A pyridine alkaloid compound in purslane and its extraction and separation method and use

Abstract

The present invention belongs to the technical field of extraction and separation of traditional Chinese medicine, and relates to the extraction, separation and identification of a pyridine alkaloid compound from Portulaca oleracea, and its extraction and separation method and use. The pyridine alkaloid compound has a molecular formula of C6H6N2O2 and is named 5-hydroxypicolinamide. The extraction and separation method of the pyridine alkaloid compound 5-hydroxypicolinamide sequentially adopts water decoction extraction, macroporous resin column chromatography, ODS column chromatography and high performance liquid chromatography for separation, purification and preparation. Its structure is determined to be the pyridine alkaloid compound 5-hydroxypicolinamide by mass spectrometry, hydrogen spectrum, carbon spectrum and two-dimensional nuclear magnetic spectrum analysis. The compound 5-hydroxypicolinamide has anti-inflammatory activity, anticholinesterase activity and antioxidant activity. The compound 5-hydroxypicolinamide and its salt or derivative 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 pyridine alkaloid compound extracted, separated and identified from a purslane medicinal material, and an extraction and separation method and use thereof. Background Art

[0002] Purslane, derived from the dried aerial parts of Portulaca oleracea L., a member of the Portulacaceae family, is designated as a wild plant with both medicinal and edible properties by my country's Ministry of Health. It is also known as longevity vegetable and five-element grass. As a traditional Chinese medicine, purslane has been used for thousands of years. Widely distributed and abundant in resources, it possesses remarkable adaptability and tenacious vitality. The 2020 edition of the Pharmacopoeia of the People's Republic of China lists the dried aerial parts of purslane as a medicinal herb. It has a sour and cold flavor 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 is used to treat heat-toxic bloody dysentery, carbuncles, furuncles, eczema, erysipelas, snake and insect bites, blood in stool, hemorrhoids, and metrorrhagia.

[0003] Portulaca oleracea contains a variety of chemical components, including alkaloids, flavonoids, organic acids, terpenes, coumarins, polysaccharides, amino acids, and minerals. Among these, purslane contains a particularly large variety of alkaloid compounds, including uracil, adenine, norepinephrine, dopamine, adenosine, N,N-dicyclohexylurea, purslanamide, allantoin, N-trans-feruloyltyramine, p-hydroxyphenylethylamine, oleracein AE, oleraindole AG, oleraisoindole, and portulaceramide A. The chemical composition of purslane is closely related to its diverse pharmacological effects. Modern pharmacological research has shown that purslane exhibits antioxidant, neuroprotective, anti-inflammatory, antibacterial, anti-tumor, hypoglycemic, hepatoprotective, and immune-enhancing properties. There are many kinds of chemical components in Purslane and their pharmacological activities are diverse. The development and separation of compounds in Purslane can provide a basis for in-depth research on Purslane. Summary of the Invention

[0004] To address the above problems, the present invention provides a pyridine alkaloid extracted from Portulaca oleracea. Studies have found that the pyridine alkaloid of the present invention has anti-inflammatory activity, anticholinesterase activity and antioxidant activity. At the same time, a simple, rapid, environmentally friendly and high-purity extraction and separation method for the compound of the present invention is provided.

[0005] To achieve the above objectives, the present invention provides a pyridine alkaloid compound isolated from the medicinal material Portulaca oleracea, with a molecular formula of C6H6N2O2, named 5-hydroxypicolinamide according to its structure, and a chemical structural formula of:

[0006]

[0007] The present invention also provides a method for extracting and separating pyridine alkaloid compounds in purslane, which specifically comprises the following steps:

[0008] Step 1: Take dried purslane, decoct in water and extract, concentrate the extract, cool to room temperature, and set aside the medicinal solution.

[0009] Step 2: The concentrated solution in step 1 is subjected to macroporous resin, and eluted with water and ethanol of different concentrations. The 30% ethanol portion is recovered under reduced pressure to obtain an extract, and the concentrate is set aside.

[0010] Step 3: Separate the concentrate in step 2 through an ODS column (Octadecylsilyl, octadecylsilane bonded silica gel filler) using a methanol-water gradient elution, detect by thin layer chromatography, develop color, combine the 50% methanol elution fractions and evaporate to dryness to obtain a concentrate for use.

[0011] Step 4: The concentrate obtained in step 3 is further separated by ODS column chromatography, using a methanol-water gradient elution, detected by thin layer chromatography, and color developed. The 10% methanol elution portion is combined and evaporated to dryness to obtain a concentrate for use.

