Anti-drug-resistant mycobacterium tuberculosis cinnamon extract I as well as preparation method and application thereof

Abstract

The invention provides an anti-drug-resistant mycobacterium tuberculosis cinnamon extract I and a preparation method and application thereof, and belongs to the technical field of traditional Chinese medicine extracts.Cinnamon is used as a raw material, and the cinnamon extract I with the anti-drug-resistant mycobacterium tuberculosis effect is obtained through moving bed extraction, countercurrent chromatography separation and distillation. The cinnamon extract I disclosed by the invention has obvious anti-mycobacterium tuberculosis activity, and has a good inhibition effect on mycobacterium tuberculosis and various drug-resistant mycobacterium tuberculosis; the cinnamon extract I has no synergistic effect with common anti-tuberculosis drugs such as rifampicin, isoniazide, ethambutol, streptomycin and levofloxacin, and has no cross resistance with rifampicin, isoniazide and levofloxacin, so that the cinnamon extract I possibly has a unique antibacterial mechanism for mycobacterium tuberculosis, and a new thought and a new method can be provided for prevention and treatment of tuberculosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine extracts, and particularly relates to a cinnamon extract I against drug-resistant Mycobacterium tuberculosis, a preparation method thereof, and an application thereof. Background Art

[0002] Tuberculosis (TB) is a serious chronic infectious disease caused by Mycobacterium tuberculosis. The emergence of drug-resistant Mycobacterium tuberculosis, especially multi-drug resistant (MDR-TB), pre-extensively drug-resistant (Pre-XDR-TB), and extensively drug-resistant (XDR-TB) bacteria, has significantly reduced the efficacy of traditional anti-tuberculosis drug treatment regimens, prolonged the treatment cycle, and the treatment success rate is not high.

[0003] Current clinical anti-tuberculosis treatment mainly relies on chemically synthesized drugs, such as isoniazid, rifampicin, etc. However, the long-term and irregular use of these drugs has led to the emergence of drug resistance in Mycobacterium tuberculosis. The drug resistance mechanism of drug-resistant Mycobacterium tuberculosis is extremely complex, and Mycobacterium tuberculosis can develop drug resistance through multiple pathways. Its gene mutation can change the drug action target, making the anti-tuberculosis drug unable to bind to it and play its efficacy. For example, a mutation in the rpoB gene will cause rifampicin to be unable to bind to RNA polymerase, losing its inhibitory effect on bacterial transcription; the efflux pump mechanism can actively expel the drug entering the bacterial cell, reducing the effective concentration of the drug inside the cell; the change in cell wall permeability will also prevent the drug from entering the bacterial body, thereby generating drug resistance. Multiple drug resistance mechanisms are intertwined, making it difficult for traditional treatment regimens to effectively inhibit the growth of drug-resistant bacteria.

[0004] Currently used drugs for treating drug-resistant tuberculosis generally have toxic and side effects. For example, aminoglycoside drugs can cause hearing loss and kidney function damage; quinolone drugs may cause gastrointestinal reactions, central nervous system symptoms, etc. Many patients are forced to interrupt treatment due to their inability to tolerate the adverse reactions of the drugs, resulting in the recurrence of the disease and further increasing the treatment difficulty.

[0005] In the journey of exploring anti-drug-resistant Mycobacterium tuberculosis, Chinese herbal medicine extracts have become a research direction that has attracted much attention. However, the current research on the treatment of drug-resistant Mycobacterium tuberculosis with Chinese herbal medicine extracts is still in its infancy and has many problems as follows: 1) The active ingredients are not clear. Most Chinese herbal medicine extracts have complex compositions, and the active ingredients that play an anti-drug-resistant tuberculosis role are often not clear. A Chinese herbal medicine extract may contain dozens or even hundreds of chemical components, making it difficult to determine which components play a key role in combating drug-resistant Mycobacterium tuberculosis. This uncertainty in components leads to the inability to accurately extract and optimize the effective components, restricting the further research and development and application of Chinese herbal medicine extracts. 2) The extraction process is not standardized. At present, the extraction process of Chinese herbal medicine extracts lacks unified specifications and standards, resulting in unstable contents and purities of the effective components in the extracts, making it difficult to ensure the consistency and stability of clinical efficacy and hindering the industrialization process of Chinese herbal medicine extracts in the field of drug-resistant tuberculosis treatment. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a cinnamon extract I against drug-resistant Mycobacterium tuberculosis, its preparation method and application; the specific cinnamon extract I extracted and separated from cinnamon by the present invention has a significant effect against drug-resistant Mycobacterium tuberculosis and can be used to prepare drugs for treating drug-resistant tuberculosis.

[0007] The present invention provides an application of a cinnamon extract I against drug-resistant Mycobacterium tuberculosis in the preparation of drugs for treating tuberculosis. The cinnamon extract I uses cinnamon as a raw material and obtains a whole-component extract by moving bed extraction; then the obtained whole-component extract is separated by countercurrent chromatography, the effluent at different time periods is collected, and distilled to obtain a refined extract, and the refined extracts with the effect of combating drug-resistant Mycobacterium tuberculosis are combined to obtain the cinnamon extract I.

[0008] Preferably, the tuberculosis includes one or more caused by Mycobacterium tuberculosis sensitive bacteria, rifampicin-resistant Mycobacterium tuberculosis, isoniazid-resistant Mycobacterium tuberculosis, ethambutol-resistant Mycobacterium tuberculosis, streptomycin-resistant Mycobacterium tuberculosis, levofloxacin-resistant Mycobacterium tuberculosis, multi-drug-resistant Mycobacterium tuberculosis, and quasi-extensively drug-resistant Mycobacterium tuberculosis.

[0009] Preferably, the tuberculosis includes cutaneous tuberculosis, lymph node tuberculosis, osteoarticular tuberculosis or pulmonary tuberculosis.

[0010] Preferably, the use concentration of the cinnamon extract I in the drug is ≥7.8 μg / mL.

[0011] The present invention provides a preparation method of the cinnamon extract I in the above application, including the following steps:

[0012] 1) Cinnamon is subjected to moving bed extraction to obtain a whole-component extract;

[0013] 2) Subject the total-component extract obtained in step 1) to countercurrent chromatography separation, and collect the effluent at different time periods;

[0014] 3) Distill the collected effluent to obtain a refined extract, and combine the refined extracts having the effect of resisting drug-resistant Mycobacterium tuberculosis to obtain cinnamomum extract I;

[0015] The extraction solvent used in the moving bed extraction in step 1) is an ethanol aqueous solution with a volume concentration of 50% to 60%; the pumping rate of the extraction solvent is 40 to 60 ml / min; the mass-volume ratio of the cinnamomum and the extraction solvent is 1 g:(8 - 12) mL.

[0016] Preferably, in the countercurrent chromatography separation in step 2), an ethanol aqueous solution with a volume concentration of 25% to 35% is used as the stationary phase, and n-hexane is used as the mobile phase; mix the ethanol aqueous solution with the total-component extract obtained in step 1) to obtain a sample solution, and the volume concentration of ethanol in the sample solution is 25% to 35%; after sample loading, pump in the mobile phase for countercurrent chromatography separation.

[0017] Preferably, the pumping rate of the mobile phase is 5 to 10 ml / min.

[0018] Preferably, the cinnamomum is cassia twig, cassia leaf or cassia bark; the cinnamomum is pulverized and sieved through a 20 - 40 mesh sieve.

