A compound extracted from magnolia officinalis, its preparation method and application

Abstract

The application relates to the field of traditional Chinese medicine against tuberculosis, in particular to a compound extracted from Magnolia officinalis, a preparation method and application of the compound, the compound is shown as formula I, the compound can effectively inhibit the growth of Mycobacterium smegmatis, and can prevent and treat Mycobacterium smegmatis infection, and the MIC of the compound on Mycobacterium smegmatis is 60 ug / mL.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine for tuberculosis treatment, specifically to a compound extracted from Magnolia officinalis, its preparation method, and its application. Background Technology

[0002] Mycobacterium smegmatis is a nontuberculous mycobacterium, an opportunistic pathogen, widely present in the natural environment. Immunocompromised individuals are more susceptible to it, and it can cause nontuberculous mycobacterial diseases, leading to infections of the skin, soft tissues, respiratory tract, bones, and joints, manifesting as local inflammation, ulcers, cough, etc. This bacterium is resistant to many anti-tuberculosis drugs, and treatment requires drug sensitivity testing for drug selection.

[0003] Magnolia officinalis, a traditional Chinese medicine, can regulate gastrointestinal function and relieve abdominal distension; it can also act as an expectorant, improving cough and excessive phlegm; and it can promote intestinal peristalsis, treating constipation. Current reports indicate that its active ingredient, magnolol, exhibits broad-spectrum antibacterial (Gram-positive bacteria, Gram-negative bacteria, fungi) and antiviral (COVID-19, dengue virus, etc.) activity. There are also reports that magnolol can inhibit the growth of Mycobacterium smegma, but its antibacterial ability is relatively weak.

[0004] In summary, there is an urgent need to provide a novel compound extracted from Magnolia officinalis that effectively inhibits the growth of Mycobacterium smegmatis, thereby preventing and treating diseases caused by Mycobacterium smegmatis infection. Summary of the Invention

[0005] This invention addresses the technical problem of providing a compound extracted from Magnolia officinalis that effectively inhibits the growth of Mycobacterium smegmatis, thereby preventing and treating diseases caused by Mycobacterium smegmatis infection.

[0006] To achieve the above objectives, the first aspect of the present invention provides a compound (hereinafter referred to as H1) extracted from Magnolia officinalis, wherein the compound is as shown in Formula I:

[0007] Formula I.

[0008] A second aspect of the present invention provides a method for preparing the above-mentioned compound extracted from Magnolia officinalis, comprising the following steps:

[0009] S1. Soak Magnolia officinalis in methanol, heat under reflux at 70°C for 1 hour, and concentrate to obtain extract;

[0010] S2. Dissolve the extract obtained in S1 in water, then extract with ethyl acetate, and concentrate under reduced pressure to obtain the ethyl acetate extract;

[0011] S3. The ethyl acetate extract obtained in S2 was subjected to macroporous resin column chromatography with first gradient elution. 50% fraction was collected and concentrated to obtain a refined extract.

[0012] S4. The purified extract obtained in S3 is subjected to normal phase silica gel column chromatography with second gradient elution to obtain 85 fractions. The 35th fraction is taken, concentrated, and crude product is obtained.

[0013] S5. Dissolve the crude product obtained in S4 in anhydrous ethanol, inject the solution into a preparative liquid chromatography system, perform a third elution, collect the eluent, and concentrate to obtain the compound.

[0014] A third aspect of the present invention provides the application of the above-described compounds extracted from Magnolia officinalis, wherein the application includes...

[0015] The application of the compound in the preparation of products that inhibit the growth of Mycobacterium smegmae;

[0016] The use of the compound in the preparation of medicaments for the treatment or prevention of Mycobacterium smegma infection;

[0017] Applications of the compound in the preparation of food, health products, food additives, and animal feed additives.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The compound provided by this invention is extracted from natural plants and can effectively inhibit the growth of Mycobacterium smegmatis and treat Mycobacterium smegmatis infection, providing a new solution and approach for the prevention and treatment of diseases caused by Mycobacterium smegmatis infection. Attached Figure Description

[0020] Figure 1 This is the high-resolution mass spectrum (HRMS) of compound H1 in the embodiments of the present invention.

[0021] Figure 2 This is the 1H NMR spectrum of compound H1 in the embodiments of the present invention. 1 H NMR spectrum.

