CN113185393B

CN113185393B - Guaiane type sesquiterpene compound, preparation method and application thereof

Info

Publication number: CN113185393B
Application number: CN202110481776.XA
Authority: CN
Inventors: 任佳丽; 钟平胜; 杨亚兰; 肖臻研; 贺雅雯; 杨桥
Original assignee: Changsha Changjun Middle School; Central South University of Forestry and Technology
Current assignee: Changsha Changjun Middle School; Central South University of Forestry and Technology
Priority date: 2021-04-30
Filing date: 2021-04-30
Publication date: 2023-06-23
Anticipated expiration: 2041-04-30
Also published as: CN113185393A

Abstract

The invention relates to the technical field of chemical medicines, in particular to a guaiane type sesquiterpene compound, a preparation method and application thereof. The structural formula of the guaiane sesquiterpene compound is shown as follows:

the guaiane sesquiterpene compound is extracted from Rumex Lactarius, can well inhibit the activity of tubercle bacillus, and particularly has excellent inhibition effect on drug-resistant tubercle bacillus or multi-drug-resistant tubercle bacillus, so that the guaiane sesquiterpene compound can be used for treating tuberculosis, particularly the existing drug-resistant tuberculosis.

Description

Guaiane type sesquiterpene compound, preparation method and application thereof

Technical Field

The invention relates to the technical field of chemical medicines, in particular to a guaiane type sesquiterpene compound, a preparation method and application thereof.

Background

Tuberculosis is the biggest killer seriously threatening human health after aids due to the increase of population flow and the generation of antibiotic resistance, and has an outbreak worldwide. WHO in 2018 indicated that the global tuberculosis increased cases up to 1000 ten thousand, with about 157 ten thousand deaths. The internationally common antitubercular drugs have the defects of large toxic and side effects and easy drug resistance, and the occurrence of multi-drug resistant strains in recent years increases the difficulty of tuberculosis treatment. Therefore, research and development of new antitubercular drugs are not slow, and natural products have infinite prospects in the development of antitubercular drugs.

The Rugu exomycorrhizal edible fungi contain various nutritional components and have high edible value. The nutritional ingredient analysis results are consistent with the nutritional ingredient analysis results of most edible mushrooms, the content of crude fat in the edible mushrooms is low, the content of crude protein and carbohydrate is high, and the content of unsaturated fatty acid in most edible mushrooms is slightly higher than that of saturated fatty acid, wherein the unsaturated fatty acid with C18 is the majority. Most edible Lactarius deliciosus have complete amino acid types, the content of which is different according to different strains, but the composition structure is basically the same, and the content of essential amino acid accounts for about 40% of the total amino acid. In addition, the edible Lactarius has histidine which is necessary for the growth and development of infants and lysine which is lacking in cereal foods, and aspartic acid and glutamic acid which provide fresh taste. The B vitamins are main vitamins in the edible Lactarius deliciosus. Modern nutrition and pharmacological research show that the Rumex fungi also contain various functional components such as sesquiterpenes, sterols, chromenes, geranium Niu Erfen, alkaloids and the like. Terpenes are hydrocarbons and derivatives thereof of the molecular formula in multiples of isoprene in nature, and are named and classified according to the number of isoprene, such as monoterpenes (10 carbons), sesquiterpenes (15 carbons), diterpenes (20 carbons), sesterterpenes (25 carbons), triterpenes (30 carbons) and tetraterpenes (35 carbons). Common terpenoids are: monoterpene borneol and camphor in camphoraceae plants, sesquiterpene artemisinin in asteraceae plants, diterpene vitamin A in animal livers, triterpene ganoderic acid in edible fungi and the like. The terpenoid has the activities of anti-inflammatory, anti-tumor, antioxidation, bacteriostasis and the like, and has great development and utilization value in the pharmaceutical industry. The biological activity of the terpenoid in the Lactarius is studied, the added value of the Lactarius product can be improved, the local economic development is promoted, and the novel preparation has great significance for the development of medicines and health care products, and provides a novel choice for the clinical treatment of tuberculosis, especially tuberculosis caused by drug-resistant tubercle bacillus.

