A Litsea cubeba extract, its preparation method, and its application in drugs for treating atopic dermatitis.

Abstract

This invention discloses a Litsea cubeba extract, its preparation method, and its applications, relating to the field of pharmaceutical compound technology. The Litsea cubeba extract has the structural formula shown in Formula I: (Formula I). ​​This Litsea cubeba extract is isolated from the Litsea cubeba plant. This novel compound can significantly inhibit NO production in an LPS-induced RAW264.7 cell inflammation model; alleviate the immune-inflammatory response in an in vitro 3D skin model of atopic dermatitis; inhibit epidermal thickening and repair skin barrier function; and reduce the inflammatory response by inhibiting the PI3K / Akt signaling pathway. It has clinical application potential in the treatment of atopic dermatitis and can be used to prepare anti-atopic dermatitis drugs, showing promising application prospects.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical compound technology, and more specifically, to a pepper extract, its preparation method, and its application in a drug for treating atopic dermatitis. Background Technology

[0002] Atopic dermatitis (AD) is a common, chronic, relapsing, inflammatory skin disease, clinically characterized by intense itching, dry skin, and eczematous lesions. Its pathogenesis is complex, involving multiple factors such as skin barrier dysfunction, genetic susceptibility, and immune response dysregulation. Epidemiological data show that AD has a high incidence rate globally and often co-occurs with other atopic diseases such as asthma and allergic rhinitis. In my country, with changes in lifestyle and environmental factors, the prevalence of AD is showing an increasing trend year by year, becoming a major public health problem affecting the quality of life of people of all ages. Current clinical treatments mainly involve topical application of glucocorticoids, calcineurin inhibitors, and antihistamines. However, long-term use may lead to secondary complications such as skin atrophy due to abnormal dermal collagen metabolism, increased susceptibility to opportunistic infections due to skin barrier damage, and decreased drug response due to receptor downregulation. New biologics and small molecule drugs, including dupilumab, utpatinib, and abuxitinib, have limited efficacy, infection risks, and high costs due to their specific targeting. There is an urgent need to develop new intervention strategies that combine long-lasting efficacy and safety.

[0003] In recent years, the development of immunomodulators based on natural products has provided new insights for the treatment of Alzheimer's disease (AD). Natural products, with their unique chemical structural diversity and broad biological activities, have played an irreplaceable role in the history of human disease prevention and treatment. Statistics show that more than half of the drugs currently used clinically are directly or indirectly derived from natural products and their structural derivatives. Plants, as an important source of natural products, produce abundant secondary metabolites such as flavonoids, terpenes, alkaloids, and phenolic acids, which exhibit various pharmacological activities, including anti-inflammatory, immunomodulatory, antioxidant, and antibacterial effects. Especially for inflammatory and immune-related skin diseases, the search for novel active molecules with multi-target regulatory effects and the ability to intervene in complex inflammatory networks from traditional medicinal plants has become an important direction in drug development.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a pepper extract, its preparation method, and its application.

[0006] This invention is implemented as follows: In a first aspect, the present invention provides a Ligustrum lucidum extract, the structural formula of which is shown in Formula I: (Formula I).

[0007] Secondly, the present invention provides a method for preparing the above-described extract of Litsea cubeba, comprising the following steps: S1. The dried Litsea cubeba medicinal material was extracted with methanol, and the solvent was recovered under reduced pressure to obtain crude extract 1; S2. Add crude extract 1 to hot water at 40~50℃ and suspend, extract with petroleum ether and recover the solvent to obtain crude extract 2; S3. Crude extract 2 was subjected to gradient elution by silica gel column chromatography with petroleum ether-ethyl acetate as the elution solvent at a volume ratio of 100:1 to 1:10. The eluent with a volume ratio of petroleum ether-ethyl acetate of 8:2 was evaporated under reduced pressure to obtain crude extract 3. S4. Crude extract 3 was subjected to Sephadex LH-20 gel chromatography with methanol-dichloromethane as the mobile phase to obtain crude extract 4. S5. Crude extract 4 was eluted by gradient elution on an ODS reverse-phase column with methanol-water as the mobile phase to obtain crude extract 5; S6. Crude extract 5 was isocratically separated by preparative high performance liquid chromatography with methanol-water as the mobile phase to obtain the compound shown in Formula I.

