A cortex moutan extract, a preparation method and application thereof

Abstract

The present application relates to a cortex moutan extract and its extraction method and application. The present application innovatively adopts "liquid nitrogen deep cold brittle fracture pretreatment + cellulase directional degradation activation + short time high temperature controllable heat shock flash explosion + medium polarity glycol synergistic low temperature dissolution", establishes a cortex moutan physical extraction method, improves its purity and biological activity, including 37 kinds of chemical components, the cortex moutan extract has good anti-inflammatory activity in the in vitro cell model, can effectively relieve the Pso-like skin symptoms of mice induced by IMQ and the AD-like skin symptoms of mice induced by Mc903+OXA, and preliminarily its potential regulation mechanism for playing a role through the "gut-skin axis". The extract of the present application has unique advantages for transdermal administration and external preparation of the drug, increases the transdermal absorption of the drug, enhances the clinical curative effect, avoids the toxic and side effects of oral and injection administration, and solves the problems of complex components, unstable curative effect of compound oral decoction.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a peony bark extract, its preparation method, and its application. Background Technology

[0002] psoriasis( Psorasis, Pso Psoriasis, commonly known as cowhide disease, is a chronic, systemic inflammatory skin disease with a genetic predisposition. Globally, there are approximately 125 million patients, with over 10 million in my country. The prevalence is increasing annually, placing a heavy burden on patients' families and the social healthcare system. This disease is often accompanied by multiple comorbidities such as psoriatic arthritis, cardiovascular and cerebrovascular diseases, mental complications, chronic kidney disease, and inflammatory bowel disease, severely impacting quality of life. Current treatments primarily focus on short-term symptom control, with a high relapse rate or rebound after discontinuation of medication, representing a clinical bottleneck. First-line topical treatments include vitamin D derivatives, retinoids, and corticosteroids, which can improve skin lesions in the short term, but their efficacy is limited, with numerous adverse reactions, high relapse rates, and difficulty in long-term control. Among traditional systemic drugs, acitretin easily causes dry skin and mucous membranes, methotrexate mainly causes bone marrow suppression and hepatotoxicity, and cyclosporine can cause liver and kidney damage and gastrointestinal reactions. In recent years, biologics such as infliximab, adalimumab, and secukinumab have become important supplements, offering advantages such as good efficacy, rapid onset of action, high lesion clearance rate, fewer overall adverse reactions, and lower dosing frequency. However, they still face challenges such as increased risk of infection, serious adverse reactions, drug resistance in some patients, and high cost. Therefore, there is an urgent need to develop safe, effective, and affordable drugs for the treatment of psoriasis.

[0003] Atopic dermatitis ( Atopic dermatitis, AD Alzheimer's disease (AD) is a common chronic relapsing inflammatory skin disease. Its pathogenesis revolves around a Th2-mediated type 2 inflammatory response, involving factors such as genetics, immune abnormalities, skin barrier disruption, allergen stimulation, and scratching. The inflammation, intense itching, and recurrent flare-ups severely impact patients' lives. Current treatment strategies primarily include controlling inflammation, repairing the skin barrier, and modulating immunity and the skin microbiome. Topical corticosteroids (TCS) are currently the first-line treatment for acute AD, providing rapid anti-inflammatory and antipruritic effects. However, long-term use can lead to infection, skin atrophy, telangiectasia, pigmentation, steroid-dependent dermatitis, and rebound phenomena. Topical calcineurin inhibitors (TCIs) are non-hormonal immunomodulators that bidirectionally regulate immunity by inhibiting calcineurin and blocking the T-cell NFAT pathway. They can be used in combination with or sequentially with TCS for maintenance therapy. Local burning, stinging, and itching are common, and sun protection is necessary. Targeted drugs (such as the IL-4Rα inhibitor dupilumab, the IL-13 monoclonal antibody trorolu, and the JAK1 / 2 inhibitor baricitinib) target key inflammatory and pruritus pathways, but biological targeted agents are expensive and have adverse reactions such as injection site reactions and conjunctivitis. A small number of patients may experience severe allergic reactions, which limits their long-term clinical use.

[0004] Moutan bark is a traditional Chinese medicine, derived from the peony root (Paeonia suffruticosa), a plant in the Paeoniaceae family. Paeonia suffruticosa The dried root bark of *Pseudomonas aeruginosa* is bitter, pungent, and slightly cold in nature, and enters the heart, liver, and kidney meridians. It has the effects of clearing heat and cooling blood, promoting blood circulation and removing blood stasis. Currently, the treatment of psoriasis (Pso) and atopic dermatitis (AD) is limited to oral administration of compound decoctions. However, the components are complex, the active substances are unclear, and the amount of drug on the skin lesions after metabolism is limited, resulting in poor efficacy and easy damage to the gastrointestinal tract, liver, and kidneys. Furthermore, the quality of the medicinal materials is uneven, and the evaluation system is flawed. There is an urgent need to elucidate its pharmacodynamic material basis and mechanism, and to develop safe, effective, and convenient treatment methods. Summary of the Invention

[0005] To address the aforementioned technical problems, the present invention aims to provide a peony bark extract, its preparation method, and its applications. This invention innovatively employs a novel physical modification extraction process: "liquid nitrogen cryogenic brittle fracture pretreatment + cellulase-directed degradation and activation + short-duration high-temperature controllable thermal shock explosion + moderately polar diol synergistic low-temperature dissolution." Through precise temporal coupling of extreme low-temperature physical modification and short-duration controllable thermal shock explosion, a physical extraction method for peony bark is established, improving its purity and bioactivity. The extract contains 37 chemical components. In in vitro cell models, the peony bark extract exhibits good anti-inflammatory activity and can effectively alleviate IMQ-induced Pso-like skin symptoms in mice and Mc903+OXA-induced AD-like skin symptoms in mice.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: The first objective of this invention is to provide a method for extracting peony bark extract, comprising the following steps: (1) Select peony bark, and use low temperature and gentle pulverization to obtain pulverized peony bark powder. Spray liquid nitrogen onto the pulverized peony bark powder to quickly cool it to an ultra-low temperature cryogenic state and keep it warm. Then, let it naturally return to room temperature to obtain pretreated peony bark material. Add water to the pretreated peony bark material and mix it evenly. Add compound cellulase to carry out constant temperature enzymatic hydrolysis to obtain activated peony bark material slurry. Filter and collect solids. Repeat the above steps 2 to 3 times for the solids. (2) The activated peony bark slurry is placed in a sealed pressure-resistant extraction reactor, and the temperature is raised to 115℃~125℃ within 90s~110s, and the pressure is simultaneously increased to 0.25 MPa~0.5 MPa, maintained for 60s~90s, and then the pressure is instantly released to atmospheric pressure within 10s~15s to obtain the initial extract; (3) Add ethanol to the initial extract and perform low-temperature dissolution 1-2 times under low-speed stirring vortex conditions, and combine the extracts; cool the extract to room temperature and filter to obtain a clear extract; concentrate the clear extract under vacuum pressure and cool to stabilize to obtain a peony bark extract with a density of 1.10-1.15.

[0007] The beneficial effects of this invention are as follows: The peony bark extraction process of this invention specifically addresses the shortcomings of existing traditional peony bark decoction, conventional alcohol extraction, and heating reflux processes. It solves the technical bottlenecks of traditional processes, such as prolonged high-temperature extraction leading to easy thermal decomposition and volatilization of the heat-sensitive core active ingredient paeonol, conventional extraction resulting in a paeonol loss rate of over 30% and extremely low activity retention rate, intact essential oil chamber structure of peony bark thin-walled cells making it difficult to fully dissolve effective components, resulting in low extraction yield, long extraction time, low production efficiency, unstable content of active ingredients in the extract, and large batch-to-batch variations. This innovative process employs liquid nitrogen cryogenic physical cell disruption combined with enzymatic hydrolysis for gentle degradation pretreatment, ensuring no thermal damage throughout the entire process. It fully preserves the thermosensitive active structure of paeonol and extracts hyrcanoside, a component not found in other patents and literature reports. Combined with a short-time, controllable thermal shock flash explosion core innovation process, it precisely breaks down the essential oil chamber to release active ingredients. The extremely short high-temperature action time eliminates the problem of over 30% thermal degradation and volatilization loss of paeonol. The entire extraction, dissolution, and concentration process utilizes a low-temperature, short-time treatment mode, significantly improving the paeonol activity retention rate compared to traditional processes. The total extraction cycle is reduced to less than 45 minutes, significantly improving production efficiency. Using an ethanol-water green extraction medium, there are no toxic reagent residues, ensuring a green, environmentally friendly, safe, and compliant production process suitable for industrial-scale mass production. The resulting peony bark extract (MDP) has a high paeonol content, intact thermosensitive activity, and stable quality, exhibiting excellent compatibility for subsequent pharmaceutical formulations and external applications, possessing extremely high clinical pharmaceutical development and industrial promotion value.

[0008] Based on the above technical solution, the present invention can be further improved as follows.

