proliferation of the keratinocyte was inhibited, and the differentiation was improved P<0.001 .

[0023] (6) The F4 / 80 and CD11b immunofluorescence results of the mouse skin tissue treated by the IMQ showed that the number of F4 / 80 and CD11b positive cells of the mouse skin tissue treated by the IMQ increased significantly, and the inflammation increased P<0.001 ). The number of F4 / 80 and CD11b positive cells of the mouse skin tissue treated by the galangin hydrogel decreased significantly, and the inflammation was reduced P<0.01 . S100a9, S100a8, Krt6, and Krt16 The gene expression level results showed that the gene expression level of the mouse skin tissue treated by the galangin hydrogel S100a9, S100a8, Krt6, and Krt16 significantly decreased, and the epidermis abnormal proliferation and anti-inflammation were inhibited P<0.01 . BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the following will briefly introduce the drawings to be used in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0025] Figure 1 It is a preparation route map of the galangin hydrogel.

[0026] Figure 2 It is a preparation hydrogel matrix map of different inulin oxidation products and chitosan quaternary ammonium salt ratios.

[0027] Figure 3 It is a characterization map of the galangin hydrogel.

[0028] Figure A is the particle size distribution of the hydrogel; Figure B is the image of the inulin / chitosan quaternary ammonium salt and berberine hydrogel; Figure C is the in vitro release curve; Figure D is the transmission electron microscopy spectrum of the inulin / chitosan quaternary ammonium salt and berberine hydrogel; Figure E is the Fourier infrared spectrum of the inulin / chitosan quaternary ammonium salt and berberine hydrogel; Figures F-G are the X-ray photoelectron spectroscopy analysis results of the inulin / chitosan quaternary ammonium salt and berberine hydrogel.

[0029] Figure 4 Figure is the test result graph of the skin condition of the IMQ-induced mouse psoriasis model.

[0030] Figure A is a schematic diagram of the IMQ-induced mouse psoriasis model; Figure B is a representative image of the back skin of the mouse; Figure C is a PASI score graph; Figure D is a representative spleen image; Figure E is a spleen index.

[0031] Figure 5 Figure is the test result graph of the skin condition of the IMQ-induced mouse psoriasis model.

[0032] Figure A is a representative image of H&E staining of the back skin section of the mouse; Figures B-C are immunohistochemical images of the back skin section of the mouse (wherein B is Ki67 and C is PCNA).

[0033] Figure 6 Figure is the immunofluorescence result graph of the stratum corneum of the IMQ-induced mouse psoriasis model improved by the berberine hydrogel.

[0034] Figure 7 Figure is the test result graph of the inhibition of the proliferation of inflammatory factors by the berberine hydrogel.

[0035] Figure A is an immunofluorescence image of F4 / 80 and CD11b; Figure B is a statistical result of the immunofluorescence staining; C is S100a9, S100a8, Krt6, and Krt16 the expression level of the isogenic gene. DETAILED DESCRIPTION

[0036] Hereinafter, the technical solutions of the present application will be described in conjunction with the examples, but the present application is not limited to the following examples. The experimental methods and detection methods described in each example are all conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified.

[0037] The berberine used in the reagents in the present application is purchased from Chengdu Pusai Biotechnology Co., Ltd., and the item number is PSD250319-725.

[0038] The instruments used in the present application are: transmission electron microscope, Hitachi, HT-7800; X-ray diffractometer, Bruker, D8 Advance; Fourier infrared spectrum analyzer, Thermo, IS50.

[0039] The technical solutions of the present application will be described clearly and completely in combination with the embodiments below. Obviously, the described embodiments are only some of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0040] Embodiment one: galangin hydrogel The present application provides a galangin hydrogel, which is composed of a polymer OI / HACC and a galangin solution; the polymer OI / HACC is formed by blending and reacting OI and chitosan quaternary ammonium salt (HACC); the galangin solution is prepared by dissolving galangin in Tween 80.

[0041] Embodiment two: preparation method of galangin hydrogel Further, the present application provides a preparation method of galangin hydrogel, which is prepared by the following steps: S1: oxidizing inulin by sodium periodate to prepare OI; S2: blending and reacting OI prepared in S1 and chitosan quaternary ammonium salt (HACC) to form a polymer OI / HACC; S3: dissolving galangin in Tween 80 to prepare a galangin solution; S4: blending the polymer OI / HACC prepared in S2 and the galangin solution prepared in S3, heating and stirring to form a gel, and obtaining the galangin hydrogel.

