A furanocoumarin compound, a preparation method and application thereof

Abstract

The application discloses a furan coumarin compound and a preparation method and application thereof, and the molecular formula of the furan coumarin compound is C 21 H 20 O6; the furan coumarin compound can reduce the inflammatory reaction of RAW264.7 macrophages induced by lipopolysaccharide (LPS), can reduce the NO production of RAW264.7 macrophages induced by LPS, and can significantly inhibit the production of inflammatory factors such as TNF-alpha, interleukin-6 (IL-6) and interleukin-1 beta (IL-1 beta).

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a furanocoumarin compound and a preparation method and application thereof. BACKGROUND

[0002] Notopterygium incisum Ting ex H.T.Chang or Notopterygium franchetii H.de Boiss. Chemical components are the material basis for the effect of traditional Chinese medicine and are also important indexes for quality control. At present, more than 400 components have been isolated from Notopterygium, and the structures are complex. Specifically, the components can be divided into volatile oil, coumarin, enyne, sesquiterpene, organic acid and organic acid ester, flavone, steroid and other types. Coumarin is the main effective component in Notopterygium and has a high content. Studies have shown that coumarin compounds have a wide range of pharmacological activities, such as anti-inflammatory, antioxidant, antiviral, antitumor, anticoagulant, antibacterial and immune function enhancement [2-3], and basic research on the anti-inflammatory activity of coumarin compounds is more. SUMMARY

[0003] The main purpose of the present application is to provide a furanocoumarin compound and a preparation method and application thereof. The compound can reduce the inflammatory response of RAW264.7 macrophages induced by lipopolysaccharide (LPS), can reduce the production of NO of RAW264.7 macrophages induced by LPS, and can significantly inhibit the production of inflammatory factors such as TNF-α, interleukin-6 (IL-6) and interleukin-1β (IL-1β).

[0004] In order to achieve the above purpose, the present application provides the following technical scheme:

[0005] A furanocoumarin compound, the structural formula of the furanocoumarin compound is:

[0006] .

[0007] As a preferred embodiment, the furanocoumarin compound is extracted from Notopterygium.

[0008] In a second aspect of the present application, a preparation method of the furanocoumarin compound is provided, comprising the following steps:

[0009] (1) Notopterygium is extracted by ethanol reflux, the extract is concentrated, and flow infusion paste treatment is performed to obtain flow infusion paste;

[0010] (2) The obtained flow infusion extract of step (1) is dispersed with distilled water, purified by D101 adsorption resin, and gradient eluted with methanol-distilled water eluent;

[0011] (3) The obtained product of step (2) is purified by silica gel column chromatography, and gradient eluted with petroleum ether-acetone;

[0012] (4) The obtained product of step (3) is purified by axial compression column, eluted with methanol-distilled water, and the eluted product is treated to obtain a furanocoumarin compound.

[0013] As a preferred embodiment of the above method for preparing a furanocoumarin compound, in step (1), the Notopterygium is crushed and extracted with 8 times the amount of 95% ethanol by refluxing; the flow infusion extract is treated by dispersing the concentrated solution with distilled water, and the dispersion is placed in a dialysis bag and soaked in distilled water for 12 days, with water changed twice a day. After soaking, the dialysis solution is concentrated to obtain the flow infusion extract.

[0014] As a preferred embodiment of the above method for preparing a furanocoumarin compound, in step (2), the methanol-distilled water gradient elution is as follows:

[0015] The elution time is 0-40 hours, the eluent is distilled water, and the eluent flow rate is 5 ml / min;

[0016] The elution time is 40-80 hours, the eluent is methanol-distilled water with a volume ratio of 30:70, the eluent flow rate is 5 ml / min, and the 30% methanol eluted product is obtained;

[0017] The elution time is 80-120 hours, the eluent is methanol-distilled water with a volume ratio of 60:40, the eluent flow rate is 5 ml / min, and the 60% methanol eluted product is obtained;

[0018] The elution time is 120-160 hours, the eluent is methanol-distilled water with a volume ratio of 90:10, the eluent flow rate is 5 ml / min, and the 90% methanol eluted product is obtained.

