A method for preparing an epoxy enynyl alcohol compound and its application

Abstract

This invention discloses a method for preparing an epoxyenynyl alcohol compound, comprising the following steps: ① Extracting Melaleuca tiliacea leaf powder with ethanol to obtain an extract, and concentrating it to obtain a Melaleuca tiliacea extract; ② Diluting the Melaleuca tiliacea extract obtained in step ① with water to form a suspension, and extracting it sequentially with petroleum ether and ethyl acetate, combining the ethyl acetate extracts and concentrating them into a paste; ③ Performing silica gel column chromatography on the ethyl acetate paste obtained in step ②, and detecting the similar fractions by TLC to separate them into 9 fractions, Fr.1–Fr.9; ④ Performing dextran gel column chromatography on fraction Fr.4 obtained in step ③, and then purifying it by high performance liquid chromatography to obtain the target product. This invention provides a new route for the extraction or preparation of this compound; and it has been found that this epoxyenynyl alcohol compound has significant and broad-spectrum antibacterial activity, and can be used in the preparation of antibiotic drugs.

Description

Technical Field

[0001] This invention relates to the field of phytochemistry, specifically to a method for preparing an epoxyenynol compound and its application. Background Technology

[0002] "Compounds from Angelica keiskei with NQO1 induction, DPPH scavenging

[0003] The article "and α-glucosidase inhibitory activities" (Food Chemistry, Liping Luo et al.) discloses the extraction of compound 23 from Angelica keiskei, with the following structural formula:

[0004] However, according to the literature, compound 23 did not exhibit NQO1-induced or DPPH-scavenging activity. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for preparing epoxy enynyl alcohol compounds and their applications.

[0006] The technical solution for achieving the first objective of this invention is a method for preparing an epoxy enynyl alcohol compound, the structural formula of which is as follows: This includes the following steps:

[0007] ① Extract the powdered branches and leaves of Melaleuca tiliacea with ethanol to obtain an extract. Concentrate the extract under reduced pressure to form a paste to obtain Melaleuca tiliacea extract.

[0008] ② Dilute the Melaleuca tiliaceus extract obtained in step ① with water to form a suspension, and extract it sequentially with petroleum ether and ethyl acetate. Combine the ethyl acetate extracts and concentrate them into an extract.

[0009] ③ The ethyl acetate extract obtained in step ② was subjected to silica gel column chromatography. The silica gel column chromatography was carried out in increasing polarity using a mixture of petroleum ether and ethyl acetate (100:0-0:100, V / V) and ethyl acetate-methanol (100:0-0:100, V / V). The fractions were collected in 500 mL increments. The similar fractions were combined by TLC and separated into 9 components, namely Fr. 1-Fr. 9.

[0010] ④ The Fr.4 fraction obtained in step ③ was first subjected to dextran gel column chromatography, and then purified by high performance liquid chromatography to obtain the target product.

[0011] During the vacuum concentration in step ① above, the temperature is 30~70℃ and the pressure is -0.06~-0.15MPa.

[0012] During concentration in step ② above, the temperature is 30~70℃ and the pressure is -0.06~-0.15MPa.

[0013] In step ③ above, during TLC detection, a chloroform-methanol mixed solvent with a chloroform volume percentage of 95% was used as the developing solvent.

[0014] In step ④ above, during dextran gel column chromatography, the eluent is a chloroform-methanol mixed solvent with a volume percentage of 50% chloroform.

[0015] In step ④ above, the eluent for high performance liquid chromatography is acetonitrile and water, with a volume ratio of 25:75.

[0016] The technical solution to achieve the second objective of this invention is the application of the above-mentioned compound in the preparation of anti-pathogenic drugs.

[0017] The pathogenic bacteria mentioned are Staphylococcus albus, Escherichia coli, Bacillus cereus, Staphylococcus aureus, or Bacillus subtilis.

[0018] This invention has positive effects:

[0019] (1) This invention is derived from Melaleuca lyrata (Latin name: Melaleuca leucadendron The following epoxide enynyl alcohol compound was extracted from L., providing a new route for the extraction or preparation of this compound.

[0020] .

[0021] (2) The present invention found that the epoxy enynyl alcohol compound has significant and broad-spectrum antibacterial activity, most of which show activity comparable to the positive control, and therefore can be used in the preparation of antibiotic drugs. Attached Figure Description

[0022] Figure 1 The image shows the H-NMR spectrum of the compound extracted in Example 1.

[0023] Figure 2 The image shows the nuclear magnetic resonance C-ray spectrum of the compound extracted in Example 1. Detailed Implementation

[0024] (Example 1)

[0025] The structural formula of the epoxy enynyl alcohol compound prepared in this embodiment is as follows:

[0026] .

