Extraction and separation method of pyridine alkaloid compound in purslane and application of pyridine alkaloid compound

Abstract

The invention relates to the technical field of traditional Chinese medicine extraction and separation, in particular to an extraction and separation method of a pyridine alkaloid compound in purslane and application of the pyridine alkaloid compound. The molecular formula of the pyridine alkaloid compound is C6H8N2O, and the pyridine alkaloid compound is named as 6-metaxyridin-3-amine. According to the method, ethyl alcohol reflux extraction, ODS column, Sephadex LH-20, a high performance liquid chromatograph and the like are adopted for separation, purification and preparation, 6-methoxyridin-3-amine is successfully extracted and separated, the method is easy to operate, the operation method is simple, convenient and rapid, ethyl alcohol and methyl alcohol are mainly adopted for extraction in the extraction and separation process, the technological method is environmentally friendly, and the method is suitable for industrial production. The purity of the compound separated by the method is higher than 99%, and research shows that the compound has an anti-inflammatory effect, so that the pyridine alkaloid compound and the salt and the derivative thereof can be used as synthesis primers of other compounds and raw materials for new drug development and pharmacological activity research, and can also be used for preparing anti-inflammatory drugs.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine extraction and separation technology, and in particular to a method for extracting and separating a pyridine alkaloid compound from purslane and its application. Background Technology

[0002] purslane( Portulaca oleracea Purslane (L.) is an annual, warm-loving, succulent herb with a wide distribution. It is commonly known as purslane in the United States and Australia, rigla in Egypt, pigweed in the United Kingdom, pourpier in France, and purslane in China. This herbaceous plant with small yellow flowers has extensive wild resources and can grow in many places. Purslane is used as a folk remedy in many countries, with good effects on fever, antibacterial properties, and anthelmintic effects. It is cool in nature and sour in taste, and is used for cooling the blood, stopping bleeding, clearing heat, and detoxifying.

[0003] The dried aerial parts of purslane can be used to treat fever, dysentery, diarrhea, lumps, eczema, and bloody stools. Purslane also contains abundant chemical components, such as alpha-linolenic acid, alpha-tocopherol, dopamine, small amounts of dopa, adenosine, uracil, adenine, N,N-dicyclohexylurea, allantoin, N-trans-feruloyltyramine, and glutathione. In addition, it provides elements including potassium, phosphorus, and iron. It is listed by the World Health Organization as one of the most commonly used medicinal plants and is known as a "global panacea."

[0004] Most of the chemical components isolated from purslane are currently known, and their structural novelty is low. Therefore, the development and isolation of new natural products from purslane is urgently needed. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a method for extracting and separating a pyridine alkaloid compound from purslane and its application. Studies have found that the pyridine alkaloid compound of this invention has anti-inflammatory effects. Furthermore, this invention provides a simple, rapid, environmentally friendly, and high-purity extraction and separation method for this pyridine alkaloid compound.

[0006] To achieve the above objectives, this invention proposes a pyridine alkaloid compound with the molecular formula C6H8N2O, named 6-methoxypyridin-3-amine, and with the following chemical structural formula: .

[0007] This invention also provides a method for extracting and separating the pyridine alkaloid compound 6-methoxypyridin-3-amine from purslane, specifically including the following steps: Step 1: Take dried purslane, extract it by reflux with ethanol, filter the ethanol extract, combine the filtrates and concentrate under reduced pressure, cool to room temperature, and obtain the medicinal liquid for later use. Step 2: After evaporating the herbal liquid from Step 1 to dryness, extract it with ethyl acetate: ethanol in different ratios. Filter the extracts and concentrate them under reduced pressure to obtain an extract for later use. Step 3: Separate the ethyl acetate:ethanol (2:1) fraction from Step 2 using an ODS (octadecylsilane-bonded silica gel) column. Elute using a methanol-water gradient to obtain several eluent fractions. Detect these fractions by thin-layer chromatography with potassium bismuth iodide as a colorimetric indicator. Combine the colored eluent fractions and concentrate them to dryness under reduced pressure to obtain A1-A. 12 ,spare; Step 4: Separate the A5 obtained in Step 3 using a pretreated dextran gel column (Sephadex LH-20) by isocratic elution with methanol to obtain several eluted fractions. These fractions are then detected by thin-layer chromatography with potassium bismuth iodide as a colorimetric indicator. Each colored eluted fraction is concentrated to dryness under reduced pressure to obtain B1-B2. 11 spare; Step 5: The pretreated ODS (octadecylsilane bonded silica gel packing) obtained in Step 4 was eluted with methanol-water gradient, detected by thin-layer chromatography, and colored with potassium bismuth iodide. The colored eluted fractions were concentrated to dryness under reduced pressure to obtain concentrates C1-C9. Step 6: The C8 obtained in Step 5 is separated and prepared by HPLC using methanol-0.1% formic acid as the mobile phase to obtain the pyridine alkaloid compound 6-methoxypyridin-3-amine.

