Diterpenoids, their extraction methods, and applications

Abstract

This invention belongs to the technical field of terpenoid compounds, and provides diterpenoid compounds, their extraction methods, and applications. The diterpenoid compounds provided by this invention have the structures shown in Formulas 1 and 2. The diterpenoid compounds provided by this invention enrich the diversity of diterpenoid compounds. Simultaneously, the diterpenoid compounds provided by this invention exhibit excellent anti-inflammatory and antibacterial properties, especially showing good inhibitory activity against Ganoderma lucidum, Fusarium solani, and Fusarium oxysporum. This invention also provides an extraction method for the diterpenoid compounds described above. The extraction method provided by this invention is simple to operate and yields diterpenoid compounds with high purity.

Description

Technical Field

[0001] This invention relates to the field of terpenoid compound technology, and more particularly to diterpenoid compounds, their extraction methods, and applications. Background Technology

[0002] Terpenes are hydrocarbons and their oxygen-containing derivatives that are multiples of isoprene units. They are a large and highly diverse class of natural products, including monoterpenes, sesquiterpenes, diterpenes, sesquiterpene cyclohexenones, triterpenes, and more. Many terpenes have been developed into important drugs for treating cancer, bacterial infections, malaria, and various other human diseases. Therefore, the synthesis of terpenes is extremely important. However, due to the incomplete elucidation of biosynthetic pathways, the non-modular structure of terpenes, and the lack of a universally applicable synthetic strategy, the source of terpenes still relies on the extraction of natural products.

[0003] According to literature reports, diterpenoids possess a variety of biological activities, including anti-inflammatory, anti-cytotoxic, anti-tumor, and antibacterial effects. Therefore, developing novel diterpenoids is of great significance in addressing the current situation of antibiotic overuse. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide diterpenoid compounds, their extraction methods, and applications. The diterpenoid compounds provided by the present invention broaden the types of terpenoid compounds, and at the same time, the diterpenoid compounds provided by the present invention possess excellent anti-inflammatory and antibacterial properties.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides two diterpenoid compounds having the structures shown in Formula 1 and Formula 2:

[0007]

[0008] This invention also provides a method for extracting diterpenoid compounds as described in the above technical solution, comprising the following steps:

[0009] The heartwood powder of the bald cypress tree trunk was mixed with an ethanol solution and then subjected to reflux extraction and vacuum evaporation to obtain a crude extract.

[0010] The crude extract was dissolved in water and then extracted with petroleum ether to obtain the petroleum ether phase.

[0011] The petroleum ether phase was concentrated under vacuum to obtain a petroleum ether extract;

[0012] The petroleum ether extract was mixed with silica gel, and then subjected to a first silica gel column chromatography elution using the first, second, third, and fourth petroleum ether-ethyl acetate systems and the first, second, third, and fourth dichloromethane-methanol systems, respectively, to obtain fractions Fr.1, Fr.2, Fr.3, Fr.4, Fr.5, Fr.6, Fr.7, and Fr.8. In the first petroleum ether-ethyl acetate system, the volume ratio of petroleum ether to ethyl acetate was 100:0, and in the second petroleum ether-ethyl acetate system, the volume ratio of petroleum ether to ethyl acetate was 65–7. The volume ratio of petroleum ether to ethyl acetate in the third petroleum ether-ethyl acetate system is 25-35:1; the volume ratio of petroleum ether to ethyl acetate in the fourth petroleum ether-ethyl acetate system is 1-5:1; the volume ratio of dichloromethane to methanol in the first dichloromethane-methanol system is 100:0; the volume ratio of dichloromethane to methanol in the second dichloromethane-methanol system is 105-95:1; the volume ratio of dichloromethane to methanol in the third dichloromethane-methanol system is 75-85:1; and the volume ratio of dichloromethane to methanol in the fourth dichloromethane-methanol system is 25-35:1.

[0013] The Fr.8 fraction was eluted by a second silica gel column chromatography using dichloromethane to obtain the crude Fr.8 fraction.

[0014] The crude Fr.8 fraction was separated by gel column chromatography using methanol to obtain Fr.8-I, Fr.8-II, Fr.8-III, and Fr.8-IV fractions.

[0015] The Fr.8-III fraction was eluted by a third silica gel column chromatography using a petroleum ether-ethyl acetate system with a volume ratio of 9:1 to obtain the Fr.8-III-1, Fr.8-III-2, Fr.8-III-3, Fr.8-III-4 and Fr.8-III-5 fractions.

[0016] The Fr.8-III-3 fraction was purified by gel column chromatography using methanol to obtain the compound with the structure shown in Formula 1.

[0017] The Fr.8-III-1 fraction was purified by silica gel column chromatography using a petroleum ether-ethyl acetate system with a volume ratio of 11:1 to obtain the compound with the structure shown in Formula 2.

