A hamelia opposita extract composition, and a preparation method and application thereof

Abstract

This invention relates to the field of traditional Chinese medicine extracts and drug preparation technology, and discloses a *Hypericum perforatum* extract composition, its preparation method, and its application, including the application of an ethanol extract of *Hypericum perforatum* or components isolated from its ethanol extract in the preparation of drugs for the prevention and / or treatment of psoriasis. In vitro and in vivo experiments have demonstrated that *Hypericum perforatum* extract can significantly improve imiquimod (IMQ)-induced psoriasis-like skin lesions (erythema, scaling, and skin thickening) in mice and reduce PASI scores. Its mechanism of action differs from single-target biological agents; instead, it simultaneously regulates the IL-23 / IL-17 inflammatory axis, corrects Th17 / Treg immune imbalance, and inhibits abnormal activation of the NF-κB signaling pathway, demonstrating the advantages of multi-component, multi-target synergistic effects of natural drugs. Furthermore, *Hypericum perforatum* is a unique plant resource in my country with a wide distribution. Compared to expensive biological agents, this invention has a significant cost advantage and is expected to be developed into a more affordable and accessible anti-psoriasis drug.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine extracts and drug preparation technology, specifically to a *Hypericum perforatum* extract composition, its preparation method, and its application. Background Technology

[0002] Psoriasis is a globally prevalent chronic, relapsing, immune-mediated inflammatory skin disease with a global prevalence of approximately 2%-3%. Its pathogenesis is complex, primarily related to abnormal activation of the IL-23 / IL-17 inflammatory axis, disruption of inflammatory signaling pathways such as NF-κB, and Th17 / Treg immune imbalance. The main pathological features are abnormal proliferation and differentiation of keratinocytes and inflammatory cell infiltration. This disease not only causes symptoms such as erythema, scaling, itching, and plaques, severely impacting skin appearance and quality of life, but can also lead to various complications such as arthritis and cardiovascular disease. Long-term, recurrent flare-ups cause immense physical and psychological suffering, and the disease's protracted course and difficulty in complete cure pose serious threats to patients' physical and mental health. Therefore, developing safe, effective, and long-term usable psoriasis treatments is of significant clinical and social value in improving patient prognosis and reducing the disease burden.

[0003] In current technologies, the treatment of psoriasis is mainly divided into two categories: traditional treatment and targeted therapy. The core focus is on "inhibiting the inflammatory response, regulating immune balance, and inhibiting abnormal proliferation of keratinocytes," attempting to alleviate symptoms and reduce recurrence. Firstly, traditional treatments primarily use drugs such as glucocorticoids and methotrexate, which exert their therapeutic effect by inhibiting the inflammatory response and cell proliferation; this is a widely used basic treatment regimen in clinical practice. Secondly, novel targeted therapies, centered on biological agents such as IL-17 inhibitors and IL-23 inhibitors, precisely target the key inflammatory axis in the pathogenesis of psoriasis, offering significant efficacy and rapid onset of action, and have gradually become an important choice for treating moderate to severe psoriasis in recent years. Thirdly, adjuvant therapies, including topical moisturizers and ultraviolet radiation, are used to relieve dry skin and itching symptoms, help control inflammation, and reduce drug side effects.

[0004] Golden-threaded half-maple lotus ( Semiliquidambar cathayensisChang is a medicinal plant unique to my country. In traditional folk applications, it is mainly used to "dispel wind and dampness, unblock meridians and relieve pain". Modern pharmacological research has further confirmed that it contains a variety of active ingredients with clear anti-inflammatory, analgesic and antioxidant biological activities. It has good potential to be developed into an anti-inflammatory and immunomodulatory drug, providing a new candidate direction for the treatment of psoriasis. However, existing technologies for the application of *Hypericum japonicum* and the treatment of psoriasis still have many significant shortcomings, making it difficult to meet clinical needs: First, existing psoriasis treatments have inherent limitations. Traditional drugs have significant side effects, are prone to drug resistance with long-term use, and have a high relapse rate. New targeted biological agents are expensive, some patients do not respond well, and long-term use may increase the risk of infection. Furthermore, there is a lack of treatment drugs that are abundant in source, have high safety, and regulate multiple targets. Second, the medicinal value of *Hypericum japonicum* has not been fully developed. Currently, there are no research reports on the use of *Hypericum japonicum* for the prevention and treatment of psoriasis, and it is unclear whether it can exert a therapeutic effect by regulating key immune-inflammatory pathways in psoriasis, such as IL-23 / IL-17. Third, the pharmacodynamic material basis of *Hypericum japonicum* is vague. The core active ingredients that exert its anti-inflammatory and immunomodulatory effects have not been identified, and there is a lack of efficient extraction and composition preparation methods, making it impossible to transform its medicinal potential into a clinical treatment for psoriasis. As a result, the resource value of this unique medicinal plant has not been fully explored. In summary, existing technologies cannot overcome the bottleneck in psoriasis treatment, nor can they fully explore the new medicinal uses of *Hypericum chinense*. There is an urgent need for an extract composition of *Hypericum chinense* and its preparation method, to clarify its anti-psoriasis activity and mechanism of action, and to provide a new safe and effective solution for the treatment of psoriasis. Summary of the Invention