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims to provide a guaiane type sesquiterpene compound, a preparation method and application thereof. The guaiane sesquiterpene compound is extracted from Rumex Lactarius, can well inhibit the activity of tubercle bacillus, and particularly has excellent inhibition effect on drug-resistant tubercle bacillus or multi-drug-resistant tubercle bacillus, so that the guaiane sesquiterpene compound can be used for treating tuberculosis, particularly the existing drug-resistant tuberculosis.

The invention is realized in the following way:

in a first aspect, the present invention provides a guaiane-type sesquiterpene compound having the structural formula:

in a second aspect, the present invention provides a method for preparing a guaiane-type sesquiterpene compound, comprising: extracting the fruiting body of Rumex Lactarius to obtain the guaiane sesquiterpene compound.

In a third aspect, the invention provides an application of a guaiane type sesquiterpene compound in preparing a medicament for treating any one of tuberculosis, single-drug-resistant tuberculosis and multi-drug-resistant tuberculosis.

In a fourth aspect, the invention provides an application of a guaiane type sesquiterpene compound in preparing an inhibitor for inhibiting any one of tubercle bacillus, single-drug-resistant tubercle bacillus and multi-drug-resistant tubercle bacillus.

The invention has the following beneficial effects: the embodiment of the invention provides a new-structure guaiane type sesquiterpene compound, which is extracted from the fruiting body of the Rumex Lactarius, and can well inhibit the activity of tubercle bacillus, particularly has an excellent inhibition effect on single-drug-resistant tubercle bacillus or multi-drug-resistant tubercle bacillus, and can be used for treating tuberculosis, particularly the existing drug-resistant tuberculosis, and further expands the types of drugs for treating tuberculosis, particularly drug-resistant tuberculosis, and also expands the application range of Rumex Lactarius extract.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a liquid chromatogram (A) and a purity analysis chromatogram (B) of a guaiane-type sesquiterpene compound prepared in example 1 of the present invention;

FIG. 2 is an ultraviolet-visible spectrum of a guaiane-type sesquiterpene compound provided by the embodiment of the invention;

FIG. 3 is an infrared spectrum of a guaiane-type sesquiterpene compound provided by the embodiment of the invention;

FIG. 4 is a mass spectrum of a guaiane-type sesquiterpene compound provided by the embodiment of the invention;

FIG. 5 shows a nuclear magnetic resonance spectrum of a guaiane-type sesquiterpene compound provided by the embodiment of the invention;

FIG. 6 shows a nuclear magnetic resonance spectrum of a guaiane-type sesquiterpene compound provided by the embodiment of the invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The embodiment of the invention provides a guaiane sesquiterpene compound, which has the following structural formula:

the invention provides a preparation method of guaiane sesquiterpene compounds, which comprises the following steps:

extracting the fruiting body of Rumex Lactarius to obtain the guaiane sesquiterpene compound. The lactarius fruit body can be any one of Rumex volvacea fruit body, rumex volvacea fruit body and Rumex aurantium fruit body or other Rumex volvacea fruit bodies containing the guaiane type sesquiterpene compound.

Firstly, preprocessing the Lactarius deliciosus fruiting body before extraction; placing the lactarius deliciosus fruiting bodies for a period of time under a medium temperature condition, then placing for a period of time under a high temperature condition, and then placing for a period of time under a low temperature condition; for example, the fresh Lactarius rupestris fruiting body is left for 4 to 6 hours (for example, for 4 to 4 hours, for 4.5 hours, for 5 hours, for 6 hours, for example, for 4 to 6 hours) under conditions of 40 to 50 ℃ (for example, for any value between 40 ℃ and 42 ℃, 44 ℃, 46 ℃, 48 ℃ and 50 ℃ and the like), and then left for 0.5 to 4 hours (for example, for 0.5 hours, 1 hour, 1.5 hours, 2 hours, 3 hours, 3.5 hours, and 4 hours) under conditions of 60 to 70 ℃ (for example, for any value between 60 ℃ and 62 ℃, 64 ℃, 66 ℃, 68 ℃ and 70 ℃, and the like), and for oxygen partial pressure less than 10% (for example, 5%) under conditions of 0.5 to 4 hours (for example, for 0.5 hours, 1 hour, 1.5 hours, 2 hours, 3 hours, 3.5 hours, and 4 hours and the like), and then left for 4 hours (for example, for 4 hours and the like) under conditions of 0 to 4 ℃ (for example, for 0 ℃ to 2 ℃,3 ℃ and 4 ℃ and the like). By adopting the mode to pretreat the Lactarius deliciosus fruiting body, the yield of the guaiane sesquiterpene compound can be effectively improved.