[0018] Preferably, the particle size of the bald cypress heartwood powder is 1 mm to 3 mm, and the volume concentration of the ethanol solution is greater than or equal to 80%.

[0019] Preferably, the ratio of the powdered heartwood of the bald cypress trunk to the ethanol solution is (30-50) g: (50-120) mL.

[0020] Preferably, the reflux extraction temperature is 55-65°C, the reflux extraction is performed 2-3 times, and the reflux extraction time for each extraction is 4-6 hours.

[0021] Preferably, the vacuum degree of the reduced pressure evaporation is 0.07-0.09 MPa and the temperature is 40-50°C.

[0022] Preferably, the mass ratio of the crude extract to the water-soluble liquid is 1:1 to 3, the volume ratio of the mixture obtained after dissolving the crude extract in water to the petroleum ether extracted by petroleum ether is 1:1, and the petroleum ether extraction is performed 2 to 4 times.

[0023] Preferably, the vacuum concentration is performed at a vacuum level of 0.07–0.09 MPa and a temperature of 40–50 °C.

[0024] Preferably, the silica gel has a particle size of 200-400 mesh, and the mass of the silica gel is 0.8-1.5 times the dry weight of the petroleum ether extract.

[0025] The present invention also provides the application of the diterpenoid compounds described in the above technical solutions or the diterpenoid compounds obtained by the extraction methods described in the above technical solutions in the preparation of antibacterial drugs or anti-inflammatory drugs.

[0026] This invention provides two diterpenoid compounds.

[0027] The diterpenoid compounds provided by this invention enrich the diversity of diterpenoid compounds. Furthermore, the diterpenoid compounds provided by this invention exhibit excellent anti-inflammatory and antibacterial properties, particularly showing good inhibitory activity against Ganoderma lucidum, Fusarium solani, and Fusarium oxysporum.

[0028] The present invention also provides a method for extracting diterpenoids as described in the above technical solution. The extraction method provided by the present invention is simple to operate and yields diterpenoids with high purity. Attached Figure Description

[0029] Figure 1 The rosinane diterpenoid compound Tengchong bald pine resin A obtained in Example 1 1 H-NMR spectrum;

[0030] Figure 2 The rosinane diterpenoid compound Tengchong bald pine resin A obtained in Example 1 13 C-NMR spectrum;

[0031] Figure 3 The rosinane diterpenoid compound Tengchong bald pine resin A obtained in Example 11 H-1H COSY spectrum;

[0032] Figure 4 The HMBC spectrum of Tengchong bald pine oleane diterpenoid compound A obtained in Example 1 is shown below.

[0033] Figure 5 The HSQC spectrum of Tengchong bald pine oleane diterpenoid compound A obtained in Example 1 is shown below.

[0034] Figure 6 The NOESY spectrum of Tengchong bald pine oleane diterpenoid compound A obtained in Example 1;

[0035] Figure 7 The terpene diterpenoid menthone compound Tengchong bald pine B obtained in Example 2 1 H-NMR spectrum;

[0036] Figure 8 The terpene diterpenoid menthone compound Tengchong bald pine B obtained in Example 2 13 C-NMR spectrum;

[0037] Figure 9 The terpene diterpenoid menthone compound Tengchong bald pine B obtained in Example 2 1 H-1H COSY spectrum;

[0038] Figure 10 The HMBC spectrum of the terpene diterpenoid menthone compound Tengchong balsamin B obtained in Example 2 is shown below.

[0039] Figure 11 The HSQC spectrum of Tengchong Taibaixin B, a diterpenoid menthone compound obtained in Example 2;

[0040] Figure 12 The NOESY spectrum of Tengchong Taishanin B, a diterpenoid menthone compound obtained in Example 2. Detailed Implementation

[0041] This invention provides two diterpenoid compounds having the structures shown in Formula 1 and Formula 2:

[0042]

[0043] In this invention, the diterpenoid compound with the structure shown in Formula 1 is a rosinane diterpenoid compound, specifically TC flousianine A.

[0044] In this invention, the diterpenoid compound with the structure shown in Formula 2 is an ice-enene diterpenoid menthone compound, specifically TC flousianine B.

[0045] This invention also provides a method for extracting diterpenoid compounds as described in the above technical solution, comprising the following steps:

[0046] The heartwood powder of the bald cypress tree trunk was mixed with an ethanol solution and then subjected to reflux extraction and vacuum evaporation to obtain a crude extract.

[0047] The crude extract was dissolved in water and then extracted with petroleum ether to obtain the petroleum ether phase.