[0019] As a preferred embodiment of the above method for preparing a furanocoumarin compound, in step (3), the 30% methanol eluted product obtained in step (2) is purified by silica gel column chromatography, and the petroleum ether-acetone gradient elution is as follows:

[0020] The elution time is 0-20 hours, the eluent is petroleum ether-acetone with a volume ratio of 30:1, and the eluent flow rate is 5 ml / min;

[0021] The elution time is 20-40 hours, the eluent is petroleum ether-acetone with a volume ratio of 25:1, and the eluent flow rate is 5 ml / min;

[0022] The elution time is 40-60 hours, the eluent is petroleum ether-acetone with a volume ratio of 20:1, and the eluent flow rate is 5 ml / min;

[0023] The elution time is 60-80 hours, the eluent is petroleum ether-acetone with a volume ratio of 15:1, and the eluent flow rate is 5 ml / min;

[0024] The elution time is 80-100 hours, the eluent is petroleum ether-acetone with a volume ratio of 10:1, and the eluent flow rate is 5 ml / min;

[0025] The elution time is 100-120 hours, the eluent is petroleum ether-acetone with a volume ratio of 5:1, and the eluent flow rate is 5 ml / min;

[0026] The elution time is 120-140 hours, the eluent is petroleum ether-acetone with a volume ratio of 1:1, and the eluent flow rate is 5 ml / min.

[0027] As a preferred embodiment of the above preparation method of the furanocoumarin compound, in step (4), the eluted product is purified by using an axial compression column with the eluent of petroleum ether-acetone with a volume ratio of 1:1;

[0028] Preferably, in step (4), when methanol-distilled water is used for elution, the volume ratio of methanol-distilled water is 65:35; the eluted part is divided into 8 segments by using an axial compression column, which facilitates subsequent passing through a normal silica gel column and preparing liquid phase, and the retention time is 20 min;

[0029] Further preferably, in step (4), the eluted product is treated by semi-preparative liquid phase to obtain the furanocoumarin compound.

[0030] As a preferred embodiment of the above preparation method of the furanocoumarin compound, the mobile phase of the semi-preparative liquid phase is a mixture of acetonitrile and water with a volume ratio of 7:13, the flow rate is 3 ml / min, the detection wavelength is 254 nm, and the column temperature is 30°C.

[0031] In a third aspect of the present application, a use of the above furanocoumarin compound or a pharmaceutically acceptable salt thereof in preparing an anti-inflammatory drug is provided.

[0032] Preferably, the anti-inflammatory drug further comprises a pharmaceutically acceptable carrier or excipient.

[0033] The furanocoumarin compound can reduce the inflammatory response of RAW264.7 macrophages induced by lipopolysaccharide (LPS), can reduce the production of NO of RAW264.7 macrophages induced by LPS, and can significantly inhibit the production of inflammatory factors such as TNF-α, interleukin-6 (IL-6), interleukin-1β (IL-1β). BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 ESI-MS spectrum of the furanocoumarin compound described in the present application;

[0035] Figure 2 UV spectrum of the furanocoumarin compound described in the present application in MeOH;

[0036] Figure 3 Infrared spectrum of the furanocoumarin compound described in the present application in MeOH;

[0037] Figure 4 1H NMR spectrum of the furanocoumarin compound described in the present application in CDCl3;

[0038] Figure 5 13C nuclear magnetic resonance spectrum of the furanocoumarin compound described in the present application in CDCl3;

[0039] Figure 6 1H-1H COSY spectrum of the furanocoumarin compound described in the present application in CDCl3;

[0040] Figure 7 HSQC spectrum of the furanocoumarin compound described in the present application in CDCl3;

[0041] Figure 8 HMBC spectrum of the furanocoumarin compound described in the present application in CDCl3;

[0042] Figure 9 Noise spectrum of the furanocoumarin compound described in the present application in CDCl3;

[0043] Figure 10 Calculated and experimental ECD spectrum of the furanocoumarin compound described in the present application in MeOH;

[0044] Figure 11 The most stable 5s-1 conformation WB97XD / DGDZVP level map in methanol is calculated by discrete Fourier transform (DFT);

[0045] Figure 12 CCK-8 method for detecting the activity of RAW264.7 macrophages;

[0046] Figure 13 Effect of the furanocoumarins compounds described in the present application on NO production of LPS-stimulated macrophages;

[0047] Figure 14 Effect of the furanocoumarins compounds described in the present application on mRNA production of LPS-stimulated macrophages. DETAILED DESCRIPTION

[0048] In order to make the person skilled in the art better understand the scheme of the present application, the technical scheme in the embodiments of the present application will be described clearly and completely in combination with cases. Obviously, the described embodiments are only a part of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by the person skilled in the art without making creative labor should belong to the protection scope of the present application.

