CN105837434A - Diterpenoids extracted from herba siegesbeckiae, and preparation method and application thereof - Google Patents
Diterpenoids extracted from herba siegesbeckiae, and preparation method and application thereof Download PDFInfo
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- 238000013375 chromatographic separation Methods 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 6
- CBOIHMRHGLHBPB-UHFFFAOYSA-N hydroxymethyl Chemical compound O[CH2] CBOIHMRHGLHBPB-UHFFFAOYSA-N 0.000 claims description 6
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- 239000012467 final product Substances 0.000 claims description 5
- 239000004599 antimicrobial Substances 0.000 claims description 4
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- 150000001875 compounds Chemical class 0.000 abstract description 17
- 241000191967 Staphylococcus aureus Species 0.000 abstract description 10
- RJQXTJLFIWVMTO-TYNCELHUSA-N Methicillin Chemical compound COC1=CC=CC(OC)=C1C(=O)N[C@@H]1C(=O)N2[C@@H](C(O)=O)C(C)(C)S[C@@H]21 RJQXTJLFIWVMTO-TYNCELHUSA-N 0.000 abstract description 4
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C61/00—Compounds having carboxyl groups bound to carbon atoms of rings other than six-membered aromatic rings
- C07C61/12—Saturated polycyclic compounds
- C07C61/125—Saturated polycyclic compounds having a carboxyl group bound to a condensed ring system
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/42—Separation; Purification; Stabilisation; Use of additives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C62/00—Compounds having carboxyl groups bound to carbon atoms of rings other than six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
- C07C62/02—Saturated compounds containing hydroxy or O-metal groups
- C07C62/06—Saturated compounds containing hydroxy or O-metal groups polycyclic
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Plant Substances (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
The invention relates to diterpenoids extracted and separated from traditional Chinese medicine herba siegesbeckiae, and a preparation method and application thereof. The diterpenoids comprise all compounds of which the structural compounds are disclosed in the specification. The preparation method comprises the following steps: pulverizing herba siegesbeckiae, extracting with an organic solvent, degreasing, and carrying out column chromatography separation purification. The diterpenoids have a growth inhibition effect on multiple microbes, especially Methicillin-resistant Staphylococcus aureus (MRSA) and fungi, and thus, are compounds having application prospects. When being used for preparing antimicrobial drugs, the diterpenoids can be used independently or jointly for preparing tablets, injections, capsules, granules, powder, suppositories, ointments, sprays, gelatinizers, lotions, films, liniments, foaming agents and other preparation formulations. The preparation method has the advantages of simple preparation process, low cost and high production benefits, and is very suitable for industrial production.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to diterpenoid compounds extracted and separated from siegesbeckia orientalis and application thereof in preparing antimicrobial drugs, especially application in preparing drugs for resisting fungi, gram-negative bacteria and gram-positive bacteria.
Background
According to the record of "Chinese pharmacopoeia" on the 2015 edition, Siegesbeckiae herba is dried aerial part of Siegesbeckiae orientalis L, Siegesbeckiae Pubescens Makino or Siegesbeckia glabrescens Makino of Compositae. Siegesbeckiae herba is pungent, bitter and cold in nature. It enters liver and kidney meridians. Has the effects of expelling wind-damp, benefiting joints and detoxifying. Can be used for treating rheumatalgia, myasthenia of tendons and bones, soreness of waist and knees, quadriplegia, hemiplegia, rubella, eczema, etc. The study of scholars at home and abroad on the pharmacological action of siegesbeckia orientalis mainly focuses on: antiinflammatory, analgesic, antiallergic, antioxidant, cytotoxic, and antithrombotic effects. At present, only a few scholars have studied the antimicrobial effect of siegesbeckia orientalis, and no scholars have reported the antifungal and gram-negative effects of diterpenoids in siegesbeckia orientalis.