[0005] The present invention aims to provide a *Hypericum perforatum* extract composition, its preparation method and application, in order to solve the technical problem of poor efficacy of existing technologies and traditional Chinese medicine preparations in the treatment of psoriasis.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: the application of a *Hymenochloa crus-galli* extract composition in the preparation of a drug for the prevention and / or treatment of psoriasis, wherein the *Hymenochloa crus-galli* extract composition is an alcoholic extract of *Hymenochloa crus-galli*.

[0007] Preferably, as an improvement, the drug comprises a unique active ingredient, a witch hazel extract composition, and pharmaceutically acceptable excipients.

[0008] Preferably, as an improvement, this solution also provides a method for preparing a *Hypericum perforatum* extract composition, comprising the following steps: Step S1: Take the roots or stems of *Ligustrum lucidum* and crush them; Step S2: Add 8 times the volume of 0-95% ethanol, and extract by heating and reflux 1-3 times; the preferred ethanol concentration is 30-95%. Step S3: Combine the extracts and concentrate under reduced pressure to obtain total extract of Hamamelis macrantha SC-Z; The resulting total extract is used as a *Hypericum perforatum* extract composition for the above-mentioned applications.

[0009] Preferably, as an improvement, the method further includes step S4, loading the total extract obtained from 60% ethanol onto an HP-20 macroporous resin column and performing gradient elution with an ethanol-water system (0%, 30%, 50%, 95%), collecting each fraction separately to obtain four fractions of different polarities: SC-W, SC-30, SC-50, and SC-95; using the SC-W, SC-30, SC-50, and SC-95 as the extract composition of *Hypericum esculentum* for the above-mentioned applications.

[0010] Preferably, as an improvement, this solution also provides a *Hamamelis japonica* extract composition, comprising the total extract prepared by the above method, or any one of SC-W, SC-30, SC-50 and SC-95 prepared by the above method, or any two or three combinations of SC-W, SC-30, SC-50 and SC-95 prepared by the above method; the *Hamamelis japonica* extract composition is used in the above applications.

[0011] Preferably, as an improvement, the *Hypericum perforatum* extract composition is a total extract or SC-95.

[0012] Preferably, as an improvement, the *Hypericum perforatum* extract composition is a total extract, wherein the total extract contains 10%-20% total polyphenols, 10%-30% total flavonoids, and 10%-30% total triterpenes.

[0013] The principles and advantages of this scheme are: 1. Clear Efficacy and Multi-Target Regulation: This invention demonstrates through in vitro and in vivo experiments that *Hypericum perforatum* extract can significantly improve imiquimod (IMQ)-induced psoriasis-like skin lesions (erythema, scaling, and skin thickening) in mice and reduce PASI scores. Its mechanism of action differs from single-target biological agents; it simultaneously regulates the IL-23 / IL-17 inflammatory axis, corrects Th17 / Treg immune imbalance, and inhibits abnormal activation of the NF-κB signaling pathway, showcasing the advantages of multi-component, multi-target synergistic effects of natural drugs.

[0014] 2. Good safety: In animal experiments, the present invention did not show any significant adverse effects on the liver and kidney function (ALT, AST, Cre, Urea) of mice at effective doses, and could even alleviate the model-related transaminase elevation, showing potential safety superior to traditional chemotherapy drugs (such as methotrexate).

[0015] 3. Clear Material Basis: This invention identified 85 chemical components in the extract of *Hypericum perforatum* using UHPLC-Q / TOF-MS technology, and determined Piceid and Piceid-6''- using chemical and biological methods. O -gallate, tarennanoside B, and oleanolic acid are its active ingredients for treating psoriasis, providing a scientific basis for subsequent drug development and quality control.

