Biomass-based tea tree essential oil slow-release application patch for treating psoriasis and preparation method thereof

Abstract

The invention discloses a biomass-based tea tree essential oil slow-release application patch for treating psoriasis and a preparation method of the biomass-based tea tree essential oil slow-release application patch. The method comprises the following steps: extracting collagen sponge from an animal connective tissue raw material, preparing a collagen solution, adding tea tree essential oil, and emulsifying to obtain a collagen Pickering emulsion; fungus strains are inoculated on a PDA solid culture medium to be cultured to obtain hyphae, then the hyphae are scraped and implanted into a PDB liquid culture medium to be cultured, and hypha membranes are obtained; finally, the mycelium membrane is coated with the collagen Pickering emulsion, so that the collagen Pickering emulsion naturally permeates into the mycelium membrane, and the biomass-based tea tree essential oil slow-release application patch is obtained after drying. The biomass-based tea tree essential oil sustained-release application patch which takes all components as biomass raw materials, has the advantages of drug sustained release, instant adhesion when encountering water and toughness when encountering water is prepared, and can be used for treating psoriasis. The sustained-release application patch has good mechanical strength, can be directly adhered to skin, has strong compliance to dynamic affected parts, and does not bring extra pain to patients.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a biomass-based tea tree oil sustained-release patch for the treatment of psoriasis and its preparation method. Background Technology

[0002] Psoriasis is a chronic inflammatory skin disease with a global prevalence of about 1% to 3%. Its clinical manifestations are characterized by erythematous scaly plaques that can affect multiple areas such as elbows, knees, and scalp. Its pathological mechanism involves complex processes such as immune cell activation, abnormal proliferation of keratinocytes, and angiogenesis. Currently, psoriasis cannot be cured, but modern medicine has a wealth of treatment methods to control symptoms and ensure patients' normal lives. It is mainly divided into systemic treatment and local treatment: (1) Systemic treatment, such as the use of traditional immunosuppressants such as methotrexate, but there is a risk of systemic toxicity; Phototherapy uses narrow-band UVB (NB-UVB) combined with psoralen and PUVA to induce cell apoptosis and inhibit inflammatory factors, which may lead to skin aging, erythema, and itching. Long-term use has a potential carcinogenic risk; (2) Local treatment: such as Baricitinib lipid preparation (BCT-OS) to achieve local high-efficiency anti-inflammatory by optimizing pH and rheological properties, and Tapinarof nanogel to enhance drug skin penetration and retention with the help of nanocarriers. Topical treatment is used by the vast majority of psoriasis patients. This method can reduce the risk of systemic poisoning, but it often uses hormonal drugs, and the side effects cannot be ignored.

[0003] In addition, commercially available topical treatments are typically creams or ointments, suitable for plaque psoriasis on most areas of the body. They provide some moisturizing and effectively deliver medication to thicker lesions. However, due to the lack of sustained-release properties, the effects of these products are short-lived. Furthermore, lacking sufficient mechanical support, creams or ointments struggle to form a persistent, locally effective layer on the skin surface, easily escaping due to gravity or friction. Moreover, the rheological properties of creams or ointments, characterized by low viscosity and weak gel strength, cannot withstand the shear forces generated by skin movement, leading to uneven drug distribution or rapid diffusion.

[0004] To address the aforementioned problems with topical treatment products, researchers have developed several novel systems, including:

[0005] (1) Hydrogel System: Utilizing hydrogels to achieve drug carrier function. Patent application CN 119280140 A discloses a methotrexate-loaded sulfonated bacterial cellulose / chitosan hydrogel for psoriasis treatment and its preparation method. However, methotrexate is a traditional immunosuppressant and poses a risk of systemic toxicity. Patent application CN 115068815 discloses a viologen hydrogel / magnesium self-powered on-demand drug delivery device and its application in psoriasis treatment. Viologen is a toxic cationic dye, and toxic organic solvents such as iodomethane, dichloromethane, and N,N-dimethylformamide (DMF) are used in the synthesis of double-bond functionalized viologen monomers. Patent CN 116270425 B discloses a long-acting gel formulation containing arsenic trioxide nanoliposomes for psoriasis treatment and its preparation method. Arsenic trioxide is a toxic substance, and the preparation process of sustained-release liposomes requires special excipients and equipment, resulting in high costs. Furthermore, none of the above technologies address the dressing properties of hydrogels; hydrogels have poor mechanical strength and suffer from durability issues when used alone as dressings.

[0006] (2) Microneedle Patch System: Microneedle patches utilize hydrogel-based patches to load drugs, while the dense microneedles on the patch facilitate skin application and drug delivery. Patent application CN 119367261 A discloses a drug-loaded microneedle patch for psoriasis treatment and its preparation method, using toxic organic solvents such as DMF (N,N-dimethylformamide) and THF (tetrahydrofuran) during the preparation process. Patent application CN 116763715 A discloses a preparation method and application of paeonol-soluble transdermal microneedles for treating psoriasis. The microneedle matrix is ​​mainly composed of soluble hyaluronic acid, which has poor mechanical strength and is prone to softening and breaking upon contact with skin moisture, affecting the puncture effect. Patent application CN 115569300 A discloses a soluble microneedle patch for psoriasis and its preparation method. In this method, the mixing of PDMS and a curing agent followed by vacuuming may generate volatile organic compounds (VOCs). The high proportion of soluble degradable materials (such as sodium hyaluronate) may accelerate patch swelling upon contact with water, leading to a rapid decrease in adhesion and preventing prolonged skin adhesion. Furthermore, this technology lacks an encapsulation and sustained-release mechanism, and the use of microneedles often causes significant stinging sensations, resulting in patient discomfort. The complex and costly preparation process of microneedle patches limits their practical application.