[0008] In the preferred preparation method, in step S3, the volume ratio of petroleum ether to ethyl acetate gradient elution varies to 100:1, 9:1; 8:2; 7:3; 6:4; 5:5; 4:6; 3:7; 1:10.

[0009] In the preferred preparation method, in step S4, the Sephadex LH-20 hydroxypropyl dextran gel is selected for the gel Sephadex LH-20 chromatography, and the volume ratio of the mobile phase methanol to dichloromethane is 1:1.

[0010] In the preferred preparation method, in step S5, the ODS reversed-phase silica column chromatography is an octadecyl bonded phase silica C18 column, specifically a YMC*GEL ODS-A-HG 12nm S-50um column, and the volume ratio of the methanol-water gradient elution varies from 50:50, 60:40, 70:30 to 80:20.

[0011] In the preferred preparation method, in step S6, the chromatographic column of the preparative high-performance liquid chromatography is a Kromasil 100-5C18, the detection wavelength is 214 nm, and the volume ratio of the mobile phase methanol to water is 80:20.

[0012] In the preferred preparation method, in step S1, the medicinal material of Litsea cubeba is the root powder of Litsea cubeba, which is extracted with methanol 2 to 4 times, and the soaking time during each extraction is 48 to 96 hours; in step S2, the ratio of crude extract 1 to hot water is 1 kg: 1 to 2 L.

[0013] Thirdly, the present invention provides an anti-atopic skin composition comprising the aforementioned pepper extract and pharmaceutically acceptable excipients.

[0014] Fourthly, the present invention provides the use of the aforementioned pepper extract in the preparation of an anti-atopic dermatitis drug.

[0015] Fifthly, the present invention provides the use of the aforementioned Ligustrum lucidum extract in the preparation of a drug capable of inhibiting LPS-induced NO production and the expression of the inflammatory protein iNOS.

[0016] Preferably, the drug can inhibit epidermal thickening and repair skin barrier function, and reduce inflammatory response by inhibiting the PI3K / Akt signaling pathway.

[0017] The present invention has the following beneficial effects: This invention is derived from Ligustrum lucidum ( Lindera glauca A sesquiterpene dimer, Linderasesqdimer B (i.e., the extract of *Litsea cubeba* with structural formula I in this application), was isolated from the plant. This novel compound significantly inhibited NO production and the expression of the inflammatory protein iNOS in an LPS-induced RAW264.7 cell inflammation model; it also alleviated the immune-inflammatory response in an in vitro 3D skin model of atopic dermatitis, inhibited epidermal thickening, and repaired skin barrier function by inhibiting the PI3K / Akt signaling pathway. It has clinical application potential in the treatment of atopic dermatitis and can be used to prepare anti-atopic dermatitis drugs, showing promising application prospects. Attached Figure Description

[0018] Figure 1 A schematic diagram illustrating the construction and culture process of a 3D skin model for atopic dermatitis; Figure 2 This is a schematic diagram of the 3D skin activity detection results; Figure 3 This is a schematic diagram of the H&E staining analysis results; Figure 4 This is a schematic diagram showing the expression results of relevant factor mRNAs in 3D skin. Figure 5 This is a schematic diagram showing the expression results of the PI3K / Akt pathway and inflammation-related proteins. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0020] This invention provides a Ligustrum lucidum extract, the structural formula of which is shown in Formula I: (Formula I).

[0021] In this application, the compound represented by Formula I is named Linderasesqdimer B, which is a sesquiterpene dimer. Experiments have confirmed that *Litsea cubeba* (…) Lindera glauca The plant-derived sesquiterpene dimer Linderasesqdimer B significantly inhibited NO production and the expression of the inflammatory protein iNOS in an LPS-induced RAW264.7 cell inflammation model; it also alleviated the immune-inflammatory response in an in vitro 3D skin model of atopic dermatitis, inhibited epidermal thickening, and repaired skin barrier function by inhibiting the PI3K / Akt signaling pathway. It has clinical application potential in the treatment of atopic dermatitis and could be used to prepare anti-atopic dermatitis drugs.

