A new pyridine alkaloid compound from Portulaca oleracea and its extraction and separation method and application

Abstract

The present invention belongs to the field of traditional Chinese medicine extraction and separation, and particularly relates to a new pyridine alkaloid compound in purslane, as well as an extraction, separation method and application thereof. The new compound has a molecular formula of C 11 H 15 NO2, and is named Olerapyridine. An extraction and separation method for the above new compound is also provided, which successively 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 by methods such as mass spectrometry, hydrogen nuclear magnetic resonance spectroscopy, carbon nuclear magnetic resonance spectroscopy and two-dimensional nuclear magnetic resonance spectroscopy, and has anti-inflammatory activity. The new compound and its salts or derivatives of the present invention 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 in particular relates to a new pyridine alkaloid 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 in 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 survival ability. It likes fertile soil, and neutral and weakly acidic soils are better. 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, carbuncle, furuncle, eczema, erysipelas, snake and insect bites, blood in the stool, hemorrhoids, metrorrhagia, etc.

[0003] Modern pharmacological research has found that there are many chemical components in purslane, including flavonoids, coumarins, volatile oils, terpenes, steroids, alkaloids, amino acids, polysaccharides, various pigments and mineral substances. Among them, alkaloids are the main chemical components of purslane. Because purslane contains rich chemical substances, it provides a material basis for the study of the pharmacological effects of purslane. Studies have found that purslane has the effects of lowering blood sugar, lowering blood lipids, anti-tumor, anti-oxidation, anti-aging, anti-browning, anti-inflammatory, anti-viral, neuroprotective, and regulating immune function. The reported components include 1-methanesulfonic acid-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, 5-hydroxy-2-carboxypyridine, tropaeoline, 3-isobutyl-6-methylpiperazine-2,5-dione, oleraciamideE, oleraciamideF, oleraciamideG, and oleracrylimideA-F.

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

[0005] In view of the above problems, the present invention provides a new pyridine alkaloid compound extracted and separated from Portulaca oleracea. Studies have shown 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] In order to achieve the above-mentioned object of the present invention, the present invention provides a new pyridine alkaloid compound, the molecular formula of which is C 11 H 15 NO7, named Olerapyridine, has the chemical formula:

[0007] .

[0008] In order to achieve the above-mentioned object of the present invention, the present invention also provides a method for extracting and separating a new pyridine alkaloid compound from Portulaca oleracea, which specifically comprises the following steps:

[0009] Step 1: Take dried purslane, extract 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, it is extracted with ethyl acetate: ethanol in different proportions, the extract is filtered, and concentrated under reduced pressure to an extract 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 filler) column, use methanol-water gradient elution to obtain several elution parts, detect by thin layer chromatography, develop color, combine the elution parts with color, and concentrate the combined elution parts to dryness under reduced pressure for use.

[0013] Step 5. The fourth fraction obtained in step 4 is further separated by chromatography on a pretreated Sephadex LH-20 column, and isocratically eluted with 70% methanol to obtain several elution fractions, which are detected by thin layer chromatography, developed in color, and the elution fractions with developed color 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 is separated by pretreated ODS column chromatography, and gradient eluted with methanol-water (10 / 90, 30 / 70, 100 / 0, v / v) (pressurized to a flow rate of 1 mL / min and room temperature) to obtain 3 fractions (i.e., 3 bottles of 120 mL each by gradient elution). The first fraction is retained and concentrated to dryness under reduced pressure below 50°C for later use.

[0015] Step 7: The first part in step 6 is further separated by pretreated dextran gel column chromatography (Sephadex LH-20), eluted with methanol, and 20 elution fractions are obtained (i.e., a total of 20 bottles, each with 50 mL), and fractions 7-11 are retained, and concentrated to dryness under reduced pressure below 50°C.

[0016] Step 8: The concentrate obtained in step 5 is separated and prepared by HPLC (high performance liquid chromatography), and methanol: 0.1% formic acid is used as a mobile phase for isocratic elution to prepare the new pyridine alkaloid compound of the present invention.

