Guaiane type sesquiterpene compound as well as preparation method and application thereof

Abstract

The invention discloses a guaiane type sesquiterpene compound as well as a preparation method and application thereof, and belongs to the technical field of chemical medicines. The invention provides a guaiane type sesquiterpene compound with a novel structure, which is extracted from lactarius deliciosus sporocarp, can well inhibit the activity of mycobacterium tuberculosis, especially has an excellent inhibition effect on single-drug-resistant mycobacterium tuberculosis and multi-drug-resistant mycobacterium tuberculosis, and can be used for preparing medicines for treating lactarius deliciosus. The guaiane type sesquiterpene compound is expected to be used for treating tuberculosis, especially drug-resistant tuberculosis, so that the variety of drugs for treating tuberculosis, especially drug-resistant tuberculosis, is expanded, and the application range of lactarius deliciosus extract is also expanded.

Description

Technical Field

[0001] This invention belongs to the field of chemical pharmaceutical technology, and more specifically relates to a guaiacolane-type sesquiterpene compound, its preparation method, and its application. Background Technology

[0002] In recent years, tuberculosis has become increasingly severe. Currently, commonly used drugs for treating tuberculosis generally have significant side effects and easily lead to drug resistance in bacteria. Furthermore, the emergence of multidrug-resistant strains in recent years has made tuberculosis treatment even more difficult. Against this backdrop, the development of novel anti-tuberculosis drugs has become an urgent priority, and natural products, with their unique advantages, show great promise in the field of anti-tuberculosis drug development.

[0003] Terpenes, as important natural products, possess a variety of biological activities, including anti-inflammatory, antitumor, antioxidant, and antibacterial properties, making them highly valuable for development and utilization in the pharmaceutical industry. The effectiveness and safety of these active compounds are closely related not only to their inherent properties but also to their purity. Currently, global pharmaceutical research faces the challenge of isolating and purifying effective compounds. This study employed multi-stage repeated silica gel column chromatography and innovatively introduced high-efficiency preparative liquid chromatography (HPLC) purification technology, increasing the product purity to over 98%. Terpenes are currently mainly extracted from plants of the Asteraceae, Apiaceae, and Lauraceae families. Research on the biological activity of terpenes extracted from edible *Lactarius deliciosus* (a type of mushroom) and their application in the preparation of anti-tuberculosis drugs remains lacking. Therefore, research on the biological activity of terpenes in *Lactarius deliciosus* is of significant importance in many aspects. It helps increase the added value of *Lactarius deliciosus* products and promotes the development of related local industries and the economy; the research results provide new ideas and options for the clinical treatment of tuberculosis, especially tuberculosis caused by drug-resistant *Mycobacterium tuberculosis*; and it is expected to promote innovative development in the field of anti-tuberculosis drugs, contributing to solving the global challenge of tuberculosis prevention and control. Summary of the Invention

[0004] The purpose of this invention is to provide a guaiac sesquiterpene compound, its preparation method, and its application to solve the problems existing in the prior art. This guaiac sesquiterpene compound is extracted from *Lactarius deliciosus* and can effectively inhibit the activity of Mycobacterium tuberculosis, especially showing excellent inhibitory effects against drug-resistant and multidrug-resistant Mycobacterium tuberculosis. Consequently, this guaiac sesquiterpene compound can be used to prepare drugs for treating tuberculosis, particularly for the preparation of existing drugs related to drug-resistant tuberculosis.

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

[0006] One of the technical solutions of this invention is to provide a method for extracting guaiacane-type sesquiterpenoid compounds, comprising the following steps:

[0007] The fruiting bodies of *Lactarius deliciosus* were extracted using a solvent system composed of chloroform and methanol. The solvent was then evaporated to obtain the primary extract.

[0008] The primary extract was extracted using a solvent system consisting of ethyl acetate and water. The organic layer was collected, concentrated, and a solid extract was obtained.

[0009] The solid extract was subjected to gradient elution using a solvent system composed of petroleum ether and acetone, the fractions were collected, evaporated, and the solid product was obtained.

