Pyrrolidone compound in purslane as well as extraction and separation method and application thereof

Abstract

The invention belongs to the technical field of extraction and separation of traditional Chinese medicines, and particularly relates to a pyrrolidone compound extracted, separated and identified from purslane as well as an extraction and separation method and application of the pyrrolidone compound. The molecular formula of the compound is C7H13N3O2, and the chemical name of the compound is N-(1-aminoethyl)-5-oxopyrrolidine-2-carboxamide, and the structural formula of the compound is shown in the description. The invention also provides an extraction and separation method of the compound. Ethanol reflux extraction, silica gel column chromatography, sephadex column chromatography and a high performance liquid chromatograph are sequentially adopted for separation, purification and preparation. The structure of the compound is determined as a new compound by adopting analysis methods such as mass spectrum, hydrogen spectrum and carbon spectrum. The compound has anti-inflammatory activity and anti-cholinesterase activity, and the compound and salts or derivatives thereof 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 a pyrrolidone compound in purslane and an extraction and separation method and use thereof, and in particular to a compound N-(1-aminoethyl)-5-oxopyrrolidine-2-carboxamide extracted, separated and identified from purslane medicinal materials and an extraction and separation method and use thereof. Background Art

[0002] Purslane comes from the dried aerial parts of Portulaca oleracea L., a plant of the Portulacaceae family. It is also known as long-life vegetable and five-element grass. Purslane is widely distributed and abundant in resources, and has very strong 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 medicine. It tastes sour and cold, and enters the liver and large intestine meridians. It has the effects of clearing away heat and detoxifying, cooling blood and stopping bleeding, and stopping dysentery. It is used for heat-toxic bloody dysentery, carbuncle, furuncle, eczema, erysipelas, snake and insect bites, blood in stool, hemorrhoids, and metrorrhagia.

[0003] Purslane contains a variety of chemical components, mainly including: alkaloids, flavonoids, organic acids, terpenes, coumarins, polysaccharides, amino acids and minerals. Among them, there are many types of alkaloid compounds in Purslane, such as uracil, adenine, norepinephrine, dopamine, adenosine, N,N-dicyclohexylurea, purslanamide, allantoin, N-trans-feruloyltyramine, p-hydroxyphenylethylamine, oleracein AE, oleraindole AG, oleraisoindole, portulaceramide A, etc. The chemical components contained in Purslane are closely related to its diverse pharmacological effects. Modern pharmacological studies have shown that Purslane has neuroprotective, anti-inflammatory, antioxidant, antibacterial, anti-tumor, hypoglycemic, liver-protecting and immune-enhancing effects. 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] In view of the above problems, the present invention provides a pyrrolidone compound extracted from Portulaca oleracea. Studies have shown that the compound of the present invention has anti-inflammatory and anticholinesterase activities. A simple, rapid, environmentally friendly and high-purity extraction and separation method for the compound of the present invention is also provided.

[0005] To achieve the above-mentioned purpose of the present invention, the present invention provides a pyrrolidone compound having a molecular formula of C7H 13N3O2, chemical name is N-(1-aminoethyl)-5-oxopyrrolidine-2-carboxamide, chemical structure formula is:

[0006]

[0007] To achieve the above-mentioned purpose of the present invention, the present invention also provides a method for extracting and separating a pyrrolidone compound N-(1-aminoethyl)-5-oxopyrrolidine-2-carboxamide, the specific steps of which include:

[0008] Step 1: Take dried purslane medicinal material, extract it with ethanol reflux, concentrate the extract, cool it to room temperature, and obtain an extract for later use;

[0009] Step 2: grind the extract in step 1, load it into a column, elute it with ethyl acetate and ethanol in different volume ratios, recover the ethyl acetate: ethanol = 1:5 part into the extract under reduced pressure, and obtain a concentrate for use;

[0010] Step 3: The concentrate in step 2 is applied to a silica gel column, eluted with ethyl acetate, and recovered under reduced pressure to obtain a concentrate for later use;

