Triterpenoids, and preparation method and application thereof

Abstract

The invention discloses a triterpenoid compound. The triterpenoid compound has a structure as shown in the specification, wherein the molecular formula of # imgabs0 # is C28H38O11. The invention also discloses a preparation method and application of the triterpenoid. The triterpenoid disclosed by the invention has remarkable antibacterial activity and can be applied to preparation of antibacterial drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of terpenoid compounds, and in particular to a triterpenoid compound, phellodendronone, and a preparation method and application thereof. Background Art

[0002] Terpenes - hydrocarbons with molecular formulas that are multiples of isoprene units and their oxygen-containing derivatives, are a large class of natural products with high diversity, including monoterpenes, sesquiterpenes, diterpenes, sesquiterpenoid cyclohexenones, triterpenes, etc. Many terpenoids have been developed as important drugs for the treatment of cancer, bacterial infections, malaria and various other human diseases. According to literature reports, triterpenoids have pharmacological activities such as hypoglycemic, anti-inflammatory, anti-tumor, antiviral and immunomodulatory. Therefore, the synthesis of terpenoids is very important. However, due to the fact that their synthetic pathways have not been fully elucidated, terpenoids have non-modular structures and there is no universal unified synthetic strategy, the source of terpenoids still depends on the extraction of natural products.

[0003] Therefore, the development of new triterpenoid compounds is of great significance to solving the current situation of antibiotic abuse. Summary of the invention

[0004] The invention provides a triterpenoid compound and a preparation method thereof; the triterpenoid compound of the invention has a novel structure and excellent antibacterial activity.

[0005] The present invention provides the following technical solutions:

[0006] The first aspect of the present invention discloses a triterpenoid compound having the following structure: The molecular formula is C 28 H 38 O 11 , the English name is phellodendronone.

[0007] The second aspect of the present invention discloses a method for preparing the triterpenoid compound, comprising the following steps:

[0008] (1) crushing the bark of Phellodendron chinense;

[0009] (2) mixing the crushed Phellodendron amurense bark with alcohol, performing reflux extraction, and concentrating under reduced pressure to obtain an extract;

[0010] (3) mixing the extract and water to obtain a suspension, adding ethyl acetate for extraction, and concentrating under low temperature and reduced pressure to obtain a crude extract of the triterpenoid compound;

[0011] (4) Purifying the obtained crude extract by chromatography to obtain the triterpenoid compound.

[0012] Preferably, the ratio of the mass of the cork bark to the volume of the organic solvent is (1.0) g: (14.0±1.0) mL; reflux extraction is performed at 65-75°C for no less than three times, each time for 4-6 hours; the ratio of the mass of the cork bark to the volume of alcohol is (1.0) g: (14.0±1.0) mL; reflux extraction is performed at 65-75°C for no less than three times, each time for 4-6 hours; the organic solvent is ethanol with a volume fraction of ≥95%.

[0013] Preferably, the temperature of reduced pressure concentration in step (2) or (3) is 40 to 60° C., and the vacuum degree is 0.07 to 0.09 MPa.

[0014] Preferably, in step (3), the mass ratio of the extract to water is 1:(2-4), the volume ratio of ethyl acetate to the suspension is (0.5-1):1, and extraction is performed 2 to 4 times.

[0015] Preferably, the chromatographic purification comprises sequentially subjecting the crude extract to silica gel column chromatography elution and gel column chromatography purification.

[0016] Preferably, the elution solvent used for silica gel column chromatography elution is chloroform-methanol and petroleum ether-acetone, and the flow rate of the eluent is 2-5 mL / min; the elution comprises the following steps: dissolving the crude extract in a mixed solution of dichloromethane and methanol to obtain a crude extract solution; mixing the crude extract solution with silica gel, removing the solvent, and sequentially using dichloromethane-methanol solutions with volume ratios of 100:0, 100:1, 70:1, 50:1, 30:1, 10:1, and 5:1. Gradient elution was performed to obtain dichloromethane-methanol solution elution portions with volume ratios of 100:0, 100:1, 70:1, 50:1, 30:1, 10:1, and 5:1, respectively; the dichloromethane-methanol solution elution portion with a volume ratio of 70:1 was taken and eluted through a silica gel column chromatography with petroleum ether-acetone with a volume ratio of 6:1 as solvent to obtain an eluent; the particle size of the silica gel was 200-400 meshes, and the mass of the silica gel was 0.8-1.5 times the mass of the crude extract.