[0048] The petroleum ether phase was concentrated under vacuum to obtain a petroleum ether extract;

[0049] The petroleum ether extract was mixed with silica gel, and then subjected to a first silica gel column chromatography elution using the first, second, third, and fourth petroleum ether-ethyl acetate systems and the first, second, third, and fourth dichloromethane-methanol systems, respectively, to obtain fractions Fr.1, Fr.2, Fr.3, Fr.4, Fr.5, Fr.6, Fr.7, and Fr.8. In the first petroleum ether-ethyl acetate system, the volume ratio of petroleum ether to ethyl acetate was 100:0, and in the second petroleum ether-ethyl acetate system, the volume ratio of petroleum ether to ethyl acetate was 65–7. The volume ratio of petroleum ether to ethyl acetate in the third petroleum ether-ethyl acetate system is 25-35:1; the volume ratio of petroleum ether to ethyl acetate in the fourth petroleum ether-ethyl acetate system is 1-5:1; the volume ratio of dichloromethane to methanol in the first dichloromethane-methanol system is 100:0; the volume ratio of dichloromethane to methanol in the second dichloromethane-methanol system is 105-95:1; the volume ratio of dichloromethane to methanol in the third dichloromethane-methanol system is 75-85:1; and the volume ratio of dichloromethane to methanol in the fourth dichloromethane-methanol system is 25-35:1.

[0050] The Fr.8 fraction was eluted by a second silica gel column chromatography using dichloromethane to obtain the crude Fr.8 fraction.

[0051] The crude Fr.8 fraction was separated by gel column chromatography using methanol to obtain Fr.8-I, Fr.8-II, Fr.8-III, and Fr.8-IV fractions.

[0052] The Fr.8-III fraction was eluted by a third silica gel column chromatography using a petroleum ether-ethyl acetate system with a volume ratio of 9:1 to obtain the Fr.8-III-1, Fr.8-III-2, Fr.8-III-3, Fr.8-III-4 and Fr.8-III-5 fractions.

[0053] The Fr.8-III-3 fraction was purified by gel column chromatography using methanol to obtain the compound with the structure shown in Formula 1.

[0054] The Fr.8-III-1 fraction was purified by silica gel column chromatography using a petroleum ether-ethyl acetate system with a volume ratio of 11:1 to obtain the compound with the structure shown in Formula 2.

[0055] This invention involves mixing powdered heartwood from the trunk of a bald cypress tree with an ethanol solution, followed by reflux extraction and vacuum evaporation to obtain a crude extract.

[0056] In this invention, the particle size of the bald cypress heartwood powder is preferably 1 mm to 3 mm. The preferred method for preparing the bald cypress heartwood powder in this invention includes the following steps: air-drying the raw bald cypress heartwood and then pulverizing it to obtain the bald cypress heartwood powder. This invention does not specifically limit the parameters for air-drying and pulverizing, as long as a particle size of 1 mm to 3 mm can be obtained.

[0057] In this invention, the volume concentration of the ethanol solution is preferably greater than or equal to 80%, more preferably 80-95%, and specifically preferably 80%, 85%, 90% or 95%.

[0058] In this invention, the preferred ratio of the powdered heartwood of the bald cypress trunk to the ethanol solution is (30-50) g:(50-120) mL, specifically preferably 30 g:50 mL, 30 g:60 mL, 30 g:90 mL, 30 g:120 mL, 40 g:60 mL, 40 g:90 mL, 50 g:50 mL, 50 g:80 mL, 50 g:90 mL, or 50 g:120 mL.

[0059] In this invention, the reflux extraction temperature is preferably 55–65°C, and more preferably 60°C. The reflux extraction is preferably performed 2–3 times, and the reflux extraction time for each extraction is preferably 4–6 hours, and more preferably 4 hours, 5 hours, or 6 hours.

[0060] After the reflux extraction, the present invention preferably further includes: filtering the obtained reflux extraction liquid, combining the filtrates, and obtaining the extract.

[0061] In this invention, the vacuum degree of the reduced pressure evaporation is preferably 0.07 to 0.09 MPa, specifically preferably 0.07 MPa, 0.08 MPa or 0.09 MPa; the temperature is preferably 40 to 50°C, specifically preferably 40°C, 45°C or 50°C.

[0062] After obtaining the crude extract, the present invention dissolves the crude extract in water and then extracts it with petroleum ether to collect the petroleum ether phase.

[0063] In this invention, the mass ratio of the crude extract to water is preferably 1:1 to 3. In this invention, the volume ratio of the mixture obtained after water dissolution of the crude extract to the petroleum ether extracted by petroleum ether is preferably 1:1. In this invention, the petroleum ether extraction is preferably performed 2 to 4 times.

[0064] After obtaining the petroleum ether phase, the present invention performs vacuum concentration on the petroleum ether phase to obtain the petroleum ether extract.

[0065] In this invention, the vacuum degree of the vacuum concentration is preferably 0.07 to 0.09 MPa, specifically preferably 0.07 MPa, 0.08 MPa or 0.09 MPa; the temperature is preferably 40 to 50°C, specifically preferably 40°C, 45°C or 50°C.

