Straight-chain diterpenoid compound as well as preparation method and application thereof

Abstract

The invention relates to the technical field of natural products, in particular to a straight-chain diterpenoid compound as well as a preparation method and application thereof. The straight-chain diterpenoid compound comprises at least one of compounds shown in the following structural formula. The straight-chain diterpenoid compound has a remarkable curative effect on alopecia, especially alopecia seborrhoeica, has an anti-inflammatory effect and can be used for treating dermatitis.

Description

Technical Field

[0001] This invention relates to the field of natural product technology, and more specifically, to linear diterpenoids, their preparation methods, and their applications. Background Technology

[0002] Plateau Tianmingjing ( Carpesium abrotanoides L.) refers to the whole herb of *L. alpineus*, a plant belonging to the genus *L.* of the Asteraceae family. It is widely distributed in Gansu, eastern Qinghai, western Sichuan, and northwestern Yunnan, growing at altitudes of 2000-3500 meters in forest edges or hillside thickets. The whole herb (called "ear-digging grass" in Tibetan) is bitter and cool in nature, possessing properties of clearing heat and detoxifying, resolving phlegm and treating malaria. It is mainly used to treat toothache, malaria, and sore throat. The fruit is bitter, pungent, and neutral, and can eliminate stagnation and kill parasites. Tibetan medicine uses it to treat stomachache, insect and snake bites, and sores. However, there is no record of its use in treating hair loss.

[0003] Seborrheic alopecia (also known as androgenetic alopecia) is a chronic hair loss disease related to genetics, hormonal imbalances, and hyperfunction of the sebaceous glands. Its typical symptoms and characteristics include: excessive sebum secretion on the scalp, thinning and hair loss, scalp itching, erythema, and folliculitis. Current treatments for seborrheic alopecia generally involve minoxidil solution, minoxidil, finasteride tablets, and spironolactone tablets. Traditional Chinese medicine or herbal extracts are rarely used to treat seborrheic alopecia.

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

[0005] The purpose of this invention is to provide a linear diterpenoid compound, its preparation method, and its application. This invention provides a linear diterpenoid compound that has a significant therapeutic effect on hair loss, especially seborrheic alopecia, and also possesses anti-inflammatory properties, enabling the treatment of dermatitis, thus further expanding the selection of drugs for treating dermatitis and seborrheic alopecia.

[0006] This invention is implemented as follows: In a first aspect, the present invention provides a linear diterpenoid compound comprising at least one of the compounds shown in the following structural formulas: .

[0007] In a second aspect, the present invention provides a method for preparing the linear diterpenoid compound described in the foregoing embodiments, comprising: extracting the alcoholic extract of gentian using an ester solvent-water to form an ester extract; The ester extract was then subjected to column chromatography, wherein the mobile phase used in the column chromatography elution process included a mixture of C1-C3 alcohol solvent and water in a volume ratio of (1-5):(5-0) to form a mixed solution and a C3-C5 ketone solvent.

[0008] In an optional embodiment, the alcohol extract is extracted using a C3-C5 ester solvent-water, and then concentrated to form an ester extract. Preferably, the method includes: extracting the alcohol extract multiple times using ethyl acetate-water, combining the organic phases formed by the multiple extractions, and concentrating them to form an ethyl acetate extract.

[0009] In an optional embodiment, the method includes: performing column chromatography on the ester extract using a macroporous adsorption resin; The column chromatography elution process includes: using C1-C3 alcohol solvents and water in volume ratios of 1:4, 1:1, 4:1, and 1:0 as the mobile phase for elution, followed by elution with C3-C5 ketone solvents, and collecting components A through E.

[0010] In an optional implementation, the method further includes: high-performance liquid chromatography separation of the C fraction formed by column chromatography elution.

[0011] In an optional embodiment, the elution process of high-performance liquid phase separation includes: mixing acetonitrile and water in a volume ratio of 70-75:30-25 to form a mixed solvent for elution.

[0012] Thirdly, the present invention provides the application of the linear diterpenoid compound described in the foregoing embodiments or the product obtained by the preparation method of the linear diterpenoid compound described in the foregoing embodiments in the preparation of a formulation for treating hair loss.

