Diterpenoid compound as well as extraction method and application thereof

Abstract

The invention belongs to the technical field of terpenoids, and provides a diterpenoid as well as an extraction method and application thereof. The diterpenoid compound provided by the invention has a structure as shown in a formula 1 and a formula 2. According to the diterpenoid compound provided by the invention, the diversity of the diterpenoid compound is enriched. Meanwhile, the diterpenoid compound provided by the invention has excellent anti-inflammatory and antibacterial properties, and particularly shows good inhibitory activity on ganoderma leuconostoc, fusarium solani and fusarium oxysporum. The invention further provides an extraction method of the diterpenoid compound according to the technical scheme, the extraction method provided by the invention is simple to operate, and the obtained diterpenoid compound is high in purity. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the technical field of terpenoid compounds, in particular to diterpenoid compounds and extraction methods and applications thereof. Background Art

[0002] Terpenes are hydrocarbons and their oxygenated derivatives that are multiples of isoprene units. They are a large class of natural products with high diversity, including monoterpenes, sesquiterpenes, diterpenes, sesquiterpenoid cyclohexenones, triterpenes, etc. Many terpenoids have been developed as important drugs for the treatment of cancer, bacterial infections, malaria and various other human diseases. Therefore, the synthesis of terpenoids is very important. However, due to the fact that the biosynthetic pathway has not been fully elucidated, terpenoids have a non-modular structure, and there is no universal unified synthetic strategy, the source of terpenoids still depends on the extraction of natural products.

[0003] According to literature reports, diterpenoids have multiple biological activities such as anti-inflammatory, anti-cytotoxic, anti-tumor and antibacterial. Therefore, the development of new diterpenoids is of great significance to solve the current situation of antibiotic abuse. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide diterpenoid compounds and extraction methods and applications thereof. 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 have excellent anti-inflammatory and antibacterial properties.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

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

[0007]

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

[0009] The powder of the heartwood of Taiwania baldness tree trunk and the ethanol solution are mixed, and then subjected to reflux extraction and reduced pressure evaporation to obtain a crude extract;

[0010] The crude extract is dissolved in water, extracted with petroleum ether, and the petroleum ether phase is collected;

[0011] The petroleum ether phase is concentrated in vacuo to obtain a petroleum ether extract;

[0012] After the petroleum ether extract and silica gel are mixed, the first, second, third and fourth petroleum ether-ethyl acetate systems and the first, second, third and fourth dichloromethane-methanol systems are used in sequence for the first silica gel column chromatography elution to obtain the Fr.1 component, the Fr.2 component, the Fr.3 component, the Fr.4 component, the Fr.5 component, the Fr.6 component, the Fr.7 component and the Fr.8 component respectively; the volume ratio of petroleum ether to ethyl acetate in the first petroleum ether-ethyl acetate system is 100:0, and the volume ratio of petroleum ether to ethyl acetate in the second petroleum ether-ethyl acetate system is 65-7 5:1, 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 component is subjected to a second silica gel column chromatography elution using dichloromethane to obtain a crude product of the Fr.8 component;

[0014] Using methanol to separate the crude product of the Fr.8 component by gel column to obtain the Fr.8-I component, the Fr.8-II component, the Fr.8-III component and the Fr.8-IV component;

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

[0016] Purifying the Fr.8-III-3 component by gel column using methanol to obtain a compound with a structure shown in Formula 1;

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

[0018] Preferably, the particle size of the Taiwania baldnessii trunk 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 usage ratio of the Taiwania bald cypress trunk heartwood powder and the ethanol solution is (30-50) g: (50-120) mL.

[0020] Preferably, the temperature of the reflux extraction is 55-65° C., the number of reflux extractions is 2-3 times, and the time of each reflux 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 water for water dissolution is 1:1-3, the volume ratio of the mixed system obtained after the crude extract is dissolved in water and the petroleum ether extracted by petroleum ether is 1:1; and the number of petroleum ether extractions is 2-4 times.

[0023] Preferably, the vacuum degree of the vacuum concentration is 0.07-0.09 MPa and the temperature is 40-50°C.

[0024] Preferably, the particle size of the silica gel is 200-400 meshes, 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 use of the diterpenoid compound described in the above technical solution or the diterpenoid compound obtained by the extraction method described in the above technical solution in the preparation of antibacterial drugs or anti-inflammatory drugs.

[0026] The invention provides two diterpenoid compounds.