[0017] Preferably, the gel column used in the gel column chromatography purification is a dextran gel column, and the solvent used is methanol.

[0018] The third aspect of the present invention discloses the use of the triterpenoid compounds in the preparation of antibacterial drugs.

[0019] Beneficial effects of the present invention:

[0020] 1. The triterpenoid compound phellodendronone of the present invention is a novel tetracyclic triterpenoid compound with strong stereoselectivity. The triterpenoid compound of the present invention exhibits obvious inhibitory activity against Fusarium solani, Fusarium oxysporum, and Sphaerotheca cucumeris, and exhibits moderate inhibitory activity against Alternaria ginseng. The triterpenoid compound of the present invention has good antibacterial activity and can be used to prepare antibacterial drugs. The triterpenoid compound of the present invention enriches the diversity of triterpenoid compounds and provides new options for the development of antibacterial drugs.

[0021] 2. The triterpenoid compound phellodendronone of the present invention is a chemical component of Phellodendron chinense, which is prepared by extracting Phellodendron chinense bark, and the preparation method has a short cycle, mild conditions, few by-products and low cost. The preparation method of the triterpenoid compound phellodendronone provided by the present invention is simple and easy to implement, and is easy to industrialize, which not only meets the needs of modern environmental protection and low-carbon economy, but also can provide a new way for the mass production of triterpenoid compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The HR-ESI-MS spectrum of the triterpenoid compound phellodendronone of the present invention is shown in FIG.

[0023] Figure 2 The triterpenoid compound phellodendronone of the present invention 1 H-NMR spectrum.

[0024] Figure 3 The triterpenoid compound phellodendronone of the present invention 13 C-NMR spectrum and DEPT spectrum.

[0025] Figure 4 The triterpenoid compound phellodendronone of the present invention 1 H- 1 H COSY spectrum.

[0026] Figure 5 This is the HMBC spectrum of the triterpenoid compound phellodendronone of the present invention.

[0027] Figure 6 This is the HSQC spectrum of the triterpenoid compound phellodendronone of the present invention.

[0028] Figure 7 The NOESY spectrum of the triterpenoid compound phellodendronone of the present invention is shown in FIG. DETAILED DESCRIPTION

[0029] The present invention is described clearly and completely below in conjunction with the technical solutions in the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Other technical solutions obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0030] The present invention provides a triterpenoid compound having the following structure: The molecular formula is C 28 H 38 O 11 , the English name is phellodendronone.

[0031] The preparation method of the triterpenoid compound of the present invention comprises the following steps:

[0032] (1) The bark of Phellodendron amurense is used as a raw material, crushed, refluxed with an organic solvent, and concentrated under low temperature and reduced pressure to obtain an extract; the organic solvent is ethanol with a volume fraction of ≥95%, the volume ratio of the Phellodendron amurense bark mass to the alcohol is 1.0 g: (14.0 ± 1.0) mL, the extraction temperature is 65-75°C, the extraction is performed 2-3 times, each time for 4-6 hours; the temperature of low temperature and reduced pressure concentration is 40-60°C, and the vacuum degree is 0.07-0.09 MPa;

[0033] (2) mixing the extract and water to obtain a suspension, adding ethyl acetate for extraction, and concentrating under low temperature and reduced pressure to obtain a crude extract of the triterpenoid compound phellodendronone; the mass ratio of the extract and water is 1:(2-4), the volume ratio of ethyl acetate to the suspension is (0.5-1):1, and the extraction is performed 2-4 times; the temperature of low temperature and reduced pressure concentration is 40-60°C, and the vacuum degree is 0.07-0.09MPa;