[0022] Therefore, this application also provides an anti-atopic dermatitis composition comprising a novel compound derived from the above-described extract of *Ligustrum lucidum*. In an optional embodiment, the anti-inflammatory composition further comprises medically acceptable pharmaceutical excipients.

[0023] Furthermore, the present invention provides the application of the pepper extract described in the foregoing embodiments in the preparation of anti-atopic dermatitis drugs.

[0024] Furthermore, the present invention provides the application of the Ligusticum chuanxiong extract described in the foregoing embodiments in the preparation of a drug capable of inhibiting LPS-induced NO production and the expression of the inflammatory protein iNOS; alleviating the immune inflammatory response of atopic dermatitis, inhibiting epidermal thickening and repairing skin barrier function, and reducing the inflammatory response by inhibiting the PI3K / Akt signaling pathway.

[0025] Furthermore, this application also provides a method for preparing the above-mentioned Ligustrum lucidum extract, which is only provided as an example, and includes: S1. Source of Litsea cubeba medicinal material: Litsea cubeba is a plant belonging to the genus Litsea in the family Lauraceae (Latin name: Litsea cubeba). Lindera glauca The root of (Sieb. et Zucc.) Bl).

[0026] S2, Coarse Extraction: 25 kg of dried Litsea cubeba was extracted with methanol 2-4 times, each time soaking for more than 48 h, and the solvent was recovered under reduced pressure to obtain 4.2 kg of crude extract 1; crude extract 1 was suspended in 6.0 L of hot water at 45℃, extracted with petroleum ether and the solvent was recovered to obtain 812.3 g of crude extract 2.

[0027] Preferably, extraction is performed with petroleum ether until the petroleum ether becomes colorless.

[0028] S3. Separation and purification: The crude extract 2 was subjected to gradient elution by silica gel column chromatography with petroleum ether-ethyl acetate in a volume ratio of 100:1 to 1:10. The eluent in a washing ratio of 8:2 was evaporated under reduced pressure to obtain 7.1 g of crude extract 3.

[0029] The crude extract 3 was subjected to Sephadex LH-20 gel chromatography with methanol-dichloromethane (v / v) as the mobile phase to obtain crude extract 4. Crude extract 4 was subjected to gradient elution on an ODS reversed-phase column with methanol-water as the mobile phase to obtain crude extract 5. Crude extract 5 was isocratically separated by preparative high-performance liquid chromatography with methanol-water (v / v) as the mobile phase to obtain the compound represented by Formula I.

[0030] Preferably, in the process of preparing crude extract 3, the volume ratio of petroleum ether-ethyl acetate gradient elution varies to 100:1, 9:1; 8:2; 7:3; 6:4; 5:5; 4:6; 3:7; 1:10, with 3 L of rinsing for each gradient.

[0031] Preferably, in the process of preparing crude extract 4, the Sephadex LH-20 hydroxypropyl dextran gel is selected for the gel Sephadex LH-20 chromatography.

[0032] Preferably, in the process of preparing crude extract 5, the ODS reversed-phase silica column chromatography is octadecyl bonded phase silica (C18) YMC*GEL ODS-A-HG 12nm S-50um, and the volume ratio of the methanol-water elution system varies to 50:50, 60:40, 70:30 and 80:20.

[0033] Preferably, the preparative high-performance liquid chromatography column used to prepare the compound shown in Formula I is a Kromasil 100-5 C18, and the detection wavelength is 214 nm.

[0034] According to the applicant's analysis, the Litsea cubeba extract represented by Formula I possesses the aforementioned anti-inflammatory efficacy due to its parent nucleus structure, which is generally represented by Formula II. It is understood that similar compounds having this parent nucleus structure are also expected to possess the same or similar anti-inflammatory activity. As an example, such similar compounds include, but are not limited to, Linderasesqdimer A, represented by Formula III, which is also isolated from Litsea cubeba.