[0010] The solid product was purified to obtain the guaiacol-type sesquiterpene compound.

[0011] Preferably, the extraction is performed 3 to 4 times; the volume ratio of chloroform to methanol is 1:1 to 2:1.

[0012] Preferably, the evaporation solvent comprises: treating the extracted product at 20–30°C and 0.08–0.085 MPa for 2–4 hours.

[0013] Preferably, the extraction process is performed 3 to 4 times; the volume ratio of ethyl acetate to water is 1:1 to 2:1.

[0014] Preferably, the gradient elution process further includes a step of dissolving the solid extract in petroleum ether before the gradient elution; the gradient elution is performed 2 to 3 times; and the volume ratio of petroleum ether to acetone during gradient elution is 98:2 to 95:5.

[0015] Preferably, the purification process further includes a step of dissolving the solid product in methanol.

[0016] Preferably, the purification conditions include: a C18 column, a mobile phase of methanol and water in a volume ratio of 95:5 to 90:10, a flow rate of 5 to 15 mL / min, and a sample loading volume of 125 to 500 μL.

[0017] Preferably, the guaiacol-type sesquiterpene compound is stored at -80°C to -20°C for later use.

[0018] The purification step is omitted, and the product purity is approximately 30%. After purification, the purity is 99.8%.

[0019] The second technical solution of the present invention: provides a guaiacane-type sesquiterpene compound extracted by the above extraction method, characterized in that the structural formula of the guaiacane-type sesquiterpene compound is:

[0020]

[0021] The third technical solution of the present invention provides the application of the above-mentioned guaiacol-type sesquiterpene compounds in the preparation of anti-tuberculosis drugs.

[0022] The present invention discloses the following technical effects:

[0023] This invention provides a novel guaiacane-type sesquiterpene compound extracted from the fruiting body of *Lactobacillus rubrum*. This compound specifically inhibits the activity of Mycobacterium tuberculosis, particularly showing excellent inhibitory effects against monodrug-resistant and multidrug-resistant Mycobacterium tuberculosis. Consequently, this guaiacane-type sesquiterpene compound can be used to treat tuberculosis, especially existing drug-resistant tuberculosis, thereby expanding the types of drugs available for treating tuberculosis, particularly drug-resistant tuberculosis, and also broadening the application scope of *Lactobacillus rubrum* extract.

[0024] The guaiacol-type sesquiterpene compounds described in this invention are isolated from Lactobacillus rubrum, with a purity of up to 99.8%. This high purity effectively increases the added value of Lactobacillus rubrum products. Attached Figure Description

[0025] Figure 1 The liquid chromatogram of the guaiacol-type sesquiterpene compound obtained in Example 1;

[0026] Figure 2 The ultraviolet spectrum of the guaiacolane-type sesquiterpene compound obtained in Example 1;

[0027] Figure 3 The mass spectrum of the guaiacolane-type sesquiterpene compound obtained in Example 1 is shown below.

[0028] Figure 4 Fourier transform infrared spectrum of the guaiacolane-type sesquiterpene compound obtained in Example 1;

[0029] Figure 5 NMR of the guaiacane-type sesquiterpene compound obtained in Example 1 1 H spectrum;

[0030] Figure 6 NMR of the guaiacane-type sesquiterpene compound obtained in Example 1 13 C spectrum;

[0031] Figure 7 The results of cytotoxicity assays on normal cells for different concentrations of the guaiacolane-type sesquiterpene compounds obtained in Example 1;

[0032] Figure 8 This is the original record of the antibacterial and antifungal properties of the guaiacol-type sesquiterpene compounds obtained in Example 1;

[0033] Figure 9The minimum inhibitory concentration (MIC) of the guaiacolane-type sesquiterpene compound obtained in Example 1 against Mycobacterium tuberculosis H37Ra is given.

