Diterpenoid compound in honeycomb as well as preparation method and application of diterpenoid compound

Abstract

The invention discloses diterpenoid compounds in honeycomb and a preparation method and application thereof.The preparation method comprises the steps that the honeycomb is dried in the air and then ground into powder, extraction is conducted through an organic reagent, purification is further conducted through macroporous resin, sephadex, normal-phase silica gel, reverse-phase silica gel and other column chromatography methods, and the diterpenoid compounds are obtained. A monomeric compound is prepared through semi-preparative high performance liquid chromatography, and finally three diterpenoid compounds with novel structures are obtained through structural identification such as one-dimensional nuclear magnetism, two-dimensional nuclear magnetism, ECD and X-single crystal diffraction. The three diterpenoid compounds provided by the invention can inhibit the generation of nitric oxide in a cell inflammation model, and the IC50 of the compound 1 is superior to that of a positive drug indometacin. In addition, the compound 1 can down-regulate gene expression of inflammatory factors IL-1beta and IL-6, has an inhibition effect stronger than that of indomethacin with the same concentration, has remarkable anti-inflammatory activity, and can be applied to development of drugs for treating gouty arthritis.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and relates to a diterpenoid compound in honeycomb and a preparation method and application thereof. BACKGROUND

[0002] Gouty arthritis is an inflammatory arthropathy caused by monosodium urate (MSU) crystals deposited in joint capsules, bursae, cartilage, bone and other tissues, and is directly related to hyperuricemia caused by purine metabolism disorder and / or decreased uric acid excretion, and is often manifested as redness, swelling, heat, pain and dysfunction of the joint, which brings great pain to patients and seriously affects their quality of life.

[0003] At present, the methods for treating gouty arthritis in clinical practice mainly include drug therapy and non-drug therapy. In terms of drug therapy, commonly used drugs include non-steroidal anti-inflammatory drugs, colchicine, glucocorticoids, uric acid-lowering drugs and the like. However, these drugs have certain limitations in use.

[0004] Although non-steroidal anti-inflammatory drugs can relieve pain and inflammation, long-term use may cause adverse reactions in the gastrointestinal tract, such as indigestion, stomach pain, gastric bleeding and the like, and may also cause damage to liver and kidney functions. Colchicine can quickly relieve the condition of patients, but after treatment, there will be adverse reactions such as liver and kidney, gastrointestinal damage and bone marrow suppression to varying degrees. Although glucocorticoids have strong anti-inflammatory effects, long-term or large-dose use can lead to water and sodium retention, hypertension, diabetes, osteoporosis, infection and other serious complications. In the initial stage of use, uric acid-lowering drugs may induce acute gout attacks due to fluctuations in blood uric acid levels, and some patients have poor response to uric acid-lowering drug therapy or drug intolerance.

[0005] Therefore, it is of important clinical application value to explore a safe and effective drug with small side effects. SUMMARY

[0006] Based on this, the purpose of the present application is to overcome the defects or deficiencies of the prior art, and to provide a diterpenoid compound in honeycomb and a preparation method and application thereof.

[0007] A diterpenoid compound in honeycomb, the structural formula of which is shown in any one of formulas 1 to 3: .

[0008] Compared with the prior art, the diterpenoid compound has anti-inflammatory activity, can exert an anti-inflammatory effect by inhibiting the generation of NO and the expression of inflammatory factors, and can be applied to the development of drugs for treating gouty arthritis.

[0009] Further, the preparation method of the diterpenoid compound in honeycomb comprises the following steps: (1) Take dried honeycomb, use organic solvent combined with ultrasonic extraction, collect the extract and concentrate it to obtain crude extract; (2) The crude extract was purified by column chromatography and high performance liquid chromatography to obtain compounds 1-3; The column chromatography includes one or more of macroporous resin column chromatography, normal-phase silica gel column chromatography, ODS reversed-phase silica gel column chromatography, and Sephadex LH-20 gel column chromatography, with the elution system being methanol, methanol-formic acid, methanol-water, methanol-water-formic acid, or petroleum ether-ethyl acetate; the high-performance liquid chromatography is prepared using a reversed-phase silica gel column, with the mobile phase being methanol-water, methanol-water-trifluoroacetic acid, acetonitrile-water, or acetonitrile-water-trifluoroacetic acid; similar fractions are combined by TLC analysis in the column chromatography, and the TLC analysis uses 1% vanillin in 10% sulfuric acid-ethanol as the colorimetric solution.

[0010] Furthermore, the high-performance liquid chromatography preparation includes using a C18 reversed-phase silica column and eluting with 30%~50% acetonitrile-water.

[0011] Further, step (2) includes: the crude extract is subjected to macroporous resin column chromatography, and eluted with an ethanol-water system with a gradient of 0:100 to 95:5. The 95% ethanol eluent is collected and concentrated to obtain fraction Fr.5. Fraction Fr.5 was subjected to normal-phase silica gel column chromatography with gradient elution using a petroleum ether-ethyl acetate system. The fraction with a gradient of 8:1 was collected and designated as Fr.5.2, and the fraction with a gradient of 3:1 was collected and designated as Fr.5.4. Compounds 1-3 were obtained by column chromatography and high performance liquid chromatography purification of fractions Fr.5.2 or Fr.5.4.

[0012] Furthermore, the fraction Fr.5.2 was prepared by column chromatography and high-performance liquid chromatography, including: Fraction Fr.5.2 was subjected to normal-phase silica gel column chromatography, eluted with a petroleum ether-ethyl acetate gradient of 98:2 to 50:50 to remove impurities. The eluent was collected and analyzed by TLC using a mixed solution of petroleum ether and ethyl acetate in a 4:1 to 3:1 ratio as the developing solvent. The R values ​​were combined. f Using a 0.5-0.6 g eluent, we obtained Fr.5.2.1. R was then combined. f The eluent was 0.3-0.4 to obtain Fr.5.2.2; the fraction Fr.5.2.1 was further purified to obtain compound 1, and Fr.5.2.2 was further purified to obtain compound 2.

[0013] Further purification of the fraction Fr.5.2.1 includes: Fr.5.2.1 being subjected to ODS reversed-phase silica gel column chromatography, eluted with methanol-water gradient of 30:70 to 100:0, and the fractions R are combined. fUsing a 0.5-0.6 gluconate solution, we obtain Fr.5.2.1.1; The fraction Fr.5.2.1.1 was purified by Sephadex LH-20 gel column chromatography with methanol elution, and the fractions R were combined. f Using a 0.5-0.6 g eluent, we obtain Fr.5.2.1.1.1; Compound 1 was prepared by HPLC using acetonitrile-water as the mobile phase and a C18 reversed-phase column in fraction Fr.5.2.1.1.1.