[0019] The present invention provides the cinnamomum extract I for resisting drug-resistant Mycobacterium tuberculosis prepared by the described preparation method, which includes coumarin, o-hydroxycinnamic acid, p-hydroxycinnamic acid, anethole, trans-cinnamaldehyde, 2'-methoxycinnamaldehyde and methyl cinnamate.

[0020] The present invention provides a drug for treating tuberculosis, which includes the cinnamomum extract I for resisting drug-resistant Mycobacterium tuberculosis described above.

[0021] Compared with the prior art, the present invention has the following beneficial effects: The cinnamomum extract I provided by the present invention uses cinnamomum as a raw material, and obtains a full-component extract through moving bed extraction; then it is separated by countercurrent chromatography, the effluent at different time periods is collected, distilled to obtain a refined extract, and the refined extracts with the effect of resisting drug-resistant mycobacterium tuberculosis are combined to obtain cinnamomum extract I. The cinnamomum extract I obtained by the present invention has obvious anti-mycobacterium tuberculosis activity, including the standard strain H37Rv of mycobacterium tuberculosis, clinically isolated sensitive mycobacterium tuberculosis, and clinically isolated drug-resistant mycobacterium tuberculosis, including rifampicin-resistant (RFP, R) mycobacterium tuberculosis, isoniazid-resistant (INH, H) mycobacterium tuberculosis, ethambutol-resistant (EMB, E) mycobacterium tuberculosis, streptomycin-resistant (Sm, S) mycobacterium tuberculosis, and levofloxacin-resistant (Lfx, L) mycobacterium tuberculosis; the present invention combines cinnamomum extract I with common anti-tuberculosis drugs (H, R, E, S, L), and the results suggest that there is no synergistic effect or cross-resistance between cinnamomum extract I and these anti-tuberculosis drugs, indicating that cinnamomum extract I may have a unique antibacterial mechanism against mycobacterium tuberculosis. The cinnamomum extract I provided by the present invention can be applied to resist mycobacterium tuberculosis, and can provide new ideas and new methods for the prevention and treatment of tuberculosis. The application scope includes cutaneous tuberculosis, scrofula, osteoarticular tuberculosis, and pulmonary tuberculosis (inhalation), etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Total ion current chromatogram of cinnamomum extract I obtained in Example 1: from top to bottom are the positive mode, negative mode, and ultraviolet absorption diagram with a wavelength of 254 nm.

[0023] Figure 2 Growth of clinically isolated mycobacterium tuberculosis on Löwenstein-Jensen medium, where 1-2 are rifampicin-resistant mycobacterium tuberculosis alone, 3-4 are isoniazid-resistant mycobacterium tuberculosis alone, 5-12 are MDR mycobacterium tuberculosis (resistant to rifampicin and isoniazid simultaneously), 13-17 are quasi-extensively drug-resistant mycobacterium tuberculosis (resistant to rifampicin, isoniazid, and levofloxacin simultaneously), and 18 is extensively drug-resistant mycobacterium tuberculosis (resistant to rifampicin, isoniazid, levofloxacin, and streptomycin simultaneously).

[0024] Figure 3 MIC test results of cinnamomum extract I against H37Rv, where the vertical axis: Rv refers to the standard strain H37Rv of mycobacterium tuberculosis, RFP refers to rifampicin, CNE I refers to cinnamomum extract I, and "negative" refers to only the medium; the numbers above the horizontal axis represent the rifampicin concentration (μg / mL), and the numbers below represent the cinnamomum extract I concentration (μg / mL).

[0025] Figure 4For the MIC detection of cinnamon extract Ⅰ against clinically isolated sensitive and drug-resistant bacteria, where the vertical axis: Rv refers to the standard strain of Mycobacterium tuberculosis H37Rv, MDR refers to multi-drug resistant bacteria, R refers to rifampicin mono-resistant bacteria, Pre-XDR refers to pre-extensively drug-resistant bacteria, L and M refer to levofloxacin- and moxifloxacin-resistant bacteria; the horizontal axis numbers represent the concentration of cinnamon extract Ⅰ.

[0026] Figure 5 For the combined antibacterial activity of cinnamon extract Ⅰ and anti-tuberculosis drugs INH, RFP, EMB, Sm, and Lfx against H37Rv; where the vertical axis represents the concentration of cinnamon extract Ⅰ, and the horizontal axis represents the concentrations of isoniazid (H), rifampicin (R), ethambutol (E), streptomycin (S), and levofloxacin (L) respectively, 100% represents 100% of the concentration of the bacterial suspension used; 1% represents 1% of the concentration of the bacterial suspension used; 0% represents no bacterial suspension.

[0027] Figure 6 For the combined antibacterial activity of cinnamon extract Ⅰ and anti-tuberculosis drugs INH, RFP, and Lfx against clinically drug-resistant bacteria. Among them, the resazurin color development diagrams of the antibacterial activities of different concentrations of cinnamon extract Ⅰ combined with INH, RFP, and Lfx against INH mono-resistant, RFP mono-resistant, Lfx mono-resistant, MDR, and Pre-XDR bacteria; in the corresponding table below the color development diagram, the yellow area is the bacteria plus various combined drugs, 0 represents only the bacterial suspension, H is isoniazid, R is rifampicin, Lfx is levofloxacin, and C1, C2, and C3 respectively represent 3.9, 7.8, and 15.6 μg / mL of cinnamon twig extract I, and the gray area is only the culture medium. Detailed implementation mode

[0028] The present invention provides an application of cinnamon extract Ⅰ against drug-resistant Mycobacterium tuberculosis in the preparation of a drug for treating tuberculosis. The cinnamon extract Ⅰ uses cinnamon as a raw material and obtains a whole-component extract through moving bed extraction; then the obtained whole-component extract is separated by countercurrent chromatography, the effluent at different time periods is collected, and distilled to obtain a refined extract, and the refined extracts with the effect of against drug-resistant Mycobacterium tuberculosis are combined to obtain cinnamon extract Ⅰ.

[0029] In the present invention, the tuberculosis includes those caused by one or more of Mycobacterium tuberculosis sensitive bacteria, rifampicin mono-resistant Mycobacterium tuberculosis, isoniazid mono-resistant Mycobacterium tuberculosis, ethambutol mono-resistant Mycobacterium tuberculosis, streptomycin mono-resistant Mycobacterium tuberculosis, levofloxacin mono-resistant Mycobacterium tuberculosis, multi-drug resistant Mycobacterium tuberculosis, and pre-extensively drug-resistant Mycobacterium tuberculosis. In the present invention, the tuberculosis includes but is not limited to cutaneous tuberculosis, lymph node tuberculosis, osteoarticular tuberculosis, or pulmonary tuberculosis.

[0030] In the present invention, the use concentration of the cinnamon extract Ⅰ in the drug is ≥7.8 μg / mL.

[0031] The present invention provides a method for preparing cinnamon extract I in the said application, comprising the following steps:

[0032] 1) Cinnamon is subjected to moving bed extraction to obtain a whole-component extract; 2) The whole-component extract obtained in step 1) is separated by countercurrent chromatography, and the effluents at different time periods are collected; 3) The collected effluents are distilled to obtain a refined extract, and the refined extracts having the effect of anti-drug-resistant Mycobacterium tuberculosis are combined to obtain cinnamon extract I.