[0066] After obtaining the petroleum ether extract, the present invention mixes the petroleum ether extract with silica gel and then performs a first silica gel column chromatography elution using the first, second, third, and fourth petroleum ether-ethyl acetate systems and the first, second, third, and fourth dichloromethane-methanol systems, respectively, to obtain fractions Fr.1, Fr.2, Fr.3, Fr.4, Fr.5, Fr.6, Fr.7, and Fr.8.

[0067] In this invention, the particle size of the silica gel is preferably 200-400 mesh. In this invention, the mass of the silica gel is preferably 0.8-1.5 times the dry weight of the petroleum ether extract, specifically preferably 0.8 times, 1 time, 1.2 times, or 1.5 times.

[0068] In this invention, the volume ratio of petroleum ether to ethyl acetate in the first petroleum ether-ethyl acetate system is 100:0.

[0069] In this invention, the volume ratio of petroleum ether to ethyl acetate in the second petroleum ether-ethyl acetate system is 65-75:1, preferably 70:1.

[0070] In this invention, the volume ratio of petroleum ether to ethyl acetate in the third petroleum ether-ethyl acetate system is 25 to 35:1, preferably 30:1.

[0071] In this invention, the volume ratio of petroleum ether to ethyl acetate in the fourth petroleum ether-ethyl acetate system is 1 to 5:1, preferably 5:1.

[0072] In this invention, the volume ratio of dichloromethane to methanol in the first dichloromethane-methanol system is 100:0.

[0073] In this invention, the volume ratio of dichloromethane to methanol in the second dichloromethane-methanol system is 105 to 95:1, preferably 100:1.

[0074] In this invention, the volume ratio of dichloromethane to methanol in the third dichloromethane-methanol system is 75-85:1, preferably 80:1.

[0075] In this invention, the volume ratio of dichloromethane to methanol in the fourth dichloromethane-methanol system is 25 to 35:1, preferably 30:1.

[0076] After obtaining the Fr.8 fraction, the present invention uses dichloromethane to elute the Fr.8 fraction with silica gel column chromatography for a second time to obtain the crude Fr.8 fraction.

[0077] The present invention does not specifically limit the parameters and process of the second silica gel column chromatography elution; any operation known to those skilled in the art can be used.

[0078] After obtaining the crude product of Fr.8, the present invention uses methanol to perform gel column separation on the crude product of Fr.8 to obtain Fr.8-I, Fr.8-II, Fr.8-III and Fr.8-IV.

[0079] The present invention does not specifically limit the process of gel column separation; any operation known to those skilled in the art can be used.

[0080] After obtaining fraction Fr.8-III, the present invention uses a petroleum ether-ethyl acetate system with a volume ratio of petroleum ether and ethyl acetate of 9:1 to perform a third silica gel column chromatography elution on fraction Fr.8-III-1, fraction Fr.8-III-2, fraction Fr.8-III-3, fraction Fr.8-III-4 and fraction Fr.8-III-5.

[0081] The present invention does not specifically limit the process of the third silica gel column chromatography elution; any operation known to those skilled in the art can be used.

[0082] After obtaining the Fr.8-III-3 component, the present invention uses methanol to perform gel column purification on the Fr.8-III-3 component to obtain the compound with the structure shown in Formula 1.

[0083] The present invention does not specifically limit the process of gel column purification; any operation known to those skilled in the art can be used.

[0084] After obtaining the Fr.8-III-1 fraction, the present invention uses a petroleum ether-ethyl acetate system with a volume ratio of petroleum ether and ethyl acetate of 11:1 to purify the Fr.8-III-1 fraction by silica gel column chromatography to obtain the compound with the structure shown in Formula 2.

[0085] The present invention does not specifically limit the process of silica gel column chromatography purification; any operation known to those skilled in the art can be used.

[0086] The present invention also provides the application of the diterpenoid compounds described in the above technical solutions or the diterpenoid compounds obtained by the extraction methods described in the above technical solutions in the preparation of antibacterial drugs or anti-inflammatory drugs.

[0087] In this invention, the antibacterial or anti-inflammatory drug is preferably present in the form of a pharmaceutical composition, wherein the content of diterpenoid compounds in the pharmaceutical composition is preferably 1 to 99 wt%.

[0088] The following detailed description, in conjunction with embodiments, illustrates the diterpenoid compounds, their extraction methods, and applications provided by this invention. However, these descriptions should not be construed as limiting the scope of protection of this invention.

[0089] Example 1

[0090] A method for extracting a rosinane diterpenoid compound with the structure shown in Formula 1, comprising the following steps:

[0091] (1) 10 kg of raw material, the heartwood of the bald cypress tree, was air-dried and pulverized to obtain bald cypress heartwood powder with a particle size of 1 mm to 3 mm; the bald cypress heartwood powder was added to an 80% ethanol solution for reflux extraction, wherein the ratio of bald cypress heartwood powder to ethanol solution was 10 g: 20 mL, the reflux extraction temperature was 60 °C, the reflux extraction was performed 3 times, and the reflux extraction time was 5 h each time; the insoluble matter was filtered and the filtrates were combined to obtain the extract; then the extract was evaporated under reduced pressure at a vacuum degree of 0.08 MPa and a temperature of 45 °C to obtain the crude extract.

