A pyridine alkaloid from purslane and its extraction and separation method and application

Abstract

The present invention belongs to the field of traditional Chinese medicine extraction and separation technology, and particularly relates to a pyridine alkaloid compound extracted and separated from Portulaca oleracea, its extraction and separation method, and application. The compound has a molecular formula of C7H9NO2 and a chemical name of 5-ethoxypyridin-2-ol. A method for extracting and separating the compound is also provided, comprising sequentially employing ethanol reflux extraction, ethyl acetate and ethanol reflux extraction in a volume ratio of 2:1, ODS medium-pressure column chromatography, Sephadex LH-20 column chromatography, and high-performance liquid chromatography for preparation. Its structure was determined by mass spectrometry, carbon spectroscopy, hydrogen spectroscopy, and two-dimensional nuclear magnetic resonance spectroscopy. The compound has anti-inflammatory and antioxidant effects. The 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 present invention belongs to the technical field of traditional Chinese medicine extraction and separation, and relates to a pyridine alkaloid in purslane, an extraction and separation method and application thereof, and in particular to a compound 5-ethoxypyridin-2-ol extracted, separated and identified from purslane, an extraction and separation method and application thereof. Background Art

[0002] Portulaca oleracea L., with its leaves often alternate or nearly opposite and cylindrical, often light brown-red stems, is an annual succulent herb from the genus Portulaca in the family Portulacaceae. First recorded in the Compendium of Materia Medica, it is a dual-purpose medicinal and edible plant. It is highly resistant to harsh environments such as drought and high salinity. With a history of thousands of years as a traditional Chinese medicine, it is not only resilient but also possesses excellent ecological restoration capabilities and strong adaptability. It is currently widely distributed in temperate and tropical regions around the world. With the development of modern Chinese medicine, Portulaca oleracea is described in the 2020 edition of the "Chinese Pharmacopoeia" as sour in taste, cold in nature, and enters the liver and large intestine meridians. It has the effects of clearing heat and detoxifying, cooling blood and stopping bleeding, and stopping dysentery. It can be used to treat heat-toxic bloody dysentery, carbuncles, furuncles, eczema, erysipelas, snake and insect bites, bloody stools, hemorrhoids, and metrorrhagia.

[0003] Modern pharmacological research on Portulaca oleracea demonstrates its anti-inflammatory, antibacterial, antiviral, antihypertensive, antilipidemic, antioxidant, anti-tumor, and immune-regulating properties. Its main chemical components include flavonoids, coumarins, terpenes, steroids, alkaloids, amino acids, various pigments, and minerals. Alkaloids are a major class of chemical components in Portulaca oleracea. Reported alkaloids include norepinephrine, dopamine, small amounts of dopa, adenosine, uracil, adenine, allantoin, and N-trans-feruloyltyramine; cyclic dipeptide alkaloids are also present.

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

[0005] To address the above problems, the present invention provides pyridine alkaloids extracted and separated from Portulaca oleracea. Studies have found that the compounds of the present invention have anti-inflammatory and antioxidant effects. At the same time, a simple, rapid, environmentally friendly, and high-purity extraction and separation method for the compounds of the present invention is provided.

[0006] To achieve the above-mentioned purpose of the present invention, the present invention provides a pyridine alkaloid with a molecular formula of C7H9NO2, named 5-ethoxypyridin-2-ol, 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 pyridine alkaloids from Portulaca oleracea, which comprises the following steps:

[0009] Step 1: Take dried purslane medicinal material, extract it with ethanol reflux, 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 medicinal liquid in step 1 is evaporated to dryness, it is extracted with ethanol and ethyl acetate, and recovered under reduced pressure to obtain an extract to obtain an extract;

[0011] Step 3, the extract in step 2 is subjected to chromatography separation on a pretreated ODS column (Octadecylsilyl, octadecylsilane bonded silica gel filler), and gradient elution with methanol-water is used to obtain several elution fractions, which are detected by thin layer chromatography and color is developed. Each elution fraction that has developed color is concentrated to dryness under reduced pressure to obtain a concentrate for use;

[0012] Step 4: The concentrate obtained in step 3 is separated by chromatography on a pretreated Sephadex LH-20 column, and isocratically eluted with 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;

[0013] Step 5: The concentrate obtained in step 4 is subjected to HPLC (high performance liquid chromatography) separation and preparation using methanol:0.1% formic acid as the mobile phase to prepare the compound of the present invention.