[0013] In an optional implementation, the hair loss includes seborrheic alopecia; Preferably, the formulation comprises the linear diterpenoid compound or a C component formed by the preparation method.

[0014] Fourthly, the present invention provides the application of the linear diterpenoid compound described in the foregoing embodiments or the product prepared by the method for preparing the linear diterpenoid compound described in the foregoing embodiments in the preparation of anti-inflammatory drugs.

[0015] Fifthly, the present invention provides the application of the linear diterpenoid compound described in the foregoing embodiments or the product prepared by the method for preparing the linear diterpenoid compound described in the foregoing embodiments in the preparation of a medicament for treating dermatitis.

[0016] This invention offers the following beneficial effects: It provides a series of novel linear diterpenoid compounds that exhibit significant therapeutic effects on hair loss, particularly seborrheic alopecia. Simultaneously, they possess anti-inflammatory effects, enabling the treatment of dermatitis and expanding the range of drugs available for treating both dermatitis and seborrheic alopecia. Furthermore, this series of linear diterpenoid compounds is derived from *Tetrandrine oleracea*, further broadening the application scope of *Tetrandrine oleracea* and enhancing its medicinal value. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a schematic diagram of the experimental procedure provided for Experimental Example 1 of the present invention; Figure 2 This is a graph showing the change in mouse body weight provided in Experiment Example 1 of the present invention; Figure 3 This is a photograph of mouse skin provided in Embodiment 1 of the present invention; Figure 4 This is a statistical chart of the number of mouse hair follicles provided in Embodiment 1 of the present invention; Figure 5 This is a schematic diagram of the experimental procedure provided in Example 3 of the present invention; Figure 6 This is a graph showing the change in mouse body weight provided in Example 3 of the present invention; Figure 7 This is a photograph of mouse skin provided in Example 3 of the present invention; Figure 8 The left image shows a statistical chart of mouse epidermal thickness, and the right image shows a statistical chart of mast cell count in each mouse, provided for Example 3 of the present invention. Figure 9 This is a schematic diagram of the experimental procedure provided in Example 4 of the present invention; Figure 10 This is a graph showing the change in mouse body weight provided in Example 4 of the present invention; Figure 11 A photograph of mouse skin provided in Example 4 of the present invention. 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] In a first aspect, the present invention provides a linear diterpenoid compound comprising at least one of the compounds shown in the following structural formulas: The compounds mentioned above are denoted as CL-1, CL-2, CL-3, CL-4, and CL-5 from left to right.

[0021] Combinations formed by any one, two, three, four, or five of the aforementioned linear diterpenoid compounds are effective in treating hair loss, especially seborrheic alopecia. They also possess anti-inflammatory effects.

[0022] The aforementioned linear diterpenoids are derived from *Gynostemma pentaphyllum*. Therefore, these linear diterpenoids can be obtained by separating the extract from *Gynostemma pentaphyllum*. The specific preparation process for these linear diterpenoids is as follows: S1, Extraction; Extraction of Tianmingjing can be carried out using existing conventional extraction methods, such as alcohol solvent heating and reflux extraction, supercritical fluid extraction, ultrasonic extraction, microwave-assisted extraction, maceration or percolation.

[0023] This invention illustrates extraction using an alcohol solvent under reflux as an example. Specifically, the air-dried whole herb of *Gynostemma pentaphyllum* is mixed with 60-80% C1-C3 alcohol solvent and extracted under reflux. The amount of C1-C3 alcohol solvent added per gram of *Gynostemma pentaphyllum* is 10-15 ml. C1-C3 alcohol solvents include, but are not limited to, methanol, ethanol, and propanol. The mixture is refluxed multiple times, each time for 1.5-2.5 hours. The mixture is then filtered, and the filtrate is concentrated to obtain the extract.