[0007] Tea tree oil (TTO), a distilled extract of Melaleuca alternifolia leaves, exhibits significant anti-psoriasis potential in its main active component, terpinen-4-ol (30%–45%), by inhibiting the release of inflammatory mediators such as TNF-α, IL-1, IL-8, and PGE2. Compared to traditional therapies, topical application of tea tree oil offers advantages such as natural low toxicity and reduced systemic adverse reactions. However, the volatile nature of tea tree oil limits its stability and duration of action, necessitating the use of novel drug delivery systems to optimize its efficacy.

[0008] Pickering emulsions, as heterogeneous systems stabilized by solid particles, exhibit unique advantages in food preservation and biomedicine due to their high stability, biocompatibility, and controlled release characteristics. Using biomass as an encapsulation material for pickering emulsions provides a green solution for sustained-release delivery of active ingredients, as its mechanical barrier function effectively inhibits droplet aggregation and is non-cytotoxic. Collagen, as a natural biomass polymer, also possesses the ability to form pickering emulsions. Combined with its excellent biological properties, collagen pickering emulsions show great potential for encapsulating and releasing tea tree oil in psoriasis treatment. However, directly using collagen pickering emulsions as sustained-release carriers for skin treatment results in the same defects as commercially available creams or ointments when applied topically due to their dispersed state. Therefore, a non-toxic, harmless, and well-adapted substrate support material should be sought to fix collagen pickering emulsions.

[0009] Mycelium is a three-dimensional fibrous network assembled from fungal hyphae. Its main components include non-toxic chitin, β-glucan, and protein. Mycelium cultivated under static conditions forms a flexible, wet membrane. After heat inactivation, it becomes a thin film, rapidly regaining its flexibility upon contact with water, thus exhibiting good biocompatibility and durability. Existing research has confirmed that by screening wild-type fungal strains and optimizing culture substrates and post-processing techniques, the porosity, hydrophilicity / hydrophobicity, and degradation rate of mycelial membranes can be precisely controlled, making them suitable for various biomedical applications.

[0010] Therefore, this invention combines mycelium, collagen Pickering emulsion, and tea tree oil to construct a novel dressing for treating psoriasis. Summary of the Invention

[0011] The purpose of this invention is to provide a biomass-based tea tree oil sustained-release patch for the treatment of psoriasis and its preparation method. The patch is composed entirely of biomass raw materials and has the characteristics of sustained drug release, adhesion upon contact with water, and toughening upon contact with water.

[0012] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0013] A method for preparing a biomass-based tea tree oil sustained-release patch for the treatment of psoriasis includes the following steps:

[0014] 1) Collagen extraction: Wash and chop fresh animal connective tissue raw materials, add acetic acid and pepsin, stir at 4℃ for 24-80h, collect the supernatant by centrifugation at 5000-15000r / min, and continuously add sodium chloride to the supernatant to precipitate collagen solid precipitate. Dissolve the collagen solid precipitate with acetic acid. After complete dissolution, dialyze the solution for 24-72h, freeze-dry the dialysate to obtain purified collagen sponge, and store the collagen sponge in a constant temperature, dry and dark place at 4℃.

[0015] 2) Preparation of collagen Pickering emulsion: Dissolve collagen sponge in deionized water to obtain a high-concentration collagen solution with a concentration of 5-10 mg / mL, and use a high-speed homogenizer to stir at high speed to assist in uniform dissolution. Add tea tree oil to the collagen solution and emulsify it using a high-speed homogenizer to obtain collagen Pickering emulsion.

[0016] 3) Culture of mycelial membrane: Inoculate the fungal strain onto PDA solid medium and statically culture it in a constant temperature and humidity incubator at 25-30℃ for 5-10 days. Then, autoclave the PDB liquid medium and inoculation tools (121℃, 30 min). Scrape the surface mycelia from the PDA solid medium and implant them into the PDB liquid medium. Statically culture it in a constant temperature and humidity incubator at 25-30℃ for 7-20 days. Take out the fully grown and uncontaminated mycelial membrane and wash it with deionized water 2-5 times. Place it in an autoclave for inactivation treatment (121℃, 30 min), dry it at 25-60℃, and store it in a dry, dark place at room temperature.

[0017] 4) Preparation of mycelium / collagen emulsion composite membrane: Collagen Pickering emulsion is coated onto the mycelium membrane using a coating machine, allowing the collagen Pickering emulsion to naturally penetrate into the mycelium membrane. After natural drying, a biomass-based tea tree oil sustained-release patch is obtained.

[0018] Furthermore, the animal connective tissue raw material mentioned in step 1) is cowhide or fish skin, etc.

[0019] Further, in step 1), the mass concentration of acetic acid is 0.1–0.7 mol / L, the mass ratio of animal connective tissue raw material to acetic acid solution is 1:20–1:60, the final concentration of pepsin is 1–3% (w / v), and the final concentration of sodium chloride is 0.4–1 mol / L.

[0020] Furthermore, the freeze-drying temperature in step 1) is -40°C, and the freeze-drying time is 48 to 96 hours.

[0021] Further, the tea tree oil described in step 2) is added to a final mass concentration of 6-10% (relative to the total mass of the emulsion).

[0022] Further, in step 2), the rotation speed of the high-speed homogenizer for uniform dissolution is 6000-15000 r / min, and the stirring time is 1-3 min. The rotation speed of the high-speed homogenizer for emulsification is 5000-20000 r / min, and the emulsification time is 2-5 min.

[0023] Furthermore, the fungal species mentioned in step 3) are Ganoderma lucidum, oyster mushroom, or king oyster mushroom, etc.

[0024] Further, the composition of the PDA solid culture medium in step 3) is as follows: PDA 13g / L.

[0025] Further, the composition of the PDB liquid culture medium in step 3) is as follows: PDB 26g / L, yeast extract 2g / L, peptone 2g / L, dipotassium hydrogen phosphate 3g / L, and magnesium sulfate heptahydrate 1.5g / L.