[0033] In the present invention, the cinnamon is cassia twig, cassia leaf or cassia bark; the properties of cinnamon extract I obtained by extracting with cassia twig, cassia leaf or cassia bark as raw materials are similar; the cinnamon is preferably crushed and sieved through a 20-40 mesh sieve. For the structure of the moving bed and the method of moving bed extraction, reference can be made to the description in Patent 202411126616.3. The extraction solvent used in the extraction of the present invention is an ethanol aqueous solution with a volume concentration of 50% - 60%, preferably 52% - 58%, and more preferably 55%; the pumping speed of the extraction solvent is preferably 40 - 60 ml / min, more preferably 45 - 55 ml / min, and most preferably 50 ml / min; the mass-volume ratio of the cinnamon to the extraction solvent is preferably 1 g:(8 - 12) mL, preferably 1 g:(9 - 11) mL, and more preferably 1 g:10 mL. In the present invention, after the moving bed extraction, the extract at the downstream end outlet is collected and its volume is concentrated to 1 / 3 of the original volume by vacuum concentration to obtain the whole-component extract.

[0034] In the present invention, the obtained whole-component extract is separated by countercurrent chromatography, and the effluents at different time periods are collected. For the device used in the countercurrent chromatography separation, reference can be made to the description in Patent 202411086627.3. The countercurrent chromatography separation of the present invention uses an ethanol aqueous solution with a volume concentration of 25% - 35% as the stationary phase, preferably 27% - 33%, and more preferably 30%; n-hexane is used as the mobile phase; specifically, the ethanol aqueous solution is mixed with the whole-component extract to obtain a sample loading solution, ensuring that the volume concentration of ethanol in the sample loading solution is 25% - 35% (i.e., loading the sample with the stationary phase); after sample loading, the mobile phase is pumped in for countercurrent chromatography separation. In the present invention, the pumping speed of the mobile phase is preferably 5 - 10 ml / min, more preferably 7 - 9 ml / min, and most preferably 8 ml / min. During the countercurrent chromatography separation process, the sample components are rapidly distributed between the two-phase solutions, and different components flow out from the outlet end rotary joint of the device successively with the mobile phase due to the difference in distribution coefficients. The effluents at different time periods are collected and labeled as components 1 - 15.

[0035] In the present invention, the collected effluent is distilled to obtain a refined extract, and the refined extracts with the effect of anti-drug-resistant Mycobacterium tuberculosis are combined to obtain cinnamon extract I. In the present invention, the temperature of the distillation is 62-98 °C, preferably 94-96 °C, more preferably 95 °C, and the time of the distillation is 2-3 h; in the present invention, the components in cinnamon are carried by steam and flow out after being cooled by a condenser tube to obtain the refined extracts of each segmented component. The refined extracts of each segmented component obtained are tested for anti-drug-resistant Mycobacterium tuberculosis, and the refined extracts with the effect of anti-drug-resistant Mycobacterium tuberculosis are combined to obtain cinnamon extract I.

[0036] The present invention provides cinnamon extract I with anti-drug-resistant Mycobacterium tuberculosis prepared by the preparation method. In the present invention, the cinnamon extract I includes coumarin, o-hydroxycinnamic acid, p-hydroxycinnamic acid, anethole, trans-cinnamaldehyde, 2'-methoxycinnamaldehyde, and methyl cinnamate. The cinnamon extract I also includes some unknown components.

[0037] The present invention also provides a drug for treating tuberculosis, including the cinnamon extract I with anti-drug-resistant Mycobacterium tuberculosis. In the present invention, the drug may also include other active ingredients against Mycobacterium tuberculosis. The present invention has no special limitation on the dosage form of the drug, and conventional dosage forms in the art can be used, including but not limited to tablets, granules, capsules, oral liquids, and inhalation preparations. In the present invention, the tuberculosis includes but is not limited to cutaneous tuberculosis, lymph node tuberculosis, osteoarticular tuberculosis, or pulmonary tuberculosis.

[0038] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0039] Example 1

[0040] Preparation and effective component detection of cinnamon extract I against Mycobacterium tuberculosis

[0041] In this example, the ramulus cinnamomi in cinnamon is used as the raw material, and the ramulus cinnamomi is pulverized and sieved through a 20-40 mesh sieve.

[0042] 1) Moving bed extraction

[0043] The device used can be referred to the description in Patent 202411126616.3;

[0044] System setup: Configure the fluid flow system into 5 zones, numbered sequentially from zone (5) to zone (1). Zone (5) is the upstream starting point, and zone (1) is the downstream end point. Each zone (i) is provided with a liquid inlet hole ZI(i) and an outlet ZO(i), and a liquid series connection path is formed through pipeline connection. The system is configured with 5 tanks, and 3 of them are used in this embodiment. Place extraction tanks with loading and unloading ports and liquid inlets and outlets in each zone, load the cinnamon twig raw materials crushed to 20 - 40 meshes into 3 extraction tanks respectively, with 2 kg of cinnamon raw materials in each extraction tank. Close the loading and unloading ports, and connect the extraction tanks to the liquid inlets and outlets of the corresponding zones.

[0045] Gradient extraction: Select ethanol with a volume concentration of 50% as the extraction solvent, pump the extraction solvent into the upstream starting point liquid inlet ZI(f) at a flow rate of 50 ml / min, so that it flows through the cinnamon twig raw materials in the extraction tanks in zones (5), (4), and (3) in sequence. Stop pumping after pumping 20 L of the predetermined volume of extraction solvent, and collect the extract at the downstream end point outlet ZO(3).

[0046] Unload the residue in the extraction tank in zone (5), load new raw materials, and move it to zone (1); move the tank in the original zone (4) to zone (5), move the tank in the original zone (3) to zone (4), install an extraction tank containing unextracted cinnamon twig raw materials in the original zone (3) and connect it;

[0047] Pump the extraction solvent into the upstream starting point liquid inlet ZI(f) again at a flow rate of 50 ml / min, so that it flows through the cinnamon twig raw materials in the extraction tanks in zones (5), (4), and (3) in sequence. Stop pumping after pumping 20 L of the predetermined volume of extraction solvent, and collect the extract at the downstream end point outlet ZO(3).

[0048] Pre - treatment of the pre - extract: The extract collected at the downstream end point outlet ZO(1) is concentrated to 1 / 3 of its original volume by vacuum concentration to obtain a whole - component extract rich in non - volatile components such as polyphenols and polysaccharides, and it is reserved for use.

[0049] 2) Counter - current chromatography separation stage

[0050] The device used is as described in Patent 202411086627.3;

[0051] Device preparation: Set up a countercurrent chromatography device, ensure that the rotating shaft (20) is firmly connected to the rotating frame (10), install 142 tubular containers (30) connected in series on the rotating frame (10) in sequence, and place a magnetic stirring member (33) in each container (30). Set the end face of the container (30) close to the rotating shaft (20) as the feed liquid inlet (31), and the end face far from the rotating shaft (20) as the feed liquid outlet (32), and complete the series connection between the containers. Install a magnetic field forming member (40) on one side of the rotating frame (10) so that the arc around the rotating frame (10) does not exceed 180°. The magnetic field can be generated by a permanent magnet or an electromagnet, and connect the inlet and outlet rotary joints as well as auxiliary equipment such as pumps and liquid storage tanks.