[0024] S2, extraction; The above-mentioned extract is extracted using an ester solvent-water mixture, wherein the volume ratio of ester solvent to water in the ester solvent-water mixture is (0.8-1.2):1. The ester solvent includes C3-C5 ester solvents, such as, but not limited to, ethyl acetate. The number of extractions is not limited, but multiple extractions are preferred, such as three extractions. The organic phase obtained from each extraction is collected and then concentrated to form an ester extract or an ethyl acetate extract.

[0025] S3, column chromatography; The ester extract was then subjected to column chromatography, specifically, column chromatography was performed using a macroporous adsorption resin; wherein, the macroporous adsorption resin used was Maclean D101.

[0026] The column chromatography elution process uses a mobile phase consisting of a mixture of C1-C3 alcohol solvents and water at a volume ratio of (1-5):(5-0) to form a mixed solution, and a C3-C5 ketone solvent. Elution is performed using C1-C3 alcohol solvents and water at volume ratios of 1:4, 1:1, 4:1, and 1:0, followed by elution with a C3-C5 ketone solvent, and fractions A through E are collected. The C1-C3 alcohol solvents include, but are not limited to, methanol and ethanol, and the C3-C5 ketone solvents include, but are not limited to, acetone.

[0027] S4, High-efficiency liquid phase separation; The C components (eluent formed by eluting C1-C3 alcohol solvents and water at a 4:1 ratio) were further separated using high-performance liquid chromatography (HPLC). The elution process included eluting with a mixed solvent of acetonitrile and water at a volume ratio of 70-75:30-25. Specifically, acetonitrile and water were eluted at a volume ratio of 70:30, forming CL-1 (16.4 min), CL-2 (16.4 min), and CL-3 (30.9 min). Then, acetonitrile and water were eluted at a volume ratio of 75:25, forming CL-4 (30.4 min) and CL-5 (32.8 min).

[0028] The present invention also provides a new use, specifically, the five straight-chain diterpenoid compounds CL-1, CL-2, CL-3, CL-4 and CL-5 and the C component prepared above all have good therapeutic effects on hair loss, especially seborrheic alopecia, and therefore can be used as a medicine for treating hair loss, especially seborrheic alopecia.

[0029] Meanwhile, the five straight-chain diterpenoid compounds CL-1, CL-2, CL-3, CL-4 and CL-5, as well as the C component prepared above, all have anti-inflammatory effects and can be used to prepare anti-inflammatory drugs, especially drugs for treating dermatitis.

[0030] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0031] Example 1 This invention provides a method for preparing a straight-chain diterpenoid compound, comprising: The whole herb of *Euphorbia hirta* was dried in the shade, pulverized, and 200 g of dry sample was obtained. It was then extracted by reflux with 3 L of 80% methanol. The reflux was repeated three times, each time for 2 hours. After filtration, the filtrate was concentrated to obtain an extract (15.2 g). This extract was then extracted three times with equal volumes of ethyl acetate (2 L) and water (2 L). The organic layer was recovered and concentrated to obtain an ethyl acetate extract (8.28 g).

[0032] The extract was separated using Maclean macroporous resin D101. Eluent was obtained by using methanol and water in volume ratios of 1:4, 1:1, 4:1, and 1:0, followed by elution with acetone, resulting in five eluent fractions, denoted as fractions A through E.

[0033] Component C was subjected to high-performance liquid-liquid phase separation via CH3CN / H2O. （v / v） Elution with a 70:30 mixed solution yielded: CL-1 (16.4 min), CL-2 (16.4 min), and CL-3 (30.9 min); then eluted with CH3CN / H2O （v / v）Elution with a mixed solvent of 75:25 yielded CL-4 (30.4 min) and CL-5 (32.8 min), thus obtaining the ae component.

[0034] Characterization The above-mentioned ae components were characterized, and the characterization results are as follows: Component A: The carbon NMR data are as follows: 112.0, CH2; 145.0, CH; 73.4, C; 41.9, CH2; 22.9, CH2; 129.1, CH; 129.7, C; 43.6, CH2; 80.2, CH; 148.3, CH; 134.0, C; 25.5, CH2; 25.9, CH2; 122.9, CH; 133.1, C; 25.8, CH3; 17.9, CH3; 173.9, C; 16.9, CH3; 28.0, CH3. Its structural formula is shown below: , denoted as CL-1.