[0049] The experimental instruments used in the embodiments of the present application are shown in Table 1:

[0050] Table 1

[0051]

[0052]

[0053] The materials and reagents used in the embodiments of the present application are shown in Table 2:

[0054] Table 2

[0055]

[0056]

[0057] The furanocoumarins compounds described in the present application are isolated from Notopterygium incisum, and the structure of the new compound is characterized by nuclear magnetic resonance, electrospray mass spectrometry (ESIMS), calculated nuclear magnetic resonance, CD, etc. The compounds described in the present application can inhibit the mRNA expression of inflammatory factors TNF-α, IL-1β and IL-6 of LPS-induced RAW264.7 macrophages by reducing the production of inflammatory factors such as NO, TNF-α, IL-1β and IL-6, and exhibit anti-inflammatory properties in vitro.

[0058] Embodiment 1

[0059] A furanocoumarins compound, the structural formula of which is:

[0060]

[0061] The furanocoumarins compounds described in the present embodiment are extracted from Notopterygium incisum.

[0062] The specific extraction method is:

[0063] (1) 100 kg of Notopterygium root is crushed and extracted with 8 times the amount of 95% ethanol by reflux. After the ethanol is removed, the concentrated solution is dispersed with distilled water, and the dispersion is placed in a dialysis bag. Distilled water is used to soak the bag for 12 days, with water changed twice a day. After soaking, the dialysate is concentrated to obtain 1 kg of a flow infusion extract;

[0064] (2) The flow infusion extract obtained in step (1) is dispersed with distilled water and loaded onto a D101 macroporous adsorption resin. Gradient elution is performed using methanol-distilled water as the eluent:

[0065] The specific elution method for methanol-distilled water is:

[0066] Elution time: 0-40 hours, eluent: distilled water, eluent flow rate: 5 ml / min;

[0067] Elution time: 40-80 hours, eluent: methanol-distilled water with a volume ratio of 30:70, eluent flow rate: 5 ml / min, to obtain a 30% methanol elution product;

[0068] Elution time: 80-120 hours, eluent: methanol-distilled water with a volume ratio of 60:40, eluent flow rate: 5 ml / min, to obtain a 60% methanol elution product;

[0069] Elution time: 120-160 hours, eluent: methanol-distilled water with a volume ratio of 90:10, eluent flow rate: 5 ml / min, to obtain a 90% methanol elution product;

[0070] (3) The 30% methanol elution product obtained in step (2) is purified using a silica gel column chromatography, and gradient elution is performed using petroleum ether-acetone:

[0071] The gradient elution method for petroleum ether-acetone is:

[0072] Elution time: 0-20 hours, eluent: petroleum ether-acetone with a volume ratio of 30:1, eluent flow rate: 5 ml / min;

[0073] Elution time: 20-40 hours, eluent: petroleum ether-acetone with a volume ratio of 25:1, eluent flow rate: 5 ml / min;

[0074] Elution time: 40-60 hours, eluent: petroleum ether-acetone with a volume ratio of 20:1, eluent flow rate: 5 ml / min;

[0075] Elution time: 60-80 hours, eluent: petroleum ether-acetone with a volume ratio of 15:1, eluent flow rate: 5 ml / min;

[0076] Elution time 80-100 hours, eluent petroleum ether-acetone (10:1, by volume), eluent flow rate 5 ml / min;

[0077] Elution time 100-120 hours, eluent petroleum ether-acetone (5:1, by volume), eluent flow rate 5 ml / min;

[0078] Elution time 120-140 hours, eluent petroleum ether-acetone (1:1, by volume), eluent flow rate 5 ml / min;

[0079] (4) The product was eluted with petroleum ether-acetone (1:1, by volume) eluent and purified by axial compression column, and eluted with methanol-distilled water (65:35, by volume), and the eluted product was treated by semi-preparative liquid phase to obtain the furan coumarin compound;

[0080] The mobile phase of the semi-preparative liquid phase was a mixture of acetonitrile and water (7:13, by volume), the flow rate was 3 ml / min, the detection wavelength was 254 nm, and the column temperature was 30°C.

[0081] Spectral data of the furan coumarin compound obtained in Example 1

[0082] Theoretical calculation of the furan coumarin compound described in Example 1 was performed using Gaussian function 161: first, conformation analysis was performed using Conflex 8, and the conformation was generated by Boltzman Jump, and then the conformation was minimized using MMFF molecular mechanics force field using Smart Minimizer.

[0083] In B3LYP / 6-31G(d,p) gas and WB97XD / DGDZVP methanol, all geometric shapes with relative energy of 0-5.0 kcal / mol were used for optimization. The room temperature equilibrium population was calculated according to the Boltzmann distribution law. The theoretical calculation of ECD was performed using TD-DFT at the CAM-B3LYP / DGDZVP level in methanol. The ECD spectrum was obtained by weighting the Boltzmann distribution rate of each geometric conformation. The single CD spectrum was summed after Boltzmann statistical weighting using SpecDis 1.712, Gaussian curve (o=0.2eV) was generated, and compared with the experimental data, and the results are shown in Figure 10 and Figure 11 .

