Benzo [g] chromone compound with activity of resisting methicillin-resistant staphylococcus aureus (MRSA) as well as preparation method and application of benzo [g] chromone compound

Abstract

The invention belongs to the technical field of food chemistry, and particularly relates to a benzo [g] chromone compound with methicillin-resistant staphylococcus aureus (MRSA) resisting activity as well as a preparation method and application of the benzo [g] chromone compound. The benzo [g] chromone compound with the activity of resisting methicillin-resistant staphylococcus aureus (MRSA) is prepared by taking a whole plant of a medicinal plant chamaecrista rotundifola as a raw material and carrying out pretreatment, extract extraction, silica gel column chromatography and high performance liquid chromatography separation, and is named as 2-(hydroxymethyl)-6-methoxy-9-methylbenzo [2, 3-g]-4H-chromone, and the structural formula of the benzo [g] chromone compound is shown in the specification. The invention relates to a high-molecular polymer, which is characterized in that the English name of the high-molecular polymer is 2-(hydroxy methyl)-6-methylox-9-methylbenz [2, 3-g]-4H-chromone, and the high-molecular polymer has the following structure: # imgabs0 #. The benzo [g] chromone compound with the antibacterial activity, disclosed by the invention, has relatively good activity of resisting drug-resistant bacteria. The compound can be used as a leading compound for research and development of drugs for resisting drug-resistant bacteria.

Description

Technical Field

[0001] The present invention belongs to the technical field of phytochemistry, and particularly relates to a benzogchromone compound with anti-methicillin-resistant Staphylococcus aureus (MRSA) activity, a preparation method thereof, and an application thereof. Background Art

[0002] Chamaecrista rotundifolia (Pers.) Greene is a semi-erect perennial herb of the legume family and the genus Cassia. Chamaecrista rotundifolia is native to Paraguay, Mexico, Brazil, Argentina and other countries in the United States. Chamaecrista rotundifolia has the advantages of acid tolerance, drought tolerance, barren tolerance, high yield, strong nitrogen fixation ability, high nutrient content, and no pests and diseases. Chamaecrista rotundifolia (Pers.) greene) is a semi-erect perennial herb of the legume family and the genus Cassia. Chamaecrista rotundifolia is native to Paraguay, Mexico, Brazil, Argentina and other countries in the United States. Chamaecrista rotundifolia has the advantages of acid tolerance, drought tolerance, barren tolerance, high yield, strong nitrogen fixation ability, high nutrient content, and no pests and diseases.

[0003] Years of cultivation have shown that: As a perennial herb, Chamaecrista rotundifolia is suitable for planting on mountains and slopes, which helps to prevent soil erosion and water loss, and also plays a significant role in maintaining ecological balance. The crude protein content of Chamaecrista rotundifolia is about 20%, and the contents of crude fat, nitrogen, phosphorus, calcium, etc. are also relatively high, while the crude fiber content is relatively low. The yield per mu reaches 1500 kg, which is equivalent to the crude protein content of 3000 kg of corn. It is a good forage crop with high economic value. In addition, Chamaecrista rotundifolia also has a wide range of medicinal values, such as clearing heat and detoxifying, promoting diuresis and relieving stranguria, reducing blood lipid, antibacterial, anti-radiation and other effects. At present, the research on the secondary metabolites of Chamaecrista rotundifolia is relatively less. Summary of the Invention

[0004] The first object of the present invention is to provide a benzogchromone compound with anti-methicillin-resistant Staphylococcus aureus (MRSA) activity; the second object is to provide a preparation method of the benzogchromone compound with anti-methicillin-resistant Staphylococcus aureus (MRSA) activity; the third object is to provide an application of the benzogchromone compound with anti-methicillin-resistant Staphylococcus aureus (MRSA) activity.

[0005] The first object of the present invention is achieved as follows. The benzogchromone compound with anti-methicillin-resistant Staphylococcus aureus (MRSA) activity is prepared from the whole plant of the medicinal plant Chamaecrista rotundifolia ( Chamaecrista rotundifolia ), through pretreatment, extract preparation, silica gel column chromatography and high performance liquid chromatography separation, and is named: 2-(hydroxymethyl)-6-methoxy-9-methylbenzo[2,3-g]-4H-chromone, with the English name: 2-(hydroxymethyl)-6-methoxy-9-methylbenzo[2,3-g]-4H-chromone, having the following structure: .