In modern society, many infectious diseases are effectively controlled with the widespread clinical use of synthetic and semi-synthetic antimicrobial drugs. However, the phenomenon of abuse of antimicrobial drugs is also becoming more serious, and the development of drug resistance has become an important issue facing the medical community at present. The abuse of antimicrobial drugs easily causes the problems of the generation of pathogenic bacteria with multiple drug resistance, the double infection of fungi, the occurrence of adverse reactions and the like. Particularly, with the continuous increase of deep fungal infection rate and the increasing of the drug resistance of the existing antifungal drugs in recent years, the clinical demand for novel antifungal drugs with broad spectrum, high efficiency and low toxicity is increasingly urgent. Some traditional Chinese medicines for clearing away heat and toxic materials have obvious antibacterial effects, active compounds which have low toxic and side effects and are not easy to generate drug resistance are searched from the traditional Chinese medicines, and the novel antibacterial drugs developed from the traditional Chinese medicines have obvious advantages.
Disclosure of Invention
The present invention provides
Diterpenoid compounds extracted from siegesbeckia orientalis are disclosed, and the chemical structure general formula of the diterpenoid compounds is as follows:
wherein,
R1is CH3Or CH2OH；
R2Is H or OH or CH3Or CH2OH；
R3Is H or CH3Or OH or CH2OH or COOH.
When R in the formula1is-CH3，R2is-H, R3When the compound is-COOH, the compound represented by the general formula I is enantiomer-16 β H-kaurane-17, 19-dicarboxylic acid.
When R in the formula1is-CH3，R2is-OH, R3is-CH3OH, the compound represented by the general formula I is enantiomer-16 α, 17-dihydroxy kaurane-19-carboxylic acid.
When R in the formula1is-CH3OH，R2is-OH, R3is-CH3OH, the compound represented by the general formula I is enantiomer-16 α,17, 18-trihydroxykaurane-19-carboxylic acid.
When R in the formula1is-CH3OH，R2is-CH3OH，R3When the formula is-H, the compound represented by the general formula I is enantiomer-17, 18-dihydroxy kaurane-16 β H-19-carboxylic acid.
The invention also provides a method for preparing diterpenoid compounds from siegesbeckia orientalis, which comprises the following steps:
step 1, extraction: crushing the siegesbeckia herb, extracting with an extracting solution, filtering, collecting filtrate, and concentrating under reduced pressure or normal pressure until the volume of the filtrate is less than 3-5% of that of the stock solution to obtain a concentrated solution;
step 2, degreasing: dissolving the concentrated solution in 0.5-1.5 times of water, extracting with n-hexane, cyclohexane or petroleum ether with the same volume, discarding the extract, and collecting the residual water solution;
step 3, separation: concentrating the residual water solution obtained in the previous step to 1-3% of the volume of the residual water solution, and then carrying out macroporous resin column chromatographic separation to obtain a separation solution; the eluent separated by the macroporous resin column chromatography is alcohol-water solutions with different concentrations, the volume ratio of alcohol to water in the alcohol-water solution is 1: 9-9: 1, and the elution method is that gradient elution is carried out in sequence from small concentration to large concentration according to the concentration of the alcohol;
and 4, purifying: purifying the separation liquid obtained in the above steps sequentially through silica gel column chromatography and Sephadex LH-20 column chromatography to obtain the final product.
The extracting solution in the step 1 is one of water, absolute ethyl alcohol, 30-95% by mass of ethyl alcohol, methanol, 30-95% by volume of ethyl alcohol aqueous solution, or methanol, 20-80% by volume of methyl alcohol aqueous solution, acetone, 30-70% by volume of acetone aqueous solution and ethyl acetate; the extraction method comprises the steps of carrying out ultrasonic wave for 15-40 minutes at 25 ℃, or carrying out heating reflux for 2 hours at 45-60 ℃ or carrying out microwave for 2-5 minutes at the power of 300-500 watts.
In the step 4, the particle size of silica gel in the silica gel column chromatography is 100-300 meshes, the eluent is a mixed solution of n-hexane, cyclohexane or petroleum ether and ethyl acetate, acetone or dichloromethane in different proportions, and the elution method is that the polarity of the eluent is eluted from small to large in sequence; the eluent of the Sephadex LH-20 column chromatography is a mixed solution of chloroform and methanol or a mixed solution of methanol and water, the elution method is based on the methanol, and the volume percentage concentration of the methanol is in the range of 30-100 percent and is eluted from small to large in sequence; the diterpenoid compounds are applied to the preparation of antimicrobial drugs.
The microorganism is a fungus or a bacterium.
The antimicrobial drug can be diterpenoid compounds or diterpenoid compound compositions and tablets, injections, capsules, granules, powder, suppositories, paste, sprays, gels, lotions, films, liniments, foams and the like prepared from pharmaceutically-acceptable auxiliary materials.