[0014] Further purification of fraction Fr.5.2.2 included: fraction Fr.5.2.2 was subjected to ODS reversed-phase silica gel column chromatography, eluted with methanol-water gradient of 40:60 to 100:0, and R was combined. f Using an elution buffer of 0.3-0.4, we obtained Fr.5.2.2.1; The fraction Fr.5.2.2.1 was purified by Sephadex LH-20 gel column chromatography with methanol elution, and the fractions R were combined. f Using a 0.3-0.4 g eluent, we obtained Fr.5.2.2.1.1; Compound 2 was prepared by HPLC using acetonitrile-water as the mobile phase and a C18 reversed-phase column in fraction Fr.5.2.2.1.1.

[0015] Furthermore, the fraction Fr5.4 was prepared by column chromatography and high-performance liquid chromatography, including: Fr5.4 was purified by normal-phase silica gel column chromatography, eluted with petroleum ether-ethyl acetate in a gradient of 10:1 to 1:2, and analyzed by TLC using a 1:1 mixture of petroleum ether and ethyl acetate as the developing solvent. Rs was then combined. f With an elution buffer of 0.5-0.6, Fr.5.4.8 was obtained; Fraction Fr.5.4.8 was eluted by a normal-phase silica gel column with a methanol-water gradient of 10:1 to 1:2, and R was combined. f Using a 0.5-0.6 gluconate solution, we obtained Fr.5.4.8.1; Fraction Fr.5.4.8.1 was purified by Sephadex LH-20 gel column chromatography with methanol elution, and the fractions R were combined. f Using a 0.5-0.6 g eluent, we obtained Fr.5.4.8.1.1; Compound 3 was prepared by HPLC using acetonitrile-water as the mobile phase and a C18 reversed-phase column in fraction Fr.5.4.8.1.1.

[0016] The present invention also provides the use of the above-mentioned diterpenoid compounds in the preparation of anti-inflammatory drugs.

[0017] Furthermore, the anti-inflammatory drugs include those used to treat gouty arthritis.

[0018] To better understand and implement this invention, the following detailed description is provided in conjunction with the accompanying drawings. Attached Figure Description

[0019] Figure 1 This is a schematic diagram illustrating the preparation methods of compounds 1-3.

[0020] Figure 2 The molecular structural formulas of compounds 1-3 are shown below.

[0021] Figure 3 For compounds 1-3 1 H- 1 H COSY and HMBC are primarily related.

[0022] Figure 4 The NOESY correlation is for compounds 1-3.

[0023] Figure 5 The image shows the X-ray single-crystal diffraction crystal structure of compound 3.

[0024] Figure 6 This is the ECD spectrum of compound 1.

[0025] Figure 7 This is the ECD spectrum of compound 2.

[0026] Figure 8 This is the ECD spectrum of compound 3.

[0027] Figure 9 The figure shows the cytotoxicity results of the crude extract and fractions Fr.1~Fr.5 on Raw264.7.

[0028] Figure 10 The inhibitory effects of crude extract and fractions Fr.1~Fr.5 on NO at 10, 20, and 40 μM were determined.

[0029] Figure 11 The graph shows the cytotoxicity results of compounds 1-3 on Raw264.7.

[0030] Figure 12 The figure shows the inhibitory effect of compound 1 on NO production induced by the combined use of LPS and MSU in Raw264.7 cells.

[0031] Figure 13 The figure shows the inhibitory effect of compound 2 on NO production induced by the combined effects of LPS and MSU in Raw264.7 cells.

[0032] Figure 14The figure shows the inhibitory effect of compound 3 on NO production induced by the combined effects of LPS and MSU in Raw264.7 cells.

[0033] Figure 15 The figure shows the effect of compound 1 on the expression of the inflammatory factor IL-1β mRNA.

[0034] Figure 16 The figure shows the effect of compound 1 on the expression of the inflammatory factor IL-6 mRNA.

[0035] Figure 17 For TLC color development and R of compounds f value. Detailed Implementation

[0036] In recent years, monomeric compounds extracted from natural Chinese herbal medicines have become a hot topic in research on the treatment of gouty arthritis, and these monomeric compounds have advantages such as low toxicity, multiple targets, and high efficacy. Therefore, this invention aims to screen compounds or compositions with high efficacy against gouty arthritis from natural Chinese herbal medicines to develop novel, especially highly effective and low-toxic, drugs for the treatment of gouty arthritis.

[0037] hive( Nidus Vespae (Also known as exposed wasp nest, it is a fruit wasp, an insect belonging to the Vespidae family.) Polistes olivaceous ( DeGeer Japanese long-legged wasp Polistes japonicus Saussure Or heterocera wasp Parapolybia varia Fabricius The honeycomb is harvested in autumn and winter, dried in the sun, or slightly steamed, removing dead bees and pupae before drying. Honeycomb possesses various medicinal properties, including detoxification, insecticidal effects, wind-dispelling and dampness-removing properties, and blood circulation-promoting and pain-relieving effects. It is mainly used to treat injuries from falls and blows, pain caused by tooth decay, acne, and mastitis. Honeycomb contains a wide variety of chemical components, including phenolic acids, flavonoids, hydrocarbons, and terpenes. Pharmacological experiments have verified that honeycomb extracts not only possess anti-inflammatory, analgesic, blood-clotting, temperature-lowering, anti-thrombotic, and blood-circulating effects, but the volatile oils in honeycomb also have the ability to treat various skin diseases, including relieving itching and reducing swelling.

[0038] In previous studies, this invention discovered that beehives can treat gouty arthritis. However, beehives contain a variety of active ingredients. Therefore, this invention conducted the following screening process to identify the active ingredients in beehives that are effective in treating gouty arthritis.

[0039] First, the honeycomb was crushed and then extracted and concentrated with ethanol using ultrasound to obtain a crude extract. Then, the crude extract was subjected to macroporous resin column chromatography with gradient elution using an ethanol-water system to obtain five fractions Fr.1 to Fr.5. The crude extract and fractions were subjected to preliminary activity screening, and it was found that fraction Fr.5 had the best anti-inflammatory activity.