[0011] Step 4: The concentrate in step 3 is loaded onto a silica gel column, eluted with ethyl acetate and methanol in different volume ratios, detected by thin layer chromatography, color developed, and the eluted portions are combined and evaporated to dryness to obtain three fractions;

[0012] Step 5: The concentrate obtained from fraction 3 in step 4 is further separated by dextran gel column chromatography, isocratically eluted with methanol, detected by thin layer chromatography, color developed, and the eluted portions are combined and evaporated to dryness to obtain four fractions;

[0013] Step 6: Separate and prepare the fraction 2 in step 5 by HPLC (high performance liquid chromatography), use methanol-0.1% formic acid (volume percentage) as the mobile phase for isocratic elution, and finally obtain the new compound described in the present invention.

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

[0015] Furthermore, in step 2, the volume ratio of ethyl acetate to ethanol is 5:1, 2:1, 1:1, 1:2, 1:5.

[0016] Furthermore, in step 4, the volume ratio of ethyl acetate to methanol is 1:0, 5:1, or 2:1.

[0017] Furthermore, in step 6, the volume ratio of methanol to 0.1% formic acid is 5:95, and the retention time of the compound is 3.680 min.

[0018] The present invention also provides a use of the N-(1-aminoethyl)-5-oxopyrrolidine-2-carboxamide separated from the purslane medicinal material in the preparation of anti-inflammatory drugs and anticholinesterase drugs.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0020] The separation and pharmacological activity research of N-(1-aminoethyl)-5-oxopyrrolidine-2-carboxamide from purslane described in the present invention has not been reported in plants; the present invention provides a pyrrolidone compound derived from purslane and an extraction and separation method for the compound of the present invention, which successively adopts ethanol reflux extraction, silica gel column chromatography, dextran gel column chromatography and high performance liquid chromatography for separation, purification and preparation, and successfully extracts and separates a pyrrolidone compound, the method has simple and rapid operation steps, and the purity of the compound separated by the method is high, greater than 90%; in addition, studies have shown that the above compounds have anti-inflammatory activity and anti-cholinesterase activity, so the new compound N-(1-aminoethyl)-5-oxopyrrolidine-2-carboxamide and its salts and derivatives can be used as raw materials for drug development and pharmacological activity research. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The compound N-(1-aminoethyl)-5-oxopyrrolidine-2-carboxamide of the present invention 1 H-NMR spectrum.

[0022] Figure 2 The compound N-(1-aminoethyl)-5-oxopyrrolidine-2-carboxamide of the present invention 13 C-NMR spectrum.

[0023] Figure 3 It is the DEPT spectrum of the compound N-(1-aminoethyl)-5-oxopyrrolidine-2-carboxamide of the present invention.

[0024] Figure 4 It is the HSQC spectrum of the compound N-(1-aminoethyl)-5-oxopyrrolidine-2-carboxamide of the present invention.

[0025] Figure 5It is the HMBC spectrum of the compound N-(1-aminoethyl)-5-oxopyrrolidine-2-carboxamide of the present invention.

[0026] Figure 6 The compound N-(1-aminoethyl)-5-oxopyrrolidine-2-carboxamide of the present invention 1 H- 1 HCOSY spectrum.

[0027] Figure 7 It is the ROESY spectrum of the compound N-(1-aminoethyl)-5-oxopyrrolidine-2-carboxamide of the present invention.

[0028] Figure 8 It is a high-resolution mass spectrum of the compound N-(1-aminoethyl)-5-oxopyrrolidine-2-carboxamide of the present invention. DETAILED DESCRIPTION

[0029] 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.

[0030] Example 1 N-(1-aminoethyl)-5-oxopyrrolidine-2-carboxamide.