After the pretreatment, the fruiting body of Lactarius is crushed and then extracted with an organic solvent, wherein the organic solvent is selected from at least one of ketone solvents (e.g. acetone), alcohol solvents (e.g. methanol) and halogen-substituted methane solvents (e.g. chloroform).

It will be appreciated that the above-mentioned acetone, methanol and chloroform are merely examples of the corresponding types of solvents, and that other solvents of the same type, such as ethanol, methylene chloride, or even other types, such as cyclohexane, are also possible, as long as the guaiane-type sesquiterpene compound can be extracted.

Specifically, acetone is mixed with the Lactarius deliciosus fruiting body for leaching for a plurality of times; wherein the temperature of each leaching is room temperature (25-30 ℃), and the time of each leaching is any value between 12 and 36 hours, such as 12 hours, 18 hours, 20 hours, 24 hours, 30 hours, 32 hours, 35 hours, 36 hours, and the like.

Mixing the mixed solvent formed by methanol and chloroform with the fruiting body of Lactarius rupestris, and extracting for multiple times; wherein the extraction temperature is room temperature (25-30deg.C), the time is 12-24 hours, such as 12-24 hours, 15-18 hours, 20-24 hours, and the like, and the volume ratio of methanol to chloroform is 1:1-1:2, such as 1:1, 1:1.2, 1:1.5, 1:1.9, and 1:2, etc., and the volume ratio is 1:1-1:2.

The (1) lactarius deliciosus fruiting body extracted with a mixed solvent of methanol and chloroform may be a new lactarius deliciosus fruiting body which has not been extracted, or may be a lactarius deliciosus fruiting body extracted with acetone as described above.

(2) The number of times of leaching and extraction can be 1 time, 2 times, 3 times or even 4 times, and the leaching or extraction times can be properly adjusted according to the requirements.

(3) The extraction with acetone leaching, methanol and chloroform mixture described above is merely an example and is not limited to this extraction solvent or method.

And combining the leaching solution obtained by leaching and the extracting solution obtained by extracting to form an extract, namely an extracting mixed solution. It will be appreciated that the extracts from each leaching may be combined, the extracts from each extraction may be combined, and then the two combined to form an extraction mixture.

Concentrating the extract, and removing organic solvents such as acetone, methanol and chloroform from the extract to obtain crude extract; and extracting the crude extract, and collecting the extracted organic solvent part.

Specifically, the step of extracting includes: extracting with mixed solvent of ester solvent and water, and collecting ester part as organic solvent; wherein the ester solvent comprises any one of ethyl acetate, methyl acetate, ethyl formate and methyl formate; the volume ratio of the ester solvent to the water is 1:1-1:2; for example, any number between 1:1 and 1:1.2, 1:1.5, 1:1.9, and 1:2, etc. 1:1 and 1:2.

Then, the ester part is subjected to column chromatography separation, and the operation steps comprise: gradient elution is carried out by using a mixed solvent of an ether solvent and an acetone solvent; specifically, gradient elution is carried out with petroleum ether and acetone in sequence at a volume ratio of 100:0 to 0:100, and petroleum ether is collected: an elution portion having a acetone volume ratio of 98:2; more specifically, petroleum ether and acetone are sequentially used in a volume ratio of 100:0, 98:2,95: 5,90: 10, 80:20, 50: gradient elution was performed at 50 and 0:100 and petroleum ether was collected: an elution fraction having a acetone volume ratio of 98:2.

Then, the petroleum ether obtained by collection was: the elution part with the acetone volume ratio of 98:2 is prepared by liquid chromatography separation and purification, and the conditions for the liquid chromatography separation and purification preparation include: the mobile phase comprises: the alcohol solvent is preferably a methanol solvent. The methanol solvent can be pure methanol solvent or mixed solvent of methanol and water with volume ratio of 95-99:5-1.