[0006] The second object of the present invention is achieved as follows. The whole plant of the medicinal plant Chamaecrista rotundifolia ( Chamaecrista rotundifolia ) is used as the raw material and prepared through pretreatment, extract preparation, silica gel column chromatography and high performance liquid chromatography separation. Specifically, it includes the following steps: A. Pretreatment: The whole plant of the raw material medicinal plant Chamaecrista rotundifolia is crushed or cut into sections to obtain material a; B. Extract preparation: An organic extraction solvent with a mass 2 - 4 times that of material a is added to material a, and it is soaked and extracted at room temperature for 3 - 5 times, with each extraction time being 24 - 72 h. The extraction solutions are combined, filtered and concentrated to obtain extract b; C. Silica gel column chromatography: 160 - 300 mesh silica gel with a weight 2 - 4 times that of extract b is added to extract b for dry column packing, and gradient elution is carried out with a chloroform - acetone solution with a volume ratio of 1:0 - 1:2. After monitoring by TLC, the same parts are combined; D. High performance liquid chromatography separation: The eluate obtained by eluting with a 7:3 ratio of chloroform - acetone solution is separated and purified by high pressure liquid chromatography to obtain the target benz[g]chromone compound with anti - methicillin - resistant Staphylococcus aureus (MRSA) activity.

[0007] The specific operations are as follows: 1. Extract preparation: The whole plant sample of Chamaecrista rotundifolia is crushed, and high - concentration methanol (w%: 80% - 100%), or high - concentration ethanol (w%: 80% - 100%), or high - concentration acetone (w%: 60% - 90%) is used as the extraction solvent. The ratio of extraction solvent to the whole plant of Chamaecrista rotundifolia (weight ratio) is 2 - 4:1. It is soaked for 24 h - 72 h, extracted 3 - 5 times, and the extraction solutions are combined, filtered and concentrated into an extract; 2. Silica gel column chromatography: The extract is dissolved with pure methanol or pure acetone with a weight ratio of 1.5 - 3 times, and then mixed with 80 - 100 mesh silica gel with a weight ratio of 0.8 - 1.2 times. Dry column packing is carried out with 160 - 300 mesh silica gel with a weight ratio of 2 - 4 times for silica gel column chromatography; Gradient elution is carried out with a chloroform - acetone solution with a volume ratio of (1:0, 20:1, 9:1, 8:2, 7:3, 6:4, 1:1 and 1:2). The same parts are combined, and each part of the eluate is collected and concentrated; 3. High performance liquid chromatography separation and purification: The 7:3 part of the column chromatography eluate is further separated and purified by high performance liquid chromatography to obtain the benz[g]chromone compound.

[0008] High performance liquid chromatography separation and purification is carried out using a C of 21.2 mm × 250 mm, 5 μm 18Chromatographic column, flow rate 12 mL / min, mobile phase 52% methanol, detection wavelength of UV detector 368 nm, 500 μL injection each time, collect chromatographic peaks for 30 min, evaporate to dryness after multiple accumulations, and then obtain the compound of the present invention after gel purification.

[0009] The structure of the benzogchromone compounds prepared by the above method was determined by the following method.

[0010] The compound of the present invention is a yellow gum. The 1 H and 13 C NMR spectra (as shown in Table 1, Figure 1 and Figure 2 ) show that it contains 16 carbons and 14 hydrogens, including a tetrasubstituted naphthalene ring (C-5 to C-13, C-4a, C-9a, H-5, H-7, H-8 and H-10), and a α,β -unsaturated carbonyl (-O-C=CH-CO-, C-2 to C-4, H-3), a methyl group (C-16 and H3-16), a hydroxymethyl group (C-15 and H2-15), and a methoxy group ( δ C 56.1 and δ H 3.81 s). Further analyzing its NMR data, α,β the -unsaturated carbonyl should be connected to the naphthalene ring to form a pyran ring, forming a benzogchromone skeleton to support the presence of 10 degrees of unsaturation in the compound, and the presence of two oxidized aromatic carbons (C-2 and C-3) in the compound. This speculation can be further confirmed by the HMBC correlations of H-3 and C-2 / C-4 / C-12, H-5 to C-4 / C-6, H-7 to C-13, H-8 and C-14, and H-10 and C-9.