[0042] Preferably, in S1, 400 mg of inulin is dissolved in 10 ml of deionized water by stirring. 200 mg of sodium periodate is dissolved in 2 ml of deionized water by stirring. The inulin solution is added to the sodium periodate solution in a dark environment, stirred, and reacted for 24 hours. An appropriate amount of ethylene glycol is added to inactivate the excess sodium periodate, and then an 8 kDa dialysis bag is used for dialysis for 3 days, with dialysis liquid changed every 12 hours. Finally, the solution is freeze-dried for 24 hours to obtain the inulin oxidation product (OI).

[0043] Preferably, in S2, OI and chitosan quaternary ammonium salt (HACC) are blended at a mass ratio of (2%-10%):(10%-2%). 800 mg of chitosan quaternary ammonium salt (HACC) is dissolved in 7 ml of deionized water, and the OI oxidation product is dissolved in 1 ml of deionized water. The HACC is added to form an OI / HACC hydrogel matrix system.

[0044] Preferably, 80 mg of the berberine reference substance is used in S3, dissolved in 10 ml of Tween 80 to obtain a berberine solution.

[0045] Preferably, 2 ml of the berberine solution prepared in S3 is added dropwise to the OI / HACC hydrogel matrix system prepared in S2 in S4, and stirred for half an hour to obtain a berberine hydrogel.

[0046] Example Three: Application of the berberine hydrogel in improving the IMQ-induced mouse psoriasis model Further, the application also provides the application of the above-mentioned berberine hydrogel in improving the IMQ-induced mouse psoriasis model.

[0047] The application is the application in reducing the Ki67 index and PCNA index in the IMQ-treated mouse skin tissue.

[0048] The application is the application in restoring the KRT14 immunofluorescence intensity in the IMQ-treated mouse skin tissue to near normal, inhibiting abnormal proliferation of keratinocytes, and improving differentiation.

[0049] The application is the application in significantly reducing the number of F4 / 80 and CD11b positive cells in the IMQ-treated mouse skin tissue, and reducing inflammation.

[0050] The application is the application in significantly reducing the gene expression level in the IMQ-treated mouse skin tissue, and inhibiting abnormal proliferation of the epidermis and anti-inflammation. S100a9, S100a8, Krt6, and Krt16 The application is the application in significantly reducing the gene expression level in the IMQ-treated mouse skin tissue, and inhibiting abnormal proliferation of the epidermis and anti-inflammation.

[0051] The application also provides a medicine for resisting psoriasis, which is prepared by taking the above-mentioned berberine hydrogel as an active ingredient.

[0052] Example Four: Preparation of the berberine hydrogel (1) Preparation of the inulin oxidation product.

[0053] Inulin 400 mg is dissolved in 10 ml of deionized water by stirring. Sodium periodate 200 mg is dissolved in 2 ml of deionized water by stirring. The inulin solution is added with the sodium periodate solution in a light-proof environment, stirred, and reacted for 24 hours. An appropriate amount of ethylene glycol is added to inactivate the excess sodium periodate, and then it is placed in a dialysis bag with a molecular weight cut-off of 8 kDa, and dialyzed for 3 days, with the dialysis solution being replaced every 12 hours. Finally, the solution is freeze-dried for 24 hours to obtain the inulin oxidation product (OI).

[0054] (2) Selection of the hydrogel matrix ratio.

[0055] Stable hydrogel matrices were selected by mixing inulin oxidation products with chitosan quaternary ammonium salt in ratios of (2%:10%, 4%:8%, 6%:6%, 8%:4%, 10%:2%). Ultimately, a stable hydrogel matrix was formed with 2% inulin oxidation products and 10% chitosan quaternary ammonium salt.

[0056] (2) Preparation of galangin solution.

[0057] Take 80 mg of galangin reference standard and dissolve it in 10 ml of Tween 80 to obtain galangin solution.

[0058] (3) Preparation of galangin hydrogel.

[0059] Dissolve 800 mg of chitosan quaternary ammonium salt (HACC) in 7 ml of deionized water, dissolve the OI oxidation product in 1 ml of deionized water, and add HACC to form an OI / HACC hydrogel matrix system. Add 2 ml of galangin solution and stir for half an hour to obtain galangin hydrogel.

[0060] The results are attached. Figure 1 The diagram shown illustrates the preparation of galangin hydrogel. (See attached image.) Figure 2 The diagram shows the preparation of hydrogel matrices with different ratios of inulin oxidation products and chitosan quaternary ammonium salts.