[0017] Furthermore, in step 1, ethanol reflux extraction is performed twice, each time for 2 hours, and the amount used is 8-16 times that of the medicinal material.

[0018] Furthermore, the mobile phase elution program used in step 2 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), ethyl acetate: ethanol (1:5), and ethanol recovery gradient elution.

[0019] Furthermore, in step 4, the volume ratio of methanol to water is 50:50, 70:30, 90:10 and 100:0 for gradient elution, and the ODS particle size is 40 to 70 μm.

[0020] Furthermore, the pretreatment process of the ODS and dextran gel is: soaking in methanol for 24 hours, loading on the column, washing with methanol until there is no turbidity when dripped into water, and then balancing with the initial mobile phase.

[0021] Furthermore, the methanol elution procedure in step 5 is 70% methanol isocratic elution.

[0022] Furthermore, in step 6, 5%, 10%, 15%, and 20% methanol are used for gradient elution.

[0023] Furthermore, the methanol elution procedure in step 7 is 5% methanol isocratic elution.

[0024] Furthermore, in step 8, the volume ratio of methanol: 0.1% formic acid water in isocratic elution is 5:95.

[0025] The novel pyridine alkaloid compound of the invention can be used for preparing anti-inflammatory drugs.

[0026] Compared with the prior art, the invention has the beneficial effects.

[0027] The separation and pharmacological activity research of the new pyridine alkaloid compound of purslane described in the present invention has not been reported by existing papers and journals; the present invention provides a new pyridine alkaloid compound derived from purslane and an extraction and separation method for the new compound of the present invention, which uses ethanol extraction, extraction with different proportions 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 fast. The extraction and separation process mainly uses ethanol extraction and methanol elution. The process method is environmentally friendly, and the purity of the compound separated by the 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 synthetic leads for 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

[0028] Figure 1 This is a high-resolution mass spectrum of the new pyridine alkaloid compound Olerapyridine of the present invention.

[0029] Figure 2 The 1H-NMR spectrum of the novel pyridine alkaloid compound Olerapyridine of the present invention is shown in FIG.

[0030] Figure 3 The 13C-NMR spectrum of the novel pyridine alkaloid compound Olerapyridine of the present invention is shown in FIG.

[0031] Figure 4 The DEPT spectrum of the novel pyridine alkaloid compound Olerapyridine of the present invention is shown in FIG.

[0032] Figure 5 This is the HMBC spectrum of Olerapyridine, a new pyridine alkaloid compound of the present invention.

[0033] Figure 6 This is the HSQC spectrum of the new pyridine alkaloid compound Olerapyridine of the present invention.

[0034] Figure 7 This is the 1H-1HCOSY spectrum of Olerapyridine, a new pyridine alkaloid compound of the present invention.

[0035] Figure 8 The ROESY spectrum of the novel pyridine alkaloid compound Olerapyridine of the present invention is shown in FIG. DETAILED DESCRIPTION

[0036] The following examples will help to understand the present invention, but these examples are only for illustrating 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.

[0037] The present invention provides a new compound, the molecular formula of which is C9H 15 N3O5, named Olerapyridine, has the chemical formula:

[0038] .

[0039] Table 1 shows the NMR data of the new pyridine alkaloid compound: 1 H-NMR and 13 C-NMR in methanol.

[0040] Table 1 NMR data of the new pyridine alkaloid Olerapyridine.

[0041]

[0042] The structural identification of the compounds of the present invention is shown in Figure 1-Figure 8 .