[0008] Furthermore, in step 1, the ethanol is refluxed twice, with an ethanol volume concentration of 50%, for 2 hours each time, and the amount of ethanol used is 8-16 times that of the medicinal material.

[0009] Furthermore, in step 2, the volume ratio of ethyl acetate to ethanol is 1:0, 5:1, 2:1, 1:1, 1:2, or 1:5.

[0010] Furthermore, in step 3, the volume ratio of methanol to water is 50:50, 70:30, 90:10, and 100:0; the ODS particle size is 40–70 μm.

[0011] Furthermore, in step 4, the methanol elution is isocratic elution with 15% methanol by volume.

[0012] Furthermore, in step 5, the volume ratio of methanol to water is 10:90, 20:80, 30:70, 50:50, or 70:30; and the ODS particle size is 40–70 μm.

[0013] Furthermore, in step 6, the volume ratio of methanol to 0.1% formic acid is 80:20, and the retention time of the compound is 11.058 min.

[0014] Furthermore, the pretreatment process for ODS and dextran gel involves soaking in methanol for 24 hours, loading onto a column, washing with methanol until no turbidity is observed when dropped into water, and then equilibrating with the initial mobile phase.

[0015] The aforementioned pyridine alkaloid compound 6-methoxypyridin-3-amine can be used in the preparation of anti-inflammatory drugs.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0017] This invention provides a pyridine alkaloid compound 6-methoxypyridin-3-amine derived from Portulaca oleracea and its extraction and separation method. The method employs ethanol reflux extraction, ODS column, Sephadex LH-20, and high-performance liquid chromatography for separation, purification, and preparation. 6-methoxypyridin-3-amine was successfully extracted and separated. This method is simple, convenient, and rapid. The extraction and separation process mainly uses ethanol and methanol, making the process environmentally friendly. Furthermore, the compounds obtained by this method have high purity, exceeding 99%. In addition, studies have shown that this compound possesses anti-inflammatory effects. Therefore, the pyridine alkaloid compounds, their salts, and derivatives of this invention can serve as lead compounds for the synthesis of other compounds, as well as raw materials for new drug development and pharmacological activity research. They can also be used to prepare anti-inflammatory drugs. Attached Figure Description

[0018] Figure 1 This is a high-resolution mass spectrum of the pyridine alkaloid compound 6-methoxypyridin-3-amine of this invention.

[0019] Figure 2 The pyridine alkaloid compound 6-methoxypyridin-3-amine of this invention 1 H-NMR spectrum.

[0020] Figure 3 The pyridine alkaloid compound 6-methoxypyridin-3-amine of this invention 13 C-NMR spectrum.

[0021] Figure 4 This is the DEPT135 spectrum of the pyridine alkaloid compound 6-methoxypyridin-3-amine of this invention.

[0022] Figure 5This is the HSQC spectrum of the pyridine bioalkaloid compound 6-methoxypyridin-3-amine of this invention.

[0023] Figure 6 This is the HMBC spectrum of the pyridine alkaloid compound 6-methoxypyridin-3-amine of this invention.

[0024] Figure 7 The pyridine alkaloid compound 6-methoxypyridin-3-amine of this invention 1 H- 1 H COSY spectrum.

[0025] Figure 8 This is the ROESY spectrum of the pyridine alkaloid compound 6-methoxypyridin-3-amine of this invention. Detailed Implementation

[0026] The following embodiments will help to understand the present invention, but these embodiments are only for illustrative purposes and the present invention is not limited thereto. The operating methods in the embodiments are all conventional operating methods in this technical field.