[0011] Table 1. 1 H NMR and 13 C NMR data (CDCl3) After the parent body of the compound is determined, the remaining substituents (methoxy group, methyl group and hydroxymethyl group) can be regarded as substituents on benzog g chromone. The methoxy group is substituted at the C-6 position, which can be determined by the methoxy hydrogen ( δ H3.81 s) and C-6 were confirmed by HMBC correlation. Through the HMBC correlation of H3-16 and C-8 / C-9 / C-14, and H-8 and C-16, it can be determined that the methyl group is substituted at the C-9 position; through the HMBC correlation of H2-15 and C-2 / C-3, and H-3 and C-15, it can be determined that the hydroxymethyl group is substituted at the C-2 position. So far, the structure of the compound of the present invention has been determined and named 2-(hydroxymethyl)-6-methoxy-9-methylbenzo[2,3-g]-4H-chromone, English name: 2-(hydroxy methyl)-6-methoxy-9-methylbenzo[2,3-g]-4H-chromone.

[0012] Infrared, UV and mass spectrometry data of the compound: UV spectrum (methanol), λ max (log ε ) 215 (4.47), 275 (4.02), 368 (3.96) nm; Infrared spectrum (potassium bromide tablet): ν max 3396, 2950, ​​1662, 1618, 1564, 1457, 1274, 1175, 1043, 938 cm -1 ; 1 H and 13 C NMR data (500 and 125 MHz, (CDCl3), see Table-1; positive ion mode ESIMS m / z 293 [M+Na] + ; Positive ion mode HRESIMS m / z 293.0789 [M+Na] + (Calculated value 293.0784, C 16 H 14 NaO 4） .

[0013] 4. In vitro antibacterial activity detection The test strain was a methicillin-resistant Staphylococcus aureus (MRSA) strain isolated from clinical specimens at Kunming Medical University. The oxacillin resistance phenotype was positive (≥4 mg / L), and the mecA gene test was positive. The microbroth dilution method recommended by the Clinical and Laboratory Standards Institute (CLSI) was used for drug sensitivity testing. The operating specifications were based on the CLSIM07-A9 standard, and the judgment criteria were based on the CLSIM100-S24 standard. The specific methods are as follows: (1) Preparation of antibacterial agent stock solution: The concentration of the prepared antibacterial agent stock solution is 2560 μg / mL. For antibacterial agents with low solubility, the concentration can be slightly lower than the above concentration. The amount of antibacterial agent solution or powder required can be calculated using a formula. The prepared antibacterial agent stock solution should be stored at a temperature below -20°C, and the storage period should not exceed 6 months.

[0014] (2) Preparation of the test bacteria: Use an inoculation loop to pick a single colony from an MH(A) culture dish incubated overnight and transfer it to an MH(B) medium. Calibrate it to a 0.5 McFarland turbidity standard, which contains approximately 1×10^8 CFU / ml of bacteria. Then dilute it 100 times to obtain a bacterial suspension containing approximately 1×10 6 CFU / mL for standby.

[0015] (3) Dilute the antibacterial agent stock solution (2560 μg / mL) 10 times respectively to obtain an antibacterial agent solution with a concentration of 256 μg / mL. Take a sterile 96-well plate. Add 200 μL of the antibacterial agent to the first well, and add 100 μL of MH broth medium to the second to tenth wells respectively. Pipette 100 μL from the first well and add it to the second well, mix well, and then pipette 100 μL to the third well, and so on. Discard 100 μL from the tenth well. At this time, the drug concentrations in each well are as follows: 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5 μg / mL. Add 100 μL of the bacterial suspension (positive control) to the eleventh well, and add 100 μL of MH(B) medium (negative control) to the twelfth well.

[0016] (4) Add 100 μL of the previously prepared bacterial suspension to each of wells 1 to 10, so that the final bacterial concentration in each tube is approximately 5×10 5 CFU / mL. The drug concentrations in wells 1 to 10 are 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 μg / mL respectively. Place the inoculated 96-well plate in an incubator at 37°C for incubation, and observe the growth of the bacterial suspension after 24 hours. At the same time, use the standard strain for quality control.

[0017] (5) Result judgment and interpretation: Before reading and reporting the MIC of the tested strain, it is necessary to check whether the bacterial growth in the growth control tube is good, and at the same time, check the subculture of the inoculum to determine whether it is contaminated, and whether the MIC value of the quality control strain is within the quality control range. Visually observe that the tube with the lowest drug concentration has no bacterial growth, which is the MIC of the tested bacteria.