[0092] (2) The crude extract was mixed with water at a mass ratio of 1:3 to form a mixed system. The mixture was extracted twice with an equal volume of petroleum ether and the petroleum ether phases were combined. The petroleum ether phases were concentrated under vacuum at a vacuum degree of 0.09 MPa and a temperature of 40 °C to obtain the petroleum ether extract.

[0093] (3) The obtained petroleum ether extract was mixed with an equal mass of silica gel and then placed in a silica gel column for chromatography. The first silica gel column chromatography was performed sequentially with the volume ratios of petroleum ether to ethyl acetate of 100:0, 70:1, 30:1, 5:1, and the volume ratios of dichloromethane to methanol of 100:0, 100:1, 80:1, and 30:1. The eluted fractions with the volume ratios of petroleum ether to ethyl acetate of 100:0, 70:1, 30:1, 5:1, and the volume ratios of dichloromethane to methanol of 100:0, 100:1, 80:1, and 30:1 were respectively Fr.1, Fr.2, Fr.3, Fr.4, Fr.5, Fr.6, Fr.7, and Fr.8.

[0094] (4) The Fr.8 fraction was eluted by silica gel column chromatography for a second time using dichloromethane to obtain the crude Fr.8 fraction.

[0095] (5) The crude product of Fr.8 was separated by gel column chromatography using methanol to obtain Fr.8-I, Fr.8-II, Fr.8-III and Fr.8-IV.

[0096] (6) The Fr.8-III fraction was eluted by silica gel column chromatography for the third time using a petroleum ether-ethyl acetate system with a volume ratio of 9:1 to obtain the Fr.8-III-1, Fr.8-III-2, Fr.8-III-3, Fr.8-III-4 and Fr.8-III-5 fractions.

[0097] (7) The Fr.8-III-3 fraction was purified by gel column chromatography using methanol to obtain the rosinane diterpenoid compound Tengchong bald pine A with the structure shown in Formula 1.

[0098] Example 2

[0099] The extraction method for the glacial diterpenoid menthone compound with the structure shown in Formula 2 includes the following steps:

[0100] (1) 10 kg of raw material, the heartwood of the bald cypress tree, was air-dried and pulverized to obtain bald cypress heartwood powder with a particle size of 1 mm to 3 mm; the bald cypress heartwood powder was added to an 80% ethanol solution for reflux extraction, wherein the ratio of bald cypress heartwood powder to ethanol solution was 10 g: 20 mL, the reflux extraction temperature was 60 °C, the reflux extraction was performed 3 times, and the reflux extraction time was 5 h each time; the insoluble matter was filtered and the filtrates were combined to obtain the extract; then the extract was evaporated under reduced pressure at a vacuum degree of 0.08 MPa and a temperature of 45 °C to obtain the crude extract.

[0101] (2) The crude extract was mixed with water at a mass ratio of 1:3 to form a mixed system. The mixture was extracted twice with an equal volume of petroleum ether and the petroleum ether phases were combined. The petroleum ether phases were concentrated under vacuum at a vacuum degree of 0.09 MPa and a temperature of 40 °C to obtain the petroleum ether extract.

[0102] (3) The obtained petroleum ether extract was mixed with an equal mass of silica gel and then placed in a silica gel column for chromatography. The first silica gel column chromatography was performed sequentially with the volume ratios of petroleum ether to ethyl acetate of 100:0, 70:1, 30:1, 5:1, and the volume ratios of dichloromethane to methanol of 100:0, 100:1, 80:1, and 30:1. The eluted fractions with the volume ratios of petroleum ether to ethyl acetate of 100:0, 70:1, 30:1, 5:1, and the volume ratios of dichloromethane to methanol of 100:0, 100:1, 80:1, and 30:1 were respectively Fr.1, Fr.2, Fr.3, Fr.4, Fr.5, Fr.6, Fr.7, and Fr.8.

[0103] (4) The Fr.8 fraction was eluted by silica gel column chromatography for a second time using dichloromethane to obtain the crude Fr.8 fraction.

[0104] (5) The crude product of Fr.8 was separated by gel column chromatography using methanol to obtain Fr.8-I, Fr.8-II, Fr.8-III and Fr.8-IV.

[0105] (6) The Fr.8-III fraction was eluted by silica gel column chromatography for the third time using a petroleum ether-ethyl acetate system with a volume ratio of 9:1 to obtain the Fr.8-III-1, Fr.8-III-2, Fr.8-III-3, Fr.8-III-4 and Fr.8-III-5 fractions.

