An alkaloid compound, and a preparation method and application thereof

Abstract

The application discloses an alkaloid compound and a preparation method and application thereof, and extracts the alkaloid compound from Mallotus apelta. The new alkaloid compound is extracted from Mallotus apelta, has a good inhibiting effect on synovial cells, has a good anti-rheumatoid arthritis effect, and can be used for preparing anti-rheumatoid arthritis drugs.

Description

Technical Field

[0001] This invention relates to the field of phytochemistry, specifically to an alkaloid compound, its preparation method, and its application. Background Technology

[0002] Guafu wood ( Fissistigma oldhamii (Hemsl.) Merr. is a plant belonging to the genus Hemsl. in the family Annonaceae. The rhizome of this plant is a well-known traditional Chinese medicine called "Zuanshanfeng," used to treat rheumatic pain, numbness in the hands and feet, and other ailments.

[0003] Chinese patent document CN (application number 201510763189.4) discloses an extract of *Citrus aurantium* var. *mongolicum*, its preparation method, and its application. The extract is prepared by the following method: *Citrus aurantium* var. *mongolicum* raw material is dried and pulverized, then extracted with alcohol under reflux. The extract is filtered, concentrated under reduced pressure to obtain a crude alcohol extract, diluted with water, and extracted with an organic solvent. The extract or the remaining aqueous solution is concentrated under reduced pressure to a paste-like state, and then vacuum dried to obtain a powder, which is the organic solvent extract or aqueous extract of *Citrus aurantium* var. *mongolicum*. The organic solvent is any one or a combination of petroleum ether, chloroform, and ethyl acetate. The document also describes the application of the *Citrus aurantium* var. *mongolicum* extract in the preparation of anti-rheumatoid arthritis drugs. The petroleum ether extract, chloroform extract, and aqueous phase fraction of this patent have good efficacy and can be used in pharmaceuticals. However, the specific chemical composition of these petroleum ether extract, chloroform extract, and aqueous phase fractions is unknown to the art.

[0004] Chinese patent document CN102241682A (application number 201110117935.4) discloses a method for preparing guafu wood alkaloid A, (1) pulverizing guafu wood medicinal material, extracting it by reflux or percolation with ethanol, concentrating the extract to a small volume, adjusting it to acidity with hydrochloric acid, filtering out insoluble matter, adjusting the acid solution to alkali, extracting it with dichloromethane, and recovering the reagent from the extract to obtain guafu wood total alkaloids; (2) separating and purifying the above guafu wood total alkaloids by high-speed countercurrent chromatography to obtain guafu wood alkaloid A. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide an alkaloid compound extracted from Guafu wood, its preparation method and application.

[0006] The technical solution for achieving the first objective of this invention is an alkaloid compound with the following structural formula:

[0007] .

[0008] The technical solution to achieve the second objective of this invention is a method for preparing alkaloid compounds as described above, comprising the following steps:

[0009] ① The dried stems of the guarful tree were extracted with ethanol to obtain an extract. The extract was then concentrated under reduced pressure to form a paste, thus obtaining the guarful tree extract.

[0010] ② After diluting the Guafu wood extract obtained in step ① into a suspension with water, extract it sequentially with petroleum ether and ethyl acetate. Combine the ethyl acetate extracts and concentrate them into an extract.

[0011] 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 with 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 approximately 1000 mL increments each time. The similar fractions were combined by TLC and separated into 8 fractions, designated as Fr. 1-8.

[0012] ④ For the fraction Fr.3 obtained in step ③, elute it by loading it onto a 300-400 mesh silica gel column and using an ethyl acetate-petroleum ether mixed solvent as the eluent; perform thin-layer chromatography using a methanol-chloroform mixed solvent as the developing solvent, and combine the fractions according to the chromatography effect to obtain three fractions, namely Fr.3-1, Fr.3-2 and Fr.3-3.