[0035] Component b: The carbon NMR data are as follows: 112.0, CH2; 145.0, CH; 73.4, C; 41.9, CH2; 22.9, CH2; 128.6, CH; 130.5, C; 45.5, CH2; 77.2, CH; 33.0, CH2; 38.6, CH; 31.0, CH2; 25.8, CH2; 123.0, CH; 133.1, C; 25.9, CH3; 17.9, CH3; 179.6, C; 16.6, CH3; 28.1, CH3. Its structural formula is shown below: , denoted as CL-2.

[0036] Faction c: The carbon NMR data are as follows: 112.0, CH2; 145.0, CH; 73.5, C; 41.9, CH2; 22.9, CH2; 128.3, CH; 130.5, C; 45.6, CH2; 77.6, CH; 35.3, CH2; 40.4, CH; 30.6, CH2; 25.8, CH2; 123.2, CH; 133.1, C; 25.9, CH3; 17.9, CH3; 179.6, C; 16.7, CH3; 28.0, CH3. Its structural formula is shown below: It is denoted as CL-3.

[0037] Component d: The carbon NMR data are as follows: 111.9, CH2; 145.0, CH; 73.8, C; 42.1, CH2; 22.8, CH2; 125.0, CH; 135.0, C; 39.3, CH2; 28.2, CH2; 144.0, CH2; 131.5, C; 34.8, CH2; 27.9, CH2; 123.7, CH; 132.3, C; 25.8, CH3; 17.9, CH3; 173.0, C; 16.1, CH3; 28.0, CH3. Its structural formula is shown below: It is denoted as CL-4.

[0038] Component e: The carbon NMR data are as follows: 112.0, CH2; 146.3, CH; 73.8, C; 43.4, CH2; 23.6, CH2; 126.0, CH; 135.7, C; 46.5, CH2; 26.7, CH; 33.1, CH2; 46.5, CH; 33.8, CH2; 27.0, CH2; 124.9, CH; 133.0, C; 25.9, CH3; 17.7, CH3; 180.7, C; 15.8, CH3; 27.6, CH3. Its structural formula is shown below: It is designated as CL-5.

[0039] Experimental Example 1: Effect of Component C on Seborrheic Alopecia (1) Animals Male C57BL / 6 mice, 6-8 weeks old, SPF grade. They were housed at a constant temperature of 20 ± 5°C and a relative humidity of 50 ± 5%, with free access to food and water, and exhibiting normal diurnal activity.

[0040] (2) Drug preparation Testosterone propionate injection: Weigh 100 mg of solid testosterone propionate using an analytical balance, dissolve it in 20 mL of solvent (oil for injection), and then bring the volume up to 40 mL.

[0041] Tincture base: 30% propylene glycol; 60% ethanol; 10% distilled water.

[0042] 5% minoxidil tincture: Dissolve 2 g of minoxidil in the tincture base, bring the volume to 40 mL, and vortex to mix well to obtain 5% minoxidil tincture.

[0043] 5% Crude Extract C Component Tincture: Dissolve 2 g of crude extract C component in the tincture matrix, then bring the volume to 40 mL with the tincture matrix, and vortex to mix well to obtain the 5% crude extract C component tincture.

[0044] (3) Indicator Measurement Mouse skin color changes: During the experimental period, the hair growth status of mice was judged based on the proportion of skin color change on the back of the mice and the duration of hair growth.

[0045] Body weight: The mice were weighed after induction on day 1, and again before administration of the drug. On day 22, the mice were weighed, then sacrificed and dissected.

[0046] Histopathological examination: On day 22, the mice were euthanized. Peripheral blood was collected by enucleation, and the skin from the shaved area was then removed, subcutaneous fat and connective tissue were excised, and the skin was placed in a tissue fixative containing 4% paraformaldehyde.

