Limonin compound Aglatestate F as well as preparation method and application thereof

Abstract

The invention relates to a limonin compound Aglatestate F as well as a preparation method and application thereof, and belongs to the field of chemical medicines. The compound is obtained by separating and extracting from branches and leaves of fruit of Japanese pedicularis (Aglaia edulis), and is simple to prepare and separate. The compound provided by the invention has a good effect of inhibiting dengue virus narrative hamster kidney (BHK-21) cell infection, has a good effect of inhibiting the dengue virus narrative hamster kidney (BHK-21) cell infection at a non-toxic concentration, has a dengue virus resisting effect EC50 of 1.290 + / -0.170, has a selection index higher than that of a positive antiviral drug ribavirin, and has a good application prospect. Therefore, the compound Aglatestate F has a good prospect of developing the anti-dengue-virus medicine, and a solid foundation is laid for the development of preparing the anti-dengue-virus medicine.

Description

Technical Field

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

[0002] Dengue fever is an acute insect-borne infectious disease caused by mosquitoes. 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 lasting for a week. Virus infection will increase the risk of internal bleeding and death. 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 is of great clinical significance. Natural products are an important source of drug discovery. Finding antiviral therapeutic agents from medicinal plants has become a research hotspot, and many active ingredients with antiviral properties have been found. Aglaia edulis is a tree of the genus Aglaia in the family Meliaceae. It has the effects of treating dizziness, fever, cough, skin inflammation, as well as promoting blood circulation, removing blood stasis, reducing swelling and relieving pain, and promoting qi and relieving depression. Modern pharmacology shows that the plant extract has multiple activities such as anti-tumor, insecticidal, antibacterial and antiviral. Research on the chemical composition of the horse kidney fruit found that its main chemical components include cyclopentanebenzofuran, limonin, diamide, steroids, flavonoids and other structural types, showing a diversity of structural types. Summary of the invention

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

[0004] The technical solutions adopted to achieve the above purpose are as follows:

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

[0006]

[0007] The compound is separated and extracted from the branches and leaves of the fruit of the genus Psoralea corylifolia.

[0008] The application of the limonoid compound Aglatestate F in the preparation of anti-dengue virus active drugs.

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

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

[0011] A pharmaceutical composition comprises the limonoid compound Aglatestate F and optionally contains a pharmaceutically acceptable carrier.

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

[0013] The dosage form of the medicine is any one of tablets, capsules and granules.

[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 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, recover the alcohol and concentrate it to obtain 5 parts of extract;

[0016] B. The crude extract was subjected to silica gel column chromatography, and a gradient elution was performed using an eluent having a volume ratio of petroleum ether to acetone of 9:1 to 1:1, with 100 portions for each gradient. Different elution fractions were detected using silica gel thin layer chromatography technology, and fractions with the same thin layer chromatography results were combined. According to the thin layer chromatography performance, seven fractions were obtained: Fr.1, Fr.2, Fr.3, Fr.4, Fr.5, Fr.6, and Fr.7;

[0017] C. Fr.6 in step B was subjected to MCI column chromatography, using a gradient elution with a volume ratio of methanol to water of 1:1 to 9:1 as the eluent, and the fractions were combined according to their thin layer chromatography performance to be divided into 4 sub-fractions: Fr.6a, Fr.6b, Fr.6c, and Fr.6d; Fr.6b was subjected to reverse column chromatography, using a gradient elution with a volume ratio of methanol to water of 1:1 to 9:1 as the eluent, to be divided into 9 sub-fractions, namely 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, using a gradient elution with a volume ratio of dichloromethane to ethyl acetate of 9:1 to 1:1 as the eluent to obtain compound 1. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the H NMR spectrum of compound Aglatestate F

[0019] Figure 2 This is the carbon NMR spectrum of compound Aglatestate F DETAILED DESCRIPTION

[0020] The present invention will be further described below in conjunction with the embodiments, but they are not intended to limit the present invention.