[0061] Example 5: Characterization of galangin hydrogel The galangin hydrogel prepared according to the method described in the above embodiments was analyzed. Its in vitro release rate was detected using UV-Vis ultraviolet spectroscopy, and the synthesis process was observed using TEM and particle size analysis. Characterization of the galangin hydrogel is shown in the appendix. Figure 3 .

[0062] Appendix Figure 3 A represents the particle size distribution of the OI / HACC@GA hydrogel. The OI / HACC@GA exhibits a very uniform particle size distribution, with a PDI value of 0.2632.

[0063] Appendix Figure 3 Image B shows the OI / HACC and OI / HACC@GA hydrogels. The addition of galangin allows for the formation of hydrogels.

[0064] Appendix Figure 3 C represents the results of in vitro drug release performance testing. In PBS (pH 7.4), a small amount of galangin was released from the OI / HACC@GA hydrogel. In PBS (pH 5.5), galangin was released from the three-dimensional structure of the hydrogel after 12 hours. After 24 hours of incubation, the release rate reached a maximum of 60%, indicating that the release efficiency of galangin is enhanced under inflammatory conditions.

[0065] AppendixFigure 3 Transmission electron microscopy (TEM) revealed that the galangin hydrogel particles were uniform in size (approximately 100 nm). The spectral analysis confirmed that the addition of galangin to the hydrogel matrix resulted in a unique coating structure, indicating the successful preparation of the galangin hydrogel system.

[0066] Appendix Figure 3 Fourier transform infrared spectra of OI / HACC and OI / HACC@GA in E show peak values ​​in the 3200-3500 cm⁻¹ range. -1 The position corresponds to the absorption associated with the stretching vibration of amino groups in OI / HACC. The peak value is between 1500 and 1750 cm⁻¹. -1 This is absorption due to the stretching vibration of the CN bond. Peak values ​​are at 1353 and 1248 cm⁻¹. -1 It is absorbed by the stretching vibration of the phenolic hydroxyl group. Comparative analysis shows that OI / HACC@GA hydrogel has been successfully synthesized.

[0067] Appendix Figure 3 X-ray photoelectron spectroscopy analysis of F showed that the X-ray diffraction of OI / HACC showed a peak at 20°, while a strong peak appeared after the addition of galangin.

[0068] The characterization results above show that this embodiment synthesized an OI / HACC hydrogel capable of releasing the drug galangin under inflammatory conditions.

[0069] Example 6: An experiment on the improvement of skin condition in an IMQ-induced psoriasis mouse model by galangin. This embodiment provides an experiment demonstrating that galangin improves the skin condition of an IMQ-induced psoriasis mouse model.

[0070] The hair on the back of the mouse tail was shaved in a 2cm × 2cm area. 5% IMQ cream was applied to the back of the tail for 7 consecutive days, once daily, at a dose of 62.5 mg each time. The control group received OI / HACC hydrogel matrix. Mice in the treatment group received 400 μl of galangin hydrogel daily in the modeling area. A schematic diagram of the experiment is attached. Figure 4 A.

[0071] Appendix Figure 4 As shown in Figure B, compared with the control group, mice treated with IMQ developed epidermal erythema, desquamation, and thickening on day 3. Mice treated with galangin hydrogel showed a significant reduction in psoriasis symptoms.

[0072] The severity of psoriatic skin condition was assessed in all animals on days 1-7 based on the clinical Psoriasis Area and Severity Index (PASI). The dorsal skin thickness was measured using a vernier caliper. Erythema and scaling were scored independently from 0 to 4 as follows: 0, none; 1, slight; 2, moderate; 3, marked; 4, very marked. The cumulative score is the sum of the three elements, indicating the severity of inflammation (total score 0-12). The severity of dorsal skin was assessed daily by thickness, scaling and erythema according to the clinical Psoriasis Area and Severity Index.

[0073] The results show that the PASI score of mice treated with galangin hydrogel is significantly reduced. Figure 4 The mice were weighed, and the spleens of the mice were taken after blood sampling, washed with normal saline to remove residual blood, and weighed after wiping off the water with filter paper. The spleen index was calculated as follows: Spleen index (mg / g) = Spleen weight (mg) / Mouse weight (g).

[0074] The results show that the spleen of the IMQ-treated mice is significantly enlarged, and the spleen index is increased compared with the control group.

