A nitrogen-containing bicyclic compound from purslane and its extraction, separation and application

Abstract

The present invention belongs to the field of extraction and separation technology of traditional Chinese medicine, and relates to an azabicyclic compound in purslane, an extraction and separation method, and an application thereof. The new compound has a molecular formula of C6H7NO3 and is named 4-ethyl-3-oxa-1-azabicyclo[2.1.1]hexane-2,5-dione according to its structure. A method for extracting and separating the new compound is also provided, which sequentially uses ethanol reflux extraction, ODS column chromatography, dextran gel chromatography, and high-performance liquid chromatography for separation, purification, and preparation. Its structure is determined to be a new compound using mass spectrometry, hydrogen spectrum, carbon spectrum, and two-dimensional nuclear magnetic spectrum analysis methods, and it has anti-inflammatory activity. The new compound of the present invention and its salts or derivatives can be used as raw materials for drug development and pharmacological activity research.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine extraction and separation, and relates to an azabicyclic compound in purslane and an extraction and separation method and application thereof. Background Art

[0002] Portulaca oleracea L. is an annual herb of the genus Portulaca of the family Portulacaceae. It is widely distributed in temperate and tropical regions. It likes high humidity, is drought-resistant and waterlogged, is sun-loving, and has strong vitality. It likes fertile soil, with neutral and slightly acidic soil being preferred. It is cultivated in all parts of north and south China. The Compendium of Materia Medica records that because its leaves are like horse teeth and are slippery like amaranth, it is named Portulaca oleracea. The 2020 edition of the Pharmacopoeia of the People's Republic of China records that Portulaca oleracea has the effects of clearing away heat and detoxifying, cooling blood and stopping bleeding, and stopping dysentery. It is often used to treat heat-toxic bloody dysentery, carbuncles, furuncles, eczema, erysipelas, snake and insect bites, blood in stool, hemorrhoids, metrorrhagia, etc.

[0003] Modern pharmacological research has revealed a diverse range of chemical components in purslane, including flavonoids, coumarins, volatile oils, terpenes, steroids, alkaloids, amino acids, polysaccharides, various pigments, and minerals. Alkaloids are the primary chemical constituents of purslane. This rich chemical content provides a foundation for research on its pharmacological effects. Studies have shown that purslane exhibits hypoglycemic and hypolipidemic properties, anti-tumor, antioxidant, anti-aging, anti-browning, anti-inflammatory, antiviral, neuroprotective, and immune-modulating effects.

[0004] Currently, most of the chemical components isolated from Portulaca oleracea are known and have low structural novelty. Therefore, the development and separation of new compounds in Portulaca oleracea are needed. Summary of the Invention

[0005] To address the above problems, the present invention provides an azabicyclic compound extracted and separated from Portulaca oleracea. Research has found that the new compound of the present invention has anti-inflammatory effects. At the same time, a simple, rapid, environmentally friendly, and high-purity extraction and separation method for the new compound of the present invention is provided.

[0006] To achieve the above-mentioned purpose of the present invention, the present invention provides an azabicyclic compound with a molecular formula of C6H7NO3, named 4-ethyl-3-oxa-1-azabicyclo[2.1.1]hexane-2,5-dione according to its structure, and a chemical structural formula of:

[0007]

[0008] To achieve the above-mentioned object of the present invention, the present invention also provides a method for extracting and separating an azabicyclic compound from Portulaca oleracea, which specifically comprises the following steps:

[0009] Step 1: Take dried purslane medicinal material, extract it with ethanol, filter the ethanol extract, combine the filtrate, concentrate under reduced pressure, and cool to room temperature to obtain a medicinal solution for use;

[0010] Step 2: After the Chinese medicine solution in step 1 is evaporated to dryness, the extracts are respectively extracted with ethyl acetate: ethanol in different proportions, the extracts are filtered, and the extracts are respectively concentrated under reduced pressure to extracts for later use;

[0011] Step 3, dissolve the ethyl acetate:ethanol (1:5) portion of the extract in step 2 with ethyl acetate:ethanol in the same ratio, and dissolve the insoluble portion with 70% methanol to obtain a medicinal solution for later use;

[0012] Step 4: Separate the Chinese medicine solution in step 3 through an ODS (octadecylsilane bonded silica gel packing) column, use a methanol-water gradient elution to obtain several elution fractions, detect by thin layer chromatography, develop color, combine the elution fractions that have developed color, and concentrate the combined elution fractions to dryness under reduced pressure for later use;

[0013] Step 5: The third fraction obtained in step 4 is passed through a pretreated Sephadex LH-20 column and isocratically eluted with 70% methanol to obtain several elution fractions, which are detected by thin layer chromatography and color developed. The colored elution fractions are combined, and the combined elution fractions are concentrated to dryness under reduced pressure for later use.