[0047] (4) Experimental methods See the experimental procedure diagram. Figure 1 Specifically, after one week of adaptive culture, the mice were anesthetized and their back hair was removed to fully expose their skin. The mice were randomly divided into four groups, each containing six individuals: (1) Control group (injected only with injectable oil and coated with tincture matrix); (2) Model group; (3) Control group 1 (5% minoxidil tincture); (4) Treatment group (5% crude extract C fraction). AGA symptoms were induced in the mice by testosterone propionate, which was dissolved in the injectable oil. The control mice were treated with the solvent only. The model was established on the first day, and the mice were injected every other day until the end of the experiment.

[0048] Starting from day 3 of the experiment, the treatment group received a uniform application of the tincture to the backs of mice at 3 PM daily. The control group received the same treatment. The model group received an equal amount of the basal tincture. Photos were taken the morning after administration to record changes in mouse skin color. Mice were euthanized on day 22 of the experiment. Skin from the backs of the mice was removed for histological examination, and blood was collected from the eyes.

[0049] Experimental results (1) The effect of component C on the skin color change on the back of AGA mice induced by testosterone propionate was observed.

[0050] The scoring details are as follows.

[0051]

[0052] The results are shown in Table 1 below. Figure 2 .

[0053] Table 1 Hair growth scoring results

[0054] according to Figure 2 The results showed that the mouse weight increased over time, indicating that component C had no effect on the mouse's diet and activity status.

[0055] (2) Results of hair growth records for each group of animals See results Figure 3 .

[0056] according to Figure 3 It was observed that on day 10, the skin of the control group mice was grayish-black, while the hair growth of the testosterone propionate-induced AGA mice was still in the resting phase (most of the skin remained pink) on day 10, and this condition persisted. From day 13 of modeling, compared with the model group, the hair of mice in both the Tianming extract C component and the control group entered the growth phase.

[0057] (3) HE staining slides of animals in each group Under a 20x magnifying glass, at least six fields of view were randomly selected from the HE-stained sections of mice, and the number of hair follicles in each field of view was counted.

[0058] See results Figure 4 , among which, among which ### p<0.001 compared to the control group; *** p<0.001 compared to the model group. According to... Figure 5 It can be seen that the number of hair follicles in mice treated with the tincture of component C of Tianming extract was significantly higher than that in the model group mice.

[0059] Experimental Example 2: CL-1 to CL-5 inhibit nitric oxide formation activity Experimental methods RAW264.7 macrophages were used for anti-inflammatory assays. Mouse RAW264.7 macrophages were seeded into 96-well plates (3 × 10⁶ cells / well). 5 Cells were cultured in wells (1 cell / well) at 37°C for 24 hours at 5% CO2. μ Cells were treated with different concentrations of the compound for 24 h in the presence of g / mL LPS. 100 g / mL LPS was used to treat cells. μ L Griess reagent (reagent A and reagent B) was added to the supernatant of cells treated with LPS or treated with LPS and the compound (n=3) to assess the amount of NO generated in each well. After incubation for 5 min, the absorbance was measured at 540 nm. Dexamethasone (Dex) was used as a positive control.

[0060] The experimental results are shown in Table 2.

[0061] Table 2. Anti-inflammatory activities of compounds CL-1 to CL-5 (IC50) 50 : μ M)

[0062] As shown in Table 2, the straight-chain diterpenoid compounds CL-1 to CL-5 provided in the embodiments of the present invention all exhibited in vitro anti-inflammatory activity.

[0063] Experimental Example 3: Component C and CL-1 attenuated the effects of DNCB-induced atopic dermatitis (AD) in mice. (1) Animals 5-6 week old male Kunming mice, SPF grade. They were housed at a constant temperature of 20 ± 5°C and a relative humidity of 50 ± 5%, with free access to food and water under a 12-hour light-dark cycle.

[0064] (2) Drug preparation 2% DNCB solution: Weigh 160 mg of solid DNCB using an analytical balance and dissolve it in 8 mL of solvent (6 mL acetone + 2 mL olive oil). 0.5% DNCB can be diluted with this solvent.