[0075] The spleen of the galangin hydrogel-treated mice returns to normal, and the spleen index decreases. Figure 4 P<0.001 The results show that the spleen of the IMQ-treated mice is significantly enlarged, and the spleen index is increased compared with the control group. P<0.001 The spleen of the galangin hydrogel-treated mice returns to normal, and the spleen index decreases.

[0076] Example Seven: H&E Staining, Ki67 Immunohistochemistry, and PCNA Immunohistochemistry Tests The dorsal skin lesions of the mice were taken. The lesions were fixed in 4% neutral buffered formaldehyde solution for 48 h. Dehydration: The fixed skin tissue was placed at 4°C for a certain period of time and then moved to 75% ethanol for repair.

[0077] The repaired tissue was dehydrated according to the following procedure: immersion in 75% alcohol solution for 4 h, immersion in 85% alcohol solution for 1.5 h, immersion in 90% alcohol solution for 1.5 h, immersion in 95% alcohol solution for 1 h, immersion in anhydrous ethanol I solution for 20 min, immersion in anhydrous ethanol II solution for 20 min, immersion in alcohol-benzene solution for 5 min, immersion in xylene I solution for 8 min, immersion in xylene II solution for 8 min, immersion in wax I for 1 h, immersion in wax II for 1 h, and immersion in wax III for 1 h. After dehydration, the skin tissue was embedded in a embedding frame with paraffin with a melting point of 55°C, and then placed in a -20°C refrigerator after embedding was completed. After the wax solidified, the wax block was taken out of the embedding frame and repaired. The wax block was placed on a paraffin sectioning machine for sectioning, and the thickness of the section was about 4 μm, which should not be too thick. Then the dorsal skin tissue was flattened on a sectioning machine and placed in a 37°C oven for several hours for baking the section. The baked section was stored at room temperature in the dark for subsequent use. When used, the section with relatively complete tissue structure was selected and placed on a glass slide.​

[0078] Put the prepared slices into xylene I for 20 min, xylene II for 20 min, anhydrous ethanol I for 10 min, anhydrous ethanol II for 10 min, 95% alcohol for 5 min, 90% alcohol for 5 min, 80% alcohol for 5 min, 70% alcohol for 5 min, and distilled water for washing.

[0079] Put all the slices into the hematoxylin dyeing solution, and take them out after 5 min of dyeing and rinsing with tap water for 1 min; then put them into 1% hydrochloric acid ethanol for about 20 s, take them out and put them into the eosin ethanol solution for 2 min of dyeing.

[0080] Put the slices into 95% alcohol I for 7 min, 95% alcohol I for 7 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, xylene I for 5 min, and xylene II for 5 min; immediately take the slices out of the xylene solution when the time is up, wipe them dry and air dry; after the slices are dried, add an appropriate amount of neutral gum to the surface of the tissue, select a suitable cover glass to seal the slices. Pay attention to not leaving bubbles under the slices, label them and make corresponding marks.

[0081] Use a microscope to take images. Use Image Pro Plus 6.0 software to measure the epidermal thickness.

[0082] Appendix Figure 5 A H&E staining showed that galangin hydrogel significantly alleviated the psoriasis-like symptoms of IMQ-induced mice P<0.001 .

[0083] Cut a small piece of mouse back skin (trim off excess hair and adipose tissue) and fix it in 4% formaldehyde; paraffin-embedded; sectioning; put the slices into a slice drying machine and bake them at 80°C for 20 min. Soak the slices in xylene for 3 min, a total of 3 times (after taking out the slices each time, replace the xylene and put the slices in again); soak the slices in anhydrous ethanol for 3 min, and then soak them in anhydrous ethanol again after replacing the anhydrous ethanol; pass through gradient alcohol (95%, 90%, 85%, 80%, 75%, 70%) for 3 min each time, and then take them out and soak them in pure water; wash the slices with PBS buffer for 5 min, a total of 3 times; add 3% H2O2 to the slice tissue and incubate it in the dark for 15 min; wash the slices with distilled water, and then wash them with PBS buffer for 5 min, a total of 3 times.

[0084] The slice is soaked in 1x citrate repair solution, heated to boiling state in the microwave oven, and after cooling, washed with PBS buffer for 5 min, 3 times. Drop 5% goat serum blocking solution on the slice tissue, block at room temperature for 1 h; add 100 μL of primary antibody and incubate at 4°C overnight. The next day, take it out and stand at room temperature for 45 min to warm up, wash the slice with PBS buffer for 5 min, 3 times; after washing, add 50 μL of secondary antibody and incubate at room temperature for 30 min; wash with PBS buffer for 3 times, 5 min each time.