[0027] Example 1.

[0028] This invention provides a pyridine alkaloid compound with the molecular formula C6H8N2O, named 6-methoxypyridin-3-amine, and with the following chemical formula: .

[0029] Table 1 shows the NMR data for 6-methoxypyridin-3-amine: 1 H-NMR and 13 C-NMR in deuterated methanol.

[0030] Table 1. NMR data of 6-methoxypyridin-3-amine of the present invention.

[0031] For the identification of the compound structure of this invention, please refer to [reference needed]. Figure 1-8 .

[0032] 6-methoxypyridin-3-amine: Yellow powder, readily soluble in methanol, slightly soluble in water. After spotting onto a silica gel thin-layer plate and spraying with potassium bismuth iodide reagent, the spots turned orange, suggesting that the compound may contain nitrogen. UHPLC-ESI-QTOF-MS showed... m / z 125.0709, [M+H] +(The calculated value is 125.0709). Combined with... 1 H-NMR, 13 Based on C-NMR and DEPT data, the molecular formula of this compound is deduced to be C6H8N2O, with an unsaturation degree of 4.

[0033] 13 The C-NMR and DEPT spectra show six carbon signals, including two quaternary carbon signals ( δ C 158.98, 166.45), three methylene signals ( δ C 139.11, 123.78, 128.11) and a methyl signal ( δ C 52.83). 1 The H-NMR spectrum showed four hydrogen signals. δ H 3.88 (3H, s), δ H 8.14(1H, d, J =2.7), δ H 7.26(1H, dd, J =2.82 / 8.64), δ H 8.0(1H, d, J =8.58).

[0034] according to 1 ¹H-NMR spectroscopy revealed that the compound contains an ABX system. Based on HMBC correlations, H-2 correlates with C-4 and C-6, H-4 correlates with C-2, C-6, and C-3, and H-5 correlates with C-3 and C-6. Furthermore, C-6 and C-2 are located at a low field, suggesting that C-2 and C-6 may be bonded to a nitrogen atom, indicating the presence of a pyridine ring. The low field location of C-3 further suggests a possible bond with an amino group, and the correlation between HMBC and H-1' and C-6 confirms the presence of a methoxy group at C-6. UHPLC-ESI-QTOF-MS confirmed its structure as 6-methoxypyridin-3-amine.

[0035] This invention also provides a method for the extraction and separation of the above-mentioned pyridine alkaloid compound 6-methoxypyridin-3-amine, the specific steps of which are as follows: Step 1: Take 250 kg of dried purslane and extract it twice with ethanol reflux, 2 hours each time, with the amount of ethanol being 10 times the volume of the purslane. Filter the ethanol extract, combine the filtrates and concentrate under reduced pressure, cool to room temperature, and use the resulting liquid for later use. Step 2: After evaporating the herbal liquid from Step 1 to dryness, extract it with ethyl acetate:ethanol in different ratios (1:0, 5:1, 2:1, 1:1, 1:2, 1:5). Filter the extract and concentrate it under reduced pressure to obtain an extract for later use. Step 3: Separate the ethyl acetate:ethanol (2:1) fraction from Step 2 using an ODS (octadecylsilane-bonded silica gel) column. The ODS particle size is 40–70 μm. Elution was performed using a methanol-water gradient (50:50, 70:30, 90:10, and 100:0) to obtain several eluent fractions. These fractions were then detected by thin-layer chromatography with potassium bismuth iodide as a colorimetric indicator. The colored eluent fractions were combined, and the combined eluent fractions were concentrated to dryness under reduced pressure to obtain Al-A. 12 ,spare; Step 4: Separate the A5 obtained in Step 3 using a pretreated dextran gel column (Sephadex LH-20) by isocratic elution with 15% methanol to obtain several eluted fractions. These fractions are then detected by thin-layer chromatography with potassium bismuth iodide as a colorimetric indicator. Each colored eluted fraction is concentrated to dryness under reduced pressure to obtain B1-B2. 11 spare; Step 5: The pretreated ODS (octadecylsilane-bonded silica gel packing) obtained in Step 4, with an ODS particle size of 40-70 μm, was eluted with a methanol-water gradient (10:90, 20:80, 30:70, 50:50, 70:30). Thin-layer chromatography was performed, with potassium bismuth iodide as the colorimetric indicator. The eluted fractions were concentrated to dryness under reduced pressure to obtain concentrates C1-C9. Step 6: The C8 obtained in Step 5 was separated and prepared by HPLC using methanol:0.1% formic acid (80:20, v / v) as the mobile phase and detection wavelengths of 210 nm and 254 nm. The compound 6-methoxypyridin-3-amine was prepared and its purity was determined to be 99.1% by normalization method.