[0014] Step 6: The product obtained in step 5 was separated by pre-treated ODS column chromatography, and eluted with methanol-water gradient to obtain 3 fractions (i.e., 3 bottles were obtained by gradient elution, each bottle was 120 mL).

[0015] Step 7: The second fraction obtained in step 6 is concentrated and then subjected to HPLC (high performance liquid chromatography) separation and preparation, and methanol:0.1% formic acid is used as the mobile phase for isocratic elution to prepare the azabicyclic compound 4-ethyl-3-oxa-1-azabicyclo[2.1.1]hexane-2,5-dione of the present invention.

[0016] Furthermore, in step 1, ethanol reflux extraction is performed twice, each time for 2 hours, and the amount of water used is 8-16 times that of the medicinal material.

[0017] Furthermore, in step 2, the mobile phase elution program used is ethyl acetate (1:0), ethyl acetate: ethanol (5:1), ethyl acetate: ethanol (2:1), ethyl acetate: ethanol (1:1), ethyl acetate: ethanol (1:2), and ethyl acetate: ethanol (1:5) gradient elution.

[0018] Furthermore, in step 4, gradient elution is performed using a volume ratio of methanol to water of 50:50, 70:30, 90:10 and 100:0, and the ODS particle size is 40 to 70 μm.

[0019] Furthermore, the pretreatment process of the ODS and dextran gel is as follows: soaking in methanol for 24 hours, loading onto the column, washing with methanol until there is no turbidity when dripping into water, and then balancing with the initial mobile phase.

[0020] Furthermore, in step 5, the methanol elution procedure is 70% methanol isocratic elution.

[0021] Furthermore, in step 6, gradient elution is performed using 5%, 10%, 15%, and 20% methanol.

[0022] Furthermore, in step 7, the volume ratio of methanol to 0.1% formic acid water in the isocratic elution is 5:95.

[0023] The azabicyclic compound 4-ethyl-3-oxa-1-azabicyclo[2.1.1]hexane-2,5-dione provided by the present invention can be used for preparing anti-inflammatory drugs or health products.

[0024] Compared with the prior art, the present invention has the following beneficial effects.

[0025] The isolation and pharmacological activity research of the purslane azabicyclic compound described in the present invention has not been reported in existing journals; the present invention provides an azabicyclic compound derived from purslane and a method for extracting and separating the new compound of the present invention, which sequentially uses ethanol extraction, extraction with different ratios of ethyl acetate and ethanol, ODS medium-pressure column, Sephadex LH-20 and high-performance liquid chromatography for separation, purification and preparation, and successfully extracts and separates the new compound. The method has only six steps, and the operation method is simple and rapid. The extraction and separation process mainly uses ethanol extraction and methanol elution, and the process method is environmentally friendly. The purity of the compound separated by this method is greater than 90%. In addition, studies have shown that this compound has anti-inflammatory effects. Therefore, the new compound of the present invention and its salts and derivatives can be used as precursors for the synthesis of other compounds, as well as raw materials for new drug development and pharmacological activity research, and can also be used to prepare anti-inflammatory drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a high-resolution mass spectrum of the azabicyclic compound 4-ethyl-3-oxa-1-azabicyclo[2.1.1]hexane-2,5-dione of the present invention.

[0027] Figure 21H-NMR spectrum of the azabicyclic compound 4-ethyl-3-oxa-1-azabicyclo[2.1.1]hexane-2,5-dione of the present invention.

[0028] Figure 3 The figure is the 13C-NMR spectrum of the azabicyclic compound 4-ethyl-3-oxa-1-azabicyclo[2.1.1]hexane-2,5-dione of the present invention.

[0029] Figure 4 The figure is a DEPT spectrum of the azabicyclic compound 4-ethyl-3-oxa-1-azabicyclo[2.1.1]hexane-2,5-dione of the present invention.

[0030] Figure 5 This is the HMBC spectrum of the azabicyclic compound 4-ethyl-3-oxa-1-azabicyclo[2.1.1]hexane-2,5-dione of the present invention.