[0052] Stationary phase filling and sample loading: Use a heavy phase solution (ethanol aqueous solution) as the stationary phase and an immiscible light phase solution (n-hexane) as the mobile phase. Mix the ethanol aqueous solution with the concentrated whole-component extract to obtain a sample loading solution, ensuring that the volume concentration of ethanol in the sample loading solution is 30 - 35%; Start the rotating drum and rotate it at a constant speed of 500 rpm. Pump 600 ml of the sample loading solution from the rotary joint at the inlet end of the device until the liquid flows out from the rotary joint at the outlet end, ensuring that all containers (30) are filled with the sample loading solution.

[0053] Sample separation: Pump the mobile phase from the rotary joint at the inlet end of the device at a speed of 8 ml / min. Under the action of the magnetic stirring member (33), the sample components are rapidly distributed between the two-phase solutions, and different components flow out from the rotary joint at the outlet end of the device successively with the mobile phase due to the difference in distribution coefficients. Collect the effluent every 50 minutes in segments and label them as component 1, component 2, component 3 - component 15. After recovering the solvent from each component obtained by countercurrent chromatography separation using a rotary evaporator, 15 separated components are obtained.

[0054] 3) Distillation stage

[0055] Raw material preparation: Transfer components 1 - 15 obtained after separation to the distillation kettle respectively, and add 50 times the volume of water according to the component characteristics and target components to fully immerse the cinnamon components.

[0056] Distillation operation: Heat the distillation kettle and raise the temperature to 95°C for steam distillation. Control the distillation temperature and time (temperature 95°C, time 2 - 3 hours) to ensure that the volatile components are fully extracted and avoid component decomposition and energy consumption waste. The steam generated carries the components in the cinnamon and flows out after being cooled by the condenser to obtain the refined extracts of components 1 - 15.

[0057] Detect the refined extracts of the above components 1 - 15 for their anti-drug-resistant Mycobacterium tuberculosis effects, and it is found that components 10, 7, and 11 have anti-drug-resistant Mycobacterium tuberculosis effects. Combine components 10, 7, and 11 to obtain cinnamon extract I.

[0058] Component detection of cinnamon extract Ⅰ using LCMS

[0059] The equipment and reagents used are shown in Tables 1 and 2 below.

[0060] Table 1 Equipment Information

[0061]

[0062] Table 2 Reagent Information

[0063]

[0064]

[0065] Experimental Procedure

[0066] Sample: Cinnamon extract Ⅰ obtained from the above extraction, Guizhi 4890629, WT202504013-01. Sample preparation: Centrifuge cinnamon extract Ⅰ directly and take the supernatant as the test solution.

[0067] Instrument Parameters and Detection Conditions

[0068] Chromatographic column: Waters ACQUITY UPLC HSS T3 2.1×100mm×1.8um (LC-226) Mobile phase: A - acetonitrile, B - 0.1% formic acid solution

[0069] Table 3 Elution Gradient

[0070] Time (min) 0 10 20 25 30 31 35 A％ 5 30 60 90 90 5 5

[0071] Flow rate: 0.3 mL / min Wavelength: 254 nm, full wavelength scan Column temperature: 35℃

[0072] Injection volume: 1 μL

[0073] Ion source: ESI

[0074] Nozzle voltage: 4 kV / 3.5 kV

[0075] Sheath gas temperature: 350℃

[0076] Sheath gas flow rate: 11 L / min

[0077] Dry gas flow rate: 8 L / min

[0078] Dry gas temperature: 325℃

[0079] Scan mode: full scan

[0080] Primary scan range: m / z 50~1700

[0081] Conical hole voltage: 100V

[0082] First-level scanning range: m / z 50 - 1700

[0083] Collision energy gradient: 10, 20, 40 Ev

[0084] The detection results are as Figure 1 shown.

[0085] Data processing: Import the raw data collected into Qualitative Analysis 10.0 software. Through its wizard settings and method templates, establish an identification process for unknown compounds and perform peak extraction on the raw data. Analyze the characteristic peaks in the sample, fit the possible molecular formulas through the obtained molecular ion chromatogram peaks and isotope peaks, and match the primary information, secondary fragments with the PCDL secondary database and the cinnamon plant database. Filter out the results with a mass deviation greater than 7.5 ppm and a matching score lower than 70 points. At the same time, use the online database SIRIUS 5.8.5 version to match the secondary fragment results, select the compounds with a matching score greater than 70 and a FingerID less than 50 to obtain the final detection results, as shown in Table 4.

[0086] Table 4 Analysis of the data results of Cinnamomum cassia 4890629, WT202504013 - 01

[0087]

[0088] Note:

[0089] 1: * indicates that there may be a peak within a peak for this peak.

[0090] 2: For the remarks showing the PCDL secondary library and the SIRIUS online library, the results are all secondary fragment matching results. For those showing plant library matching, they are all primary plant component library matching;

[0091] 3: The retention time in the table is based on the 254 nm chromatogram in the positive mode. In the negative mode, there is a retention time deviation of 0.1 - 0.3 minutes due to the acquisition time interval compared to the negative mode, but it does not affect the judgment;

[0092] 4: "-" indicates no matching result, and only the possible molecular weight of this peak is speculated;

[0093] Example 2

[0094] Application of the anti - Mycobacterium tuberculosis cinnamon extract Ⅰ (Cinnamon Extract Ⅰ, CNEⅠ) prepared in Example 1

[0095] 1. Experimental strains: Mycobacterium tuberculosis standard strain H37Rv, from Guizhou Provincial Center for Disease Control and Prevention; clinically isolated Mycobacterium tuberculosis strains, from the Affiliated Hospital of Zunyi Medical University, specifically rifampicin-resistant (RFP, R) Mycobacterium tuberculosis, isoniazid-resistant (INH, H) Mycobacterium tuberculosis, ethambutol-resistant (EMB, E) Mycobacterium tuberculosis, streptomycin-resistant (Sm, S) Mycobacterium tuberculosis, and levofloxacin-resistant Mycobacterium tuberculosis (Lfx, L).

[0096] 2. Main consumables: ultrasonic dispersion tubes, 1.5 mL Eppendorf tubes, centrifuge tubes (15 / 50 mL specifications), glass bottles (250 / 500 / 800 mL specifications), pipette tips (10 / 100 / 1000 μL specifications), U-bottom 96-well plates, black 96-well plates, sterile rubber gloves, bacterial preservation tubes, spreading rods, three-pronged plates, sealing films, inoculation loops, sterile filter tips, etc.

[0097] 3. Culture of Mycobacterium tuberculosis standard strain H37Rv

[0098] 3.1 Preparation of neutral Lowenstein-Jensen medium

[0099] 3.1.1 Preparation of fresh chicken egg liquid: Prepare fresh eggs (about 50 mL each), thoroughly clean the eggshell surface with soapy water, then rinse with clean water and dry with sterile gauze. Next, disinfect the eggshell and the body of the electric egg beater with 75% alcohol. After disinfection, place the eggs and the egg beater in a laminar flow hood and perform ultraviolet irradiation sterilization for 30 minutes. Then, break the egg liquid into a pre-sterilized stainless steel bucket and use the electric egg beater to stir it evenly. The stirring time is controlled within 5 - 10 seconds. If it is not evenly mixed, the time can be appropriately increased. Then filter the egg liquid through sterile gauze into a 2000 mL beaker, pay attention to the scale line, and make up the volume to 1250 mL.

