New compound and preparation method and anti-inflammatory application thereof

Abstract

The invention discloses a novel compound as well as a preparation method and anti-inflammatory application thereof, and relates to the technical field of medicines. The invention comprises the novel compound, a preparation method of the novel compound and application of the novel compound in preparation of anti-inflammatory drugs. In the invention, Caragana stenocarpus belongs to a leguminous Caragana plant, a plurality of components related to flavonoids, stilbenes and triterpenes are found through phytochemical research on the Caragana plant in the early stage, and the components are mainly closely related to pharmacological activities such as anti-inflammatory activity, anti-tumor activity, antibacterial activity, anti-injury activity, anti-oxidation activity, anti-diabetes activity and the like, so that the caragana stenocarpus belongs to the plant of the leguminous caragana plant. The method comprises the following steps: systematically separating an ethyl acetate extract of 80% ethanol extract of caragana angustifolia, and separating to obtain a new flavone compound with anti-inflammatory activity under the guidance of in-vitro activity evaluation of lipopolysaccharide stimulated mouse peritoneal macrophages (raw264.7).

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, in particular to a new compound and a preparation method and anti-inflammatory use thereof. Background Art

[0002] Inflammation is the body's defensive response to injury, infection, or damaging factors, aimed at eliminating harmful factors and repairing tissues.

[0003] At present, the main methods for treating inflammation include drug therapy, physical therapy and surgical treatment. Drug therapy is one of the most common methods for managing inflammation. This includes the use of nonsteroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen and aspirin, hormone drugs such as glucocorticoids, and antibiotics such as penicillin and cephalosporin. Physical therapy is also an important part of inflammation management. It mainly includes hot and cold compresses, massage and physical therapy. Surgical treatment is necessary in some cases. For example, surgical drainage may be required in severe infection or abscess formation;

[0004] However, there are some shortcomings in the treatment of inflammation, one of which is that it is prone to recurrence. Despite the continuous advancement of modern medical technology, the treatment of inflammation still faces challenges, and more research and development of new treatments are needed to improve the cure rate;

[0005] In view of the above problems, the inventors proposed a new compound and its preparation method and anti-inflammatory use to solve the above problems. Summary of the invention

[0006] In order to solve the problem of poor anti-inflammatory effect of inflammatory drugs, the purpose of the present invention is to provide a new compound and its preparation method and anti-inflammatory use.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solution: a new compound, the chemical structure of which is as follows:

[0008]

[0009] A method for preparing a new compound comprises the following steps:

[0010] S1, the aerial part of Caragana angustifolia was separated and first extracted with 80% ethanol, then extracted with petroleum ether, ethyl acetate, dichloromethane and n-butanol in sequence, and then the extract was concentrated under reduced pressure to obtain a crude extract;

[0011] S2, the ethyl acetate extract was subjected to column chromatography on silica gel, and gradient elution was performed with dichloromethane / methanol 10:1, 5:1, 2:1, 1:1, 1:2, 1:5, 1:10 to obtain fractions F1-F7, which were concentrated for later use;

[0012] S3, loading fraction F4 onto a Sephadex LH-20 column and eluting with 80% methanol aqueous solution to obtain fraction FF1, which was concentrated for later use;

[0013] S4. The obtained product is further separated by semi-preparative HPLC. The conditions of HPLC are C18 chromatographic column, 70% methanol as mobile phase, detection wavelength is 254nm, main peak eluate is collected, and dissolved to obtain the target compound.

[0014] Preferably, in S2, the mesh size of the silica gel is 100 to 200 meshes.

[0015] A method for preparing a new compound and application of the obtained new compound in preparing anti-inflammatory drugs.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] In the present invention, Caragana stenophylla Pojark. belongs to the genus Caragana of the Leguminosae family. Previous phytochemical studies on the genus Caragana have found a variety of components related to flavonoids, stilbenes and triterpenes, which are closely related to pharmacological activities such as anti-inflammatory, anti-tumor, antibacterial, anti-injury, antioxidant and anti-diabetic. A new flavonoid compound with anti-inflammatory activity is isolated by systematically separating the ethyl acetate extract of the 80% ethanol extract of Caragana stenophylla Pojark. and under the guidance of in vitro activity evaluation of mouse peritoneal macrophages (raw264.7) stimulated by lipopolysaccharide. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 It is a scatter plot of the logarithmic concentration inhibition rate of the compounds of the present invention.