[0022] Figure 3 This is the carbon NMR spectrum of compound H1 in the embodiments of the present invention. 13 C10 NMR spectrum.

[0023] Figure 4 This is the heteronuclear single quantum correlation (HSQC) spectrum of compound H1 in the embodiments of the present invention.

[0024] Figure 5 This is the heteronuclear multibond correlation spectrum (HMBC) of compound H1 in the embodiments of the present invention.

[0025] Figure 6 This is the hydrogen-hydrogen correlation spectrum of compound H1 in the embodiments of the present invention. 1 H- 1 H COSY spectrum.

[0026] Figure 7 This is a graph showing the effects of compounds H1 and magnolol H2 on the growth of Mycobacterium smegma in the embodiments of this application. In the graph, Figure A shows the optical density values ​​(OD) of Mycobacterium smegma after treatment with H1 and H2. 600 Figure B shows the CFU count of Mycobacterium smegmatis after treatment with H1 and H2. The "1", "2", "3" and "4" on the plate represent the CFU count results after spotting the samples after dilutions of 1:10, 1:100, 1:1000 and 1:10000, respectively.

[0027] Figure 8 These are microscopic observation results of Mycobacterium smegmatis after treatment with compound H1 and magnolol H2 in the embodiments of this application. In the figure, from left to right, there are Mycobacterium smegmatis after treatment with DMSO (control), H1, and H2. The top row is observed at 30,000x magnification, and the bottom row is observed at 100,000x magnification.

[0028] Figure 9 This is a graph showing the minimum inhibitory concentration (MIC) results of compounds H1 and magnolol H2 against Mycobacterium smegma in the embodiments of this application. In the graph, Figure A and Figure B are the MIC test results of different concentrations of H1 and H2 against Mycobacterium smegma, respectively. Pre represents the bacterial CFU count before treatment, and the rest represent the bacterial CFU count after treatment. "2" on the plate represents the CFU count result after spotting the sample after a 1:100 dilution.

[0029] Figure 10 This is a graph showing the effect of compound H1 and magnolol H2 combined with first-line anti-tuberculosis drugs on the growth of Mycobacterium smegma in the embodiments of this application. In the graph, Figure A and Figure B show the effect of H1 and H2 combined with isoniazid (Figure A) and rifampin (Figure B) on the growth of Mycobacterium smegma, respectively. On the plate, "1", "2", "3", "4" and "5" represent the CFU count results after spotting the sample after dilution by 1:10, 1:100, 1:1000, 1:10000, and 1:100000, respectively. Detailed Implementation

[0030] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0031] In the prior art, although magnolol has a certain inhibitory effect on Mycobacterium smegmatis, its inhibitory effect as a potential inhibitor of Mycobacterium smegmatis is not ideal. However, the inventors have discovered that a new compound can be obtained from Magnolia officinalis using a specific isolation method. This compound can effectively inhibit the growth of Mycobacterium smegmatis.

[0032] Therefore, in a first aspect, the present invention provides a compound extracted from Magnolia officinalis, wherein the compound is as shown in Formula I:

[0033] Formula I.

[0034] A second aspect of the present invention provides a method for preparing the above-mentioned compound extracted from Magnolia officinalis, comprising the following steps:

[0035] S1. Soak Magnolia officinalis in methanol, heat under reflux at 70°C for 1 hour, and concentrate to obtain extract;

[0036] S2. Dissolve the extract obtained in S1 in water, then extract with ethyl acetate, and concentrate under reduced pressure to obtain the ethyl acetate extract;

[0037] S3. The ethyl acetate extract obtained in S2 was subjected to macroporous resin column chromatography with first gradient elution. 50% fraction was collected and concentrated to obtain a refined extract.

[0038] S4. The purified extract obtained in S3 is subjected to normal phase silica gel column chromatography with second gradient elution to obtain 85 fractions. The 35th fraction is taken, concentrated, and crude product is obtained.

[0039] S5. Dissolve the crude product obtained in S4 in anhydrous ethanol, inject the solution into a preparative liquid chromatography system, perform a third elution, collect the eluent, and concentrate to obtain the compound.

[0040] According to the present invention, in step S1, the mass ratio of Magnolia officinalis to extract is 20:1;

[0041] The mass ratio of Magnolia officinalis to methanol is 1:10.