[0106] (7) The fraction Fr.8-III-1 was purified by silica gel column chromatography using a petroleum ether-ethyl acetate system with a volume ratio of 11:1 to obtain the icyne diterpenoid menthone compound Tengchong bald pine B with the structure shown in Formula 2.

[0107] Characterization and performance testing

[0108] (I) Structural Identification

[0109] The diterpenoid compounds obtained in Examples 1 and 2 were identified by 1D / 2DNMR (one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy) and HR-ESI-MS (high-resolution electrospray ionization mass spectrometry). The results are as follows: Figures 1-12 As shown.

[0110] Figure 1 The rosinane diterpenoid compound Tengchong bald pine resin A obtained in Example 1 1 H-NMR spectrum, Figure 2 The rosinane diterpenoid compound Tengchong bald pine resin A obtained in Example 1 13 C-NMR spectrum, Figure 3 The rosinane diterpenoid compound Tengchong bald pine resin A obtained in Example 1 1 H-1H COSY spectrum, Figure 4 The HMBC spectrum of Tengchong bald pine oleanol A, a diterpenoid compound obtained in Example 1, is shown below. Figure 5 The HSQC spectrum of the rosinane diterpenoid compound Tengchong bald pine A obtained in Example 1 is shown below. Figure 6 The NOESY spectrum of the rosinane diterpenoid compound Tengchong bald pine A obtained in Example 1 is shown below. Figure 7 The terpene diterpenoid menthone compound Tengchong bald pine B obtained in Example 2 1 H-NMR spectrum, Figure 8 The terpene diterpenoid menthone compound Tengchong bald pine B obtained in Example 2 13 C-NMR spectrum, Figure 9 The terpene diterpenoid menthone compound Tengchong bald pine B obtained in Example 2 1 H-1H COSY spectrum, Figure 10 The HMBC spectrum of the terpene diterpenoid menthone compound Tengchong balsamin B obtained in Example 2 is shown below. Figure 11 The HSQC spectrum of the terpene diterpenoid menthone compound Tengchong pachycarboxin B obtained in Example 2 is shown below. Figure 12 The NOESY spectrum of Tengchong Taishanin B, a diterpenoid menthone compound obtained in Example 2.

[0111] The chemical shifts δ of H and C atoms of diterpenoids can be obtained from HSQC spectroscopy combined with carbon spectroscopy, as shown in Table 1.

[0112] Table 1. Diterpenoids 13 C(150MHz) and 1 H (600MHz) NMR data

[0113]

[0114] According to Table 1, the HR-ESI-MS m / z of Tengchong bald pine oleic acid A is [M+H]. + The quasi-molecular ion peak is at 317.2112 (C). 20 H 29 O3[M+H] + Calculated value: 317.2111), indicating that its molecular formula is C. 20 H28 O3 contains 7 degrees of unsaturation. Through analysis of... Figure 1 , Figure 2 and Figure 5 Analysis revealed that rosinane diterpenoids contain 5 methyl groups [δ]. H 0.99(H-19),1.07(H-18),1.10(H-20),1.20(H-16),1.22(H-17); δ C 17.3(C-19), 29.5(C-18), 15.9(C-20), 23.5(C-16), 23.5(C-17)], 3 methylene groups [δ H 2.41(H-2),1.86(H-6),2.80(H-7); δ C 34.3(C-2); 19.3(C-6), 26.4(C-7)], 5 methines [δ H 3.75(H-1),3.53(H-3),1.24(H-5),6.57(H-14); 3.25(C-15); δ C 96.6 (C-1, oxygenated), 81.0 (C-3, oxygenated), 47.0 (C-5), 118.5 (C-14, aromaticized), 27.4 (C-15)] and 7 quaternary carbons [δ C 40.2(C-4),122.1(C-8,aromatized),40.6(C-10),143.9(C-11,aromatized),136.7(C-12,oxygenated),136.1(C-13,oxygenated)].

[0115] The planar structure of Tengchong bald pine pine A was determined by comparison of NMR data and 2D NMR experiments, H16 (δH1.20s) / H15 (δ H 3,23-3.27m) / H17(δ H The 1H-1H COSY correlation of 1.22m) combined with the HMBC correlation of H15 / C-12, C-14, C-16, C-17 confirmed that a benzene ring is linked to an isopropyl group, and the hydroxyl group is linked to C12. H5(δ) H 1.23-1.25m) / H6(δ H 1.83-1.89m) / H7(δ H 2.59-2.68m, 2.92-2.98m) and H1 (δ H 3.73-3.77m) / H2(δ H 2.31-2.48m) / H3(δ HThe 1H-1H COSY-related binding of H7 / C-5,C-6,C-8,C-9; H6 / C-4,C-5,C-7,C-8,C-10; H20 / C-1,C-5,C-9,C-10; H1 / C-3,C-5,C-9,C-10,C-20; H2 / C-1,C-3,C-4,C-10; H3 / C-1,C-2,C-4,C-18,C-19; H18 / C-3,C-4,C-5,C-19; and H19 / C-3,C-4,C-4,C-18 was confirmed. Therefore, the structure of this compound was fully confirmed, and it was named Tengchong bald cypressin A.