[0016] 4. Lower cost: Golden winged schefflera is a plant resource unique to my country and widely distributed. Compared with expensive biological agents, this invention has a significant cost advantage and is expected to be developed into an anti-psoriasis drug with a more affordable price and higher accessibility. Attached Figure Description

[0017] Figure 1 This is an HPLC chromatogram of the separation of the extract of *Hypericum perforatum* in an embodiment of the present invention.

[0018] Figure 2 The results show the safety of four semi-maple extracts obtained by different extraction methods in Experiment Example 1 of this invention.

[0019] Figure 3 The results of anti-inflammatory experiments on four extracts of *Liriope muscari* obtained by different extraction methods in Experiment Example 1 of this invention are shown.

[0020] Figure 4 The effect of different polarity sites of *Ficus pumila* on the viability of RAW264.7 cells in Experimental Example 2 of this invention (n=3).

[0021] Figure 5 The effect of *Hypericum perforatum* extract and different parts on NO release from RAW264.7 cells in Experimental Example 2 of this invention (n = 3).

[0022] Figure 6 The extracts of *Hypericum perforatum* and different parts thereof in Experimental Example 2 of this invention have effects on cellular inflammatory factors (n = 3).

[0023] Figure 7 The effect of *Hypericum perforatum* extract on IMQ-induced psoriatic dermatitis in mice (n = 6) in Experiment Example 3 of this invention (A, morphological image of the back of the mouse on day 7; B, erythema score; C, thickness score; D, desquamation score; E, total PASI score; values ​​are calculated as Means ± SEM; ### p<0.001 VS control group; p<0.001, p<0.05 (VSmodel group).

[0024] Figure 8 The effect of *Hypericum perforatum* extract on body weight and spleen changes in IMQ-induced psoriasis-like mice in Experiment Example 3 of this invention (n = 6) (A, Body weight change graph of psoriasis mice; B, Spleen index of psoriasis mice; C, Spleen morphology graph of psoriasis mice; Values ​​are calculated as Means ± SEM; ###p<0.001 VS control group; p<0.001, p < 0.01 (VS model group).

[0025] Figure 9 The effect of the extract of *Hypericum perforatum* on the pathological changes of the skin lesions on the back and spleen tissue of psoriasis-like mice in Experiment Example 3 of this invention (A, back skin section of mice; B, spleen section of mice).

[0026] Figure 10 The effect of *Hypericum perforatum* ethanol extract on peripheral blood leukocyte subsets and NLR in IMQ-induced psoriasis mice in Experiment Example 3 of this invention (n = 6) (A. Bar chart of absolute neutrophil count in peripheral blood of mice in each group; B. Bar chart of absolute lymphocyte count in peripheral blood of mice in each group; C. Bar chart of NLR in peripheral blood of mice in each group; values ​​are calculated as Means ± SEM; ###p<0.001 VS control group; p<0.001 VS model group.

[0027] Figure 11 The effects of *Hypericum perforatum* extract on liver and kidney function indicators in IMQ-induced psoriasis mice in Experiment Example 3 of this invention (n = 3) (A. Effect of liver function ALT; B. Effect of liver function AST; C. Effect of kidney function Cre; D. Effect of kidney function Urea; values ​​are calculated as Means ± SEM).

[0028] Figure 12 The effect of *Symplocos henryi* on the expression of inflammation-related cytokines in psoriasis-like mice in Experiment Example 3 of this invention (n = 6) (A, IL-17A release; B, IL-23 release; C, IL-22 release; values ​​are calculated as Means ± SEM; ###p<0.001 VS control group; p<0.001 VS model group.

[0029] Figure 13 The effect of *Hypericum perforatum* extract on CD4+ in the spleen of psoriasis-like mice in Experiment Example 3 of this invention. + The effect of T cell proportion (n = 4) (A. Flow cytometry gating analysis plot; B. Spleen CD4 count in each group) + T cell proportion statistics; values ​​are calculated as mean ± SEM; ###p<0.001 VS control group; p<0.001, p<0.01, p<0.05 (VSmodel group).