[0014] Furthermore, in step 1, 50% ethanol reflux extraction is performed twice, each time for 2 hours, and the volume of 50% ethanol used is 10 times that of the medicinal material.

[0015] Furthermore, in step 2, the volume ratio of ethyl acetate to ethanol is 2:1, reflux extraction is performed twice, each time for 2 hours, and the amount of ethyl acetate and ethanol used is 10 times that of the medicinal material.

[0016] Furthermore, in step 3, the volume ratio of methanol to water in the gradient elution is 40:60, 50:50, 70:30, and 100:0.

[0017] Furthermore, in step 5, the volume ratio of methanol to 0.1% formic acid is 30:70, and the retention time of the compound is 14.693 min.

[0018] Furthermore, the pretreatment process of the ODS and dextran gel is to soak them in methanol for 24 hours, load them onto the column, wash them with methanol until there is no turbidity when dripping into water, and then balance them with the initial mobile phase.

[0019] Compared with the prior art, the present invention has the following beneficial effects.

[0020] The isolation and pharmacological activity research of 5-ethoxypyridin-2-ol from purslane of the present invention has not been reported in existing paper journals; the present invention provides pyridine alkaloids derived from purslane and a method for extracting and separating the compound of the present invention, which sequentially adopts ethanol reflux extraction, ethanol and ethyl acetate extraction in a volume ratio of 1:2, ODS medium-pressure column, Sephadex LH-20 and high-performance liquid chromatography for separation, purification and preparation, and successfully extracts and separates the compound. The method has only five operating steps and is simple and rapid. The extraction and separation process mainly adopts ethanol and ethyl acetate extraction and methanol elution, and the process method is environmentally friendly. The purity of the compound separated by the method is relatively high, both greater than 90%. In addition, studies have shown that the compound has anti-inflammatory and antioxidant effects. Therefore, the 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 and antioxidant drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The compound 5-ethoxypyridin-2-ol of the present invention 1 H-NMR spectrum.

[0022] Figure 2 The compound 5-ethoxypyridin-2-ol of the present invention 13 C-NMR spectrum.

[0023] Figure 3 This is the DEPT spectrum of the compound 5-ethoxypyridin-2-ol of the present invention.

[0024] Figure 4 This is the HSQC spectrum of the compound 5-ethoxypyridin-2-ol of the present invention.

[0025] Figure 5 This is the HMBC spectrum of the compound 5-ethoxypyridin-2-ol of the present invention.

[0026] Figure 6 The compound 5-ethoxypyridin-2-ol of the present invention 1 H- 1 H COSY spectrum.

[0027] Figure 7 The figure is the ROESY spectrum of the compound 5-ethoxypyridin-2-ol of the present invention.

[0028] Figure 8 This is a high-resolution mass spectrum of the compound 5-ethoxypyridin-2-ol of the present invention. DETAILED DESCRIPTION

[0029] 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.

[0030] Example 1.

[0031] The present invention provides a pyridine alkaloid with a molecular formula of C7H9NO2, named 5-ethoxypyridin-2-ol, and a chemical structural formula of:

[0032]

[0033] Table 1 shows the NMR data of 5-ethoxypyridin-2-ol. The solvent used in NMR is deuterated methanol.

[0034] Table 1 NMR data of the compound 5-ethoxypyridin-2-ol of the present invention

[0035]

[0036]