[0116] HR-ESI-MS m / z of Tengchong bald pine phosphate B: [M+H] + The quasi-molecular ion peak is at 329.1755 (C). 20 H 25 O4[M+H] + Calculated value: 329.1753), indicating that its molecular formula is C. 20 H 24 O4 contains 9 degrees of unsaturation. Through analysis of... Figure 7 , Figure 8 and Figure 11 Analysis revealed that the glacial diterpenoid menthone compounds contain four methyl groups [δ-]. H 1.26(H-17),1.24(H-18),1.17(H-19),1.16(H-20); δ C 22.9(C-17); 22.7(C-18), 29.3(C-19), 20.6(C-20)], 2 methylene groups [δ H 2.46(H-6α), 1.70(H-6β), 2.93(H-14α), 2.74(H-14β); δ C 34.4(C-6); 31.4(C-14)], 6 methines [δ H 7.32(H-1),5.94(H-2),2.69(H-5),5.13(H-7,),6.45(H-9),3.11(H-16); δ C 152.5(C-1), 128.4(C-2), 78.4(C-7, oxygenated), 111.6(C-9), 27.3(C-16)] and 7 quaternary carbons [δ C206.3(C-3,ketone),43.8(C-4),136.3(C-8,aromatized),132.3(C-10,aromatized),142.0(C-11,oxygenated),152.5(C-12,oxygenated)].

[0117] The planar structure of Tengchong bald pine pine B was determined by comparison of NMR data and 2DNMR experiments. H16(δ H 3.07-3.14s) / H17, 18(δ H The 1H-1H COSY correlation (1.24–1.26 s) combined with the HMBC correlation of H16 / C-9, C-10, C-17, and C-18 confirms that a benzene ring is linked to an isopropyl group, and two hydroxyl groups are linked to C11 and C12, respectively. H7(δ) H 5,12-5,15d) / H6(δ H 1.66-1.73m, 2.43-2.50m) and H1 (δ H 7.31-7.33d) / H2(δ H The 1H-1H COSY-related binding of H9 / C-7,C-11,C-13,C / 16; H7 / C-5,C-9,C-13,C-15; H6 / C-4,C-5,C-7,C-8,C-15; H1 / C-5; and H2 / C-4,C-15 (5.93-5.95d) was confirmed. Therefore, the structure of this compound was fully confirmed, and it was named Tengchong bald cypressin B.

[0118] (II) Antibacterial activity of diterpenoids

[0119] (1) PDB (Potato Dextrose Broth) medium: Wash and peel potatoes, weigh 200g and cut them into cubes. Boil them in water until they can be pierced with a glass rod (10-15 min). Filter the filtrate with gauze. Heat the filtrate and add 20g of glucose. Stir until the glucose is completely dissolved. Add water to 1000mL. Dispense into 250mL Erlenmeyer flasks, seal them, and sterilize them in a high-temperature sterilizer at 120℃ for 30 min. After cooling, store for later use.

[0120] (2) Activation of fungi: Fungi such as Cucumber Glomerella, Alternaria ginseng, Fusarium solani, Fusarium oxysporum, Boletus globosum, and Ganoderma lucidum were inoculated into PDB medium and cultured at 28°C for 3 days.

[0121] (3) The antibacterial test was performed using the two-fold dilution method: the diterpenoid compound to be tested and the positive control drug (fungus: Nystatin) were dissolved in DMSO to prepare a working solution of 10.24 mg / mL and 5 μL was added to a 96-well plate. Each sample was set up with 3 replicates. Then, 85 μL of blank culture medium (fungus: PDB medium) was added. The samples were diluted to different concentrations (512, 256, 128, 64, 32, 16, 84, 2, 1, 0.5, 0.25, 0.125 μg / mL) using the two-fold dilution method. 200 μL of the activated bacterial solution was diluted to 10 mL with the corresponding culture medium. 10 μL of the diluted bacterial solution was added to the sample wells and control wells to make the final volume of each well 100 mL. No bacterial solution was added to the blank group. After the bacterial culture was thoroughly mixed, the 96-well plate was placed in a constant temperature shaker for incubation (fungi: 28℃, 3d). The absorbance of each well under visible light was measured using an ELISA reader. The concentration of the compound in the well corresponding to the absorbance value compared with the blank control was 1. The results are shown in Table 2.

