A limonoid compound Aglatestate F, a preparation method and purposes thereof

Abstract

The application relates to a limonoid compound Aglatestate F, a preparation method and application thereof, and belongs to the field of chemical medicines. The compound is obtained by separation and extraction from branches and leaves of Aglaia edulis, and preparation and separation are simple. The compound has a good inhibiting effect on dengue virus BHK-21 cell infection, and the compound has a good inhibiting effect on dengue virus BHK-21 cell infection at a non-toxic concentration, the anti-dengue virus effect EC 50 = 1.290+ / -0.170, and the selectivity index is higher than that of the positive drug antiviral drug ribavirin, so that the compound Aglatestate F has a good development prospect of anti-dengue virus drugs, and lays a solid foundation for preparation of anti-dengue virus drug development.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pharmaceutical chemistry, and particularly relates to a limonoid compound Aglatestate F, a preparation method and use thereof. BACKGROUND

[0002] Dengue fever is an acute arboviral disease transmitted by mosquitoes, and about 400 million people are infected with dengue virus every year. It is estimated that about 96 million infected people will have severe fever, rash, muscle and joint pain for a week, and the risk of internal bleeding and death will increase if infected with the virus. There is currently no specific drug to treat the disease. Therefore, the research and development of new anti-dengue virus with low toxicity or minimal (or no) side effects have important clinical significance. Natural products are an important source of drug discovery. Searching for antiviral therapeutic agents from medicinal plants has become a research hotspot, and a variety of active ingredients with antiviral activity have been found from them. Aglaia edulis is a tree of the family Meliaceae and genus Aglaia. It has the effects of treating dizziness, fever, cough, skin inflammation, promoting blood circulation to remove blood stasis, relieving swelling and pain, and regulating qi and relieving depression. Modern pharmacology shows that the plant extract has various activities such as anti-tumor, insecticidal, antibacterial and antiviral. Chemical composition research on Aglaia edulis shows that its main chemical components include cyclopentane benzofuran, limonoids, bisamides, steroids, flavonoids and other structural types, showing structural diversity. SUMMARY

[0003] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of the present application is to provide a limonoid compound Aglatestate F, a preparation method and use thereof.

[0004] The technical scheme adopted to achieve the above-mentioned purpose is as follows:

[0005] 1. A limonoid compound Aglatestate F, having the structural formula of formula (1):

[0006]

[0007] The compound is isolated and extracted from the branches and leaves of Aglaia edulis.

[0008] The limonoid compound Aglatestate F is used in the preparation of an anti-dengue virus active drug.

[0009] The limonoid compound Aglatestate F is used in the preparation of an anti-dengue virus active drug, and the compound Aglatestate F shows anti-dengue virus activity in a dose-dependent manner.

[0010] The compound Aglatestate F has good inhibitory effect on dengue virus infection of Syrian hamster kidney cells.

[0011] A pharmaceutical composition comprising the limonoid compound Aglatestate F, optionally comprising a pharmaceutically acceptable carrier.

[0012] The pharmaceutical composition comprising the limonoid compound Aglatestate F and a pharmaceutically acceptable carrier, which contains 0.1-99% of the limonoid compound Aglatestate F by mass fraction, and the rest is a pharmaceutical carrier or excipient.

[0013] The dosage form of the medicine is any one of tablet, capsule and granule.

[0014] The preparation method of the limonoid compound Aglatestate F comprises the following steps:

[0015] A. Take 50 parts of the branches and leaves of Euptelea pleiosandra, crush, and extract 4 times at room temperature with 200 parts of 95% industrial methanol, each time for 5 days, and recover the alcohol to obtain 5 parts of extract;

[0016] B. The extract of the crude extract is subjected to silica gel column chromatography, gradient elution is performed with eluent of petroleum ether and acetone in a volume ratio of 9:1-1:1, 100 parts per gradient, and thin layer chromatography technology is used to detect different elution fractions, and the fractions with the same thin layer chromatography results are combined, and seven fractions are obtained according to the thin layer chromatography performance: Fr.1, Fr.2, Fr.3, Fr.4, Fr.5, Fr.6, Fr.7;

[0017] C. Fr.6 in step B is subjected to MCI column chromatography, gradient elution is performed with eluent of methanol and water in a volume ratio of 1:1-9:1, and is combined according to the thin layer chromatography performance, and is divided into 4 sub-fractions: Fr.6a, Fr.6b, Fr.6c, Fr.6d; Fr.6b is subjected to reverse column chromatography, gradient elution is performed with eluent of methanol and water in a volume ratio of 1:1-9:1, and is divided into 9 sub-fractions: Fr.6b.1, Fr.6b.2, Fr.6b.3, Fr.6b.4, Fr.6b.5, Fr.6b.6, Fr.6b.7, Fr.6b.8 and Fr.6b.9; Fr.6b.7 is subjected to normal phase silica gel column chromatography, gradient elution is performed with eluent of dichloromethane / ethyl acetate in a volume ratio of 9:1-1:1, and compound 1 is obtained. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Nuclear magnetic resonance hydrogen spectrum of the compound Aglatestate F

[0019] Figure 2 The carbon NMR spectrum of compound Aglatestate F Detailed Implementation

[0020] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0021] Example 1: Preparation of compound Aglatestate F

[0022] A method for preparing a limonene compound includes the following steps:

[0023] (1) Take 50 parts of the branches and leaves of the horse kidney fruit, crush them, use 200 parts of 95% industrial methanol, extract them 4 times at room temperature, each time for 5 days, and recover the alcohol and concentrate to obtain 5 parts of extract.