[0031] Figure 6 This is the HSQC spectrum of the azabicyclic compound 4-ethyl-3-oxa-1-azabicyclo[2.1.1]hexane-2,5-dione of the present invention.

[0032] Figure 7 This is the 1H-1HCOSY spectrum of the azabicyclic compound 4-ethyl-3-oxa-1-azabicyclo[2.1.1]hexane-2,5-dione of the present invention.

[0033] Figure 8 This is the ROESY spectrum of the azabicyclic compound 4-ethyl-3-oxa-1-azabicyclo[2.1.1]hexane-2,5-dione of the present invention. DETAILED DESCRIPTION

[0034] The following examples will help to understand the present invention, but these examples are only for illustration of the present invention and the present invention is not limited to these contents. The operating methods in the examples are all conventional operating methods in the art.

[0035] The present invention provides a new compound with a molecular formula of C6H7NO3, a chemical formula of 4-ethyl-3-oxa-1-azabicyclo[2.1.1]hexane-2,5-dione, and a chemical formula of:

[0036]

[0037] Table 1 shows the NMR data of the new alkaloid compound: 1H-NMR and 13C-NMR in methanol.

[0038] Table 1 NMR data of new alkaloids.

[0039]

[0040] Reference for structural identification of invented compounds Figures 1-8 .

[0041] New alkaloid: Yellow oil, easily soluble in methanol, slightly soluble in water. After spotting on a silica gel thin layer plate, spraying with dilute potassium bismuth iodide test solution resulted in an orange-yellow spot, indicating that the compound is an alkaloid component. UHPLC-ESI-Q-TOF-MS gave a quasi-molecular ion peak of m / z: 140.0352 [MH]- (calculated value [C6H6NO3]- is 140.0352). 1 H-NMR, 13 Based on the C-NMR and DEPT data, it is speculated that the possible molecular formula of the compound is C6H7NO3, and the degree of unsaturation is 3. The 13C-NMR spectrum and DEPT spectrum show 6 carbon signals, namely 1 methyl carbon (δC: 7.69), 2 methylene carbons (δC: 44.27; δC: 33.62), 1 quaternary carbon (δC: 76.54), and 2 carbonyl carbons (δC: 178.28; δC: 174.86).

[0042] 1 The H-NMR spectrum revealed one methyl signal at δH 0.76 (1H, t, H-2′) and two methylene signals at δH 2.45 and 2.25 (2H, m, H-6), and δH 1.53 and 1.62 (2H, m, H-7). The HH COSY spectrum showed correlations between H-1′ and H-2′, indicating the presence of a methyl group linked to a methine group. The HMBC spectrum showed correlations between H-6 and C-2, C-4, and C-1′, indicating a link between two ring structures. H-2′ correlated with C-1 (δC: 56.99), and H-1′ correlated with C-2, confirming the ethyl group's attachment to C-1.

[0043] at the same time 13 The C-NMR spectrum shows that C-1, C-2, and C-4 (δC: 151.13) are located downfield, indicating that the compound contains heteroatoms, and the C-4 chemical shift is even lower, indicating that C-4 is connected to two heteroatoms, N and O. Therefore, based on the above information, it can be confirmed that this new compound has the above structure.

[0044] The present invention also provides a method for extracting and separating the new alkaloid, which comprises the following specific steps:

[0045] Step 1: Weigh 250 kg of dried purslane medicinal materials, extract with ethanol, the amount of ethanol is 10 times the amount of medicinal materials, extract twice, each time for 2 hours, filter the water extract, combine the filtrate, concentrate under reduced pressure to 250 L, cool to room temperature, and obtain the medicinal solution for use.

[0046] Step 2: After the Chinese medicine solution in step 1 is evaporated to dryness, it is extracted with ethyl acetate: ethanol in different proportions. The extract is filtered to obtain 7 parts in total, which are concentrated under reduced pressure to obtain extracts.

[0047] Step 3: Dissolve the ethyl acetate:ethanol (1:5) portion of the extract in step 2 with ethyl acetate:ethanol in the same ratio, dissolve the insoluble portion with 70% methanol and separate it with a medium- and low-pressure ODS column, wherein the filler particle size is 40-70 μm, and use methanol-water (50 / 50, 70 / 30, 90 / 10, 100 / 0, v / v) gradient elution to obtain a total of 10 parts, detect by thin layer chromatography, develop color, combine the same parts, and concentrate the combined third part to dryness under reduced pressure below 75°C for standby use.