[0034] Figure 10 The results of the Mycobacterium tuberculosis H37Ra proliferation curve inhibition test;

[0035] Figure 11 This is an original record of the growth status of clinical isolates of Mycobacterium tuberculosis on LJ medium slant. Detailed Implementation

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0038] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0039] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0040] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0041] It should be noted that any aspects not described in detail in this invention are conventional practices in the field and are not the focus of this invention.

[0042] Unless otherwise specified, all raw materials used in this invention are commercially available products, and the source of these commercially available products does not affect the technical effect of this invention.

[0043] Unless otherwise specified, the room temperature involved in this invention is 25±5℃.

[0044] Example 1

[0045] This embodiment provides the preparation of the guaiacol-type sesquiterpene compound, and the specific steps are as follows:

[0046] The fruiting bodies of *Lactarius rubra* were dried at 60°C, ground into powder, and stored at -20°C until use. At room temperature, 100 g of the *Lactarius rubra* fruiting body powder was extracted three times using a solvent system of 500–1000 mL CHCl3 and MeOH (1:1, v / v). The resulting mixture was then evaporated at 0.085 MPa at 30°C for 2 h to obtain a dark brown syrupy primary extract. Subsequently, 10 g of the primary extract was dissolved in 150 mL of ethyl acetate, and 150 mL of water was added. The primary extract was separated into layers under thorough stirring, and this process was repeated until the aqueous layer was clear. The organic layer was collected and concentrated at 0.085 MPa at 30°C for 4 h to obtain a black solid extract. Then, 10 g of the black solid extract was dissolved in 20 mL of petroleum ether and eluted with a petroleum ether-acetone mixture (98:2–98:5, v / v) gradient for silica gel column chromatography. This gradient elution process was repeated three times to further remove impurities. The gradient elution program was set to 0–60 min, 98:2; 60–120 min, 98:3; 120–240 min, 98:5. The collected purple fraction was evaporated at 0.085 MPa and 30 °C for 2 h to obtain a purple solid. The purple solid was then dissolved in 10 mL of methanol and eluted using a C18 column. Purification was performed using a preparative HPLC system (Prep C18, 19×250mm, 5μm, Waters Corporation, USA). The chromatographic conditions for the preparative HPLC were as follows: mobile phase: MeOH / H2O (95:5, v / v); flow rate: 15 mL / min; sample loading: 500 μL; detection was performed using a photodiode array detector (2998, Waters Corporation, USA) at 312 nm. The target peak was collected, combined, and evaporated at 0.085 MPa and 30 °C for 2 h to obtain the deep purple compound, which was the guaiacolane-type sesquiterpene compound. The sample was then lyophilized and stored at -20 °C for later use.

[0047] Performance testing:

[0048] I. Characterization:

[0049] Figure 1 The liquid chromatogram of the guaiacol-type sesquiterpene compound obtained in Example 1 is shown.

[0050] 1. The guaiacane-type sesquiterpene compound obtained in Example 1 was dissolved in 1 mL of methanol and subjected to ultraviolet spectroscopy (UV-2600, Shimadzu). The results are as follows: Figure 2 As shown.

[0051] Figure 2 The image shows the UV spectrum of the guaiacolane-type sesquiterpene compound obtained in Example 1. Figure 2 The ultraviolet absorption spectrum shows a maximum value at 312 nm and a second maximum value at 241 nm, indicating that the compound has conjugated double bonds. The maximum detection wavelength for chromatographic purity identification can be set to 312 nm.

[0052] 2. Purity analysis:

[0053] The purity of the guaiacol-type sesquiterpenes obtained in Example 1 was analyzed by HPLC (LC-20A, Shimadzu). A Sepax Bio-C18 column (4.6 × 250 mm, 5 μm) was used, with MeOH / H2O (95:5, v / v), a flow rate of 1 mL / min, and a detection wavelength of 313 nm. The purity of the guaiacol-type sesquiterpenes obtained in Example 1 was determined to be 99.8%, which was verified by total ion current (TIC) chromatogram of LC-MS (Agilent 1260-6120). The results are as follows. Figure 3 As shown. The protonated molecular ion has m / z 213([M+H]). + The result indicates that the relative molecular mass of the compound is 212.