[0027] The diterpenoid compounds provided by the present invention enrich the diversity of diterpenoid compounds. At the same time, the diterpenoid compounds provided by the present invention have excellent anti-inflammatory and antibacterial properties, especially showing good inhibitory activity against white-fleshed Ganoderma lucidum, Fusarium solani and Fusarium oxysporum.

[0028] The present invention also provides a method for extracting the diterpenoid compounds described in the above technical solution. The extraction method provided by the present invention is simple to operate and the obtained diterpenoid compounds have high purity. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The abietane diterpenoid compound Tengchong tsutsugasu A obtained in Example 1 1 H-NMR spectrum;

[0030] Figure 2 The abietane diterpenoid compound Tengchong tsutsugasu A obtained in Example 1 13 C-NMR spectrum;

[0031] Figure 3 The abietane diterpenoid compound Tengchong tsutsugasu A obtained in Example 11 H-1H COSY spectrum;

[0032] Figure 4 This is the HMBC spectrum of the abietane diterpenoid compound Tengchong Alopecuroides A obtained in Example 1;

[0033] Figure 5 This is the HSQC spectrum of the abietane diterpenoid compound Tengchong tsutsugamushi A obtained in Example 1;

[0034] Figure 6 This is the NOESY spectrum of the abietane diterpenoid compound Tengchong Alopecuroides A obtained in Example 1;

[0035] Figure 7 The glaucoma diterpenoid menthol compound Tengchong tsutsugasu B obtained in Example 2 1 H-NMR spectrum;

[0036] Figure 8 The glaucoma diterpenoid menthol compound Tengchong tsutsugasu B obtained in Example 2 13 C-NMR spectrum;

[0037] Fig. 9 The glaucoma diterpenoid menthol compound Tengchong tsutsugasu B obtained in Example 2 1 H-1H COSY spectrum;

[0038] Fig.10 This is the HMBC spectrum of the glaucoma diterpenoid menthone compound Tengchong safflower oil B obtained in Example 2;

[0039] Fig.11 This is the HSQC spectrum of the borneol diterpenoid menthone compound Tengchong tsutsugamushi B obtained in Example 2;

[0040] Fig.12 This is the NOESY spectrum of the glacial diterpenoid menthone compound Tengchong alopecuroides B obtained in Example 2. DETAILED DESCRIPTION

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

[0042]

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

[0044] In the present invention, the diterpene compound with the structure shown in Formula 2 is a bornene diterpene menthone compound, specifically TC flousianine B.

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

[0046] The powder of the heartwood of Taiwania baldness tree trunk and the ethanol solution are mixed, and then subjected to reflux extraction and reduced pressure evaporation to obtain a crude extract;

[0047] The crude extract is dissolved in water, extracted with petroleum ether, and the petroleum ether phase is collected;

[0048] The petroleum ether phase is concentrated in vacuo to obtain a petroleum ether extract;

[0049] After the petroleum ether extract and silica gel are mixed, the first, second, third and fourth petroleum ether-ethyl acetate systems and the first, second, third and fourth dichloromethane-methanol systems are used in sequence for the first silica gel column chromatography elution to obtain the Fr.1 component, the Fr.2 component, the Fr.3 component, the Fr.4 component, the Fr.5 component, the Fr.6 component, the Fr.7 component and the Fr.8 component respectively; the volume ratio of petroleum ether to ethyl acetate in the first petroleum ether-ethyl acetate system is 100:0, and the volume ratio of petroleum ether to ethyl acetate in the second petroleum ether-ethyl acetate system is 65-7 5:1, 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 component is subjected to a second silica gel column chromatography elution using dichloromethane to obtain a crude product of the Fr.8 component;

[0051] Using methanol to separate the crude product of the Fr.8 component by gel column to obtain the Fr.8-I component, the Fr.8-II component, the Fr.8-III component and the Fr.8-IV component;

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

[0053] Purifying the Fr.8-III-3 component by gel column using methanol to obtain a compound with a structure shown in Formula 1;

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

[0055] The invention mixes the Taiwania bald cypress trunk heartwood powder and ethanol solution, performs reflux extraction and vacuum evaporation in sequence to obtain a crude extract.