[0012] Step 5: The concentrate obtained in step 4 is separated and prepared by HPLC (high performance liquid chromatography), and isocratic elution is performed using methanol-0.1% formic acid (volume percentage) as the mobile phase to finally obtain the 5-hydroxypicolinamide of the present invention.

[0013] Furthermore, in step 1, the extraction is performed twice with water decoction, each time for 2 hours, and the amount of water used is 8 to 16 times the amount of the medicinal material.

[0014] Furthermore, in step 2, the macroporous resin is AB-8 macroporous resin, and the volume ratio of ethanol to water is 30:70, 50:50, 70:30 and 100:0 for gradient elution.

[0015] Furthermore, the volume ratio of methanol and water used in step 3 is 10:0, 30:70, 50:50, 70:30 and 100:0 for gradient elution; and the ODS particle size is 40-70 μm.

[0016] Furthermore, in step 4, the volume ratio of methanol to water is 10:0, 30:70, 50:50, 70:30 and 100:0 for gradient elution; and the ODS particle size is 40-70 μm.

[0017] Furthermore, the ODS pretreatment process in step 3 and step 4 is: soaking in methanol for 24 hours, after loading on the column, washing with methanol until there is no turbidity when dripping into water, and then balancing with the initial mobile phase.

[0018] Furthermore, in step 5, the volume ratio of methanol to 0.1% formic acid is 7:93, and the retention time of the compound is 3.472 min.

[0019] The present invention also provides a use of the 5-hydroxypicolinamide separated from the purslane medicinal material in the preparation of anti-inflammatory drugs, anticholinesterase drugs and antioxidant drugs.

[0020] Compared with the prior art, the present invention has the following beneficial effects.

[0021] The isolation and pharmacological activity research of 5-hydroxypicolinamide from purslane described in the present invention has not been reported in plants. The present invention provides a pyridine alkaloid compound derived from purslane and a method for extracting and separating the compound of the present invention. The method sequentially adopts water decoction extraction, macroporous resin column chromatography, ODS column chromatography and high performance liquid chromatography for separation, purification and preparation, and successfully extracts and separates a pyridine alkaloid compound. The method has only five operating steps, and the operation method is simple and rapid. The extraction is carried out with water, and the process method is environmentally friendly. The compound separated by the method has a high purity of greater than 90%. In addition, studies have shown that the above compound has anti-inflammatory activity, anticholinesterase activity and antioxidant activity. Therefore, the new natural product 5-hydroxypicolinamide and its salts and derivatives can be used as raw materials for drug development and pharmacological activity research. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 5-hydroxypicolinamide of the present invention 1 H-NMR spectrum.

[0023] Figure 2 5-hydroxypicolinamide of the present invention 13 C-NMR spectrum.

[0024] Figure 3 This is the DEPT spectrum of 5-hydroxypicolinamide of the present invention.

[0025] Figure 4 HSQC spectrum of 5-hydroxypicolinamide of the present invention.

[0026] Figure 5 This is the HMBC spectrum of 5-hydroxypicolinamide of the present invention.

[0027] Figure 6 5-hydroxypicolinamide of the present invention 1H- 1 H COSY spectrum.

[0028] Figure 7 The figure is the ROESY spectrum of 5-hydroxypicolinamide of the present invention.

[0029] Figure 8 This is a high-resolution mass spectrum of 5-hydroxypicolinamide of the present invention. DETAILED DESCRIPTION

[0030] 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.

[0031] Example 1.

[0032] The present invention provides a pyridine alkaloid compound with a molecular formula of C6H6N2O2, named 5-hydroxypicolinamide, and a chemical structural formula of:

[0033]

[0034] Table 1 shows the NMR data of 5-hydroxypicolinamide. The solvent used for NMR is CD3OD.

[0035] Table 1 NMR data of 5-hydroxypicolinamide

[0036]

[0037] 5-hydroxypicolinamide: pink powder, easily soluble in methanol, slightly soluble in water. After being spotted on a silica gel thin layer plate, the spots will appear orange when sprayed with dilute potassium bismuth iodide test solution, indicating that the compound is an alkaloid component. 1 H-NMR, 13 C-NMR and HR-ESI-TOF-MS signals indicate that the possible molecular formula of the compound is C6H6N2O2, with an unsaturation degree of 5. HR-ESI-TOF-MS gives m / z 137.0356 [MH] - The quasi-molecular ion peak of is 137.0351. 1 In H-NMR, δ H 8.06 (1H, d, J = 8.4 Hz, H-3), δ H 7.34 (1H, d, J = 8.4 Hz, H-4), δ H8.19 (1H, s, H-6) suggests the presence of a 1,3,4-trisubstituted benzene ring structure. HMBC shows that H-3 and C-5 (δ C 158.91), H-4 and C-5, C-6 (δ C 138.36), C-2(δ C 140.13), H-6 is related to C-5, C-4 (δ C 127.91), C-3(δ C 124.24) and C-2, indicating the presence of a pyridine ring structure in the structure. C-5 is located in the low field region, so the C-5 position is substituted with a hydroxyl group. In addition, HMBC shows that H-3 and C-7 (δ C 167.59), suggesting a carbonyl substitution at the C-2 position. Combined with the molecular weight from the HR-ESI-TOF-MS spectrum, we infer the presence of an amino group. Based on this information, we can confirm that this new natural compound has the aforementioned structure.