[0009] Furthermore, the particle size of the low-temperature and mild pulverization treatment in step (1) is 20 mesh to 40 mesh, and the temperature is 10℃ to 25℃; The liquid nitrogen spraying rate is 1.5L / kg to 2.5L / kg, the rapid cooling time is 30s to 60s, the ultra-low temperature cryogenic state temperature is -120℃ to -150℃, and the heat preservation time is 3min to 5min.

[0010] The beneficial effects of adopting the above-mentioned further scheme are as follows: This invention utilizes the low-temperature penetration effect of liquid nitrogen to form an ice crystal expansion force in the gaps between the thin-walled cells and essential oil chamber cells of peony bark, gently inducing the formation of micro-cracks in the protective structure of the thin-walled cell walls and essential oil chambers of peony bark. The entire process is heat-free, with no volatilization or loss of paeonol, and does not damage the molecular structure of heat-sensitive active ingredients. After the cryogenic insulation is completed, the material naturally returns to room temperature, completing a single-stage cryogenic brittle fracture pretreatment specifically for peony bark, precisely destroying the cell wall protective barrier, while completely preserving the basic structure of heat-sensitive active substances such as paeonol. The particle size is controlled to 20-40 mesh, avoiding premature volatilization and loss of heat-sensitive paeonol caused by strong high-temperature pulverization.

[0011] Subsequently, by utilizing an extremely short period of high temperature and high pressure, the sealed structure of the essential oil chambers storing paeonol inside the peony bark is precisely destroyed, promoting the rapid and efficient dissolution of heat-sensitive paeonol. After the steady-state extraction is completed, the pressure is instantly released to atmospheric pressure, forming a violent cavitation shear turbulence effect, which further breaks down the thin-walled cells and residual essential oil chambers of the peony bark, completely releasing the residual active ingredients of paeonol in the cells, achieving efficient dissolution of heat-sensitive components with zero volatilization and zero degradation.

[0012] Furthermore, the mass ratio of the compound cellulase, the pretreated peony bark material, and the water in step (1) is 0.1~0.5:1:8~15.

[0013] Further, the complex cellulase in step (1) includes endocellulase, exocellulase, and β-glucosidase; the enzyme activity ratio of the endocellulase, the exocellulase, and the β-glucosidase is 1~2:2~4:1~2; The isothermal enzymatic hydrolysis time is 30 minutes and the temperature is 37°C.

[0014] The beneficial effects of adopting the above-mentioned further scheme are: constant temperature enzymatic hydrolysis softens and loosens the cell wall tissue structure, expands the microscopic permeation channels, enhances the extraction and permeation capacity of the subsequent extraction medium for the heat-sensitive active ingredient of paeonol in the cells, and ensures the uniformity and stability of the extract components; after alternating cycles of this step, multiple modifications and cell wall disruptions of peony bark thin-walled cells can be achieved, which not only improves the cell wall disruption effect, but also ensures low-temperature and gentle treatment throughout the process, and prevents the degradation and loss of heat-sensitive components.

[0015] The complex cellulase comprises endocellulase, exocellulase, and β-glucosidase, which work synergistically in an enzyme activity ratio of 1-2:2-4:1-2. The endocellulase randomly cleaves the inner layers of cellulose molecules. β -1,4 glycosidic bonds create new reducing ends; exonuclease cleaves the cellulose chain from the ends to produce cellobiose; β-glucosidase hydrolyzes cellobiose into glucose, simultaneously eliminating product feedback inhibition. These three enzymes have distinct functions and are indispensable, forming a complete cellulose degradation chain. Using any one enzyme alone or in combination of any two cannot achieve effective cell wall degradation: using an endonuclease alone leads to cellobiose accumulation and feedback inhibition; using an exonuclease alone results in extremely low degradation efficiency due to the lack of the reducing ends created by the endonuclease; using β-glucosidase alone cannot directly act on cellulose itself; when endonucleases and exonucleases are used in combination but β-glucosidase is lacking, cellobiose cannot be further degraded, which also inhibits enzyme activity.

[0016] This process utilizes liquid nitrogen cryogenic brittle fracture pretreatment to create microscopic cracks in the thin-walled cell walls and essential oil chamber protective structures of peony bark, providing preferential attack sites for the complex enzyme and significantly improving enzyme accessibility and degradation efficiency. After enzymatic hydrolysis, the cell wall structure becomes looser, and the microscopic permeability channels expand, allowing subsequent flash explosion treatment to more thoroughly disrupt the sealed structure of the essential oil chambers. The low-temperature dissolution medium also more easily penetrates into the cells, achieving efficient dissolution of heat-sensitive components such as paeonol. From liquid nitrogen cryogenic treatment (-120℃~-150℃) to isothermal enzymatic hydrolysis (37℃), then to low-temperature dissolution (20℃~30℃) and low-temperature concentration (≤45℃), the entire process forms a complete low-temperature protection chain, effectively avoiding the thermal degradation and volatilization loss of paeonol caused by traditional thermal extraction methods (60℃~100℃). Through a gradual cell wall disruption process coupled with physical-biological coupling, the dissolution rate of paeonol is significantly improved, the activity retention rate is greatly enhanced, and the extract components are uniform and stable with minimal batch-to-batch variation.

[0017] Furthermore, in step (3), the time for the low-speed stirring vortex is 10 min to 15 min, and the stirring speed is 120 rpm; the temperature for the low-temperature dissolution is; and the temperature for the vacuum decompression concentration is ≤45℃ and the vacuum degree is ≤-0.08MPa.

[0018] The beneficial effects of adopting the above-mentioned further scheme are: relying on the micro-crack structure of the early stage to achieve rapid low-temperature dissolution of heat-sensitive components, avoiding the oxidation and inactivation of paeonol caused by prolonged soaking; low-temperature dissolution can be repeated 1-2 times as needed, maximizing the total yield and activity retention of paeonol. Strict control of concentration temperature and vacuum degree, low-temperature low-negative-pressure concentration is carried out to prevent secondary volatilization and degradation of paeonol caused by high-temperature concentration; after concentration to a relative density of 1.10-1.15, and cooling to stabilize, a peony bark-specific extract with high heat-sensitive activity and high paeonol content is obtained.

[0019] The second objective of this invention is to provide a peony bark extract prepared by the aforementioned method, wherein the peony bark extract comprises cisplatin, paeoniflorin, 6-O-veratrol, paeonol, and paeonol.

[0020] The beneficial effects of this invention are as follows: The peony bark extract prepared by the extraction method of this invention yielded 37 chemical components through component identification, including previously unreported cetirizine. This method significantly improves the purity and bioactivity of the extract. Unlike traditional oral administration, the peony bark extract can directly act on the skin lesions, achieving local drug accumulation and rapid onset of action. It also avoids systemic side effects such as first-pass metabolism in the liver and gastrointestinal irritation, making it more suitable for the long-term topical medication needs of psoriasis patients. This invention elucidates that peony bark can inhibit the expression levels of RORC mRNA and key receptors in the IL-17 signaling pathway, thereby exerting an anti-inflammatory effect on Psoriasis. Simultaneously, it improves abnormal systemic immune responses by regulating immune cell subsets such as Th17 and corrects intestinal flora imbalance, utilizing the "gut-skin axis" to synergistically improve the treatment of Psoriasis. This invention is the first to develop peony bark extract into a topical preparation, filling the technological gap in the field of local treatment of psoriasis with this medicinal material. It is also easy to prepare various topical dosage forms such as gels, creams, and microemulsions, and has good industrialization prospects and clinical translation value.

[0021] A third object of the present invention is to provide a medicament for treating skin diseases, the medicament comprising the aforementioned peony bark extract.

[0022] The beneficial effects of the present invention are: the peony bark extract MDP of the present invention can achieve the following unexpected technical effects: (1) inhibiting the abnormal proliferation of HaCaT cells; (2) downregulating RORC mRNA expression and inhibiting the IL-17 inflammatory pathway; (3) regulating the balance of Th17, Treg, Th1, Th2 and Th22 immune cells and restoring local immune homeostasis; (4) improving IMQ-induced skin lesions, splenomegaly and intestinal dysfunction in mice; (5) regulating the structure of intestinal flora and affecting the bidirectional regulation of the "skin-gut axis".

[0023] Furthermore, the mass content of the peony bark extract in the drug is 5% to 15%.

[0024] Furthermore, the skin condition is atopic dermatitis and / or psoriasis.

[0025] Furthermore, the dosage form of the drug includes at least one of the following: aerosol, powder, solution, suspension, emulsion, lipid preparation, transdermal preparation, lyophilized powder for injection, tincture, face cream, lotion, and emulsion.

[0026] Furthermore, the extract of the present invention can also be used as a cosmetic ingredient in the preparation of cosmetics.

[0027] The beneficial effects of adopting the above-mentioned further solutions are: (1) Compared with existing oral decoctions, this invention uses a topical administration method, which has the following advantages that oral preparations do not have at all: it can form a high drug concentration at the lesion site, directly acting on the diseased tissue, with a more direct and precise effect; it avoids the digestion of the drug through the gastrointestinal tract and the first-pass effect, thereby reducing the metabolic inactivation of active ingredients and improving bioavailability; the drug hardly enters the systemic blood circulation, avoiding systemic distribution and related toxic side effects; it does not require long-term oral administration, significantly improving patient medication compliance, and is convenient to use, and can be applied locally at any time and as needed. The above advantages make this invention particularly suitable for skin-related diseases that require long-term management and local intervention, and it is fundamentally different from oral decoctions in terms of administration route, efficacy mechanism, and safety.