[0085] Prepare DAB working solution in advance, drop on the slice tissue, stand for color development for 10 min, and rinse with distilled water. Stain with hematoxylin for 2 min; dehydrate by gradient alcohol (70%, 75%, 80%, 85%, 90%, 95%), 3 min each time; after dehydration, place the slice in xylene for 3 min, 3 times (after taking out the slice each time, replace the xylene and put the slice in again). Use neutral gum to mount the slice, observe the number of Ki67 and PCNA positive cells under the microscope, and count the positive expression rate of Ki67 and PCNA cells.

[0086] Appendix Figure 5 B The results of Ki67 immunohistochemistry showed that the Ki67 index in the skin tissue of IMQ-treated mice increased, indicating that the keratinocyte proliferation was vigorous, and the epidermis was obviously abnormally thickened. P<0.001 The Ki67 index in the skin tissue of rhodosperma hydrogel-treated mice decreased, indicating that it could improve keratinocyte proliferation. P<0.01

[0087] Appendix Figure 5 C The results of PCNA immunohistochemistry showed that the PCNA index in the skin tissue of IMQ-treated mice increased, indicating that the proliferation activity of keratinocytes increased. P<0.001 The PCNA index in the skin tissue of rhodosperma hydrogel-treated mice decreased, indicating that it could improve the proliferation activity of keratinocytes. P<0.001

[0088] Example Eight: KRT14 Immunofluorescence Test ​​The mouse frozen skin tissue was taken out from the -80 °C refrigerator and placed in the freezing microtome for 10 min. After the frozen skin tissue was balanced with the temperature of the microtome, the tissue was cut into 10 μm thick and immediately attached to an anti-static glass slide, and stored in a -20 °C refrigerator for standby. Take out the frozen sections of each group of skin tissue, recover at room temperature for 15 min, then fix with 4% paraformaldehyde for 15 min. After the section is fixed, rinse with PBS for 3 times, 5 min / time; After permeation with 0.5% Triton X-100 for 15 min, use an oil pen to draw a circle, and block at room temperature for 1 h with blocking solution. After blocking, rinse with PBS for 3 times, 5 min / time; co-stain the sections with purified anti-mouse / human KRT14 (keratinocyte 14) antibody (1 / 400 dilution), rabbit anti-mouse F4 / 80 (1 / 200 dilution), and purified anti-mouse / human CD11b (CD11 antigen-like family member B) antibody (1 / 400 dilution), and incubate overnight at 4 °C. The next day, take out the sections, rinse with PBS for 3 times, 5 min / time; then incubate with immunofluorescence secondary antibody at room temperature for 1 h in the dark, rinse with PBS for 3 times, 5 min / time; and stain the cell nuclei with DAPI at room temperature for 45 min, rinse with PBS for 3 times, 5 min / time; finally, mount with anti-fluorescence quenching agent. After staining, observe and take pictures under a 20x lens using a laser confocal ultra-high resolution microscope. Use Image J to statistically analyze the fluorescence intensity of the photographed images.

[0089] Appendix Figure 6 The KRT14 immunofluorescence results show that the KRT14 immunofluorescence intensity in the skin tissue of the IMQ-treated mice increases, the epidermis thickens ( P<0.001 ). The KRT14 immunofluorescence intensity in the skin tissue of the morindin dihydrochloride hydrogel-treated mice is restored to near normal, inhibiting the abnormal proliferation of keratinocytes and improving differentiation ( P<0.001 ).

[0090] Example Nine: F4 / 80, CD11b immunofluorescence, S100a9, S100a8, Krt6, and Krt16 Test of gene expression level Appendix Figure 7 The F4 / 80 and CD11b immunofluorescence results of A, 7B show that the number of F4 / 80 and CD11b positive cells in the skin tissue of the IMQ-treated mice increases significantly, and the inflammation increases ( P<0.001 ). The number of F4 / 80 and CD11b positive cells in the skin tissue of the morindin dihydrochloride hydrogel-treated mice is significantly reduced, and the inflammation is reduced ( P<0.01 ).

[0091] Total RNA was extracted from the dorsal skin of mice using TRIzol reagent. RNA was reverse transcribed into cDNA using HiScript III RT SuperMix (+gDNA wiper) and quantified by ChamQ Universal SYBR qPCR MasterMix with QuantStudio™ 5 system. The 2 -ΔΔCT Gene expression was calculated relative to the housekeeping gene Hprt1. All primers listed in Table 1 were synthesized by GenScript Biotech Corporation.