The embodiment of the invention also provides application of the guaiane sesquiterpene compound, which can effectively inhibit tubercle bacillus, drug-resistant tubercle bacillus and multi-drug-resistant tubercle bacillus, and can be used for inhibiting the pathogens and treating diseases caused by the pathogen infection. And wherein the drug resistant tuberculosis or the drug resistant in the single resistant tubercle bacillus comprises any one of rifampicin, ethambutol, kanamycin, amikacin, cursorin, ofloxacin and pyrazinamide; the drug resistant in the multi-drug resistant tuberculosis or multi-drug resistant tubercle bacillus comprises at least 2 of rifampicin, ethambutol, kanamycin, amikacin, cursorine, ofloxacin and pyrazinamide.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

Example 1

The embodiment of the invention provides a preparation method of a guaiane sesquiterpene compound, which comprises the following steps:

(1) Pretreatment: the fresh russula fruiting body was left at 45℃for 6 hours, then at 60℃for 3 hours with an oxygen partial pressure of 5%, then at 0℃for 24 hours.

(2) Mincing 1.3Kg of the pretreated fruiting body of Rumex Lactarius with tissue crusher, extracting with acetone (0.5L) at 25deg.C for 3 times each for 24 hr, and collecting extractive solutions obtained by combining 3 times of extraction. Next, methanol was used with chloroform=1:1 _(V:V) Extracting the extracted fruiting body of Rumex ruri with mixed solvent for 24 hr for 2 times, and collecting and mixing the 2 times of extractive solutions; mixing the above extractive solutions, vacuum filtering with gauze, and vacuum rotary evaporating to remove organic solvents such as acetone, methanol and chloroform to obtain crude extract. Dissolving the crude extract with ethyl acetate, adding water with the same volume into the ethyl acetate solution for extraction for 3 times, discarding the water phase, and evaporating the ethyl acetate phase in vacuum to obtain ethyl acetate extract.

(3) Weighing 200-300 mesh silica gel with 40 times of sample loading amount, adding petroleum ether, stirring to obtain uniform bubble-free homogenate, adding the silica gel homogenate into a glass chromatographic column with an opened piston, continuously flushing with petroleum ether after the silica gel subsides until the liquid level at the top of the column is stable and the chromatographic column is bubble-free, and loading the sample. Dissolving the ethyl acetate extract in the step (2) by using petroleum ether, dripping the ethyl acetate extract into the top of a silica gel column by using a dropper, and sequentially using petroleum ether and acetone from the gradient of 100:0 and 98:2,95: 5,90: 10, 80:20, 50:50 and 0:100 (volume ratio), and collecting petroleum ether: acetone=98:2 _(V:V) Is a red fraction of (c).

(4) The red fraction was dissolved in methanol and purified by preparative HPLC. The preparation conditions of the liquid phase are as follows: the mobile phase is methanol: water=95:5 solution, flow rate 10mL/min, sample concentration: 10mg/mL, sample injection amount of 2mL, detection wavelength of 241nm, to obtain red compound, see FIG. 1A.

And (3) purity detection: the pure methanol was used as a mobile phase, and the analytical column was an ODS C18 column (4.6 mm. Times.250 mm,5 μm), 241nm in wavelength and 1mL/min in flow rate. Purity was 95% as measured, see B in fig. 1.

The yield of this compound was 1.3% (on a dry basis).

