Callicarpa bodinieri diterpenoid compound as well as extraction method and application thereof

Abstract

The invention relates to a callicarpa bodinieri diterpenoid compound as well as an extraction method and application thereof, and relates to the technical field of compound extraction. The diterpenoid compound extracted from the callicarpa bodinieri comprises at least one of a compound 1, a compound 2, a compound 3 and a compound 4. The invention also discloses an extraction method of the compounds, and the extracted compounds 1, 2, 3 and 4 have obvious anti-inflammatory activity, are safe, effective and easy to obtain when being used for preparing skin anti-inflammatory medicines, and have wide application prospects.

Description

Technical Field

[0001] This invention relates to the field of compound extraction technology, specifically to a diterpenoid compound of Callicarpa balsamina and its extraction method and application. Background Technology

[0002] As the largest organ in the human body, the skin protects internal organs from external stimuli. Inflammation can be triggered by external stimuli such as mechanical damage, ultraviolet radiation, allergen exposure, and infection, or by internal factors such as autoimmune responses and genetic mutations, or by the complex interaction of internal and external factors. There are more than twenty types of chronic non-infectious inflammatory skin diseases, including atopic dermatitis, psoriasis, chronic spontaneous urticaria, nodular prurigo, scleroderma, and cutaneous lupus erythematosus, with a prevalence rate as high as 20-25%. Atopic dermatitis (10-30% in children, 2-10% in adults) and psoriasis (2-3%) have the highest prevalence. Inflammatory skin diseases impose a heavy burden on patients and society due to their high incidence, difficulty in diagnosis, incurability, and numerous complications.

[0003] Treatment options for inflammatory skin diseases include topical therapy, systemic therapy, and phototherapy. Topical medications mainly include corticosteroids, calcineurin inhibitors, and moisturizers. Systemic medications mainly include immunosuppressants, corticosteroids, and biologics. Phototherapy involves irradiating the damaged skin with ultraviolet light. However, corticosteroids are prone to side effects such as osteoporosis and hypertension; immunosuppressants can cause liver and kidney toxicity, bone marrow suppression, and impaired immune function; and biologics are often expensive and carry the risk of inducing non-melanoma skin cancer. These side effects limit the long-term use of these drugs. Therefore, developing safe, effective, and readily available treatments for skin inflammation remains of practical significance.

[0004] The diversity and structural complexity of natural products make them the best choice for finding novel drugs or active templates. Natural product molecules that can be used as drugs or drug lead compounds are usually secondary metabolites from plants, and exhibit different activity tendencies due to differences in their chemical structures: for example, phenolic compounds typically have antioxidant and damage-protective effects; alkaloids can be developed into psychotropic drugs and poisons; and terpenes exhibit anti-inflammatory, wound-healing, and antibacterial effects. Numerous studies have shown that natural product molecules effectively regulate the immune system. Different types of natural product molecules target and bind to specific receptors on immune cells, triggering intracellular signaling pathways for the treatment of various immune and inflammatory diseases. Therefore, continuing to search for active lead compounds or drug candidates with anti-inflammatory properties from traditional medicinal plants remains of practical significance.

[0005] White-haired long-leaved purple beauty ( Callicarpa longifolia Lamk. var. flakySchauer is a plant belonging to the genus Callicarpa in the Lamiaceae family. In my country, it is mainly distributed in Guangxi, Sichuan, and Guizhou provinces, while in other countries, it is found in India, Singapore, Indonesia, and the Philippines. According to "Research on Guizhou Traditional Chinese Medicine Resources," the medicinal part is its leaves, which have the effects of dispelling wind and dampness, and reducing inflammation. It is used to treat rheumatic pain, arthritis, and otitis media.

[0006] Currently, there is no clear record of finding active lead compounds or drug candidates for anti-skin inflammation from *Callicarpa glabra*. Summary of the Invention

[0007] To address the aforementioned technical problems, the present invention aims to provide a diterpenoid compound of *Callicarpa pubescens*, its extraction method, and its application. The diterpenoid compound extracted from *Callicarpa pubescens* includes compound 1, compound 2, compound 3, and compound 4. The present invention also discloses the extraction method for the above compounds. The extracted compounds 1, 2, 3, and 4 exhibit significant anti-inflammatory activity and are safe, effective, and readily available for use in the preparation of anti-inflammatory drugs for the skin, showing broad application prospects.