[0092] Table 1 RT-qPCR primer sequences

[0093] Appendix Figure 7 C S100a9, S100a8, Krt6, and Krt16 The results of gene expression levels showed that the skin tissue of mice treated with galangin hydrogel had significantly lower expression levels of S100a9, S100a8, Krt6, and Krt16 genes, inhibited epidermal abnormal proliferation and anti-inflammatory P<0.01 ).

[0094] The above results show that the OI / HACC@GA hydrogel provided in Example 1 has good anti-inflammatory and inhibitory effects on abnormal proliferation of keratinocytes, and has better anti-psoriasis effect than other hydrogels in the prior art.

[0095] As can be seen from the above examples and experimental examples, the present application provides a composite hydrogel, which is equipped with galangin and has multiple biological activities such as anti-inflammatory, inhibition of epidermal thickening and inhibition of epidermal abnormal proliferation, and has a good promoting effect on the treatment of psoriasis.

[0096] The above-described examples are only some of the embodiments of the present application, not all the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the claimed application, but only represents the preferred embodiments of the present application. All other embodiments obtained by related deduction and replacement of persons skilled in the art under the condition of the concept of the present application, without making creative labor, belong to the scope of protection of the present application.

Claims

1. A galangin hydrogel, characterized in that, The galangin hydrogel is composed of a polymer OI / HACC and a galangin solution; the polymer OI / HACC is formed by the co-reaction of OI and chitosan quaternary ammonium salt (HACC); the galangin solution is prepared by dissolving galangin in Tween 80.

2. The method for preparing galangin hydrogel as described in claim 1, characterized in that, The preparation method is as follows: S1: Inulin is oxidized with sodium periodate to produce OI; S2: The OI obtained in S1 is blended with chitosan quaternary ammonium salt (HACC) and reacted to form polymer OI / HACC; S3: Dissolve galangin in Tween 80 to prepare a galangin solution; S4: Mix the polymer OI / HACC obtained in S2 with the galangin solution obtained in S3, heat and stir to form a gel, and then obtain the final product.

3. The method for preparing galangin hydrogel as described in claim 2, characterized in that, S1 was prepared by dissolving 400 mg of inulin in 10 ml of deionized water with stirring. 200 mg of sodium periodate was dissolved in 2 ml of deionized water with stirring. The sodium periodate solution was added to the inulin solution in a light-protected environment, stirred, and reacted for 24 hours. An appropriate amount of ethylene glycol was added to inactivate excess sodium periodate. The solution was then dialyzed for 3 days using an 8 kDa dialysis bag, with the dialysate changed every 12 hours. Finally, the solution was freeze-dried for 24 hours to obtain the inulin oxidation product (OI).

4. The method for preparing galangin hydrogel as described in claim 2, characterized in that, In S2, OI and chitosan quaternary ammonium salt (HACC) are blended at a mass ratio of (2%-10%):(10%-2%). 800 mg of chitosan quaternary ammonium salt (HACC) is dissolved in 7 ml of deionized water, the OI oxidation product is dissolved in 1 ml of deionized water, and HACC is added to form an OI / HACC hydrogel matrix system.

5. The method for preparing galangin hydrogel as described in claim 2, characterized in that, In S3, 80 mg of galangin reference standard was dissolved in 10 ml of Tween 80 to obtain a galangin solution.

6. The method for preparing galangin hydrogel as described in claim 2, characterized in that, In S4, 2 ml of the galangin solution prepared in S3 was added dropwise to the OI / HACC hydrogel matrix system prepared in S2, and stirred for half an hour to obtain galangin hydrogel.

7. The application of the galangin hydrogel as described in any one of claims 1 to 6, or the galangin hydrogel prepared by the method described in claims 1 to 6, in improving the IMQ-induced mouse psoriasis model.

8. The application of galangin hydrogel as described in 7 or the galangin hydrogel prepared by the same method in improving an IMQ-induced mouse psoriasis model, characterized in that... The applications include the reduction of Ki67 and PCNA indices in IMQ-treated mouse skin tissue; restoration of KRT14 immunofluorescence intensity to near normal; and a significant reduction in the number of F4 / 80 and CD11b positive cells. S100a9, S100a8, Krt6 and Krt16 One or more of the following: significantly reduced gene expression levels.

9. A drug for treating psoriasis, characterized in that, The drug for treating psoriasis is made with galangin hydrogel prepared by any one of claims 1 to 6 as the active ingredient.

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