[0043] Olerapyridine: Colorless oil, easily soluble in methanol, slightly soluble in water. After spotting on a silica gel thin layer plate, the spot appears orange-yellow when sprayed with dilute potassium bismuth iodide test solution, indicating that the compound is an alkaloid component. UHPLC-ESI-Q-TOF-MS gives m / z: 110.0242 [M-C6H 11 O5] - The quasi-molecular ion peak, (calculated value [C5H4NO2] - =110.0242). Combined 1 H-NMR, 13 C-NMR and DEPT data suggest that the possible molecular formula of the compound is C 11 H 15 NO7, unsaturation is 12. 13 The C-NMR spectrum and DEPT spectrum showed 11 carbon signals, including 1 methylene carbon (δ C :62.50)、6 methyl carbons(δ C :71.26;74.94;77.92;78.08;103.63;118.50;119.41;126.21)、2 quaternary carbons(δ C : 151.13; 158.15).

[0044] The 1H-NMR spectrum shows two CH3 signals and one methylene signal as δ H 3.63, 3.80 (2H, dd,J =10.80, 11.58, Hz, H-7ʹ), 7 methyl signals are δH3.31 (1H, m, H-4ʹ), δ H 3.32 (1H, m, H-5ʹ), δH3.35 (1H, m, H-6ʹ), δ H 4.69 (1H, d, J =7.08Hz, H-2 ʹ ),δ H 6.74 (1H, d, J =8.70Hz, H-3), δ H 7.16 (1H, d, J =8.70Hz, H-4), δ H 7.52 (1H, s, H-6). By comparison 13 C-NMR, DEPT data and δ H 4.69 (1H, d, J =7.08Hz, H-2 ʹ ), it can be inferred that the compound contains β-D-glucose. 13 C-NMR spectrum shows that C-6 (δ C :158.15)、C-2(δ C :119.41) is located in the low field, indicating that there is a nitrogen between the two and a hydroxyl group on C-6. The HMBC spectrum shows that H-2 (δ H 7.52, 1H, s) and C-4 (δ C :126.21)、C-6(δ C :158.15) related; H-4 (δ H 7.16, 1H, d) and C-2 (δ C :119.41)、C-6(δ C :158.15) related; H-5 (δ H 6.74, 1H, d) and C-3 (δ C : 151.13) related; indicating that there is a pyridine ring in the structure. At the same time, the HMBC spectrum shows that H-2 ʹ (δ H 4.69,1H,d, J =7.08Hz) and C-5 (δ C :151.13), indicating that glucose is connected to the pyridine ring. Therefore, based on the above information, it can be determined that this new compound has the above structure.

[0045] The present invention also provides a method for extracting and separating the above-mentioned pyridine alkaloid compound, which specifically comprises the following steps:

[0046] 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.

[0047] Step 2: After the Chinese medicine solution in step 1 is evaporated to dryness, it is extracted with ethyl acetate: ethanol in different proportions, and the extract is filtered to obtain 7 parts in total, which are concentrated under reduced pressure to extracts.

[0048] 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, where 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 10 fractions in total, detect by thin layer chromatography, develop color, combine the same fractions, and concentrate the combined 4th fraction to dryness under reduced pressure below 75°C for later use.

[0049] Step 4: The obtained product in step 3 is separated by pre-treated dextran gel column chromatography (Sephadex LH-20), eluted with methanol, and 6 elution parts are obtained (i.e., 6 bottles are obtained, each bottle is 50 mL), detected by thin layer chromatography, color is developed, and the 6 colored parts are retained, and concentrated to dryness under reduced pressure below 50°C for standby use. The pretreatment process of ODS and dextran gel is to soak in methanol for 24 hours, load the column, wash with methanol until there is no turbidity when dripped into water, and then balance with the initial mobile phase.

[0050] Step 5: The product obtained in step 4 is further separated by pretreated ODS medium-pressure column chromatography, wherein the filler particle size is 40-60 μm, and gradient elution is performed with methanol-water (5:95, 10:90, 15:85, 20:80, v / v) (pressurized to a flow rate of 1 mL / min, and the temperature is room temperature) to obtain 3 fractions (i.e., 3 bottles are obtained by gradient elution, each bottle has 120 mL), retain the first fraction, and concentrate to dryness under reduced pressure below 50°C for later use.