[0065] Cream base: white petrolatum (7 g), glyceryl monostearate (1 g), cetyl alcohol (5 g), SDS (0.5 g), glycerin (4.5 g), and pure water (to 50 g). Heat to 80°C to dissolve and maintain the temperature while stirring for 30 min.

[0066] Dexamethasone cream: Dissolve 7.5 mg of dexamethasone in 1000 ml of water. μ In L's DMSO, take 200 later. μ The dexamethasone cream is obtained by mixing L solution with 2 g cream base.

[0067] 1% Component C Cream: Dissolve 100 mg of component C in 1000 ml of water. μ In L DMSO, then take 200 μ Mix L solution with 2g of melted cream base, and after cooling, you will have a 1% C component cream.

[0068] 2% CL-1 / C component cream: Dissolve 200 mg of CL-1 / C component in 1000 ml of water. μ In L DMSO, then take 200 μ Mix L solution with 2g of melted cream base, and after cooling, you will have a 2% CL-1 / C component cream.

[0069] Cream base used in the model group: 200 μ Mix L DMSO with 2 g of melted cream base, cool, and set aside.

[0070] (3) Indicator Measurement Epidermal dermatitis scoring: During the experimental period, dermatitis scores were assessed based on the following three symptoms: erythema / bleeding, abrasion / erosion, and induration / desquamation. Each symptom was graded from 0 to 5, ranging from asymptomatic to severe, and the final score was calculated by summing.

[0071] Body weight: The mice were weighed after induction on day 1, and again before applying 0.2% DNCB. On day 22, the mice were weighed, then sacrificed and dissected.

[0072] Histopathological examination: On day 22, the mice were euthanized. Peripheral blood was collected by enucleation, and the skin from the shaved area was then removed, subcutaneous fat and connective tissue were excised, and the skin was placed in a tissue fixative containing 4% paraformaldehyde.

[0073] (4) Experimental methods The experimental procedure is as follows Figure 5 As shown: After anesthetizing the mice, the hair on the backs of the mice was removed to fully expose the skin. The mice were randomly divided into 6 groups, each containing 6 individuals: (1) blank (only the solvent was applied, acetone: olive oil = 3:1) (denoted as Control), (2) model (denoted as Modle), (3) positive control (dexamethasone) (denoted as Dex), (4) high-dose group compound 1 (2%) (denoted as H-CL), (5) high-dose group component C (2%) (denoted as H-extract), (6) low-dose group component C (1%) (denoted as L-extract). AD-like skin lesions in mice were induced by DNCB, which was dissolved in a solvent (acetone: olive oil = 3:1). The blank mice were treated with the solvent only. For the first three days, except for the blank group, 150 mg of DNCB was applied to the shaved area of ​​each group of mice. µ Mice were sensitized with 2% DNCB. Starting on day six, 50 mg of DNCB was applied to the back of the mice every other day. μ 8 times with 0.5% DNCB solution.

[0074] In the treatment group, an equal amount of cream was applied at a fixed time every morning starting from day 6. The positive control group (dexamethasone) received the same treatment as the treatment group, while the model group received an equal amount of basal cream containing the same volume of DMSO. Photos were taken the morning after administration, and dermatitis scores and weights were recorded (see figure for details). Mice were euthanized on day 22 of the experiment. Skin from the back of the mice was removed for histological examination, and blood was collected from the eyeballs.

[0075] Experimental results (1) Results of epidermatitis score and weight change The effects of CL-1 and crude extract C on dorsal skin lesions and dermatitis scores in DNCB-induced AD mice were observed.

[0076] See results Figure 6 And Table 3.

[0077] Table 3 Mouse Dermatitis Scoring Table

[0078] According to Table 3 and Figure 6 It was found that DNCB-induced AD mice exhibited severe inflammatory responses, such as erythema, induration, and scaling, on day 7 of the experiment, and these symptoms persisted. From day 10 of modeling, compared with the model group, the above symptoms were significantly improved in all treatment groups with different doses of the drug, and the improvement in dermatitis symptoms was more pronounced with increasing dose (Table 3). Except for the positive control group, the body weight of mice in other groups increased over time, indicating that CL-1 and C components had no effect on the mice's diet and activity level. Figure 6 ).