[0040] Fr.5 was then separated and purified by normal-phase silica gel column chromatography with gradient elution using a petroleum ether-ethyl acetate system. Similar fractions were combined based on thin-layer chromatography analysis, resulting in six fractions (Fr.5.1-Fr.5.6). The fractions Fr.5.2 and Fr.5.4, which showed stronger anti-inflammatory activity, were further purified by normal-phase silica gel column chromatography with gradient elution using a petroleum ether-ethyl acetate system. Based on thin-layer chromatography analysis, fraction Fr.5.2 yielded two major fractions, Fr.5.2.1 and Fr.5.2.2, and fraction Fr.5.4 yielded the major fraction Fr.5.4.8.

[0041] Fraction Fr. 5.2.1 was subjected to ODS reversed-phase silica gel column chromatography, Sephadex LH-20 gel column chromatography, and preparative reversed-phase high-performance liquid chromatography sequentially to obtain compound 1; fraction Fr. 5.2.2 was subjected to ODS reversed-phase silica gel column chromatography, Sephadex LH-20 gel column chromatography, and preparative reversed-phase high-performance liquid chromatography sequentially to obtain compound 2; fraction Fr. 5.4.8 was subjected to normal-phase silica gel column chromatography, Sephadex LH-20 gel column chromatography, and preparative reversed-phase high-performance liquid chromatography sequentially to obtain compound 3. The structural formulas of compounds 1-3 are as follows: Figure 2 As shown.

[0042] Activity verification of compounds 1-3 revealed that they significantly inhibited nitric oxide production in RAW264.7 mouse macrophages induced by LPS combined with MSU, with compound 1 exhibiting a more significant inhibitory effect. Furthermore, compound 1 dose-dependently downregulated the mRNA expression of two key inflammatory cytokines, IL-1β and IL-6, and at a concentration of 20 μM, its effect was superior to indomethacin. This indicates that compounds 1-3 possess the potential for developing drugs to treat gouty arthritis.

[0043] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that these descriptions are merely exemplary and not intended to limit the scope of the invention. Furthermore, descriptions of well-known structures and technologies are omitted in the following description to avoid unnecessarily obscuring the concepts of the invention.

[0044] Unless otherwise specified, the experimental methods described in the following embodiments of the present invention are generally performed under conventional conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the embodiments are commercially available products.

[0045] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0046] The following embodiments further describe the present invention, but these embodiments are not intended to limit the scope of protection of the present invention.

[0047] Example 1 This embodiment provides a method for preparing compound 1, which includes the following steps.

[0048] S1. Take dried honeycomb, extract it using organic solvents combined with ultrasound, collect the extract and concentrate it to obtain crude extract.

[0049] Specifically, 4 kg of dried honeycomb was crushed and passed through a 20-mesh sieve to obtain honeycomb powder. Then, 15 L of 60% ethanol was added, stirred thoroughly, and cold-soaked for 24 h. After that, it was ultrasonically extracted at 180 W for 1 h. The extraction was repeated 3 times, and the extracts were combined and concentrated to obtain crude extract.

[0050] S2. The crude extract was subjected to macroporous resin column chromatography and eluted using an ethanol-water gradient system with a gradient of 0:100 to 95:5. The 95% ethanol eluent was collected and concentrated to obtain fraction Fr.5.

[0051] Specifically, the crude extract was subjected to macroporous resin column chromatography with gradient elution using an ethanol-water system. Elution gradients were 0:100, 20:80, 40:60, 60:40, and 95:5, with four column volumes eluted for each gradient. The eluents of each ratio were concentrated to obtain five fractions (Fr.1–Fr.5). The crude extract and the five fractions underwent initial activity screening. Cytotoxicity was first assessed using the CCK-8 assay, followed by NO inhibition activity detection using the Griess assay. The results showed that Fr.5 exhibited the highest NO inhibition rate and the best anti-inflammatory effect, demonstrating potential for the preparation of anti-inflammatory drugs.

[0052] S3. Perform normal-phase silica gel column chromatography on fraction Fr.5, using a gradient elution system of petroleum ether-ethyl acetate. Collect the fraction with a gradient of 8:1, denoted as Fr.5.2, and collect the fraction with a gradient of 3:1, denoted as Fr.5.4.

[0053] Specifically, fraction Fr.5 was selected and subjected to normal-phase silica gel column chromatography with gradient elution using a petroleum ether-ethyl acetate system. The elution gradients were 10:1, 8:1, 5:1, 3:1, 1:1, and 2:1, with four column volumes eluted for each gradient. Each 30 mL eluent was collected into a test tube, and each test tube was analyzed sequentially by TLC. Samples showing red or purple spots on TLC with the same Rf value were enriched to obtain two target fractions, Fr.5.2 and Fr.5.4. The eluents from the 8:1 elution gradient were combined to obtain fraction Fr.5.2, and the eluents from the 3:1 elution gradient were combined to obtain fraction Fr.5.4.

[0054] S4-1 and fraction Fr.5.2 were purified by normal-phase silica gel column chromatography using a petroleum ether-ethyl acetate gradient of 98:2 to 50:50. TLC analysis was performed using a 4:1 mixture of petroleum ether and ethyl acetate as the developing solvent. Rs was combined. f The eluent was 0.5-0.6, yielding Fr.5.2.1. The fraction Fr.5.2.1 was subjected to ODS reversed-phase silica gel column chromatography, eluted with a methanol-water gradient of 30:70–100:0. R was then combined. f The eluent was 0.5-0.6, yielding Fr.5.2.1.1. The fraction Fr.5.2.1.1 was purified by Sephadex LH-20 gel column chromatography with methanol elution. The fractions R and R were then combined. f The eluent was 0.54, yielding fraction Fr.5.2.1.1.1. Compound 1 was prepared in fraction Fr.5.2.1.1.1 using acetonitrile-water as the mobile phase via HPLC with a C18 reversed-phase column. The TLC analysis used 1% vanillin in 10% sulfuric acid-ethanol as the developing solution. Step S4-1 specifically includes steps S4-1A to S4-1D.

[0055] S4-1A: Fraction Fr.5.2 was subjected to normal-phase silica gel column chromatography with gradient elution using a petroleum ether-ethyl acetate system. The elution gradients were 98:2, 96:4, 94:6, 92:8, 90:10, 88:12, 86:14, and 50:50, with four column volumes eluted per gradient. Each 30 mL eluent was collected in a test tube, and each tube was analyzed sequentially by TLC. Based on TLC spot analysis, two main fractions, Fr.5.2.1 and Fr.5.2.2, were identified. The R of Fr.5.2.1... f The value is 0.5-0.6, corresponding to an elution gradient of 98:2~96:4, and the R of Fr.5.2.2 is... f The value is 0.3-0.4, and the corresponding elution gradient is 94:6~92:8.