[0054] Figure 3 The mass spectrum of the guaiacolane-type sesquiterpene compound obtained in Example 1 is shown. Figure 3 The relative molecular mass of the guaiacol-type sesquiterpene compound obtained in Example 1 was confirmed to be 212, thus confirming the molecular mass and purity.

[0055] 3. Between 500 and 4000 cm -1 The Fourier transform infrared spectra (Tracer-100, Shimadzu) of the guaiacolane-type sesquiterpene compounds obtained in Example 1 were tested within the specified range, and the results are as follows: Figure 4 As shown.

[0056] Figure 4 The image shows the Fourier transform infrared spectrum of the guaiacol-type sesquiterpene compound obtained in Example 1. Figure 4 The key functional group was identified: 1726.2cm -1 The carbonyl group (C=O) at the position extends to 2924.0 cm. -1 and 2852.7cm -1 Aliphatic CH extension at the location, 1645.2cm -1and 1612.4cm -1 Two cyclic intraolefins at 1402.2 cm -1 The methyl group (CH) at that location undergoes bending vibration.

[0057] Figure 5 NMR of the guaiacane-type sesquiterpene compound obtained in Example 1 1 H spectrum.

[0058] 1 H-NMR(DMSO-d6,500MHz):10.30(1H,s,H-15),9.59(1H,d,J=1.8Hz,H-8),8.24(1H,d,J=4.3Hz,H-2),7.91(1H,dd,J=10.7,1.8Hz,H- 6),7.65(1H,d,J=10.7Hz,H-5),7.37(1H,d,J=4.3Hz,H-3),3.22(1H,m,H-11),2.91(3H,s,H-14),1.35(6H,d,J=6.9Hz,H-12,H-13).

[0059] Figure 6 NMR of the guaiacane-type sesquiterpene compound obtained in Example 1 13 C-spectrum.

[0060] 13 C-NMR(DMSO-d6,125MHz):186.2(d,C-15),148.9(s,C-7),148.6(s,C-4),143.3(s,C-10),140.6(d,C-2),139.0(s,C-9),138 .2(d,C-8),136.1(d,C-6),131.5(d,C-5),125.2(s,C-1),115.8(d,C-3),37.7(d,C-11),24.3(q,C-12,C-13),24.2(q,C-14).

[0061] Figure 5 and Figure 6 Obtained in deuterated DMSO using a Bruker AVANCE III 500MHz spectrometer (Bruker, Germany). 1 H and 13 The C chemical shift is consistent with the structure of the guaiacol-type sesquiterpene compound obtained in Example 1.

[0062] The above test results confirm that the present invention has successfully isolated the fruiting body of *Lactarius deliciosus* with the structural formula […]. Guaiacane-type sesquiterpenoid compounds.

[0063] II. Cytotoxicity test on normal cells:

[0064] Normal cell lines, RAW 264.7, HUVEC, BEAS-2B, and Lo 2, were used to assess cytotoxicity. Cell viability after treatment with the guaiacol-type sesquiterpenes obtained in Example 1 was detected using the MTS assay, with absorbance values ​​recorded at 490 nm using a multi-functional microplate reader (SpectraMax i3X, Molecular Devices Instruments Co., Ltd., USA).