[0100] 3.1.2 Components and preparation method of neutral medium: Weigh 3.0 g of KH2PO4, 0.3 g of MgSO4·7H2O, 0.75 g of magnesium citrate, 9 g of sodium glutamate, and 15 mL of glycerol and add them to 750 mL of distilled water. Mix well and autoclave at 121 °C for 30 minutes. Cool to room temperature to obtain 765 mL of the basic medium solution. Add 765 mL of the basic solution to the filtered 1250 mL of fresh chicken egg liquid, and then add 25 mL of 2% malachite green. Mix well to obtain 2040 mL of the original medium solution. Use a pipette to add 2040 mL of the original medium solution to sterile culture tubes at 7 mL per tube, tighten the bottle caps, place them in an 85 °C steam incubator for 50 minutes, take out the culture tubes, cool to room temperature, and store them in a 4 °C refrigerator for later use.

[0101] 3.1.3 Aliquot the mixed solution into sterile culture tubes at 7 mL / tube. During aliquoting, perform the action at a uniform and slow speed to avoid generating bubbles as much as possible. Strictly pay attention to sterility during the operation process. After aliquoting, place the culture tubes uniformly on a 30° inclined bracket for fixation, and transfer the entire inclined bracket to a steam incubator for baking at 85°C for 50 min.

[0102] 3.1.4 For the medium that has cooled and solidified at room temperature, randomly select 2 tubes and place them in a 37°C constant temperature incubator for incubation for 24 h for sterility detection.

[0103] 3.1.5 After passing the sterility verification (no microbial growth in the culture tubes), aliquot and seal them, and store them in the dark at 4°C for standby.

[0104] 3.2 MIC detection of cinnamaldehyde extract Ⅰ against Mycobacterium tuberculosis standard strain H37Rv

[0105] 3.2.1 Inoculation and culture: Take out the cryopreserved stock tube of Mycobacterium tuberculosis standard strain H37Rv from the -80°C ultra-low temperature refrigerator, about 1 mL per tube. After thawing, mix well, pipette 100 μL of the bacterial suspension, and evenly smear it on the slope of Lowenstein-Jensen medium. A total of about 2 - 3 tubes are inoculated. After covering the bottle cap, mark the name of the inoculated bacteria, the operator, and the inoculation date on the tube body and the bottle cap. Then place it in a 37°C constant temperature incubator for culture. Observe the growth of Mycobacterium tuberculosis every 1 day. Obvious colony growth can be seen in about 2 - 4 weeks. The colonies are yellow granules, clumps, or cauliflower-like.

[0106] 3.2.2 Preparation of cinnamaldehyde extract Ⅰ: Mark the extracted cinnamaldehyde extract Ⅰ (storage concentration 1 mg / ml) clearly and store it in a -20°C refrigerator for standby.

[0107] 3.2.3 MIC operation steps

[0108] 3.2.3.1 Preparation of items: Put cinnamaldehyde extract Ⅰ, rifampicin, 7H9 + 10% OADC medium, U-bottom 96-well plate, centrifuge tube, inoculation loop, ultrasonic dispersion tube, pipette tips, sample addition trough, etc. into the biosafety cabinet, sterilize them by ultraviolet irradiation for 30 min, and preheat the ultrasonic disperser for 15 min.

[0109] 3.2.3.2 Cinnamon extract Ⅰ dilution: Take 748.8 μl of 1 mg / ml cinnamon extract Ⅰ into a 5-ml EP tube containing 2251.2 μl of culture medium, and pipette to mix well to obtain a total volume of 3 ml of cinnamon extract Ⅰ with a concentration of 249.6 μg / mL. Next, perform serial dilution. Add 1 mL of the 249.6 μg / mL cinnamon extract I solution to a new EP tube, and then add 1 mL of the culture medium to obtain a cinnamon extract I solution with a concentration of 124.8 μg / mL. Repeat this operation to obtain cinnamon extract I solutions with the following concentrations: 0.975 μg / mL, 1.95 μg / mL, 3.9 μg / mL, 7.8 μg / mL, 15.6 μg / mL, 31.2 μg / mL, 62.4 μg / mL, 124.8 μg / mL, 249.6 μg / mL.

[0110] 3.2.3.3 Rifampicin dilution: The operation is the same as that of cinnamon extract Ⅰ dilution. The storage concentration of rifampicin is 10000 μg / mL. First, dilute it tenfold with the culture medium, and then perform serial dilution to obtain the following concentrations of RFP solutions: 0.0098 μg / mL, 0.0195 μg / mL, 0.039 μg / mL, 0.078 μg / mL, 0.156 μg / mL, 0.312 μg / mL, 0.624 μg / mL, 1.248 μg / mL, 2.496 μg / mL.

[0111] 3.2.3.4 Preparation of H37Rv bacterial solution: Select well-grown H37Rv from the Lowenstein-Jensen medium, add 1 mL of 7H9 + 10% OADC medium to the ultrasonic dispersion tube, and then use an inoculation loop to scrape an appropriate amount of colonies from the surface of the Lowenstein-Jensen medium and add them to it. Treat with an ultrasonic disperser for 40 s, and then let it stand for 10 - 20 min. Aspirate the supernatant into a new ultrasonic dispersion tube, add 7H9 + 10% OADC medium (the minimum detectable volume of the ultrasonic disperser is 1 mL), and use turbidimetry to detect the Mcf value. By adjusting the ratio of the bacterial solution to the culture medium, make the turbidity concentration of the bacterial solution reach 2.5 Mcf (1 Mcf = 2×10 7 CFU / mL), and then dilute it 50 times to obtain 1.0×10 6 CFU / mL. Method of adding samples to a 96-well plate: Add 100 μl of the bacterial solution + 100 μl of cinnamon extract I to each well, which is equivalent to a 2-fold dilution, and obtain a target concentration of the bacterial solution of 5×10 5 CFU / mL.

[0112] 3.2.4 Sample addition: Use a pipette to separately aspirate 100 μL of the diluted cinnamon extract I solution and the RFP dilution solution, and sequentially add them to each well of a U-bottom 96-well plate. Subsequently, add 100 μL of the adjusted bacterial solution to each well (Note: Add the medicinal solution first, and then the bacterial solution). The final concentrations of cinnamon extract I in the 96-well plate are successively: 0.4875 μg / mL, 0.975 μg / mL, 1.95 μg / mL, 3.9 μg / mL, 7.8 μg / mL, 15.6 μg / mL, 31.2 μg / mL, 62.4 μg / mL, 124.8 μg / mL. Add 200 μL of the culture medium to the negative control wells, and add 100 μL of the bacterial solution and 100 μL of 7H9 culture medium to the positive control wells. Finally, use a sealing film to seal around the edges of the 96-well plate in two circles, then place it in a transparent sealed bag, and then put it into a 37°C constant temperature incubator for cultivation.

[0113] 3.2.5 Result observation and interpretation:

[0114] On the second day of cultivation, take a photo to observe the situation of the 96-well plate. If a white turbid area appears, it indicates contamination by common bacteria. After that, take a photo every other day to record the growth of Mycobacterium tuberculosis. When obvious colony differences appear in the 96-well plate (about 9 - 11 days), then add 20 μL of the diluted resazurin indicator to each well, and observe the resazurin color change 1 - 2 days later. Result determination criteria: In the 96-well plate, if a round white precipitate appears in the well, it indicates that Mycobacterium tuberculosis can grow at this drug concentration, and as the cultivation time extends, the white precipitate will gradually increase; while the black area indicates that this drug concentration inhibits the growth of Mycobacterium tuberculosis and will not change with the extension of the cultivation time. After adding the resazurin indicator, the appearance of a pink color in the well indicates that Mycobacterium tuberculosis can survive and reproduce at this drug concentration, while the blue area indicates that this drug concentration inhibits the growth of Mycobacterium tuberculosis.