[0056] S4-1B: Fraction Fr.5.2.1 was subjected to ODS reversed-phase silica gel column chromatography with gradient elution using a methanol-water system. The elution gradients were 30:70, 35:65, 40:60, 45:55, 50:50, 53:47, 56:44, 59:41, 61:39, 64:36, 67:33, and 70:30. Four column volumes were eluted for each gradient, and 30 mL of eluent was collected in one test tube. Each test tube was then analyzed by TLC. Based on the TLC analysis, the major fraction Fr.5.2.1.1 was obtained, with a corresponding elution gradient of 50:50 to 70:30.

[0057] S4-1C. Fraction Fr.5.2.1.1 was subjected to Sephadex LH-20 gel column chromatography, eluted with methanol to remove impurities. Each 2 mL eluent was collected into a test tube, and each test tube was analyzed by TLC sequentially to obtain the main fraction Fr.5.2.1.1.1 (the eluent from tubes 20 to 35 is fraction Fr.5.2.1.1.1).

[0058] S4-1D and fraction Fr.5.2.1.1.1 were prepared and purified by HPLC. The mobile phase was 43% acetonitrile-water. The mixture was purified by C18 reversed-phase chromatography column. The peak at tR=21.9 min was collected to obtain compound 1 (tR 21.9 min).

[0059] Compound 1 was systematically identified using spectroscopic methods, and the results are as follows: white powder, [α] D 25 = +113.3 (c=0.01, MeOH); HRESIMS gives a quasi-molecular ion peak [M+H] + Peak m / z 287.2006 (Calculated C) 19 H 27 The molecular weight of O2 is 287.2011. 1 H-NMR and 13 C1-NMR spectra confirmed its molecular formula to be C10. 19 H 26 O2, the calculated degree of unsaturation is 7.

[0060] As shown in Table 1, 1 In H-NMR (600 MHz, CDCl3), δ H 1.34 (s, 3H), δ H 1.04–1.02 (m, 6H) represents three methyl hydrogen signals. δ H 5.65 (dd, J = 17.5, 10.7 Hz, 1H), δ H 4.91 (d, J =10.8 Hz, 1H) and δ H 4.82 (d, J = 17.5 Hz, 1H) represents the three olefin hydrogen signals. 13 C10 NMR (150 MHz, CDCl3) showed 19 carbon signals. δ C 210.7, δC 197.6 represents two independent carbonyl signals. δ C 163.1, δ C 144.9, δ C 130.5, δ C 112.0 represents four olefin carbon signals.

[0061] The direct correlation signals between carbon and hydrogen were fully assigned using HSQC spectroscopy. 1 H- 1 ¹H COSY spectroscopy revealed correlated signals for H-1 / H-2, H-4 / H-5 / H-6 / H-18, H-11 / H-12, and H-15 / H-16. HMBC spectroscopy showed H-1 correlated with C-3, H-6 with C-7, and H-19 with C-2 / C-5 / C-11. Combining these correlated signals, the structure was identified as a Labdane-type diterpene.

[0062] Table 1: Compounds 1-3 1 H-NMR and 13 C-NMR (CDCl3) data

[0063] Boltzmann distribution analysis was performed on the geometry of compound 1 using the relative thermal free energy (AG) of B3LYP / 631G(d) PCM / MeOH. Geometric optimization and frequency calculations were conducted under the B3LYP / 6-31+G condition. The ECD spectrum of (CAM-B3LYP / TZVP)(-B3LYP / 6-311G(d,p)) was calculated (see...). Figure 6 The relative configuration of compound 1 was determined to be 4. S 5 S 10 S 13 S Named (4) S 5 S 10 S ,13 S )-4,13-dimethyl-labdane-8(9),15(16)-diene-3,7-dione, with the following structural formula: .

[0064] Example 2 This embodiment provides a method for preparing compound 2, which includes the following steps, wherein steps S1-S3 are the same as in Example 1, and will not be repeated in this embodiment.

[0065] S1. Take dried honeycomb, extract it using organic solvents combined with ultrasound, collect the extract and concentrate it to obtain crude extract.

[0066] S2. The crude extract was subjected to macroporous resin column chromatography and eluted using an ethanol-water gradient system with a gradient of 0:100 to 95:5. The 95% ethanol eluent was collected and concentrated to obtain fraction Fr.5.

[0067] S3. Perform normal-phase silica gel column chromatography on fraction Fr.5, using a gradient elution system of petroleum ether-ethyl acetate. Collect the fraction with a gradient of 8:1, denoted as Fr.5.2, and collect the fraction with a gradient of 3:1, denoted as Fr.5.4.

[0068] Normal-phase silica gel column chromatography (S4-2, Fr.5.2) was performed. Impurities were removed by elution with petroleum ether-ethyl acetate in a gradient of 98:2 to 50:50. TLC analysis was then performed using a 3:1 mixture of petroleum ether and ethyl acetate as the developing solvent. Rs was combined. f The eluent was 0.3-0.4 g to obtain Fr.5.2.2. The fraction Fr5.2.2 was subjected to ODS reversed-phase silica gel column chromatography, eluted with methanol-water gradients of 40:60 to 100:0. R was combined. f The eluent was 0.3-0.4 g / mL, yielding Fr.5.2.2.1. Fr5.2.2.1 was then subjected to Sephadex LH-20 gel column chromatography with methanol elution. The fractions R were combined. f The eluent was 0.35, yielding Fr.5.2.2.1.1. Compound 2 was prepared by HPLC using acetonitrile-water as the mobile phase and a C18 reversed-phase column in the fraction Fr.5.2.2.1.1. The TLC analysis used 1% vanillin in 10% sulfuric acid-ethanol as the colorimetric solution. Step S4-2 specifically includes steps S4-2A to S4-2D.