[0008] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: The first objective of this invention is to provide a diterpenoid compound of *Callicarpa pubescens*, comprising compound 1, compound 2, compound 3, and compound 4; wherein the structural formula of compound 1 is shown in formula ①; the structural formula of compound 2 is shown in formula ②; the structural formula of compound 3 is shown in formula ③; and the structural formula of compound 4 is shown in formula ④. .

[0009] The beneficial effects of this invention are: the diterpenoid compounds extracted from *Callicarpa spicata* include at least one of compound 1, compound 2, compound 3, and compound 4; this invention also discloses the extraction method of the above compounds, and the extracted compounds 1, 2, 3, and 4 have significant anti-inflammatory activity. Their use in the preparation of anti-inflammatory drugs for the skin is safe, effective, and readily available, and has broad application prospects.

[0010] Based on the above technical solution, the present invention can be further improved as follows.

[0011] The second objective of this invention is to provide a method for extracting diterpenoid compounds from *Callicarpa spicata*, comprising the following steps: (1) The dried branches and leaves of white hair long-leaved purple beauty are crushed and extracted with ethanol under reflux to obtain an extract. The extract is concentrated under reduced pressure to obtain an extract. The extract is dispersed in water and then extracted with petroleum ether, dichloromethane, ethyl acetate and n-butanol in sequence to obtain petroleum ether extract and dichloromethane extract. (2) The petroleum ether extract was subjected to silica gel column chromatography with a petroleum ether-ethyl acetate gradient elution to obtain fractions Fr.1, Fr.2, Fr.3, Fr.4, Fr.5, Fr.6, and Fr.7. Fr.6 was further separated by MCI column chromatography with a methanol-water gradient elution to obtain Fr.6.1, Fr.6.2, Fr.6.3, and Fr.6.4. Fr.6.2 was purified by reversed-phase high-performance liquid chromatography to obtain compound 4. (3) The dichloromethane extract was subjected to silica gel column chromatography gradient elution to obtain fractions Fr.1a, Fr.2a, Fr.3a, Fr.4a, Fr.5a, Fr.6a, Fr.7a, and Fr.8a. Fr.2a was eluted with silica gel column chromatography gradient to obtain Fr.2.1a, Fr.2.2a, Fr.2.3a, Fr.2.4a, Fr.2.5a, and Fr.2.6a. Fr.2.5a was eluted with MCI column chromatography gradient to obtain Fr.2.5.1a and Fr.8a. Fr.2.5.2a, Fr.2.5.3a, Fr.2.5.4a, Fr.2.5.5a, and Fr.2.5.6a; Fr.2.5.2a was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) to obtain Fr.2.5.2.1a and Fr.2.5.2.2a; Fr.2.5.2.1a was purified by RP-HPLC to obtain compound 2 and compound 3; Fr.2.5.2.2a was purified by RP-HPLC to obtain compound 1.

[0012] The beneficial effects of this invention are: the extraction method of this invention is simple and easy to operate, and the extracted compounds 1, 2, 3, and 4 have significant anti-inflammatory activity. When used in the preparation of anti-inflammatory drugs for the skin, they are safe, effective, and readily available, and have broad application prospects.

[0013] Based on the above technical solution, the present invention can be further improved as follows.

[0014] Furthermore, the ethanol reflux extraction in step (1) specifically involves reflux extraction with 95% ethanol at a liquid-to-solid ratio of 8:1 three times, each time for 2 hours.

[0015] The beneficial effect of adopting the above-mentioned further scheme is that it can increase the extraction rate.

[0016] Furthermore, the eluent for silica gel column chromatography in step (2) is petroleum ether-ethyl acetate eluent, wherein the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate eluent is 20:1 to 1:1.