[0115] 3.3 Detection of the MIC of cinnamon extract I against clinically isolated sensitive and drug-resistant strains of Mycobacterium tuberculosis

[0116] The clinical strains are from the Affiliated Hospital of Zunyi Medical University. The sputum / alveolar lavage fluid specimens of pulmonary tuberculosis patients are identified by the traditional proportion method and drug sensitivity test. After digestion and centrifugation, the supernatant is discarded, and the precipitate is evenly inoculated on an acidic culture medium and placed in a 37°C constant temperature incubator for growth for 4 - 8 weeks. Granular, clumpy, and cauliflower-like yellow colonies can be seen growing on the slant culture medium, as Figure 2As shown, the clinically isolated Mycobacterium tuberculosis strains with positive culture were identified for species and drug susceptibility tested by the traditional proportion method according to the "Tuberculosis Laboratory Testing Procedures" in 2015 to obtain clinically isolated sensitive and drug-resistant Mycobacterium tuberculosis strains. Clinically isolated Mycobacterium tuberculosis strains were screened from the identified specimens for subculture and preservation. When the growth status of the subcultured Mycobacterium tuberculosis was good, the MIC of cinnamon extract I was detected.

[0117] 3.3.1 Bacterial suspension dilution: Add 1 mL of 7H9 + 10% OADC medium to a labeled ultrasonic dispersion tube. Use an inoculation loop to scrape an appropriate amount of colonies from the surface of the Lowenstein-Jensen medium and add them thereto. After treatment with an ultrasonic disperser for 40 s, let it stand for 10 - 20 min. Subsequently, use a pipette to aspirate an appropriate amount of supernatant (try to avoid aspirating the precipitate below) into another labeled ultrasonic dispersion tube, add an appropriate amount of medium (the minimum detection volume of the ultrasonic disperser is 1 mL), and use the turbidimetric method to measure the Mcf value. Adjust the ratio of the bacterial suspension to the medium according to the steps in 3.23.4 to make the turbidity reach 2.5 Mcf (1 Mcf = 2×107 CFU / mL). Finally, dilute the bacterial suspension 50 times to obtain 1.0×10 6 CFU / mL. Method for adding samples to a 96-well plate: Add 100 μl of bacterial suspension + 100 μl of cinnamon extract I to each well, which is equivalent to a 2-fold dilution, ensuring that the final concentration of the bacterial suspension in the 96-well plate is 5×10 5 CFU / mL.

[0118] 3.3.2 Dilution of cinnamon extract I: Add 100 μL of 7H9 + 10% OADC medium to columns 2 - 10 of a 96-well plate. Obtain a cinnamon extract I concentration of 249.6 μg / mL according to the steps in 3.2.3.2 above. After mixing the mixture in each well in column 11 evenly with a multichannel pipette, aspirate 100 μL and serially dilute it forward to column 3 (discard 100 μL aspirated from column 3). Make the final concentrations of cinnamon extract I in the 96-well plate after adding 100 μL of bacterial suspension be: 0.49 μg / mL, 0.98 μg / mL, 1.95 μg / mL, 3.9 μg / mL, 7.8 μg / mL, 15.6 μg / mL, 31.2 μg / mL, 62.4 μg / mL, 124.8 μg / mL in sequence.

[0119] 3.3.3 Sample addition: Use a pipette to aspirate 100 μL of the correspondingly adjusted bacterial suspension and add it to each well of a U-bottom 96-well plate in sequence. Add 200 μL of medium to the negative wells. Finally, use a sealing film to seal around the 96-well plate twice, then put it into a transparent sealed bag, and then place it in an incubator at 37°C for incubation.

[0120] 3.3.4 Result observation and interpretation:

[0121] On the second day of cultivation, take a photo to observe the situation of the 96-well plate. If a white turbid area appears, it indicates contamination by common bacteria. After that, take a photo every other day to record the growth of Mycobacterium tuberculosis. When obvious colony differences appear in the 96-well plate (about 9 - 11 days), then add 20 μL of diluted resazurin indicator to each well, and observe the resazurin color development 1 - 2 days later. Result judgment criteria: In the 96-well plate, if a round white precipitate appears in the well, it indicates that Mycobacterium tuberculosis can grow at this drug concentration, and as the cultivation time extends, the white precipitate will gradually increase; while the black area indicates that this drug concentration inhibits the growth of Mycobacterium tuberculosis and will not change with the extension of the cultivation time. After adding the resazurin indicator, the appearance of a pink color in the well indicates that Mycobacterium tuberculosis can survive and reproduce at this drug concentration, while the blue area indicates that this drug concentration inhibits the growth of Mycobacterium tuberculosis.

[0122] 3.4 Combined antibacterial activity of cinnamaldehyde extract Ⅰ with INH, RFP, EMB, Sm and Lfx against H37Rv

[0123] 3.4.1 Select H37Rv with good growth status from the Lowenstein-Jensen medium.

[0124] 3.4.2 The preparation of the bacterial solution and the dilution of cinnamaldehyde extract Ⅰ are the same as in 3.3.2. The final concentration of the H37Rv bacterial solution in the 96-well plate is 5×105 CFU / mL. After adding 100 μL of the bacterial solution, the final concentrations of cinnamaldehyde extract Ⅰ in the 96-well plate are 1.95 μg / mL, 3.9 μg / mL, 7.8 μg / mL, 15.6 μg / mL, 31.2 μg / mL, 62.4 μg / mL, and 124.8 μg / mL.

[0125] 3.4.3 Dilute isoniazid, rifampicin, ethambutol, streptomycin, and levofloxacin to corresponding concentrations. The dilution principle is the same as that of cinnamaldehyde extract Ⅰ.

[0126] 3.4.5 Sample addition: Pipette 50 μL of the diluted cinnamaldehyde extract Ⅰ and 50 μL of the diluted anti-tuberculosis drug into the corresponding positions in the 96-well plate according to the concentration, and then add 100 μL of the diluted bacterial solution; seal the 96-well plate along the edge with a sealing film, then put it into a sealed bag and transfer it to an incubator at 37 °C for cultivation.

[0127] 3.4.5 Result Observation: On the second day of culture, take a photo to observe the situation of the 96-well plate. If a white turbid area appears, it indicates contamination by common bacteria. Then, take photos every other day to record the growth of Mycobacterium tuberculosis. When obvious colony differences appear in the 96-well plate (about 9 - 11 days), then add 20 μL of diluted resazurin indicator to each well and observe the resazurin color development 1 - 2 days later. Result Judgment Criteria: In the 96-well plate, if a round white precipitate appears in the well, it indicates that Mycobacterium tuberculosis can grow at this drug concentration, and as the culture time extends, the white precipitate will gradually increase; while the black area indicates that this drug concentration inhibits the growth of Mycobacterium tuberculosis and will not change with the extension of the culture time. After adding the resazurin indicator, the appearance of a pink color in the well indicates that Mycobacterium tuberculosis can survive and reproduce at this drug concentration, while the blue area indicates that this drug concentration inhibits the growth of Mycobacterium tuberculosis.