[0069] S4-2A: Fraction Fr.5.2 was subjected to normal-phase silica gel column chromatography with gradient elution using a petroleum ether-ethyl acetate system. The elution gradients were 98:2, 96:4, 94:6, 92:8, 90:10, 88:12, 86:14, and 50:50, with four column volumes eluted for each gradient. Each 30 mL eluent was collected in a test tube, and each tube was analyzed sequentially by TLC. Based on the TLC spot analysis, two main fractions, Fr.5.2.1 and Fr.5.2.2, were obtained. S4-2B, the fraction Fr.5.2.2 was subjected to ODS reversed-phase silica gel column chromatography with gradient elution using a methanol-water system. The elution gradients were 40:60, 43:57, 46:54, 49:51, 52:48, 55:45, 58:42, 61:39, 64:36, 67:33, and 70:30, with four column volumes eluted per gradient. Each 30 mL eluent was collected in a test tube, and each tube was analyzed sequentially by TLC to obtain R. f The eluent with a concentration of 0.3-0.4 (corresponding to an elution gradient of 40:60~70:30) was collected as the major fraction Fr.5.2.2.1; S4-2C, the fraction Fr.5.2.2.1 was subjected to Sephadex LH-20 gel column chromatography, eluted with methanol to remove impurities, and 2 mL of eluent was collected in each test tube. Each test tube was then subjected to TLC analysis to obtain the major fraction Fr.5.2.2.1.1 (the eluent from tubes 20 to 40 is fraction Fr.5.2.1.1.1). S4-2D, the fraction Fr.5.2.2.1.1 was prepared and purified by HPLC. The mobile phase was 35% acetonitrile-water. The mixture was purified by C18 reversed-phase chromatography column. The peak at tR=27.2 min was collected to obtain compound 2 (tR 27.2 min).

[0070] Compound 2 was systematically identified using spectroscopic methods, and the results are as follows: yellow powder, [α] D 25 = -94.2 (c=0.01, MeOH); HRESIMS gives a quasi-molecular ion peak [M+H] + Peak m / z 303.1591 (Calculated C) 18 H 23 The molecular weight of O4 is 302.1518. 1 H NMR and 13 C NMR spectra determined its molecular formula to be C. 18 H 22 O4, the calculated degree of unsaturation is 5.

[0071] As shown in Table 1, 1 In H-NMR (600 MHz, CDCl3), δ H 12.95 (s, 1H) represents the hydroxyl hydrogen signal on the carboxyl group. δ H 2.21 (s, 3H), δ H 1.35 (s, 3H),δ H 1.24 (s, 3H) represents the three methyl hydrogen signals. 13 C18 NMR (150MHz, CDCl3) showed 18 carbon signals. δ C 205.0 is an independent carbonyl signal. δ C 181.9 represents the carbonyl group signal on a carboxylic acid.

[0072] The direct correlation signals between carbon and hydrogen were fully assigned using HSQC spectroscopy. 1 H- 1 ¹H COSY spectroscopy revealed correlated signals for H-1 / H-2, H-5 / H-6, H-11 / H-12, and H-15 / H-16. HMBC spectroscopy showed H-1 correlated with C-7 / C-18, H-11 with C-13, H-16 with C-5 / C-6 / C-17, and H-15 with C-8 / C-11 / C-13. Based on these correlated signals, the structure was identified as a Podocarpane-type diterpene.

[0073] Boltzmann distribution analysis was performed on the geometry of compound 2 using the relative thermal free energy (AG) of B3LYP / 631G(d) PCM / MeOH. Geometric optimization and frequency calculations were conducted under the B3LYP / 6-31+G condition. The ECD spectrum of (CAM-B3LYP / TZVP)(-B3LYP / 6-311G(d,p)) was calculated (see...). Figure 7 The relative configuration of compound 2 was determined to be 4. R 5 R 9 S Named (4) R 5 R 10 S )-13-hydroxy-7-oxo-4,10,14-trimethyl-podocarpa-8,11,13-trien-17-oic acid, with the following structural formula: .

[0074] Example 3 This embodiment provides a method for preparing compound 3, which includes the following steps, wherein steps S1-S3 are the same as in Example 1, and will not be repeated in this embodiment.

[0075] S1. Take dried honeycomb, extract it using organic solvents combined with ultrasound, collect the extract and concentrate it to obtain crude extract.

[0076] S2. The crude extract was subjected to macroporous resin column chromatography and eluted using an ethanol-water gradient system with a gradient of 0:100 to 95:5. The 95% ethanol eluent was collected and concentrated to obtain fraction Fr.5.

[0077] S3. Perform normal-phase silica gel column chromatography on fraction Fr.5, using a gradient elution system of petroleum ether-ethyl acetate. Collect the fraction with a gradient of 8:1, denoted as Fr.5.2, and collect the fraction with a gradient of 3:1, denoted as Fr.5.4.

[0078] S4-3 and Fr5.4 were purified by normal-phase silica gel column chromatography, eluted with petroleum ether-ethyl acetate in a gradient of 10:1 to 1:2, and analyzed by TLC using a 1:1 mixture of petroleum ether and ethyl acetate as the developing solvent. The R values ​​were combined. f The eluent was 0.5-0.6, yielding Fr.5.4.8; the fraction Fr.5.4.8 was passed through a normal-phase silica gel column and eluted with a methanol-water gradient of 10:1 to 1:2, and the fractions R were combined. f The eluent was 0.5-0.6, yielding Fr.5.4.8.1; the fraction Fr.5.4.8.1 was purified by Sephadex LH-20 gel column chromatography with methanol elution, and the fractions were combined. f The eluent was 0.53, yielding Fr.5.4.8.1.1; compound 3 was prepared by HPLC using acetonitrile-water as the mobile phase and a C18 reversed-phase column in fraction Fr.5.4.8.1.1. The TLC analysis used 1% vanillin in 10% sulfuric acid-ethanol as the chromogenic solution. Step S4-3 specifically includes steps S4-3A to S4-3D.