[0048] Step 4: The product obtained in step 3 is further separated by pre-treated Sephadex column chromatography (Sephadex LH-20), eluted with methanol, and 6 elution fractions are obtained (i.e., a total of 6 bottles, each 50 mL). The elution fractions are detected by thin layer chromatography, color is developed, and the 6 colored fractions are retained and concentrated to dryness under reduced pressure below 50°C for later use. The ODS and the Sephadex pretreatment process are as follows: soaking in methanol for 24 hours, loading the column, washing with methanol until no turbidity is observed when dripping into water, and then equilibration with the initial mobile phase.

[0049] Step 5: The product obtained in step 4 was further separated by pretreated ODS medium-pressure column chromatography, wherein the filler particle size is 40-60 μm, and gradient elution was performed with methanol-water (5:95, 10:90, 15:85, 20:80, v / v) (pressurized to a flow rate of 1 mL / min and room temperature) to obtain 3 fractions (i.e., 3 bottles were obtained by gradient elution, each bottle having 120 mL). The second fraction was retained and concentrated to dryness under reduced pressure below 50°C for later use.

[0050] Step 6: The second fraction obtained in step 5 was concentrated and then subjected to HPLC (high performance liquid chromatography) separation and preparation. Methanol: 0.1% formic acid water (5:95, v / v) was used as the mobile phase for isocratic elution. The detection wavelengths were 210 nm and 254 nm. The azabicyclic compound 4-ethyl-3-oxa-1-azabicyclo[2.1.1]hexane-2,5-dione of the present invention was separated and prepared. The purity was 90-99% as determined by normalization method.

[0051] Example 2.

[0052] The invention discloses an anti-inflammatory effect of the azabicyclic compound 4-ethyl-3-oxa-1-azabicyclo[2.1.1]hexane-2,5-dione.

[0053] 1. Main materials.

[0054] 1.1 Drugs and Reagents: The azabicyclic compounds used in the experiments were prepared as described above with a purity of 90-99%. They were accurately weighed and diluted with DMSO to the desired solution for each dose group as described below. DMEM high-glucose medium and fetal bovine serum were obtained from Hyclone (USA); penicillin and streptomycin were obtained from Hangzhou Sijiqing Biotechnology Co., Ltd.; LPS was obtained from Sigma (USA); ELISA kits for IL-1β and TNF-α were obtained from Cayman Islands (USA); and cell lysis buffer and Griess reagent were obtained from Beyotime Biotechnology Co., Ltd.

[0055] 1.2 Cell line: RAW264.7 macrophages (ATCC cell bank, USA).

[0056] 1.3 Grouping: Divide into control group, LPS group and experimental group, one group each.

[0057] 2. Experimental method.

[0058] 2.1 Cell culture: DMEM high-glucose medium was added with 10% fetal bovine serum and 1% antibiotics (100 U / mL penicillin and 100 μg / mL streptomycin) and cultured in a 37.5°C, CO2 incubator.

[0059] 2.2 CCK-8 assay for cell viability: The three groups of RAW264.7 macrophages in logarithmic growth phase were inoculated into 96-well culture plates at a cell density of 1×10 4 After overnight incubation at 37°C with 5% CO2, the experimental group was treated with different concentrations of the compound of the present invention (5μM to 50μM). After incubation for 1 hour, LPS at a concentration of 1μg / mL was added to the LPS group and the experimental group, respectively. A zero adjustment group (culture medium containing DMSO solvent) was also set up. Each group had three replicate wells to investigate the effects of drug addition on the cells. After 24 hours of incubation, 10μL of CCK-8 was added to each well of the cells. After an additional 2 hours of incubation at 37°C with 5% CO2, the absorbance of each well was measured at a wavelength of 450nm using a microplate reader.

[0060] 2.3 ELISA assay for inflammatory cytokines IL-1β and TNF-α: RAW264.7 macrophages in the logarithmic growth phase were seeded in 24-well culture plates at a cell density of 1×10 5Cells were incubated overnight at 37°C in 5% CO2 with 1 mL per well. The experimental group was treated with the azabicyclic compound of the present invention (1-20 μM). After 1 hour of incubation, LPS (final concentration of 1 μg / mL) was added to each well and incubated for 24 hours. Each treatment was repeated in triplicate. The levels of IL-1β and TNF-α secreted by RAW264.7 macrophages after treatment with the purslane-derived azabicyclic compound were determined by ELISA.

[0061] 3. Experimental results.