[0128] 3.5 Combined Bacteriostatic Activity of Cinnamon Extract Ⅰ with INH, RFP, and Lfx against Clinically Drug-Resistant Mycobacterium tuberculosis

[0129] 3.5.1 Randomly select clinically drug-resistant bacteria with drug sensitivity phenotypes of mono-resistance to INH, mono-resistance to RFP, mono-resistance to Lfx, MDR, and Pre-XDR.

[0130] 3.4.2 Drug Concentration Selection: For INH, RFP, and Lfx, the drug concentrations are selected according to the breakpoint concentrations recommended by WHO using the microplate method, which are 0.125 μg / mL, 0.5 μg / mL, and 1 μg / mL respectively. For Cinnamon Extract Ⅰ, three concentrations of 3.9 μg / mL, 7.8 μg / mL, and 15.6 μg / mL are selected.

[0131] 3.4.3 Bacterial Solution Preparation and Drug Dilution are the same as 3.3.1, and the final concentration of the bacterial solution in the 96-well plate is 5×105 CFU / mL.

[0132] 3.4.4 Sample Addition: Combine Cinnamon Extract Ⅰ with each of the three anti-tuberculosis drugs alone, in pairs, or in combinations of three or more. Then, pipette 100 μL of the drug mixture into the corresponding positions in the 96-well plate, and then pipette 100 μL of the diluted bacterial solution into the 96-well plate. Seal the 96-well plate along the edges with a sealing film, then place it in a sealed bag and transfer it to an incubator at 37°C for culture.

[0133] 3.4.5 Result Observation and Interpretation:

[0134] On the second day of cultivation, take a photo to observe the situation of the 96-well plate. If a white turbid area appears, it indicates contamination by common bacteria. Then, take a photo every other day to record the growth of Mycobacterium tuberculosis. When obvious colony differences appear in the 96-well plate (about 9 - 11 days), then add 20 μL of diluted resazurin indicator to each well, and observe the resazurin color development 1 - 2 days later. Result judgment criteria: In the 96-well plate, if a round white precipitate appears in the well, it indicates that Mycobacterium tuberculosis can grow at this drug concentration, and as the cultivation time extends, the white precipitate will gradually increase; while the black area indicates that this drug concentration inhibits the growth of Mycobacterium tuberculosis and will not change with the extension of the cultivation time. After adding the resazurin indicator, a pink color in the well indicates that Mycobacterium tuberculosis can survive and reproduce at this drug concentration, while the blue area indicates that this drug concentration inhibits the growth of Mycobacterium tuberculosis.

[0135] 3.5 Detection Results

[0136] 3.5.1 MIC Detection of Cinnamon Extract Ⅰ against Mycobacterium tuberculosis Standard Strain H37Rv

[0137] To understand the activity of Cinnamon Extract Ⅰ against Mycobacterium tuberculosis, the MIC detection of Cinnamon Extract Ⅰ against Mycobacterium tuberculosis was carried out, and it was found that Cinnamon Extract Ⅰ had antibacterial activity against Mycobacterium tuberculosis ( Figure 3 ), showing that Cinnamon Extract Ⅰ had good antibacterial activity against H37Rv, and its MIC value was 7.8 μg / mL.

[0138] To further understand whether Cinnamon Extract Ⅰ has a bactericidal effect on Mycobacterium tuberculosis, its bactericidal activity was evaluated. The results showed that the MBC of Cinnamon Extract Ⅰ against Mycobacterium tuberculosis standard strain H37Rv was 7.8 - 15.6 μg / mL, the MIC was 7.8 μg / mL, and MBC ≤ 4 times MIC, indicating that Cinnamon Extract Ⅰ had good antibacterial and bactericidal activities against Mycobacterium tuberculosis.

[0139] 3.5.2 MIC Detection of Cinnamon Extract Ⅰ against Clinically Isolated Sensitive and Resistant Strains of Mycobacterium tuberculosis

[0140] To understand the antibacterial activity and drug resistance of Cinnamon Extract Ⅰ against clinically isolated strains of Mycobacterium tuberculosis, the MIC screening of Cinnamon Extract Ⅰ against clinically isolated strains was carried out. Its MIC value was 7.8 μg / mL, showing that Cinnamon Extract Ⅰ had good antibacterial activity against both clinically isolated sensitive and resistant strains ( Figure 4 ), and currently no clinically resistant strains to Cinnamon Extract Ⅰ were found, indicating that Mycobacterium tuberculosis is not likely to develop resistance to Cinnamon Extract Ⅰ.

[0141] 3.5.3 Combined Antibacterial Activity of Cinnamon Extract Ⅰ and Antituberculosis Drugs INH, RFP, EMB, and Lfx against H37Rv

[0142] To understand the effects of cinnamon extract Ⅰ on commonly used anti-tuberculosis drugs and their mutual relationships, the checkerboard test was used to observe the combined antibacterial activities of cinnamon extract Ⅰ combined with INH, RFP, EMB, Sm, and Lfx against H37Rv respectively. The results showed that there was no synergistic effect between cinnamon extract Ⅰ and the above-mentioned commonly used anti-tuberculosis drugs( Figure 5 , Table 5).

[0143] Table 5. Statistical table of MIC values and FIC indices of the combination of cinnamon extract Ⅰ with INH, RFP, EMB, Sm, and Lfx against H37Rv

[0144]

[0145] 3.5.4 Combined antibacterial activities of cinnamon extract Ⅰ combined with anti-tuberculosis drugs INH, RFP, and Lfx against clinically drug-resistant bacteria

[0146] Currently, the clinical treatment of tuberculosis is the combined use of multiple drugs, and the interaction between drugs is crucial for the treatment effect. To simulate clinical drug use, cinnamon extract Ⅰ was combined with anti-tuberculosis drugs, and the antibacterial activities against clinically isolated bacteria with different drug-resistant phenotypes (mono-resistant to INH, mono-resistant to RFP, mono-resistant to Lfx, MDR, and Pre-XDR) were detected. The results are shown in( Figure 6 ). The MIC of using anti-tuberculosis drugs alone confirmed that the clinical bacteria were indeed drug-resistant bacteria. However, the antibacterial activities of cinnamon extract Ⅰ alone and in combination with INH, RFP, and Lfx against clinically drug-resistant bacteria were consistent. There was no cross-resistance between cinnamon extract Ⅰ and these commonly used anti-tuberculosis drugs, further suggesting that cinnamon extract Ⅰ may have a new and unique antibacterial mechanism.

[0147] 3.5.5 Acute oral toxicity test of cinnamon extract Ⅰ

[0148] 1. Test substance: Cinnamon extract Ⅰ, colorless and transparent liquid, plastic bottle.

[0149] 2. Strain: KM mice, source: Liaoning Changsheng Biotechnology Co., Ltd., 10 male mice and 10 female mice, SPF grade, 18 - 20 g;

[0150] 3. Feed source: Liaoning Changsheng Biotechnology Co., Ltd., SPF-grade maintenance feed for mice and rats, batch number 19080311.

[0151] 4. Test conditions: Breeding room number A010, temperature: 20.7 - 24.0 °C, relative humidity: 42 - 67%. The experimental animal use license number is SYXK(Liao)2015 - 0006.

[0152] Method

[0153] Grouping: The animals were divided into 2 groups according to gender: 10 animals in each group, and they were fed separately in cages. Before the experiment, the animals were generally fasted overnight without restricting water intake.