[0035] (Formula II); (Formula III); In this case, the H atom in the aforementioned general formula II can be replaced by OCH3, OH, substituted or unsubstituted alkyl groups, substituted or unsubstituted alkenyl groups, etc.

[0036] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0037] Example 1 This embodiment provides a Ligustrum lucidum extract, the structural formula of which is shown in Formula I: (Formula I).

[0038] Its preparation method includes the following steps: (1) Source of Litsea cubeba medicinal material: Litsea cubeba is a plant belonging to the genus Litsea in the family Lauraceae (Latin name: Litsea cubeba). Lindera glauca The root of (Sieb. et Zucc.) Bl).

[0039] (2) Coarse extraction: 25 kg of dried Litsea cubeba was extracted with methanol 2-4 times, each time soaking for 48 h, and the solvent was recovered under reduced pressure to obtain 4.2 kg of crude extract 1; crude extract 1 was suspended in 6.0 L of hot water at 45 °C, extracted with petroleum ether and the solvent was recovered to obtain 812.3 g of crude extract 2.

[0040] Preferably, extraction is performed with petroleum ether until the petroleum ether becomes colorless.

[0041] (3) Separation and purification: The crude extract 2 was subjected to gradient elution using silica gel column chromatography with petroleum ether-ethyl acetate as the eluent. The volume ratio gradients were 100:1, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, and 1:10, with 3 L of eluent used for each gradient. The eluent fraction with an 8:2 ratio was evaporated to dryness under reduced pressure to obtain 7.1 g of crude extract 3. The crude extract 3 was then subjected to Sephadex LH-20 gel chromatography with methanol-dichloromethane (volume ratio 1:1) as the mobile phase to obtain crude extract 4. Crude extract 4 was then subjected to gradient elution using an ODS reversed-phase column with methanol-water (volume ratios of 50:50, 60:40, 70:30, and 80:20) as the mobile phase to obtain crude extract 5. Crude extract 5 was isocratically separated by preparative high performance liquid chromatography using a Kromasil 100-5 C18 column, with a detection wavelength of 214 nm and a mobile phase of methanol-water (volume ratio 80:20) to obtain the compound shown in Formula I.

[0042] Experimental Example 1 The structure of the new compound Linderasesqdimer B was determined by structural analysis, and the following experimental data were obtained: New compound Linderasesqdimer B:C 30 H 44 O4, (-)-HRESIMS m / z 467.3163 [M - H] - (Calculated value: 467.3155).

[0043] The NMR data of compound Linderasesqdimer B are shown in Table 1.

[0044] Table 1. NMR data of compound Linderase sqdimer B Experimental Example 2: Anti-inflammatory cell screening model of compound Linderasesqdimer B Experimental methods The new compound Linderasesqdimer B or the positive control drug indomethacin was dissolved in DMSO and prepared into different drug concentrations using culture medium. Each concentration was prepared in triplicate, and model control wells (containing only LPS) and blank control wells (containing cells and culture medium) were also prepared.

[0045] RAW264.7 cells in the logarithmic growth phase were selected and cultured at a rate of 2 × 10⁻⁶ cells. 4Cells were seeded at a density of 1 / well in 96-well plates and co-cultured for 12 h. Different concentrations of the drug were then added to the corresponding plates. Two h later, LPS at a final concentration of 1 μg / mL was added to induce macrophage differentiation into an inflammatory state. After 48 h of culture, 50 μL of cell supernatant was collected, and 50 μL each of Griess Reagent I and Griess Reagent II from a nitric oxide assay kit were added. The NO content was determined using the Griess method.

[0046] Inhibition rate = [1-(NO treat -NO control ) / (NO model -NO control )]×100% Experiment Example 3: Effects of Compounds on Cell Viability MTT can be reduced to water-insoluble blue crystals by succinate dehydrogenase in the mitochondria of living cells and deposited in living cells, while dead cells do not have this function.