[0028] (2) The peony bark extract (MDP) extracted in this invention was systematically evaluated for its in vitro and in vivo pharmacological activity in a Pso-like inflammation model. In the in vitro study, the expression of inflammation-related mRNAs in an M5-induced HaCaT cell model was detected by RT-PCR. The results showed that RORC mRNA expression was significantly upregulated in the model group compared with the normal group, while MDP intervention significantly downregulated RORC mRNA expression compared with the model group. Immunofluorescence staining results showed that MDP could inhibit M5-induced IL-17RA protein expression, verifying its regulatory role in key inflammatory signaling pathways.

[0029] (3) In vivo studies showed that MDP effectively improved skin inflammation in mice with IMQ-induced Pso-like dermatitis, manifested as a decrease in PASI score, reduction in skin lesions, and a decrease in epidermal thickness. It also significantly repaired systemic immune abnormalities (splenomegaly) and intestinal barrier damage (colon shortening). H&E staining showed that, compared with the model group, MDP significantly reduced neutrophil infiltration and improved pathological changes such as epidermal structural disorder, tissue edema, and abnormal keratinization. Flow cytometry analysis of the spleen showed that the proportion of pro-inflammatory Th1, Th17, and Th22 cells in the skin immune microenvironment of the model group mice was significantly increased, while MDP intervention (250, 1250 mg / kg) significantly inhibited the differentiation of these pro-inflammatory cells. Simultaneously, the proportion of anti-inflammatory Treg and Th2 cells decreased in the model group, while the high-dose MDP group significantly increased their proportion. In summary, MDP can significantly inhibit IMQ-induced skin inflammation in mice by regulating immune cell balance through multiple targets. (4) No clinical efficacy data or patent literature on the use of peony bark alone in the treatment of atopic dermatitis and psoriasis has been found in the prior art. Although CN112741884A, CN102727826A, CN102335366A, CN112891416A, etc. disclose compositions containing peony bark, they are all in the form of compound oral preparations, and the active ingredients are extracted by traditional water decoction. The prescriptions are complicated, and there are problems such as obvious first-pass effect and poor patient compliance. None of them provide any pharmacodynamic experimental data on the treatment of psoriasis with peony bark alone. Therefore, it is impossible to measure the specific contribution of peony bark to the treatment of the disease, and it is impossible to confirm whether it constitutes an active ingredient with therapeutic effect. The present invention develops peony bark extract into a topical preparation that acts directly on the skin lesion site. It has good anti-inflammatory activity and can effectively relieve IMQ-induced Pso-like skin symptoms in mice and Mc903+OXA-induced AD-like skin symptoms in mice. It preliminarily explores the potential regulatory mechanism of its function through the "gut-skin axis". The extract of this invention is administered transdermally, and the topical formulation has unique advantages over the drug, increasing transdermal absorption, enhancing clinical efficacy, and avoiding the toxic side effects of oral and injectable administration. Attached Figure Description

[0030] Figures 1-2 These are the 37 chemical components of the peony bark extract in Example 5 of this invention; Figure 3 This is the extracted ion chromatogram at m / z 495.1508 in negative ion mode in Example 5 of the present invention; Figure 4 This is the second-order mass spectrum of m / z 495.1508 in negative ion mode in Example 5 of the present invention; Figure 5 In the negative ion mode of Embodiment 5 of the present invention, C 10 Schematic diagram of the pyrolysis pathway; Figure 6 This is the extracted ion chromatogram at m / z 505.1563 in negative ion mode in Example 5 of the present invention; Figure 7 This is the second-order mass spectrum of m / z 505.1563 in negative ion mode in Example 5 of the present invention; Figure 8 In the negative ion mode of Embodiment 5 of the present invention, C 14 Schematic diagram of the pyrolysis pathway; Figure 9 This is the extracted ion chromatogram at m / z 525.1614 in the negative ion mode of Example 5 of the present invention; Figure 10 This is a secondary mass spectrum of m / z 525.1614 in negative ion mode in Example 5 of the present invention; Figure 11In the negative ion mode of Embodiment 5 of the present invention, C 18 Schematic diagram of the pyrolysis pathway; Figure 12 This is the extracted ion chromatogram with m / z 629.1876 in negative ion mode in Example 5 of the present invention; Figure 13 This is a secondary mass spectrum of m / z 629.1876 in negative ion mode in Example 5 of the present invention; Figure 14 In the negative ion mode of Embodiment 5 of the present invention, C 57 Schematic diagram of the pyrolysis pathway; Figure 15 The results of cell experiments of the present invention are shown below; (a) is the chromatogram of the base peak of MDP extract in negative ion mode analyzed by UPLC-Q-TOF-MS technology; (b) is the effect of RT-PCR on RORC mRNA expression in M5-stimulated HaCaT cells; (c) is the fluorescence representative image of M5, Dex, and M5+MDP at 24 h detected by immunofluorescence; and (d) is the fluorescence intensity statistics of M5, Dex, and M5+MDP at 24 h. Figure 16 This is a representative image of the skin of each group of Pso mice induced by MDP and IMQ in Example 8 of the present invention; Figure 17 Examples 8 of this invention show the changes in various indicators during the establishment and treatment of Pso model mice; (a) is the PASI score of Pso mice induced by MDP on IMQ 10 days after administration, calculated by combining erythema, scaling, and hypertrophy scores; (b) is the change in skin thickness in each group during the establishment of IMQ-induced Pso mice; (c) is a representative image of the spleen of Pso mice induced by MDP on IMQ 10 days after administration; (d) is the spleen index of Pso mice induced by MDP on IMQ 10 days after administration; (e) is the colon length of Pso mice induced by MDP on IMQ 10 days after administration; and (f) is the colon index of Pso mice induced by MDP on IMQ 10 days after administration. Figure 18 This document describes the treatment of Pso model mice in Example 8 of the present invention; (a) is a representative H&E staining diagram of each group of Pso model mice on day 10 of the experiment; (b) is a schematic diagram of the maximum (E-Tmax) and minimum (E-Tmin) epidermal thickness of mice; (c) is a statistical graph of E-Tmax epidermal thickness of each group of Pso model mice on day 10 of the experiment after H&E staining; (d) is a statistical graph of E-Tmin epidermal thickness of each group of Pso model mice on day 10 of the experiment after H&E staining; and (e) is a statistical graph of neutrophils in each group of Pso model mice on day 10 of the experiment. Figure 19This diagram illustrates the regulatory effect of MDP on the immune microenvironment of Pso model mice in Example 8 of the present invention; where (a) represents different Th17 groups; (b) represents different Treg groups; (c) represents different Th1 groups; (d) represents different Th2 groups; (e) shows the proportion of different Th17 cell populations; (f) shows the proportion of different Treg cell populations; (g) shows the proportion of different Th1 cell populations; and (h) shows the proportion of different Th2 cell populations. Figure 20 This is a diagram illustrating the regulatory effect of MDP on the immune microenvironment of Pso model mice in Example 8 of the present invention; where (a) is a representative diagram of different Th22 groups; and (b) is the proportion of different Th22 cell populations. Figure 21 The effect of MDP on the gut microbiota of Pso mice in Example 9 of this invention α、β Diversity analysis; where (a) is the dilution curve; (b) is the Chao index; (c) is the Ace index; (d) is the Shannon index; (e) is the Sobs index; (f) is the PCoA analysis; and (g) is the NMDS analysis. Figure 22 This is an analysis of the effects of MDP on the intestinal flora composition and species differences in Pso mice in Example 9 of the present invention; (a) is a bar chart of community composition at the phylum level; (b) is a bar chart of community composition at the genus level; (c) is a bar chart of Kruskal-Wallis H test at the genus level; (d) is the abundance change of Bacteroides; and (e) is the abundance change of Prevotellaceae_UCG-001.

