Natural sesquiterpene lactone compound and application thereof in preparation of medicine for treating or preventing inflammatory diseases

Abstract

The invention discloses a natural sesquiterpene lactone compound and application thereof in preparation of a medicine for treating or preventing inflammatory diseases, and the structural formula of the natural sesquiterpene lactone compound is shown as campetolide A (1) and campetolide B. According to the natural sesquiterpene lactone compound, the new compounds campetolide A (1) and campetolide B (2) are extracted from branches and leaves of golden camellia, and the two compounds are found to have a remarkable inhibiting effect on NO generation; the compound can be used for preparing an NO production inhibitor, has a good application prospect in preparation of drugs for treating or preventing NO-mediated inflammatory diseases including immune system inflammation, respiratory system inflammation, digestive system inflammation and the like, and can be used as a lead compound for preparing drugs or drugs for treating or preventing NO-mediated inflammatory diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to a natural sesquiterpene lactone compound and its application in the preparation of a drug for treating or preventing inflammatory diseases. BACKGROUND

[0002] Inflammation often accompanies the occurrence of various diseases, and at the same time promotes the further development of diseases. To some extent, preventing or relieving inflammation is crucial to maintaining health. At present, various inflammations have been found, different forms caused by various stimuli, and regulated by various mechanisms (Wen et al., Front. Pharmacol. 2022, 13: 1040163). According to the speed of occurrence and clinical course of inflammation, it can be divided into two categories: acute inflammation and chronic inflammation. Acute inflammation has the characteristics of acute course and short duration, and is an early response of the body to the sudden onset of inflammatory factors (Soares et al., Biomed. Pharmacother. 2023, 168: 115764). The main pathological features of acute inflammation are exudative changes centered on blood, changes in blood flow, increased vascular permeability, and white blood cell exudation, and a variety of cytokines, chemokines, vasoactive amines, etc. If the acute inflammatory response fails to eliminate pathogens, the inflammatory process continues and is replaced in a new form, T cells and macrophages replace neutrophil infiltration, and chronic inflammation state will appear if it continues to develop. Chronic inflammation is related to many diseases (Kong et al., Signal. Transduct. Target Ther. 2022, 7: 131), such as atherosclerosis, obesity, type 2 diabetes, asthma, inflammatory bowel disease, neurodegenerative disease, rheumatoid arthritis and cancer, etc. Chronic inflammation is not only the main cause of these diseases, but also the main driving factor of the disease. In general, as an adaptive response to restore balance in the body, regulated inflammatory response is beneficial, but if the regulation is out of balance or the damage and anti-damage capacity tend to be balanced, the resulting persistent chronic inflammation not only cannot restore the balance of the body, but also can aggravate the damage to the body (Herrero-Cervera et al., Cell Mol. Immunol. 2022, 19: 177-191).

[0003] Nitric oxide (NO) is a gaseous free radical synthesized by numerous cells involved in immunity and inflammation. It also regulates the functional activity, growth, and death of macrophages, T lymphocytes, antigen-presenting cells, neutrophils, and NK cells, thereby influencing the occurrence and development of inflammation (Lundberg et al., Cell, 2022, 185:2853-2878). NO is a metabolic byproduct of nitric oxide synthase (NOS) in the conversion of L-arginine to L-citrulline. To date, three NOS subtypes have been identified (nNOS, eNOS, and iNOS). eNOS and nNOS release relatively low amounts of NO, while iNOS can synthesize large amounts of NO over a prolonged period and plays a dominant role in extended inflammatory responses. NO regulates the expression of endothelial cell adhesion factors, leukocyte-endothelial cell interactions, and the infiltration of activated leukocytes into inflammatory sites through its interaction with NF-κB, and is an important regulator of the inflammatory cascade (Seim et al., Nat. Chem. Biol. 2023, 19:265-274).

[0004] Therefore, it is of great significance to find NO production inhibitors that are highly efficient, selective, and have good pharmacokinetic properties, and they have broad application prospects for the treatment of NO-mediated inflammation-related diseases. Summary of the Invention

[0005] This application involves the protective harvesting of Camellia chrysantha branches and leaves, from which new compounds campetelolide A (1) and campetelolide B (2) were extracted. It was found that these two compounds have a significant inhibitory effect on NO production and can be used to prepare NO production inhibitors. They have good application prospects in the preparation of drugs for treating NO-mediated inflammatory diseases, including immune system inflammation, respiratory system inflammation, and digestive system inflammation. They can be used as lead compounds for the preparation of drugs for treating or preventing NO-mediated inflammatory diseases.