[0065] RAW264.7 and BEAS-2B cell lines were cultured in DMEM medium (Gibco, Thermo Fisher Scientific, USA), while HUVEC and Lo 2 cell lines were cultured in RPMI 1640 medium (Gibco). Additionally, 20% fetal bovine serum (Gibco) and 1% penicillin-streptomycin bispecific antibiotic solution were added to the medium. Cells cultured to the logarithmic growth phase at 37°C and 5% CO2 in an incubator (BB150 CO2 incubator, Thermo Fisher Scientific, USA) were washed with phosphate-buffered saline (PBS), digested with trypsin, and the digestion reaction was terminated by adding culture medium. The cells were then dispersed into a homogeneous cell suspension and the cell density was adjusted to 10T. 6 CFU / mL. 100 μL of cell suspension was seeded into 96-well plates (purchased from Corning Incorporated, USA) and cultured for 12 h. When the cell density reached 70%, the old culture medium was aspirated and replaced with drug-containing culture medium containing different concentrations of the guaiacol-type sesquiterpene compounds obtained in Example 1, with a dimethyl sulfoxide (DMSO) concentration not exceeding 0.1%. Cells were cultured continuously, and cell growth was observed under a microscope every 2 h. After 8–12 h of culture, 10 μL of LMTS reagent (CellTiter 96RAQueousOne Solution cell proliferation assay, Promega, USA) was added to each well, and the cells were incubated at 37°C for 30 min. The absorbance at 490 nm was measured and the viability was calculated.

[0066] Survival rate = (A x -A2) / (A0-A2)×100%;

[0067] Among them: A x A1 represents the absorbance of the experimental wells (containing cells, DMSO, culture medium, and different concentrations of guaiacol-type sesquiterpenoid compounds obtained in Example 1); A2 represents the absorbance of the control wells (containing cells, DMSO, and culture medium); and A3 represents the absorbance of the blank wells (containing DMSO and culture medium).

[0068] The culture medium was discarded, and the cells were observed and photographed under an inverted fluorescence microscope (Nikon Ti2-E, Nikon Corporation, Japan). The results are shown below. Figure 7 .

[0069] Figure 7 The results show the cytotoxicity of different concentrations of the guaiacol-type sesquiterpene compounds obtained in Example 1 on normal cells.

[0070] Depend on Figure 7 It can be seen that when the concentration is <500μg / mL (<4×MIC), (MIC is defined as the lowest concentration at which the bacterial growth inhibition rate reaches more than 99%), no obvious cytotoxicity was observed.

[0071] III. Specific inhibition of Mycobacterium tuberculosis H37Ra (Mycobacterium tuberculosis H37Ra is preserved by Hunan Provincial Chest Hospital):

[0072] Microbial culture: Gram-positive (G+) and Gram-negative (G-) bacteria were revived from slant culture and cultured in LB broth at 37°C until they reached the exponential growth phase. The cells were collected by centrifugation (5000×g, 10 min), washed twice with PBS, and resuspended in PBS buffer. Cell density was adjusted to OD0.05. 600 =0.2 for downstream assays. Fungal isolates were activated in PDA broth at 30–37°C for 72 h and then treated in the same way as bacteria.

[0073] All strains of Mycobacterium tuberculosis were in - Activate the culture by incubating on Jensen (LJ) slant at 37°C. After the colonies have reached the logarithmic growth phase, scrape off colonies to form sputum culture bottles and grind them with glass beads in PBS buffer for 5 minutes. Afterward, collect the homogeneous supernatant suspension and adjust it to various concentrations as needed for subsequent applications.

[0074] The antibacterial activity of the guaiacol-type sesquiterpenoid compounds obtained in Example 1 against Gram-positive bacteria (G+), Gram-negative bacteria (G-), and fungi was determined using the agar disk diffusion method. The bacterial and fungal spore suspensions were adjusted to OD0.05. 600 =0.2. Subsequently, 100 μL of the bacterial suspension was inoculated onto LB agar plates or PDA agar plates and spread evenly. The guaiacol-type sesquiterpene compound obtained in Example 1 was dissolved in DMSO (66.7%) to prepare a stock solution with a concentration of 2 mg / mL, and serially diluted as required. 10 μL of drug solutions of different concentrations were spread onto circular filter paper and then affixed to the agar surface. 1 mg / mL ciprofloxacin was used as a positive control, and 66.7% DMSO was used as a negative control. All microorganisms, including bacteria and fungi, were cultured at 30–37°C for 24–72 h.