[0031] Figure 23 Example 9 of this invention shows the MDP analysis of LEfSe in the gut microbiota of Pso mice; where (a) is a LEfSe abundance difference graph; and (b) is an LDA bar chart. Figure 24 Pearson correlation analysis of gut microbiota and Pso biochemical indicators in Example 9 of this invention; Figure 25 The changes in various indicators of AD model mice under MDP during modeling and treatment in Example 10 of the present invention are shown; (a) is a representative map of dorsal skin lesions at the end of modeling; (b) is the ADI score of skin lesion severity; and (c) is the spleen index. Figure 26 This is a diagram showing the regulatory effect of MDP on the immune microenvironment of AD model mice in Example 10 of the present invention; where (a) is a representative diagram of different Th1 groups; (b) is a representative diagram of different Th2 groups; (c) is the proportion of different Th17 cell populations; (d) is the proportion of different Th1 cell populations; (e) is the proportion of different Th2 cell populations; and (f) is the proportion of different Th17 cell populations. Detailed Implementation

[0032] The principles and features of the present invention are described below. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0033] The experimental reagents and instruments are shown in Tables 1 and 2: Table 1 Experimental Reagents Table 2 Experimental Instruments and Consumables Example 1: (1) Select high-quality peony bark raw materials that are dry, clean, free from mold, and have the required content of main characteristic components (such as cisplatin, paeoniflorin, 6-O-veratrol, and paeonol J), and pulverize them using a CM-450B cryogenic pulverizer to obtain peony bark powder with a particle size of 30 mesh. Spread the pulverized peony bark powder evenly (8-12 mm thick) in a sealed, corrosion-resistant stainless steel extraction tray, and extract at a rate of 1 L / min·m 2 ~3L / min·m 2 The liquid nitrogen spraying rate and the liquid-to-material ratio of liquid nitrogen to pulverized peony bark powder were 3L:1kg. High-purity liquid nitrogen was sprayed at a uniform speed using a quantitative and precise spraying method, and the temperature drop rate was strictly controlled to ensure that the overall temperature of the peony bark material dropped to -135℃ in 45 seconds to an ultra-low temperature cryogenic state. This cryogenic constant temperature stable state was maintained for 4 minutes. After the cryogenic heat preservation was completed, the material naturally returned to room temperature, and the pretreated peony bark material was obtained. The pretreated peony bark material was put into an MJG series enzymatic hydrolysis tank, and purified water was added at a ratio of 8L~15L:1kg to make a uniform slurry. Then, a compound enzyme (endo-cellulase, exo-cellulase, β-glucosidase, and compound cellulase) was added at a ratio of 1~2:2~4:1~2. The enzymatic hydrolysis temperature was precisely controlled at 37℃ and the pH value was neutral. The enzymatic hydrolysis was carried out at a constant temperature for 30 minutes to obtain the activated peony bark material slurry. The above steps were repeated 3 times. (2) The activated peony bark slurry was quickly and sealed and transferred to a sealed pressure-resistant extraction reactor. The entire process was sealed with no volatilization loss. The heating and pressurization rate was precisely controlled. The temperature was rapidly increased to 120°C within 100s, and the pressure was simultaneously increased to 0.35 MPa. The short-term steady state was maintained for 75s. After extraction, the pressure was instantly released to atmospheric pressure in 13s to form a violent cavitation shear turbulence effect, which broke the thin-walled cells and residual essential oil chambers of peony bark in a secondary manner, and completely released the residual paeonol active ingredients in the cells, so as to achieve efficient dissolution of heat-sensitive components, zero volatilization, and zero degradation, and obtain the primary extract. (3) Add room temperature anhydrous ethanol to the primary extract to obtain a mixture. Adjust the volume fraction of ethanol in the mixture to 55 vol% as the diol aqueous solution extraction medium, which is suitable for the moderately polar solubility of paeonol. Strictly control the solid-liquid ratio of the primary extract to anhydrous ethanol to 1 g: 10 mL. Stir and vortex at 120 rpm for 13 minutes. Repeat the low-temperature dissolution twice. After extraction, cool the system to room temperature, filter to remove solid residue, collect and combine the clear extract. Transfer to a spherical vacuum concentrator. Strictly control the concentration temperature to ≤45℃ and the vacuum degree to ≤-0.08 MPa for low-temperature and low-pressure concentration. Concentrate to a relative density of 1.13. After cooling and stabilization, the peony bark extract MDP with high thermosensitive activity and high paeonol content is obtained.

[0034] Example 2: (1) Select high-quality peony bark raw materials that are dry, clean, free from mold, and have the required content of main characteristic components (such as cisplatin, paeoniflorin, 6-O-veratrol, and paeonol J) and pulverize them using a CM-450B cryogenic pulverizer to obtain peony bark powder with a particle size of 20 mesh. Spread the pulverized peony bark powder evenly in a sealed, corrosion-resistant stainless steel extraction tray and extract it at a rate of 1 L / min·m 2 ~3L / min·m 2 The liquid nitrogen spraying rate and the liquid-to-material ratio of liquid nitrogen to pulverized peony bark powder were 3L:1kg. High-purity liquid nitrogen was sprayed at a uniform speed using a quantitative and precise spraying method, and the temperature drop rate was strictly controlled to ensure that the overall temperature of the peony bark material dropped to -120℃ in 30 seconds to an ultra-low temperature cryogenic state. This cryogenic constant temperature stable state was maintained for 3 minutes. After the cryogenic heat preservation was completed, the material naturally returned to room temperature, and the pretreated peony bark material was obtained. The pretreated peony bark material was put into an MJG series enzymatic hydrolysis tank, and purified water was added at a ratio of 8L~15L:1kg to make a uniform slurry. Then, a compound enzyme (endo-cellulase, exo-cellulase, β-glucosidase, and compound cellulase) was added at a ratio of 1~2:2~4:1~2. The enzymatic hydrolysis temperature was precisely controlled at 37℃ and the pH value was neutral. The enzymatic hydrolysis was carried out at a constant temperature for 30 minutes to obtain the activated peony bark material slurry. The above steps were repeated twice. (2) The activated peony bark slurry was quickly and sealed and transferred to a sealed pressure-resistant extraction reactor. The entire process was sealed with no volatilization loss. The heating and pressurization rate was precisely controlled. The temperature was rapidly increased to 115°C within 90s, and the pressure was simultaneously increased to 0.25 MPa. The short-term steady state was maintained for 90s. After extraction, the pressure was instantly released to atmospheric pressure in 10s to form a violent cavitation shear turbulence effect, which broke the thin-walled cells and residual essential oil chambers of peony bark in a secondary manner, and completely released the residual paeonol active ingredients in the cells, so as to achieve efficient dissolution of heat-sensitive components, zero volatilization, and zero degradation, and obtain the primary extract. (3) Add room temperature anhydrous ethanol to the primary extract to obtain a mixture. Adjust the volume fraction of ethanol in the mixture to 40 vol% as the diol aqueous solution extraction medium, which is suitable for the moderately polar solubility of paeonol. Strictly control the solid-liquid ratio of the primary extract to anhydrous ethanol to 1 g: 8 mL. Stir and vortex at 120 rpm for 10 minutes. Repeat the low-temperature dissolution twice. After extraction, cool the system to room temperature, filter to remove solid residue, collect and combine the clear extract. Transfer to a spherical vacuum concentrator. Strictly control the concentration temperature to ≤45℃ and the vacuum degree to ≤-0.08 MPa for low-temperature and low-pressure concentration. Concentrate to a relative density of 1.10. After cooling and stabilization, the peony bark extract MDP with high thermosensitive activity and high paeonol content is obtained.

[0035] Example 3: (1) Select high-quality peony bark raw materials that are dry, clean, free from mold, and have the required content of main characteristic components (such as cisplatin, paeoniflorin, 6-O-veratrol, and paeonol J) and pulverize them using a CM-450B cryogenic pulverizer to obtain peony bark powder with a particle size of 40 mesh. Spread the pulverized peony bark powder evenly in a sealed, corrosion-resistant stainless steel extraction tray and extract it at a rate of 1 L / min·m 2 ~3L / min·m 2 The liquid nitrogen spraying rate and the liquid-to-material ratio of liquid nitrogen to pulverized peony bark powder were 3L:1kg. High-purity liquid nitrogen was sprayed at a uniform speed using a quantitative and precise spraying method, and the temperature drop rate was strictly controlled to ensure that the overall temperature of the peony bark material dropped to -150℃ in 60 seconds to an ultra-low temperature cryogenic state. This cryogenic constant temperature stable state was maintained for 5 minutes. After the cryogenic heat preservation was completed, the material naturally returned to room temperature, and the pretreated peony bark material was obtained. The pretreated peony bark material was put into an MJG series enzymatic hydrolysis tank, and purified water was added at a ratio of 8L~15L:1kg to make a uniform slurry. Then, a compound cellulase (endo-cellulase, exo-cellulase, β-glucosidase, and compound enzyme) was added at a ratio of 1~2:2~4:1~2. The enzymatic hydrolysis temperature was precisely controlled at 37℃ and the pH value was neutral. The enzymatic hydrolysis was carried out at a constant temperature for 30 minutes to obtain the activated peony bark material slurry. The above steps were repeated 3 times. (2) The activated peony bark slurry was quickly and sealed and transferred to a sealed pressure-resistant extraction reactor. The entire process was sealed with no volatilization loss. The heating and pressurization rate was precisely controlled. The temperature was rapidly increased to 125°C within 110s, and the pressure was simultaneously increased to 0.5 MPa. The short-term steady state was maintained for 60s. After extraction, the pressure was instantly released to atmospheric pressure in 15s to form a violent cavitation shear turbulence effect, which was used to break the thin-walled cells and residual essential oil chambers of peony bark in a secondary manner, and to completely release the residual paeonol active ingredients in the cells. This achieved efficient dissolution of heat-sensitive components, zero volatilization, and zero degradation, and the initial extract was obtained. (3) Add room temperature anhydrous ethanol to the primary extract to obtain a mixture. Prepare an extraction medium with an ethanol volume fraction of 70 vol% diol aqueous solution to match the moderately polar solubility of paeonol. Strictly control the solid-liquid ratio of the primary extract to anhydrous ethanol to 1 g: 12 mL. Stir and vortex at 120 rpm for 15 minutes. Repeat the low-temperature dissolution twice. After extraction, cool the system to room temperature, filter to remove solid residue, collect and combine the clear extract. Transfer to a spherical vacuum concentrator. Strictly control the concentration temperature ≤ 45℃ and vacuum degree ≤ -0.08 MPa for low-temperature and low-pressure concentration. Concentrate to a relative density of 1.15. After cooling and stabilization, a peony bark extract MDP with high thermosensitive activity and high paeonol content is obtained.