[0006] The compounds campetelolide A(1) and campetelolide B(2) involved in this application were extracted and isolated from the branches and leaves of Camellia chrysantha, and have the following chemical structural formulas:

[0007]

[0008] This invention reveals that compounds campetelolide A and B significantly inhibit NO production in LPS-induced RAW264.7 cells during LPS-induced inflammation suppression experiments, without affecting cell viability. Therefore, they have significant potential applications in the pharmaceutical field, particularly in the preparation of drugs for the prevention and / or treatment of NO-mediated inflammation-related diseases.

[0009] This application also provides the use of a natural sesquiterpene lactone compound in the preparation of a drug for the prevention or treatment of NO-mediated inflammatory diseases, wherein the natural sesquiterpene lactone compound is a compound represented by formula (1) or / and formula (2), that is, the natural sesquiterpene lactone compound of formula (1) or formula (2) can be used alone or in combination.

[0010] Optionally, the inflammatory disease includes inflammation of the immune system, respiratory system, or digestive system.

[0011] Inflammation of the immune system includes, but is not limited to, rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, allergic rhinitis, allergic asthma, and eczema. Inflammation of the respiratory system includes, but is not limited to, rhinitis, sinusitis, pharyngitis, tonsillitis, tracheitis, bronchitis, pneumonia, and pleurisy. Inflammation of the digestive system includes, but is not limited to, gastritis, enteritis, colitis, hepatitis, pancreatitis, and cholecystitis.

[0012] This application also provides the use of a natural sesquiterpene lactone compound in the preparation of a NO production inhibitor, wherein the natural sesquiterpene lactone compound is a compound of formula (1) or / and formula (2).

[0013] This application also provides a pharmaceutical composition comprising a therapeutically effective amount of a natural sesquiterpene lactone compound as described in formula (1) or / and formula (2).

[0014] The compounds described in this application can be used alone or in combination with other active ingredients, or combined with pharmaceutically acceptable carriers or excipients, and can be formulated into oral or non-oral dosage forms for the treatment of NO-mediated inflammatory diseases using conventional methods, preferably in the form of tablets, capsules, granules or injections.

[0015] This application also provides a method for preparing the sesquiterpene lactone compound, wherein the preparation method involves extraction from natural plants, comprising:

[0016] S1: Dry the branches and leaves of Camellia chrysantha at room temperature, pulverize them, and extract them once or multiple times at room temperature with a methanol-water solution of 70% or higher concentration. Concentrate the extract, suspend it in water, and extract it sequentially with equal volumes of petroleum ether, ethyl acetate and n-butanol to obtain four components: petroleum ether extract, ethyl acetate extract, n-butanol extract and water.

[0017] S2: After the ethyl acetate extract was concentrated under reduced pressure, it was sequentially subjected to silica gel column chromatography and MCI column chromatography with gradient elution solvent, and then sequentially separated by Sephadex LH-20 gel and reversed-phase semi-preparative high performance liquid chromatography to obtain the compound with the structural formula shown in formula (1).

[0018] Optionally, the methanol-water solution is a 90% methanol-water solution, i.e., a mixed solution with a methanol-water volume ratio of 90:10.

[0019] Optionally, the methanol-water solution in S1 can be extracted at room temperature 3 times or more, up to 5 times. There is no special limit to the extraction time at room temperature, which can be 12 hours or more per extraction.

[0020] Optionally, when the ethyl acetate extract is concentrated under reduced pressure and then passed through silica gel column chromatography, the elution gradient is set as follows: petroleum ether-ethyl acetate volume ratio 15:1 → 10:1 → 5:1 → 3:1 → 1:1 → 0:1, to obtain four components Fr.1 to Fr.4.