[0026] Furthermore, the coating thickness of the collagen Pickering emulsion described in step 4) is 1–3 mm.

[0027] When using the biomass-based tea tree oil sustained-release patch, cut it to the size of the affected area, spray a small amount of water mist onto the emulsion surface of the patch with a small sprayer, or drop 1-2 drops of water onto the emulsion surface of the patch with a dropper, and immediately apply the emulsion surface to the psoriasis affected area.

[0028] This invention prepares a biomass-based tea tree oil sustained-release patch, composed entirely of biomass raw materials, possessing drug sustained release properties, and exhibiting adhesiveness and toughening properties upon contact with water, for the treatment of psoriasis. This invention has the following beneficial effects:

[0029] (1) All raw materials used in the sustained-release patch of the present invention have excellent biocompatibility, do not use toxic substances, are green and environmentally friendly in preparation process, have no toxic substance residues, are simple in preparation process, and are suitable for mass production.

[0030] (2) The present invention uses collagen picklein emulsion to achieve dual functions: drug encapsulation and sustained release, enhancing the therapeutic effect; it is sticky when it comes into contact with water, making it easy to use, not easy to fall off, and causing no secondary damage to the patient's skin.

[0031] (3) The present invention uses mycelial membrane as a base to achieve flexibility when exposed to water, providing mechanical support and improving the dynamic compliance of the affected area.

[0032] (4) The mycelium / collagen emulsion double-layer combination technology of the present invention is simple and feasible, and has strong practical large-scale production feasibility: the penetration and filling effect of the emulsion into the mycelium pores results in strong bonding force at the double-layer interface and no stratification occurs.

[0033] (5) The sustained-release patch prepared by the present invention has good mechanical strength and can be directly adhered to the skin. It has strong conformity to dynamic lesions and does not bring additional pain to the patient. Attached Figure Description

[0034] Figure 1 SEM results of the mycelium / collagen / tea tree oil composite membrane prepared in step 4; a) Collagen emulsion surface of the mycelium / collagen / tea tree oil composite membrane; b) Mycelium surface of the mycelium / collagen / tea tree oil composite membrane; c) Cross section of the mycelium / collagen / tea tree oil composite membrane.

[0035] Figure 2 Drug release curves of mycelium / collagen / tea tree essential oil composite membranes with different essential oil concentrations prepared in Examples 1, 4, and 7 in PBS buffer solution.

[0036] Figure 3 The mechanical properties of the mycelium membrane (MYC) and the mycelium / collagen / tea tree oil composite membrane (MYC / COL / TTO) prepared in Example 4 are shown in diagrams; a is the stress-strain curves of the mycelium membrane and the composite membrane in dry and wet states; b is the fracture energy of the mycelium membrane and the composite membrane; c is the torsion test diagram of the material in dry and wet states; d is the skin-adhesion effect of the composite membrane in different states of wrist and elbow.

[0037] Figure 4 Figure 1 shows the animal experimental characterization; a) shows the mouse treatment procedure; b) shows the effect of 5-day treatment in mice; c) shows tissue staining under different treatment methods; d) shows the skin thickness of mice under different treatment methods. Detailed Implementation

[0038] In the following examples, the compositions of the PDA solid culture medium and the PDB liquid culture medium remain constant, as follows:

[0039] PDA solid culture medium: PDA 13g / L

[0040] PDB liquid medium: PDB 26 g / L, yeast extract 2 g / L, peptone 2 g / L, dipotassium hydrogen phosphate 3 g / L, magnesium sulfate heptahydrate 1.5 g / L

[0041] Example 1

[0042] A method for preparing a biomass-based tea tree oil sustained-release patch for the treatment of psoriasis includes the following steps:

[0043] (1) Fresh cowhide was washed and cut into small pieces. 0.1 mol / L acetic acid and 2% (w / v) pepsin were added at a material-to-liquid mass ratio of 1:50. After stirring at 4°C for 24 h, the supernatant was collected by centrifugation at 5000 r / min. Sodium chloride was continuously added to the supernatant to precipitate collagen, with a final sodium chloride concentration of 2 mol / L. The collagen precipitate was dissolved in 0.3 mol / L acetic acid. After complete dissolution, the solution was dialyzed for 72 h. Finally, the purified collagen sponge was obtained by freeze-drying and stored at 4°C in a dry, dark place.

[0044] (2) Dissolve the collagen sponge in deionized water to obtain a high-concentration collagen solution with a concentration of 5 mg / mL. Stir the solution at high speed for 1 min using a high-speed homogenizer to aid in uniform dissolution. Add tea tree oil to the collagen solution until the final mass concentration is 10%, and emulsify it at high speed for 2 min using a high-speed homogenizer at a speed of 15000 r / min to obtain a collagen-based Pickering emulsion.

[0045] (3) Inoculate the fungal strain (oyster mushroom) onto PDA solid medium and statically culture it in a constant temperature and humidity incubator at 30℃ for 8 days. Then, autoclave the PDB liquid medium and inoculation tools (121℃, 30 min). Scrape the surface hyphae on the solid medium and implant them into the liquid medium. Static culture it in a constant temperature and humidity incubator at 30℃ for 13 days. Take out the intact and uncontaminated hyphae membrane and wash the hyphae membrane twice with deionized water. Put it into an autoclave for inactivation treatment (121℃, 30 min). After drying at 55℃, store it in a dry and dark place at room temperature.

[0046] (4) Collagen Pickering emulsion was coated onto the mycelium membrane using a coating machine with a coating thickness of 1 mm, allowing the emulsion to naturally penetrate into the mycelium membrane and dry naturally to obtain a biomass-based tea tree oil sustained-release patch (mycelium / collagen / tea tree oil composite membrane).