[0031] The present invention provides a new pyrrolidone compound, the molecular formula of which is C7H 13 N3O2, chemical name is N-(1-aminoethyl)-5-oxopyrrolidine-2-carboxamide, chemical structure formula is:

[0032]

[0033] Table 1 shows the NMR data of N-(1-aminoethyl)-5-oxopyrrolidine-2-carboxamide. The solvent used for NMR is CD3OD.

[0034] Table 1 NMR data of N-(1-aminoethyl)-5-oxopyrrolidine-2-carboxamide of the present invention

[0035]

[0036] N-(1-aminoethyl)-5-oxopyrrolidine-2-carboxamide: white powder, easily soluble in methanol, slightly soluble in water. After spotting on a silica gel thin layer plate, spraying with dilute potassium bismuth iodide solution resulted in an orange-yellow spot, indicating that the compound is an alkaloid component. UHPLC-ESI-Q-TOF-MS gives m / z: 172.1082 [M+H] + The quasi-molecular ion peak (calculated value [C7H 14 N3O2] + =172.1081). Combined 1 H-NMR, 13 C-NMR and DEPT data suggest that the possible molecular formula of the compound is C7H 13 N3O2, unsaturation is 2. Combined 13 C-NMR and DEPT spectra showed seven carbon signals, including two carbonyl carbons (δ C 181.13,δ C 175.94), two methines (δ C 30.46,δ C 26.11), two methylene groups (δ C 57.09,δ C 49.54), one methyl group (δ C 17.61). 1 H-NMR showed two methine hydrogen signals (δ H 4.25,m,δ H 4.36, m), two methylene hydrogen signals (δ H 2.33,m,δ H 2.48, m), a methyl hydrogen signal (δ H 1.37, d, J = 7.5 Hz). HMBC spectrum showed that H-5 (δ H 4.25,m) and C-2(δ C 181.13), C-3(δ C 30.46), C-4(δ C 26.11) related, H-3(δ H 2.33,m) and C-2, C-5 (δ C 57.09), where C-2 and C-5 are located in the low field, suggesting that there is a N atom between them. 1 H- 1 H COSY spectrum shows that H-3 and H-4 (δ H 2.48,m), H-4 is related to H-5, and based on the above information, it is speculated that the structure may contain a 2-pyrrolidone structure. The HMBC spectrum shows that H-4 is related to C-1′(δ C175.94), indicating that there is a carbonyl group at the C-5 position. H 4.36, m) is related to C-1′, and H-3′ is located in the low field region, suggesting that C-3′ (δ C 49.54) may be linked to C-1′ via NH; 1 H- 1 H COSY showed that H-3′ and H-4′ (δ H 1.37), indicating that C-4′(δ C 17.61) is connected to C-3′; according to the mass spectrometry molecular weight, it is speculated that NH2 exists in the structure. Based on the above information, it is speculated that the structure is N-(1-aminoethyl)-2-oxopyrrolidine-1-carboxamide.

[0037] The present invention also provides a method for extracting and separating N-(1-aminoethyl)-2-oxopyrrolidine-1-carboxamide, which comprises the following specific steps:

[0038] Step 1: Weigh 250 kg of dried purslane medicinal material, use ethanol reflux extraction, the amount of which is 10 times that of the medicinal material, extract twice, each time for 2 hours, combine the extracts, heat and concentrate, cool to room temperature, and obtain the medicinal solution for use.

[0039] Step 2: evaporate the drug solution obtained in step 1 to dryness, grind it, load it into a column, and use ethyl acetate-ethanol (5:1, 2:1, 1:1, 1:2 and 1:5, v:v) gradient elution to collect the ethyl acetate:ethanol = 1:5 part, and recover it under reduced pressure to obtain an extract to obtain a concentrate for use.

[0040] Step 3: Separate the concentrate in step 2 through a silica gel column, elute with ethyl acetate, and recover under reduced pressure to obtain a concentrate for later use.