[0021] Optionally, the eluent fraction Fr.4 with a petroleum ether-ethyl acetate volume ratio of 3:1 is separated by MCI column chromatography with an elution gradient of methanol-water volume ratio of 70:30→80:20→90:10→100:0. Based on the colorimetric analysis of thin-layer chromatography (TLC), fractions 70:30, 80:20, and 90:10 are combined to obtain three fractions Fr.4A to Fr.4C. The fraction Fr.4B with a methanol-water volume ratio of 80:20 is eluted with Sephadex LH-20 using methanol as the mobile phase. Based on the colorimetric analysis of TLC and the Rf value, four subfractions Fr.4B1 to Fr.4B4 are combined. The subfraction Fr.4B1 with a TLC ratio of CH2Cl2:CH3OH, 15:1 v / v, and an Rf value of 0.2–0.3 is then subjected to semi-preparative high-performance liquid chromatography (HPLC) with a methanol-water volume ratio of 65:35. t R =7.1 min, the component is obtained, and the compound with the structure shown in formula (1) is obtained.

[0022] Collect t R = 14.6 min, the component is obtained, and the compound with the structure shown in formula (1) is obtained.

[0023] The compounds described in this invention can be obtained by isolation and purification from plants; they can also be synthesized by chemical methods well known to those skilled in the art.

[0024] Compared with the prior art, this application has the following beneficial effects:

[0025] This application provides campetelolide A and B, compounds with novel chemical structures, for the treatment of NO-mediated inflammatory diseases. Campetelolide A and B exhibit significant anti-inflammatory activity by inhibiting LPS-induced NO production in RAW264.7 cells, and show promise for applications in NO-mediated inflammatory diseases, especially chronic inflammatory diseases. Attached Figure Description

[0026] Figure 1 It is Campelolide A(1) 1 1H NMR spectrum (in CD3OD, 400M).

[0027] Figure 2 It is Campelolide A(1) 13 C10 NMR spectrum (in CD3OD, 100M).

[0028] Figure 3 It is Campelolide B(2) 1 1H NMR spectrum (in CD3OD, 400M).

[0029] Figure 4 It is Campelolide B(2) 13 C10 NMR spectrum (in CD3OD, 100M). Detailed Implementation

[0030] The technical solution of this application will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0032] Natural products, characterized by structural complexity and diversity, are an important source of new drug discovery. The unique chemical structures of natural products and their derivatives endow them with advantages such as high efficacy, high selectivity for specific targets, and potentially unique mechanisms of action. Therefore, the search for and development of novel and highly effective LPS-induced inflammation inhibitors in RAW264.7 cells from natural active ingredients holds significant research value.

[0033] Golden camellia (scientific name: *Camellia petelotii*) is an evergreen shrub belonging to the genus *Camellia* in the family Theaceae. It is a rare and endangered ornamental plant, currently a Class I protected plant in China. The tree grows to a height of 2–5 meters, with sparse branches, pale grayish-yellow bark, and dark green, narrowly oblong leaves. The flowers are golden yellow. Golden camellia is a common traditional medicine and is also considered a novel food. This plant, used for both medicinal and edible purposes, exhibits various pharmacological effects, including antioxidant, anticancer, antibacterial, hypoglycemic, hypolipidemic, immunomodulatory, anti-allergic, anti-anxiety, and antidepressant effects (Wu et al., Foods 2023, 12:3010). Currently, only a few triterpenoid saponins with α-glucosidase inhibitory activity have been reported in *Camellia petelotii* (Cuc et al., Chem. Biodivers. 2023, 20:e202300093), and its chemical composition requires further research. This application involves the protective harvesting of Camellia chrysantha branches and leaves, and the isolation of compounds campetelolide A and B from a 90% methanol extract of the branches and leaves. Multiple pharmacological studies have shown that these compounds possess significant anti-inflammatory activity in inhibiting LPS-induced NO production in RAW264.7 cells.

[0034] In another embodiment, the compound can also be synthesized by conventional chemical methods.