[0047] When using, cut the patch to size according to the affected area, spray a small amount of water mist onto the lotion side of the patch with a small sprayer, and immediately apply the lotion side to the psoriasis affected area.

[0048] Example 2

[0049] A method for preparing a biomass-based tea tree oil sustained-release patch for the treatment of psoriasis includes the following steps:

[0050] (1) Fresh fish skin was washed and cut into small pieces. 0.1 mol / L acetic acid and 2% (w / v) pepsin were added at a material-to-liquid mass ratio of 1:50. After stirring at 4℃ for 48 h, the supernatant was collected by centrifugation at 10000 r / min. Sodium chloride was continuously added to the supernatant to precipitate collagen solids, with a final sodium chloride concentration of 2 mol / L. The collagen solid precipitate was dissolved in 0.3 mol / L acetic acid. After complete dissolution, the solution was dialyzed for 72 h. Finally, the purified collagen sponge was obtained by freeze-drying and stored at 4℃ in a dry, dark place.

[0051] (2) Dissolve the collagen sponge in deionized water to obtain a high-concentration collagen solution with a concentration of 15 mg / mL. Stir the solution at high speed for 1 min using a high-speed homogenizer to aid in uniform dissolution. Add tea tree oil to the collagen solution until the final mass concentration is 8%, and emulsify it at high speed for 2 min using a high-speed homogenizer with a speed of 15000 r / min to obtain a collagen-based Pickering emulsion.

[0052] (3) Inoculate the fungal strain (Ganoderma lucidum) onto PDA solid medium and statically culture it in a constant temperature and humidity incubator at 30℃ for 10 days. Then, autoclave the PDB liquid medium and inoculation tools (121℃, 30 min). Scrape the surface hyphae on the solid medium and implant them into the liquid medium. Static culture it in a constant temperature and humidity incubator at 30℃ for 13 days. Take out the intact and uncontaminated hyphal membrane and wash it twice with deionized water. Put it into an autoclave for inactivation treatment (121℃, 30 min). After drying at 50℃, store it in a dry and dark place at room temperature.

[0053] (4) Collagen Pickering emulsion was coated onto the mycelial membrane using a coating machine with a coating thickness of 1.5 mm, allowing the emulsion to naturally penetrate into the mycelial membrane and dry naturally to obtain a biomass-based tea tree oil sustained-release patch (mycelium / collagen / tea tree oil composite membrane).

[0054] When using, cut the patch to size according to the affected area, drop 1-2 drops of water onto the lotion side of the patch with a dropper, and immediately apply the lotion side to the psoriasis affected area.

[0055] Example 3

[0056] A method for preparing a biomass-based tea tree oil sustained-release patch for the treatment of psoriasis includes the following steps:

[0057] (1) Fresh fish skin was washed and cut into small pieces. 0.5 mol / L acetic acid and 3% (w / v) pepsin were added at a material-to-liquid mass ratio of 1:50. After stirring at 4℃ for 80 h, the supernatant was collected by centrifugation at 5000 r / min. Sodium chloride was continuously added to the supernatant to precipitate collagen, with a final sodium chloride concentration of 0.7 mol / L. The collagen precipitate was dissolved in 0.1 mol / L acetic acid. After complete dissolution, the solution was dialyzed for 72 h. Finally, the purified collagen sponge was obtained by freeze-drying and stored at 4℃ in a dry, dark place.

[0058] (2) Dissolve the collagen sponge in deionized water to obtain a high-concentration collagen solution with a concentration of 10 mg / mL. Stir the solution at high speed for 1 min using a high-speed homogenizer to aid in uniform dissolution. Add tea tree oil to the collagen solution until the final mass concentration is 6%, and emulsify it at high speed for 2 min using a high-speed homogenizer at a speed of 20,000 r / min to obtain a collagen-based Pickering emulsion.

[0059] (3) Inoculate the fungal strain (oyster mushroom) onto PDA solid medium and statically culture it in a constant temperature and humidity incubator at 27℃ for 10 days. Then, autoclave the PDB liquid medium and inoculation tools (121℃, 30 min). Scrape the surface hyphae on the solid medium and implant them into the liquid medium. Static culture it in a constant temperature and humidity incubator at 27℃ for 15 days. Take out the intact and uncontaminated hyphae membrane and wash it twice with deionized water. Put it into an autoclave for inactivation treatment (121℃, 30 min). After drying at 25℃, store it in a dry and dark place at room temperature.

[0060] (4) Collagen Pickering emulsion was coated onto the mycelial membrane using a coating machine with a coating thickness of 2 mm. The emulsion was allowed to penetrate the mycelial membrane naturally and then dried naturally to obtain a biomass-based tea tree oil sustained-release patch (mycelium / collagen / tea tree oil composite membrane).

[0061] When using, cut the patch to size according to the affected area, drop 1-2 drops of water onto the lotion side of the patch with a dropper, and immediately apply the lotion side to the psoriasis affected area.

[0062] Example 4

[0063] A method for preparing a biomass-based tea tree oil sustained-release patch for the treatment of psoriasis includes the following steps:

[0064] (1) Fresh cowhide was washed and cut into small pieces. 0.5 mol / L acetic acid and 3% (w / v) pepsin were added at a material-to-liquid mass ratio of 1:50. After stirring at below 10°C for 72 h, the supernatant was collected by centrifugation at 10000 r / min. Sodium chloride was continuously added to the supernatant to precipitate collagen, with a final sodium chloride concentration of 0.7 mol / L. The collagen precipitate was dissolved in 0.1 mol / L acetic acid. After complete dissolution, the solution was dialyzed for 72 h. Finally, the purified collagen sponge was obtained by freeze-drying and stored at 4°C in a dry, dark place.