[0030] Figure 14 The effect of the ethanol extract of *Hypericum perforatum* on the Th17 / Treg balance in the spleen of psoriasis-like mice in Experiment Example 3 of this invention (n = 4) (A, IL-17A) + CD4 + T cell (Th17) flow cytometry scatter plot; B, CD25 + FoxP3 + T cell (Treg) flow cytometry scatter plot; C, IL-17A + CD4 + T cell count statistics; D, CD25 + FoxP3 + T cell count statistics; E and Th17 / Treg ratio statistics. Values ​​are calculated as mean ± SEM; ###p<0.001 VS control group; p<0.001, p<0.05 VS model group.

[0031] Figure 15This invention relates to the effect of *Hypericum perforatum* extract on the expression of NF-κB signaling pathway-related proteins in skin lesions of psoriasis-like mice in Experiment Example 3 (n = 3). The results included: (A) Western blot analysis of p-p65, p65, p-IκBα, and IκBα protein expression levels in skin lesions of mice in each group, with β-actin as an internal reference; (B) Quantitative analysis of p-p65 / p65 grayscale values; (C) Quantitative analysis of p-IκBα / IκBα grayscale values; values ​​are calculated as Means ± SEM; ###p<0.001, ##p<0.01 vs control group;) p<0.001, p<0.01, p<0.05 vs model group.

[0032] Figure 16 The BPI diagrams (A, BPI) of the *Liriope muscari* extract under positive and negative ion modes in Experimental Example 4 of this invention are shown. + Positive ion mode; B, BPI - (Negative ion mode).

[0033] Figure 17 This is the standard curve for the determination of total polyphenols, total flavonoids and total triterpenoids in Experimental Example 4 of the present invention.

[0034] Figure 18 The effect of monomeric compounds derived from the extract of *Hypericum perforatum* on LPS-induced RAW264.7 cells in Experimental Example 5 of this invention (n = 3) (values ​​are calculated as Means ± SEM; ###P<0.001 VS control group; P<0.001, P < 0.01 (VS model group). Detailed Implementation

[0035] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used can all be obtained commercially.

[0036] Example 1 This solution provides a method for preparing a *Hypericum perforatum* extract composition, comprising the following steps: Step S1: Take 800g of roots and stems of *Liriope muscari* and crush them.

[0037] Step S2: Add 8 times the volume of 60% ethanol, heat and reflux to extract twice, 2 hours each time.

[0038] Step S3: Combine the two extracts and concentrate under reduced pressure (rotary evaporator) to obtain SC-Z, a total extract of 60% ethanol from *Hypericum esculentum* (i.e., the effective fraction extract, totaling 1423.1 g).

[0039] The total extract can be used directly or further suspended in pure water for animal gavage experiments.

[0040] Step S4, Separation: To further enrich the active ingredients, the total extract can be loaded onto an HP-20 macroporous resin column and eluted with an ethanol-water system (0%, 30%, 50%, 95%). Each fraction is collected separately, yielding four fractions of different polarities: SC-W, SC-30, SC-50, and SC-95. The HPLC chromatograms of each fraction are shown below. Figure 1 As shown in Table 1, the yields of the four extracts were calculated based on the total ethanol extract.

[0041] Table 1. Yields of the four extracts

[0042] This solution also provides a *Hamamelis japonica* extract, prepared by the above method, comprising total extract or SC-95%.

[0043] This solution also provides the application of *Hypericum perforatum* extract in the preparation of a drug for treating psoriasis.

[0044] Experiment Example 1: Experiments on Ethanol Extracts of Different Concentrations and Their Safety (1) Experimental method for extract preparation: The *Hylocereus undatus* herb was pulverized into powder. Four portions of the powder, each 200 g, were placed in round-bottom flasks. Eight times the volume (v / w) of water, 30% ethanol, 60% ethanol, and 95% ethanol were added, and the mixtures were heated under reflux for two times, each time for 2 hours. The filtrates from each extraction were combined and concentrated under reduced pressure using a rotary evaporator to obtain the corresponding extracts, named W-SC, 30%-SC, 60%-SC, and 95%-SC, respectively. The yields of W-SC, 30%-SC, 60%-SC, and 95%-SC were 4.6±1.2%, 6.3±1.3%, 6.5±1.5%, and 2.1±1.2%, respectively.

[0045] (2) The steps of the safety test are as follows: RAW264.7 cells were seeded in 96-well plates, and different concentrations of witch hazel extract were added. After culturing for 24 h, CCK-8 reagent was added, and absorbance at 450 nm was measured to calculate cell viability.