[0020] Figure 2 It is the regression curve diagram of the final concentration inhibition rate of the compound of the present invention.

[0021] Figure 3 The NMR of the new compound of the present invention 1 H-NMR spectrum (600 MHz, deuterated methanol).

[0022] Figure 4 The NMR of the new compound of the present invention 13C-NMR spectrum (600 MHz, deuterated methanol).

[0023] Figure 5 The following is the nuclear magnetic resonance HSQC spectrum (600 MHz, deuterated methanol) of the novel compound of the present invention.

[0024] Figure 6 The nuclear magnetic resonance HMBC spectrum (600 MHz, deuterated methanol) of the novel compound of the present invention is shown in FIG. DETAILED DESCRIPTION

[0025] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] Example: Figure 1-6 As shown, the present invention provides a new compound, the chemical structure of which is as follows:

[0027]

[0028] The present invention also provides a method for preparing the new compound, comprising the following steps:

[0029] S1, the aerial part of Caragana angustifolia was separated and first extracted with 80% ethanol, then extracted with petroleum ether, ethyl acetate, dichloromethane and n-butanol in sequence, and then the extract was concentrated under reduced pressure to obtain a crude extract;

[0030] S2, the ethyl acetate extract was subjected to column chromatography on 100-200 mesh silica gel, and gradient eluted with dichloromethane / methanol 10:1, 5:1, 2:1, 1:1, 1:2, 1:5, 1:10 to obtain fractions F1-F7, which were concentrated for later use;

[0031] S3, loading fraction F4 onto a Sephadex LH-20 column and eluting with 80% methanol aqueous solution to obtain fraction FF1, which was concentrated for later use;

[0032] S4. The obtained product is further separated by semi-preparative HPLC. The conditions of HPLC are C18 chromatographic column, 70% methanol as mobile phase, detection wavelength is 254nm, main peak eluate is collected, and dissolved to obtain the target compound.

[0033] The present invention also provides a method for preparing a new compound and application of the new compound prepared in the preparation of anti-inflammatory drugs.

[0034] Structural confirmation of new compounds:

[0035] Caracanflovanone (1) is a yellow amorphous powder. 1 H NMR data (see Table 1) show the presence of two ABX spin systems [δ H 7.63(1H,d,J=2.1Hz,H-2"'), 7.54(1H,dd,J=8.5,2.1Hz,H-6"'), 6.91(1H,d,J=8.5Hz,H-5"'); δ H 7.40 (1H, dd, J = 8.5, 2.1 Hz, H-6′), 7.38 (1H, d, J = 2.1 Hz, H-2′), 6.90 (1H, d, J = 8.5 Hz, H-5′)], two AB spin systems, δ H The aromatic proton signals at 6.45 (1H, d, J = 2.0 Hz, H-8) and 6.21 (1H, d, J = 2.0 Hz, H-6), as well as δ H 6.40 (1H, d, J = 1.9 Hz, H-8") and 6.20 (1H, d, J = 1.9 Hz, H-6"). In δ H There is a single aromatic proton signal at 6.55 (1H, s, H-3), which is attributed to the compound at the C-3 position. H The presence of methoxyl group was found at 3.78 (3H, s). 13 The C NMR spectrum showed 31 carbon resonances, which, combined with the DEPT and HSQC data, indicated that Caracanf l avanone (1) was a biflavonoid composed of one flavonoid unit and one molecular flavonol unit;

[0036] The methoxy group is located at C-10, which is due to the δ H 3.78 and δ C 139.5 related. H-3(δ H 6.55) and C-10 (δ C 105.3), C-1′(δ C 123.7), H-2′(δ H 7.38) and C-2(δ C 166.4), H-6′(δ H 7.40) and C-2(δ C The presence of the flavonoid unit was confirmed by the long-range correlation of the HMBC spectrum with the C-3′-OC-7″ bond from the H-2′ (δ H 7.38) and C-7"(δ C 166.0), H-6"(δH 6.20) and C-7"(δ C 166.0), H-8"(δ H 6.40) and C-7"(δ C 166.0). Therefore, the planar structure of Caracanflovanone (1) is as follows Figure 3-Figure 6 As shown, it represents a new biflavonoid derivative;