[0027] The preparation method of the above-mentioned compound includes the following steps:

[0028] ① 5.0 kg of Melaleuca tiliaceus leaf powder was extracted four times with 30%~95% (v / v) ethanol (75% ethanol, v / v in this example) for 3 days each time to obtain an extract. The extract was concentrated under reduced pressure into a paste to obtain 560 g of Melaleuca tiliaceus extract.

[0029] During vacuum concentration, the temperature is 30~70℃ and the pressure is -0.06~-0.15MPa; in this embodiment, the temperature is 45℃ and the pressure is -0.095 MPa.

[0030] ② Dilute the Melaleuca truncata extract obtained in step ① with water to form a suspension, and extract it sequentially with petroleum ether (3L × 3 times, i.e., extract with petroleum ether 3 times, each time using 3L of petroleum ether) and ethyl acetate (3L × 3 times, i.e., extract with ethyl acetate 3 times, each time using 3L of ethyl acetate). Combine the ethyl acetate extracts and concentrate them into an extract.

[0031] The concentration temperature is 30~70℃ and the pressure is -0.06~-0.15MPa; in this embodiment, the temperature is 45℃ and the pressure is -0.095 MPa.

[0032] ③ The ethyl acetate extract obtained in step ② was subjected to silica gel column chromatography. The column was loaded with 200-300 mesh silica gel and chromatographically analyzed using a petroleum ether-ethyl acetate mixture (100:0–0:100, V / V) and ethyl acetate-methanol mixture (100:0–0:100, V / V) in increasing polarity. Approximately 500 mL fractions were collected each time. TLC analysis separated similar fractions into nine groups, Fr. 1–Fr. 9. A chloroform-methanol mixture with 95% chloroform (v / v) was used as the developing solvent for TLC analysis.

[0033] ④ The Fr.4 fraction obtained in step ③ was first subjected to dextran gel column chromatography. In this embodiment, Sephadex LH-20 column chromatography was used, and the eluent was a chloroform-methanol mixed solvent with a volume percentage of 50% chloroform. Then, it was purified by high performance liquid chromatography, and the eluent for high performance liquid chromatography was acetonitrile and water (volume ratio of 25:75). 12 mg of the target product was obtained, which was named compound 1.

[0034] The NMR spectrum of this compound is shown below. Figure 1 The nuclear magnetic resonance C-ray spectrum is shown below. Figure 2 .

[0035] The identification results of the compounds obtained above are as follows:

[0036] Appearance: Colorless paste.

[0037] 1 H NMR (400 MHz, CDCl3) δ 5.64, 5.63, 5.62, 5.60, 5.58, 5.54, 5.52,5.49, 5.45, 5.21, 5.19, 4.40, 4.38, 4.37, 2.13, 2.11, 2.10, 2.08, 1.76, 1.76,1.74, 1.73, 1.72, 1.27, 1.25, 1.03, 1.01, 0.99, 0.90, 0.88, 0.86.

[0038] 13 C NMR (101 MHz, CDCl3) δ 134.65, 127.74, 80.24, 79.14, 68.96, 64.08,58.62, 31.80, 30.63, 29.28, 29.17, 29.11, 27.69, 22.65, 14.11, 9.30.

[0039] Optical rotation is + 76.8 (c = 0.2, MeOH), HR-ESI-MS m / z 265.1808 [M - H] - , combined 1 H-NMR and 13 According to C18NMR, the molecular formula is C18N2. 16 H 26 O3.

[0040] according to 1 H-NMR, 13 C-NMR and DEPT analysis revealed that the compound contains one carbon-carbon triple bond, one carbon-carbon double bond, one epoxy functional group, and two hydroxyl groups.

[0041] according to 1 H, 13 According to CNMR data and the literature (Liping Luo, Ruihan Wang, Xiaojun Wang, Zhongjun Ma, Ning Li. 2012. Compounds from Angelica keiskei with NQO1 induction, DPPH scavenging and α-glucosidase inhibitory activities. FoodChemistry. 131: 992-998), this compound is compound 23 from that literature.

[0042] (Experimental examples, pharmacological activity experiments)

[0043] Test method: microdilution method, which is often used to determine the inhibitory activity of pathogenic bacteria against sensitive drugs or new drugs.

[0044] Use a 96-well microdilution plate with a U-shaped bottom and a capacity of 0.20–0.30 mL per well. Dilute the prepared drug solution two-fold in test tubes using MH medium, and then fill each tube into a 96-well plate. Leave one row of wells as a control, containing only culture medium and bacterial suspension. After the experiment, mix thoroughly with a micro-stirrer, and then incubate the 96-well plate at 28°C or 37°C for 18–48 h. Measure the absorbance at 630 nm using a microplate reader.