[0122] Table 2. MIC values ​​of Tengchong bald lichen A, Tengchong bald lichen B, and Nystatin

[0123]

[0124] Table 2 shows that, in the screening test of diterpenoid compounds against in vitro antibacterial activity (positive control: nystatin), Tengchong typhaein A and Tengchong typhaein B exhibited good inhibitory activity against Ganoderma lucidum and Fusarium oxysporum. Therefore, the diterpenoid compounds provided by this invention have research value as lead compounds for the development of antibacterial and anti-inflammatory drugs.

[0125] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A diterpenoid compound, characterized in that, It has the structure shown in Equation 1 and Equation 2: Formula 1, Formula 2.

2. The method for extracting diterpenoid compounds according to claim 1, characterized in that, Includes the following steps: The heartwood powder of the bald cypress tree trunk was mixed with an ethanol solution and then subjected to reflux extraction and vacuum evaporation to obtain a crude extract. The crude extract was dissolved in water and then extracted with petroleum ether to obtain the petroleum ether phase. The petroleum ether phase was concentrated under vacuum to obtain a petroleum ether extract. The petroleum ether extract was mixed with silica gel, and then subjected to a first silica gel column chromatography elution using the first, second, third, and fourth petroleum ether-ethyl acetate systems and the first, second, third, and fourth dichloromethane-methanol systems, respectively, to obtain fractions Fr.1, Fr.2, Fr.3, Fr.4, Fr.5, Fr.6, Fr.7, and Fr.

8. In the first petroleum ether-ethyl acetate system, the volume ratio of petroleum ether to ethyl acetate was 100:0, and in the second petroleum ether-ethyl acetate system, the volume ratio of petroleum ether to ethyl acetate was 65-7. The volume ratio of petroleum ether to ethyl acetate in the third petroleum ether-ethyl acetate system is 25-35:1; the volume ratio of petroleum ether to ethyl acetate in the fourth petroleum ether-ethyl acetate system is 1-5:1; the volume ratio of dichloromethane to methanol in the first dichloromethane-methanol system is 100:0; the volume ratio of dichloromethane to methanol in the second dichloromethane-methanol system is 105-95:1; the volume ratio of dichloromethane to methanol in the third dichloromethane-methanol system is 75-85:1; and the volume ratio of dichloromethane to methanol in the fourth dichloromethane-methanol system is 25-35:

1. The Fr.8 fraction was eluted by a second silica gel column chromatography using dichloromethane to obtain the crude Fr.8 fraction. The crude Fr.8 fraction was separated by gel column chromatography using methanol to obtain Fr.8-I, Fr.8-II, Fr.8-III, and Fr.8-IV fractions. The Fr.8-III fraction was eluted by a third silica gel column chromatography using a petroleum ether-ethyl acetate system with a volume ratio of 9:1 to obtain the Fr.8-III-1, Fr.8-III-2, Fr.8-III-3, Fr.8-III-4 and Fr.8-III-5 fractions. The Fr.8-III-3 fraction was purified by gel column chromatography using methanol to obtain the compound with the structure shown in Formula 1. The Fr.8-III-1 fraction was purified by silica gel column chromatography using a petroleum ether-ethyl acetate system with a volume ratio of 11:1 to obtain the compound with the structure shown in Formula 2.

3. The extraction method according to claim 2, characterized in that, The particle size of the heartwood powder from the bald cypress trunk is 1 mm to 3 mm, and the volume concentration of the ethanol solution is greater than or equal to 80%.

4. The extraction method according to claim 3, characterized in that, The ratio of the powdered heartwood of the bald cypress trunk to the ethanol solution is (30~50)g:(50~120)mL.

5. The extraction method according to claim 2, characterized in that, The reflux extraction temperature is 55~65℃, the reflux extraction is performed 2~3 times, and the reflux extraction time is 4~6 hours each time.

6. The extraction method according to claim 2, characterized in that, The vacuum degree of the vacuum drying process is 0.07~0.09MPa, and the temperature is 40~50℃.

7. The extraction method according to claim 2, characterized in that, The mass ratio of the crude extract to the water-soluble liquid is 1:1 to 3, and the volume ratio of the mixture obtained after dissolving the crude extract in water to the petroleum ether extracted by petroleum ether is 1:1; the petroleum ether extraction is performed 2 to 4 times.

8. The extraction method according to claim 2, characterized in that, The vacuum concentration is performed at a vacuum level of 0.07~0.09 MPa and a temperature of 40~50℃.

9. The extraction method according to claim 2, characterized in that, The silica gel has a particle size of 200-400 mesh, and the mass of the silica gel is 0.8-1.5 times the dry weight of the petroleum ether extract.

10. The use of the diterpenoid compound of claim 1 or the diterpenoid compound obtained by the extraction method of any one of claims 2 to 9 in the preparation of antibacterial drugs, wherein the antibacterial drugs are used to inhibit Cucumber Coccidioides, Ginseng Alternaria, Fusarium solani, and Fusarium oxysporum.

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