The diterpenoid compounds in the herba siegesbeckiae can be used as antimicrobial drugs, in particular to the application of methicillin-resistant Staphylococcus aureus (MRSA), Candida albicans (Candida albicans), Candida tropicalis (Candida tropicalis), Staphylococcus aureus (Staphylococcus aureus), Pseudomonas aeruginosa (Pseudomonas aeruginosa) and Escherichia coli (Escherichia coli).
The diterpenoid compounds in the siegesbeckia orientalis can be used for preparing antimicrobial drugs or lead compounds, and particularly can be used for preparing drugs or lead compounds of drugs for resisting methicillin-resistant Staphylococcus aureus (MRSA), Candida albicans (Candida albicans), Candida tropicalis (Candida tropicalis), Staphylococcus aureus (Staphylococcus aureus), Pseudomonas aeruginosa (Pseudomonas aeruginosa) and Escherichia coli (Escherichia coli).
The diterpenoid compounds in the siegesbeckia orientalis can be obtained by separating and purifying from plants; or may be synthetically obtained via chemical modification methods well known to those skilled in the art.
The diterpenoid compound can be used alone or used as a pharmaceutical composition when preparing an antimicrobial drug, and can be used for preparing dosage forms such as tablets, injections, capsules, granules, powder, suppositories, paste, sprays, gels, lotions, films, liniments, foams and the like.
The invention has simple preparation, low cost and high production benefit, and is very suitable for industrial production.
Detailed Description
Example 1:
this example provides a method for preparing the compound ent-16 β H-kaurane-17, 19-dicarboxylic acid and a method and results for determining its antibacterial activity.
The chemical structural formula is as follows:
step 1, extraction: crushing herba siegesbeckiae, soaking in absolute ethanol, performing ultrasonic extraction for 3 times at 25 ℃ for 15 minutes, 30 minutes and 45 minutes respectively, filtering, collecting filtrate, and concentrating under reduced pressure or normal pressure until the volume of the filtrate is less than 3-5% of that of the stock solution to obtain concentrated solution;
step 2, degreasing: dissolving the concentrated solution in 0.5-1.5 times of water, extracting with n-hexane, cyclohexane or petroleum ether with the same volume, discarding the extract, and collecting the residual water solution;
step 3, separation: concentrating the residual water solution obtained in the previous step to 1-3% of the volume of the residual water solution, and then carrying out macroporous resin column chromatographic separation to obtain a separation solution; the eluent separated by the macroporous resin column chromatography is alcohol-water solutions with different concentrations, the volume ratio of alcohol to water in the alcohol-water solution is 1: 9-9: 1, and the elution method is that gradient elution is carried out in sequence from small concentration to large concentration according to the concentration of the alcohol;
and 4, purifying, namely purifying the separation liquid obtained in the step sequentially through silica gel column chromatography and Sephadex (Sephadex LH-20) column chromatography to obtain the product.
In the step 4, the particle size of silica gel in the silica gel column chromatography is 100-200 meshes, the eluent is a mixed solution of petroleum ether and ethyl acetate, and the elution method comprises the step of gradient elution of the two solutions in a ratio of 10: 1-1: 1 in sequence;
the eluent of the Sephadex LH-20 column chromatography is a mixed solution of chloroform and methanol, and the elution method is that the chloroform and the methanol are sequentially subjected to gradient elution according to the ratio of 2: 1-1: 2;
the method for measuring the antibacterial activity comprises the following steps: dissolving the compound in PBS buffer solution to prepare stock solution, inoculating test bacteria and fungi by a two-fold dilution method, culturing the bacteria at a constant temperature of 37 ℃ for 24 hours, culturing the fungi at a constant temperature of 28 ℃ for 48 hours, and observing the result to find out the lowest drug concentration for preventing the bacteria from growing, namely the Minimum Inhibitory Concentration (MIC). On the basis of MIC, the bacterial liquid is continuously inoculated on a sterile culture medium for culture, the number of bacterial colonies is calculated by a viable count method, and the drug concentration with the average number of bacterial colonies being less than 5 is the Minimum Bactericidal Concentration (MBC).
Antibacterial activity assay results: see table 1.