[0062] The experimental results show that the azabicyclic compound of the present invention has no effect on the proliferation of LPS-induced macrophage RAW264.7 at 20 μM, is safe and non-toxic; and can effectively inhibit the excessive production of inflammatory cytokines IL-1β and TNF-α by LPS-induced macrophage RAW264.7 in a concentration-dependent manner.

[0063] The relative survival rate experimental results of the azabicyclic compounds of the present invention are shown in Table 2.

[0064] Table 2 Relative survival rates of azabicyclic compounds.

[0065]

[0066] Note: * P<0.05 compared with the control group, # P<0.05 compared with the LPS group.

[0067] The results of ELISA determination of inflammatory factors IL-1β and TNF-α are shown in Table 3.

[0068] Table 3 Effects of the azabicyclic compounds of the present invention on the levels of IL-1β and TNF-α secreted by RAW264.7 cells induced by LPS (mean ± standard deviation, n=3).

[0069]

[0070]

[0071] Note: * P<0.05 compared with the control group, # P<0.05 compared with the LPS group.

[0072] In summary, the present invention provides an azabicyclic compound and an extraction and separation method thereof, which uses ethanol extraction, ODS medium-pressure column, Sephadex LH-20 and high-performance liquid chromatography for separation, purification and preparation, and successfully separates and obtains this new compound. The method is simple, rapid and environmentally friendly, and the compound separated by the method has a high purity. Since the obtained azabicyclic compound has a unique chemical structure and is extracted from the commonly used traditional Chinese medicine Portulaca oleracea, it has an anti-inflammatory effect. Therefore, the azabicyclic compound of the present invention and its salts and derivatives can be used as natural products to develop new traditional Chinese medicine drugs, which has broad prospects.

Claims

1. An azabicyclic compound in Portulaca oleracea, characterized in that: The molecular formula is C6H7NO3, and the chemical structure is: 。 2. The method for extracting and separating an azabicyclic compound from Portulaca oleracea according to claim 1, wherein: The specific steps include: Step 1: Take dried purslane medicinal material, extract it with ethanol, filter the ethanol extract, combine the filtrate, concentrate under reduced pressure, and cool to room temperature to obtain a medicinal solution for use; Step 2, after evaporating the Chinese medicinal solution in step 1, extract with different ratios of ethyl acetate: ethanol, filter the extract, and concentrate under reduced pressure to obtain an extract for later use; the mobile phase elution program used is ethyl acetate, ethyl acetate: ethanol = 5:1, ethyl acetate: ethanol = 2:1, ethyl acetate: ethanol = 1:1, ethyl acetate: ethanol = 1:2, ethyl acetate: ethanol = 1:5, and recover ethanol for gradient elution; Step 3, dissolve the extract in step 2 in a ratio of ethyl acetate to ethanol = 1:5 with ethyl acetate to ethanol in the same ratio, and dissolve the insoluble part with 70% methanol to obtain a medicinal solution for later use; Step 4: Separate the Chinese medicine solution in step 3 through an ODS column, use methanol-water gradient elution to obtain several elution fractions, detect by thin layer chromatography, develop color, combine the elution fractions with color, and concentrate the combined elution fractions to dryness under reduced pressure for later use; the volume ratio of methanol to water used for gradient elution is 50:50, 70:30, 90:10 and 100:0, and the ODS particle size is 40 to 70 μm; Step 5: The third fraction obtained in step 4 is subjected to a pretreated dextran gel chromatography column and isocratically eluted with 70% methanol to obtain several elution fractions, which are detected by thin layer chromatography and color developed. The color developed elution fractions are combined, and the combined elution fractions are concentrated to dryness under reduced pressure for later use; Step 6: The product obtained in step 5 was further separated by pre-treated ODS column chromatography, using a methanol-water gradient elution to obtain three fractions; wherein the gradient elution was performed using 5%, 10%, 15%, and 20% methanol; Step 7: The second fraction obtained in step 6 was concentrated and subjected to HPLC separation and preparation, and isocratic elution was performed using methanol:0.1% formic acid as the mobile phase. The volume ratio of methanol-0.1% formic acid used was 5:95, and the retention time of the compound was 9.614 min; the azabicyclic compound was obtained.

3. The extraction and separation method according to claim 2, wherein In the step 1, ethanol reflux extraction is performed twice, each time for 2 hours, and the amount of water used is 8 to 16 times the amount of the medicinal material.

4. The azabicyclic compound according to claim 1, wherein The azabicyclic compound is used for preparing anti-inflammatory drugs.

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