[0017] Furthermore, the eluent for the MCI column chromatography in step (2) is a methanol-water eluent; the volume ratio of methanol to water in the methanol-water eluent is 1:1 to 1:0.

[0018] The advantage of adopting the above-mentioned further scheme is that it enables more precise separation of compounds.

[0019] Further, the step of purifying Fr.6.2 to obtain compound 4 by reverse high performance liquid chromatography in step (2) is as follows: Fr.6.2 is purified by reverse high performance liquid chromatography to obtain Fr.6.2.4; Fr.6.2.4 is purified by reverse high performance liquid chromatography to obtain compound 4; When purifying Fr.6.2, the eluent used is methanol-0.1% formic acid water eluent, and the volume ratio of methanol to 0.1% formic acid water is 1:1.

[0020] When purifying Fr.6.2.4, the eluent used is methanol-0.1% formic acid water eluent, and the volume ratio of methanol to 0.1% formic acid water is 55:45.

[0021] The advantage of adopting the above-mentioned further scheme is that the separation effect is optimal under these conditions.

[0022] Furthermore, the eluent used in step (3) for purifying the dichloromethane extract by silica gel column chromatography is a dichloromethane-methanol eluent; the volume ratio of dichloromethane to methanol in the dichloromethane-methanol eluent is 100:1, 50:1, 25:1, 15:1, 10:1, 5:1, 3:1, or 1:1. The eluent used in the silica gel column chromatography purification of Fr.2a is a petroleum ether-ethyl acetate eluent; the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate eluent is 20:1, 12:1, 8:1, 5:1, 3:1, or 1:1.

[0023] The advantage of adopting the above-mentioned further scheme is that the separation effect is good under these conditions.

[0024] Furthermore, in step (3), the eluent for purifying Fr2.5a by MCI column chromatography is a methanol-water eluent, and the volume ratio of methanol to water in the methanol-water eluent is 5:5, 6:4, 7:3, 8:2, 9:1, or 1:0.

[0025] Furthermore, in step (3), when purifying Fr.2.5.2a, the elution solution used is methanol-0.1% formic acid water elution solution, and the volume ratio of methanol to 0.1% formic acid water is 15:85; When purifying Fr.2.5.2.1a, the eluent used is methanol-0.1% formic acid water eluent, and the volume ratio of methanol to 0.1% formic acid water is 40:60. When purifying Fr.2.5.2.2a, the eluent used is methanol-0.1% formic acid water eluent, and the volume ratio of methanol to 0.1% formic acid water is 40:60.

[0026] The advantage of adopting the above-mentioned further scheme is that the separation effect is optimal under these conditions.

[0027] A third object of the present invention is to provide an application of the diterpenoid compound of *Callicarpa pubescens*, using the composition of claim 1 in the preparation of anti-inflammatory drugs and / or cosmetics.

[0028] The beneficial effects of this invention are: the compounds 1, 2, 3, and 4 extracted by this invention have significant anti-inflammatory activity, and their use in the preparation of anti-inflammatory drugs for the skin is safe, effective, and readily available, with broad application prospects. Detailed Implementation

[0029] The principles and features of the present invention are described below. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0030] Example 1: (1) The dried branches and leaves of *Callicarpa spicata* were crushed and then extracted three times with 95% ethanol at a liquid-to-solid ratio of 8:1, each time for 2 h. The extracts were combined and concentrated under reduced pressure to obtain an extract. The extract was then mixed with an appropriate amount of water and stirred until it became a uniform slurry without lumps. It was then extracted sequentially with petroleum ether, dichloromethane, ethyl acetate and n-butanol. The solvents were recovered by concentration under reduced pressure to obtain five extracts with different polarities: petroleum ether extract, dichloromethane extract, ethyl acetate extract and n-butanol extract. (2) The petroleum ether extract was subjected to silica gel column chromatography and eluted with petroleum ether-ethyl acetate at a rate of 40 mL / min. The volume ratios of petroleum ether to ethyl acetate during elution were 20:1, 15:1, 10:1, 8:1, 5:1, 3:1, and 1:1, respectively, yielding Fr.1, Fr.2, Fr.3, Fr.4, Fr.5, Fr.6, and Fr.7. Fr.6 was further separated by MCI column chromatography with a methanol-water gradient elution (the volume ratios of methanol to water during elution were 5:5, 7:3, 8:2, 9:1, and 1:0, respectively) at a rate of 6 mL / min. Fr.6.1, Fr.6.2, Fr.6.3, and Fr.6.4 were obtained by elution at a flow rate of L / min. Fr.6.2 was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) with methanol-0.1% formic acid aqueous solution at a rate of 3 mL / min and a methanol-0.1% formic acid aqueous solution volume ratio of 50:50, yielding Fr.6.2.1, Fr6.2.2, Fr6.2.3, Fr6.2.4, Fr6.2.5, and Fr.6.2.6. Fr.6.2.4 was purified by reversed-phase high-performance liquid chromatography with methanol-0.1% formic acid aqueous solution at a rate of 3 mL / min and a methanol-0.1% formic acid aqueous solution volume ratio of 55:45, yielding compound 4.