[0036] Example 4: The peony bark extract MDP from Example 1 was dissolved in 50% to 70% ethanol to prepare a clear liquid, which is a topical tincture with a peony bark extract MDP concentration of 5% to 15%.

[0037] The tincture in Example 4 has good external application effect, increases transdermal absorption of drugs, enhances clinical efficacy, avoids the toxic side effects of oral and injection administration, and solves the problems of complex composition and unstable efficacy of compound oral decoctions.

[0038] Comparative Example 1: Compared with Example 1, the only difference in this comparative example is that step (1) was not rapidly cooled to an ultra-low temperature cryogenic state, while the other steps and conditions were the same as in Example 1.

[0039] Comparative Example 2: The only difference between this comparative example and Example 1 is that step (1) was not performed with isothermal enzymatic hydrolysis, while the other steps and conditions are the same as in Example 1.

[0040] Comparative Example 3: Compared with Example 1, the only difference of this comparative example is that step (1) is omitted, while the other steps and conditions are the same as those of Example 1.

[0041] Comparative Example 4: The only difference between this comparative example and Example 1 is that step (2) is omitted, while the remaining steps and conditions are the same as in Example 1.

[0042] Compared to Comparative Examples 1-4, Examples 1-3 contain more heat-sensitive components while fully preserving the basic structure of heat-sensitive active substances such as paeonol; achieving efficient dissolution, zero volatilization, and zero degradation of heat-sensitive components. Isothermal enzymatic hydrolysis eliminates degradation and loss of heat-sensitive components. The extraction methods of Examples 1-3 form micro-cracks in the thin-walled cell walls and essential oil chamber protective structures of peony bark through liquid nitrogen cryogenic brittle fracture pretreatment, which greatly improves the accessibility of enzymes and degradation efficiency. After enzymatic hydrolysis, the cell wall structure is loose and the micro-permeability channels are expanded, and the subsequent flash explosion treatment can more thoroughly destroy the sealed structure of the essential oil chamber, achieving efficient dissolution of heat-sensitive components such as paeonol. However, Comparative Example 1 did not undergo rapid cooling to an ultra-low temperature cryogenic state, so micro-cracks could not be formed; step (1) of Comparative Example 2 did not undergo isothermal enzymatic hydrolysis, and the role of enzymes was mainly to dissolve more effective components; Comparative Examples 3 and 4 omitted steps (1) and (2) respectively, and could not effectively improve the dissolution rate of paeonol and significantly improve the activity retention rate.

[0043] Comparative Example 5: The only difference between this comparative example and Example 1 is that step (3) was not performed by low-temperature dissolution, while the other steps and conditions were the same as in Example 1.

[0044] Comparative Example 6: The only difference between this comparative example and Example 1 is that step (3) was not performed with vacuum decompression concentration, while the other steps and conditions were the same as in Example 1.

[0045] Compared to Comparative Examples 5 and 6, Examples 1-3 showed significantly improved total yield and activity of paeonol. Firstly, compared to Comparative Example 5, step (3) in Examples 1-3, combined with the pre-existing microcrack structure, enabled rapid low-temperature dissolution of the heat-sensitive component, avoiding prolonged soaking that could lead to oxidative failure of paeonol. Repeated low-temperature dissolution 1-2 times maximized the total yield and activity retention of paeonol. Furthermore, compared to Comparative Example 6, Examples 1-3 strictly controlled the concentration temperature and vacuum level, performing low-temperature, low-negative-pressure concentration to prevent secondary volatilization and degradation of paeonol caused by high-temperature concentration.

[0046] Comparative Example 7: (1) Select high-quality peony bark raw materials that are dry, clean, free from mold, and have the required content of main characteristic components (such as cisplatin, paeoniflorin, 6-O-veratrol, and paeonol J) and pulverize them using a CM-450B cryogenic pulverizer to obtain 10-mesh pulverized peony bark powder. Spread the pulverized peony bark powder evenly in a sealed, corrosion-resistant stainless steel extraction tray and extract it at a rate of 1 L / min·m 2 ~3L / min·m 2The liquid nitrogen spraying rate and the liquid-to-material ratio of liquid nitrogen to pulverized peony bark powder were 3L:1kg. High-purity liquid nitrogen was sprayed at a uniform speed using a quantitative and precise spraying method, and the temperature drop rate was strictly controlled to ensure that the overall temperature of the peony bark material dropped to -100℃ in 10 seconds to an ultra-low temperature cryogenic state. This cryogenic constant temperature stable state was maintained for 1 minute. After the cryogenic heat preservation was completed, the material naturally returned to room temperature, and the pretreated peony bark material was obtained. The pretreated peony bark material was put into an MJG series enzymatic hydrolysis tank, and purified water was added at a ratio of 8L~15L:1kg to make a uniform slurry. Then, a compound cellulase (endo-cellulase, exo-cellulase, β-glucosidase, and compound cellulase) was added at a ratio of 1~2:2~4:1~2. The enzymatic hydrolysis temperature was precisely controlled at 40℃ and the pH value was neutral. The enzymatic hydrolysis was carried out at a constant temperature for 15 minutes to obtain the activated peony bark material slurry. The above steps were repeated twice. (2) The activated peony bark slurry was quickly and sealed and transferred to a sealed pressure-resistant extraction reactor. The entire process was sealed with no volatilization loss. The heating and pressurization rate was precisely controlled. The temperature was rapidly increased to 90°C within 70 seconds, and the pressure was simultaneously increased to 0.20 MPa. The short-term steady state was maintained for 80 seconds. After extraction, the pressure was instantly released to atmospheric pressure in 8 seconds to obtain the initial extract. (3) Add room temperature anhydrous ethanol to the primary extract to prepare an extraction medium of 30 vol% diol aqueous solution of ethanol, which is suitable for the moderate polarity solubility of paeonol; strictly control the solid-liquid ratio of primary extract to anhydrous ethanol to 1 g: 6 mL, stir and vortex at 100 rpm for 10 minutes, repeat the low temperature dissolution twice, after extraction, cool the system to room temperature, filter to remove solid residue, collect and combine the clear extract; transfer to a spherical vacuum concentrator, strictly control the concentration temperature ≤45℃ and vacuum degree ≤-0.08MPa for low temperature and low negative pressure concentration, concentrate to a relative density of 1.10, and after cooling and stabilization, the peony bark extract MDP with high thermosensitive activity and high paeonol content is obtained.

[0047] Comparative Example 8: (1) Select high-quality peony bark raw materials that are dry, clean, free from mold, and have the required content of main characteristic components (such as cisplatin, paeoniflorin, 6-O-veratrol, and paeonol J) and pulverize them using a CM-450B cryogenic pulverizer to obtain 80-mesh pulverized peony bark powder. The pulverized peony bark powder is then evenly spread in a sealed, corrosion-resistant stainless steel extraction tray, and extracted at a rate of 1 L / min·m 2 ~3L / min·m 2The liquid nitrogen spraying rate and the liquid-to-material ratio of liquid nitrogen to pulverized peony bark powder were 3L:1kg. High-purity liquid nitrogen was sprayed at a uniform speed using a quantitative and precise spraying method, and the temperature drop rate was strictly controlled to ensure that the overall temperature of the peony bark material dropped to -150℃ in 100 seconds to an ultra-low temperature cryogenic state. This cryogenic constant temperature stable state was maintained for 10 minutes. After the cryogenic heat preservation was completed, the material naturally returned to room temperature, and the pretreated peony bark material was obtained. The pretreated peony bark material was put into an MJG series enzymatic hydrolysis tank, and purified water was added at a ratio of 8L~15L:1kg to make a uniform slurry. Then, a compound cellulase (endo-cellulase, exo-cellulase, β-glucosidase, and compound cellulase) was added at a ratio of 1~2:2~4:1~2. The enzymatic hydrolysis temperature was precisely controlled at 37℃ and the pH value was neutral. The enzymatic hydrolysis was carried out at a constant temperature for 45 minutes to obtain an activated peony bark slurry. The above steps were repeated 3 times. (2) The activated peony bark slurry was quickly and sealed and transferred to a sealed pressure-resistant extraction reactor. The entire process was sealed with no volatilization loss. The heating and pressurization rate was precisely controlled. The temperature was rapidly increased to 130°C within 200s, and the pressure was simultaneously increased to 0.7 MPa. The short-term steady state was maintained for 80s. After extraction, the pressure was instantly released to atmospheric pressure in 25s to obtain the initial extract. (3) Add room temperature anhydrous ethanol to the primary extract to prepare an 80 vol% diol aqueous solution extraction medium, which is suitable for the moderately polar solubility of paeonol; strictly control the solid-liquid ratio of the primary extract to anhydrous ethanol to 1 g: 20 mL, stir and vortex at 120 rpm for 20 minutes, repeat the low-temperature dissolution twice, cool the system to room temperature after extraction, filter to remove solid residue, collect and combine the clear extract; transfer to a spherical vacuum concentrator, strictly control the concentration temperature ≤ 55℃ and vacuum degree ≤ -0.01 MPa for concentration, concentrate to a relative density of 1.15, cool and stabilize to obtain paeonol extract MDP.