[0041] Step 4: The concentrate in step 3 is separated again by silica gel column, and gradient eluted with ethyl acetate-methanol (1:0, 5:1, 2:1, v:v), detected by thin layer chromatography, and color developed. The eluted parts are combined and evaporated to dryness to obtain 3 parts.

[0042] Step 5: The concentrate obtained from fraction 3 in step 4 is separated by dextran gel column chromatography, isocratically eluted with methanol, detected by thin layer chromatography, and color developed. The eluted portions are combined and evaporated to dryness to obtain 4 fractions.

[0043] Step 6: The fraction 2 obtained in step 5 was separated and prepared by HPLC, using methanol and 0.1% formic acid in a volume ratio of 5:95 as the mobile phase and detection wavelengths of 210 and 254 nm to separate and prepare the compound of the present invention. The purity determined by normalization method was 90%.

[0044] Example 2 Anti-inflammatory effect of N-(1-aminoethyl)-5-oxopyrrolidine-2-carboxamide of the present invention.

[0045] 1 Main Materials

[0046] 1.1 Drugs and reagents: The new compounds used in the experiment were prepared by the above method with a purity of 90%. DMEM high-glucose medium, fetal bovine serum (Hyclone, USA); penicillin and streptomycin (Hangzhou Sijiqing Company); LPS (Sigma, USA); IL-1β ELISA kit (Cayman, USA); cell lysate.

[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 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 the compound of the present invention N-(1-aminoethyl)-2-oxopyrrolidine-1-carboxamide (5μM~50μM) 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, and 3 duplicate wells were set up in each group to investigate the effect of the addition of drugs on the cells. After the above-mentioned groups of cells were cultured for 24h, 10μL of CCK-8 was added to the cells in each well, and after further incubation for 4h at 37℃, 5% CO2, the absorbance value of each well was measured at a wavelength of 450nm by an enzyme marker.

[0052] 2.3ELISA method for determination of inflammatory factor IL-1β: RAW264.7 macrophages in the logarithmic growth phase were inoculated in 24-well culture plates at a cell density of 1×10 5 / mL, 1mL per well, cultured overnight at 37°C, 5% CO2, the experimental group added the compound of the present invention N-(1-aminoethyl)-2-oxopyrrolidine-1-carboxamide (1μM~20μM) and cultured for 1h, then LPS (final concentration of 1μg / mL) was added to each well, and incubated for 24h, and each group was treated with 3 wells. The content of IL-1β was determined 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 the compounds of the present invention on the relative survival rate of RAW264.7 macrophages.

[0057]

[0058]

[0059] The results of ELISA determination of inflammatory factor IL-1β are shown in Table 3.

[0060] Table 3 Effects of the compounds of the present invention on the IL-1β content secreted by RAW264.7 cells induced by LPS (mean±standard deviation, n=3).

[0061]

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

[0063] Example 3 Anticholinesterase effect of N-(1-aminoethyl)-5-oxopyrrolidine-2-carboxamide.

[0064] 1 Main Materials

[0065] 1.1 Drugs and reagents: The new compounds used in the experiment were prepared by the above method with a purity of 90%. Physostigmine (Shanghai Hanxiang Biotechnology Co., Ltd.), acetylcholine iodide (ATCI) and acetylcholinesterase (AChE) (Dalian Meilun Biotechnology Co., Ltd.), dithiodinitroformic acid (DTNB) (Shanghai Jinshui Biotechnology Co., Ltd.), disodium hydrogen phosphate and sodium dihydrogen phosphate (Shanghai Sinopharm Reagent Co., Ltd.).