[0038] The present invention also provides a method for extracting and separating 5-hydroxypicolinamide, which comprises the following steps:

[0039] Step 1: Weigh 250 kg of dried purslane medicinal materials, use water decoction to extract, the amount of which is 10 times that of the medicinal materials, extract twice, each time for 2 hours, combine the extracts, heat and concentrate, cool to room temperature, and obtain the medicinal solution for use.

[0040] Step 2: The medicinal solution obtained in step 1 is evaporated to dryness and separated by chromatography on an AB-8 macroporous resin column using an ethanol-water (30:70, 50:50, 70:30 and 100:0, v:v) gradient elution, collecting the 30% ethanol portion, and recovering it under reduced pressure to obtain an extract to obtain a concentrate for later use.

[0041] Step 3: The concentrate in step 2 was separated by a pretreated ODS column with a filler particle size of 40 to 70 μm, and gradient elution was performed using methanol-water (10:0, 30:70, 50:50, 70:30 and 100:0, v:v). The mixture was detected by thin layer chromatography and color was developed. The 50% methanol elution fractions were combined, concentrated to dryness under reduced pressure, and set aside.

[0042] Step 4: The product obtained in step 3 is further separated by pre-treated ODS column chromatography using a filler with a particle size of 40-70 μm. The product is eluted with a gradient of methanol-water (10:0, 30:70, 50:50, 70:30, and 100:0, v / v). Thin layer chromatography is performed for detection and color development. The 10% methanol elution fractions are combined and concentrated to dryness under reduced pressure for later use. The ODS pre-treatment process is as follows: soaking in methanol for 24 hours. After loading the column, the column is washed with methanol until there is no turbidity when dripped into water, and then equilibrated with the initial mobile phase.

[0043] Step 5: The fraction obtained in step 4 was separated and prepared by HPLC using methanol and 0.1% formic acid in a volume ratio of 7:93 as the mobile phase and detection wavelengths of 210 and 254 nm to obtain the new natural product of the present invention. The purity was determined to be 96% by normalization method.

[0044] Example 2 Anti-inflammatory effect of 5-hydroxypicolinamide.

[0045] 1 Main materials.

[0046] 1.1 Drugs and Reagents: The new natural product used in this experiment was prepared by the above-mentioned method with a purity of 96%. DMEM high-glucose medium and fetal bovine serum were obtained from Hyclone (USA); penicillin and streptomycin were obtained from Hangzhou Sijiqing Company; LPS was obtained from Sigma (USA); an IL-1β ELISA kit was obtained from Cayman (USA); and cell lysate was used.

[0047] 1.2 Cell line: RAW264.7 macrophages (ATCC cell bank, USA).

[0048] 1.3 Grouping: Divided into control group, LPS group and experimental group.

[0049] 2 Experimental methods.

[0050] 2.1 Cell culture: Add 10% fetal bovine serum and 1% antibiotics (100 U / mL penicillin and 100 μg / mL streptomycin) to DMEM high-glucose medium and store at 4°C.

[0051] 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 / mL, 100μL per well, 37 ℃, 5% CO2 conditions after overnight incubation, the experimental group added different concentrations of the compound of the present invention 5-hydroxypicolinamide, (5μM ~ 50μM), incubation for 1h, LPS at a concentration of 1μg / mL was added to the LPS group and experimental group, respectively, and a zero adjustment group (culture medium 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 group, 10μL of CCK-8 was added to each well of cells, 37 ℃, 5% CO2 conditions continued to incubate for 4h, and the absorbance of each well was measured at a wavelength of 450nm by a microplate reader.

[0052] 2.3 ELISA assay for inflammatory factor IL-1β: 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 this invention, 5-hydroxypicolinamide (1 μM to 20 μM), for 1 hour. LPS (final concentration 1 μg / mL) was then added to each well and incubated for 24 hours. Each treatment was repeated in triplicate. IL-1β levels were measured by ELISA.