[0031] (3) The dichloromethane extract was subjected to silica gel column chromatography. The volume ratio of dichloromethane to methanol during elution was 100:1, 50:1, 25:1, 15:1, 10:1, 5:1, 3:1, and 1:1, respectively. The elution rate was 40 mL / min. Eight fractions were obtained: Fr.1a, Fr.2a, Fr.3a, Fr.4a, Fr.5a, Fr.6a, Fr.7a, and Fr.8a. Fr.2a was further separated by silica gel column chromatography, eluted with a petroleum ether-ethyl acetate gradient at a rate of 10 mL / min, with the following volume ratios of petroleum ether to ethyl acetate: 20:1, 12:1, 8:1, 5:1, 3:1, and 1:1, yielding Fr.2.1a, Fr.2.2a, Fr.2.3a, Fr.2.4a, Fr.2.5a, and Fr.2.6a. Fr.2.5a was purified by MCI column chromatography, eluted with methanol-water at a rate of 6 mL / min, with the following volume ratios of methanol to water: 5:5, 6:4, 7:3, 8:2, 9:1, and 1:0, yielding Fr.2.51a, Fr.2.5.2a, Fr.2.5.3a, Fr.2.5.4a, Fr.2.5.5a, and Fr.2.5.6a. Fr.2.5.2a was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) using acetonitrile-0.1% formic acid aqueous solution as elution at a rate of 3 mL / min and a volume ratio of acetonitrile to 0.1% formic acid aqueous solution of 15:85, yielding Fr.2.5.2.1a and Fr.2.5.2.2a. Fr.2.5.2.1a was purified by RP-HPLC using methanol-0.1% formic acid aqueous solution as elution at a rate of 3 mL / min and a volume ratio of methanol to 0.1% formic acid aqueous solution of 40:60, yielding compounds 2 and 3. Fr.2.5.2.2a was purified by RP-HPLC using methanol-0.1% formic acid aqueous solution as elution at a rate of 3 mL / min and a volume ratio of methanol to 0.1% formic acid aqueous solution of 40:60, yielding compound 1.

[0032] Experimental Example 1: Structural identification of compounds 1-4: Structural identification of compound 1: Pale yellow amorphous powder, m / z 329.2097 [M+Na] + The molecular formula is C 19 H 30 O3, 1 H-NMR and 13 C-NMR data are shown in Table 1, and the structural formula is shown in Equation ①: .

[0033] Table 1 Compound 1 1H-NMR and 13 C-NMR data (deuterated chloroform) (2) Structural identification of compound 2: White amorphous powder, m / z 293.2129 [M + H] + The molecular formula is C 18 H 28 O3, 1 H-NMR and 13 C-NMR data are shown in Table 2, and the structural formula is shown in Equation ②: .

[0034] Table 2 Compound 2 1 H-NMR and 13 C-NMR data (deuterated methanol) 3. Structural identification of compound 3: White amorphous powder, m / z 289.1776 [M + Na] + The molecular formula is C 16 H 26 O3, 1 H-NMR and 13 C-NMR data are shown in Table 3, and the structural formula is shown in Equation ③: .