[0075] The guaiacol-type sesquiterpene compound solution obtained in Example 1 was dissolved in LJ medium to prepare a solid slant containing a drug concentration of 500 μg / mL. 100 μL of Mycobacterium tuberculosis bacterial suspension was spread onto the drug-containing LJ slant and incubated at 37°C for 5 weeks. The growth of Mycobacterium tuberculosis was recorded during this period. All operations were performed in a sterile workbench and biosafety cabinet at Hunan Provincial Chest Hospital. For the negative control, except that the guaiacol-type sesquiterpene compound obtained in Example 1 was not added, all other steps were performed as described above. Uninoculated culture medium served as a blank control to ensure that the culture medium remained uncontaminated.

[0076] MIC determination of Mycobacterium tuberculosis: The MIC of the guaiacol-type sesquiterpene compound obtained in Example 1 against Mycobacterium tuberculosis was determined by resazurin microtitration (REMA). 0.025 g of the guaiacol-type sesquiterpene compound obtained in Example 1 was dissolved in 0.05 mL of DMSO and then transferred to 4.95 mL of Middlebrook 7H9 broth with added OADC. A series of concentrations from 500 μg / mL to 15.6 μg / mL were obtained using a twofold dilution method, ensuring that the final DMSO content was ≤1%. The H37Ra type Mycobacterium tuberculosis suspension was adjusted to McFarland 1.0 (equivalent to OD500). 600 =0.185), then diluted with 7H9-OADC broth at a ratio of 1:20 (v / v). An equal volume (100 μL) of the drug-containing medium and bacterial suspension was added to a 96-well plate and mixed. The culture was incubated at 37°C for 10 days, then 30 μL of resalicylate solution (0.02%) was added to observe bacterial growth. The plates were incubated for another 48 hours until a colorimetric change (blue to pink) was observed in the control wells. Fluorescence (E) was measured using a microplate reader. x / E m Quantification was performed at 560 / 590 nm. The results are as follows: Figure 9 As shown. The same protocol was used for the determination of MIC for first-line drugs.

[0077] The selective antibacterial activity results of the guaiacolane-type sesquiterpene compounds obtained in Example 1 are shown in Table 1.

[0078] Table 1. Antibacterial activity of the guaiacolane-type sesquiterpene compounds obtained in Example 1 against G+, G- and fungi.

[0079]

[0080]

[0081] In the table, Dihydrolactaroviolin: dihydrolactaroviolin, the guaiacane-type sesquiterpene compound obtained in Example 1; Cip.: full name of ciprofloxacin; *ns indicates not significant, ND indicates not detected.

[0082] The MIC of anti-tuberculosis mycobacterium H37Ra was determined using the REMA method.

[0083] Figure 8 This is the original record of the antibacterial and antifungal properties of the guaiacol-type sesquiterpene compounds obtained in Example 1.

[0084] In the diagram, A: *E. coli* ATCC 25922; B: *E. coli* O157:H7 CMCC 44102; C: *Pseudomonas fluorescens* O157:H7 CMCC 44102; D: *Salmonella enteritidis*; E: *Salmonella typhimurium* ATCC 14028; F: *S. aureus* ATCC 29213; G: *S. aureus* ATCC 25923. H: Pseudomonas aeruginosa ATCC 9027; I: Bacillus cereus ATCC 14597; J: Listeria monocytogenes ATCC 13932; K: Aspergillus purpureus ATCC 16426; L: Aspergillus flavus ATCC 11489; M: Aspergillus niger ATCC 16404; N: Saccharomyces cerevisiae ATCC 204218.

[0085] The black letters on the plate represent: A: DMSO, B: Ciprofloxacin, C-F: 2.0, 1.0, 0.5, and 0.25 mg / mL of the guaiacane-type sesquiterpene compound obtained in Example 1. The red letters on the plate represent: A-D: 2.0, 1.0, 0.5, and 0.25 mg / mL of the guaiacane-type sesquiterpene compound obtained in Example 1, and E: DMSO.

[0086] The results showed that the guaiacol-type sesquiterpene compounds obtained in Example 1 had no antibacterial activity against the selected G+, G- and fungi, but specifically inhibited Mycobacterium tuberculosis.