[0065] (2) Dissolve the collagen sponge in deionized water to obtain a high-concentration collagen solution with a concentration of 10 mg / mL. Stir the solution at high speed for 1 min using a high-speed homogenizer to aid in uniform dissolution. Add tea tree oil to the collagen solution until the final mass concentration is 8%, and emulsify it at high speed for 2 min using a high-speed homogenizer at a speed of 20,000 r / min to obtain a collagen-based Pickering emulsion.

[0066] (3) Inoculate the fungal strain (oyster mushroom) onto PDA solid medium and statically culture it in a constant temperature and humidity incubator at 25℃ for 7 days. Then, autoclave the PDB liquid medium and inoculation tools (121℃, 30 min). Scrape the surface hyphae on the solid medium and implant them into the liquid medium. Static culture it in a constant temperature and humidity incubator at 25℃ for 20 days. Take out the intact and uncontaminated hyphae membrane and wash it three times with deionized water. Put it into an autoclave for inactivation treatment (121℃, 30 min). After drying at 60℃, store it in a dry and dark place at room temperature.

[0067] (4) Collagen Pickering emulsion was coated onto the mycelial membrane using a coating machine with a coating thickness of 1.5 mm, allowing the emulsion to naturally penetrate into the mycelial membrane and dry naturally to obtain a biomass-based tea tree oil sustained-release patch (mycelium / collagen / tea tree oil composite membrane).

[0068] When using, cut the patch to size according to the affected area, spray a small amount of water mist onto the lotion side of the patch with a small sprayer, and immediately apply the lotion side to the psoriasis affected area.

[0069] Figure 1 The SEM results of the mycelium / collagen / tea tree oil composite membrane prepared in step 4 were obtained. The morphology of different surfaces and cross-sections of the composite membrane was observed using scanning electron microscopy (SEM). Figure 1(a, b, c) The mycelial surface of the composite membrane exhibits a distinct mycelial network structure, with interwoven hyphae forming numerous pores and a relatively loose structure. Image J analysis shows that the porosity of the mycelial membrane is 45%, and the average diameter of the hyphal fibers is 1.2 ± 0.2 μm. The emulsion-modified side of the composite membrane is relatively smooth, exhibiting a dense morphology. Figure 1 As can be seen from the cross-sectional diagram of the composite membrane, the emulsion penetrates into the hyphae to a certain distance, filling part of the network pores of the hyphae structure. This penetration changes the original loose structure of the hyphae surface. The emulsion penetrates from the hyphae surface into the network structure of the hyphae, making the structure of the penetrated hyphae more compact. After drying, the emulsion and hyphae combine to form a complete composite membrane.

[0070] Example 5

[0071] A method for preparing a biomass-based tea tree oil sustained-release patch for the treatment of psoriasis includes the following steps:

[0072] (1) Fresh cowhide was washed and cut into small pieces. 0.1 mol / L acetic acid and 2% (w / v) pepsin were added at a material-to-liquid mass ratio of 1:50. After stirring at 4℃ for 48 h, the supernatant was collected by centrifugation at 15000 r / min. Sodium chloride was continuously added to the supernatant to precipitate collagen, with a final sodium chloride concentration of 0.7 mol / L. The collagen solid precipitate was dissolved using 0.3 mol / L acetic acid. After complete dissolution, the solution was dialyzed for 72 h. Finally, the purified collagen sponge was obtained by freeze-drying and stored at 4℃ in a dry, dark place.

[0073] (2) Dissolve the collagen sponge in deionized water to obtain a high-concentration collagen solution with a concentration of 10 mg / mL. Stir the solution at high speed for 1 min using a high-speed homogenizer to aid in uniform dissolution. Add tea tree oil to the collagen solution until the final mass concentration is 8%, and emulsify it at high speed for 2 min using a high-speed homogenizer at a speed of 15000 r / min to obtain a collagen-based Pickering emulsion.

[0074] (3) Inoculate the fungal strain (King Oyster Mushroom) onto PDA solid medium and statically culture it in a constant temperature and humidity incubator at 25℃ for 10 days. Then, autoclave the PDB liquid medium and inoculation tools (121℃, 30 min). Scrape the surface hyphae on the solid medium and implant them into the liquid medium. Static culture it in a constant temperature and humidity incubator at 25℃ for 10 days. Take out the intact and uncontaminated hyphae membrane and wash it three times with deionized water. Put it into an autoclave for inactivation treatment (121℃, 30 min). After drying at 45℃, store it in a dry and dark place at room temperature.

[0075] (4) Collagen Pickering emulsion was coated onto the mycelial membrane using a coating machine with a coating thickness of 1.5 mm, allowing the emulsion to naturally penetrate into the mycelial membrane and dry naturally to obtain a biomass-based tea tree oil sustained-release patch (mycelium / collagen / tea tree oil composite membrane).

[0076] When using, cut the patch to size according to the affected area, spray a small amount of water mist onto the lotion side of the patch with a small sprayer, and immediately apply the lotion side to the psoriasis affected area.

[0077] Example 6

[0078] A method for preparing a biomass-based tea tree oil sustained-release patch for the treatment of psoriasis includes the following steps:

[0079] (1) Fresh fish skin was washed and cut into small pieces. 0.1 mol / L acetic acid and 2% (w / v) pepsin were added at a material-to-liquid mass ratio of 1:30. After stirring at 4°C for 80 h, the supernatant was collected by centrifugation at 10000 r / min. Sodium chloride was continuously added to the supernatant to precipitate collagen, with a final sodium chloride concentration of 0.7 mol / L. The collagen precipitate was dissolved in 0.3 mol / L acetic acid. After complete dissolution, the solution was dialyzed for at least 72 h. Finally, the purified collagen sponge was obtained by freeze-drying and stored at 4°C in a dry, dark place.