[0051] Step 6: The product obtained in step 5 is further separated by pretreated dextran gel column chromatography (Sephadex LH-20), eluted with methanol to obtain 20 elution fractions (i.e., a total of 20 bottles, each with 50 mL), detected by thin layer chromatography, color developed, and the colored fractions 7-11 are retained, and concentrated to dryness under reduced pressure below 50°C.

[0052] Step 7: The obtained product in the step was separated and prepared by HPLC, with methanol: 0.1% formic acid water (5:95, v / v) as the mobile phase for isocratic elution, and the detection wavelength was 210nm and 254nm, to separate and prepare the new pyridine alkaloid compound of the present invention, and the purity determined by the normalization method was 90-99%.

[0053] Example 2.

[0054] The anti-inflammatory effect of the new pyridine alkaloid compound of the present invention.

[0055] 1. Main materials.

[0056] 1.1 Drugs and reagents: The new pyridine alkaloid compounds used in the experiment were prepared by the above method with a purity of 90-99%. They were accurately weighed and diluted with DMSO to the required solution for each dose group as follows: DMEM high-glucose medium, fetal bovine serum (Hyclone, USA); penicillin and streptomycin (Hangzhou Sijiqing Company); LPS (Sigma, USA); IL-1 β TNF- α ELISA kit (Cayman, USA); cell lysate, Griess reagent (Biyuntian Biotechnology Co., Ltd.).

[0057] 1.2 Cell line: RAW264.7 macrophages (ATCC cell bank, USA).

[0058] 1.3 Grouping: The mice were divided into control group, LPS group and experimental group, each with one group.

[0059] 2. Experimental method.

[0060] 2.1 Cell culture: DMEM high-glucose medium, added with 10% fetal bovine serum, 1% antibiotics (100 U / mL penicillin and 100 μg / mL streptomycin), cultured in a 37.5°C, CO2 incubator.

[0061] 2.2 CCK-8 assay for cell viability: The three groups of RAW264.7 macrophages in logarithmic growth phase were inoculated in 96-well culture plates at a cell density of 1×10 4 / mL, 100μL per well, 37℃, 5% CO2, after overnight culture, different concentrations of Olerapyridine (5μM~50μM) of the compound of the present invention were added to the experimental group, and after incubation for 1h, LPS with a concentration of 1μg / mL was added to the LPS group and the experimental group, and a zero adjustment group (culture solution containing DMSO solvent) was set up. Each group had 3 replicates to investigate the effect of the addition of drugs on the cells. After the cells in the above groups were cultured for 24h, 10μL of CCK-8 was added to the cells in each well, and the cells were incubated for another 2h at 37℃ and 5% CO2, and the absorbance of each well was measured at a wavelength of 450nm by an enzyme marker.

[0062] 2.3 ELISA for determination of inflammatory factor IL-1 β 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 5 / mL, 1mL per well, cultured overnight at 37°C and 5% CO2, the experimental group added the new pyridine alkaloid compound Olerapyridine (1-20μM) of the present invention, cultured for 1h, and then LPS (final concentration of 1μg / mL) was added to each well, incubated for 24h, and each group was treated with 3 wells. ELISA method was used to determine the secretion of IL-1 by RAW264.7 macrophages after treatment with the new alkaloid compound from Purslane β and TNF- α The content.

[0063] 3. Experimental results.

[0064] The experimental results show that the new pyridine alkaloid 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 inflammatory cytokine IL-1 produced by LPS-induced macrophage RAW264.7 β and TNF- α , and was concentration-dependent.

[0065] The relative survival rate experimental results of the new pyridine alkaloid compounds of the present invention are shown in Table 2.

[0066] Table 2 Relative survival rates of new pyridine alkaloid compounds.

[0067]

[0068] Note: * P<0.05 compared with the control group. # P<0.05 compared with LPS group.

[0069] ELISA method for determination of inflammatory factor IL-1β and TNF- α The results are shown in Table 3.