[0035] The following is a description using specific examples:

[0036] Golden flower tea leaves were harvested from Pingbian County, Yunnan Province, and dried in the shade before being pulverized into powder. Specific rotation was measured using an SGW-1 polarimeter at 25℃. Ultraviolet (UV) spectral data were obtained using a Shimadzu PharmaSpec-1800 UV spectrometer. High-resolution mass spectrometry (HR-ESI-MS) was performed using a Thermo Fisher Q-Exactive mass spectrometer, employing an ESI ion source to simultaneously obtain both positive and negative ion modes. NMR was obtained using a Bruker Avance II 400 nuclear magnetic resonance spectrometer, with chemical shifts referenced to the undeuterated residual solvent peaks, expressed as δ (ppm). Thin-layer chromatography (TLC) plates were purchased from Qingdao Ocean Chemical Co., Ltd., and UV (λ: 254nm, 365nm) and sulfuric acid-vanillin solution were used for color development. Column chromatography primarily used MCI microporous resin CHP 20P (Mitsubishi Chemical Industries, 75-150μm) and Sephadex LH-20 gel (GE Healthcare BioSciences). AB); The Shimadzu CBMA-20A analytical and semi-preparative liquid chromatography system is equipped with an SPD-20A photodiode array detector (PDA) and a Cosmosil Packed Column (10×250mm, 5μm, 5C18-MS-II or πNAP).

[0037] Example 1: Preparation of compounds Campelolide A and Campelolide B

[0038] 8.6 kg of dried Camellia chrysantha branches and leaves were thoroughly pulverized and extracted four times with 10 L of 90% methanol at room temperature for 24 hours each time. The extracts were combined and concentrated under reduced pressure to obtain 700 g of total extract. The extract was dispersed in 2 L of water and extracted three times each with equal volumes of petroleum ether, ethyl acetate, and n-butanol to obtain four fractions: petroleum ether, ethyl acetate, n-butanol, and water. The ethyl acetate fraction, totaling 50.1 g, was eluted by silica gel column chromatography with a petroleum ether-ethyl acetate gradient (15:1→10:1→5:1→3:1→1:1→0:1, v / v) to obtain four fractions (Fr.1~Fr.4).

[0039] Fraction Fr.4 (15.5 g, 3:1 eluent) was separated by MCI column chromatography using a methanol-water gradient elution (70:30→80:20→90:10→100:0, v / v). Based on the colorimetric results of thin-layer chromatography, the 70:30, 80:20, and 90:10 fractions were combined to obtain three fractions (Fr.4A to Fr.4C). Fr.4B (80:20 eluent) was purified using Sephadex LH-20 (methanol as mobile phase), and then combined based on the colorimetric results of thin-layer chromatography and Rf values ​​to obtain four subfractions Fr.4B1 to Fr.4B4. Fr.4B1 (thin-layer chromatography CH2Cl2:CH3OH, 15:1, v / v, Rf value 0.2~0.3) was further separated by semi-preparative high performance liquid chromatography (methanol-water, 65:35) to finally obtain compound Campelolide A(1) (7.2mg, flow rate: 3mL / min, t R =7.1min) and compound Campelolide B(2) (3.7mg, flow rate: 3mL / min, t R =14.6min), the structure is shown in the figure below.

[0040]

[0041] The physicochemical data of the compound are as follows:

[0042] Campetelolide A(1), its NMR and physicochemical data are as follows:

[0043] White, amorphous powder; [α] 25 D -54.8(c 0.11,MeOH); UV(MeOH)λ max (logε):207(3.80),251(3.60)nm; ECD(MeOH)λ max (Δε)244(-10.5),340(+1.3)nm; 1 H NMR (in CD3OD, 400MHz): δ H3.26(1H,dd,J=16.8,4.2Hz,H-2α),3.13(1H,d,J=16.8Hz,H-2β),3.89(1H,d,J=4.2Hz,H-3),3.23(1H,m,H-5),4.48(1H,t,J=10.6Hz,H-6),3.42(1H,dtd,J=10.6,3.2,1.7Hz,H-7),5.78(1H,dt,J=5.9,1.7Hz,H-8),2.15(1H,dd,J=15.6,7.8Hz,H-9α),3.62(1H,dd,J=15.6,5.9Hz,H-9β),5.56(1H,d,J=3.3Hz,H-13a),6.15(1H,d,J=3.3Hz,H-13b),9.94(1H,s,H-14),1.61(3H,s,H-15),6.00(1H,dd,J=7.2,1.5Hz,H-3′),1.81(3H,dd,J=7.2,1.5Hz,H3-4′),1.69(3H,t,J=1.5Hz,H3-5′); 13 13C NMR(in CD3OD,100MHz):δ C 168.2(C-1),38.5(C-2),80.1(C-3),82.1(C-4),57.6(C-5),77.0(C-6),54.9(C-7),65.7(C-8),28.2(C-9),133.2(C-10),137.4(C-11),170.8(C-12),120.2(C-13),193.2(C-14),22.9(C-15),168.1(C-1′),128.8(C-2′),139.0(C-3′),16.0(C-4′),20.6(C-5′);HRESIMSm / z 399.1418[M+Na] + ,(calcd for C 20 H 24 O7Na,399.1420).