[0048] Compared to Comparative Examples 7 and 8, Examples 1-3 showed significantly improved total yield and activity of paeonol. This is because Examples 1-3, from liquid nitrogen cryogenics to isothermal enzymatic hydrolysis, followed by low-temperature dissolution and concentration, formed a complete low-temperature protection chain of ≤45℃, effectively avoiding thermal degradation and volatilization loss of paeonol caused by high temperatures. Therefore, compared to Comparative Examples 7 and 8, Examples 1-3, through a gradual cell-wall disruption process involving physical-biological coupling, significantly improved the dissolution rate of paeonol, greatly enhanced the activity retention rate, and resulted in uniform and stable extract components with minimal batch-to-batch variation. Furthermore, the extracts of Examples 1-3 of this invention not only contain conventionally disclosed extractable components but also previously unreported components such as cicatricialoside.

[0049] Comparative Example 9: Peony bark was ground into powder and passed through a No. 3 sieve to obtain fine powder. Approximately 2 g of the fine powder was accurately weighed and placed in a stoppered conical flask. 100 mL of 70% methanol was accurately added and weighed. The mixture was ultrasonically treated at 240 W at 40 kHz for 30 min, cooled, and weighed again. The weight loss was made up with 70% methanol, shaken well, filtered, and the filtrate was filtered through a 0.22 μm microporous membrane to obtain the peony bark extract.

[0050] Comparative Example 10: Peony bark was ground into powder and passed through a No. 3 sieve to obtain fine powder. Approximately 2 g of the fine powder was accurately weighed and placed in a stoppered conical flask. 100 mL of 70% ethanol was accurately added and weighed. The mixture was ultrasonically treated at 240 W at 40 kHz for 30 min, cooled, and weighed again. The weight loss was made up with 70% ethanol, shaken well, and filtered. The filtrate was then filtered through a 0.22 μm microporous membrane to obtain the peony bark extract.

[0051] Compared with the four traditional extraction methods reported in Wang Li, Hou Shixiang, Hu Ping, et al. (2005) "Study on the Optimal Extraction Process of Peony Bark": alcohol extraction, distillation and decoction, supercritical CO2 fluid extraction, and traditional decoction (total extract 7.28%~13.51%, paeonol 0.31%~2.45%), as shown in Table 3, the new physical modification extraction process of this invention, which uses "liquid nitrogen cryogenic brittle fracture + cellulase directional degradation + short-time high-temperature heat shock explosion + low-temperature dissolution of moderately polar diol", increases the total extract yield to 20%~30% and the paeonol yield to 4%~6% through liquid nitrogen brittle fracture of cell walls, cellulase hydrolysis to release intracellular components, heat shock explosion to disintegrate tissues, and low-temperature selective dissolution of 55% ethanol. This is significantly improved compared with the best method in the literature. At the same time, it avoids the volatilization loss of paeonol and the problem of organic solvent residue caused by high-temperature heating, demonstrating the high efficiency of the process of this invention.

[0052] Table 3 Example 5: (1) The peony bark extract of Example 1 was analyzed using a Waters UPLC-Q-TOF-MS liquid chromatography-mass spectrometry system under the following conditions: Adopting ACQUITY UPLCTMBEH C 18A chromatographic column (100 mm × 2.1 mm, 1.8 μm) was used; the mobile phase was 0.1% formic acid-water (A) / acetonitrile (B), with gradient elution (0–26 min, 95–83% A; 26–40 min, 83–82% A; 40–44 min, 82–73% A; 44–50 min, 73–65% A; 50–56 min, 65–60% A; 56–61 min, 60–40% A; 61–62 min, 40–10% A; 62–64 min, 10–95% A); the flow rate was 0.3 mL / min; the column temperature was 40 ℃; and the injection volume was 2.0 μL.

[0053] The SCIEX Triple-TOF 6600+ mass spectrometry system employed an electrospray ionization (ESI) source in negative ion mode. Mass spectrometry conditions were as follows: acquisition range 50–1200 °C; nebulizer gas (Gas1): 344.74 kPa; heating gas (Gas2): 344.74 kPa; curtain gas (CUR): 241.32 kPa; temperature 550 °C; source ejection voltage (ISVF): 5500 V for positive ion mode and -4500 V for negative ion mode; declustering voltage (DP): ±80 V; collision energy (CE): 40 ± 20 eV; accumulation time 100 ms. Information-dependent acquisition (IDA) was performed: MS2 acquisition of the 10 strongest response peaks exceeding 50 cps, with dynamic background subtraction (DBS) enabled. Automatic calibration (CDS) was used throughout the experiment. Drying gas (N2) pressure: 100 kPa.

[0054] (2) The chemical components of the peony bark extract prepared in Example 1 were analyzed using UPLC-Q-TOF-MS technology, and the collected data were processed using MsssLynx 4.2 software. The negative ion mode mass spectrometry data were analyzed, and the precise molecular mass was determined based on the measured quasi-molecular ion peaks. The molecular composition was calculated using mass spectrometry analysis software, and the theoretical and measured values ​​were compared. Combined with relevant literature data, fragment ions in the secondary mass spectrometry of the compounds were analyzed, and the structure of the compounds was inferred by searching SciFinder. A total of 37 chemical components were finally identified. Among them, oxypaeoniflorin (C64- ... 10 C 23 H 27 O 12 ), 6-O-Veratroylcatalpol (6-O-veratrol, C 18 C 24 H 29 O 13 Paeonoside J (Paeonoside J, C)57 C 31 H 33 O 14 Unreported ingredients include Hyrcanoside (Cycasoside, C...). 14 C 21 H 29 O 14 )wait.

[0055] The base peak chromatogram of peony bark extract in negative ion mode is shown below. Figure 15 (a) The liquid chromatography-mass spectrometry (LC-MS) data and identification results for each component are shown in Table 4. Figures 3-14 The specific structures of each component are shown in the figure. Figures 1-2 .

[0056] Table 4. Liquid chromatography-mass spectrometry data and identification results of compounds in Paeonia suffruticosa under negative ion mode. Table 3 Figures 3-14 It can be seen that the extracts of this invention contain highly responsive components such as paeoniflorin, 6-O-veratrol, and paeonol J. In addition, the MDP of this invention also contains cisanoside, a component not found in other patents and literature reports. Studies have found that cisanoside has immunomodulatory and antitumor effects (Rimpelová S, Zimmermann T, Drašar PB, et al. Steroid Glycosides Hyrcanoside and Deglucohyrcanoside:On Isolation, Structural Identification, and Anticancer Activity [J]. Foods, 2021, 10(1).), and paeoniflorin has anti-inflammatory and immunomodulatory effects (Wang H, Yu W, Wang T, et al. Therapeutic potential and pharmacological insights of total glucosides of paeony in dermatologic diseases: a comprehensive review [J]. Front Pharmacol, 2024, 15: 1423717.).

[0057] Example 6: RT-PCR determination of mRNA expression of relevant cellular inflammatory factors HaCaT cells in the logarithmic growth phase were harvested and their concentration adjusted to 2 × 10⁻⁶ cells / year. 5 Cells were seeded at 2 mL / well in 6-well plates and cultured for 24 h. Cells were divided into normal control group, model group, positive control group, and MDP administration groups at different concentrations. The normal control group was given complete medium containing 10% FBS and 1% P / S; the model group was given complete medium containing 2.5 ng / mL M5; the positive control group was given 2.5 μg / mL dexamethasone (Dex) in addition to M5 stimulation; and the MDP administration groups were given M5 stimulation with final concentrations of 50, 100, and 200 μg / mL MDP, respectively. After another 24 h of culture, total RNA was extracted from each group of cells according to the mRNA extraction kit instructions, and the mRNA expression levels of inflammation-related cytokines were detected by RT-PCR. Results are shown below. Figure 15 As shown in (b): The result is Figure 15 (b) We can obtain: RT-PCR results showed that the expression level of RORC mRNA in the model group was significantly increased compared with the normal group, confirming the successful construction of the M5-induced HaCaT cell inflammation model. After intervention with MDP (50, 100, 200 μg / mL), the expression level of RORC mRNA was significantly downregulated, indicating that the MDP extract has good anti-inflammatory activity in the in vitro cell model.