[0036] Furthermore, the pretreatment process of the ODS and dextran gel involves soaking in methanol for 24 hours, loading onto a column, washing with methanol until no turbidity is observed when dropped into water, and then equilibrating with the initial mobile phase.

[0037] Example 2: Anti-inflammatory effect of 6-methoxypyridin-3-amine of the present invention.

[0038] 1. Main Materials 1.1 Drugs and Reagents: The novel natural products used in the experiments were prepared by the above method, with a purity greater than 99%. Accurately weighed, they were diluted with DMSO to the required solutions for each of the following dosage groups. DMEM high-glucose culture medium, fetal bovine serum (Hyclone, USA); penicillin, streptomycin (Hangzhou Sijiqing Pharmaceutical Group Co., Ltd.); LPS (Sigma-Aldrich, USA); TNF-α αIL-1 β ELISA kit (Cayman, USA); cell lysis buffer (Beyotime Biotechnology Co., Ltd.).

[0039] 1.2 Cell line: RAW 264.7 macrophages (ATCC Cell Bank, USA).

[0040] 1.3 Grouping: The group was divided into a control group, an LPS group, and an experimental group, with one group for each group.

[0041] 2. Experimental methods.

[0042] 2.1 Cell Culture: DMEM high-glucose medium was added with 10% fetal bovine serum and 1% antibiotics (100 U / mL penicillin and 100 U / mL). μ (g / mL streptomycin) was placed in a 37.5% CO2 incubator for incubation.

[0043] 2.2 MTT assay for cell viability: Logarithmically growing RAW 264.7 macrophages from the above three groups were seeded in 96-well culture plates at a cell density of 1 × 10⁻⁶ cells / well. 4 100 cells / mL, 100 cells / well μ After overnight incubation at 37°C and 5% CO2, the experimental groups were supplemented with different concentrations of the novel natural product 6-methoxypyridin-3-amine (1-100 g / L). μ M), after incubation for 1 hour, add a final concentration of 1 to the LPS group and the experimental group respectively. μ A zero-adjustment group (culture medium containing DMSO) was set up, with 3 replicates per group, to investigate the effect of drug addition on cells. After culturing cells for 24 h in all groups, 5 mg / mL MTT20 was added to each well. μ After incubating at 37℃ and 5% CO2 for 4 hours, the culture was terminated, the liquid in the wells was aspirated, 100 μL of dimethyl sulfoxide (DMSO) was added to each well, and the mixture was shaken for 10 min to fully dissolve the intracellular crystals. The absorbance of each well was measured at 570 nm using a microplate reader.

[0044] 2.3 ELISA method for measuring inflammatory factor TNF-α α、 IL-1 β Log-phase RAW 264.7 macrophages were seeded in 24-well culture plates at a cell density of 1 × 10⁻⁶ cells / well. 5 1 mL / mL, 1 mL / well, cultured overnight at 37°C with 5% CO2. The experimental group was treated with the alkaloid compound 6-methoxypyridin-3-amine (1-50 g / mL) of this invention. μAfter incubation for 1 hour, LPS (final concentration 1 μg / mL) was added to each well, and incubation was continued for 24 hours. Each treatment was repeated in triplicate. ELISA was used to determine TNF-α secreted by RAW 264.7 macrophages treated with purslane-derived alkaloids. α、 IL-1 β The content of.

[0045] 3. Experimental results.

[0046] Experimental results show that the novel natural product of this invention has no effect on LPS-induced proliferation of RAW 264.7 macrophages, is safe and non-toxic; and can effectively inhibit the excessive production of the inflammatory cytokine IL-1 by RAW 264.7 macrophages induced by LPS. β and TNF- α And it is concentration-dependent.