[0034] (3) The crude extract of the triterpenoid compound phellodendronone was separated by silica gel column chromatography, and gradient elution was performed using dichloromethane-methanol solutions with volume ratios of 100:0, 100:1, 70:1, 50:1, 30:1, 10:1, and 5:1, respectively, to obtain dichloromethane-methanol solution elution fractions with volume ratios of 100:0, 100:1, 70:1, 50:1, 30:1, 10:1, and 5:1, and the volume ratios were taken. The eluted part is eluted by silica gel column chromatography with a volume ratio of 70:1 dichloromethane-methanol solution and a solvent of petroleum ether-acetone with a volume ratio of 6:1 to obtain an eluent; the mass of the silica gel is 0.8 to 1.5 times the dry weight of the crude extract; the triterpenoid compound phellodendronone is obtained; the particle size of the silica gel is preferably 200 to 400 meshes; the mass of the silica gel is preferably 0.8 to 1.5 times the dry weight of the crude extract, and more preferably 1 to 1.2 times. The present invention has no special limitation on the process of mixing the crude extract solution with the silica gel, and it can be carried out according to the process well known in the art.

[0035] The gel column used for gel column chromatography purification is a dextran gel column, and the solvent used is methanol. After the gel column chromatography purification is completed, a triterpenoid compound phellodendrononee with antibacterial activity is obtained. The present invention has no special limitation on the specific process of the drying, and it can be carried out according to the process well known in the art.

[0036] The invention relates to the use of the triterpenoid compound phellodendronone prepared by the above preparation method in the preparation of antibacterial drugs.

[0037] Example 1: Preparation of triterpenoid compound phellodendronone, the steps are as follows:

[0038] (1) 10 kg of Phellodendron amurense bark was air-dried and crushed, and then added to 95% ethanol by volume for extraction. Ethanol was added in a ratio of 1.0 g of Phellodendron amurense bark to 14.0 mL of ethanol by volume. The extraction reflux temperature was 70° C., and the extraction reflux was repeated 3 times for 3 hours each time. The insoluble matter was filtered and the filtrate was combined. Then, the extract was evaporated to dryness under low temperature and reduced pressure at a vacuum degree of 0.08 MPa and a temperature of 50° C.;

[0039] (2) the obtained extract and water were mixed uniformly at a mass ratio of 1:3 to prepare a suspension, extracted three times with equal volumes of ethyl acetate, the extracts were combined, and evaporated to dryness under reduced pressure at a vacuum degree of 0.09 MPa and a temperature of 45° C. to obtain an ethyl acetate extract phase;

[0040] (3) The obtained ethyl acetate extract phase is mixed with an equal mass of silica gel, and then placed in a silica gel column for chromatography, and gradient elution is performed using dichloromethane-methanol solutions with volume ratios of 100:0, 100:1, 70:1, 50:1, 30:1, 10:1, and 5:1 in sequence, to obtain dichloromethane-methanol solution elution portions with volume ratios of 100:0, 100:1, 70:1, 50:1, 30:1, 10:1, and 5:1, respectively; the dichloromethane-methanol solution elution portion with a volume ratio of 70:1 is taken, and eluted by silica gel column chromatography using petroleum ether-acetone with a volume ratio of 6:1 as solvent to obtain an eluate, which is dried; and then purified by methanol gel column chromatography using a dextran gel column solvent to obtain the triterpene compound phellodendronone.

[0041] Characterization and performance testing:

[0042] (A) Structural identification

[0043] The prepared compound was taken and its structure was identified by 1D / 2DNMR (one-dimensional nuclear magnetic resonance spectroscopy and two-dimensional nuclear magnetic resonance spectroscopy) and HR-ESI-MS (high resolution electrospray ionization mass spectrometry). Figures 1 to 7 shown.

[0044] From the HSQC spectrum combined with the carbon spectrum, it can be obtained that the chemical shift δ assignments of H and the C connected to the triterpenoid compounds are shown in Table 1.

[0045] Table 1 The prepared compounds 13 C-NMR (100 MHz) and 1 H-NMR (400 MHz) data, CDC13 as solvent.