[0035] Table 3 Compound 3 1 H-NMR and 13 C-NMR data (deuterated chloroform) (4) Structural identification of compound 4: White amorphous powder, m / z 331.1909 [M + H] + The molecular formula is C 20 H 26 O4, 1 H-NMR and 13 C-NMR data are shown in Table 4, and the structural formula is shown in Equation ④: .

[0036] Table 1 Compound 1 1 H-NMR and 13 C-NMR data (deuterated methanol) Experimental Example 2: Determination of Anti-inflammatory Activity The inhibitory effects of compounds 1-4 at 25 μM on LPS-induced NO release from RAW264.7 mouse macrophages were investigated, as follows: (1) NO inhibitory activity: RAW264.7 cells in logarithmic growth phase (Chinese Academy of Sciences Cell Bank) were used with 1.5 × 10⁻⁶ NO inhibitory agents. 4 Cells were seeded at a density of 1 / 2 well in 96-well plates and cultured for 24 h. Afterward, the culture medium (89% DMEM (Dalian Meilun Biotechnology Co., Ltd.) + 10% fetal bovine serum (Gibco) + 1% penicillin-drug antibody (Beijing Solarbio Science & Technology Co., Ltd.)) was discarded, and the cells were divided into a control group, an LPS group, and a drug-treated group. Compounds 1-4 from Example 1 were prepared as a 10 mM stock solution using DMSO, and then diluted to a final concentration of 25 μM with culture medium. The control and LPS groups were treated with 0.1 mL of culture medium containing 0.25% DMSO, while the drug-treated group was treated with 0.1 mL of drug-containing culture medium (the prepared 10 mM stock solution of compounds 1-4). After pretreatment for 2-4 h, LPS at a final concentration of 0.1 μg / mL was added for induction for 24 h. 50 μL of cell supernatant from each group was taken, and 50 μL of Griess reagent I was added, followed by 50 μL of Griess reagent II. The absorbance at 540 nm was measured using a Tecan microplate reader. The NO inhibition rate is calculated using the formula: NO inhibition rate (%) = [1 - (C 给药 -C 对照 ) / (C LPS -C 对照 )]×100%; the results are shown in Table 5.

[0037] Table 5. Inhibitory effects of compounds 1-4 on LPS-induced NO production in RAW264.7 cells. (2) Inflammatory factor assay: RAW264.7 cells in logarithmic growth phase were used to measure 3×10⁻⁶ inflammatory factors. 5The cells were seeded at a density of 1 / 2 well in 6-well plates and cultured for 24 h. The culture medium (89% DMEM medium (Dalian Meilun Biotechnology Co., Ltd.) + 10% fetal bovine serum (Gibco) + 1% penicillin antibiotics (Beijing Solarbio Technology Co., Ltd.)) was then discarded. Compound 1 was prepared into a stock solution with a concentration of 10 mM using DMSO, and then diluted with culture medium to the final concentrations (6.25, 12.5, 25 μM) to obtain the 6.25 μM group, 12.5 μM group, and 25 μM group. Dexamethasone was prepared into a stock solution with a concentration of 40 mM using DMSO, and then diluted with culture medium to 10 μM to obtain the dexamethasone group. The blank control group and LPS group were treated with 2 mL of medium containing 0.25% DMSO. The drug treatment groups (6.25 μM group, 12.5 μM group, 25 μM group, and dexamethasone group) were pretreated with 2 mL of drug-containing medium for 2 h, and then induced with 0.1 μg / mL LPS for 24 h. The cell supernatant was collected, and the contents of IL-6 and TNF-α in the supernatant were measured according to the instructions of the ELISA kit (Wuhan Yilairuit Biotechnology Co., Ltd.). The results are shown in Table 6. Table 6. Effects of compound 1 on LPS-induced inflammatory factors IL-6 and TNF-α in RAW264.7 cells. ### P <0.001 vs. Blank group, *** P <0.001 vs. LPS Group Table 6 shows that compared with the blank group, the levels of inflammatory factors IL-6 and TNF-α in the LPS group were significantly increased, indicating that the in vitro inflammation model was successfully constructed. Compared with the LPS group, the low, medium and high doses of compound 1 significantly reduced the levels of inflammatory factors IL-6 and TNF-α, indicating that it has significant in vitro anti-inflammatory activity.