[0047] Experimental methods RAW264.7 cells in the logarithmic growth phase were harvested and divided into two groups of 2 × 10⁶ cells per well. 4 Cells were seeded at a density of 1 / well in 96-well plates and incubated at 37 ℃ in a 5% CO2 incubator for 12 h to allow them to adhere. Then, Linderasesqdimer B or indomethacin, the test compound, was added to each well at different concentrations (100, 50, 25, 12.5, 6.25, 3.125, 1.563 μM), dissolved in DMSO and diluted with culture medium, with three replicates for each concentration. Blank control wells (containing only cells and culture medium) were also included. After 48 h of incubation, 10 μL of MTT solution (5 mg / mL) was added to each well, and incubation continued for 4 h. The supernatant was discarded, and 150 μL of DMSO was added to each well, shaking to fully dissolve the purple formazan crystals. The absorbance (A) of each well was measured at 570 nm using a microplate reader. Cell viability reflects the inhibitory effect of the drug on cell proliferation; a higher viability indicates lower drug toxicity. The formula for calculating cell viability is as follows: Survival rate (%) = [(A1 – A0) / (A2 – A0)] × 100%, where A0 is the absorbance value of the blank control, A1 is the absorbance value of the sample, and A2 is the absorbance value of the positive control well.

[0048] Five concentrations (200 μM, 100 μM, 50 μM, 25 μM, and 12.5 μM) of the sample were measured, and dose-inhibition rate curves were plotted to determine their CC (correlation function). 50 Values. Each sample was measured three times. The results of Experiments 2 and 3 are shown in Table 2.

[0049] Table 2. Inhibition rate of NO by compound Linderasesqdimer B The results showed that compound Linderasesqdimer B exhibited strong anti-NO production activity, with an IC50 value of [missing information]. 50 It is 4.35 ± 0.35 M has stronger activity than the positive control indomethacin (IC50). 50 = 24.1 ± 2.1 M). And this compound is at 45.0 ± 1.42 At M concentration, no cytotoxic activity was observed, indicating that it has great potential for further preclinical studies.

[0050] Experimental Example 4: Effects and Mechanisms of Compound Linderasesqdimer B on an In Vitro 3D Atopic Dermatitis Skin Model S1. Experimental Protocol 1. Establishment and drug administration of a 3D skin model of atopic dermatitis Human primary epidermal keratinocytes (NHEK), primary dermal fibroblasts (HDF), and human acute monocytic leukemia cells (THP-1) were extracted from healthy skin samples discarded during surgery. The extracted HDF and THP-1 cells were resuspended in extracellular matrix, with an HDF density of 2 × 10⁻⁶ cells / mL. 6 cells / mL, THP-1 cell density was 4 × 10⁻⁶ 5 cells / mL, take 100 μL of extracellular matrix-cell suspension and carefully add it dropwise into the upper chamber of a 24-well Transwell plate. Figure 1The cells were cultured at 37°C in a 5% CO2 incubator for 30 min. After the matrix solidified, 600 μL of culture medium (a 1:1 mixture of DMEM complete medium and CnT-BM.1 medium) was added to each well in the lower chamber to form the dermis of the in vitro 3D skin. Primary KC cell suspension was seeded on top of the dermis and cultured at 37°C in a 5% CO2 incubator. After the primary KC cells adhered, they formed the epidermis. On day 4, the culture medium above the epidermis was aspirated to create an air-liquid interface to induce epidermal differentiation and stratum corneum formation. Culture continued until day 10, when the number of cells in the epidermis and dermis increased significantly. The cells were then divided into a control group (Control), an atopic dermatitis group (AD), and Linderasesqdimer B (low, medium, and high doses, 5 μM, 10 μM, and 20 μM, respectively; μM is the industry-standard unit for drug concentration). Each group had three biological replicates. On day 11, IL-4 and IL-13 (both at a final concentration of 50 ng / mL) were added to the culture medium in the lower chamber to simulate an AD-like inflammatory microenvironment. The medium was then completely replaced (500 μL) every 24 hours for 5 days of induction. On day 15, appropriate drug treatment was initiated, while IL-4 and IL-13 were continuously administered in combination to maintain inflammatory stimulation. On day 20, culture medium samples from the lower chamber and skin tissue samples from the upper chamber were collected for subsequent relevant marker detection.