[0087] Figure 9 The minimum inhibitory concentration (MIC) of the guaiacol-type sesquiterpene compound obtained in Example 1 against Mycobacterium tuberculosis H37Ra is given.

[0088] Depend on Figure 9 It is known that the MIC of the guaiacol-type sesquiterpene compound obtained in Example 1 against Mycobacterium tuberculosis H37Ra is 125 μg / mL.

[0089] The results of the tuberculosis H37Ra proliferation curve inhibition test are as follows: Figure 10 As shown.

[0090] Depend on Figure 10 It can be seen that the guaiacol-type sesquiterpene compound obtained in Example 1 showed a dose-dependent inhibition of the growth of Mycobacterium tuberculosis. When the MIC value was 2.0×MIC and 1.0×MIC, the growth of Mycobacterium tuberculosis H37Ra completely stopped, while when the MIC value was 0.5×MIC, the growth of Mycobacterium tuberculosis H37Ra partially stopped. Compared with the control group, DMSO had no significant effect on bacterial growth.

[0091] IV. Antibacterial spectrum analysis of the guaiacane-type sesquiterpenoid compounds obtained in Example 1 on clinically isolated Mycobacterium tuberculosis strains:

[0092] Clinically isolated Mycobacterium tuberculosis strains were obtained from Hunan Provincial Chest Hospital. Twenty-five clinical isolates were cultured in vitro, and 16 well-growing strains were selected for MIC determination. The method was consistent with that described in point three. Each experiment was performed in triplicate (n=3). Detailed information on drug resistance of Mycobacterium tuberculosis was determined in advance using a commercial automated culture system MGIT960 (Becton, Dickinson, New Jersey, USA), and the results are shown in Table 2.

[0093] Table 2. MICs of the guaiacol-type sesquiterpene compounds obtained in Example 1 against clinically isolated Mycobacterium tuberculosis strains.

[0094]

[0095] In the table, S indicates sensitive; R indicates resistant. SM: Streptomycin; INH: Isoniazid; RIF: Rifampin; EMB: Ethambutol; PZA: Pyrazinamide; AMK: Amikacin; LVF: Levofloxacin; LZD: Linezolid; BDQ: Bedaquiline; CFZ: Clofazimine; MOX: Moxifloxacin; KM: Kanamycin.

[0096] Table 2 shows that the guaiacol-type sesquiterpene compounds obtained in Example 1 have specific inhibitory effects on Mycobacterium tuberculosis (including drug-resistant strains), indicating that none of the clinically isolated strains showed pre-existing resistance to the guaiacol-type sesquiterpene compounds obtained in Example 1. The MIC of the guaiacol-type sesquiterpene compounds obtained in Example 1 against the standard control strains H37Ra and H37Rv was 125 μg / mL. In contrast, the lowest MIC value of the clinically isolated drug-resistant strains was only 7.8 μg / mL.

[0097] Furthermore, the antibacterial activity was further validated using the LJ medium culture method. The specific test procedure was as follows: 0.025 g of the guaiacol-type sesquiterpene compound obtained in Example 1 was first dissolved in 0.05 mL of DMSO, and then dispersed in LJ medium. The resulting mixture was sterilized at 85°C for 80 min to create a drug-containing LJ medium slant. The final concentration of DMSO in all media did not exceed 1.0%, which had no significant effect on the growth of Mycobacterium tuberculosis. Therefore, DMSO (concentration 1.0%) was designated as a negative control. To ensure suitable growth of Mycobacterium tuberculosis on standard LJ medium, a control group (basal medium culture group without any added drug or solvent, Control) was set up. In addition, an uninoculated blank group was included to confirm the absence of contamination. The experimental group was Dihydro-LAC. Tables 3-1, 3-2, and 3-3 show the growth of clinical isolates on LJ medium slants containing 0.5 mg / mL of the guaiacol-type sesquiterpene compound obtained in Example 1.

[0098] Figure 11 This is an original record of the growth status of clinical isolates of Mycobacterium tuberculosis on LJ medium slant.