[0013] ⑤ For Fr.3-2 obtained in step ④, perform Sephadex LH-20 column chromatography with chloroform-methanol mixed solvent as eluent to remove pigments and obtain component Fr.3-2-1. Fr.3-2-1 is purified by high performance liquid chromatography to obtain the alkaloid compound of claim 1.

[0014] During step ①, when concentrating under reduced pressure, the temperature is 30~70℃ and the pressure is -0.06~-0.15MPa.

[0015] During concentration in step ②, the temperature is 30~70℃ and the pressure is -0.06~-0.15MPa.

[0016] In step ④, an ethyl acetate-petroleum ether mixed solvent with a volume percentage of 20% was used as the eluent for elution, and a methanol-chloroform mixed solvent with a volume percentage of 5% was used as the developing solvent for thin-layer chromatography.

[0017] In step ⑤, Sephadex LH-20 column chromatography was performed using a chloroform-methanol mixed solvent with a chloroform volume percentage of 50% as the eluent.

[0018] In step ⑤, when Fr.3-2-1 is analyzed and purified by high performance liquid chromatography, the eluent is acetonitrile and water in a volume ratio of 65:35.

[0019] The technical solution to achieve the third objective of this invention is the application of the above-mentioned compound in the preparation of a drug for treating rheumatoid arthritis.

[0020] The present invention has positive effects: the present invention extracts a new alkaloid compound from the guarana tree, which has a good inhibitory effect on synovial cells and has good anti-rheumatoid arthritis efficacy, and can be used to prepare anti-rheumatoid arthritis drugs. Attached Figure Description

[0021] Figure 1 The image shows the H-NMR spectrum of the compound extracted in Example 1.

[0022] Figure 2 The image shows the nuclear magnetic resonance C-ray spectrum of the compound extracted in Example 1.

[0023] Figure 3 The image shows the HMBC spectrum of the compound extracted in Example 1. Detailed Implementation

[0024] (Example 1)

[0025] In this embodiment, the alkaloids were extracted from *Calamus spp.*, and the extraction process is as follows:

[0026] ① 8 kg of dried stems of *Gnaphalium affine* were extracted three times with ethanol at 30-95% v / v (75% v / v in this example) for 7 days each time to obtain an extract. The extract was then concentrated under reduced pressure into a paste to obtain 1000 g of *Gnaphalium affine* extract.

[0027] 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.

[0028] ② Dilute the Guafumu extract obtained in step ① with 3L of distilled water to form a suspension, and extract it sequentially with petroleum ether (3L x 3 times, i.e., 3 extractions, 3L each time) and ethyl acetate (3L x 3 times, i.e., 3 extractions, 3L each time). Combine the ethyl acetate extracts and concentrate them into an extract of about 256g.

[0029] 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.

[0030] ③ Perform silica gel column chromatography on the ethyl acetate extract obtained in step ②. Use 200-300 mesh silica gel to load the column and perform silica gel column chromatography with petroleum ether-ethyl acetate mixed solvent (100:0-0:100, V / V) and ethyl acetate-methanol (100:0-0:100, V / V) in increasing polarity. Collect the fraction in approximately 1000 mL increments each time.

[0031] Similar fractions were separated into eight components (Fr. 1–8) by TLC analysis. Thin-layer chromatography was performed using a methanol-chloroform mixture with a methanol volume percentage of 5% as the developing solvent.

[0032] ④ For the fraction Fr.3 obtained in step ③, the column was loaded with 300~400 mesh silica gel and eluted with a 20% (v / v) ethyl acetate-petroleum ether mixed solvent as the eluent. Thin-layer chromatography was performed with a 5% (v / v) methanol-chloroform mixed solvent as the developing solvent. Based on the chromatographic effect, the fractions were combined to obtain three fractions, namely Fr.3-1, Fr.3-2 and Fr.3-3.