[0079] S4-3A: Fraction Fr.5.4 was subjected to normal-phase silica gel column chromatography with gradient elution using a petroleum ether-ethyl acetate system. The elution gradients were 10:1, 8:1, 5:1, 4.5:1, 4:1, 3.5:1, 3.1:1, 2.5:1, 2:1, 1:1, and 1:2, with each gradient eluting for 4 column volumes. Each 30 mL eluent was collected in a test tube, and each tube was analyzed sequentially by TLC. According to the TLC analysis, the fraction Fr.5.4.8, which showed a purple color, had a corresponding elution gradient of 3:1 to 1:1. S4-3B: Fraction Fr.5.4.8 was subjected to normal-phase silica gel column chromatography with gradient elution using a petroleum ether-ethyl acetate system. The elution gradients were 10:1, 5:1, 3:1, 2.5:1, 2:1, 1.5:1, 1:1, and 1:2, with 4 column volumes eluted for each gradient. Each 30 mL eluent was collected in a test tube, and each tube was analyzed sequentially by TLC. The main fraction Fr.5.4.8.1, which appeared purple, was obtained, with a corresponding elution gradient of 3:1 to 1:1. S4-3C, fraction Fr.5.4.8.1 was subjected to Sephadex LH-20 gel column chromatography, eluted with methanol. 2 mL of eluent was collected in each test tube, and each tube was analyzed sequentially by TLC. The main fraction Fr.5.4.8.1.1, which appeared purple, was obtained (the eluent from tubes 20 to 40 was fraction Fr.5.4.8.1.1). S4-3D and fraction Fr.5.4.8.1.1 were prepared and purified by HPLC using 43% acetonitrile-water as the mobile phase and purified by C18 reversed-phase chromatography. The peak at tR=35.3 min was collected to obtain compound 3 (tR35.3 min).

[0080] Compound 3 was systematically identified using spectroscopic methods, and the results are as follows: white powder, [α] D 25 = +183.0 (c=0.01, MeOH); HRESIMS gives a quasi-molecular ion peak [M+H] + Peak m / z 289.2162 (Calculated C) 19 H 29 The molecular weight of O2 is 289.2168. 1 H NMR and 13 C NMR spectra determined its molecular formula to be C. 19 H 30 O3, the calculated degree of unsaturation is 5.

[0081] As shown in Table 1, 1 In H-NMR (600 MHz, CDCl3), δ H 1.85 (d, J = 1.6 Hz, 3H), δ H 0.78 (s, 3H), δ H 0.77 (s, 3H) represents the signal from three methyl hydrogen atoms. δ H 3.12 (d, J = 10.9 Hz, 1H) and δ H 3.12 (d, J =10.9 Hz, 1H) is the hydroxymethyl signal, δ H 6.82 (t, J = 6.4 Hz, 1H), δ H 4.82 (d,J = 1.9 Hz, 1H) and δ H 4.38 (d, J = 1.8 Hz, 1H) represents 3 olefin hydrogen signals. 13 C10 NMR (150 MHz, CDCl3) showed 19 carbon signals. δ C 172.3 is the carbonyl group signal on a carboxylic acid. δ C 148.2, δ C 146.5, δ C 126.4, δ C 107.9 represents a signal from four olefin carbons. δ C 72.2 is the signal of the secondary carbon attached to the hydroxyl group.

[0082] The direct correlation signals between carbon and hydrogen were fully assigned using HSQC spectroscopy. 1 H- 1 1H COSY spectroscopy revealed correlations between H-1 / H-2, H-6 / H-7 / H-8, and H-9 / H-11 / H-12. Similar spectral signals indicated that the above data had similar chemical structures to 15-nor-14-oxo-8(17) and 12-labdadiene-18-ol, differing only in the substitution of the ketone carbonyl group at C-14, where C-14 is a carboxyl group, thus confirming a Labdane-type diterpene structure.

[0083] Using the method of X-ray diffraction of a single crystal with a copper target (see...) Figure 5 The geometry of compound 3 was analyzed by Boltzmann distribution analysis using the relative thermal free energy (AG) of B3LYP / 631G(d) PCM / MeOH. Geometric optimization and frequency calculations were performed under the B3LYP / 6-31+G condition. The ECD spectrum of (CAM-B3LYP / TZVP)(-B3LYP / 6-311G(d,p)) was calculated (see...). Figure 8 The relative configuration of compound 3 was determined to be 4. R 5 R 9 S 10 R Named (4) R 5 R 9 S 10 R)-4-(hydroxymethyl)-4,10,13-trimethyl-labdane-8(16),(12Z)-diene-14-oic acid, with the following structural formula: .

[0084] Test Example 1 Compounds 1-3 from Examples 1-3 were used as test samples in an in vitro experiment to inhibit nitric oxide release from RAW264.7 mouse macrophages induced by lipopolysaccharide combined with sodium urate (MSU). Indomethacin (a synthetic anti-inflammatory drug) was used as a positive control, and no sample treatment was used as a blank control. The samples were dissolved in DMSO, and the concentration of DMSO in the system was controlled within the range that would not affect the experimental detection. The cytotoxicity of the samples on RAW264.7 mouse macrophages was tested using the CCK-8 assay. The inhibitory effect of the samples on LPS / MSU-induced NO release in RAW264.7 mouse macrophages was determined using the Griess assay. The effect on the expression of inflammatory factor mRNA was detected using RT-qPCR.

[0085] I. Cell Culture RAW264.7 mouse macrophages were cultured in DMEM medium (containing 10% FBS, 1% penicillin, and 1% streptomycin) and placed in a 37°C, 5% CO2 incubator.

[0086] II. Cytotoxicity Evaluation (CCK8 Assay) (1) Plating: Dilute the RAW264.7 cell suspension to 200,000 / mL, add 100 μL of cell suspension (20,000 cells) to each well of a 96-well plate, and incubate at 37°C and 5% CO2 for 24 h.

[0087] (2) Once the cells have adhered, the drug can be administered. The stock solution of the compound (10 mM) was serially diluted with DMEM to set up six concentration gradients from 6.25 to 200 μM, and a control group without the drug was also set up. The original culture medium was discarded, and 100 μL of the corresponding concentration of the drug solution was added to each well, and the cells were cultured for another 24 hours.

[0088] (3) Cell viability was assessed to evaluate drug toxicity using a CCK-8 assay kit. The 96-well plate was removed, the supernatant was discarded, and 100 μL of DMEM solution containing 5% CCK-8 was added to each well in the dark. A blank control group (containing no cells) was also included. The plate was incubated at 37°C in the dark for 30 minutes.

[0089] (4) After incubation, the OD value of each well was measured at a wavelength of 450 nm using a multi-functional microplate reader. Cell viability (%) = (OD value of each well) 给药组 -OD空白对照组 ) / (OD 对照组 -OD 空白对照组 () × 100%. The median lethal concentration (LD50) and cell viability were calculated using GraphPad Prism software to determine the concentration for subsequent cell administration.

[0090] III. Griess method for detecting the inhibitory activity of compounds against NO (1) Plating: Dilute the cell suspension to 500,000 cells / mL and add 100 μL to each well of a 96-well plate, which is 50,000 cells. To reduce edge effects, add PBS buffer to the outermost ring of the wells and incubate at 37°C with 5% CO2 for 24 h.