[0046]

[0047] According to Table 1, the HR-ESI-MS m / z of the compound: [M+Na] + The quasi-molecular ion peak is 573.2302 (C 28 H 38 O 11 Na,[M+Na] + Calculated value: 573.2306) indicates that its molecular formula is C 28 H 38 O 11 , containing 10 unsaturated degrees. 1 H-NMR( Figure 2 ), 13 C-NMR( Figure 3 ) and HSQC( Figure 6 ) Spectral analysis, 1H-NMR showed that the compound had five characteristic methyl groups δ H 1.37, 1.49, 1.45, 1.49, and 1.23, 1 double bond group δ H 6.56 (d, J = 11.8, 1H) and δ H 5.99 (d, J = 11.8, 1H), 13 C-NMR showed signals of 28 C atoms, and combined with DEPT and HSQC, it showed two ester groups, δ C 156.59, 167.3 and a keto carbonyl delta C 207.8, the compound contains 7 methyl groups H 1.23(H-30),1.37(H-18),1.45(H-28),1.49(H-19),1.49(H-29),3.36(OCH3-21),3.46(OCH3-23); δ C 17.52 (C-30), 22.83 (C-18), 32.15 (C-28), 16.46 (C-19), 26.87 (C-29), 54.46 (OCH3-21), 56.78 (OCH3-23), 3 methylene δ H 1.78(H-12α),2.12(H-12β),1.89(H-11),2.28(H-6α),2.94(H-6β); δ C 29.17 (C-12), 19.38 (C-11), 40.5 (C-6), 9 methines δ H 2.23(H-9),2.61(H-5),3.68(H-15),4.41(H-22),4.72(H-21),4.84(H-23),4.95(H-17),5.99(H-2),6.56(H-1); δ C 48.7 (C-9), 57.1 (C-5), 53.63 (C-15), 80.43 (C-22), 107.55 (C-21), 109.22 (C-23), 82.44 (C-17), 122.92 (C-2), 156.59 (C-1) and 9 quaternary carbon δ C 167.07 (C-3), 84.06 (C-4), 207.8 (C-7), 52.64 (C-8), 43.38 (C-10), 38.85 (C-13), 65.76 (C-14), 167.3 (C-16), 81.99 (C-20). These spectral characteristics show that the compound is very similar to the compound kihadanin C reported in the literature, except that the δ C 80.43 (C-22) and δC 109.22 (C-23) carbon signal, if the compound is kihadanin C, the carbon chemical shift on the five-membered ring should be δ C 76.2 (C-22) and δ C 112.0 (C-23), the configuration is 20S, 21S, 22S, 23R, therefore, the configuration of the compound may be 20R*, 21R*, 22R*, 23S*, and the combination of the two possible configurations and DP4+ probability analysis shows that the probability of 20R, 21R, 22R, 23S-1 is 99.97%, which is consistent with the NOE correlation. The structure of the compound was fully confirmed, and the triterpenoid compound was named phellodendronone.

[0048]

[0049] DP4+ probability analysis of compounds

[0050] Example 2: Screening of antibacterial activity of triterpenoid compound phellodendronone

[0051] 1. Antibacterial activity of triterpenoid compound phellodendronone:

[0052] (1) PDB (Potato Dextrose Broth) medium: Wash and peel potatoes, weigh 200 g and cut into cubes, add water and boil until they can be pierced with a glass rod (10-15 minutes), filter the filtrate with gauze, heat the filtrate and add 20 g of glucose, stir evenly until the glucose is completely dissolved, add water to 1000 mL, dispense into 250 mL conical flasks, seal, place in a high temperature sterilizer at 121°C for 30 min, remove, cool and store for later use;

[0053] (2) Activation of fungi: Sphaerotheca cucumeris, Alternaria ginseng, Fusarium solani, and Fusarium oxysporum were inoculated into PDB medium respectively and cultured at 28° C. for 3 days;