[0037] 5-ethoxypyridin-2-ol: Pale yellow powder, freely soluble in methanol, insoluble or slightly soluble in water. Spotted on a silica gel thin layer plate, the spot appears cyan when sprayed with ferric chloride solution, indicating the compound contains a phenolic hydroxyl group. UHPLC-ESI-QTOF-MS yields an m / z of 138.0559 [MH]. - The quasi-molecular ion peak of β-hydroxybenzoic acid is 138.0559. 1 H-NMR, 13 Based on C-NMR and DEPT data, it was speculated that the possible molecular formula of the compound was C7H9NO2, with an unsaturation degree of 4. 13 The C-NMR spectrum and DEPT spectrum showed 7 carbon signals, including 1 methyl carbon (δ14.59), 1 methylene carbon (δ62.47), 3 methine carbons (δ139.08; 123.76; 128.02), and 2 quaternary carbons (δ165.99; 158.92). 1 H-NMR spectrum showed one CH3 signal as δ H 1.34 (3H, t, J = 7.14); 1 methylene signal is δ H4.34 (2H, q, J = 6.78, 14.22); 3 methine signals are δ H 8.12 (1H, d, J = 2.7), δ H 7.98 (1H, d, J = 8.58) and δ H 7.25 (1H, dd, J = 2.82, 8.64). HMBC spectrum showed that H-3 and C-2 (δ C 158.92), C-5(δ C 165.99); H-4 is related to C-2, C-6 (δ C 139.08); H-6 is related to C-2, C-4 (δ C 128.02) related, 1 H- 1 The H COSY spectrum shows that H-3 and H-4 are related, and it is speculated that the core structure of this structure is a pyridine ring with a para-substituted structure. Since the chemical shift of C-2 is in the downfield, it can be judged that C-2 is connected to a hydroxyl group. The HMBC shows that H-2′(δ H 4.34) and C-3′(δ C 14.59), C-5(δ C 165.99) related, H-3′(δ H 1.34) and C-2′(δ C 62.47). And the chemical shift of C-6 is in the downfield, so it is speculated that C-6 is connected to C-2′ through an oxygen atom. In addition, 1 H- 1 The H COSY spectrum showed that H-2′ and H-3′ were correlated, further confirming the branched structure. Therefore, based on the above information, this compound was identified as 5-ethoxypyridin-2-ol.

[0038] The present invention also provides a method for extracting and separating the above-mentioned compound, which specifically comprises the following steps:

[0039] Step 1: Weigh 150 kg of dried purslane medicinal materials, use 50% ethanol reflux extraction, the amount of 50% ethanol used (v / v) is 10 times that of the medicinal materials, reflux extraction twice, each time for 2 hours, filter the alcohol extract, combine the filtrate, and concentrate under reduced pressure to 200 L, cool to room temperature, and obtain the medicinal solution for use.

[0040] Step 2: Evaporate the medicinal solution obtained in step 1 to dryness and extract it with ethyl acetate and ethanol in a volume ratio of 2:1. The extracted medicinal solution is decompressed below 40° C. to recover the extract to obtain an extract.

[0041] Step 3: The extract in step 2 was separated by pretreated ODS medium-pressure column chromatography, wherein the filler particle size was 20-40 μm, and gradient elution was performed with methanol-water (40 / 60, 50 / 50, 70 / 30, 100 / 0, v / v) (pressurized to a flow rate of 1 mL / min and room temperature) to obtain 14 fractions (i.e., 32 bottles were obtained by gradient elution, each bottle was 80 mL), which were detected by thin-layer chromatography and color developed. The colored 12-18 fractions were retained and concentrated to dryness under reduced pressure below 50°C for later use.

[0042] Step 4: The product obtained in step 3 was 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 40 mL), detected by thin layer chromatography, and color was developed. The 10-15 colored fractions were retained and concentrated to dryness under reduced pressure below 50°C for later use.

[0043] Step 5: The product obtained in step 4 was separated and prepared by HPLC using methanol:0.1% formic acid (30:70, v / v) as the mobile phase and detection wavelengths of 210 nm and 254 nm to separate and prepare the compound of the present invention. The purity was determined by normalization method to be 90-99%.

[0044] 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 no turbidity occurs when dripped into water, and then balancing with the initial mobile phase.

[0045] Example 2 Anti-inflammatory effect of 5-ethoxypyridin-2-ol.

[0046] 1 Main Materials

[0047] 1.1 Drugs and Reagents: The compounds used in the experiments were prepared as described above with a purity of 90%. 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-6, TNF-α, and PGE2 were obtained from Cayman Islands (USA); and cell lysate and Griess reagent were obtained from Beyotime Biotechnology Co., Ltd.

[0048] 1.2 Cell line: RAW264.7 macrophages (ATCC cell bank, USA).

[0049] 1.3 Grouping: Divided into control group, LPS group and experimental group.