[0056] In the present invention, the particle size of the Taiwania bald cypress trunk heartwood powder is preferably 1 mm to 3 mm. In the present invention, the method for preparing the Taiwania bald cypress trunk heartwood powder preferably comprises the following steps: air-drying the Taiwania bald cypress trunk heartwood raw material, and then crushing it to obtain the Taiwania bald cypress trunk heartwood powder; the present invention does not specifically limit the parameters of the air-drying and crushing, as long as the Taiwania bald cypress trunk heartwood powder with a particle size of 1 mm to 3 mm can be obtained.

[0057] In the present 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 the present invention, the dosage ratio of the Taiwania bald cypress trunk heartwood powder and the ethanol solution is preferably (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 the present invention, the temperature of the reflux extraction is preferably 55-65° C., and more preferably 60° C. In the present invention, the number of reflux extractions is preferably 2-3 times, and the time of each reflux 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 comprises: filtering the obtained reflux extraction liquid, combining the filtrate, and obtaining an extract.

[0061] In the present invention, the vacuum degree of the reduced pressure evaporation is preferably 0.07-0.09 MPa, specifically preferably 0.07 MPa, 0.08 MPa or 0.09 MPa; the temperature is preferably 40-50°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, extracts it with petroleum ether, and collects the petroleum ether phase.

[0063] In the present invention, the mass ratio of the crude extract to the water for water dissolution is preferably 1:1 to 3. In the present invention, the volume ratio of the mixed system obtained after the crude extract is dissolved in water to the petroleum ether extracted with petroleum ether is preferably 1:1. In the present invention, the number of times of petroleum ether extraction is preferably 2 to 4 times.

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

[0065] In the present invention, the vacuum degree of the vacuum concentration is preferably 0.07-0.09 MPa, specifically preferably 0.07 MPa, 0.08 MPa or 0.09 MPa; the temperature is preferably 40-50°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 and silica gel and sequentially uses the first, second, third, and fourth petroleum ether-ethyl acetate systems and the first, second, third, and fourth dichloromethane-methanol systems to perform the first silica gel column chromatography elution to obtain the Fr.1 component, the Fr.2 component, the Fr.3 component, the Fr.4 component, the Fr.5 component, the Fr.6 component, the Fr.7 component, and the Fr.8 component, respectively.

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

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

[0069] In the present 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 the present invention, the volume ratio of petroleum ether to ethyl acetate in the third petroleum ether-ethyl acetate system is 25-35:1, preferably 30:1.

[0071] In the present 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 the present invention, the volume ratio of dichloromethane to methanol in the first dichloromethane-methanol system is 100:0.

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

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

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

[0076] After obtaining the Fr.8 component, the present invention uses dichloromethane to elute the Fr.8 component for a second time through a silica gel column to obtain a crude product of the Fr.8 component.

[0077] The present invention does not specifically limit the parameters and process of the second silica gel column chromatography elution, and operations familiar to those skilled in the art may be used.

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

[0079] The present invention does not specifically limit the gel column separation process, and operations well known to those skilled in the art may be used.

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

[0081] The present invention does not specifically limit the process of the third silica gel column chromatography elution, and operations well known to those skilled in the art may 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 a compound with a structure shown in Formula 1.

[0083] The present invention does not specifically limit the gel column purification process, and operations familiar to those skilled in the art may be used.

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

[0085] The present invention does not specifically limit the process of silica gel column chromatography purification, and operations familiar to those skilled in the art may be used.

[0086] The present invention also provides the use of the diterpenoid compound described in the above technical solution or the diterpenoid compound obtained by the extraction method described in the above technical solution in the preparation of antibacterial drugs or anti-inflammatory drugs.

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

[0088] The diterpenoid compounds and extraction methods and applications provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0089] Example 1

[0090] A method for extracting abietane diterpenoid compounds having a structure shown in Formula 1 comprises the following steps:

[0091] (1) 10 kg of raw material Taiwania baldwinii trunk heartwood is air-dried and crushed to obtain Taiwania baldwinii trunk heartwood powder with a particle size of 1 mm to 3 mm; the Taiwania baldwinii trunk heartwood powder is added to an ethanol solution with a volume fraction of 80% for reflux extraction, wherein the amount ratio of Taiwania baldwinii trunk heartwood powder to ethanol solution is 10 g:20 mL, the reflux extraction temperature is 60° C., the number of reflux extractions is 3 times, and the time of each reflux extraction is 5 hours; insoluble matter is filtered and the filtrate is combined to obtain an extract; and then the extract is evaporated to dryness under reduced pressure at a vacuum degree of 0.08 MPa and a temperature of 45° C. to obtain a crude extract.