[0024] (2) The crude extract was subjected to silica gel column chromatography with a gradient elution of petroleum ether to acetone in a volume ratio of 9:1 to 1:1. 100 fractions of each gradient were eluted. Silica gel thin-layer chromatography was used to detect the different eluted fractions. Fractions with the same thin-layer chromatographic results were combined to obtain seven fractions based on their thin-layer chromatographic performance: Fr.1, Fr.2, Fr.3, Fr.4, Fr.5, Fr.6, and Fr.7.

[0025] (3) Fr.6 was subjected to MCI column chromatography with a methanol to water volume ratio of 1:1 to 9:1 as an eluent gradient. The fractions were combined according to their thin-layer chromatographic properties and were divided into four subfractions: Fr.6a, Fr.6b, Fr.6c, and Fr.6d. Fr.6b was subjected to reverse column chromatography with a methanol to water volume ratio of 1:1 to 9:1 as an eluent gradient and was divided into nine subfractions: Fr.6b.1, Fr.6b.2, Fr.6b.3, Fr.6b.4, Fr.6b.5, Fr.6b.6, Fr.6b.7, Fr.6b.8, and Fr.6b.9. Fr.6b.7 was subjected to normal-phase silica gel column chromatography with a dichloromethane / ethyl acetate volume ratio of 9:1 to 1:1 as an eluent gradient to obtain compound Aglatestate F.

[0026] Example 2: Structural identification of compound Aglatestate F

[0027] The compound was analyzed by high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy. 1 H NMR, 13 A comprehensive analysis of data from C NMR, 2D-NMR, ultraviolet spectroscopy, infrared spectroscopy, and optical rotation was conducted to determine the structural formula of compound 1 (Aglatestate F), as shown below:

[0028]

[0029] Compound Aglatestate F: yellowish oil, molecular formula is C 28 H 34 O 10 (c 0.47, MeOH); UV (MeOH) λ max (logε) 201 (4.24) nm; IR (KBr) v max 3446, 2928, 1726, 1393, 1374, 1283, 1222, 1166, 1116, 1084 and 1027 cm -1 ; HR-ESI-MS m / z 553.20442 [M+Na] + (calcd. for C 28 H 34 O 10 Na, 553.21520); NMR data as shown in Table 1.

[0030] Table 1: NMR data of Compound Aglatestate F, 1 H (600 MHz) and 13 C (150 MHz) NMR Data of 1 in CD3Cl.

[0031]

[0032] Example 3: Anti-dengue virus infection activity verification

[0033] In order to further verify the beneficial effects of the compounds described in the present application, the dengue virus inhibitory activity of the compound Aglatestate F on Syrian hamster kidney (BHK-21) cells was studied, and the specific experiment was as follows:

[0034] (1) MTT method was used to detect the cytotoxicity of the drug: the Syrian hamster kidney cell (BHK-21) line was inoculated into 96-well microplates (5 x 10 3 ​cells), 37℃, 5% CO2 for 24 hours. After the cells grow into a monolayer, the culture supernatant is discarded, and DMEM medium containing a gradient dilution of the test compound is added, with three replicate wells for each concentration, and normal cell controls, solvent controls, and blank controls are set up. After 2 days of culture, 10 μL of 5 mg / mL MTT is added to each well, and incubation is continued in a 37℃ constant-temperature incubator for 4 hours. 100 μL of supernatant is discarded, 160 μL of DMSO solution is added, and shaking is continued at 37℃ for 15 minutes. After the crystals are completely dissolved, the OD value is detected by a Bio-TEK enzyme label meter (detection wavelength 490 nm). A dose-response curve is plotted according to the experimental results, and the 50% inhibition of cell growth concentration (CC 50 ) value is calculated using the Reed & Muench method.

[0035] (2) Virus plaque assay to detect the effect of the compound on the virus plaque: BHK-21 cells are plated at 2×10 5 cells / well in a 12-well plate and incubated overnight at 37℃ in a 5% CO2 incubator. The virus-free control group is added with DMEM medium, the drug group and the virus group are added with DENV-2 (MOI=0.01), and after 2 hours of adsorption at 37℃, the supernatant is removed and washed twice with PBS. The drug group is added with 1 mL of maintenance medium containing aglaiastatin (0.25, 0.5, 1, 2, 4 μM), and the virus group is added with 1 mL of maintenance medium containing DMSO. After 48 hours of culture, the supernatant is removed and stored at -80℃. BHK-21 cells are plated at 2×10 5 cells / well in a 12-well plate and incubated overnight at 37℃ in a 5% CO2 incubator. The supernatant is discarded, and the cells are washed with PBS. 300 μL of the supernatant sample is added to each well. After 2 hours of adsorption at 37℃ in an incubator, the supernatant is removed and washed with PBS. 1 mL of mixed medium of 4% FBS DMEM medium and 2% low-melting-point agarose is added to each well. After solidification, the plate is placed in a 37℃ incubator and incubated for 6-7 days. 4% paraformaldehyde is used for fixation for 30 minutes, and the agarose cover is discarded by inversion. 1% crystal violet staining solution is used for staining for 20 minutes, and the plate is washed with flowing water. The plate is air-dried at room temperature. The number of plaques is counted by enzyme-linked plaque assay (CTL), and a dose-response curve is plotted according to the experimental results. The EC 50 of the test sample is calculated.