[0050] 2 Experimental methods

[0051] 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% CO2 incubator.

[0052] 2.2 MTT colorimetric assay for cell viability: RAW264.7 macrophages in the logarithmic growth phase were taken from the three groups and inoculated into 96-well culture plates at a cell density of 1×10 4 / mL, 100μL per well, temperature 37 ℃, 5% CO2 after overnight incubation, the experimental group added different concentrations of the compound of the present invention 5-ethoxypyridin-2-ol (10-100μM), incubated for 1h, LPS with a final concentration of 1μg / mL was added to the LPS group and the experimental group, respectively, 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 24h of cell culture in each of the above groups, 20μL of 5mg / mL MTT was added to the cells in each well, and the temperature was 37℃, 5% CO2 was continued to incubate for 4h, the culture was terminated, the liquid in the well was discarded, 100μL of dimethyl sulfoxide (DMSO) was added to each well, and the cells were shaken for 10min to fully dissolve the crystals in the cells. The absorbance of each well was measured at a wavelength of 570nm using a microplate reader.

[0053] 2.3 The inhibitory effect of the compounds of the present invention on LPS-induced NO production in RAW264.7 mouse macrophages was investigated by measuring NO content using the Griess method. RAW264.7 mouse macrophages were passaged and cultured in DMEM (DMEM), a high-glucose cell culture medium supplemented with 10% fetal bovine serum. Experimental groups were treated with varying concentrations of the compound of the present invention, 5-ethoxypyridin-2-ol (5-50 μM). After incubation at 37°C, 5% CO₂ for 1 hour, an inflammatory response was induced with LPS (final concentration of 1 μg / mL). Supernatants were collected 24 hours later. Each treatment was repeated in triplicate. The NO content in the cell supernatant was determined by the Griess method. The effect of varying concentrations of the compounds of the present invention on LPS-induced NO release from RAW264.7 cells was used to reflect NO levels.

[0054] 2.4 ELISA determination of inflammatory factors IL-6, TNF-α and inflammatory mediator PGE2: 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 compound of the present invention, 5-ethoxypyridin-2-ol (5-50 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. ELISA was used to determine the levels of IL-6, TNF-α, and PGE2 secreted by RAW264.7 macrophages after treatment with the purslane-derived compound.

[0055] 3 Experimental results

[0056] The experimental results show that the compound of the present invention has no effect on the proliferation of LPS-induced macrophage RAW264.7, is safe and non-toxic; and can effectively inhibit the excessive inflammatory cytokines IL-6, TNF-α and inflammatory mediators NO and PGE2 produced by LPS-induced macrophage RAW264.7 in a concentration-dependent manner.

[0057] The results of the relative cell survival rate experiment are shown in Table 2.

[0058] Table 2 Effect of the present invention on the relative survival rate of RAW264.7 macrophages

[0059]

[0060] Note: * P<0.05 compared with the control group (the high concentration group had a significant difference)

[0061] The results of the Griess method for determining NO content are shown in Table 3.

[0062] Table 3 Effects of the present invention on LPS-induced NO release in RAW264.7 cells (mean ± standard deviation, n=3)

[0063]

[0064] Note: * P<0.05 compared with the control group, # P<0.05 compared with the LPS group.

[0065] The results of ELISA determination of inflammatory factors IL-6, TNF-α and inflammatory mediator PGE2 are shown in Table 4.

[0066] Table 4 Effects of the present invention on the levels of IL-6, TNF-α and PGE2 secreted by RAW264.7 cells induced by LPS (mean ± standard deviation, n=3)

[0067]

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

[0069] Example 3 Antioxidant effect of 5-ethoxypyridin-2-ol.

[0070] 1 Main Materials

[0071] 1.1 Drugs and Reagents: The compounds used in the experiments were prepared as described above with a purity of 90%. They were accurately weighed and diluted with methanol to the desired solution for each dose group as described below. DPPH (1,1-diphenyl-2-picrylhydrazyl free radical) was obtained from Sigma-Fluka; BHA (tert-butylhydroxyanisole) was obtained from Shanghai Xiangrui Technology Co., Ltd.; and methanol, chromatographic grade, was obtained from Changtai Xingye Co., Ltd.

[0072] 1.2 Grouping: Divided into control group, experimental group and blank group.