Table 1: MIC (mg/mL) and MBC (mg/mL) for ent-16. beta. H-kaurane-17, 19-dicarboxylic acid for each strain
Example 2:
this example provides a method for preparing the compound enantiomer-16 α, 17-dihydroxykaurane-19-carboxylic acid, and a method and results for determining its antibacterial activity.
The chemical structure is as follows:
the preparation method comprises the following steps:
step 1, extraction: crushing herba siegesbeckiae, heating and refluxing the crushed herba siegesbeckiae for 2 hours at 45-60 ℃ by using an ethanol water solution with the volume percentage concentration of 85-95%, extracting for 3 times, filtering, collecting filtrate, and concentrating the filtrate under reduced pressure or normal pressure until the volume of the filtrate is less than 3-5% of that of the stock solution to obtain a concentrated solution;
step 2, degreasing: dissolving the concentrated solution in 0.5-1.5 times of water, extracting with n-hexane, cyclohexane or petroleum ether with the same volume, discarding the extract, and collecting the residual water solution;
step 3, separation: concentrating the residual water solution obtained in the previous step to 1-3% of the volume of the residual water solution, and then carrying out macroporous resin column chromatographic separation to obtain a separation solution; the eluent separated by the macroporous resin column chromatography is alcohol-water solutions with different concentrations, the volume ratio of alcohol to water in the alcohol-water solution is 1: 9-9: 1, and the elution method is that gradient elution is carried out in sequence from small concentration to large concentration according to the concentration of the alcohol;
and 4, purifying: purifying the separation liquid obtained in the above steps sequentially through silica gel column chromatography and Sephadex LH-20 column chromatography to obtain the final product.
In the step 4, the particle size of silica gel in the silica gel column chromatography is 200-300 meshes, the eluent is a mixed solution of cyclohexane and ethyl acetate, and the elution method is that the ratio of the cyclohexane to the ethyl acetate is 8: 1-4: 1, and gradient elution is carried out in sequence;
the eluent of the Sephadex LH-20 column chromatography is a mixed solution of chloroform and methanol, and the elution method is that the two are sequentially subjected to gradient elution according to the ratio of 3: 1-1: 1;
the method for measuring the antibacterial activity comprises the following steps: dissolving the compound in PBS buffer solution to prepare stock solution, inoculating test bacteria and fungi by a two-fold dilution method, culturing the bacteria at a constant temperature of 37 ℃ for 24 hours, culturing the fungi at a constant temperature of 28 ℃ for 48 hours, and observing the result to find out the lowest drug concentration for preventing the bacteria from growing, namely the Minimum Inhibitory Concentration (MIC). On the basis of MIC, the bacterial liquid is continuously inoculated on a sterile culture medium for culture, the number of bacterial colonies is calculated by a viable count method, and the drug concentration with the average number of bacterial colonies being less than 5 is the Minimum Bactericidal Concentration (MBC).
Antibacterial activity assay results: see table 2.
Table 2: MIC (mg/mL) and MBC (mg/mL) of ent-16 α, 17-dihydroxy kaurane-19-carboxylic acid for each strain
Example 3:
this example provides a method for preparing compound enantiomer-16 α,17, 18-trihydroxykaurane-19-carboxylic acid and a method and results for determining its antibacterial activity.
The chemical structure is as follows:
the preparation method comprises the following steps:
step 1, extraction: crushing herba siegesbeckiae, heating and refluxing the crushed herba siegesbeckiae for 2 hours at 45-60 ℃ by using an anhydrous methanol solution, extracting for 3 times, filtering, collecting filtrate, and concentrating the filtrate under reduced pressure or normal pressure until the volume of the filtrate is less than 3-5% of the volume of the stock solution to obtain a concentrated solution;
step 2, degreasing: dissolving the concentrated solution in 0.5-1.5 times of water, extracting with n-hexane, cyclohexane or petroleum ether with the same volume, discarding the extract, and collecting the residual water solution;
step 3, separation: concentrating the residual water solution obtained in the previous step to 1-3% of the volume of the residual water solution, and then carrying out macroporous resin column chromatographic separation to obtain a separation solution; the eluent separated by the macroporous resin column chromatography is alcohol-water solutions with different concentrations, the volume ratio of alcohol to water in the alcohol-water solution is 1: 9-9: 1, and the elution method is that gradient elution is carried out in sequence from small concentration to large concentration according to the concentration of the alcohol;
and 4, purifying: purifying the separation liquid obtained in the above steps sequentially through silica gel column chromatography and Sephadex LH-20 column chromatography to obtain the final product.