[0042] According to the present invention, in step S2, the mass ratio of the extract to the volume ratio of water is 1:5;

[0043] The volume ratio of water to ethyl acetate is 1:1;

[0044] The mass ratio of the extract to the ethyl acetate extract is 5:1.

[0045] According to the present invention, in step S3, the conditions for the first gradient elution include: using methanol and water gradient elution as the first eluent, with a total of 4 gradients, in each gradient the volume ratio of methanol to water is 0:100, 30:70, 60:40, and 100:0 respectively, and the elution volume of each gradient is 5 times the column volume.

[0046] According to the present invention, in step S3, the mass ratio of ethyl acetate extract to refined extract is 10:3.

[0047] According to the present invention, in step S4, the conditions for the second gradient elution include: using petroleum ether and ethyl acetate as the second eluent, with a total of 6 gradients, in each gradient the volume ratio of petroleum ether to ethyl acetate is 100:0, 70:30, 50:50, 30:70, 20:80, 0:100, and the elution volume of each gradient is 10 times the column volume.

[0048] According to the present invention, in step S4, the mass ratio of the refined extract to the crude product is 30:1.

[0049] According to the present invention, in step S5, the conditions for the third gradient elution include: using methanol and water as the mobile phase, with one gradient, the volume ratio of methanol to water being 60:40, and the elution volume being 30 times the column volume.

[0050] The mass ratio of the crude product to the compound is 1000:9.

[0051] A third aspect of the present invention provides the application of the above-described compounds extracted from Magnolia officinalis, wherein the application includes...

[0052] The application of the compound in the preparation of products that inhibit the growth of Mycobacterium smegmae;

[0053] The use of the compound in the preparation of medicaments for the treatment or prevention of Mycobacterium smegma infection;

[0054] Applications of the compound in the preparation of food, health products, food additives, and animal feed additives.

[0055] The present invention will be described in detail below through embodiments.

[0056] Test instructions:

[0057] Materials and Instruments

[0058] Instruments: Clean bench (Suzhou Antai Air Technology Co., Ltd., model: SW-CJ-2); Incubator (Shanghai Fuma Experimental Equipment Co., Ltd., model: DDX-9505213-1); Shaker (Shanghai Fuma Experimental Equipment Co., Ltd., model: KYC 100B); Microplate reader (Thermo Scientific, model: Multiscan FC).

[0059] Materials: LBT liquid medium (1% Tryptone, 0.5% Yeast Extract, 1% NaCl, 0.05% Tween 80); LB solid medium (1% Tryptone, 0.5% Yeast Extract, 1% NaCl, 1.5% Agar); electron microscopy fixative (Wuhan Saiweier Biotechnology Co., Ltd.); rifampicin (RFP) and isoniazid (INH).

[0060] Unless otherwise specified in the following examples and comparative examples, all conditions were performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments used, unless otherwise specified, are all commercially available products.

[0061] Example: Extraction, separation and structural identification of compound H1

[0062] S1. Soak 200 kg of Magnolia officinalis in 2000 L of methanol, heat under reflux at 70 °C for 1 h, and concentrate to obtain 10 kg of extract.

[0063] S2. Dissolve the extract obtained in S1 in 50L of water, then extract with ethyl acetate and concentrate to obtain 2kg of ethyl acetate extract. The volume ratio of water to ethyl acetate is 1:1.

[0064] S3. The ethyl acetate extract obtained in S2 was subjected to macroporous resin column chromatography with first gradient elution. 50% fraction was collected and concentrated to obtain 600g of purified extract. The conditions for the first gradient elution included: using methanol and water as the first eluent, with a total of 4 gradients. In each gradient, the volume ratio of methanol to water was 0:100, 30:70, 60:40, and 100:0, respectively. The elution volume of each gradient was 5 times the column volume.

[0065] S4. The purified extract obtained in S3 was subjected to normal-phase silica gel column chromatography with a second gradient elution to obtain 85 fractions. The 35th fraction was taken and concentrated to obtain 20g of crude product. The conditions for the second gradient elution included: using petroleum ether and ethyl acetate as the second eluent, with a total of 6 gradients. In each gradient, the volume ratio of petroleum ether to ethyl acetate was 100:0, 70:30, 50:50, 30:70, 20:80, 0:100, and 0:100, respectively. The elution volume of each gradient was 10 times the column volume.