[0036] (3) Anti-DENV efficacy evaluation: The therapeutic index (TI) is the ratio of the 50% inhibition concentration of the drug on cells (CC 50 ) to the 50% effective concentration of the drug on viruses (EC 50 ), which represents the safety of the drug. The larger the value, the safer the drug.

[0037] (4) The experimental results show that the compound has good inhibitory effect on dengue virus BHK-21 cell infection at non-toxic concentration, and the anti-dengue virus effect EC 50 = 1.290 ± 0.170 and the selectivity index is higher than that of the positive drug ribavirin (antiviral drug), so the compound Aglatestate F has good prospects for developing anti-dengue virus drugs.

[0038] The cytotoxicity and anti-dengue virus activity of the compound (Table 2) are as follows:

[0039] Table 2: Cytotoxicity and anti-dengue virus activity of the compound

[0040]

[0041] Note: Ribavirin positive control ribavirin

[0042] The above description is only a preferred embodiment of the present application, and is not intended to limit the other forms of the present application. Any person skilled in the art can use the disclosed technical content to make changes or modifications to equivalent embodiments. However, any simple modification, equivalent change and modification of the above embodiments without departing from the technical solution of the present application, according to the technical essence of the present application, still belongs to the protection scope of the technical solution of the present application.

Claims

1. Use of a limonoid compound Aglatestate F in the preparation of a drug for resisting dengue virus, the structural formula of the limonoid compound Aglatestate F is formula (1): ###0001### Formula (1) The compound Aglatestate F shows anti-dengue virus activity in a dose-dependent manner. 。 2. The use of limonoids Aglatestate F according to claim 1 for the preparation of an antiviral drug against dengue virus, characterized in that, The compound Aglatestate F has good inhibitory effect on dengue virus infection of Syrian hamster kidney cells.

3. The use of limonoids Aglatestate F according to claim 1 for the preparation of an antiviral drug against dengue virus, characterized in that, The pharmaceutical composition comprises the limonoid compound Aglatestate F, and optionally comprises a pharmaceutically acceptable carrier, the structural formula of the limonoid compound Aglatestate F is formula (1): ###0001### Formula (1) 4. A pharmaceutical composition, characterized by, 5. The pharmaceutical composition of claim 4, comprising the limonoid compound Aglatestate F and a pharmaceutically acceptable carrier, the pharmaceutical composition contains 0.1-99% of the limonoid compound Aglatestate F by mass fraction, and the rest is a pharmaceutical carrier. 。 The dosage form of the drug is any one of a tablet, a capsule, and a granule.

6. The pharmaceutical composition of claim 4, wherein, The method comprises the following steps:

7. A method for preparing a limonoid compound Aglatestate F, characterized by: A. Take 50 parts of equisetum arvense, crush, and extract 4 times at room temperature using 95% industrial methanol 200 parts, each time for 5 days, and recover the alcohol to obtain 5 parts of extract; B. The extract is subjected to silica gel column chromatography, gradient elution is performed using petroleum ether and acetone in a volume ratio of 9:1~1:1, 100 parts per gradient, and thin layer chromatography is used to detect different elution fractions, and the fractions with the same thin layer chromatography results are combined, and seven fractions are obtained according to the thin layer chromatography: Fr. 1, Fr. 2, Fr. 3, Fr. 4, Fr. 5, Fr. 6, and Fr. 7; C. Fr. 6 in step B is subjected to MCI column chromatography, gradient elution is performed using methanol and water in a volume ratio of 1:1~9:1, and according to the thin layer chromatography, it is combined into 4 sub-fractions: Fr. 6a, Fr. 6b, Fr. 6c, and Fr. 6d; Fr. 6b is subjected to reverse C18 column chromatography, gradient elution is performed using methanol and water in a volume ratio of 1:1~9:1, and it is divided into 9 sub-fractions: Fr. 6b.1, Fr. 6b.2, Fr. 6b.3, Fr. 6b.4, Fr. 6b.5, Fr. 6b.6, Fr. 6b.7, Fr. 6b.8, and Fr. 6b.9; Fr. 6b.7 is subjected to normal phase silica gel column chromatography, gradient elution is performed using dichloromethane / ethyl acetate in a volume ratio of 9:1~1:1, and compound Aglatestate F is obtained, the structural formula of the limonoid compound Aglatestate F is formula (1): ###0001### Formula (1) ​ 。

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