[0079] (2) Photographs of skin inflammation in animals in each experimental group See results Figure 7 .according to Figure 7 It is understood that component C and CL-1 provided in the embodiments of the present invention can improve skin inflammation.

[0080] (3) Stained sections of animals in each experimental group Under a 20x magnifying glass, randomly select at least 6 fields of view from the HE-stained sections of mice and count the epidermal thickness of the mice in each field of view. Randomly select at least 6 fields of view from the TB-stained sections of mice and count the number of mast cells of the mice in each field of view.

[0081] See results Figure 8 ,according to Figure 8 It was found that the administration of CL-1 and component C significantly reduced the epidermal thickness and mast cell number in DNCB-induced AD mice. ### p<0.001 compared to the control group; * p<0.05, ** p<0.01, *** p<0.001 compared to the model group.

[0082] Experiment 4: Component C and CL-1 attenuated the effect of imiquimod (IMQ) in inducing psoriasis-like lesions in mouse skin. (1) Animals 5-6 week old male Kunming mice, SPF grade. They were housed at a constant temperature of 20 ± 5°C and a relative humidity of 50 ± 5%, with free access to food and water under a 12-hour light-dark cycle.

[0083] (2) Drug preparation Solvent matrix: Weigh 500 mg of CMC-Na powder using an analytical balance, dissolve it in 80 mL of physiological saline, and then bring the volume up to 100 mL to obtain a 0.5% CMC-Na solution.

[0084] 0.04% Dex suspension: Weigh 16 mg of Dex powder using an analytical balance, dissolve it in 2 mL of DMSO, and take 500 ml of the solution. μ Dissolve L in 8 mL of solvent matrix, vortex mix, and then bring the volume to 10 mL to obtain 0.04% Dex suspension.

[0085] 0.04% CL-1 suspension: Weigh 16 mg of compound 1 using an analytical balance, dissolve it in 2 mL of DMSO, and take 500 ml of the solution. μ Dissolve L in 8 mL of solvent matrix, vortex mix, and then bring the volume to 10 mL to obtain 0.04% CL-1 suspension.

[0086] 1.2% Component C suspension: Weigh 480 mg of component C using an analytical balance, dissolve it in 2 mL of DMSO, and take 500... μ Dissolve L in 8 mL of solvent matrix, vortex mix, and then bring the volume to 10 mL to obtain 1.2% Dex suspension.

[0087] Blank group suspension: Take 500 μ Add L DMSO to 8 mL of solvent matrix, vortex mix, and then bring the volume to 10 mL.

[0088] (3) Indicator Measurement Epidermal scoring: During the experimental period, epidermal scores were based on the following three symptoms: erythema, psoriasis, and induration. Each symptom was graded from 0 to 5, ranging from asymptomatic to severe, and the final score was calculated by summing the scores.

[0089] Body weight: Weigh the mice after induction on day 1, and weigh them again before applying IMQ. On day 6, weigh the mice, then euthanize and dissect them.

[0090] Histopathological examination: On day 6, the mice were euthanized. Peripheral blood was collected by enucleation, and the skin from the shaved area was then removed, subcutaneous fat and connective tissue were excised, and the skin was placed in a tissue fixative containing 4% paraformaldehyde.

[0091] (4) Experimental methods The experimental procedure is as follows Figure 9As shown: After anesthetizing the mice, the hair on their backs was removed to fully expose the skin. The mice were randomly divided into 5 groups, each containing 6 individuals: (1) Control (only Vaseline was applied, and the control suspension was administered by gavage), (2) Model (3) Positive control (62.5 mg IMQ was applied to the back; 0.04% dexamethasone suspension was administered by gavage), (4) CL-1 treatment group (62.5 mg IMQ was applied to the back; 0.40% CL-1 suspension was administered by gavage), (5) Component C treatment group (62.5 mg IMQ was applied to the back; 1.2% component C suspension was administered by gavage). Psoriasis-like skin lesions were induced in mice by IMQ. Vaseline was applied to the backs of the control mice, and the control suspension was administered by gavage. Except for the control group, mice in each group were sensitized by applying 62.5 mg of IMQ cream to the shaved area from day 1 to day 5. From day 1 to day 5, mice were administered a suspension of the treatment drug (Dex and CL-1: 3 mg / Kg; component C: 100 mg / Kg) by gavage daily.