[0053] 3 Experimental results.

[0054] The experimental results show that the compound of the present invention has no effect on the proliferation of LPS-induced macrophages RAW264.7; it can effectively inhibit the secretion of excessive inflammatory cytokine IL-1β produced by LPS-induced macrophages RAW264.7 in a concentration-dependent manner.

[0055] The results of the relative cell survival rate experiment are shown in Table 2.

[0056] Table 2 Effects of compound 5-hydroxypicolinamide on the relative survival rate of RAW264.7 macrophages.

[0057]

[0058] The results of ELISA determination of inflammatory factor IL-1β are shown in Table 3.

[0059] Table 3 Effects of compound 5-hydroxypicolinamide on the IL-1β content secreted by RAW264.7 cells induced by LPS (mean ± standard deviation, n = 3).

[0060]

[0061]

[0062] Note: * P<0.05 compared with the control group, # P<0.05 compared with the LPS group.

[0063] Example 3 Anticholinesterase Effect of 5-hydroxypicolinamide.

[0064] 1 Main materials.

[0065] 1.1 Drugs and Reagents: The new natural products used in this study were prepared by the above-mentioned method with a purity of 96%. Physostigmine was obtained from Shanghai Hanxiang Biotechnology Co., Ltd., acetylcholine iodide (ATCI) and acetylcholinesterase (AChE) were obtained from Dalian Meilun Biotechnology Co., Ltd., dithiodinitroformic acid (DTNB) was obtained from Shanghai Jinshui Biotechnology Co., Ltd., and sodium dihydrogen phosphate and sodium dihydrogen phosphate were obtained from Shanghai Sinopharm Reagent Co., Ltd.

[0066] 1.2 Experimental instruments and equipment: HBS-1096A 96-well microplate reader (Nanjing Detie Experimental Equipment Co., Ltd.), 1 / 100,000 balance (METTLER, Switzerland), HH-4 digital display constant temperature water bath (Jiangsu Jintan Ronghua Instrument Manufacturing Co., Ltd.).

[0067] 2 Experimental methods.

[0068] In this experiment, the anticholinesterase activity of each compound was determined using a modified Ellman method. 5-hydroxypicolinamide and physostigmine were accurately weighed and prepared in methanol to prepare five sample solutions at concentrations of 31.25 μM, 62.5 μM, 125 μM, 250 μM, and 500 μM. The following steps were performed: 140 μL of phosphate buffered saline (0.1 M, pH 8.0, containing 0.1 mol / L sodium dihydrogen phosphate and sodium dihydrogen phosphate), 20 μL of sample solution, and 15 μL of AChE (0.2 U / mL) were added to a 96-well microplate. The mixture was incubated at 37°C for 10 minutes. Then, 10 μL of ATCI (4 mmol / L) and 10 μL of DTNB (15 mmol / L) were added. After incubation at 37°C for 20 minutes, the 96-well plate was placed in a microplate reader, and the absorbance of each sample was measured at a wavelength of 405 nm. The blank group was treated with methanol instead of the sample solution, and the positive control group was treated with physostigmine instead of the sample solution. The cholinesterase inhibition rate of each compound was calculated according to the following formula (A represents absorbance):

[0069] Inhibition rate (%) = (A 空白 -A 样品 ) / A 空白 ×100%

[0070] 3 Experimental results.

[0071] The experimental results show that the compound 5-hydroxypicolinamide of the present invention exhibits certain anticholinesterase activity, and the inhibitory effect on cholinesterase increases with the increase of the compound concentration, showing a dose-dependent trend.

[0072] The anticholinesterase activity of the compound 5-hydroxypicolinamide of the present invention is shown in Table 4.

[0073] Table 4 Anticholinesterase activity of compound 5-hydroxypicolinamide.

[0074]

[0075] Example 4 Antioxidant effect of 5-hydroxypicolinamide.

[0076] 1 Main materials.

[0077] 1.1 Drugs and Reagents: The new natural product used in the experiment was prepared by the above method with a purity of 96%. 1,1-Diphenyl-2-picrylhydrazyl free radical (DPPH) was obtained from Sigma-Aldrich, USA; butylated hydroxyanisole (BHA) was obtained from Shanghai Xiangrui Biological Co., Ltd.; and methanol (chromatographically pure, Tianjin Kaixin Chemical Industry Co., Ltd.) was used.

[0078] 1.2 Experimental instruments and equipment: Hitachi UV-3010 ultraviolet-visible spectrophotometer (Hitachi, Japan), 1 / 100,000 balance (METTLER, Switzerland).