[0037]

[0038] Table 1 NMR data of Caracanflavanone (1) in deuterated methanol

[0039] Effects of new compounds on inflammatory cells:

[0040] 1. Materials

[0041] 1.1 Test compounds

[0042] The highest dose of each sample was 100uM or 10uM (final concentration), and the samples were diluted in a 4-fold decreasing ratio for a total of five concentrations;

[0043] 1.2 Main Reagents

[0044] Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS, HyClone), bacterial lipopolysaccharide (LPS, SIGMA), CCK-8 (Tongren Chemical), thioglycollate (WAKO), Griess solution (1% sulfonamide / 0.1% naphthylethylenediamine / 2.5% H3PO4), and the rest were domestic analytical grade reagents;

[0045] 1.3 Cell lines

[0046] Mouse RAW 264.7 mononuclear macrophage cell line was purchased from the American Type Culture Collection (ATCC);

[0047] 1.4 Methods

[0048] 1.4.1 Determination of macrophage survival rate

[0049] RAW264.7 cells were cultured in a 37°C, 5% CO2 incubator with DMEM containing 10% inactivated fetal bovine serum (FBS), 100U / ml penicillin and 100μg / ml streptomycin, and subcultured every 3 days. Raw264.7 cells were seeded in a 96-well plate at 1×105 / well. Placed in an incubator to adhere for 4 hours. Add compounds at different concentrations and set up corresponding cell controls. Cultured in a 37°C, 5% CO2 incubator for 24 hours. Determination of macrophage survival rate (CCK-8 method): 2 hours before the end of culture, add 20μl of CCK-8 to each well and continue culturing in the incubator. At the end of culture, measure the OD value at 450nM (reference 650nM) on an enzyme reader;

[0050] 1.4.2 Determination of NO release

[0051] Raw264.7 cells were seeded in 96-well plates at 1×105 cells / well. They were placed in an incubator to adhere for 4 hours. Compounds of different concentrations were added, LPS (final concentration 10 μg / ml) was added, and corresponding cell controls and stimulation controls (RAW264.7 cells were added with the above concentration of LPS) were set up. They were cultured in a 37°C, 5% CO2 incubator for 24 hours;

[0052] At the end of the culture, the supernatant was carefully pipetted into a 1.5 ml centrifuge tube, incubated at 5000 rpm for 10 min, pipetted into a 1.5 ml centrifuge tube again, and frozen for testing. The release of NO was tested using the Griess method;

[0053] The experimental results showed that the compound had no cytotoxicity and its IC50 was 1.94ng / ml. Figure 1 and Figure 2 ;

[0054] The above results indicate that Caracanf l avanone(1), a new compound isolated from Caragana angustifolia, has anti-inflammatory uses.

[0055] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A new compound, characterized in that The chemical structure is as follows:

2. A method for preparing the new compound according to claim 1, characterized in that: The following steps are involved: S1, the aerial part of Caragana angustifolia was separated and first extracted with 80% ethanol, then extracted with petroleum ether, ethyl acetate, dichloromethane and n-butanol in sequence, and then the extract was concentrated under reduced pressure to obtain a crude extract; S2, the ethyl acetate extract was subjected to column chromatography on silica gel, and gradient elution was performed with dichloromethane / methanol 10:1, 5:1, 2:1, 1:1, 1:2, 1:5, 1:10 to obtain fractions F1-F7, which were concentrated for later use; S3, loading fraction F4 onto a Sephadex LH-20 column and eluting with 80% methanol aqueous solution to obtain fraction FF1, which was concentrated for later use; S4. The obtained product is further separated by semi-preparative HPLC. The conditions of HPLC are C18 chromatographic column, 70% methanol as mobile phase, detection wavelength is 254nm, main peak eluate is collected, and dissolved to obtain the target compound.

3. The method for preparing a novel compound according to claim 2, characterized in that: In S2, the mesh size of the silica gel is 100 to 200 meshes.

4. Use of a new compound obtained by the method for preparing a new compound as claimed in any one of claims 1 to 3 in the preparation of an anti-inflammatory drug.

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