The prepared compound is characterized, and the characterization result is shown in figures 2-6, wherein figure 2 is an ultraviolet visible spectrum; FIG. 3 is an infrared spectrum; FIG. 4 is a mass spectrum; FIG. 5 is a nuclear magnetic resonance hydrogen spectrum; FIG. 6 is a nuclear magnetic resonance spectrum. As can be seen from fig. 2-6, the characterization data are as follows: 1406cm ^-1 There is an infrared characteristic absorption peak indicating that the compound contains a c=c double bond at 1651cm ^-1 The absorption peak was found to contain-CHO. m/z:211[ M+H ]]Molecular weight of 210, molecular formula C is presumed ₁₅ H ₁₄ O。 ¹ H-NMR(DMSO-d ₆ ,400MHz):10.31(1H,s,H-15),9.77(1H,d,J＝2.1Hz,H-8),8.28(1H,d,J＝4.3Hz,H-2),8.07(1H,dd,J＝10.9,2.1Hz,H-6),7.66(1H,d,J＝10.9Hz,H-5),7.43(1H,d,J＝4.3Hz,H-3),5.50(1H,br s,H-13a),5.39(1H,br s,H-13b),2.94(3H,s,H-14),2.30(3H,br s,H-12)； ¹³ C-NMR(DMSO-d ₆ ,100MHz):186.7(d,C-15),149.7(s,C-4),145.7(s,C-11),143.2(s,C-10),141.5(d,C-2),141.2(s,C-7),137.9(s,C-9),136.8(d,C-8),135.1(d,C-6),131.0(d,C-5),126.4(s,C-1),116.8(d,C-3),116.7(t,C-13),24.3(q,C-14),22.6(q,C-12)。

And finally determining that the red compound is guaiane sesquiterpene, the monomer is red solid, and the compound is soluble in organic solvents such as methanol petroleum ether and the like, and the molecular formula is C ₁₅ H ₁₄ O has the following structural formula:

comparative example 1: the guaiane-type sesquiterpene compounds were extracted by the method of example 1, except that: the fresh russula fruiting body is not treated, namely 1.3Kg of the fresh russula fruiting body is directly minced by a tissue crusher and then extracted. This comparative example was identical to example 1 except that no pretreatment was performed, and the yield of the guaiane-type sesquiterpene compound was 0.03% (on a dry basis).

Experimental example

Acidic rogowski medium: the basic acid Roche medium contains L-sodium glutamate, magnesium sulfate, magnesium citrate, potassium dihydrogen phosphate, potato starch, malachite green and the like, 52.4g of the basic acid Roche medium is weighed and dissolved in 600mL of distilled water, 12mL of glycerol and 24mL of tween-80 are respectively added for uniform mixing, the mixture is boiled in a boiling water pot for 30-40min (continuous shaking and stirring during the period) to form paste, and when the mixture is cooled to about 50 ℃, 1000mL of sterile fresh whole egg liquid is added for preparation of the medicine-containing medium after uniform mixing.

Preparing a medicine-containing culture medium: the guaiane-type sesquiterpene compound prepared in example 1 was dissolved in DMSO, added to the above-mentioned acidic Roche medium containing 1.5% Tween-80 to prepare a medium having a drug concentration of 9.5. Mu. Mol/mL, and the medium was diluted twice to prepare a medium having a concentration of 4.8. Mu. Mol/mL, 2.4. Mu. Mol/mL, 1.2. Mu. Mol/mL, 0.6. Mu. Mol/mL, 0.3. Mu. Mol/mL and 0.15. Mu. Mol/mL, each having 3 parallel positive controls of streptomycin, and negative controls of a blank medium having no drug, solidified at 80-90℃for 90min, and subjected to aseptic test at 37℃for 24 hours after cooling, to inoculate H37Ra.

Inoculation of H37 Ra: vortex grinding the cultured H37Ra culture with glass beads in 0.5% Tween-80 physiological saline, and adjusting absorbance to concentration of bacterial suspension of 10 ⁶ cfu/mL. Taking 0.1mL of the prepared H37Ra bacterial suspension, inoculating to a positive culture medium, a negative culture medium and a drug-containing culture medium with different concentrations respectively under aseptic conditions, culturing at a constant temperature of 37 ℃, continuously observing for 4 weeks, and recording the growth condition of tubercle bacillus on the surface of the culture medium weekly.

Judging experimental results: no colonies or fewer than 20 are-; less than 100 but not more than 1/4 of the area of the bevel is +;100-200 colonies occupy 1/2 of the area of the inclined plane, which is++; most of the fusion fragments (200-500 colonies) occupy 3/4 of the slant area, which is++; fusion plates (greater than 500 colonies), 3/4 of the area of the inclined plane, ++, were used as effective results for control group growth, and the concentration was the lowest inhibitory concentration MIC when the ratio of colony numbers of the fourth week negative control and drug-containing medium was close to 1%. The experiment was set up in 3 replicates and the results averaged 3 times.

The results are shown in Table 1.