[0018] (6) The test results show that compound (1) has prominent antibacterial activity and has obvious antibacterial advantages compared with the antibacterial agents currently used clinically to treat infections caused by Gram-positive bacteria. The MIC 90It was 52.84 ± 4.2 μg / mL, with significant antibacterial activity, approaching the MIC of levofloxacin 90 value (> 50.84 ± 5.5 μg / mL).

[0019] Compared with the prior art, the present invention has the following outstanding advantages: (1) The compound of the present invention uses the whole plant of Chamaecrista rotundifolia as the raw material. In order to control soil and water loss, Chamaecrista rotundifolia has been widely planted artificially in barren wastelands and slopes. It has a large biological yield, a very wide raw material source, low cost, and easy separation and preparation. (2) The extraction method of the compound of the present invention is simple and easy to separate. (3) The preparation method combining conventional column chromatography and high performance liquid chromatography is adopted. The compound preparation operation process is simple, the purity of the compound of the present invention obtained is high, and subsequent industrial production is easy to achieve. (4) The compound of the present invention has low toxicity to animals, is safe to use, exhibits good anti-drug-resistant bacteria activity, and can be used as a leading compound for the development of anti-drug-resistant bacterial drugs. Description of the Drawings

[0020] Figure 1 13C NMR of the antibacterial active benz[g]chromone compound of the present invention; 13 C NMR); Figure 2 1H NMR of the antibacterial active benz[g]chromone compound of the present invention; 1 H NMR); Figure 3 Key HMBC correlation diagram of the antibacterial active benz[g]chromone compound of the present invention. Detailed Embodiments

[0021] The following further illustrates the present invention in conjunction with examples and drawings, but does not limit the present invention in any way. Any transformation or replacement based on the teachings of the present invention belongs to the protection scope of the present invention.

[0022] The benz[g]chromone compound with anti-methicillin-resistant Staphylococcus aureus (MRSA) activity described in the present invention uses the whole plant of the medicinal plant Chamaecrista rotundifolia Chamaecrista rotundifolia as the raw material, and is prepared by pretreatment, extract extraction, silica gel column chromatography and high performance liquid chromatography separation, named: 2-(hydroxymethyl)-6-methoxy-9-methylbenzo[2,3-g]-4H-chromone, and its English name is: 2-(hydroxy methyl)-6-methoxy-9-methylbenzo[2,3-g]-4H-chromone, with the following structure: .

[0023] The preparation method of the benz[g]chromone compound with anti-methicillin-resistant Staphylococcus aureus (MRSA) activity described in the present invention uses the whole plant of the medicinal plant Chamaecrista rotundifolia ( Chamaecrista rotundifolia ) as the raw material, and is prepared by pretreatment, extract extraction, silica gel column chromatography and high performance liquid chromatography separation. The specific steps are as follows: A. Pretreatment: The whole plant of the raw material medicinal plant Chamaecrista rotundifolia is crushed or cut into sections to obtain material a; B. Extract extraction: Add an organic extraction solvent 2-4 times the mass of material a to material a, soak and extract at room temperature for 3-5 times, with each extraction time being 24-72 h, combine the extraction liquids, filter and concentrate to obtain extract b; C. Silica gel column chromatography: Add silica gel with a mesh size of 160-300, 2-4 times the weight of extract b, to the dry column of extract b, and perform gradient elution with a chloroform-acetone solution with a volume ratio of 1:0-1:2. Monitor by TLC and combine the same parts; D. High performance liquid chromatography separation: The eluate obtained by eluting with a chloroform-acetone solution with a ratio of 7:3 is separated and purified by high pressure liquid chromatography to obtain the target benz[g]chromone compound with anti-methicillin-resistant Staphylococcus aureus (MRSA) activity.

[0024] The organic extraction solvent described in step B is an aqueous methanol solution with a mass concentration of 80%-100%, an aqueous ethanol solution with a mass concentration of 80%-100%, or an aqueous acetone solution with a mass concentration of 60%-90%.

[0025] Before loading the column in step C, it also includes a sample mixing step of dissolving with an organic solvent 1.5-3 times the weight of extract b and adding silica gel with a mesh size of 80-100, 0.8-1.2 times the weight of extract b.

[0026] The organic solvent is pure methanol, pure ethanol or pure acetone.

[0027] The volume ratio of the chloroform-acetone solution described in step C is 1:0, 20:1, 9:1, 8:2, 7:3, 6:4, 1:1 and 1:2.