[0066] S5. Dissolve the crude product obtained in S4 in 80% anhydrous ethanol and inject it into a preparative liquid chromatography system for third elution. The conditions for third gradient elution include: using methanol and water as mobile phases, with one gradient, a methanol-to-water volume ratio of 60:40, and an elution volume of 30 times the column volume. Collect the eluent from the third elution and concentrate the eluents obtained at different time points to obtain 180 mg of white powder compound H1 and 350 mg of compound H2.

[0067] Compounds H1 and H2 were characterized and, according to HR-ESIMS, showed a quasi-molecular ion peak [M+H] at m / z 283.1318. + Its calculated value is 282.1256, combined with 1 H-NMR and 13 C1-NMR data determined its molecular formula to be C10. 18 H 18 O3, degree of unsaturation Ω = 10.

[0068] pass 1 H NMR and 13 2D NMR analysis showed that H1 data was similar to that of honokiol, except that the chemical shift of H1's C-3' carbon spectrum shifted to a lower field, and the mass spectrum of H1 was 16 ppm higher than that of H2, suggesting that H1 is a hydroxyl-substituted compound. In the HMBC spectrum, a long-range correlation was observed between the H-2' proton and the C-3' carbon atom, thus suggesting that the C-3' hydrogen proton is substituted by a hydroxyl group. This hypothesis was confirmed by 2D NMR. Compound H1 is a new compound, named 3'-hydroxyhonokiol, abbreviated as H1.

[0069] Compound H2, a white powder, was identified by NMR data comparison with known compounds and honokiol, abbreviated as H2.

[0070] Table 1 shows the compounds H1. 1 H and 13 C10 NMR spectrum data.

[0071] Table 1

[0072]

[0073] Test Example 1: Effects of compounds H1 and H2 on the growth of Mycobacterium smegma

[0074] Compounds H1 and H2 were added to Mycobacterium smegmatis mc 2 155) In the culture medium, the effects of H1 and H2 on the growth of Mycobacterium smegmatis were analyzed using bacterial density detection and colony formation unit (CFU) experiments.

[0075] Specifically, select the resurgent M. smegmatis mc 2 155 single bacterial colonies were added to 3 mL of LBT medium and incubated at 37°C with shaking for 72 hours. 70 μL of this fresh bacterial suspension was then added to 100 mL of LBT medium, mixed well, and the OD was measured. 600 Approximately 0.05 μL was dispensed into sterile test tubes at 3 mL per tube. A control group and an experimental group were set up, with three parallel tubes in each group. The control group received 6 μL of DMSO solution, and the experimental group received 6 μL of 5 mg / mL H1 or H2, bringing the final concentration to 10 μg / mL. After incubation at 37°C with shaking for 24 hours, photographs were taken for observation. 200 μL of bacterial culture from each group was collected and its OD value was recorded. 600 Values ​​were determined. Simultaneously, 10 μL of bacterial suspension from both the experimental and control groups were diluted 1:10, 1:100, 1:1000, and 1:10000. 5 μL of each dilution was then inoculated onto LB agar plates for CFU counting. Statistical analysis was performed using GraphPad Prism 9 software.

[0076] The results are as shown in the attached instruction manual. Figure 7 As shown, in a 3 mL Mycobacterium smegmatis culture system, the control group was treated with DMSO (a solvent to dissolve H1 and H2), while the experimental group was treated with either H1 or H2 to a final concentration of 10 μg / mL. Bacterial density was measured 24 hours after inoculation, and bacterial CFU counts were performed. (See attached instruction manual.) Figure 7 The results showed that, compared with the control group, the bacterial culture with added H1 and H2 was clearer, and the OD... 600 The value was significantly reduced, and the number of bacterial colonies was significantly decreased, indicating that H1 and H2 could significantly inhibit the growth of Mycobacterium smegmatis.

[0077] Test Example 2: Microscopic observation of the morphology of Mycobacterium smegmatis by compounds H1 and H2

[0078] Compounds H1 and H2 were added to M. smegmatis mc2 The effects of H1 and H2 on the morphology of Mycobacterium smegmatis were observed using scanning electron microscopy in culture medium 155.