[0033] ⑤ For Fr.3-2 obtained in step ④, Sephadex LH-20 column chromatography was performed using a chloroform-methanol mixed solvent (50% chloroform by volume) as the eluent to remove the pigment, yielding fraction Fr.3-2-1. Fr.3-2-1 was analyzed and purified by high-performance liquid chromatography (HPLC) using acetonitrile and water (volume ratio 65:35) as the eluent, resulting in a new alkaloid compound, named Stepharine A. The alkaloid compound obtained in this example is a white powder.

[0034] The NMR spectrum of this compound is shown below. Figure 1 The nuclear magnetic resonance C-ray spectrum is shown below. Figure 2 HMBC spectrum Figure 3 .

[0035] High-resolution mass spectrometry shows [M+H] + 282.1128, molecular formula is C 17 H 15 NO3.

[0036] 1 H NMR (400 MHz, MeOD- d4) δ7.20, 7.17, 7.05, 7.03, 6.68, 6.41, 6.39,6.29, 6.27, 6.14, 4.56, 4.54, 4.52, 3.81, 3.61, 3.59, 3.57, 3.56, 3.30, 3.28,3.26, 3.24, 2.93, 2.92, 2.48, 2.46, 2.45, 2.43, 2.42, 2.40, 2.37, 2.34.

[0037] 13 C NMR (101 MHz, MeOD-d4)) δ187.94, 155.48, 152.07, 146.93, 145.29,133.01, 128.57, 128.40, 128.34, 127.52, 113.07, 100.92, 57.63, 52.14, 46.72,44.75, 25.24.

[0038] according to 1 HNMR, 13 CNMR analysis revealed that this compound possesses the skeleton of the stepharine alkaloid. The -OCH2O group in the HMBC spectrum correlates with carbons at positions 1 and 2. Comparison with data from the known compound stepharine showed that, except for the absence of two methoxy groups at positions 1 and 2, the structure was identical. Therefore, the structure of this compound can be determined as follows, and it is named stepharine A.

[0039] The structural identification results of Stepharine A are as follows:

[0040] .

[0041] (Experimental Example)

[0042] I. Experimental Materials and Instruments

[0043] Cells: Synovial cells (primary rat synovial cells).

[0044] Cell culture medium: primary rat synovial cell culture medium.

[0045] Reagents: Thiazol blue (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide, MTT, Sigma). Lactate dehydrogenase (LDH) kit (purchased from Beyotime Biotechnology). Methotrexate (MTX, Shanghai Xinyi Pharmaceutical Co., Ltd., batch number 20140307); Stepharine A extracted in Example 1.

[0046] Instruments: 96-well cell culture plate; Infinite 200Pro multi-functional microplate reader.

[0047] II. Experimental Methods

[0048] (1) MTT assay to determine the inhibitory effect of drugs on synovial cells

[0049] Synovial cells in the logarithmic growth phase were seeded into 96-well culture plates (1×10⁻⁶). 4 Cells were divided into three groups (10, 50, and 100 µg / mL) and wells. The groups were designated as a blank group (no drug administration), a methotrexate group (administered 1 µg / mL methotrexate), and drug-treated groups (administered 10, 50, and 100 µg / mL of drug). Cells were incubated overnight before use in experiments. After culturing cells at different drug concentrations for 48 h, 10 µL of 5 mg / mL MTT was added to each well. Cells were cultured for another 4 h, and the supernatant was carefully aspirated. 100 µL of DMSO was added to each well to dissolve the formazan crystals. The cells were shaken at room temperature for 5 min until completely dissolved. The optical density (OD value) was measured at 570 nm using a microplate reader. Cell viability was calculated based on the OD value.

[0050] .

[0051] (2) Effect of LDH activity assay on synovial cell viability

[0052] Synovial cells in the logarithmic growth phase were seeded into 96-well culture plates (1×10⁻⁶). 4 Cells per well were divided into three groups: a control group (no drug administration), a methotrexate group (administered 1 µg / mL methotrexate), and drug administration groups (administered drug concentrations of 10, 50, and 100 µg / mL). Cells were incubated overnight. After culture, the cell supernatant was collected, and LDH activity was measured using an LDH assay kit. LDH activity was calculated using the following formula:

[0053] .