[0028] In step D, the high pressure liquid chromatography separation and purification is carried out using a C column of 21.2 mm × 250 mm, 5 μm 18 chromatographic column, the flow rate is 12 mL / min, the mobile phase is 52% methanol, the detection wavelength of the ultraviolet detector is 368 nm, 500 μL is injected each time, the chromatographic peaks for 30 min are collected, and after multiple accumulations, it is evaporated to dryness to obtain the target antibacterial active benz[g]chromone compound.

[0029] The application described in the present invention is the application of the benz[g]chromone compound having anti-methicillin-resistant Staphylococcus aureus (MRSA) activity in the preparation of a drug against methicillin-resistant Staphylococcus aureus (MRSA).

[0030] The following specific examples using Cassia rotundifolia raw materials from different producing areas in Yunnan further illustrate the present invention: Example 1

[0031] Preparation of benz[g]chromone compound C 16 H 14 O4, including extract extraction, silica gel column chromatography, and high-performance liquid chromatography separation. The specific steps are as follows: 1. Extract extraction: Crush the whole plant of Cassia rotundifolia, and use high-concentration methanol (w%: 95%), high-concentration ethanol (w%: 95%), or high-concentration acetone (w%: 70%) as the extraction solvent. The ratio of the extraction solvent to the whole plant of Cassia rotundifolia (by weight) is 3:5. Soak for 54 h, extract 4 times, combine the extraction solutions, filter and concentrate to obtain an extract.

[0032] 2. Silica gel column chromatography: Dissolve the extract with 2.5 times the weight of pure methanol, pure ethanol, or pure acetone, then mix the sample with 1.2 times the weight of 80-100 mesh silica gel, and dry-load a column with 3 times the weight of 250 mesh silica gel for silica gel column chromatography; use a chloroform-acetone solution with a volume ratio of (1:0, 20:1, 9:1, 8:2, 7:3, 6:4, 1:1, and 1:2) for gradient elution, combine the same parts, collect the eluates of each part and concentrate.

[0033] 3. High-performance liquid chromatography separation: The 7:3 part of the column chromatography eluate is further separated and purified by high-performance liquid chromatography to obtain the benz[g]chromone compound. The high-performance liquid chromatography separation and purification is carried out using a C 18 chromatographic column of 21.2 mm × 250 mm, 5 μm, with a flow rate of 12 mL / min, a mobile phase of 52% methanol, a detection wavelength of 368 nm for the ultraviolet detector, inject 500 μL each time, collect the chromatographic peaks for 30 min, and evaporate to dryness after multiple accumulations.

[0034] For the substance separated and purified by high-performance liquid chromatography, a preferred post-treatment scheme is to dissolve the obtained compound with pure methanol again, and then use gel column chromatography with pure methanol as the mobile phase for further separation and purification.

[0035] The whole plant of Cassia rotundifolia used in the present invention is not restricted by regions and varieties, and the present invention can be realized. The following uses the whole plant of Cassia rotundifolia from different producing areas in Yunnan to further illustrate the present invention: Example 2

[0036] The whole plant of Chamaecrista rotundifolia is from Yuanjiang, Yunnan. 2.0 kg of the whole plant of Chamaecrista rotundifolia was sampled and crushed, extracted 5 times with 95% methanol for 24 h each time. The extracts were combined, filtered, and concentrated under reduced pressure to obtain an extract, yielding 82.5 g of the extract. The extract was dissolved in 2.0 times the weight of pure methanol and mixed with 120 g of 100-mesh coarse silica gel. 0.6 kg of 160-mesh silica gel was packed into a column for silica gel column chromatography, and gradient elution was carried out with chloroform-acetone with volume ratios of 1:0, 20:1, 9:1, 8:2, 7:3, 6:4, 1:1, and 1:2. The same parts were monitored by TLC and combined to obtain 8 parts. Among them, the part eluted with chloroform-acetone with a volume ratio of 7:3 was separated by Agilent 1100 semi-preparative high performance liquid chromatography. Using 52% methanol as the mobile phase, Zorbax SB-C18 (21.2 × 250 mm, 5 μm ） The preparative column was used as the stationary phase, the flow rate was 12 mL / min, the detection wavelength of the ultraviolet detector was 368 nm, 200 μL of sample was injected each time, the chromatographic peaks for 30 min were collected, and after multiple accumulations, it was evaporated to dryness; the obtained product was dissolved again with pure methanol, and then using pure methanol as the mobile phase, it was separated by Sephadex LH-20 gel column chromatography to obtain the new compound.