[0046] The results are as follows Figure 2 As shown, none of the four extracts obtained from ethanol extraction at concentrations ≤100 μg / mL exhibited significant cytotoxicity. Therefore, based on the aforementioned safety results, the total extract obtained from 60% ethanol extraction was selected for subsequent activity experiments.

[0047] (3) Anti-inflammatory experiment like Figure 3 As shown in the NO results, all four extracts of *Hypericum perforatum* exhibited anti-inflammatory activity in the concentration range of 50–100 μg / mL.

[0048] To further evaluate the inhibitory effects of the four extracts on inflammatory factors, PCR was used for detection. The results showed that all four extracts exhibited varying degrees of inhibition of inflammatory factor activity.

[0049] Experimental Example 2: Screening Model and Method for In Vitro Anti-inflammatory Activity Model: Lipopolysaccharide (LPS) induced inflammation model of mouse RAW264.7 macrophages.

[0050] method: (1) Cytotoxicity assay (CCK-8 assay): RAW264.7 cells were seeded in 96-well plates and different concentrations (3.125-200 μg / mL) of *Hydrangea macrophylla* extract or different parts were added. After culturing for 24 h, CCK-8 reagent was added and absorbance at 450 nm was measured to calculate cell viability.

[0051] The results are as follows Figure 4 As shown in Table 2, considering the cell viability results and the requirements for subsequent activity evaluation, this protocol selects 12.5-50.0 μg / mL as the drug concentration for subsequent in vitro anti-inflammatory activity screening of each fraction.

[0052] Table 2. Effects of different polarity fractions of *Leymus chinensis* on the survival rate of RAW264.7 cells.

[0053] (2) NO release detection (Griess method): Cells were seeded in 96-well plates and divided into a control group, an LPS model group (0.5 μg / mL), a drug administration group (LPS + different concentrations of samples), and a positive control group (LPS + hydrocortisone). After culturing for 24 h, the supernatant was collected, Griess reagent was added, and the absorbance at 540 nm was measured.

[0054] The results are as follows Figure 5 As shown in Table 3, the extracts and individual fractions of *Hypericum perforatum* significantly inhibited LPS-induced NO release, with the total extract showing an inhibition rate of 32.5% and the 95% ethanol fraction showing an inhibition rate of 92.2%. This indicates that the anti-inflammatory effect of *Hypericum perforatum* extract is not dominated by a single fraction, but rather by the synergistic effect of multiple components from multiple fractions. The overall activity of the total extract (SC-Z) was superior to any single fraction, demonstrating good anti-inflammatory potential.

[0055] Table 3. Effects of extracts and different fractions of *Hypericum perforatum* on NO release from RAW264.7 cells.

[0056] (3) Detection of mRNA expression of inflammatory factors (q-PCR method): Cells were seeded in 6-well plates, grouped as above, and total RNA was extracted after 24 h of treatment. After reverse transcription, q-PCR was performed. Primers included IL-6, TNF-α, and IL-1β.

[0057] The results are as follows Figure 6 As shown in Table 4, the extract of *Hypericum perforatum* can significantly downregulate the mRNA expression levels of pro-inflammatory factors such as IL-6 and IL-1β.

[0058] Table 4. Effects of different polarity sites on the relative expression of inflammatory factors induced by LPS in RAW264.7 cells.

[0059] Experimental Example 3: In vivo evaluation model and method for anti-psoriasis drug efficacy Model: Psoriasis-like dermatitis model in C57BL / 6J mice induced by 5% imiquimod (IMQ).

[0060] (1) Animal grouping: 60 female C57 mice were randomly divided into 6 groups (n=10): blank control group (Control), model group (Model), low, medium and high dose groups of witch hazel extract (25, 50, 100 mg / kg), and positive drug methotrexate group (MTX, 1.3 mg / kg).

[0061] (2) Administration regimen: After hair removal on the backs of mice, except for the blank control group, the other groups were treated with 62.5 mg of 5% IMQ cream daily for 7 consecutive days. At the same time, the treatment groups were administered the corresponding dose of the drug by gavage daily, while the control group and the model group were administered an equal volume of pure water by gavage.