[0044] Campetelolide B(2), its nuclear magnetic and physical and chemical data are as follows:

[0045] White, amorphous powder; [α] 25 D -23.4(c 0.10,MeOH); UV(MeOH)λ max (logε):207(3.76),256(3.51)nm; ECD(MeOH)λ max(Δε)217(-25.1)nm; 1 H NMR (in CD3OD, 400MHz): δ H 2.49(1H,m,H-2α),3.05(1H,m,H-2β),4.10(1H,m,H-3),2.93(1H,m,H-5),4.42(1H,t,J=10.6Hz,H-6),3.25( 1H,m,H-7),5.73(1H,m,H-8),2.46(1H,dd,J=15.6,7.5Hz,H-9α),2.97(1H,dd,J=15.6,5.9Hz,H-9β),5.58(1 H,d,J=3.3Hz,H-13a),6.17(1H,d,J=3.3Hz,H-13b),3.79(1H,d,J=12.3Hz,H-14a),4.00(1H,d,J=12.3Hz,H- 14b),1.38(3H,s,H-15),6.09(1H,m,H-3′),1.86(3H,dd,J=7.2,1.5Hz,H3-4′),1.77(3H,t,J=1.5Hz,H3-5′); 13 CNMR (in CD3OD, 100MHz): δ C 132.8(C-1),39.3(C-2),66.5(C-3),81.6(C-4),57.7(C-5),78.5(C-6),54.3(C-7),66.5(C-8),35.4(C-9),133.2(C-10),137.2(C-11 ),170.7(C-12),120.6(C-13),65.4(C-14),17.3(C-15),168.3(C-1′),128.9(C-2′),139.3(C-3′),16.2(C-4′),20.8(C-5′); HRESIMS m / z 419.1239[M+CH3CN] + ,(calcd for C 22 H 29 NO3, 419.1240).

[0046] Campetelolide A(1) 1 H NMR spectrum (in CD3OD, 400M) as shown Figure 1 As shown, Campelolide A(1) 13 C NMR spectrum (in CD3OD, 100M) as shown Figure 2 As shown; Campelolide B(2) 1H NMR spectrum (in CD3OD, 400M) as shown Figure 3 As shown, Campelolide B(2) 13 C NMR spectrum (in CD3OD, 100M) as shown Figure 4 As shown.

[0047] Example 2: Anti-inflammatory activity test of compounds as shown in formulas (1) and (2)

[0048] Experimental methods:

[0049] 1. Cells and their culture

[0050] RAW264.7 cells were purchased from Beyotime Biotech Ltd. Cells were cultured in DMEM medium (Gibco, USA) containing 10% fetal bovine serum (inactivated at 55°C for 30 min, PAN, Germany) and 1% penicillin-streptomycin solution (Gibco, USA) at 37°C with 5% CO2.

[0051] 2. NO detection

[0052] RAW264.7 cells were seeded at a density of 10,000 cells / well in 96-well plates and incubated overnight. Subsequently, different concentrations of the test compound were added for pretreatment for 1 h. Then, 0.5 μg / mL of LPS (E. coli O111:B4, Sigma, Shanghai) was added for further stimulation for 14 h. Afterward, 50 μL of the supernatant was collected, and 50 μL of Griess Reagent I and II (Beyotime, Shanghai) were added respectively. The cells were incubated at room temperature for 2 min, and the absorbance was measured at 562 nm. The NO content was calculated by substituting the absorbance into the NO standard curve.

[0053] 3. Cell viability assay

[0054] After NO assay, the culture medium was removed from the cells, and 100 μL of fresh DMEM medium containing 10% CCK8 reagent (APExBio, Houston, USA) was added to each well. The cells were incubated at 37°C for 1 h. The absorbance was measured at 450 nm, and the relative cell viability compared to the LPS stimulation group was calculated (% = 100% * OD value / average OD of the LPS stimulation group).