[0079] Specifically, take M. smegmatis mc 2 15 μL of fresh bacterial culture was added to 20 mL of LBT medium, mixed well, and OD was measured. 600 The concentration was 0.05. A control group and an experimental group were set up. The control group was treated with 160 μL of DMSO solution, and the experimental group was treated with 160 μL of 5 mg / mL H1 and H2, bringing the final concentration to 40 μg / mL. After incubation at 37℃ with shaking for 24 hours, the bacterial cultures from both groups were collected by centrifugation at 5000×g for 10 min. The cultures were washed twice with PBS, resuspended in electron microscopy fixative, and sent to Wuhan Saiwei Biotechnology Co., Ltd. to prepare scanning electron microscopy smears. Observation and photography were performed using a HITACHI Regulus 8100 scanning electron microscope.

[0080] The results are as shown in the attached instruction manual. Figure 8 As shown, the instruction manual is attached. Figure 8 The results showed that in the control group with added DMSO, Mycobacterium smegmatis was long and rod-shaped with a smooth surface; in the experimental groups with added H1 and H2, the morphology of Mycobacterium smegmatis was significantly abnormal, the bacteria became longer, possibly due to restricted normal division, and some bacteria completely ruptured.

[0081] Test Example 3: Determination of the minimum inhibitory concentration (MIC) of compounds H1 and H2 against Mycobacterium smegma

[0082] To M. smegmatis mc 2 Different amounts of compounds H1 and H2 were added to the culture medium at 155, and the MICs of compounds H1 and H2 against Mycobacterium smegmatis were determined by CFU counting analysis.

[0083] Specifically, take M. smegmatis mc 2 Add 6 μL of 155% fresh bacterial culture to 10 mL of LBT medium, mix well, and OD... 600The value was 0.05. 10 μL of bacterial suspension was taken and diluted 1:10, 1:100, 1:1000, and 1:10000. 5 μL of each dilution was then inoculated onto LB agar for pre-treatment CFU counting. Simultaneously, 100 μL of this bacterial suspension was aliquoted into each well of a sterile 96-well plate, and H1 or H2 was added to final concentrations of 20, 40, 60, and 100 μg / mL. After incubation at 37°C for 24 hours, 10 μL of bacterial suspension was taken from each well and diluted 1:10, 1:100, 1:1000, and 1:10000. 5 μL of each dilution was then inoculated onto LB agar for post-treatment CFU counting. Statistical analysis was performed using GraphPad Prism 9 software.

[0084] The results are as shown in the attached instruction manual. Figure 9 As shown, compared with the bacterial CFU before treatment, after 24 hours of treatment with different concentrations of H1 and H2, the MIC of the compound provided by this invention against Mycobacterium smegmatis was significantly better than that of magnolol. At concentrations of 60 μg / mL H1 and 100 μg / mL H2, Mycobacterium smegmatis showed almost no growth. This indicates that the MICs of H1 and H2 against Mycobacterium smegmatis were 60 μg / mL and 100 μg / mL, respectively, indicating that H1 had a stronger ability to inhibit the growth of Mycobacterium smegmatis than H2.

[0085] Test Example 4: Effects of compounds H1 and H2 on the growth of Mycobacterium smegma when used in combination with first-line anti-tuberculosis drugs.

[0086] Compounds H1 and H2 were added simultaneously with the first-line anti-tuberculosis drugs rifampin (RFP) and isoniazid (INH) to M. smegmatis mc 2 In 155 culture medium, the effects of H1, H2, and combination therapy with first-line anti-tuberculosis drugs on the growth of Mycobacterium smegma were investigated.

[0087] Specifically, take M. smegmatis mc 2 Add 6 μL of 155% fresh bacterial culture to 10 mL of LBT medium, mix well, and OD... 600The value was 0.05. 100 μL of this bacterial culture was aliquoted into each well of a sterile 96-well plate. Control and experimental groups were set up. The control group received 1 μL of ddH2O or DMSO, while the experimental group received 1 μL of NIH dissolved in ddH2O (final concentration 0.5 μg / mL), or RFP dissolved in DMSO (final concentration 1 μg / mL), or 1 μL of H1 to a final concentration of 30 μg / mL and 1 μL of H2 to a final concentration of 50 μg / mL, in addition to NIH and RFP. After incubation at 37℃ for 24 hours, 10 μL of bacterial culture was taken from each well and diluted 1:10, 1:100, 1:1000, and 1:10000. 5 μL of each dilution was then inoculated onto LB agar for CFU counting. Statistical analysis was performed using GraphPadPrism 9 software.