[0062] (3) Evaluation Indicators and Results: Skin Lesion Appearance and PASI Score: Erythema, scaling, and thickness of the back skin were observed and recorded daily, and PASI scores were assigned from 0 to 4. Results are as follows: Figure 7As shown, compared with the model group, the severity of skin lesions and PASI scores of mice in each treatment group of *Hypericum perforatum* were significantly reduced, and the decrease was dose-dependent; it was determined that *Hypericum perforatum* extract can effectively alleviate IMQ-induced psoriasis-like skin lesions in mice.

[0063] (4) Spleen Index: After sacrifice, the spleen was harvested and weighed, and the spleen index (spleen weight / body weight) was calculated. Results are as follows: Figure 8 As shown, the spleen index in the model group was significantly increased, while the spleen index in the treatment group was significantly decreased (e.g., the high-dose group decreased from 12.80 to 6.10), indicating that it can inhibit systemic immune activation.

[0064] (5) Histopathology (H&E staining): Skin and spleen tissue were taken from the back, fixed, embedded, sectioned, stained with H&E, and examined under a microscope. Results are as follows: Figure 9 As shown, the model group exhibited significant epidermal thickening, abnormal keratinization, and dermal inflammatory cell infiltration; the drug-treated group showed a significant reduction in these pathological changes.

[0065] (6) Peripheral blood indicators: Blood samples were collected to test complete blood count (neutrophils Neu), lymphocytes Lym) and serum biochemistry (alanine aminotransferase ALT, aspartate aminotransferase AST, creatinine Cre, urine Urea). Results are as follows: Figure 10 , Figure 11 As shown in Tables 5 and 6, the drug-treated group could correct the model-induced increase in neutrophil / lymphocyte ratio (NLR) and had no significant toxicity to liver and kidney function. The combined results of ALT, AST, Cre, and Urea indicators indicate that at the dosage and administration period in this experiment, *Hypericum perforatum* extract not only did not aggravate IMQ-induced liver and kidney damage in mice, but also improved the model-related transaminase elevation and renal function abnormalities to a certain extent, providing a basis and evidence for subsequent mechanistic and long-term intervention studies.

[0066] Table 5. Quantitative analysis of peripheral blood routine tests of *Hypericum perforatum* ethanol extract in LPS-induced IMQ model mice.

[0067] Table 6. Effects of *Hypericum perforatum* extract on liver and kidney function in IMQ-induced psoriasis mice.

[0068] (7) Detection of key inflammatory factors (q-PCR / ELISA): Detection of the expression of IL-17A, IL-23, and IL-22 in skin tissue. Results are as follows: Figure 12As shown, *Hypericum perforatum* extract can significantly downregulate the expression of the above-mentioned factors and inhibit the IL-23 / IL-17 inflammatory axis. Specifically, under model conditions, *Hypericum perforatum* can downregulate the abnormally elevated levels of IL-17A, IL-23, and IL-22 to varying degrees, especially at medium and high doses, where the inhibitory effect on the Th17 / IL-23 / IL-22 axis is more significant, further supporting its anti-inflammatory effect in psoriatic diseases.

[0069] (8) Immunocellular analysis (flow cytometry): A single-cell suspension was prepared from the spleen and CD4 was detected by flow cytometry. + Th17 (CD4) cells in T cells + IL-17A + ) and Treg (CD4) + CD25 + FoxP3 + The proportion of ). The results are as follows Figure 13 As shown, the Th17 / Treg ratio was significantly increased in the model group, while the drug-treated group effectively reduced this ratio, correcting the immune imbalance. Specifically, IMQ-induced modeling significantly reduced CD4+. + The proportion of T cells suggests an imbalance in peripheral immunity; the extract of *Hypericum perforatum* (witch hazel) has an effect on model-related CD4... + The decrease in T cells was corrected, and the effect was more pronounced and stable at higher doses; from an immunological perspective, this supports its potential for immunomodulation / anti-inflammatory, providing a basis for further analysis of its impact on downstream Th17 / Treg cell subset imbalance. Furthermore, such as... Figure 14 As shown, the IMQ-induced psoriasis-like model exhibited abnormally high Th17 cell counts, relative Treg cell insufficiency, and a significantly increased Th17 / Treg ratio. Intervention with *Hypericum perforatum* extract could inhibit the Th17 / Treg imbalance to varying degrees and promote the restoration of immune balance, with the high-dose group showing the most significant effect.