[0092] (2) The obtained crude extract was mixed with water in a mass ratio of 1:3 to prepare a mixed system, and extracted twice with petroleum ether having an equal volume to the mixed system, and the petroleum ether phases were combined; the petroleum ether phases were vacuum concentrated at a vacuum degree of 0.09 MPa and a temperature of 40° C. to obtain a 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 elution was carried out using a volume ratio of petroleum ether to ethyl acetate of 100:0, 70:1, 30:1, 5:1, and a volume ratio of dichloromethane to methanol of 100:0, 100:1, 80:1, and 30:1, respectively, to obtain elution portions with a volume ratio of petroleum ether to ethyl acetate of 100:0, 70:1, 30:1, 5:1, and a volume ratio of dichloromethane to methanol of 100:0, 100:1, 80:1, and 30:1, which were respectively the Fr.1 component, the Fr.2 component, the Fr.3 component, the Fr.4 component, the Fr.5 component, the Fr.6 component, the Fr.7 component, and the Fr.8 component.

[0094] (4) The Fr.8 component is subjected to a second silica gel column chromatography elution using dichloromethane to obtain a crude product of the Fr.8 component.

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

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

[0097] (7) The Fr.8-III-3 component was purified by gel column using methanol to obtain Tengchong tauropside A, a rosinane diterpene compound with a structure shown in Formula 1.

[0098] Example 2

[0099] The method for extracting the glacial diterpenoid menthone compound of the structure shown in Formula 2 comprises the following steps:

[0100] (1) 10 kg of raw material Taiwania baldwinii trunk heartwood is air-dried and crushed to obtain Taiwania baldwinii trunk heartwood powder with a particle size of 1 mm to 3 mm; the Taiwania baldwinii trunk heartwood powder is added to an ethanol solution with a volume fraction of 80% for reflux extraction, wherein the amount ratio of Taiwania baldwinii trunk heartwood powder to ethanol solution is 10 g:20 mL, the reflux extraction temperature is 60° C., the number of reflux extractions is 3 times, and the time of each reflux extraction is 5 hours; insoluble matter is filtered and the filtrate is combined to obtain an extract; and then the extract is evaporated to dryness under reduced pressure at a vacuum degree of 0.08 MPa and a temperature of 45° C. to obtain a crude extract.

[0101] (2) The obtained crude extract was mixed with water in a mass ratio of 1:3 to prepare a mixed system, and extracted twice with petroleum ether having an equal volume to the mixed system, and the petroleum ether phases were combined; the petroleum ether phases were vacuum concentrated at a vacuum degree of 0.09 MPa and a temperature of 40° C. to obtain a 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 elution was carried out using a volume ratio of petroleum ether to ethyl acetate of 100:0, 70:1, 30:1, 5:1, and a volume ratio of dichloromethane to methanol of 100:0, 100:1, 80:1, and 30:1, respectively, to obtain elution portions with a volume ratio of petroleum ether to ethyl acetate of 100:0, 70:1, 30:1, 5:1, and a volume ratio of dichloromethane to methanol of 100:0, 100:1, 80:1, and 30:1, which were respectively the Fr.1 component, the Fr.2 component, the Fr.3 component, the Fr.4 component, the Fr.5 component, the Fr.6 component, the Fr.7 component, and the Fr.8 component.

[0103] (4) The Fr.8 component is subjected to a second silica gel column chromatography elution using dichloromethane to obtain a crude product of the Fr.8 component.

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

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

[0106] (7) The Fr.8-III-1 component was purified by silica gel column chromatography using a petroleum ether-ethyl acetate system with a volume ratio of petroleum ether to ethyl acetate of 11:1 to obtain Tengchong safflower oil B, a glaucoma diterpenoid menthol compound with a structure shown in Formula 2.