[0058] Example 7: Immunofluorescence observation of intracellular fluorescent expression of protein IL-17RA HaCaT cells in logarithmic growth phase were digested with 0.25% Trypsin-EDTA, centrifuged, and resuspended. The cells were then treated with 1×10⁻⁶... 5 Cells were seeded at a density of [number] cells / mL in 96-well plates and cultured for 24 h. According to the experimental design, cells were divided into a normal group, a model group, a Dex-positive control group, and an MDP-treated group. The normal group was given complete medium containing 10% FBS and 1% P / S; the model group was given medium containing 2.5 ng / mL M5; the Dex-positive control group and the MDP-treated group were co-incubated with 2.5 μg / mL Dex or 100 μg / mL MDP (Example 1), respectively, in addition to M5 stimulation. After 24 h of culture, the supernatant was discarded, and the cells were washed with TBST, fixed with 4% paraformaldehyde, treated with a permeabilizer, and blocked with 1.5% BSA. Cells were then incubated sequentially with Anti-IL-17RA primary antibody (4°C overnight) and Alexa Fluor 568-labeled secondary antibody (room temperature, protected from light, for 1 h) to label the target protein, and the nuclei were counterstained with DAPI. Finally, the fluorescence intensity of each group was analyzed using a high-content imaging system to evaluate the effect of the drug on IL-17RA protein expression.

[0059] The results are as follows Figure 15 (c) Figure 15 As shown in (d): The results showed that intervention with 100 μg / mL MDP significantly inhibited the fluorescence intensity of IL-17RA in M5-induced HaCaT cells. This indicates that MDP can effectively downregulate the expression of key receptors in the IL-17 signaling pathway, providing direct visual evidence at the protein level for MDP intervention in IL-17-mediated inflammatory responses.

[0060] Example 8: MDP effectively alleviates IMQ-induced inflammation in Pso mice (1) Pso mouse model construction: Healthy female Bablc mice weighing (20±2) g and aged 7-8 weeks (purchased from the Institute of Medical Biology, Chinese Academy of Medical Sciences) were selected and acclimatized for 1 week. One day before the experiment, the mice were anesthetized by intraperitoneal injection, and hair was removed from their backs (2cm×3cm) with depilatory cream. Seven mice were set up in each group: normal group, model group, Dex positive drug group, low-dose (250 mg / kg) and high-dose (1250 mg / kg) MDP group. 62.5 mg of 5% IMQ cream was applied topically daily, and the corresponding drugs were applied according to the group for 10 consecutive days. PASI scores were also performed. The scoring criteria were based on the Pso Area and Severity Index (PASI) objective scoring system, from 0 to 4 (0 points for none, 1 point for mild, 2 points for moderate, 3 points for severe, and 4 points for very severe). Three different parameters (i.e., erythema, scaling, and thickness) were scored independently, and then summed to obtain the total severity score throughout the treatment process.

[0061] (2) After the experiment, the mice were sacrificed on day 10, and various samples were immediately collected for further analysis. The collected samples included back skin, colon tissue, feces, whole blood, and spleen for further experiments.

[0062] The result is Figures 16-18 We can obtain: (1) MDP significantly improved IMQ-induced Pso-like skin lesions in mice. During modeling and administration, the skin on the back was photographed every other day. Results showed no significant changes in the skin of the normal group mice; the model group showed obvious white scales, erythema, and raised inflammatory infiltration, presenting typical Pso-like lesions. Compared with the model group, the back lesions of mice in each MDP dose group showed varying degrees of improvement on day 10, manifested as reduced erythema, decreased scales, and a smoother, more even epidermis. Figure 16 (a) indicates that MDP can effectively alleviate IMQ-induced Pso-like skin symptoms in mice.

[0063] (3) The PASI score was used to evaluate the therapeutic effect of MDP on IMQ-induced Pso mice. The results showed that from day 7 after modeling, compared with the model group, the PASI scores of all MDP dose groups (250, 1250 mg / kg) showed a decreasing trend, with the high-dose group showing a more significant decrease. Figure 17 (a)). Measurements of back skin thickness showed that, starting from day 7, the skin thickness in each MDP dose group continuously decreased compared to the model group, indicating that MDP can effectively inhibit IMQ-induced epidermal hyperplasia. Figure 17 (b)).

[0064] MDP also has a regulatory effect on Pso-related systemic and intestinal pathologies. Spleen index data showed that the spleen was significantly enlarged in the model group, and high doses of MDP could significantly reduce its size. Figure 17 (c) and (d). Colon length analysis showed that the colon in the model group was significantly shortened, while the colon length in each MDP dose group was significantly restored. Figure 17 (e) and (f) suggest that MDP has a repair effect on Pso-related intestinal structural damage.

[0065] (4) The effect of MDP on the amelioration of skin lesions on the back of psoriasis model mice. H&E staining was performed on skin tissue from mice in each group. The results showed that the epidermis of the normal group was thinner, structurally intact, and without subcutaneous hemorrhage or significant inflammatory cell infiltration. The model group showed dyskeratosis, subcutaneous hemorrhage, epidermal thickening, extensive inflammatory cell infiltration, and a nail-like appearance of the epidermis, indicating successful modeling. The MDP group showed significant improvement in epidermal structure, tissue edema, and keratinization abnormalities, and a significant reduction in neutrophil infiltration, such as... Figure 18 As shown in (a).

[0066] Epidermal thickness statistics showed that the model group was significantly thicker than the normal group, while the high-dose MDP group was significantly thinner. Figure 18 As shown in (b), (c), and (d), dermal neutrophil counts showed a significant increase in the model group compared to the normal group, and a significant decrease in the high-dose MDP group. Figure 18 (e) This indicates that MDP can effectively improve skin pathological damage and reduce local inflammation in Pso model mice.

[0067] (6) MDP has a regulatory effect on the immune microenvironment. The results showed that compared with the normal group, the proportions of pro-inflammatory Th17, Th1, and Th22 cells in the spleen of mice in the model group were significantly increased. After MDP intervention, the proportions of the above-mentioned pro-inflammatory immune cell subsets were significantly reduced. Figure 19 (a), (c), (e), (g), Figure 20 (a), (b). Meanwhile, the proportions of anti-inflammatory Treg and Th2 cells in the model group showed a decreasing trend, while high-dose MDP intervention significantly increased the proportions of Treg and Th2 cells. Figure 19(b), (d), (f), (h). The above immunomodulatory effects are highly consistent with the improvement of skin pathological damage and clinical symptoms (reduced PASI score, thinned skin, restored colon length, etc.) by MDP, suggesting that MDP can regulate the balance of immune cells through multiple targets and inhibit IMQ-induced skin inflammatory response in mice.

[0068] Example 9: The effect of MDP on the gut-skin axis (1) Fecal samples from the normal group, model group, and high-dose MDP group were selected for 16S rRNA sequencing. Total genomic DNA of the microbial community was extracted using the FastPure Stool DNA Isolation Kit. The integrity was detected by 1% agarose gel electrophoresis, and the concentration and purity were determined by NanoDrop2000. PCR amplification was performed using barcode-specific primers, and the products were purified by agarose gel electrophoresis. Libraries were constructed using the NEXTFLEX® Rapid DNA-Seq Kit, quantified by Synergy HTX, and finally sequenced on the Illumina NextSeq 2000 PE300 platform.

[0069] (2) All data analyses were conducted on the MajorBio Cloud Platform (https: / / cloud.majorbio.com). The α-diversity index (Chao, Shannon, etc.) was calculated using Mothur software, and the Wilcoxon rank-sum test was used to analyze inter-group differences in β-diversity. Principal coordinate analysis (PCoA) was performed based on Bray-Curtis distance, and PERMANOVA was used to assess the significance of differences in community structure between groups. LEfSe analysis (linear discriminant analysis of effect size) was used to screen for bacterial communities with significant differences in abundance from the phylum to the genus level. Finally, Pearson correlation analysis was used to assess the correlation between key differentially expressed bacterial communities and psoriasis (Pso) disease indicators.

[0070] The result is Figures 21-24 We can obtain: (1) MDP increases the species richness of gut microbiota and improves its structural composition. Through comprehensive comparative analysis, this invention selected a normal group, a model group, and a high-dose MDP group for fecal intestinal flora analysis. Alpha diversity analysis showed that the dilution curve tended to flatten, confirming sufficient sequencing depth. The Ace, Shannon, Chao, and Sobs indices in the model group were significantly lower than those in the normal group, suggesting that Pso can lead to a significant decrease in intestinal flora diversity and abundance; after MDP treatment, all indices showed an upward trend, indicating that MDP can partially reverse the Pso-induced intestinal microecological imbalance, such as... Figure 21(a), 21 (b), 21 (c), 21 (d), 21 (e). In the β-diversity analysis, PCoA and NMDS results showed that the normal group, model group, and MDP group each exhibited significant clustering and inter-group differentiation; the MDP group was distributed between the normal group and the model group and was closer to the normal group, suggesting that MDP can partially correct the Pso-induced dysbiosis of the gut microbiota structure. Figure 21 As shown in (f) and 21(g).