[0051] The extracellular matrix was prepared as follows: The extracellular matrix mainly consisted of rat tail collagen type I and bovine fibrinogen. The final concentration of rat tail collagen type I was 4 mg / mL (coagulation conditions: pH was adjusted to about 7 using sodium hydroxide solution), and the final concentration of bovine fibrinogen was 2 mg / mL (coagulation conditions: thrombin was added to make the final thrombin concentration of the mixed extracellular matrix 3 U / mL). The extracellular matrix was prepared on ice and quickly mixed with cells and seeded in 24-well Transwell chambers.

[0052] S2. Experimental Results 1.3D Skin Activity Detection To assess the viability of Linderasesqdimer B in in vitro 3D skin, we performed cell viability staining on each group of organoids on day 20 of culture. The results showed that, at day 20, cell viability in the in vitro 3D skin remained at a high level in all groups, with cell viability >95%, and no significant differences between groups. ns P >0.05 ( Figure 2 ).

[0053] 2. H&E staining analysis The pathological changes in an in vitro 3D skin model of atopic dermatitis (AD) were assessed using HE staining. Results showed that after treatment with 50 ng / mL IL-4 combined with IL-13, the epidermis in the model group exhibited significant thickening, accompanied by typical atopic dermatitis-like pathological features such as hyperkeratosis and parakeratosis. Figure 3 After intervention with Linderasesqdimer B, the above pathological manifestations were significantly improved, and the improvement was in a good dose-dependent manner.

[0054] Figure 3 In this study, the Control group consisted of normal in vitro 3D skin, the AD group was treated with 50 ng / mL IL-4 / IL-13, and the other groups were treated with Linderasesqdimer B in the AD group. P <0.05,** P <0.01,*** P <0.001 vs Control group; # P <0.05, ## P <0.01, ### P <0.001 vs AD group.

[0055] Expression of related factor mRNA in 3D skin 3.1 Design primers. The following primers were synthesized by Beijing Pantech Co., Ltd., as shown in Table 3.

[0056] Table 3 Primer sequence listing like Figure 4 The results showed that, compared with the control group, IL-4 / IL-13 induction significantly increased the mRNA expression of inflammatory factors IL-1β, TNF-α, IL-4, IFN-γ, and IL-13, while significantly decreasing the relative mRNA expression of TGF-β. This indicates that an in vitro 3D model of Alzheimer's disease (AD) was successfully constructed, consistent with the clinical manifestations of AD patients. Compared with the AD group, the Linderasesqdimer B treatment group showed significantly downregulated mRNA expression of inflammatory factors IL-1β, TNF-α, IL-4, IFN-γ, and IL-13, while also increasing the mRNA expression of TGF-β. This suggests that Linderasesqdimer B can reduce the expression of type 2 inflammatory factors in in vitro 3D skin of AD patients and enhance skin barrier function.

[0057] Figure 4 In this study, the Control group consisted of normal in vitro 3D skin, the AD group was treated with 50 ng / mL IL-4 / IL-13, and the other groups were treated with Linderasesqdimer B in the AD group.P <0.05,** P <0.01,*** P <0.001 vs Control group; # P <0.05, ## P <0.01, ### P <0.001 vs AD group.

[0058] 4.3 Expression of p-PI3K / PI3K, p-Akt / Akt, iNOS and Arg-1 proteins in skin To further explore the mechanism, we examined the expression of the PI3K / Akt pathway and inflammation-related proteins. The results showed that the expression of p-PI3K, p-Akt, iNOS, and ARG-1 was significantly increased in the AD model group; after administration of Linderasesqdimer B, the phosphorylation level of the PI3K / Akt pathway and the expression of iNOS and ARG-1 decreased in a dose-dependent manner. Figure 5 This suggests that Linderasesqdimer B may improve AD-related inflammatory responses by inhibiting the activation of the PI3K / Akt signaling pathway.

[0059] Figure 5 In this study, the Control group consisted of normal in vitro 3D skin, the AD group was treated with 50 ng / m³ LIL-4 / IL-13, and the other groups were treated with Linderasesqdimer B in the AD group. P <0.05,** P <0.01,*** P <0.001 vs Control group; # P <0.05, ## P <0.01, ### P <0.001 vs AD group.