[0054] III. Statistical Methods

[0055] SPSS 22.0 statistical software was used to analyze the experimental data. Results are expressed as mean ± standard deviation (SD). This indicates that one-way ANOVA was used to analyze differences between groups. P A value <0.05 is considered statistically significant.

[0056] IV. Experimental Results

[0057] (1) MTT assay to determine the inhibitory effect of drugs on synovial cells

[0058] The cell viability rates of the blank group (no drug administration), the methotrexate group (administered 1 µg / mL methotrexate), and the drug administration groups (administered drug concentrations of 10, 50, and 100 µg / mL) were 97.18%, 31.13%, 48.12%, 41.33%, and 34.25%, respectively.

[0059] IC50 of methotrexate and Stepharine A 50 The values ​​are 1.2 µM and 9.8 µM, respectively.

[0060] The results showed that the compound stepharine A had a good inhibitory effect on synovial cells.

[0061] (2) Effect of LDH activity assay on synovial cell viability

[0062] Cell viability was detected by LDH activity assay after culturing with different concentrations of drugs for 48 h.

[0063] The effects of LDH on cell viability in the blank group (no drug administration), the methotrexate group (administered 1 µg / mL methotrexate), and the drug administration groups (administered drug concentrations of 10, 50, and 100 µg / mL) were 98.2%, 94.16%, 95.38%, 91.12%, and 89.28%, respectively.

[0064] The results showed that the LDH activity in the cell culture medium did not vary significantly after treatment with different concentrations of the drug.

[0065] The above results indicate that the alkaloid compound stepharine A extracted in this invention has a good inhibitory effect on synovial cells and has little effect on LDH activity in cell culture medium. Experiments show that alkaloid compounds have good anti-rheumatoid arthritis efficacy and can be used in the preparation of anti-rheumatoid arthritis drugs.

Claims

1. An alkaloid compound with the following structural formula: 。 2. A method for preparing the alkaloid compound as described in claim 1, characterized in that... Includes the following steps: ① The dried stems of the guarful tree were extracted with ethanol to obtain an extract. The extract was then concentrated under reduced pressure to form a paste, thus obtaining the guarful tree extract. ② After diluting the Guafumu extract obtained in step ① into a suspension with water, 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. The silica gel column chromatography was carried out in increasing polarity with a mixed solvent of petroleum ether and ethyl acetate at a volume ratio of 100:0 to 0:100 and a mixed solvent of ethyl acetate and methanol at a volume ratio of 100:0 to 0:

100. The fractions were collected in 1000 mL increments each time. The similar fractions were combined by TLC and separated into 8 fractions, which were designated as Fr.1-8. ④ For fraction Fr.3 obtained in step ③, the column was loaded with 300-400 mesh silica gel and eluted with a 20% (v / v) ethyl acetate-petroleum ether mixed solvent as the eluent; thin-layer chromatography was performed with a 5% (v / v) methanol-chloroform mixed solvent as the developing solvent. Based on the chromatographic results, the fractions were combined to obtain three fractions, namely Fr.3-1, Fr.3-2, and Fr.3-3. ⑤ For Fr.3-2 obtained in step ④, use a chloroform-methanol mixed solvent with 50% chloroform (v / v) as the eluent for Sephadex LH-20 molecular sieve column chromatography to remove pigments and obtain component Fr.3-2-1. Fr.3-2-1 is purified by high performance liquid chromatography to obtain the alkaloid compound described in claim 1. When Fr.3-2-1 is analyzed and purified by high performance liquid chromatography, the eluent is acetonitrile and water in a volume ratio of 65:

35.

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

4. The method for preparing alkaloid compounds according to claim 2, characterized in that: During concentration in step ②, the temperature is 30~70℃ and the pressure is -0.06~-0.15MPa.

5. The use of the compound of claim 1 in the preparation of a medicament for treating rheumatoid arthritis.

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