[0084] Yellow powder: C 21 H 20 O6; [α] D 20 +1(c 0.3, MeOH); IR (KBr) vmax 3443,2921,1726,1623,1454,1130,1069, cm -1 ; 1 H NMR (600 MHz, CDC13) δ: 6.29 (1H, d, J = 9.9 Hz, H-3), 8.13 (1H, d, J = 9.7 Hz, H-4), 7.17 (1H, s, H-8), 7.61 (1H d, H-9), 6.94 (1H, dd, J = 2.3, 1.0 Hz, H-10), 4.99 (2H, d, J = 6.7 Hz, H-1'a, H-1'b), 5.71 (1H t, J = 6.4 Hz, H-2'), 2.40 (1H dd, J = 13.7, 8.8 Hz, H-4'a), 2.34 (1H, dd, J = 13.7, 4.5 Hz, H-4'b), 4.80 (1H, td, J = 8.3, 4.5 Hz, H-5'), 6.41 (1H, dq, J = 7.9, 1.4 Hz, H-6'), 1.81 (3H s, H-1"), 1.80 (3H s, H-2");13C NMR (151 MHz, CDC13) δ: 161.3 (C-2), 113.0 (C-3), 139.4 (C-4), 148.7 (C-5), 114.1 (C-6), 158.3 (C-7), 94.6 (C-8), 145.3 (C-9), 105.0 (C-10), 107.5 (C-4a), 152.8 (C-8a), 69.3 (C-1'), 123.8 (C-2'), 137.8 (C-3'), 46.5 (C-4'), 66.5 (C-5'), 153.4 (C-6'), 139.0 (C-7'), 194.9 (C-8'), 17.1 (C-1"), 9.8 (C-2");

[0085] ESI HRMS m / z 369.1330 [M+H] + (C 21 H 21 O6+[M+H] + , 369.1333).

[0086] 1. CCK-8 method to detect cell viability

[0087] Take the logarithmic growth period RAW264.7 macrophages, 1 x 10 4The RAW264.7 macrophages were seeded in 96-well cell culture plates at a density of 2×10

[0088] The test results are shown in Table 1: Figure 12

[0089] As can be seen from Table 1, the furanocoumarin compounds of the present application have no toxic effect on macrophages at different concentrations. Figure 12 2. Determination of NO production

[0090] The RAW264.7 macrophages in logarithmic growth phase were seeded in 6-well cell culture plates at a density of 2×10 5 After 24 h of culture in a cell incubator, a certain amount of the furanocoumarin compound of Example 1 of the present application was added to the cell culture medium to obtain a final concentration of 2.5, 5 and 7.5 μM, and a positive control group was set up with a dexamethasone concentration of 30 μM. The solvent volume was adjusted with DMSO. After 2 h of pretreatment of the cells, LPS (1 μg / mL) was added. After 24 h of culture after administration, the cell culture plates were removed for processing. 50 μL of the supernatant of each group was taken to a 96-well plate, and three replicate wells were prepared for each group. 50 μL of Griess Reagent I and 50 μL of Griess Reagent II were added to each well, and the absorbance was detected at 540 nm using an enzyme marker.

[0091] The test results are shown in Table 2:

[0092] As can be seen from Table 2, the furanocoumarin compounds of the present application can inhibit the production of NO in RAW264.7 macrophages induced by LPS in a concentration-dependent manner. Figure 13 Figure 13 3. Reverse transcription-polymerase chain reaction (RT-PCR) analysis

[0093] The RAW264.7 macrophages were seeded in 6-well cell culture plates at a density of 2×10

[0094] ​​RAW264.7 macrophages in logarithmic growth phase were harvested at a rate of 2 × 10⁻⁶. 5 Cells were seeded at a density of cells / well in 6-well cell culture plates and cultured for 24 h in a cell culture incubator. A certain amount of the furanocoumarin compound described in this application was added to the cell culture medium to achieve final concentrations of 2.5, 5, and 7.5 μM. A positive control group was set up, and dexamethasone was added at a concentration of 15 μM. The solvent volume was balanced with DMSO, and after pretreatment for 2 h, LPS (1 μg / mL) was added. After 12 h of culture following drug administration, the cell culture plates were removed and processed. 1 mL of Trizol reagent was added to each well to extract RNA. The purity and concentration of RNA were detected using an ultraviolet spectrophotometer. The RNA was reverse transcribed into cDNA using the RT EasyTMII (WithgDNase) kit, and then the cDNA was amplified using the Real-Time PCR EasyTM-SYBR Green I kit. The gene levels of TNF-α, IL-1β, and IL-6 in the cells were measured, and the gene sequences are shown in Table 3.