In the step 4, the particle size of silica gel in the silica gel column chromatography is 150-300 meshes, the eluent is a mixed solution of normal hexane and acetone, and the elution method is that the two are sequentially subjected to gradient elution according to the ratio of 4: 1-1: 2;
the eluent of the Sephadex LH-20 column chromatography is sequentially subjected to gradient elution with methanol and water in a ratio of 9: 1-7: 1;
the method for measuring the antibacterial activity comprises the following steps: dissolving the compound in PBS buffer solution to prepare stock solution, inoculating test bacteria and fungi by a two-fold dilution method, culturing the bacteria at a constant temperature of 37 ℃ for 24 hours, culturing the fungi at a constant temperature of 28 ℃ for 48 hours, and observing the result to find out the lowest drug concentration for preventing the bacteria from growing, namely the Minimum Inhibitory Concentration (MIC). On the basis of MIC, the bacterial liquid is continuously inoculated on a sterile culture medium for culture, the number of bacterial colonies is calculated by a viable count method, and the drug concentration with the average number of bacterial colonies being less than 5 is the Minimum Bactericidal Concentration (MBC).
Antibacterial activity assay results: see table 3.
Table 3: MIC (mg/mL) and MBC (mg/mL) of ent-16 α,17, 18-trihydroxykaurane-19-carboxylic acid for each strain
Example 4:
this example provides a method for preparing the compound enantiomer-17, 18-dihydroxy-kaurane-16 β H-19-carboxylic acid, and a method and results for determining its antibacterial activity.
The chemical structure is as follows:
the preparation method comprises the following steps:
step 1, extraction: crushing herba siegesbeckiae, soaking in 75-90 vol% methanol aqueous solution, performing microwave extraction for 3 times at power of 300-500W for 3 min, 4 min and 5 min, filtering, collecting filtrate, and concentrating under reduced pressure or normal pressure until the volume of the filtrate is less than 3-5 vol% of the stock solution to obtain concentrated solution;
step 2, degreasing: dissolving the concentrated solution in 0.5-1.5 times of water, extracting with n-hexane, cyclohexane or petroleum ether with the same volume, discarding the extract, and collecting the residual water solution;
step 3, separation: concentrating the residual water solution obtained in the previous step to 1-3% of the volume of the residual water solution, and then carrying out macroporous resin column chromatographic separation to obtain a separation solution; the eluent separated by the macroporous resin column chromatography is alcohol-water solutions with different concentrations, the volume ratio of alcohol to water in the alcohol-water solution is 1: 9-9: 1, and the elution method is that gradient elution is carried out in sequence from small concentration to large concentration according to the concentration of the alcohol;
and 4, purifying: purifying the separation liquid obtained in the above steps sequentially through silica gel column chromatography and Sephadex LH-20 column chromatography to obtain the final product.
In the step 4, the particle size of silica gel in the silica gel column chromatography is 100-150 meshes, the eluent is a mixed solution of petroleum ether and dichloromethane, and the elution method comprises the step of gradient elution of the two solutions in a ratio of 7: 1-1: 1;
the eluent of the Sephadex LH-20 column chromatography is sequentially subjected to gradient elution with the ratio of methanol to water being 10: 1-8: 1;
the method for measuring the antibacterial activity comprises the following steps: dissolving the compound in PBS buffer solution to prepare stock solution, inoculating test bacteria and fungi by a two-fold dilution method, culturing the bacteria at a constant temperature of 37 ℃ for 24 hours, culturing the fungi at a constant temperature of 28 ℃ for 48 hours, and observing the result to find out the lowest drug concentration for preventing the bacteria from growing, namely the Minimum Inhibitory Concentration (MIC). On the basis of MIC, the bacterial liquid is continuously inoculated on a sterile culture medium for culture, the number of bacterial colonies is calculated by a viable count method, and the drug concentration with the average number of bacterial colonies being less than 5 is the Minimum Bactericidal Concentration (MBC).
Antibacterial activity assay results: see table 4.