[0091] (2) After cell adhesion, an inflammation model was induced and drugs were applied. The maximum non-toxic concentration determined by the CCK-8 assay was used as the starting concentration, and each compound was serially diluted using DMEM basal medium containing 100 ng / mL LPS. A 50 / 50 dilution method was used to set up five concentration gradients for each compound. The experimental setup included: a pure DMEM control group, a DMEM+LPS model group, and drug treatment groups at various concentrations, with four replicates per group. The pure DMEM control group was treated with DMEM basal medium, the model group was treated with LPS, and the drug treatment groups at various concentrations were treated with both LPS and the compound.

[0092] (3) After preparation, take out the 96-well plate, discard the old culture medium, and add the prepared culture medium.

[0093] (4) After culturing for 2 h, discard the old culture medium. The method is the same as step (2). The difference is that the inducing agent LPS in the model group and the drug administration group is replaced with MSU, and each well is replaced with fresh culture medium containing 200 μg / mL MSU and the corresponding concentration of drug.

[0094] (5) After culturing for 12 hours, cell viability was detected using the Griess reagent kit. 50 μL of supernatant was aspirated from each well of the original 96-well plate and transferred to a new 96-well plate. Under light-protected conditions, equal volumes of Griess reagent I and II were added sequentially, and the mixture was gently shaken to mix. Simultaneously, blank control wells (containing only 50 μL each of Griess reagent I and II) were set up. Immediately after addition, the OD values ​​of each well were measured at 570 nm using a multi-mode microplate reader. NO inhibition rate (%) = [1 - (OD 给药组 -OD 空白对照组 ) / (OD 模型组 -OD 空白对照组 () × 100%. Based on the results, the IC50 of each compound was calculated using GraphPad Prism 8 software. 50 .

[0095] IV. RT-qPCR (1) Cell plating and drug administration RAW264.7 cells were planted at a density of 1 × 10⁶ cells per well. 6 Individual samples were seeded at a density of 100 μg / mL in 6-well plates and incubated at 37°C with 5% CO2 for 24 h. Control group, model group (100 ng / mL LPS + 200 μg / mL MSU), compound 1 (5, 10, 20 μM) groups, and positive control group (indomethacin, 20 μM) were also included.

[0096] The administration route involved simultaneous administration of the LPS / MSU inducer and the drug. Before treatment, the supernatant was discarded, the sample was washed twice with PBS, and then replaced with a culture medium containing the corresponding drug and stimulant. The samples were then incubated together for 6 hours.

[0097] (2) Trizol method for RNA collection Six hours after drug treatment, the culture medium was discarded, and the cells were washed twice with pre-cooled PBS. 1 mL of Trizol was added to each well to lyse the cells, and the cells were repeatedly pipetted to lyse them and detach them from the culture plate surface. After the cells were fully lysed, the lysate was transferred to RNase-free 1.5 mL centrifuge tubes for later use.

[0098] (3) RNA extraction Add 200 μL of chloroform to the Trizol lysis buffer, vortex, and incubate in a pre-chilled centrifuge at 4°C for 3 min. Then centrifuge at 12,000 rpm for 15 min at 4°C. After centrifugation, transfer 200–400 μL of the colorless supernatant to a new RNase-free centrifuge tube. Add an equal volume of isopropanol to the new tube and mix by inverting. Incubate at 4°C for 10 min, then centrifuge at 12,000 rpm for 15 min at 4°C.

[0099] After centrifugation, the white precipitate is RNA. Remove the supernatant, add 800 μL of pre-chilled 75% ethanol (prepared with DEPC water) to each tube, and manually invert the tube back and forth. Centrifuge at 12,000 rpm for 5 min at 4°C. Discard the supernatant again, retaining the precipitate. Invert the centrifuge tubes onto clean filter paper and allow them to air dry at room temperature until the RNA precipitate becomes clear. Depending on the RNA yield, resuspend the precipitate in an appropriate amount of RNase-free DEPC water and incubate on ice for 30 min.

[0100] (4) Reverse transcription reaction RNA concentration was determined using an ultra-micro spectrophotometer. Targeting 2000 ng RNA per reaction system, the required RNA solution volume and RNase-free DEPC-free water volume were calculated and brought to a final volume of 16 μL. The calculated RNA solution, DEPC-free water, and 4 μL of 5×RT Master Mix (Prime Script) were added sequentially to the PCR tube, bringing the total volume to 20 μL. After gentle mixing and brief centrifugation, the reaction tube was placed in the PCR instrument, and the program was set (37℃, 15 min; 85℃, 5 sec; 4℃, ∞) for reverse transcription.

[0101] (5)Quantitative Real-Time PCR Dilute the cDNA template 6-fold for later use. Perform qPCR using a 10 μL reaction system, with each well containing 4 μL of cDNA and 6 μL of premix (including SYBR Green Master Mix and forward and reverse primers). Seal the PCR plate with a sealing film and centrifuge using a microplate centrifuge (2500 rpm, 3 min). Run the program on a real-time quantitative PCR instrument, setting the reaction program as follows: pre-denaturation 95℃ for 30 s; PCR reaction 95℃ for 5 s, 60℃ for 30 s; repeat the above cycle 40 times. After the reaction, perform data analysis using GraphPad software.

[0102] The results obtained using the above method are shown below. Figures 11-16 Table 2 shows the inhibitory activity of compounds 1-3 on NO in LPS-MSU-induced RAW264.7 cells and their cytotoxicity to RAW264.7 cells; IC50 50 Indicates the half-maximal inhibitory concentration (IC50) of NO; ​​CC 50 This indicates the half-maximal inhibitory concentration (IC50) for cell viability.

[0103] Table 2. NO inhibitory activity and RAW264.7 cytotoxicity of compounds 1-3

[0104] Pharmacological activity studies showed that compounds 1-3 of this invention significantly inhibited nitric oxide production in RAW264.7 mouse macrophages induced by LPS combined with MSU, compared to the IC50 of indometacin. 50 The IC50 value was 55.03 ± 2.4 μM, and the values ​​for compounds 1 and 3 were... 50 All were lower than indometacin, exhibiting inhibitory activity, with compound 1 showing a more significant inhibitory effect, and its IC50 value for LPS combined with MSU-induced NO in RAW264.7 cells was lower. 50The concentration was 28.88 ± 1.8 μM. Further studies showed that in the inflammation model, the mRNA expression levels of cytokines IL-1β and IL-6 were significantly upregulated, while compound 1 dose-dependently downregulated the mRNA expression of these two key inflammatory factors, with a superior effect compared to indomethacin at a concentration of 20 μM. In summary, these results reveal that compound 1 exerts its anti-inflammatory effect by inhibiting NO production and inflammatory factor expression, demonstrating its potential as a lead compound for the treatment of gouty arthritis.