[0060] In summary, this invention relates to *Litsea cubeba* (a type of wild pepper). Lindera glauca A novel sesquiterpene dimer, Linderasesqdimer B, was isolated from plants. This new compound significantly inhibited NO production in an LPS-induced RAW264.7 cell inflammation model; reduced the immune-inflammatory response in an in vitro 3D skin model of atopic dermatitis; inhibited epidermal thickening; and repaired skin barrier function. It also alleviated the inflammatory response by inhibiting the PI3K / Akt signaling pathway. It has clinical application potential in the treatment of atopic dermatitis and can be used to prepare anti-atopic dermatitis drugs, showing promising application prospects.

[0061] The applicant declares that the above specific embodiments are preferred embodiments described to facilitate understanding of the present invention, but the present invention is not limited to the above embodiments, that is, it does not mean that the present invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvement to the present invention, equivalent substitution of the raw materials used in the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A pepper extract, characterized in that, Its structural formula is shown in Formula I: (Equation I).

2. A method for preparing the Litsea cubeba extract as described in claim 1, characterized in that, Includes the following steps: S1. The dried Litsea cubeba medicinal material was extracted with methanol, and the solvent was recovered under reduced pressure to obtain crude extract 1; S2. Add crude extract 1 to hot water at 40~50℃ and suspend, extract with petroleum ether and recover the solvent to obtain crude extract 2; S3. Crude extract 2 was subjected to gradient elution by silica gel column chromatography with petroleum ether-ethyl acetate as the elution solvent at a volume ratio of 100:1 to 1:

10. The eluent with a volume ratio of petroleum ether-ethyl acetate of 8:2 was evaporated under reduced pressure to obtain crude extract 3. S4. Crude extract 3 was subjected to Sephadex LH-20 gel chromatography with methanol-dichloromethane as the mobile phase to obtain crude extract 4. S5. Crude extract 4 was eluted by gradient elution on an ODS reverse-phase column with methanol-water as the mobile phase to obtain crude extract 5; S6. Crude extract 5 was isocratically separated by preparative high performance liquid chromatography with methanol-water as the mobile phase to obtain the compound shown in Formula I.

3. The method for preparing the Litsea cubeba extract according to claim 2, characterized in that, In step S3, the volume ratio of petroleum ether to ethyl acetate gradient elution changes to 100:1, 9:1; 8:2; 7:3; 6:4; 5:5; 4:6; 3:7; 1:

10.

4. The method for preparing the Litsea cubeba extract according to claim 2, characterized in that, In step S4, the Sephadex LH-20 hydroxypropyl dextran gel is selected for the gel Sephadex LH-20 chromatography, and the mobile phase methanol-dichloromethane volume ratio is 1:

1.

5. The method for preparing the Litsea cubeba extract according to claim 2, characterized in that, In step S5, the ODS reversed-phase silica column chromatography is an octadecyl bonded phase silica C18 column, and the volume ratio of the methanol-water gradient elution varies from 50:50, 60:40, 70:30 to 80:

20.

6. The method for preparing the Litsea cubeba extract according to claim 2, characterized in that, In step S6, the preparative high-performance liquid chromatography column is a Kromasil 100-5 C18, the detection wavelength is 214 nm, and the volume ratio of the mobile phase methanol to water is 80:

20.

7. The method for preparing the Litsea cubeba extract according to claim 2, characterized in that, In step S1, the medicinal material of Litsea cubeba is the root powder of Litsea cubeba, which is extracted with methanol 2 to 4 times, and the soaking time during each extraction is 48 to 96 hours; in step S2, the ratio of crude extract 1 to hot water is 1 kg: 1 to 2 L.

8. A composition for treating atopic dermatitis, characterized in that, It includes the pepper extract as described in claim 1 and pharmaceutically acceptable excipients.

9. The use of the pepper extract according to claim 1 in the preparation of an anti-atopic dermatitis drug.

10. The use of the Litsea cubeba extract according to claim 1 in the preparation of a drug capable of inhibiting LPS-induced NO production and the expression of the inflammatory protein iNOS.

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