[0079] 2 Experimental methods.

[0080] This experiment uses the DPPH free radical scavenging method to determine the antioxidant activity of each compound. Accurately weigh 5-hydroxypicolinamide and BHA, and use methanol to prepare five series of sample solutions with concentrations of 12.5μM, 25μM, 50μM, 100μM and 200μM. In addition, the DPPH solution is prepared as needed, and an appropriate amount of DPPH is accurately weighed and used to prepare a solution with a concentration of 80μM with methanol. The whole process is protected from light. The specific operation is as follows: 1mL of sample solution is fully mixed with 1mL of DPPH solution, and placed at room temperature in a light-proof environment for 10 minutes. After setting the detection wavelength of the ultraviolet spectrophotometer to 517nm, the absorbance value of the sample is measured. Among them, methanol is used instead of the sample solution as the blank group, BHA is used instead of the sample solution as the positive control group, and a mixed solution of 1mL methanol and 1mL sample solution is used as the control group. The DPPH scavenging rate of each compound is calculated according to the following formula (A represents absorbance):

[0081] DPPH clearance rate (%) = (1-(A 样品 -A 对照 ) / A 空白 )×100%

[0082] 3 Experimental results.

[0083] The experimental results show that the compound 5-hydroxypicolinamide of the present invention exhibits certain antioxidant activity, and the antioxidant effect is enhanced with the increase of the compound concentration, showing a dose-dependent trend.

[0084] The antioxidant activity of the compound 5-hydroxypicolinamide of the present invention is shown in Table 5.

[0085] Table 5 Antioxidant activity of compound 5-hydroxypicolinamide.

[0086]

[0087] In summary, the present invention provides the compound 5-hydroxypicolinamide and its extraction and isolation method. The method employs water decoction extraction, macroporous resin column chromatography, ODS column chromatography, and high-performance liquid chromatography for separation, purification, and preparation, successfully extracting and isolating two pyridine alkaloid compounds. This method is simple, rapid, and environmentally friendly, and the compounds isolated by this method are of high purity. Because the resulting compound is extracted from the commonly used traditional Chinese medicine Portulaca oleracea, it exhibits anti-inflammatory, anticholinesterase, and antioxidant activities. Therefore, the novel natural product 5-hydroxypicolinamide, its salts, and derivatives, as well as its salts and derivatives, can provide insights into drug development and have broad development prospects.

Claims

1. A method for extracting and separating pyridine alkaloid compounds from Portulaca oleracea, characterized in that: The specific steps include: Step 1: Take dried purslane, decoct in water, concentrate the extract, and cool to room temperature to obtain a medicinal solution for later use; Step 2: The concentrated solution in step 1 is subjected to a macroporous resin, and eluted with water and ethanol of different concentrations. The 30% ethanol portion is recovered under reduced pressure to obtain an extract, and the concentrate is prepared for use; the macroporous resin is AB-8 macroporous resin, and the volume ratio of ethanol to water is 30:70, 50:50, 70:30 and 100:0 for gradient elution; Step 3: The concentrate in step 2 is separated by ODS column, eluted with methanol-water gradient, detected by thin layer chromatography, and color developed. The 50% methanol elution portion is combined and evaporated to dryness to obtain a concentrate for use; the volume ratio of methanol to water is 10:0, 30:70, 50:50, 70:30 and 100:0 for gradient elution; the ODS particle size is 40-70 μm; Step 4: The concentrate obtained in step 3 is further separated by ODS column chromatography, eluted with a methanol-water gradient, detected by thin layer chromatography, and developed. The 10% methanol elution fraction is combined and evaporated to dryness to obtain a concentrate for use; the volume ratio of methanol to water is 10:0, 30:70, 50:50, 70:30 and 100:0 for gradient elution; the ODS particle size is 40-70 μm; Step 5: The concentrate obtained in step 4 was separated and prepared by HPLC, with methanol-0.1% formic acid as the mobile phase for isocratic elution, the volume ratio of methanol-0.1% formic acid was 7:93, and the retention time of the compound was 3.472 minutes; finally, the pyridine alkaloid compound was obtained; The chemical structural formula of the pyridine alkaloid compound is .

2. The extraction and separation method according to claim 1, wherein In step 1, the extraction is performed by decocting in water twice, each time for 2 hours, and the dosage is 8 to 16 times of the medicinal material.

3. A method for separating pyridine alkaloid compounds from Portulaca oleracea according to claim 1, characterized in that: The alkaloid compound is used for preparing anti-inflammatory drugs, anticholinesterase drugs and antioxidant drugs.

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