[0088] The results are as shown in the attached instruction manual. Figure 10 As shown, add 0.5 μg / mL rifampicin or 1 μg / mL isoniazid alone to Mycobacterium smegmatis, or add 30 μg / mL H1 or 50 μg / mL H2 to the mixture of rifampicin and isoniazid. See the instruction manual. Figure 10 The results showed that the growth of Mycobacterium smegmatis was more inhibited when treated with rifampicin or isoniazid alone, when treated with H1 or H2. This indicates that H1 and H2 can enhance the antibacterial ability of rifampicin and isoniazid against Mycobacterium smegmatis, and H1 enhances the antibacterial ability of rifampicin and isoniazid against Mycobacterium smegmatis more strongly.

[0089] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be included within the scope of protection of the present invention. In particular, it should be noted that the addition or subtraction of traditional Chinese medicine compound and fermentation strain, as well as all similar substitutions and modifications, are obvious to those skilled in the art and are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate changes and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

Claims

1. A compound extracted from Magnolia officinalis, characterized in that, The compound is shown in Formula I: Formula I.

2. The method for preparing the compound extracted from Magnolia officinalis according to claim 1, characterized in that, Includes the following steps: S1. Soak Magnolia officinalis in methanol, heat under reflux at 70°C for 1 hour, and concentrate to obtain extract; S2. Dissolve the extract obtained in S1 in water, then extract with ethyl acetate, and concentrate under reduced pressure to obtain the ethyl acetate extract; S3. The ethyl acetate extract obtained in S2 was subjected to macroporous resin column chromatography with first gradient elution. 50% fraction was collected and concentrated to obtain a purified extract. S4. The purified extract obtained in S3 was subjected to normal phase silica gel column chromatography with second gradient elution to obtain 85 fractions. The 35th fraction was taken, concentrated, and the crude product was obtained. S5. Dissolve the crude product obtained in S4 in anhydrous ethanol, inject the solution into a preparative liquid chromatography system, perform a third elution, collect the eluent, and concentrate to obtain the compound.

3. The method for preparing the compound extracted from Magnolia officinalis according to claim 2, characterized in that, In step S1, the mass ratio of Magnolia officinalis to extract is 20:1; The mass ratio of Magnolia officinalis to methanol is 1:

10.

4. The method for preparing the compound extracted from Magnolia officinalis according to claim 2, characterized in that, In step S2, the mass ratio of the extract to the volume ratio of water is 1:5; The volume ratio of water to ethyl acetate is 1:1; The mass ratio of the extract to the ethyl acetate extract is 5:

1.

5. The method for preparing the compound extracted from Magnolia officinalis according to claim 2, characterized in that, In step S3, the conditions for the first gradient elution include: using methanol and water gradient elution as the first eluent, with a total of 4 gradients. In each gradient, the volume ratio of methanol to water is 0:100, 30:70, 60:40, and 100:0, respectively, and the elution volume of each gradient is 5 times the column volume.

6. The method for preparing the compound extracted from Magnolia officinalis according to claim 2, characterized in that, In step S3, the mass ratio of ethyl acetate extract to refined extract is 10:

3.

7. The method for preparing the compound extracted from Magnolia officinalis according to claim 2, characterized in that, In step S4, the conditions for the second gradient elution include: using petroleum ether and ethyl acetate as the second eluent, with a total of 6 gradients. In each gradient, the volume ratio of petroleum ether to ethyl acetate is 100:0, 70:30, 50:50, 30:70, 20:80, and 0:100, respectively, and the elution volume of each gradient is 10 times the column volume.

8. The method for preparing the compound extracted from Magnolia officinalis according to claim 2, characterized in that, In step S4, the mass ratio of the refined extract to the crude product is 30:

1.

9. The method for preparing the compound extracted from Magnolia officinalis according to claim 2, characterized in that, In step S5, the conditions for the third gradient elution include: using methanol and water as the mobile phase, with one gradient, the volume ratio of methanol to water being 60:40, and the elution volume being 30 times the column volume. The mass ratio of the crude product to the compound is 1000:

9.

10. The application of the compound extracted from Magnolia officinalis according to claim 1, characterized in that, The applications include: The application of the compound in the preparation of products that inhibit the growth of Mycobacterium smegmae; The use of the compound in the preparation of medicaments for the treatment or prevention of Mycobacterium smegma infection; Applications of the compound in the preparation of food, health products, food additives, and animal feed additives.

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