[0107] Characterization and performance testing

[0108] (I) Structural identification

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

[0110] Figure 1 The abietane diterpenoid compound Tengchong tsutsugasu A obtained in Example 1 1 H-NMR spectrum, Figure 2 The abietane diterpenoid compound Tengchong tsutsugasu A obtained in Example 1 13 C-NMR spectrum, Figure 3 The abietane diterpenoid compound Tengchong tsutsugasu A obtained in Example 1 1 H-1H COSY spectrum, Figure 4 This is the HMBC spectrum of the abietane diterpenoid compound Tengchong Alopecuroides A obtained in Example 1. Figure 5 This is the HSQC spectrum of the abietane diterpenoid compound Tengchong tsutsugamushi A obtained in Example 1. Figure 6 This is the NOESY spectrum of the abietane diterpenoid compound Tengchong Alopecuroides A obtained in Example 1. Figure 7 The glaucoma diterpenoid menthol compound Tengchong tsutsugasu B obtained in Example 2 1 H-NMR spectrum, Figure 8 The glaucoma diterpenoid menthol compound Tengchong tsutsugasu B obtained in Example 2 13 C-NMR spectrum, Fig. 9 The glaucoma diterpenoid menthol compound Tengchong tsutsugasu B obtained in Example 2 1 H-1H COSY spectrum, Fig.10 This is the HMBC spectrum of the glaucoma diterpenoid menthone compound Tengchong tsutsugamushi B obtained in Example 2. Fig.11 This is the HSQC spectrum of the glaucoma diterpenoid menthone compound Tengchong tsutsugamushi B obtained in Example 2. Fig.12 This is the NOESY spectrum of the glacial diterpenoid menthone compound Tengchong alopecuroides B obtained in Example 2.

[0111] From the HSQC spectrum combined with the carbon spectrum, it can be obtained that the chemical shift δ assignments of H and the C connected to the diterpenoid compounds are shown in Table 1.

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

[0113]

[0114] According to Table 1, HR-ESI-MS m / z of Tengchong tauropside A: [M+H] + The quasi-molecular ion peak is 317.2112 (C 20 H 29 O 3 [M+H] + Calculated value: 317.2111), indicating that its molecular formula is C20 H 28 O 3 , containing 7 degrees of unsaturation. Figure 1 , Figure 2 and Figure 5 The analysis shows that the abietane 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, aromatized), 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 baldwood extract A was determined by NMR data comparison and 2D NMR experiments. H 3,23-3.27m) / H17(δ H 1.22m) combined with the HMBC correlation of H15 / C-12, C-14, C-16, and C-17 confirmed that a benzene ring was connected to the isopropyl group and the hydroxyl group was connected to C12. 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 H2.31-2.48m) / H3(δ H 3.51-3.55m) of 1H-1H COSY correlation with 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 of HMBC. Therefore, the structure of the compound was fully confirmed and it was named Tengchong baldwood extract A.

[0116] HR-ESI-MS m / z of Tengchong tauropside B:[M+H] + The quasi-molecular ion peak is 329.1755 (C 20 H 25 O 4 [M+H] + Calculated value: 329.1753), indicating that its molecular formula is C 20 H 24 O 4 , containing 9 degrees of unsaturation. Figure 7 , Figure 8 and Fig.11 The analysis showed that the menthone compounds of the glacial diterpenoids contain 4 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 baldwood extract B was determined by NMR data comparison and 2D NMR experiments. H 3.07-3.14s) / H17, 18(δ H 1.24-1.26s) 1H-1H COSY correlation combined with HMBC correlation of H16 / C-9, C-10, C-17, C-18 confirmed that a benzene ring is connected to an isopropyl group, and two hydroxyl groups are connected to C11 and C12 respectively. 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 5.93-5.95d) of 1H-1H COSY correlation with 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; H2 / C-4, C-15 of HMBC. Therefore, the structure of the compound was fully confirmed and it was named Tengchong baldwood extract B.

[0118] (II) Antibacterial activity of diterpenoids

[0119] (1) PDB (Potato Dextrose Broth) medium: Wash and peel potatoes, weigh 200 g and cut into cubes, add water and boil until they can be pierced with a glass rod (10-15 min), filter the filtrate with gauze, heat the filtrate and add 20 g of glucose, stir evenly until the glucose is completely dissolved, add water to 1000 mL, dispense into 250 mL conical flasks, seal the flasks, place in a high-temperature sterilizer at 120°C for 30 min, remove from the flasks, cool and store for later use.

[0120] (2) Activation of fungi: The fungi Sphaerotheca cucumeris, Alternaria ginseng, Fusarium solani, Fusarium oxysporum, Dictyostelium truncatum, and Ganoderma lucidum were inoculated into PDB medium respectively and cultured at 28°C for 3 days.