[0092] The mice were photographed the morning after drug administration, and their skin condition was scored and they were weighed (see figures for details). The mice were euthanized on the sixth day of the experiment. Skin from the mice's backs was removed for histological examination, and blood was collected from their eyes.

[0093] Experimental results (1) Results of skin score and weight change The effects of CL-1 and C components on IMQ-induced psoriatic lesions and dermatitis scores on the back of mice were observed. Results are shown in Table 4. Figure 10 .

[0094] Table 4 Mouse Dermatitis Scoring Table

[0095] according to Figure 10 As shown in Table 4, in the experiment, IMQ-induced psoriasis-like lesions in mice showed an inflammatory response starting on day 3. Symptoms persisted and gradually worsened over time. Starting on day 4 of drug administration, as shown in the table, the different treatment groups all alleviated the inflammatory response to varying degrees. CL-1 showed a similar effect to Dex, while component C showed a more significant improvement in dermatitis symptoms (Table 4). Except for the positive control group, the body weight of mice in other groups increased over time, indicating that CL-1 and component C had no effect on the mice's diet and activity level. Figure 10 ).

[0096] (2) Photographs of skin inflammation in animals in each experimental group See results Figure 11 .according to Figure 11 It is known that the linear diterpenoid compounds provided in the embodiments of the present invention can significantly improve skin inflammation.

[0097] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A linear diterpene compound, characterized by, It comprises at least one of the compounds shown in the following structural formula: 。 2. The method for preparing the linear diterpene compound according to claim 1, characterized in that, It comprises: The alcohol extract of the tuber fleeceflower root is extracted by using ester solvent-water to form an ester extract; Then, the ester extract is subjected to column chromatography, wherein the mobile phase used in the elution process of the column chromatography comprises a mixed solution formed by mixing C1-C3 alcohol solvent and water in a volume ratio of (1-5):(5-0) and C3-C5 ketone solvent.

3. The production method according to claim 2, characterized by, It comprises: The alcohol extract is extracted by using C3-C5 ester solvent-water, and the ester extract is formed after extraction and concentration; Preferably, it comprises: the alcohol extract is extracted by using ethyl acetate-water multiple times, the organic phases formed by multiple extractions are combined, and the ethyl acetate layer extract is formed after concentration.

4. The production method according to claim 2, characterized by, It comprises: The ester extract is subjected to column chromatography by using macroporous adsorption resin; The elution process of the column chromatography comprises: elution by using C1-C3 alcohol solvent and water in a volume ratio of 1:4, 1:1, 4:1, 1:0 as the mobile phase and elution by using C3-C5 ketone solvent, and collection of components A-E.

5. The production method according to claim 4, characterized by, It further comprises: The component C formed by the elution of the column chromatography is subjected to high-performance liquid separation.

6. The production method according to claim 5, wherein The elution process of the high-performance liquid separation comprises: elution by mixing acetonitrile and water in a volume ratio of 70-75:30-25 to form a mixed solvent.

7. Use of a product prepared by the preparation method of the linear diterpenoid compound of claim 1 or the linear diterpenoid compound of claim 2 in the preparation of a preparation for treating alopecia.

8. Use according to claim 7, characterized in that, The alopecia comprises seborrheic alopecia; Preferably, the preparation comprises the linear diterpenoid compound or the component C formed by the preparation method.

9. Use of a product prepared by the preparation method of the linear diterpenoid compound of claim 1 or the linear diterpenoid compound of claim 2 in the preparation of an anti-inflammatory drug.

10. Use of a product prepared by the preparation method of the linear diterpenoid compound of claim 1 or the linear diterpenoid compound of claim 2 in the preparation of a drug for treating dermatitis.

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