Table 4: MIC (mg/mL) and MBC (mg/mL) of enantiomer-17, 18-dihydroxykaurane-16. beta. H-19-carboxylic acid for each strain
The experimental results show that: the diterpenoid compounds in the siegesbeckia orientalis can inhibit Candida albicans (Candida albicans), Candida tropicalis (Candida tropicalis), Staphylococcus aureus (Staphylococcus aureus), Pseudomonas aeruginosa (Pseudomonas aeruginosa) and Escherichia coli (Escherichia coli), especially can inhibit methicillin-resistant Staphylococcus aureus (MRSA), so the diterpenoid compounds in the siegesbeckia orientalis can be used for preparing antibacterial drugs or lead compounds of the antibacterial drugs.
The obtained diterpenoid compound or diterpenoid compound composition can be mixed with pharmaceutically acceptable adjuvants to make into tablet, injection, capsule, granule, powder, suppository, unguent, spray, gel, lotion, pellicle, liniment, foam, liniment, etc.
The above embodiments are merely preferred embodiments of the present invention, which are provided for illustration and not for limitation.
Claims (7)
1. Diterpenoid compounds extracted from herba siegesbeckiae are characterized in that: the general chemical structure of the diterpenoid compound is as follows:
wherein,
R1is CH3Or CH2OH；
R2Is H or OH or CH3Or CH2OH；
R3Is H or CH3Or OH or CH2OH or COOH.
2. A method for extracting diterpenoid compounds according to claim 1 from Siegesbeckiae herba, which comprises: the method comprises the following steps:
step 1, extraction: crushing the siegesbeckia herb, extracting with an extracting solution, filtering, collecting filtrate, and concentrating under reduced pressure or normal pressure until the volume of the filtrate is less than 3-5% of that of the stock solution to obtain a concentrated solution;
step 2, degreasing: dissolving the concentrated solution in 0.5-1.5 times of water, extracting with n-hexane, cyclohexane or petroleum ether with the same volume, discarding the extract, and collecting the residual water solution;
step 3, separation: concentrating the residual water solution obtained in the previous step to 1-3% of the volume of the residual water solution, and then carrying out macroporous resin column chromatographic separation to obtain a separation solution; the eluent separated by the macroporous resin column chromatography is alcohol-water solutions with different concentrations, the volume ratio of alcohol to water in the alcohol-water solution is 1: 9-9: 1, and the elution method is that gradient elution is carried out in sequence from small concentration to large concentration according to the concentration of the alcohol;
and 4, purifying: purifying the separation liquid obtained in the above steps sequentially through silica gel column chromatography and Sephadex LH-20 column chromatography to obtain the final product.
3. The extraction method according to claim 2, characterized in that: the extracting solution in the step 1 is one of water, absolute ethyl alcohol, 30-95% ethanol water solution by volume percentage concentration, methanol, 20-80% methanol water solution by volume percentage concentration, acetone, 30-70% acetone water solution by volume percentage concentration and ethyl acetate; the extraction method comprises the steps of carrying out ultrasonic wave for 15-40 minutes at 25 ℃, or carrying out heating reflux for 2 hours at 45-60 ℃ or carrying out microwave for 2-5 minutes at the power of 300-500 watts.
4. The extraction method according to claim 2, characterized in that: in the step 4, the particle size of silica gel in the silica gel column chromatography is 100-300 meshes, the eluent is a mixed solution of n-hexane, cyclohexane or petroleum ether and ethyl acetate, acetone or dichloromethane in different proportions, and the elution method is that the polarity of the eluent is eluted from small to large in sequence; the eluent of the Sephadex LH-20 column chromatography is a mixed solution of chloroform and methanol or a mixed solution of methanol and water, the elution method is based on the methanol, and the methanol volume percentage concentration is in the range of 30-100% and is eluted from small to large in sequence.
5. Use of the diterpenoid of claim 1 for the preparation of an antimicrobial medicament.
6. The use of claim 5, wherein: the microorganism is a fungus or a bacterium.
7. The use of claim 5, wherein: the antimicrobial drug can be diterpenoid compounds or diterpenoid compound compositions and tablets, injections, capsules, granules, powder, suppositories, paste, sprays, gels, lotions, films, liniments, foams and paints prepared from the diterpenoid compounds or diterpenoid compound compositions and pharmaceutically acceptable auxiliary materials.
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