[0066] 1.2 Experimental instruments and equipment: HBS-1096A96-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 according to the modified Ellman method. N-(1-aminoethyl)-5-oxopyrrolidine-2-carboxamide and physostigmine were accurately weighed and prepared into five series of sample solutions with concentrations of 31.25 μM, 62.5 μM, 125 μM, 250 μM and 500 μM using methanol. The specific operation is as follows: add 140 μL of phosphate buffered saline (0.1M, pH=8.0, containing 0.1 mol / L disodium hydrogen phosphate and sodium dihydrogen phosphate), 20 μL of sample solution, 15 μL of AChE (0.2U / mL) to a 96-well ELISA plate, incubate at 37°C for 10 minutes, add 10 μL of ATCI (4mmol / L) and 10 μL of DTNB (15mmol / L), incubate at 37°C for 20 minutes, place the 96-well plate in an ELISA instrument, and measure the absorbance of each group of samples at a wavelength of 405nm. Among them, methanol was used instead of the sample solution as the blank group, and physostigmine was used instead of the sample solution as the positive control group. 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 new natural product of the present invention exhibits certain anti-cholinesterase activity, and the inhibitory effect on cholinesterase increases with the increase of the compound concentration, showing a dose-dependent trend.

[0072] The anticholinesterase activities of the novel compounds of the present invention are shown in Table 4.

[0073] Table 4 Anticholinesterase activities of the compounds of the present invention.

[0074]

[0075] In summary, the compound N-(1-aminoethyl)-2-oxopyrrolidine-1-carboxamide and its extraction and separation method and application provided by the present invention are separated, purified and prepared by ethanol reflux extraction, silica gel column chromatography, dextran gel column chromatography and high performance liquid chromatography in sequence, and the compound is successfully extracted and separated. The operation method is simple, rapid and environmentally friendly, and the compound separated by the method has a high purity. Since the obtained compound is extracted from the commonly used traditional Chinese medicine Portulaca oleracea and has anti-inflammatory activity and anticholinesterase activity, the new compound N-(1-aminoethyl)-2-oxopyrrolidine-1-carboxamide and its salts and derivatives can provide ideas for drug development and have broad development prospects.

Claims

1. A pyrrolidone compound isolated from Portulaca oleracea, characterized in that: The molecular formula is: C7H 13 N3O2, chemical name is N-(1-aminoethyl)-5-oxopyrrolidine-2-carboxamide, chemical structure formula is: 。 2. A method for extracting and separating pyrrolidone compounds separated from the purslane medicinal material according to claim 1, characterized in that: The specific steps include: Step 1: Take dried purslane medicinal material, extract it with ethanol reflux, concentrate the extract, cool it to room temperature, and obtain the medicinal solution for use; Step 2: grind the extract in step 1, load it into a column, elute it with ethyl acetate and ethanol in a volume ratio of 5:1, 2:1, 1:1, 1:2, 1:5, recover the ethyl acetate: ethanol = 1:5 part to the extract under reduced pressure, and obtain a concentrate for use; Step 3: The concentrate in step 2 is applied to a silica gel column, eluted with ethyl acetate, and recovered under reduced pressure to obtain a concentrate for later use; Step 4: The concentrate in step 3 is loaded onto a silica gel column and eluted with ethyl acetate and methanol in a volume ratio of 1:0, 5:1, and 2:

1. The mixture is detected by thin layer chromatography and color is developed. The eluted portions are combined and evaporated to dryness to obtain three fractions. Step 5: The concentrate obtained from fraction 3 in step 4 is further separated by dextran gel column chromatography, isocratically eluted with methanol, detected by thin layer chromatography, color developed, and the eluted portions are combined and evaporated to dryness to obtain four fractions; Step 6: Fraction 2 in step 5 is separated and prepared by HPLC, and isocratic elution is performed using methanol-0.1% formic acid in a volume ratio of 5:95 as the mobile phase to finally obtain the compound described in the present invention.

3. The extraction and separation method according to claim 2, characterized in that: In the step 1, 50% ethanol is refluxed and extracted twice, each time for 2 hours, and the amount used is 10 times that of the medicinal material.

4. Use of the pyrrolidone compounds separated from the Portulaca oleracea medicinal material according to claim 1 in the preparation of anti-inflammatory drugs and anticholinesterase drugs.

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