[0070] (9) Signaling pathway protein detection (Western Blot): The expression of key NF-κB pathway proteins p-p65, p65, p-IκBα, and IκBα in skin lesions was detected. Results Figure 15As shown, the p-p65 / p65 and p-IκBα / IκBα ratios were increased in the model group, while these ratios were significantly decreased in the treatment groups, indicating that it can inhibit the activation of the NF-κB signaling pathway. Specifically, compared with the control group, the p-p65 / p65 and p-IκBα / IκBα ratios were significantly increased in the model group, suggesting that IMQ induction can significantly activate the NF-κB signaling pathway. After intervention with *Hymenochloa crus-galli* extract, the above indicators decreased to varying degrees in all treatment groups, with the SC-M and SC-H groups showing more significant decreases, suggesting that medium and high doses of intervention have a stronger inhibitory effect on the NF-κB signaling pathway. The above results indicate that *Hymenochloa crus-galli* extract alleviates psoriasis-like inflammatory responses by inhibiting IκBα phosphorylation and NF-κB p65 activation.

[0071] Experiment Example 4: Basic Research Methods and Results of Chemical Substances (1) UHPLC-Q / TOF-MS analysis: The chemical composition of *Hypericum perforatum* extract was systematically analyzed using UHPLC-Q / TOF-MS. Specific conditions were as follows: an ACQUITY BEH C18 column was used with a gradient elution of 0.1% formic acid-water-0.1% formic acid-acetonitrile mobile phase; mass spectrometry was performed using an ESI ion source in both positive and negative ion modes.

[0072] result Figure 16 As shown, full scan and MS / MS data of the samples were acquired in both positive and negative ion modes. Combined with the seven standards obtained later in this chapter, characteristic ion filtering, precise relative molecular mass, isotope distribution, retention time, and comparison with literature and databases, a total of 85 compounds were identified. These mainly include tannins, resveratrol and its derivatives, ellagic acid and its derivatives, lignans and their glycosides, catechins and triterpenoids, and a small number of other types of components. Among them, the tannins were mainly galloyl glucose series with different substitution sites, and there were also many types of resveratrol derivatives, ellagic acid derivatives, catechins, and lignans. The above results clarify the main chemical component types and fragmentation characteristics of the *Hamamelis japonica* extract, providing a research basis for subsequent quantitative analysis of components.

[0073] (2) Determination of total polyphenols, total flavonoids and total triterpenes: Experimental methods: The content of target compounds in the samples was determined using the Folin-Ciocalteu colorimetric method, the NaNO2-AlCl3-NaOH colorimetric method, and the vanillin-perchloric acid colorimetric method, respectively. The content of the corresponding compounds in the samples was calculated using standard curves of standard references (gallic acid, rutin, and oleanolic acid) for all three methods. The correlation coefficient (R0) of the standard curves was used. 2 The values ​​are all close to 1, showing a good linear relationship. Figure 17 ).

[0074] The experimental results showed that the extract contained abundant amounts of the three classes of active ingredients, with an average total flavonoid content of 18.42%, total triterpenes of 17.31%, and total polyphenols of 13.02%. The relative standard deviations (RSDs) of the determination results for each component were 4.28%, 3.75%, and 5.51%, respectively, indicating that the established analytical method has good repeatability and precision.

[0075] Experimental Example 5: Research Methods and Results on the Material Basis of Pharmacodynamics Experimental methods: Based on previous UPLC-Q / TOF-MS chemical characterization results, nine representative candidate chemical components (2',5-di-) were selected from the extract of *Hypericum perforatum*. O -galloyl-hamamelose (1), Apocynin (2), 4-(4'-hydroxy-3'-methoxyphenyl)-2-butanone (3), Coniferaldehyde (4), Piceid (5), Piceid-6''- O Activity evaluation was conducted on gallate (6), tarennanoside B (7), stigmasterol (8), and oleanolic acid (9).

[0076] Model: Lipopolysaccharide (LPS) induced inflammation model of mouse RAW264.7 macrophages.

[0077] method: (1) NO release detection (Griess method): Cells were seeded in 96-well plates and divided into control group, LPS model group (0.5 μg / mL), drug administration group (LPS + different concentrations of sample, 25, 50, 100 μM), and positive control group (LPS + hydrocortisone). After culturing for 24 h, the supernatant was collected, Griess reagent was added, and the absorbance at 540 nm was measured.

[0078] The results are as follows Figure 18 As shown, compared with the LPS model group, different compounds exhibit certain differences in their inhibitory effects on NO formation. Among them, Piceid and Piceid-6''- O -gallate, tarennanoside B, and oleanolic acid showed significant inhibitory effects on LPS-induced NO release, and their overall effects were better than those of the other tested compounds, suggesting that they have good in vitro anti-inflammatory activity.