[0080] (2) Dissolve the collagen sponge in deionized water to obtain a high-concentration collagen solution with a concentration of 5 mg / mL. Stir the solution at high speed for 1 min using a high-speed homogenizer to aid in uniform dissolution. Add tea tree oil to the collagen solution until the final mass concentration is 8%. Emulsify the solution at high speed for 2 min using a high-speed homogenizer at a speed of 15000 r / min to obtain a collagen-based Pickering emulsion.

[0081] (3) Inoculate the fungal strain (oyster mushroom) onto PDA solid medium and statically culture it in a constant temperature and humidity incubator at 27℃ for 8 days. Then, autoclave the PDB liquid medium and inoculation tools (121℃, 30 min). Scrape the surface hyphae on the solid medium and implant them into the liquid medium. Static culture it in a constant temperature and humidity incubator at 25℃ for 10 days. Take out the intact and uncontaminated hyphae membrane and wash it three times with deionized water. Put it into an autoclave for inactivation treatment (121℃, 30 min). After drying at 50℃, store it in a dry and dark place at room temperature.

[0082] (4) Collagen Pickering emulsion was coated onto the mycelial membrane using a coating machine with a coating thickness of 3 mm. The emulsion was allowed to penetrate the mycelial membrane naturally and then dried naturally to obtain a biomass-based tea tree oil sustained-release patch (mycelium / collagen / tea tree oil composite membrane).

[0083] When using, cut the patch to size according to the affected area, drop 1-2 drops of water onto the lotion side of the patch with a dropper, and immediately apply the lotion side to the psoriasis affected area.

[0084] Example 7

[0085] A method for preparing a biomass-based tea tree oil sustained-release patch for the treatment of psoriasis includes the following steps:

[0086] (1) Fresh cowhide was washed and cut into small pieces. 0.5 mol / L acetic acid and 2% (w / v) pepsin were added at a material-to-liquid mass ratio of 1:60. After stirring at 4℃ for 72 h, the supernatant was collected by centrifugation at 9000 r / min. Sodium chloride was continuously added to the supernatant to precipitate collagen, with a final sodium chloride concentration of 0.5 mol / L. The collagen precipitate was dissolved in 0.3 mol / L acetic acid. After complete dissolution, the solution was dialyzed for at least 72 h. Finally, the purified collagen sponge was obtained by freeze-drying and stored at 4℃ in a dry, dark place.

[0087] (2) Dissolve the collagen sponge in deionized water to obtain a high-concentration collagen solution with a concentration of 10 mg / mL. Stir the solution at high speed for 1 min using a high-speed homogenizer to aid in uniform dissolution. Add tea tree oil to the collagen solution until the final mass concentration is 6%, and emulsify it at high speed for 2 min using a high-speed homogenizer at a speed of 15000 r / min to obtain a collagen-based Pickering emulsion.

[0088] (3) Inoculate the fungal strain (King Oyster Mushroom) onto PDA solid medium and statically culture it in a constant temperature and humidity incubator at 25℃ for 7 days. Then, autoclave the PDB liquid medium and inoculation tools (121℃, 30 min). Scrape the surface hyphae on the solid medium and implant them into the liquid medium. Static culture it in a constant temperature and humidity incubator at 27℃ for 10 days. Take out the intact and uncontaminated hyphae membrane and wash it three times with deionized water. Put it into an autoclave for inactivation treatment (121℃, 30 min). After drying at 60℃, store it in a dry and dark place at room temperature.

[0089] (4) Collagen Pickering emulsion was coated onto the mycelial membrane using a coating machine with a coating thickness of 2 mm. The emulsion was allowed to penetrate the mycelial membrane naturally and then dried naturally to obtain a biomass-based tea tree oil sustained-release patch (mycelium / collagen / tea tree oil composite membrane).

[0090] When using, cut the patch to size according to the affected area, drop 1-2 drops of water onto the lotion side of the patch with a dropper, and immediately apply the lotion side to the psoriasis affected area.

[0091] Example 8

[0092] A method for preparing a biomass-based tea tree oil sustained-release patch for the treatment of psoriasis includes the following steps:

[0093] (1) Fresh fish skin was washed and cut into small pieces. 0.1 mol / L acetic acid and 3% (w / v) pepsin were added at a material-to-liquid mass ratio of 1:50. After stirring at 4℃ for 48 h, the supernatant was collected by centrifugation at 10000 r / min. Sodium chloride was continuously added to the supernatant to precipitate collagen, with a final sodium chloride concentration of 0.7 mol / L. The collagen precipitate was dissolved in 0.1 mol / L acetic acid. After complete dissolution, the solution was dialyzed for at least 72 h. Finally, the purified collagen sponge was obtained by freeze-drying and stored at 4℃ in a dry, dark place.

[0094] (2) Dissolve the collagen sponge in deionized water to obtain a high-concentration collagen solution with a concentration of 10 mg / mL. Stir the solution at high speed for 1 min using a high-speed homogenizer to aid in uniform dissolution. Add tea tree oil to the collagen solution until the final mass concentration is 8%, and emulsify it at high speed for 2 min using a high-speed homogenizer at a speed of 15000 r / min to obtain a collagen-based Pickering emulsion.

[0095] (3) Inoculate the fungal strain (Ganoderma lucidum) onto PDA solid medium and statically culture it in a constant temperature and humidity incubator at 25℃ for 10 days. Then, autoclave the PDB liquid medium and inoculation tools (121℃, 30 min). Scrape the surface hyphae on the solid medium and implant them into the liquid medium. Static culture it in a constant temperature and humidity incubator at 27℃ for 14 days. Take out the intact and uncontaminated hyphal membrane and wash it with deionized water 5 times. Put it into an autoclave for inactivation treatment (121℃, 30 min). After drying at 45℃, store it in a dry and dark place at room temperature.