[0021] Example 1: Preparation of Compound Aglatestate F

[0022] A method for preparing a limonoid compound comprises the following steps:

[0023] (1) 50 parts of the branches and leaves of the spatholobus spatholobus were taken, crushed, and extracted with 200 parts of 95% industrial methanol at room temperature for 4 times, each time for 5 days, and the alcohol was recovered and concentrated to obtain 5 parts of an extract;

[0024] (2) The crude extract was subjected to silica gel column chromatography, using a gradient elution with a volume ratio of petroleum ether to acetone of 9:1 to 1:1, with 100 parts per gradient. Different elution fractions were detected using silica gel thin layer chromatography technology, and fractions with the same thin layer chromatography results were combined to obtain seven fractions according to the thin layer chromatography 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, using a gradient elution with a volume ratio of methanol to water of 1:1 to 9:1 as the eluent, and was combined according to the thin layer chromatography performance to be divided into 4 sub-fractions: Fr.6a, Fr.6b, Fr.6c, and Fr.6d; Fr.6b was subjected to reverse column chromatography, using a gradient elution with a volume ratio of methanol to water of 1:1 to 9:1 as the eluent, and was 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 was subjected to normal phase silica gel column chromatography, using a gradient elution with a volume ratio of dichloromethane to ethyl acetate of 9:1 to 1:1 as the eluent, to obtain compound Aglatestate F.

[0026] Example 2: Structural Identification of Compound Aglatestate F

[0027] The compounds were characterized by high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy ( 1 H NMR, 13 C NMR, 2D-NMR), UV spectrum, infrared spectrum, optical rotation and other data were comprehensively analyzed to determine the structural formula of compound 1 (Aglatestate F), as shown below:

[0028]

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

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

[0031]

[0032] Example 3: Verification of anti-dengue virus infection activity

[0033] In order to further verify the beneficial effects of the compound of the present invention, the compound Aglatestate F was used to study the dengue virus inhibition activity in Syrian hamster kidney (BHK-21) cells. The specific experiment is as follows:

[0034] (1) MTT assay for drug cytotoxicity: Syrian hamster kidney cells (BHK-21) were inoculated into 96-well microplates (5 × 10 3cells), cultured at 37°C, 5% CO2 for 24 hours. After the cells grow into a monolayer, the culture supernatant is discarded, and DMEM medium containing gradient dilutions of the test compound is added. Three replicate wells are set for each concentration, and normal cell control, solvent control, and blank control are set. After culturing for 2 days, 10 μL of 5 mg / mL MTT is added to each well, and the cells are incubated in a 37°C constant temperature incubator for 4 hours. 100 μL of supernatant is discarded, and 160 μL of DMSO solution is added. The cells are shaken at 37°C for 15 minutes. After the crystals are completely dissolved, the OD value is detected by Bio-TEK microplate reader (detection wavelength 490 nm); a dose-response curve is drawn based on the experimental results, and the 50% inhibition of cell growth concentration (CC 50 )value.

[0035] (2) Virus plaque assay to detect the effect of compounds on virus plaques: BHK-21 cells were cultured at 2×10 5 100 μL / well were plated in a 12-well plate and cultured overnight in a 37°C, 5% CO2 incubator; DMEM medium was added to the virus-free control group, and DENV-2 (MOI = 0.01) was added to the drug group and the virus group. After adsorption at 37°C for 2 hours, the supernatant was removed and washed twice with PBS. 1 mL of maintenance medium containing aglaiastatin (0.25, 0.5, 1, 2, 4 μM) was added to the drug group, and 1 mL of maintenance medium containing DMSO was added to the virus group. After 48 hours of culture, the supernatant was frozen at -80°C. BHK-21 cells were cultured at 2×10 5 100 μL / well of the plate was plated in a 12-well plate and cultured in a 37°C, 5% CO2 incubator overnight; the supernatant was discarded, washed with PBS, and 300 μL of the supernatant sample was added to each well. Placed in an incubator at 37°C for adsorption for 2 hours; the supernatant was discarded and washed with PBS, and 1 mL of a mixed culture medium of 4% FBSDMEM culture medium and 2% low-melting-point agarose was added to each well. After solidification, it was placed in a 37°C incubator and cultured for 6 to 7 days; 4% paraformaldehyde was used for fixation for 30 minutes, and the agarose covering was discarded upside down; 1% crystal violet staining solution was used for staining for 20 minutes, and the plate was washed with running water; the plate was dried at room temperature. The number of plaques was counted by enzyme-linked fluorescent spot analyzer (CTL), and a dose-response curve was drawn according to the experimental results to calculate the EC of the sample to be tested. 50 .