[0105] It should be noted that the specific features, structures, materials or characteristics described in this specification can be combined in any way. For the sake of brevity, not all possible combinations of the various technical features in the above embodiments have been described. Without contradiction, those skilled in the art can combine and integrate the different embodiments and features described in this specification.

[0106] The embodiments described above are merely illustrative of several implementations of the present invention, 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 the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A diterpenoid compound found in beehives, characterized in that, The structural formula is shown in any one of the following 1 to 3: 。 2. The method for preparing diterpenoid compounds from honeycomb according to claim 1, characterized in that, Includes the following steps: (1) Take dried honeycomb, use organic solvent combined with ultrasonic extraction, collect the extract and concentrate it to obtain crude extract; (2) The crude extract was purified by column chromatography and high performance liquid chromatography to obtain compounds 1-3; The column chromatography includes one or more of macroporous resin column chromatography, normal-phase silica gel column chromatography, ODS reversed-phase silica gel column chromatography, and Sephadex LH-20 gel column chromatography, with the elution system being methanol, methanol-formic acid, methanol-water, methanol-water-formic acid, or petroleum ether-ethyl acetate; the high-performance liquid chromatography is prepared using a reversed-phase silica gel column, with the mobile phase being methanol-water, methanol-water-trifluoroacetic acid, acetonitrile-water, or acetonitrile-water-trifluoroacetic acid; similar fractions are combined by TLC analysis in the column chromatography, and the TLC analysis uses 1% vanillin in 10% sulfuric acid-ethanol as the colorimetric solution.

3. The preparation method according to claim 2, characterized in that, The high-performance liquid chromatography preparation includes using a C18 reversed-phase silica column and eluting with 30%~50% acetonitrile-water.

4. The preparation method according to claim 2, characterized in that, The step (2) includes: the crude extract is subjected to macroporous resin column chromatography, and eluted with an ethanol-water system with a gradient of 0:100 to 95:

5. The 95% ethanol eluent is collected and concentrated to obtain fraction Fr.

5. Fraction Fr.5 was subjected to normal-phase silica gel column chromatography with gradient elution using a petroleum ether-ethyl acetate system. The fraction with a gradient of 8:1 was collected and designated as Fr.5.2, and the fraction with a gradient of 3:1 was collected and designated as Fr.5.

4. Compounds 1-3 were obtained by column chromatography and high performance liquid chromatography purification of fractions Fr.5.2 or Fr.5.

4.

5. The preparation method according to claim 4, characterized in that, The preparation of fraction Fr.5.2 by column chromatography and high-performance liquid chromatography includes: Fraction Fr.5.2 was subjected to normal-phase silica gel column chromatography, eluted with a petroleum ether-ethyl acetate gradient of 98:2 to 50:50 to remove impurities. The eluent was collected and analyzed by TLC using a mixed solution of petroleum ether and ethyl acetate in a 4:1 to 3:1 ratio as the developing solvent. The R values ​​were combined. f Using a 0.5-0.6 g eluent, we obtained Fr.5.2.

1. R was then combined. f The eluent was 0.3-0.4 to obtain Fr.5.2.2; the fraction Fr.5.2.1 was further purified to obtain compound 1, and Fr.5.2.2 was further purified to obtain compound 2.

6. The preparation method according to claim 5, characterized in that, Further purification of the fraction Fr.5.2.1 includes: Fr.5.2.1 being subjected to ODS reversed-phase silica gel column chromatography, eluted with methanol-water gradient of 30:70 to 100:0, and the fractions R... f Using a 0.5-0.6 gluconate solution, we obtain Fr.5.2.1.1; The fraction Fr.5.2.1.1 was purified by Sephadex LH-20 gel column chromatography with methanol elution, and the fractions R were combined. f Using a 0.5-0.6 g eluent, we obtain Fr.5.2.1.1.1; Compound 1 was prepared by HPLC using acetonitrile-water as the mobile phase and a C18 reversed-phase column in fraction Fr.5.2.1.1.

1.

7. The preparation method according to claim 5, characterized in that, Further purification of fraction Fr.5.2.2 included: fraction Fr.5.2.2 was subjected to ODS reversed-phase silica gel column chromatography, eluted with a methanol-water gradient of 40:60 to 100:0, and R was combined. f Using an elution buffer of 0.3-0.4, we obtained Fr.5.2.2.1; The fraction Fr.5.2.2.1 was purified by Sephadex LH-20 gel column chromatography with methanol elution, and the fractions R were combined. f Using a 0.3-0.4 g eluent, we obtained Fr.5.2.2.1.1; Compound 2 was prepared by HPLC using acetonitrile-water as the mobile phase and a C18 reversed-phase column in fraction Fr.5.2.2.1.

1.

8. The preparation method according to claim 4, characterized in that, The preparation of fraction Fr.5.4 by column chromatography and high-performance liquid chromatography included: Fr.5.4 The sample was purified by normal-phase silica gel column chromatography, eluted with petroleum ether-ethyl acetate in a gradient of 10:1 to 1:2, and analyzed by TLC using a 1:1 mixture of petroleum ether and ethyl acetate as the developing solvent. The R values ​​were then combined. f With an elution buffer of 0.5-0.6, Fr.5.4.8 was obtained; Fraction Fr.5.4.8 was eluted by a normal-phase silica gel column with a methanol-water gradient of 10:1 to 1:2, and R was combined. f Using a 0.5-0.6 gluconate solution, we obtained Fr.5.4.8.1; Fraction Fr.5.4.8.1 was purified by Sephadex LH-20 gel column chromatography with methanol elution, and the fractions R were combined. f Using a 0.5-0.6 g eluent, we obtained Fr.5.4.8.1.1; Compound 3 was prepared by HPLC using acetonitrile-water as the mobile phase and a C18 reversed-phase column in fraction Fr.5.4.8.1.

1.

9. The use of the diterpenoid compound according to claim 1 in the preparation of anti-inflammatory drugs.

10. The application according to claim 9, characterized in that, The anti-inflammatory drugs include those used to treat gouty arthritis.

Images