[0045] Preparation of pathogenic strains:

[0046] Five activated pathogenic bacteria were inoculated into sterilized LB medium and then placed in a constant temperature shaker at 28°C for 24 hours.

[0047] Initial screening of compounds:

[0048] The cultured pathogenic bacteria were diluted with culture medium to a dilution ratio of 1:500-1:1000. The diluted bacterial culture medium was then quantitatively added to each well of a 96-well plate. A 1 mg / mL solution of the sample (the compound extracted in the previous example, referred to as compound 1) was prepared and quantitatively added to each well. After addition, the plates were incubated at 28°C for 48 hours, and then the absorbance of each well was measured at 630 nm using a microplate reader.

[0049] The experimental results showed that the tested compound had significant inhibitory activity against five pathogenic bacteria (Staphylococcus albus, Escherichia coli, Bacillus cereus, Staphylococcus aureus, or Bacillus subtilis).

[0050] Minimum inhibitory concentration (MIC) test:

[0051] The cultured pathogenic bacteria were diluted with culture medium to a dilution ratio of 1:500-1:1000. 96 μL of the diluted bacterial culture medium was added to each well in the first row of a 96-well plate. Then, 4 μL of 1 mg / mL sample was added to each well in the first row. Next, 100 μL of the diluted bacterial culture medium was added to each well in the 96-well plate. 100 μL of the mixture was then added to each well in the second row, and so on, until the last well was reached. Finally, diluted bacterial culture medium was added to ensure each well contained 200 μL of the mixture. After addition, the plate was incubated at 28°C for 48 hours. The absorbance of each well was then measured at 630 nm using a microplate reader. Ciprofloxacin was used as a positive control.

[0052] The results are shown in Table 1.

[0053] Table 1. Minimum inhibitory concentrations of epoxy enynyl alcohol compounds in Melaleuca truncatum against five pathogenic bacteria.

[0054]

[0055] The experimental results showed that the epoxy enynyl alcohol compounds possess significant and broad-spectrum antibacterial activity. Compound 1 exhibited broad-spectrum antibacterial activity and showed activity comparable to the positive control, making it suitable for the preparation of antibiotic drugs.

Claims

1. A method for preparing an epoxy enynyl alcohol compound, the structural formula of which is as follows: Its characteristics Includes the following steps: ① Extract the powdered branches and leaves of Melaleuca tiliacea with ethanol to obtain an extract. Concentrate the extract under reduced pressure to form a paste to obtain Melaleuca tiliacea extract. ② Dilute the Melaleuca tiliaceus extract obtained in step ① with water to form a suspension, and extract it sequentially with petroleum ether and ethyl acetate. Combine the ethyl acetate extracts and concentrate them into an extract. ③ The ethyl acetate extract obtained in step ② was subjected to silica gel column chromatography using a mixture of petroleum ether and ethyl acetate (volume ratio 100:0–0:100) and ethyl acetate and methanol (volume ratio 100:0–0:100) in increasing polarity, with 500 mL fractions collected each time. Similar fractions were combined and separated into 9 components (Fr. 1–Fr. 9) by TLC analysis. ④ The Fr.4 fraction obtained in step ③ was first subjected to dextran gel column chromatography, and then purified by high performance liquid chromatography to obtain the target product. During dextran gel column chromatography, the eluent was a chloroform-methanol mixed solvent with 50% chloroform by volume. The eluent for high performance liquid chromatography was acetonitrile and water in a volume ratio of 25:

75.

2. The method for preparing the epoxy enynyl alcohol compound according to claim 1, characterized in that: During step ①, when concentrating under reduced pressure, the temperature is 30~70℃ and the pressure is -0.06~-0.15MPa.

3. The method for preparing the epoxy enynyl alcohol compound according to claim 1, characterized in that: During concentration in step ②, the temperature is 30~70℃ and the pressure is -0.06~-0.15MPa.

4. The method for preparing the epoxy enynyl alcohol compound according to claim 1, characterized in that: In step ③, during TLC detection, a chloroform-methanol mixed solvent with a chloroform volume percentage of 95% was used as the developing solvent.

5. The use of the compound prepared by the method of claim 1 in the preparation of antimicrobial drugs.

6. In the application according to claim 5, the pathogenic bacteria are Staphylococcus albus, Escherichia coli, Bacillus cereus, Staphylococcus aureus, or Bacillus subtilis.

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