[0038] The target study group and the model group processed by LPS were compared. ### P <0.001 vs. Blank group, *** P <0.001 vs. The LPS group indicates that the results are statistically significant.

[0039] Experimental Example 3: Method for Assaying Anti-Skin Inflammatory Activity HaCaT cells in logarithmic growth phase (Chinese Academy of Sciences Cell Bank) were used at a concentration of 1×10⁻⁶. 5Cells were seeded at a density of 1 / 2 well in 6-well plates and cultured for 24 h, after which the culture medium was discarded. Compound 1 was prepared as a 10 mM stock solution using DMSO, and then diluted with culture medium to final concentrations (6.25, 12.5, 25 μM). Andrographolide was prepared as a 40 mM stock solution using DMSO, and then diluted with culture medium to 10 μM. The blank control group and the IL-17A+TNF-α group were treated with 2 mL of culture medium containing 0.25% DMSO. The drug-treated groups (6.25 μM, 12.5 μM, 25 μM, and andrographolide groups) were pretreated with 2 mL of drug-containing culture medium for 2 h, followed by the addition of IL-17A and TNF-α for 24 h. Cell supernatants were collected, and the contents of IL-6 and CCL-20 in the supernatant were measured according to the instructions of the ELISA kit (Wuhan Yilairuit Biotechnology Co., Ltd.). The results are shown in Table 7.

[0040] Table 7. Effects of Compound 1 on IL-17A and TNF-α-induced inflammatory cytokine IL-6 and chemokine CCL-20 in HaCaT cells. ### P <0.001 vs. Blank group, ** P <0.01, *** P <0.001 vs. IL-17A+TNF-α group Table 7 shows that compared with the blank group, the levels of IL-6 and CCL-20 in the IL-17A+TNF-α group were significantly increased, indicating that the psoriasis-like inflammation model was successfully constructed. Compared with the IL-17A+TNF-α group, the low, medium and high doses of compound 1 significantly reduced the levels of inflammatory factor IL-6 and chemokine CCL-20, indicating its anti-psoriasis potential.

[0041] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A diterpenoid compound of *Callicarpa spp.*, characterized in that, The compound includes at least one of compound 1, compound 2, compound 3, and compound 4; wherein the structural formula of compound 1 is shown in formula ①; the structural formula of compound 2 is shown in formula ②; the structural formula of compound 3 is shown in formula ③; and the structural formula of compound 4 is shown in formula ④. 。 2. The method for extracting diterpenoid compounds from *Callicarpa spp.* according to claim 1, characterized in that, Includes the following steps: (1) The dried branches and leaves of white hair long-leaved purple beauty are crushed and extracted with ethanol under reflux to obtain an extract. The extract is concentrated under reduced pressure to obtain an extract. The extract is dispersed in water and then extracted with petroleum ether, dichloromethane, ethyl acetate and n-butanol in sequence to obtain petroleum ether extract and dichloromethane extract. (2) The petroleum ether extract was subjected to silica gel column chromatography with a petroleum ether-ethyl acetate gradient elution to obtain fractions Fr.1, Fr.2, Fr.3, Fr.4, Fr.5, Fr.6, and Fr.

7. Fr.6 was further separated by MCI column chromatography with a methanol-water gradient elution to obtain Fr.6.1, Fr.6.2, Fr.6.3, and Fr.6.