[0037] Example 3

[0038] The whole plant sample of Chamaecrista rotundifolia is from Hekou, Yunnan. 3.5 kg of the whole plant of Chamaecrista rotundifolia was sampled and chopped, extracted 4 times with 95% ethanol for 48 h each time. The extracts were combined, filtered, and concentrated under reduced pressure to obtain an extract, yielding 250 g of the extract. The extract was dissolved in 2.0 times the weight of pure methanol and mixed with 236 g of 80-mesh coarse silica gel. 1.2 kg of 200-mesh silica gel was packed into a column for silica gel column chromatography, and gradient elution was carried out with chloroform-acetone with volume ratios of 1:0, 20:1, 9:1, 8:2, 7:3, 6:4, 1:1, and 1:2. The same parts were monitored by TLC and combined to obtain 8 parts. Among them, the part eluted with chloroform-acetone with a volume ratio of 7:3 was separated by Agilent 1100 semi-preparative high performance liquid chromatography. Using 52% methanol as the mobile phase, Zorbax SB-C18 (21.2 × 250 mm, 5 μm) preparative column was used as the stationary phase, the flow rate was 12 mL / min, the detection wavelength of the ultraviolet detector was 368 nm, 200 μL of sample was injected each time, the chromatographic peaks for 30 min were collected, and after multiple accumulations, it was evaporated to dryness; the obtained product was dissolved again with pure methanol, and then using pure methanol as the mobile phase, it was separated by Sephadex LH-20 gel column chromatography to obtain the new compound.

[0039] Example 4

[0040] The whole plant sample of Chamaecrista rotundifolia was sourced from Mengding, Yunnan. 5 kg of the whole plant of Chamaecrista rotundifolia was sampled and crushed, and ultrasonic extraction was carried out 3 times with 75% acetone for 72 h each time. The extracts were combined, filtered, and concentrated under reduced pressure to obtain an extract, yielding 339 g of the extract. The extract was dissolved in pure methanol at a weight ratio of 1.6 times, and then mixed with 400 g of 90-mesh coarse silica gel. 2.4 kg of 180-mesh silica gel was packed into a column for silica gel column chromatography, and gradient elution was performed with chloroform-acetone with volume ratios of 1:0, 20:1, 9:1, 8:2, 7:3, 6:4, 1:1, 1:2. The same parts were monitored by TLC and combined to obtain 8 parts. Among them, the part eluted with chloroform-acetone with a volume ratio of 7:3 was separated by Agilent 1100 semi-preparative high performance liquid chromatography. Using 52% methanol as the mobile phase, a Zorbax SB-C18 (21.2 × 250mm, 5 μm) preparative column as the stationary phase, the flow rate was 12 ml / min, the detection wavelength of the ultraviolet detector was 368 nm, 200 μL was injected each time, the chromatographic peaks for 30 min were collected, and after multiple accumulations, it was evaporated to dryness; the obtained product was dissolved in pure methanol again, and then separated by Sephadex LH-20 gel column chromatography with pure methanol as the mobile phase to obtain the new compound.

[0041] Example 5

[0042] Identification of the compound structure Take the compounds prepared in Examples 1-4, and the structures of the benz[g]chromone compounds prepared by the above method were determined by the following method. The compound of the present invention is a light yellow gum, and the compound of the present invention is a yellow gum. The 1 H and 13 C NMR spectra (as shown in Table 1, Figure 1 and Figure 2 ) showed that it contains 16 carbons and 14 hydrogens, including a tetrasubstituted naphthalene ring (C-5~C-13, C-4a, C-9a, H-5, H-7, H-8 and H-10), and a α,β α,β-unsaturated carbonyl (-O-C=CH-CO-, C-2~C-4, H-3), a methyl group (C-16 and H3-16), a hydroxymethyl group (C-15 and H2-15), and a methoxy group ( δ C 56.1 and δ H 3.81 s). Further analysis of its NMR data, α,β- The unsaturated carbonyl group should be connected to the naphthalene ring to form a pyran ring, resulting in a benzo[g]chromone skeleton, which supports the presence of 10 degrees of unsaturation in the compound and two oxidized aromatic carbons (C-2 and C-3) in the compound. This speculation can be further confirmed by the HMBC correlations of H-3 with C-2 / C-4 / C-12, H-5 to C-4 / C-6, H-7 to C-13, H-8 with C-14, and H-10 with C-9.