[0096] (4) Collagen Pickering emulsion was coated onto the mycelial membrane using a coating machine with a coating thickness of 1.5 mm, allowing the emulsion to naturally penetrate into the mycelial membrane and dry naturally to obtain a biomass-based tea tree oil sustained-release patch (mycelium / collagen / tea tree oil composite membrane).

[0097] When using, cut the patch to size according to the affected area, spray a small amount of water mist onto the lotion side of the patch with a small sprayer, and immediately apply the lotion side to the psoriasis affected area.

[0098] Example 9

[0099] Tea tree oil sustained-release curve of mycelium / collagen / tea tree oil composite membrane in PBS buffer solution

[0100] The mycelial membranes and composite membranes prepared in Examples 1, 4, and 7 were used to simulate the human physiological environment using PBS buffer solution (with 0.8% Tween-80 added to promote essential oil dissolution). Approximately 0.2g of sample (the mycelial / collagen / tea tree essential oil composite membrane prepared in Examples 1, 4, and 7) was weighed and placed in 100mL of PBS buffer solution. The solution was shaken at 37℃ and 100rpm / min. The absorbance (265nm) was measured at set time points. The drug release rate was calculated using the following formula:

[0101]

[0102] In the above formula, CTR represents the drug release rate; c t This represents the essential oil concentration, in mg / mL; v is the volume of PBS buffer, in mL; w t This is the amount of essential oil in the sample, in grams.

[0103] Figure 2 The study demonstrates the changes in tea tree oil release rates over time for systems containing different mass fractions of TTO (6%, 8%, and 10%). The results show that the release rates of all systems initially increased rapidly over time before stabilizing. The 6% TTO system exhibited a relatively faster release rate, reaching nearly 65% ​​at approximately 4 hours, followed by a slow increase and maintenance at around 70%. The 8% TTO system showed a slightly slower release process, with a release rate of approximately 55% at 4 hours, eventually stabilizing in the 55%–60% range. The 10% TTO system showed a slow initial release, with only about 30% at 4 hours, later maintaining a range of 25%–30%.

[0104] Example 10

[0105] Mechanical property characterization of mycelium / collagen / tea tree oil composite membrane

[0106] The mycelial membrane and composite membrane prepared in Example 4 were tested under dry and wet conditions (by spraying a small amount of water mist onto any side of the mycelial membrane or the emulsion surface of the composite membrane with a small sprayer, or by dripping 1-2 drops of water onto any side of the mycelial membrane or the emulsion surface of the composite membrane with a dropper).

[0107] Figure 3 a presents the stress-strain curves of different membrane materials (wet mycelium membrane, dry composite membrane, wet composite membrane, dry mycelium membrane). Figure 3 b represents the fracture energy data for the corresponding material. From the stress-strain curves, the dry composite membrane exhibits a rapid initial stress increase followed by quick fracture, with a strain of only 2%, demonstrating typical brittle fracture characteristics. The wet composite membrane, on the other hand, shows a continuous increase in stress with increasing strain, reaching nearly 10%, exhibiting superior deformation capacity. Comparing the dry and wet mycelial membranes, the wet mycelial membrane shows better strain range and stress bearing capacity in its stress-strain curve. The fracture energy diagram further corroborates this, showing a fracture energy of approximately 4.2 MJ / m² for the wet mycelial membrane. 3 The concentration of the mycelium is higher than that of the dry mycelium membrane (approximately 3.8 MJ / m). 3 The fracture energy of the wet composite membrane is approximately 10 MJ / m². 3 It is also far superior to dry composite membranes (approximately 2.5 MJ / m). 3 The toughness has increased by 400%. Figure 3 As shown in Figure c, the composite membrane cracked upon 180° twisting in a dry state. This indicates that the stress concentration within the material is difficult to effectively disperse during twisting, resulting in poor torsional resistance. In stark contrast, the composite membrane in a wet state remained relatively smooth upon 180° twisting, without significant damage; even after 360° twisting, only slight wrinkles appeared, and the overall structure remained intact. In summary, the combination of mycelial membrane and collagen Pickering emulsion significantly optimizes the mechanical properties in a wet state, demonstrating a prominent toughening effect. The excellent compliance and fracture energy resistance of the composite membrane provide mechanical performance support for its application in dynamic affected areas. Figure 3 As shown in Figure d, the composite film has no adhesion to the skin in a dry state. After wetting, the collagen Pickering emulsion surface of the composite film can adhere tightly to the skin, and the composite film does not fall off with limb movement. It can also deform in accordance with the movement of the limb, indicating that it has good adhesion ability.

[0108] Example 11

[0109] Animal experiments

[0110] The mycelial membrane and composite membrane prepared in Example 4 were used to induce psoriasis in mice by continuously applying 5% imiquimod (IMQ). Specifically, 6-8 week old Balb / c mice were anesthetized, and their backs were shaved. After removing hair with depilatory cream, the backs of the mice were cleaned, ensuring complete removal of hair. Before the experiment, a certain amount of IMQ ointment was weighed and placed on smooth membrane paper, then evenly applied to the backs of the mice. This was repeated every 24 hours for 7 consecutive days to successfully induce psoriasis-like skin lesions. On day 8, the material was applied to the backs of the mice to begin treatment. The mice were divided into a blank group, a control group, a MYC / COL (mycelial / collagen) treatment group, and a MYC / COL / TTO (mycelial / collagen / tea tree oil composite membrane) treatment group, with three replicates in each group. After treatment, the mice were euthanized, and the skin tissue in the psoriasis-inducing areas was carefully observed and excised. After fixation, sectioning, staining, and other steps, the tissue was observed under a microscope.