4. Fr.6.2 was purified by reversed-phase high-performance liquid chromatography to obtain compound 4. (3) The dichloromethane extract was subjected to silica gel column chromatography gradient elution to obtain fractions Fr.1a, Fr.2a, Fr.3a, Fr.4a, Fr.5a, Fr.6a, Fr.7a, and Fr.8a. Fr.2a was eluted with silica gel column chromatography gradient to obtain Fr.2.1a, Fr.2.2a, Fr.2.3a, Fr.2.4a, Fr.2.5a, and Fr.2.6a. Fr.2.5a was eluted with MCI column chromatography gradient to obtain Fr.2.5.1a and Fr.8a. Fr.2.5.2a, Fr.2.5.3a, Fr.2.5.4a, Fr.2.5.5a, and Fr.2.5.6a; Fr.2.5.2a was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) to obtain Fr.2.5.2.1a and Fr.2.5.2.2a; Fr.2.5.2.1a was purified by RP-HPLC to obtain compound 2 and compound 3; Fr.2.5.2.2a was purified by RP-HPLC to obtain compound 1.

3. The method for extracting diterpenoids from *Callicarpa spp.* according to claim 2, characterized in that, In step (1), the ethanol reflux extraction is specifically performed by using 95% ethanol at a liquid-to-solid ratio of 8:1 for reflux extraction three times, each time for 2 hours.

4. The method for extracting diterpenoid compounds from *Callicarpa spp.* according to claim 2, characterized in that, The eluent for silica gel column chromatography in step (2) is petroleum ether-ethyl acetate eluent, and the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate eluent is 20:1, 15:1, 10:1, 8:1, 5:1, 3:1, or 1:

1.

5. The method for extracting diterpenoid compounds from *Callicarpa spp.* according to claim 2, characterized in that, The eluent for the MCI column chromatography in step (2) is a methanol-water eluent; the volume ratio of methanol to water in the methanol-water eluent is 5:5, 7:3, 8:2, 9:1, or 1:

0.

6. The method for extracting diterpenoid compounds from *Callicarpa spp.* according to claim 2, characterized in that, The step of purifying Fr.6.2 to obtain compound 4 by reverse high performance liquid chromatography in step (2) is as follows: Fr.6.2 is purified by reverse high performance liquid chromatography to obtain Fr.6.2.4; Fr.6.2.4 is purified by reverse high performance liquid chromatography to obtain compound 4; When purifying Fr.6.2, the eluent used is methanol-0.1% formic acid water eluent, and the volume ratio of methanol to 0.1% formic acid water is 1:

1. When purifying Fr.6.2.4, the eluent used is methanol-0.1% formic acid water eluent, and the volume ratio of methanol to 0.1% formic acid water is 55:

45.

7. The method for extracting diterpenoid compounds from *Callicarpa spp.* according to claim 2, characterized in that, The eluent used in step (3) for purifying the dichloromethane extract by silica gel column chromatography is a dichloromethane-methanol eluent; the volume ratio of dichloromethane to methanol in the dichloromethane-methanol eluent is 100:1, 50:1, 25:1, 15:1, 10:1, 5:1, 3:1, or 1:

1. The eluent used in the silica gel column chromatography purification of Fr.2a is a petroleum ether-ethyl acetate eluent; the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate eluent is 20:1, 12:1, 8:1, 5:1, 3:1, or 1:

1.

8. The method for extracting diterpenoid compounds from *Callicarpa spp.* according to claim 2, characterized in that, The eluent for purifying Fr2.5a by MCI column chromatography in step (3) is a methanol-water eluent, and the volume ratio of methanol to water in the methanol-water eluent is 5:5, 6:4, 7:3, 8:2, 9:1, or 1:

0.

9. The method for extracting diterpenoid compounds from *Callicarpa spp.* according to claim 2, characterized in that, In step (3), when purifying Fr.2.5.2a, the elution solution used is methanol-0.1% formic acid water elution solution, and the volume ratio of methanol to 0.1% formic acid water is 15:

85. When purifying Fr.2.5.2.1a, the eluent used is methanol-0.1% formic acid water eluent, and the volume ratio of methanol to 0.1% formic acid water is 40:

60. When purifying Fr.2.5.2.2a, the eluent used is methanol-0.1% formic acid water eluent, and the volume ratio of methanol to 0.1% formic acid water is 40:

60.

10. The application of a diterpenoid compound of *Callicarpa spp.*, characterized in that, The composition of claim 1 may be used in the preparation of anti-inflammatory drugs and / or cosmetics.

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