[0099] Table 3-1 Growth of clinical isolates on LJ medium slant containing 0.5 mg / mL of the guaiacane-type sesquiterpene compound obtained in Example 1.

[0100]

[0101] Table 3-2

[0102]

[0103] Table 3-3

[0104]

[0105]

[0106] In Tables 3-1, 3-2, and 3-3, Dihydrolactaroviolin is the guaiacol-type sesquiterpene compound obtained in Example 1. S indicates sensitivity; R indicates resistance. SM: Streptomycin; INH: Isoniazid; RIF: Rifampin; EMB: Ethambutol; RFT: Rifapentine; PZA: Pyrazinamide; KM: Kanamycin; LVF: Levofloxacin; CPM: Capreomycin; AMK: Amikacin; TH1321: Prothionamide. Colony count: ++++ (covering the entire slant, or >500 colonies); +++ (3 / 4 of the slant, or 200–500 colonies); ++ (100–200 colonies); + (50–100 colonies). Each group is replicated. / : Slant contamination, including slant #2 and #11.

[0107] From Table 3-1, Table 3-2, Table 3-3 and Figure 11 It is evident that the guaiacol-type sesquiterpene compound obtained in Example 1 exhibits antibacterial activity against all clinical isolates selected in this assay. This indicates that the guaiacol-type sesquiterpene compound obtained in Example 1, isolated from the fruiting body of *Lactobacillus rubrum*, can specifically inhibit *Mycobacterium tuberculosis*, including drug-resistant clinical isolates.

[0108] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0109] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for extracting guaiacane-type sesquiterpenoid compounds, characterized in that, Includes the following steps: Fresh Lactarius deliciosus fruiting bodies are dried at 60°C, then ground into powder and stored at -20°C until use; The dried fruiting body powder of *Lactarius deliciosus* was extracted using a solvent system composed of chloroform and methanol. The solvent was then evaporated to obtain the primary extract. The primary extract was further extracted using a two-phase solvent system consisting of ethyl acetate and water. The organic layer was collected, concentrated, and a solid extract was obtained. The solid extract was subjected to gradient elution by silica gel column chromatography using a solvent system composed of petroleum ether and acetone. The fractions were collected, evaporated, and the solid product was obtained. The solid product was further purified by preparative HPLC to obtain the guaiacol-type sesquiterpene compound.

2. The extraction method according to claim 1, characterized in that, The extraction is performed 3 to 4 times; the volume ratio of chloroform to methanol is 1:1 to 2:

1.

3. The extraction method according to claim 1, characterized in that, The evaporation solvent includes: treating the extracted product at 20–30°C and 0.08–0.085 MPa for 2–4 hours.

4. The extraction method according to claim 1, characterized in that, The extraction process is repeated 3 to 4 times; the volume ratio of ethyl acetate to water is 1:1 to 2:

1.

5. The extraction method according to claim 1, characterized in that, The gradient elution process includes a step of dissolving the solid extract in petroleum ether before the gradient elution; the gradient elution is performed 2 to 3 times; and / or, the volume ratio of petroleum ether to acetone during gradient elution is 98:2 to 95:

5.

6. The extraction method according to claim 1, characterized in that, The purification process also includes a step of dissolving the solid product in methanol.

7. The extraction method according to claim 1, characterized in that, The purification conditions include: a C18 column, a mobile phase of methanol and water in a volume ratio of 95:5 to 90:10, a flow rate of 5 to 15 mL / min, and a sample loading volume of 125 to 500 μL.

8. The extraction method according to claim 1, characterized in that, The guaiacol-type sesquiterpene compounds are stored at -80℃ to -20℃ for future use.

9. The guaiacol-type sesquiterpene compound extracted by the extraction method according to any one of claims 1 to 8, characterized in that, The structural formula of the guaiacolane-type sesquiterpene compound is:

10. The use of the guaiacol-type sesquiterpene compound of claim 9 in the preparation of anti-tuberculosis drugs.

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