[0111] To investigate the therapeutic effect of the MYC / COL / TTO composite membrane on psoriasis in animals, the specific operational procedure is as follows: Figure 4 As shown in Figure a. After treatment, the therapeutic effects of the MYC / COL composite film and the MYC / COL / TTO composite film were assessed by observing and recording changes in skin lesions. (See Figure a.) Figure 4 As shown in b, after 5 days of treatment, compared with the control group without any treatment, the skin lesions in the MYC / COL / TTO treatment group were significantly improved. Specifically, the scaling was reduced and the abnormal proliferation of the skin surface was significantly alleviated. Compared with the control group, the scaling in the MYC / COL treatment group was slightly reduced, but the abnormal proliferation of the skin surface remained severe. The repair of psoriasis was assessed by H&E staining as follows: Figure 4 As shown in Figure c, the skin structure of the control group mice was intact, with uniform distribution of epidermal cells and clear layers. In contrast, the IMQ-induced control group showed significant epidermal thickening, dilation of the stratum spinosum, and downward extension of epidermal protrusions. Furthermore, the intercellular spaces in this group were significantly increased, and capillary proliferation and extensive inflammatory cell infiltration were observed in the superficial epidermis. Numerous incompletely keratinized cells were also visible in the stratum corneum. In comparison, the epidermal thickness of mice treated with MYC / COL and MYC / COL / TTO was significantly reduced, and capillary proliferation and inflammatory cell infiltration were significantly alleviated. The improvement was particularly pronounced in the MYC / COL / TTO treatment group. Figure 4As shown in Figure d, the Blank group (blank group) served as the baseline, with a relatively low skin thickness of 26 μm. The Control group (control group) showed significantly higher skin thickness than the Blank group, indicating that after IMQ treatment, the skin of the psoriasis model mice exhibited obvious pathological thickening characteristics. Analysis of the data from the MYC / COL group and the MYC / COL / TTO group revealed that the skin thickness of both was lower than that of the Control group, indicating that both MYC / COL and MYC / COL / TTO treatments could improve the skin thickening symptoms in psoriasis mice. Furthermore, the skin thickness of the MYC / COL / TTO group was significantly lower than that of the MYC / COL group, demonstrating the optimal therapeutic effect of the MYC / COL / TTO group on mice. In conclusion, the MYC / COL / TTO composite membrane showed the best performance in alleviating skin symptoms in psoriasis mice and can be considered a potentially effective strategy for the treatment of psoriasis.

Claims

1. A method for preparing a biomass-based tea tree oil sustained-release patch for the treatment of psoriasis, characterized in that, Includes the following steps: 1) Collagen extraction: Wash and chop fresh animal connective tissue raw materials, add acetic acid and pepsin, stir at 4 ℃ for 24~80 h, centrifuge to collect supernatant, and continuously add sodium chloride to supernatant to precipitate collagen solid precipitate. Dissolve collagen solid precipitate with acetic acid. After complete dissolution, dialyze the solution for 24~72 h, freeze dry the dialysate to obtain purified collagen sponge; 2) Preparation of collagen Pickering emulsion: Dissolve collagen sponge in water to obtain a collagen solution with a concentration of 5~10 mg / mL, and stir with a high-speed homogenizer to help uniform dissolution. Then add tea tree oil to the collagen solution and emulsify with a high-speed homogenizer to obtain collagen Pickering emulsion. 3) Culture of mycelial membrane: The fungal strain was inoculated on PDA solid medium and statically cultured in a constant temperature and humidity incubator at 25-30 ℃ for 5-10 days. Then, the surface mycelia on the PDA solid medium were scraped and implanted into PDB liquid medium and statically cultured in a constant temperature and humidity incubator at 25-30 ℃ for 7-20 days. The fully grown and uncontaminated mycelial membrane was taken out and then washed, inactivated and dried in sequence. 4) Preparation of mycelium / collagen emulsion composite membrane: Collagen Pickering emulsion is coated onto the mycelium membrane using a coating machine, allowing the collagen Pickering emulsion to naturally penetrate into the mycelium membrane. After natural drying, a biomass-based tea tree oil sustained-release patch is obtained.

2. The method for preparing a biomass-based tea tree oil sustained-release patch for the treatment of psoriasis according to claim 1, characterized in that, The animal connective tissue raw material mentioned in step 1) is cowhide or fish skin.

3. The method for preparing a biomass-based tea tree oil sustained-release patch for the treatment of psoriasis according to claim 1, characterized in that, In step 1), the acetic acid has a mass concentration of 0.1~0.7 mol / L, the mass ratio of animal connective tissue raw material to acetic acid is 1:20~1:60, the final concentration of pepsin is 1~3% (w / v), the final concentration of sodium chloride is 0.4~1 mol / L, the freeze-drying temperature is -40 ℃, and the freeze-drying time is 48~96 h.

4. The method for preparing a biomass-based tea tree oil sustained-release patch for the treatment of psoriasis according to claim 1, characterized in that, Step 2) involves adding tea tree oil to achieve a final mass concentration of 6-10%.

5. A method for preparing a biomass-based tea tree oil sustained-release patch for the treatment of psoriasis according to claim 1, characterized in that, Step 2) Use a high-speed homogenizer to assist in uniform dissolution at a speed of 6000~15000 r / min and a stirring time of 1~3 min. Use a high-speed homogenizer to emulsify at a speed of 5000~20000 r / min and an emulsification time of 2~5 min.

6. The method for preparing a biomass-based tea tree oil sustained-release patch for the treatment of psoriasis according to claim 1, characterized in that, The fungal species mentioned in step 3) are Ganoderma lucidum, oyster mushroom or king oyster mushroom.

7. A method for preparing a biomass-based tea tree oil sustained-release patch for the treatment of psoriasis according to claim 1, characterized in that, The coating thickness of the collagen Pickering emulsion in step 4) is 1~3 mm.

8. A biomass-based tea tree oil sustained-release patch for the treatment of psoriasis, prepared by the method according to any one of claims 1 to 7.

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