[0036] (3) Evaluation of anti-DENV efficacy: The therapeutic index (TI) is the half inhibitory concentration CC of the drug on cells. 50 and the half effective concentration (EC) of the virus 50 The ratio represents the safety of the drug. The larger the value, the safer it is.

[0037] (4) The experimental results showed that at non-toxic concentrations, the compound had a good inhibitory effect on dengue virus infection in Syrian hamster kidney (BHK-21) cells, and its 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 compound Aglatestate F has good prospects for developing anti-dengue virus drugs.

[0038] The cytotoxicity and anti-dengue virus activity of the compounds (Table 2) are shown below:

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

[0040]

[0041] Note: Ribavirin positive control ribavirin

[0042] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims

1. A limonoid compound Aglatestate F, having the structural formula (1):

2. The limonoid compound Aglatestate F according to claim 1, characterized in that: The compound is separated and extracted from the branches and leaves of the fruit of the genus Psoralea corylifolia.

3. Use of the limonoid compound Aglatestate F described in claim 1 in the preparation of anti-dengue virus drugs.

4. The use of the limonoid compound Aglatestate F according to claim 3 in the preparation of anti-dengue virus drugs, characterized in that: The compound Aglatestate F exhibits anti-dengue virus activity in a dose-dependent manner.

5. The use of the limonoid compound according to claim 3 in anti-dengue virus drugs, characterized in that: The compound Aglatestate F has a good inhibitory effect on dengue virus infection of Syrian hamster kidney cells.

6. A pharmaceutical composition, characterized in that The limonoid compound Aglatestate F according to claim 1 optionally comprises a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, comprising the limonoid compound Aglatestate F and a pharmaceutically acceptable carrier, wherein the mass fraction of the limonoid compound Aglatestate F is 0.1-99%, and the rest is a pharmaceutical carrier or excipient.

8. A pharmaceutical composition according to claim 6, characterized in that: The dosage form of the medicine is any one of tablets, capsules and granules.

9. A method for preparing the limonoid compound Aglatestate F according to claim 1, characterized in that: The following steps are involved: A. 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, recover the alcohol and concentrate it to obtain 5 parts of extract; B. The crude extract was subjected to silica gel column chromatography, and a gradient elution was performed using an eluent having a volume ratio of petroleum ether to acetone of 9:1 to 1:1, with 100 portions for each gradient. Different elution fractions were detected using silica gel thin layer chromatography technology, and fractions with the same thin layer chromatography results were combined. According to the thin layer chromatography performance, seven fractions were obtained: Fr.1, Fr.2, Fr.3, Fr.4, Fr.5, Fr.6, and Fr.7; C. Fr.6 in step B was subjected to MCI column chromatography, using a gradient elution with a volume ratio of methanol to water of 1:1 to 9:1 as the eluent, and was combined according to the thin layer chromatography performance to be divided into 4 sub-fractions: Fr.6a, Fr.6b, Fr.6c, and Fr.6d; Fr.6b was subjected to reverse C18 column chromatography, using a gradient elution with a volume ratio of methanol to water of 1:1 to 9:1 as the eluent, and was divided into 9 sub-fractions, namely 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, using a gradient elution with a volume ratio of dichloromethane to ethyl acetate of 9:1 to 1:1 as the eluent, to obtain compound Aglatestate F.

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