[0079] (2) IL-6 and NF κB mRNA expression detection (q-PCR method): Cells were seeded in 6-well plates, grouped as above, and total RNA was extracted after 24 h of treatment. After reverse transcription, q-PCR was performed for detection.

[0080] To validate four candidate compounds (Piceid, Piceid-6''- O The anti-inflammatory effects and pathways of IL-6, NF-κB, and oleanolic acid (IL-6, NAFLD, IL-6, NF-κB) in RAW264.7 cells stimulated with LPS were investigated using q-PCR. Expression levels of κB mRNA. Results are as follows: Figure 18 As shown, compared with the control group, IL-6 mRNA expression was significantly increased in the LPS model group (P<0.001). Treatment with the four compounds resulted in varying degrees of downregulation of IL-6 mRNA levels, with Piceid and Piceid-6''- being the most significantly downregulated. O The inhibitory effects of β-gallate and oleanolic acid were particularly significant, with statistically significant differences compared to the model group (P<0.001), indicating that these compounds can inhibit the production of the key pro-inflammatory factor IL-6 at the transcriptional level. Compared with the control group, the LPS model group showed significantly reduced NF κB mRNA expression was significantly increased (P<0.001). After treatment with four compounds, NF... κB mRNA levels decreased in a concentration-dependent manner, with Piceid-6''- O α-gallate and oleanolic acid showed significant inhibitory effects at 25 μM, with even more pronounced inhibitory effects at 50 μM and 100 μM (P<0.001), indicating that these compounds can inhibit NF at the transcriptional level. Overactivation of the κB pathway.

[0081] The above results indicate that Piceid, Piceid-6''- O -gallate, tarennanoside B, and oleanolic acid can inhibit NF at the transcriptional level. The overactivation of the κB pathway, consistent with its reduced NO release and downregulation of IL-6 mRNA, further supports the view that these compounds work by blocking NF-κB pathway. The κB signaling pathway plays an anti-inflammatory role.

[0082] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. The use of a *Hypericum perforatum* extract composition in the preparation of a medicament for the prevention and / or treatment of psoriasis, characterized in that: The extract composition of *Hydrangea macrophylla* is an alcoholic extract of *Hydrangea macrophylla* or a component separated from an alcoholic extract of *Hydrangea macrophylla*.

2. The application according to claim 1, characterized in that: The drug comprises a unique active ingredient, a combination of witch hazel extract and pharmaceutically acceptable excipients.

3. A method for preparing a *Ligustrum lucidum* extract composition, characterized in that: Includes the following steps: Step S1: Take the roots or stems of *Ligustrum lucidum* and crush them; Step S2: Add 8 times the volume of 0-95% ethanol and heat under reflux to extract 1-3 times; Step S3: Combine the extracts and concentrate under reduced pressure to obtain total extract of Hamamelis macrantha SC-Z; The resulting total extract is a *Hypericum perforatum* extract composition, used in the application described in claim 1.

4. The preparation method according to claim 3, characterized in that: The method also includes step S4, in which the total extract obtained from 60% ethanol is loaded onto an HP-20 macroporous resin column and eluted using an ethanol-water system (0%, 30%, 50%, 95%), and each fraction is collected to obtain four fractions of different polarities: SC-W, SC-30, SC-50, and SC-95; the SC-W, SC-30, SC-50, and SC-95 are used as the extract composition of *Hypericum esculentum* for the application described in claim 1.

5. A composition for extracting *Ligustrum lucidum*, characterized in that: The extract includes the total extract prepared by the method of claim 3, or any one of SC-W, SC-30, SC-50 and SC-95 prepared by the method of claim 4, or any two or three combinations of SC-W, SC-30, SC-50 and SC-95 prepared by the method of claim 4; the extract composition is used in the application described in claim 1.

6. The extract composition of *Hypericum perforatum* according to claim 5, characterized in that: The extract composition of *Hypericum perforatum* is a total extract or SC-95.

7. The extract composition of *Hypericum perforatum* according to claim 6, characterized in that: The extract composition of *Hymenochloa crus-galli* is a total extract, wherein the total polyphenol content is 10-20%, the total flavonoid content is 10-30%, and the total triterpenoid content is 10-30%.

Images