[0054] (3) The antibacterial test was conducted using the two-fold dilution method: the triterpenoid compound to be tested and the positive control drug (fungus: nystatin) were dissolved in DMSO to prepare a 10.24 mg / ml working solution, and 5 uL was added to a 96-well plate. Three replicate wells were set for each sample, and then 85 uL of blank culture medium (fungus: PDB culture medium) was added. The samples were diluted to different concentrations (512, 256, 128, 64, 32, 16, 84, 2, 1, 0.5, 0.25, 0.125 ug / ml) using the two-fold dilution method. 200 uL of the activated bacterial solution was diluted to 10 mL with the corresponding culture medium, and 10 uL of the diluted bacterial solution was added to the sample wells and control wells to make the final volume of each well 100 mL. No bacterial solution was added to the blank group. After the bacterial solution was fully mixed, the 96-well plate was placed in a constant temperature shaker for culture (fungi: 28°C, 3 days). The absorbance of each well under visible light was measured using an enzyme marker. The concentration of the compound in the well corresponding to the ratio of the absorbance value to the blank control value of 1 was the minimum inhibitory concentration (MIC) of the sample for the corresponding strain. The results are shown in Table 2.

[0055] Table 2

[0056]

[0057] The results in Table 2 show that in the in vitro antibacterial activity screening test of the triterpenoid compound phellodendronone (positive control: Nystatin), the triterpenoid compound phellodendronone showed good inhibitory activity against Sphaerotheca cucumeris, Fusarium solani and Fusarium oxysporum, and showed moderate inhibitory activity against Alternaria ginseng. Therefore, the triterpenoid compound phellodendronone prepared by the present invention has research value as a lead compound for the development of antibacterial and anti-inflammatory drugs.

[0058] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A triterpenoid compound, characterized in that: Has the following structure: The molecular formula is C 28 H 38 O 11 .

2. The method for preparing triterpenoid compounds according to claim 1, characterized in that: The following steps are involved: (1) crushing the bark of Phellodendron chinense; (2) mixing the crushed Phellodendron amurense bark with an organic solvent, performing reflux extraction, and concentrating under reduced pressure to obtain an extract; (3) mixing the extract and water to obtain a suspension, adding ethyl acetate for extraction, and concentrating under low temperature and reduced pressure to obtain a crude extract of the triterpenoid compound; (4) Purifying the obtained crude extract by chromatography to obtain the triterpenoid compound.

3. The preparation method according to claim 2, characterized in that: The mass ratio of the cork bark to the volume of the organic solvent is 1.0 g: (14.0±1.0) mL; reflux extraction is performed at 65-75° C. for no less than three times, each time for 4-6 hours; and the organic solvent is ethanol with a volume fraction of ≥95%.

4. The preparation method according to claim 2, characterized in that: The temperature of reduced pressure concentration in step (2) or (3) is 40 to 60° C., and the vacuum degree is 0.07 to 0.09 MPa.

5. The preparation method according to claim 2, characterized in that: In step (3), the mass ratio of the extract to water is 1:(2-4), the volume ratio of ethyl acetate to the suspension is (0.5-1):1, and extraction is performed 2 to 4 times.

6. The preparation method according to claim 2, characterized in that: The chromatographic purification in step (4) comprises sequentially subjecting the crude extract to silica gel column chromatography elution and gel column chromatography purification.

7. The preparation method according to claim 6, characterized in that: The elution solvents used for silica gel column chromatography elution are dichloromethane-methanol and petroleum ether-acetone, and the flow rate of the eluent is 2-5 mL / min; the elution comprises the following steps: dissolving the crude extract in a mixed solution of dichloromethane and methanol to obtain a crude extract solution; mixing the crude extract solution with silica gel, removing the solvent, and gradient elution with dichloromethane-methanol solutions with volume ratios of 100:0, 100:1, 70:1, 50:1, 30:1, 10:1, and 5:1 in sequence. The method comprises the steps of: eluting the mixture with a volume ratio of 100:0, 100:1, 70:1, 50:1, 30:1, 10:1 and 5:1 to obtain dichloromethane-methanol solution elution portions respectively; taking the dichloromethane-methanol solution elution portion with a volume ratio of 70:1, and eluting it through a silica gel column chromatography with petroleum ether-acetone with a volume ratio of 6:1 as solvent to obtain an eluent; the particle size of the silica gel is 200-400 meshes, and the mass of the silica gel is 0.8-1.5 times the mass of the crude extract.

8. The preparation method according to claim 6, characterized in that: The gel column used in the gel column chromatography purification is a dextran gel column, and the solvent used is methanol.

9. Use of the triterpenoid compound according to claim 1 in the preparation of antibacterial drugs.

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