TABLE 1 inhibition results of the H37Ra of Bacillus tuberculosis by guaiane-type sesquiterpene compounds at different concentrations

As is clear from Table 1, as the concentration of the guaiane-type sesquiterpene compound increases, the inhibition effect on H37Ra of Bacillus tuberculosis is stronger, and when the concentration of the compound is lower than 0.6. Mu. Mol/mL, a small amount of colony of H37Ra of Bacillus tuberculosis on the inclined surface can be observed in the first week; at compound concentrations of 2.4 and 1.2. Mu. Mol/mL, a small number of M.tuberculosis H37Ra colonies on the slope could be observed in the third week; when the compound concentration was higher than 4.8. Mu. Mol/mL, the growth of H37Ra colonies on the slope was not observed even by the fourth week, and the growth of tubercle bacillus colonies was not observed in the following fifth to eighth weeks. The result shows that the guaiane type sesquiterpene compound with the concentration of 4.8 mu mol/mL has obvious antibacterial effect on the tubercle bacillus H37Ra.

Similarly, the guaiane type sesquiterpene compound and the crude extract thereof prepared in example 1 were dissolved in DMSO, and added to an acidic Roche medium containing 1.5% Tween-80, the crude extract and the monomer were prepared into 9.5. Mu. Mol/mL medium, respectively, and the blank medium was a non-medicated blank medium, and the medicated medium and the blank medium were coagulated at 80-90℃for 90min, and after cooling, subjected to a sterile test at 37℃for 24 hours, and used for inoculation of drug-resistant tubercle bacillus.

Inoculation of clinical strains: the culture of H37Rv and clinical tubercle bacillus B1, B8, B25 and B30 is ground and mixed with Tween-80 physiological saline of 0.5%, and the mixture is mixed with a Mitsubishi turbidimetric tube to prepare a bacterial suspension with the wet weight of 1 mg/mL. 1mg/mL of the fungusThe suspension is diluted to 10 times ^-2 mg/mL, 0.1mL of bacterial liquid is inoculated on a guaiane type sesquiterpene compound and a crude extract drug-containing culture medium thereof and a blank culture medium inclined plane, and the results are observed after the culture is carried out at the constant temperature of 37 ℃ for 4 weeks.

Reporting the results: -indicating that the medium slope is free of tubercle bacillus colony growth; + represents the number of colonies which occupies about 1/4 of the area of the inclined plane; ++ means that the colony count occupies about 1/2 of the area of the inclined plane; ++ indicates that the colony count is approximately equal to 3/4 of the area of the inclined plane; ++ indicates that the colony count grows like a lawn, and the colony count is reported when the colony count on the drug-containing medium is below 20.

The results are shown in Table 2.

Meanwhile, a BACTEC MGIT 960 drug resistance analysis system is adopted to test the drug resistance, and the result is shown in Table 3.

TABLE 2 guaiane sesquiterpene Compounds and crude extracts thereof (9.5. Mu. Mol/mL) inhibiting tubercle bacillus H37Rv and drug-resistant, multi-resistant tubercle bacillus results

As is clear from Table 2, no colony growth was observed on LB slopes containing 9.5. Mu. Mol/mL of the guaiane-type sesquiterpene compound and the crude extract thereof, indicating that the extracted compound and the crude extract thereof had a good inhibitory effect on clinical tubercle bacillus at a concentration of 9.5. Mu. Mol/mL. The drug resistance of the B1, B3, B8 and B25 strains used in the experiment was determined by the same method, and the bacterial types were identified by an immune colloidal gold rapid assay, and the results are shown in Table 3.

Table 3 quality control results of H37Rv, B1, B8, B25, and B30

Wherein AST is a drug sensitivity test by adopting a conventional SM; r is drug resistance; s, sensitivity; MPB64+ is immune colloidal gold rapid detection method detection result is tubercle bacillus.

As can be seen from table 3, the quality control strain H37Rv was sensitive to antitubercular drugs isoniazid, rifampicin, ethambutol, kanamycin, amikacin, cursorcin, ofloxacin and pyrazinamide; b1 is tolerant to six of the eight drugs tested, isoniazid, rifampin, ethambutol, kanamycin, amikacin, and pyrazinamide; b8 has tolerance to isoniazid; b25 is resistant to isoniazid, rifampicin, kanamycin and pyrazinamide; b30 is resistant to frizzled and ofloxacin. From the above, B8 is a single resistant strain, and B1, B25 and B30 are multiple resistant strains.