[0043] After determining the parent structure of the compound, the remaining substituents (methoxy, methyl, and hydroxymethyl) can be regarded as substituents on benzo[g]chromone. The methoxy group is substituted at the C-6 position, which can be confirmed by the HMBC correlation between the methoxy hydrogen ( δ H 3.81 s) and C-6. The methyl group is substituted at the C-9 position, which can be determined by the HMBC correlations of H3-16 with C-8 / C-9 / C-14 and H-8 with C-16; the hydroxymethyl group is substituted at the C-2 position, which can be determined by the HMBC correlations of H2-15 with C-2 / C-3 and H-3 with C-15. Thus, the structure of the compound of the present invention is determined and named 2-(hydroxymethyl)-6-methoxy-9-methylbenzo[2,3-g]-4H-chromone, and its English name is: 2-(hydroxy methyl)-6-methoxy-9-methylbenzo[2,3-g]-4H-chromone.

[0044] Example 6

[0045] Take the compound prepared in Example 3, which is a light yellow gum. The determination method is the same as that in Example 5, and it is confirmed that the compound prepared in Example 3 is the above-mentioned benzo[g]chromone compound - the name of this compound is: 2-(hydroxymethyl)-6-methoxy-9-methylbenzo[2,3-g]-4H-chromone.

[0046] Example 7

[0047] Take the compound prepared in Example 4, which is a yellow gum. The determination method is the same as that in Example 5, and it is confirmed that the compound prepared in Example 4 is the above-mentioned compound, and the name of this compound is: 2-(hydroxymethyl)-6-methoxy-9-methylbenzo[2,3-g]-4H-chromone.

[0048] Example 8

[0049] Anti - drug - resistant bacteria experiments were conducted on the compounds of the present invention. The test strains were methicillin - resistant Staphylococcus aureus (MRSA) strains isolated from clinical specimens by Kunming Medical University. The oxacillin - resistant phenotype was positive (≥4 mg / L), and the mecA gene test was positive. The micro - broth dilution method recommended by the Clinical and Laboratory Standards Institute (CLSI) of the United States was used for the drug sensitivity test. The operation specifications were referred to the CLSI M07 - A9 standard, and the judgment criteria were referred to the CLSI M100 - S24 standard. The specific method is as follows: (1) Preparation of antibacterial drug stock solution: The concentration of the antibacterial drug stock solution prepared was 2560 μg / mL. For antibacterial drugs with low solubility, the concentration could be slightly lower than the above - mentioned concentration. The required amount of antibacterial drug solution or powder dose could be calculated by a formula. The prepared antibacterial drug stock solution should be stored in an environment below - 20°C, and the storage period should not exceed 6 months.

[0050] (2) Preparation of the test bacteria: Use an inoculation loop to pick a single colony on the MH(A) culture dish after overnight culture into the MH(B) medium, calibrate it to a 0.5 McFarland turbidity standard, which contains approximately 1×108 CFU / ml of bacteria, and then dilute it 100 times, that is, a bacterial solution containing approximately 1×10 6 CFU / mL was obtained and reserved for use.

[0051] (3) Dilute the antibacterial drug stock solution mother liquor (2560 μg / mL) by 10 times respectively to obtain an antibacterial drug solution with a concentration of 256 μg / mL. Take a sterile 96 - well plate, add 200 μL of the antibacterial drug to the first well, add 100 μL of MH broth medium to the second to tenth wells respectively. Pipette 100 μL from the first well and add it to the second well, mix well, and then pipette 100 μL to the third well, and so on. Discard 100 μL from the tenth well. At this time, the drug concentrations in each well are as follows: 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5 μg / mL. Add 100 μL of the bacterial solution (positive control) to the eleventh well and 100 μL of MH(B) medium (negative control) to the twelfth well.

[0052] (4) Add 100 μL of the previously prepared bacterial solution to each of the first to tenth wells, so that the final bacterial solution concentration in each tube is approximately 5×10 5 CFU / mL. The drug concentrations in the first to tenth wells are 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 μg / mL respectively. Place the inoculated 96 - well plate in an incubator at 37°C for culture, and observe the growth of the bacterial solution after 24 h. At the same time, use the standard strain for quality control.

[0053] (5) Result judgment and interpretation: Before reading and reporting the MIC of the tested strains, it is necessary to check whether the bacterial growth in the growth control tube is good. At the same time, the subculture of the inoculum should also be checked to determine whether it is contaminated, and whether the MIC value of the quality control strain is within the quality control range. Visually observed, the tube with the lowest drug concentration without bacterial growth is the MIC of the tested bacteria.