[0070] Table 3 Effect of the novel pyridine alkaloid compounds of the present invention on IL-1 secretion by RAW264.7 cells induced by LPS β and TNF- α The influence of content (mean ± SD, n = 3).

[0071]

[0072] Note: * P<0.05 compared with the control group. # P<0.05 compared with LPS group.

[0073] In summary, the present invention provides a new pyridine alkaloid 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 the new compound. The method is simple, rapid and environmentally friendly, and the compound separated by the method has a high purity. Since the obtained new pyridine alkaloid 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 new pyridine alkaloid compound and its salts and derivatives of the present invention can be used as natural products to develop new traditional Chinese medicines, and have broad prospects.

Claims

1. A new pyridine alkaloid compound from Portulaca oleracea, characterized in that: The molecular formula is C 11 H 15 NO7, named Olerapyridine, has the chemical structure: 。 2. A method for extracting and separating a new pyridine alkaloid compound from Portulaca oleracea, characterized in that: The specific steps include: Step 1, take dried purslane, extract with ethanol, filter the ethanol extract, combine the filtrate, concentrate under reduced pressure, cool to room temperature, and obtain a medicinal solution for use; Step 2, after the Chinese medicine solution in step 1 is evaporated to dryness, it is extracted with different proportions of ethyl acetate: ethanol, the extract is filtered, and the extract is concentrated under reduced pressure to an extract for standby use; Step 3, dissolve the extract in step 2 in a ratio of ethyl acetate to ethanol of 1:5 with ethyl acetate to ethanol in the same ratio, and dissolve the insoluble part with 70% methanol to obtain a liquid medicine for standby use; Step 4, separating the Chinese medicine solution in step 3 through an ODS column, using a methanol-water gradient elution to obtain a number of elution fractions, detecting by thin layer chromatography, developing color, combining the elution fractions with color, and concentrating the combined elution fractions to dryness under reduced pressure for later use; Step 5, the obtained product in step 4 is further separated by pre-treated dextran gel column chromatography, isocratically eluted with 70% methanol to obtain several elution fractions, detected by thin layer chromatography, color developed, combined the color developed elution fractions, and concentrated the combined elution fractions to dryness under reduced pressure for standby use; Step 6: The product obtained in step 5 is separated by pre-treated ODS column chromatography, and eluted with a methanol-water v / v gradient of 10 / 90, 30 / 70, and 100 / 0 to obtain three fractions, and the first fraction is retained and concentrated to dryness under reduced pressure below 50° C. for standby use; Step 7: The product obtained in step 6 is separated by pre-treated dextran gel column chromatography, eluted with methanol, and 20 elution fractions are obtained, detected by thin layer chromatography, and color is developed, and the color-developed fractions 7-11 are retained, and concentrated to dryness under reduced pressure below 50°C; Step 8: The concentrate obtained in step 5 is separated and prepared by HPLC, and methanol:0.1% formic acid is used as the mobile phase for isocratic elution to prepare the new pyridine alkaloid compound as claimed in claim 1.

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

4. The extraction and separation method according to claim 2, characterized in that: The mobile phase elution program used in step 2 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, ethyl acetate:ethanol 1:5, and ethanol recovery gradient elution.

5. The extraction and separation method according to claim 2, characterized in that: The volume ratio of water to methanol used in step 4 is 100:0, 90:10, 70:3 and 50:50 for gradient elution; the particle size of ODS is 40-70 μm.

6. The extraction and separation method according to claim 2, characterized in that: In step 6, 5%, 10%, 15%, and 20% methanol were used for gradient elution.

7. The extraction and separation method according to claim 2, characterized in that: The methanol elution procedure in step 7 is 5% methanol isocratic elution.

8. The extraction and separation method according to claim 2, characterized in that: The volume ratio of methanol to 0.1% formic acid used in step 8 is 5:95, and the retention time of the compounds is 11.346 min.

9. Use of a new pyridine alkaloid compound from Portulaca oleracea as claimed in claim 1 in the preparation of anti-inflammatory drugs.

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