[0154] Dose setting: One dose group was set according to the maximum limit test at a dose of 5000 mg / kg. The animals were exposed to the test substance by gastric gavage according to the standard, and the dosing volume was 0.2 mL / 10 g. After exposure, the toxic manifestations, the number of deaths and the time of death of the animals were observed. Autopsies were performed on the dead animals and the animals sacrificed at the end of the observation period. Macroscopic observations were made, and if any abnormal tissues or organs were found, further histopathological examinations were required. The observation period was 14 days.

[0155] Results: Autopsies were performed on the animals sacrificed at the end of the observation period, and no abnormalities were found upon macroscopic observation.

[0156] Conclusion: The results of the acute oral toxicity test of the test substance on mice showed it was actually non-toxic.

[0157] 3.5.6 One complete skin irritation test

[0158] I. Materials and animals

[0159] 1. Test substance: Cinnamon extract I, which is a colorless and transparent liquid in a plastic bottle.

[0160] During the experiment, 0.5 mL of the test substance was taken from each rabbit as the test article.

[0161] 2. Animals: 3 Japanese white rabbits provided by Xinglong Experimental Animal Breeding Factory in Haidian District, Beijing, ordinary grade, female, weighing 1.7 - 2.1 kg. The production license number of the experimental animals is SCXK(Jing)2016 - 0003.

[0162] 3. Animal feed: Provided by Liaoning Changsheng Biotechnology Co., Ltd., and the production license number is SCXK(Liao)2015 - 0003.

[0163] 4. Test conditions: Ordinary animal laboratory, temperature 21 ± 5°C, humidity 40% ± 70%. II. Methods

[0164] 1. 24 hours before the experiment, the hair on both sides of the spine on the back of the rabbits was removed with a hair clipper without damaging the skin. The hair removal area was about 3 cm × 3 cm on the left and right sides respectively.

[0165] 2. On the next day, directly drop the test substance [0.5 ml of the stock solution] onto the intact depilated skin on one side with an area of 2.5 cm x 2.5 cm, and apply the solvent on the other side as a control. At 4 h after application, wash with water or a suitable non-irritating solvent, and observe the local skin reaction at 1 h, 24 h, and 48 h after removing the residue, and score according to Table 2-11 in Section 2.3.3 of the "Disinfection Technical Specifications" (2002 Edition). For the convenience of applying the test substance and observing the results, hair should be clipped if necessary. The treatment method of the control area is the same as that of the test area.

[0166] 3. At each observation time point, score the formation of skin erythema and edema in animals according to Table 2-11 in the "Disinfection Technical Specifications", and add up the scores of 3 animals at each time point and divide by the number of animals to obtain the mean value of skin irritation reaction scores (irritation index) at different time points. Take the highest skin irritation index among them, and evaluate the level of skin irritation intensity of the test substance to animals according to Table 2-12 in the "Disinfection Technical Specifications". During the test, the highest skin irritation index of 3 rabbits was 0.3, and the result of the single complete skin irritation test of the test substance on rabbits was non-irritating.

[0167] As can be seen from the above examples, the cinnamomum extract Ⅰ provided by the present invention has strong antibacterial and bactericidal activities against mycobacterium tuberculosis. It not only has antibacterial activity against mycobacterium tuberculosis, but also has a bactericidal effect; it can not only resist mycobacterium tuberculosis sensitive bacteria, but more importantly, it can also resist mycobacterium tuberculosis drug-resistant bacteria. Moreover, it has no synergistic effect with the commonly used anti-tuberculosis drugs rifampicin, isoniazid, ethambutol, streptomycin-resistant and levofloxacin, and has no cross-resistance with rifampicin, isoniazid and levofloxacin, indicating that the cinnamomum extract Ⅰ has a new antibacterial mechanism against mycobacterium tuberculosis. The application of cinnamomum extract Ⅰ can provide new ideas and methods for the prevention and treatment of tuberculosis, and the application scope includes the treatment of skin tuberculosis, lymph node tuberculosis, osteoarticular tuberculosis and pulmonary tuberculosis (inhalation), etc.

[0168] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. Use of cinnamon extract I against drug-resistant Mycobacterium tuberculosis in the preparation of a medicament for treating tuberculosis, characterized in that the cinnamon extract I uses cinnamon as the raw material, and a whole-component extract is obtained through moving bed extraction; then the obtained whole-component extract is separated by countercurrent chromatography, the effluents at different time periods are collected, distilled to obtain a refined extract, and the refined extracts with the effect of against drug-resistant Mycobacterium tuberculosis are combined to obtain cinnamon extract I.

2. The application according to claim 1, characterized in that, The tuberculosis includes that caused by one or more of Mycobacterium tuberculosis sensitive bacteria, rifampicin-monoresistant Mycobacterium tuberculosis, isoniazid-monoresistant Mycobacterium tuberculosis, ethambutol-monoresistant Mycobacterium tuberculosis, streptomycin-monoresistant Mycobacterium tuberculosis, levofloxacin-monoresistant Mycobacterium tuberculosis, multi-drug resistant Mycobacterium tuberculosis and quasi-extensively drug-resistant Mycobacterium tuberculosis.

3. The application according to claim 1, wherein The tuberculosis includes cutaneous tuberculosis, scrofula, osteoarticular tuberculosis or pulmonary tuberculosis.

4. The application according to claim 1, characterized in that, The using concentration of the cinnamon extract I in the medicament is ≥7.8 μg / mL.

5. The preparation method of cinnamon extract I in the application according to any one of claims 1 to 4, characterized in that, It includes the following steps: 1) Cinnamon is subjected to moving bed extraction to obtain a whole-component extract; 2) The whole-component extract obtained in step 1) is separated by countercurrent chromatography, and the effluents at different time periods are collected; 3) The collected effluents are distilled to obtain a refined extract, and the refined extracts with the effect of against drug-resistant Mycobacterium tuberculosis are combined to obtain cinnamon extract I; For the moving bed extraction in step 1), the extraction solvent used is an ethanol aqueous solution with a volume concentration of 50% - 60%; the pumping speed of the extraction solvent is 40 - 60 ml / min; the mass-volume ratio of the cinnamon to the extraction solvent is 1 g : (8 - 12) mL.

6. The preparation method according to claim 5, characterized in that, For the countercurrent chromatography separation in step 2), an ethanol aqueous solution with a volume concentration of 25% - 35% is used as the stationary phase, and n-hexane is used as the mobile phase; the ethanol aqueous solution is mixed with the whole-component extract obtained in step 1) to obtain a sample solution, and the volume concentration of ethanol in the sample solution is 25% - 35%; after sample loading, the mobile phase is pumped in for countercurrent chromatography separation.

7. The preparation method according to claim 6, characterized in that, The pumping speed of the mobile phase is 5 - 10 ml / min.

8. The preparation method according to claim 5, characterized in that, The cinnamon is cassia twig, cassia leaf or cassia bark; the cinnamon is crushed and passed through a 20 - 40 mesh sieve.

9. The cinnamon extract I against drug-resistant Mycobacterium tuberculosis obtained by the preparation method according to any one of claims 5 to 8, characterized in that It includes coumarin, o-hydroxycinnamic acid, p-hydroxycinnamic acid, anethole, trans-cinnamaldehyde, 2'-methoxycinnamaldehyde and methyl cinnamate.

10. A drug for treating tuberculosis, characterized in that, It includes the cinnamon extract I against drug-resistant Mycobacterium tuberculosis as described in claim 9.

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