[0054] (6) The test results show that compound (1) has outstanding antibacterial activity and obvious antibacterial advantages compared with the antibacterial drugs currently used clinically for treating infections caused by Gram-positive bacteria. The MIC 90 is 52.84 ± 4.2 μg / mL, with significant antibacterial activity, approaching the MIC 90 value (>50.84 ± 5.5 μg / mL) of levofloxacin.

[0055] Example 9

[0056] The toxicological evaluation of the compound of the present invention was carried out. The mouse bone marrow micronucleus test, Ames test and TK gene mutation test all proved that the compound of the present invention is non-toxic to animals and safe to use.

Claims

1. A benzog]chromone compound having anti-methicillin-resistant Staphylococcus aureus (MRSA) activity, characterized in that, The benzogchromone compound with anti-methicillin-resistant Staphylococcus aureus (MRSA) activity is prepared from the whole plant of the medicinal plant Chamaecrista rotundifolia through pretreatment, extract preparation, silica gel column chromatography, and high-performance liquid chromatography separation, and is named 2-(hydroxymethyl)-6-methoxy-9-methylbenzo[2,3-g]-4H-chromone, with the following structure: 。 2. A method for preparing a benz[g]chromone compound having anti-Methicillin-resistant Staphylococcus aureus (MRSA) activity as described in claim 1, characterized in that, It is prepared from the whole plant of the medicinal plant Chamaecrista rotundifolia through pretreatment, organic solvent extraction, silica gel column chromatography, and high-performance liquid chromatography separation, specifically including the following steps: A. Pretreatment: The whole plant of the raw material medicinal plant Chamaecrista rotundifolia is crushed or cut into sections to obtain material a; B. Organic solvent extraction: Add an organic extraction solvent 2 to 4 times the mass of material a to material a, soak and extract at room temperature for 3 to 5 times, with each extraction time being 24 to 72 h, combine the extraction solutions, filter and concentrate to obtain extract b; C. Silica gel column chromatography: Add silica gel with a mesh size of 160 to 300, 2 to 4 times the weight of extract b, to the dry column of extract b, and perform gradient elution with a chloroform-acetone solution with a volume ratio of 1:0 to 1:

2. Monitor by TLC and combine the same parts; D. High-performance liquid chromatography separation: The eluate obtained by eluting with a chloroform-acetone solution with a ratio of 7:3 is separated and purified by high-pressure liquid chromatography to obtain the target benzogchromone compound with anti-methicillin-resistant Staphylococcus aureus activity.

3. The preparation method according to claim 2, characterized in that, The organic extraction solvent described in step B is an aqueous methanol solution with a mass concentration of 80% to 100%, an aqueous ethanol solution with a mass concentration of 80% to 100%, or an aqueous acetone solution with a mass concentration of 60% to 90%.

4. The preparation method according to claim 2, characterized in that, Before loading the column in step C, it also includes a sample mixing step of dissolving with an organic solvent 1.5 to 3 times the weight of extract b and adding silica gel with a mesh size of 80 to 100, 0.8 to 1.2 times the weight of extract b.

5. The preparation method according to claim 2, characterized in that, The organic extraction solvent is pure methanol, pure ethanol, or pure acetone.

6. The preparation method according to claim 2, characterized in that, The volume ratio of the chloroform-acetone solution described in step C is 1:0, 20:1, 9:1, 8:2, 7:3, 6:4, 1:1, and 1:

2.

7. The preparation method according to claim 2, wherein In step D, high performance liquid chromatography separation and purification was carried out using a C chromatographic column with dimensions of 21.2 mm × 250 mm and a particle size of 5 μm. 18 The flow rate was 12 mL / min, the mobile phase was 52% methanol, the detection wavelength of the ultraviolet detector was 368 nm, 500 μL of sample was injected each time, the chromatographic peaks were collected for 30 min, and after multiple accumulations and evaporation to dryness, the target antibacterial active benz[g]chromone compounds were obtained.

8. Use of a benzogchromone compound having anti-Methicillin-resistant Staphylococcus aureus (MRSA) activity as described in claim 1, characterized in that, Application of the benzogchromone compound with anti-methicillin-resistant Staphylococcus aureus activity in the preparation of anti-methicillin-resistant Staphylococcus aureus drugs.

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