[0095] Table 3

[0096]

[0097] from Figure 14 As can be seen, the furanocoumarin compounds described in this application significantly inhibited the expression of inflammatory factors TNF-α, IL-1β and IL-6 mRNA in LPS-induced RAW264.7 macrophages (****P<0.0001; ***P<0.001; **P<0.01; *P<0.05 compared with the LPS group).

[0098] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A furanocoumarin compound, characterized in that, The structural formula of the furanocoumarin compound is: The preparation method of the furanocoumarin compound includes the following steps: (1) The extract of Notopterygium incisum was extracted by ethanol reflux, the extract was concentrated, and the extract was treated to obtain a fluid extract. (2) The fluid extract obtained in step (1) is dispersed in distilled water, purified by D101 adsorption resin, and eluted by gradient elution with methanol-distilled water eluent. The methanol-distilled water gradient elution is as follows: Elution time: 0-40 hours; eluent: distilled water; eluent flow rate: 5 ml / min. The elution time was 40-80 hours, the eluent was methanol-distilled water with a volume ratio of 30:70, the flow rate of the eluent was 5 ml / min, and a 30% methanol eluent product was obtained. The elution time was 80-120 hours, the eluent was methanol-distilled water with a volume ratio of 60:40, the flow rate of the eluent was 5 ml / min, and 60% methanol elution product was obtained. The elution time was 120-160 hours, the eluent was methanol-distilled water with a volume ratio of 90:10, the flow rate of the eluent was 5 ml / min, and 90% methanol elution product was obtained. (3) The 30% methanol elution product obtained in step (2) was purified by silica gel column chromatography and eluted with petroleum ether-acetone gradient. The gradient elution of petroleum ether-acetone is as follows: Elution time: 0-20 hours; eluent: petroleum ether-acetone (volume ratio: 30:1); eluent flow rate: 5 ml / min. The elution time is 20-40 hours, the eluent is petroleum ether-acetone with a volume ratio of 25:1, and the eluent flow rate is 5 ml / min. The elution time is 40-60 hours, the eluent is petroleum ether-acetone with a volume ratio of 20:1, and the eluent flow rate is 5 ml / min. The elution time is 60-80 hours, the eluent is petroleum ether-acetone with a volume ratio of 15:1, and the eluent flow rate is 5 ml / min. The elution time is 80-100 hours, the eluent is petroleum ether-acetone with a volume ratio of 10:1, and the eluent flow rate is 5 ml / min. The elution time is 100-120 hours, the eluent is petroleum ether-acetone with a volume ratio of 5:1, and the eluent flow rate is 5 ml / min. The elution time is 120-140 hours, the eluent is petroleum ether-acetone with a volume ratio of 1:1, and the eluent flow rate is 5 ml / min. (4) The product obtained in step (3) is purified by axial compression column and eluted with methanol-distilled water. The eluted product is then processed to obtain furanocoumarin compounds.

2. The furanocoumarin compound according to claim 1, characterized in that, In step (1), the Notopterygium root is crushed and extracted by reflux with 8 times the amount of 95% ethanol; the fluid extract is processed by dispersing the concentrated solution with distilled water, and then soaking the dispersion in a dialysis bag in distilled water for 12 days, changing the water twice a day. After soaking, the dialysis solution is concentrated to obtain the fluid extract.

3. The furanocoumarin compound according to claim 1, characterized in that, In step (4), the elution product of the petroleum ether-acetone eluent with a volume ratio of 1:1 is purified by axial compression column.

4. The furanocoumarin compound according to claim 1, characterized in that, In step (4), when eluting with methanol-distilled water, the volume ratio of methanol to distilled water is 65:

35.

5. The furanocoumarin compound according to claim 1, characterized in that, In step (4), the elution product is processed by semi-preparative liquid phase to obtain furanocoumarin compounds.

6. The furanocoumarin compound according to claim 5, characterized in that, The mobile phase of the semi-preparative liquid phase was a mixture of acetonitrile and water with a volume ratio of 7:13, the flow rate was 3 ml / min, the detection wavelength was 254 nm, and the column temperature was 30 °C.

7. The use of a furanocoumarin compound or a pharmaceutically acceptable salt thereof as described in any one of claims 1-6 in the preparation of an anti-inflammatory drug.

8. The application according to claim 7, characterized in that, The anti-inflammatory drugs also include pharmaceutically acceptable carriers or excipients.

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