[0071] (2) To clarify the effect of MDP on the composition of fecal microbiota, analyses were conducted at the phylum and genus levels. At the phylum level, Bacteroides (46.9%) was the dominant phylum in the normal group, while Firmicutes (73.6%) was the dominant phylum in the model group. After MDP intervention, Bacillota decreased to 63.5%, while Bacteroidota increased to 14.5%, effectively reversing the phylum-level dysbiosis. At the genus level, Ligilactobacillus (18.8%), Muribaculaceae (14.0%), and Bacteroides (14.3%) were enriched in the normal group; in the model group, the three decreased to 11.5%, 7.2%, and 0.5%, respectively, while Lactobacillus abnormally increased to 21.8%. After MDP treatment, the abundance of the three core bacteria increased, and Lactobacillus decreased to 1.2%. Figure 22 As shown in (a) and 22(b), differential bacterial analysis revealed that the abundance of beneficial bacteria (Bacteroides, Odoribacter, Prevotellaceae_UCG-001, Alistipes) was significantly reduced in the model group, while the abundance of pathogenic bacteria Staphylococcus was significantly increased. MDP could inhibit Staphylococcus proliferation, increase the abundance of the aforementioned beneficial bacteria, and effectively reverse the Pso-related bacterial community disorder, such as... Figure 22(c), 22(d), 22(e) are shown. (3) LEfSe multi-level species discrimination analysis (LDA>3) further clarified the key biomarkers of each group: at the phylum level, the normal group is centered on Bacteroidota, and the model group is characterized by Bacillota; at the family level, the normal group is mainly composed of Prevotellaceae, the model group is represented by Erysipelotrichaceae, etc., and the MDP group is mainly composed of Ruminococaceae and Sutterellaceae; at the genus level, the normal group is marked by Bacteroides, Prevotellaceae_UCG-001, Odoribacter, etc., the model group is characterized by Staphylococcus as the key pathogen, and the MDP group is characterized by Turicimonas as the key differential fecal flora, such as Figure 23 (a) Figure 23 As shown in (b).

[0072] (4) To explore the association between key differentially expressed bacterial groups and Pso indicators, Pearson analysis was performed on the top 15 differentially expressed bacterial genera. The results showed that *Odoribacter*, *Prevotellaceae_UCG-001*, and *Bacteroides* were negatively correlated with PASI score, spleen index, pro-inflammatory factors such as IL-17A, and the Th1 / Th17 / Th22 cell ratio, and positively correlated with colon length and the Th2 cell ratio. *Lactobacillus* showed the opposite correlation with the above indicators. This indicates that these bacterial groups are closely related to psoriasis immune parameters and may play an important role in the treatment of MDP, such as... Figure 24 As shown in (a).

[0073] Example 10: Establishment of a mouse model of atopic dermatitis (AD) and evaluation of the effect of MDP in treating AD Fifteen healthy (hFcRn / TNF-α) female mice weighing (20±2) g and aged 6-8 weeks were selected. One day before the experiment, the mice were anesthetized by intraperitoneal injection, and hair was removed from the backs of the mice (2cm×3cm) with hair removal cream. The mice were randomly divided into four groups: normal group (3 mice), model group (4 mice), Dex positive drug group (4 mice), and MDP group (4 mice).

[0074] Mc903 80 μmol / L was prepared from 100% ethanol; 5% and 0.1% OXA (4:1 acetone:olite) were also used. In the model group, 80 μL of Mc903 was applied topically to the skin on the back of mice daily for 15 consecutive days. Six hours after Mc903 administration, OXA (4:1 acetone:olite) was applied again: 200 μL of 5% OXA on day 1, and 200 μL of 0.1% OXA every other day from day 8 onwards, for a total of 15 days, to establish the AD mouse model. At the end of the experiment, mice were sacrificed, and the skin, spleen, and other major organs on the back of the mice were harvested. The remaining tissues were rapidly frozen in liquid nitrogen and stored at -80°C.

[0075] After establishing the Dex-positive drug group and MDP group, mice in each group were given Mc903+OXA topically for half an hour each day, followed by the application of the corresponding drug (MDP 500mg / kg or Dex 30mg) twice a day for 15 consecutive days.

[0076] Skin lesion severity scoring: The inflammatory phenotype of mice was assessed using the Atopic Dermatitis Index (ADI). Three different parameters (erythema, erosion, scaling, and edema) were independently scored from 0 to 3 (0 for none, 1 for mild, 2 for moderate, and 3 for severe). The sum of these scores was then used as the dermatitis index, as shown in Table 7. The weight of each mouse was recorded daily, and the skin on the back was photographed every three days. After euthanizing the mice, the spleen was extracted, weighed, and the changes in the proportion of immune cell subsets were analyzed by flow cytometry.

[0077] The results are as follows Figure 25 , 26 As shown: (1) No significant changes were observed in the back skin of mice in the normal group throughout the experiment. Compared with the normal group, the backs of mice were photographed every 3 days during the establishment of the AD model. Compared with the normal group, the backs of mice in the model group showed obvious scaling, erythema, edema, epidermal peeling, and splenomegaly. Compared with the model group, the back skin lesions of the MDP group were improved at the end of the experiment, with reduced erythema, reduced scaling, thinning of the epidermis, and smaller spleen. The ADI score results showed that from the 10th day of modeling, the ADI score of the model group gradually increased compared with the normal group, reaching a peak on the 13th day. Compared with the model group, the ADI score of the MDP group showed a decreasing trend. The above results indicate that the MDP group can effectively alleviate the AD-like skin symptoms induced by Mc903+OXA in mice.

[0078] (2) The results showed that, compared with the normal group, the proportion of Th1 and Th17 cells was significantly increased and the proportion of Th2 cells was significantly decreased in the AD group, indicating that the model was successfully established. Compared with the model group, the proportion of Th1 and Th17 cells was significantly decreased and the proportion of Th2 cells was significantly increased in the MDP group. This suggests that MDP can inhibit the skin inflammatory response induced by Mc903+OXA in AD mice by regulating the balance of immune cells.

[0079] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for extracting peony bark extract, characterized in that, Includes the following steps: (1) Select peony bark, and use low temperature and gentle pulverization to obtain pulverized peony bark powder. Spray liquid nitrogen onto the pulverized peony bark powder to quickly cool it to an ultra-low temperature cryogenic state and keep it warm. Then, let it naturally return to room temperature to obtain pretreated peony bark material. Add water to the pretreated peony bark material and mix it evenly. Add compound cellulase to carry out constant temperature enzymatic hydrolysis to obtain activated peony bark material slurry. Filter and collect solids. Repeat the above steps 2 to 3 times for the solids. (2) The activated peony bark slurry is placed in a sealed pressure-resistant extraction reactor, and the temperature is raised to 115℃~125℃ within 90s~110s, and the pressure is simultaneously increased to 0.25 MPa~0.5 MPa, maintained for 60s~90s, and then the pressure is instantly released to atmospheric pressure within 10s~15s to obtain the initial extract; (3) Add ethanol to the initial extract and perform low-temperature dissolution 1-2 times under low-speed stirring vortex conditions, and combine the extracts; cool the extract to room temperature and filter to obtain a clear extract; concentrate the clear extract under vacuum pressure and cool to stabilize to obtain a peony bark extract with a density of 1.10-1.

15.

2. The method for extracting peony bark extract according to claim 1, characterized in that, The particle size of the low-temperature and mild pulverization treatment in step (1) is 20 mesh to 40 mesh, and the temperature is 10℃ to 25℃; The liquid nitrogen spraying rate is 1.5L / kg to 2.5L / kg, the rapid cooling time is 30s to 60s, the ultra-low temperature cryogenic state temperature is -120℃ to -150℃, and the heat preservation time is 3min to 5min.

3. The method for extracting peony bark extract according to claim 1, characterized in that, The mass ratio of the compound cellulase, the pretreated peony bark material, and the water in step (1) is 0.1~0.5:1:8~15.

4. The method for extracting peony bark extract according to claim 2, characterized in that, The complex cellulase mentioned in step (1) includes endocellulase, exocellulase, and β-glucosidase; the enzyme activity ratio of the endocellulase, exocellulase, and β-glucosidase is 1~2:2~4:1~2; The isothermal enzymatic hydrolysis time is 30 minutes and the temperature is 37°C.

5. The method for extracting peony bark extract according to claim 1, characterized in that, In step (3), the low-speed stirring vortex time is 10 min to 15 min, and the stirring speed is 120 rpm; the low-temperature dissolution temperature is 20℃ to 30℃; and the vacuum concentration temperature is ≤45℃ and the vacuum degree is ≤-0.08MPa.

6. A peony bark extract, characterized in that, The peony bark extract is prepared by the preparation method according to any one of claims 1 to 5, and the peony bark extract includes cisplatin, paeoniflorin, 6-O-veratrol, paeonol, and paeonol.

7. A medicine for treating skin diseases, characterized in that, The drug includes the peony bark extract as described in claim 6.

8. The medicament for treating skin diseases according to claim 7, characterized in that, The mass content of the peony bark extract in the drug is 5% to 15%.

9. A medicament for treating skin diseases according to claim 8, characterized in that, The skin condition is atopic dermatitis and / or psoriasis.

10. A medicament for treating skin diseases according to any one of claims 7 to 9, characterized in that, The dosage form of the drug is a topical dosage form, including at least one of aerosol, powder, solution, suspension, emulsion, lipid preparation, transdermal preparation, lyophilized powder injection, tincture, face cream, lotion, and emulsion.

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