[0121] (3) The antibacterial test was conducted using the two-fold dilution method: the diterpenoid compounds to be tested and the positive control drug (fungus: nystatin) were dissolved in DMSO to prepare a 10.24 mg / mL working solution, and 5 μL was added to a 96-well plate. Three replicate wells were set for each sample, and then 85 μL of blank culture medium (fungus: PDB culture 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, and 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 solution was fully mixed, the 96-well plate was placed in a constant temperature shaker for culture (fungi: 28°C, 3d), and 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 ratio of the absorbance value to the blank control value of 1 was the minimum inhibitory concentration (MIC) of the sample for the corresponding strain. The results are shown in Table 2.

[0122] Table 2 MICs values ​​of Tengchong baldwood extract A, Tengchong baldwood extract B and Nystatin

[0123]

[0124] The results in Table 2 show that in the in vitro antibacterial activity screening test of diterpenoid compounds (positive control: nystatin), Tengchong balsamina A and Tengchong balsamina B showed good inhibitory activity against white ganoderma and Fusarium oxysporum. Therefore, the diterpenoid compounds provided by the present invention have research value as lead compounds for the development of antibacterial drugs and anti-inflammatory drugs.

[0125] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

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

2. The method for extracting diterpenoids according to claim 1, characterized in that: The following steps are involved: The powder of the heartwood of Taiwania baldness tree trunk and the ethanol solution are mixed, and then subjected to reflux extraction and reduced pressure evaporation to obtain a crude extract; The crude extract is dissolved in water, extracted with petroleum ether, and the petroleum ether phase is collected; The petroleum ether phase is concentrated in vacuo to obtain a petroleum ether extract; After the petroleum ether extract and silica gel are mixed, the first, second, third and fourth petroleum ether-ethyl acetate systems and the first, second, third and fourth dichloromethane-methanol systems are used in sequence for the first silica gel column chromatography elution to obtain the Fr.1 component, the Fr.2 component, the Fr.3 component, the Fr.4 component, the Fr.5 component, the Fr.6 component, the Fr.7 component and the Fr.8 component respectively; the volume ratio of petroleum ether to ethyl acetate in the first petroleum ether-ethyl acetate system is 100:0, and the volume ratio of petroleum ether to ethyl acetate in the second petroleum ether-ethyl acetate system is 65-7 5:1, 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 component is subjected to a second silica gel column chromatography elution using dichloromethane to obtain a crude product of the Fr.8 component; Using methanol to separate the crude product of the Fr.8 component by gel column to obtain the Fr.8-I component, the Fr.8-II component, the Fr.8-III component and the Fr.8-IV component; The Fr.8-III component was eluted by silica gel column chromatography for the third time using a petroleum ether-ethyl acetate system with a volume ratio of petroleum ether to ethyl acetate of 9:1 to obtain a Fr.8-III-1 component, a Fr.8-III-2 component, a Fr.8-III-3 component, a Fr.8-III-4 component and a Fr.8-III-5 component; Purifying the Fr.8-III-3 component by gel column using methanol to obtain a compound with a structure shown in Formula 1; The Fr.8-III-1 component was purified by silica gel column chromatography using a petroleum ether-ethyl acetate system with a volume ratio of petroleum ether to ethyl acetate of 11:1 to obtain a compound with a structure shown in Formula 2.

3. The extraction method according to claim 2, characterized in that The particle size of the Taiwania baldnessii trunk heartwood powder 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 dosage ratio of the Taiwania bald cypress trunk heartwood powder and the ethanol solution is (30-50) g: (50-120) mL.

5. The extraction method according to claim 2, characterized in that The temperature of the reflux extraction is 55-65° C., the number of reflux extractions is 2-3 times, and the time of each reflux extraction is 4-6 hours.

6. The extraction method according to claim 2, characterized in that The vacuum degree of the reduced pressure evaporation is 0.07-0.09 MPa, and the temperature is 40-50°C.

7. The extraction method according to claim 2, characterized in that The mass ratio of the crude extract to water for water dissolution is 1:1-3, the volume ratio of the mixed system obtained after the crude extract is dissolved in water to the petroleum ether extracted with petroleum ether is 1:1; and the number of times of petroleum ether extraction is 2-4 times.

8. The extraction method according to claim 2, characterized in that The vacuum degree of the vacuum concentration is 0.07-0.09 MPa, and the temperature is 40-50°C.

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

10. Use of the diterpenoid compound according to claim 1 or the diterpenoid compound obtained by the extraction method according to any one of claims 2 to 9 in the preparation of antibacterial drugs or anti-inflammatory drugs.

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