[0047] The results of the relative cell viability experiment are shown in Table 2.

[0048] Table 2. Effect of 6-methoxypyridin-3-amine of the present invention on the relative survival rate of RAW264.7 macrophages. Note: * P<0.05 compared with the control group (the high concentration group showed a significant difference).

[0049] ELISA method for measuring inflammatory factor IL-1 β and TNF- α The results are shown in Table 3. Experiments have demonstrated that this compound is effective at 10... μ M can effectively downregulate the inflammatory factor IL-1 β and TNF- α At medium levels, it exhibits good anti-inflammatory activity.

[0050] Table 3. Effects of 6-methoxypyridin-3-amine of the present invention on LPS-induced IL-1 secretion in RAW264.7 cells. β and TNF- α Effect of content (mean ± standard deviation, n=3) Note: # P<0.05 compared with the control group, * P<0.05 compared with the LPS group, mean ± SD, n=3.

Claims

1. A pyridine alkaloid compound isolated from Portulaca oleracea Linn, characterized in that, The molecular formula is C6H8N2O, and the chemical structural formula is: 。 2. The extraction and separation method of pyridine alkaloid compounds extracted and separated from Portulaca oleracea according to claim 1, characterized in that, Specifically comprising the following steps: Step 1, taking the dried purslane medicinal material, using ethanol reflux extraction, filtering the alcohol extract, combining the filtrate, reducing pressure concentration, and cooling to room temperature to obtain a medicinal liquid for standby; Step 2, the medicinal liquid in step 1 is evaporated and extracted with different proportions of ethyl acetate and ethanol, the extract is filtered, and the filtrate is reduced pressure concentrated to extract, standby; Step 3, the ethyl acetate:ethanol 2:1 fraction from Step 2 was separated on an ODS column using a methanol-water gradient to elute several fractions, which were tested by thin layer chromatography with bismuth potassium iodide visualization. The visualized fractions were combined and concentrated to dryness under reduced pressure to give A1-A 12 , standby; Step 4, the A5 obtained in step 3 was separated by pre-treatment Sephadex gel column chromatography, eluted with methanol isocratic, to obtain several elution parts, detected by thin layer chromatography, and developed with bismuth potassium iodide, each developed elution part was concentrated to dryness under reduced pressure, to obtain B1-B 11 Standby; Step 5, the B8 obtained in step 4 is pretreated ODS, eluted with methanol-water gradient, detected by thin layer chromatography, colored with bismuth potassium iodide, and the colored elution part is reduced pressure concentrated to dryness to obtain a concentrate C1-C9; Step 6, C8 obtained in step 5 is separated by HPLC to prepare a pyridine alkaloid compound.

3. The method of claim 2, wherein, In step 1, the ethanol reflux extraction is performed twice, the volume concentration of ethanol is 50%, each time is 2 hours, and the amount of ethanol is 8-16 times of the medicinal material.

4. The method of claim 2, wherein, In step 2, the volume ratio of ethyl acetate and ethanol is 1:0, 5:1, 2:1, 1:1, 1:2 and 1:

5.

5. The method of claim 2, wherein the step of extracting and separating is characterized by, In step 3, the volume ratio of methanol and water is 50:50, 70:30, 90:10 and 100:0; the ODS particle size is 40-70 μm.

6. The method of claim 2, wherein, In step 4, the methanol elution is 15% methanol isocratic elution.

7. The extraction and separation method according to claim 2, characterized in that, In step 5, the volume ratio of methanol and water is 10:90, 20:80, 30:70, 50:50 and 70:30; the ODS particle size is 40-70 μm.

8. The method of claim 2, wherein, In step 6, the volume ratio of methanol and 0.1% formic acid is 80:20, and the compound retention time is 11.058 min.

9. The method of claim 2, wherein, The pretreatment process of ODS and dextran gel is methanol immersion for 24 hours, column, washing with methanol until no turbidity is added into water, and then equilibration with the initial mobile phase.

10. The pyridine alkaloid compound of claim 1 in the preparation of an anti-inflammatory drug.

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