[0055] The test results showed that both compounds exhibited significant inhibitory activity against LPS-induced RAW264.7 cells, and no obvious cytotoxic effect was observed at a concentration of 20 μM (see table below for details). A positive control was 1400w (N-[3-(aminomethyl)benzyl]acetamidine), IC50... 50 The value is 1.05±0.56μM.

[0056] Table 1. Inhibitory activity of RAW 264.7 cells against LPS-stimulated NO production.

[0057]

[0058] These data are expressed as the mean ± SEM of three replicate experiments. b Positive control.

[0059] The above results indicate that the compounds described in this application can be used to prepare drugs for treating NO-mediated inflammation or as lead compounds for such drugs.

[0060] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. The use of natural sesquiterpene lactone compounds in the preparation of drugs for the prevention or treatment of NO-mediated inflammatory diseases, characterized in that, The natural sesquiterpene lactone compound is a compound of formula (1) or / and formula (2):

2. The use according to claim 1, characterized in that, The inflammatory diseases mentioned include inflammation of the immune system, respiratory system, or digestive system.

3. The use of natural sesquiterpene lactone compounds in the preparation of NO production inhibitors, characterized in that, The natural sesquiterpene lactone compound is a compound of formula (1) or / and formula (2):

4. A pharmaceutical composition, characterized in that, Including therapeutically effective amounts of natural sesquiterpene lactone compounds as described in formula (1) and / or formula (2):

5. The pharmaceutical composition according to claim 4, characterized in that, The natural sesquiterpene lactone compounds are combined with excipients to form tablets, capsules, granules or injections.

6. A natural sesquiterpene lactone compound, characterized in that, Its structural formula is shown in equation (1):

7. The method for extracting natural sesquiterpene lactone compounds as described in claim 6, characterized in that, include: S1: Dry the branches and leaves of Camellia chrysantha at room temperature, pulverize them, and extract them once or multiple times at room temperature with a methanol-water solution of 70% or higher concentration. Concentrate the extract, suspend it in water, and extract it sequentially with equal volumes of petroleum ether, ethyl acetate and n-butanol to obtain four components: petroleum ether extract, ethyl acetate extract, n-butanol extract and water. S2: After the ethyl acetate extract was concentrated under reduced pressure, it was sequentially subjected to silica gel column chromatography and MCI column chromatography with gradient elution solvent, and then sequentially separated by Sephadex LH-20 gel and reversed-phase semi-preparative high performance liquid chromatography to obtain the compound with the structural formula shown in formula (1).

8. The extraction method according to claim 7, characterized in that, The methanol-water solution is a 90% methanol-water solution.

9. The extraction method according to claim 7, characterized in that, After the ethyl acetate extract was concentrated under reduced pressure, the elution gradient was set as follows for silica gel column chromatography: petroleum ether-ethyl acetate volume ratio 15:1 → 10:1 → 5:1 → 3:1 → 1:1 → 0:1, yielding four fractions Fr.1 to Fr.

4.

10. The extraction method according to claim 9, characterized in that, The eluent fraction Fr.4, with a petroleum ether-ethyl acetate volume ratio of 3:1, was separated by MCI column chromatography with an elution gradient of methanol-water volume ratio of 70:30→80:20→90:10→100:

0. Based on the colorimetric results of thin-layer chromatography (TLC), the 70:30, 80:20, and 90:10 fractions were combined to obtain three fractions Fr.4A to Fr.4C. The fraction Fr.4B, with a methanol-water volume ratio of 80:20, was eluted with Sephadex LH-20 using methanol as the mobile phase. Based on the colorimetric results of TLC and the Rf value, four subfractions Fr.4B1 to Fr.4B4 were combined. The subfraction Fr.4B1, with a TLC ratio of CH2Cl2:CH3OH, 15:1 v / v, and an Rf value of 0.2–0.3, was further separated by semi-preparative high-performance liquid chromatography (HPLC) with a methanol-water volume ratio of 65:

35. The t R =7.1 min, the component is obtained, and the compound with the structure shown in formula (1) is obtained.

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