[0073] 2 Experimental methods

[0074] The ability to eliminate DPPH free radicals was determined by colorimetry: the experimental group took 1 mL of DPPH solution (80 μM) and added it to a cuvette, and then added 1 mL of 5-ethoxypyridin-2-ol (6.25-100 μM) of different concentrations; the control group took 1 mL of methanol solution and added it to a cuvette, and then added 1 mL of sample solution of different concentrations; the blank group took 1 mL of DPPH solution and added it to a cuvette, and then added 1 mL of methanol solution. All three groups were thoroughly mixed, kept at room temperature in the dark for 10 minutes, and the absorbance was measured at 517 nm. Each sample was measured three times on average and the average value was taken. The positive control was a BHA solution of different concentrations (6.25-100 μM). The scavenging rate of the sample for DPPH free radicals was calculated according to the following formula, and its free radical scavenging rate IC was further calculated. 50 value.

[0075] DPPH clearance rate (%) = 1-(A1-A2) / A0×100%

[0076] Among them, A0 is the absorbance value of the blank group; A1 is the absorbance value of the sample group; A2 is the absorbance value of the control group.

[0077] 3 Experimental results

[0078] The experimental results show that the compounds of the present invention have a scavenging effect on DPPH free radicals, and the scavenging rate increases significantly with the increase of drug concentration.

[0079] The compounds of the present invention have an IC value for DPPH free radicals. 50 See Table 5 for values.

[0080] Table 5 Scavenging effect of the compounds of the present invention on DPPH free radicals

[0081] Group <![CDATA[IC 50 (μM)]]> BHA 58.68 5-ethoxypyridin-2-ol 52.45

[0082] In summary, the present invention provides the compound 5-ethoxypyridin-2-ol, its extraction and isolation methods, and applications. The compound was successfully isolated and purified using ethanol reflux extraction, extraction with ethyl acetate and ethanol in a 2:1 volume ratio, ODS medium-pressure column chromatography, dextran gel column chromatography, and high-performance liquid chromatography. This method is simple, rapid, and environmentally friendly, and the resulting compound is of high purity. Because the resulting compound is extracted from the commonly used traditional Chinese medicine Portulaca oleracea and exhibits anti-inflammatory and antioxidant effects, the compound, its salts, and derivatives of the present invention can provide an experimental basis for drug development and have broad prospects.

Claims

1. A method for extracting and separating pyridine alkaloids from Portulaca oleracea, characterized in that: The specific steps include: Step 1: Take dried purslane, extract with ethanol reflux, 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 the Chinese medicine solution in step 1 is evaporated to dryness, it is extracted with ethyl acetate and ethanol, and recovered under reduced pressure to obtain an extract; Step 3: Evaporate the drug solution from step 2 to dryness and apply it to an ODS column. Elute with a gradient of methanol and water in a volume ratio of 40:60, 50:50, 70:30, and 100:

0. Detect by thin layer chromatography and develop color. Concentrate each colored elution fraction to dryness under reduced pressure to obtain a concentrate for later use. Step 4: The extract obtained in step 3 is separated by pre-treated dextran gel column chromatography, isocratically eluted with methanol, detected by thin layer chromatography, color developed, and the colored elution fractions are combined, and the combined elution fractions are concentrated to dryness under reduced pressure; Step 5: The concentrate obtained in step 4 is subjected to HPLC separation and preparation, using methanol:0.1% formic acid in a volume ratio of 30:70 as the mobile phase to prepare the pyridine alkaloids; The chemical structural formula of the pyridine alkaloids is .

2. The extraction and separation method according to claim 1, wherein In the step 1, 50% ethanol reflux extraction is performed twice, each time for 2 hours, and the volume of 50% ethanol used is 10 times that of the medicinal material.

3. The extraction and separation method according to claim 1, wherein In the step 2, the volume ratio of ethyl acetate to ethanol is 2:1, and reflux extraction is performed twice, each time for 2 hours. The amount of ethyl acetate and ethanol used is 10 times that of the medicinal material.

4. Use of the pyridine alkaloids isolated from the Portulaca oleracea medicinal material according to claim 1 in the preparation of anti-inflammatory drugs and antioxidant drugs.

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