In conclusion, the guaiane type sesquiterpene compound provided by the embodiment of the invention has good activity of resisting tubercle bacillus H37Ra, and the minimum antibacterial concentration is 4.8 mu mol/mL, which is equivalent to that of streptomycin with positive control of 100 mu g/mL; the inhibition experiment of clinical drug-resistant strain shows that the compound has strong inhibition effect on H37Rv, single-drug-resistant tubercle bacillus and multi-drug-resistant tubercle bacillus, and has extremely high patent drug value, and the compound is expected to become a new antitubercular drug, further expands the type of the antitubercular drug and expands the application range of the Lactarius rupestris.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The application of the guaiane type sesquiterpene compound in preparing an inhibitor for inhibiting any one of single-drug-resistant tubercle bacillus and multi-drug-resistant tubercle bacillus is characterized in that the guaiane type sesquiterpene compound has the following structural formula:

；

the drug resistant in the single-drug resistant tubercle bacillus is selected from any one of isoniazid, rifampicin, ethambutol, kanamycin, amikacin, curcin, ofloxacin and pyrazinamide;

the drug resistant in the multi-drug resistant tubercle bacillus is selected from at least 2 of isoniazid, rifampicin, ethambutol, kanamycin, amikacin, cursorine, ofloxacin and pyrazinamide.

2. The use according to claim 1, characterized in that the process for the preparation of guaiane-type sesquiterpene compounds comprises: placing the fruiting body of Lactarius in 40-50deg.C for 4-6 hr, placing at 60-70deg.C for 0.5-4 hr with oxygen partial pressure less than 10%, placing at 0-4deg.C for more than 4 hr, extracting the fruiting body with organic solvent, concentrating, extracting, column chromatography, and liquid chromatography to obtain extract;

wherein the organic solvent is selected from at least one of ketone solvents, alcohol solvents and halogen-substituted methane solvents;

the extraction steps include: extracting with a mixed solvent of an ester solvent and water, and collecting an ester part;

the step of column chromatography comprises: gradient elution is carried out by using a mixed solvent of an ether solvent and an acetone solvent;

the conditions for liquid chromatography separation and purification preparation include: the mobile phase is an alcohol solvent.

3. The use according to claim 2, wherein the step of obtaining the extract comprises: extracting the fruiting body of Lactarius rupestris with at least one solvent selected from acetone, methanol and chloroform.

4. The use according to claim 2, wherein the step of obtaining the extract comprises: extracting the lactarius deliciosus fruiting body by using acetone, and then mixing the extracted lactarius deliciosus fruiting body by using a mixed solvent of methanol and chloroform for multiple times; and then combining the extracted liquid with the extracted liquid.

5. The use according to claim 4, wherein the volume ratio of methanol to chloroform is 1:1 to 1:2.

6. The use according to claim 2, wherein the ester solvent is selected from any one of ethyl acetate, methyl acetate, ethyl formate and methyl formate; the volume ratio of the ester solvent to the water is 1:1-1:2.

7. The use according to claim 2, wherein the step of column chromatography comprises: sequentially carrying out gradient elution by using petroleum ether and acetone according to the volume ratio of 100:0 to 0:100, and collecting petroleum ether: the volume ratio of acetone is 98:2.

8. The use according to claim 2, wherein the step of column chromatography comprises: sequentially carrying out gradient elution by using petroleum ether and acetone according to the volume ratio of 100:0, 98:2, 95:5, 90:10, 80:20, 50:50 and 0:100, and collecting an elution part of which the volume ratio of petroleum ether to acetone is 98:2.

9. The use according to claim 2, wherein the conditions for the preparation of the liquid chromatography separation and purification comprise: the mobile phase is methanol solvent.

10. The use according to any one of claims 2-9, wherein the lactarius fruit body is selected from any one of a russula vinosa fruit body, a lactarius pinnatifida fruit body and a lactarius aurantiacus fruit body.