A pyrrolidone compound from purslane and its extraction and separation method and use

Abstract

The present invention belongs to the field of traditional Chinese medicine extraction and separation technology, and in particular relates to a pyrrolidone compound extracted, separated and identified from purslane and its extraction and separation method and use. The compound has the molecular formula C7H 13 N3O2, chemically known as N-(1-aminoethyl)-5-oxopyrrolidine-2-carboxamide. A method for extracting and isolating the compound is also provided, employing ethanol reflux extraction, silica gel column chromatography, dextran gel column chromatography, and high-performance liquid chromatography for separation, purification, and preparation. Its structure was confirmed as a new compound using analytical methods such as mass spectrometry, hydrogen spectroscopy, and carbon spectroscopy. The compound exhibits anti-inflammatory and anticholinesterase activity. 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 extraction and separation of traditional Chinese medicines, and relates to a pyrrolidone compound in purslane, an extraction and separation method and use thereof, and in particular to the compound N-(1-aminoethyl)-5-oxopyrrolidine-2-carboxamide extracted, separated and identified from purslane, an extraction and separation method and use thereof. Background Art

[0002] Purslane, also known as longevity herb and five-element grass, originates from the dried aerial parts of Portulaca oleracea L., a plant of the Portulacaceae family. Widely distributed and abundant, it possesses exceptionally strong adaptability and tenacious vitality. The 2020 edition of the Pharmacopoeia of the People's Republic of China lists the dried aerial parts of Portulaca oleracea 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 relieving 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 high variety of alkaloid compounds, such as 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 neuroprotective, anti-inflammatory, antioxidant, 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 pyrrolidone compound extracted from Portulaca oleracea. Studies have found that the compound of the present invention has anti-inflammatory and anticholinesterase activities. 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] In order to achieve the above-mentioned purpose of the present invention, the present invention provides a pyrrolidone compound having the molecular formula C7H 13N3O2, chemical name is N-(1-aminoethyl)-5-oxopyrrolidine-2-carboxamide, chemical structure is:

[0006]

[0007] To achieve the above-mentioned object 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, which specifically comprises the following steps:

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

[0009] Step 2: The extract in step 1 is crushed, loaded into a column, and eluted with ethyl acetate and ethanol in different volume ratios. The ethyl acetate: ethanol = 1:5 part is recovered under reduced pressure to add to the extract to 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 was loaded onto a silica gel column and eluted with ethyl acetate and methanol in different volume ratios. The mixture was detected by thin layer chromatography and color was developed. The eluted fractions were 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, and color developed. The eluted fractions are combined and evaporated to dryness to obtain four fractions;

[0013] Step 6: Fraction 2 in step 5 was separated and prepared by HPLC (high performance liquid chromatography), and isocratic elution was performed using methanol-0.1% formic acid (volume percentage) as the mobile phase to finally obtain the new compound of the present invention.

[0014] Furthermore, in step 1, 50% ethanol reflux extraction is performed 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, or 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 isolated from the purslane medicinal material in the preparation of anti-inflammatory drugs and anticholinesterase drugs.

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

[0020] The isolation and pharmacological activity research of N-(1-aminoethyl)-5-oxopyrrolidine-2-carboxamide from purslane described in the present invention have not been reported in plants. The present invention provides a pyrrolidone compound derived from purslane and a method for extracting and separating the compound of the present invention. The method sequentially 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 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 and anticholinesterase activity. Therefore, 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 This is the DEPT spectrum of the compound N-(1-aminoethyl)-5-oxopyrrolidine-2-carboxamide of the present invention.

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

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

[0028] Figure 8 This 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 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 N-(1-aminoethyl)-5-oxopyrrolidine-2-carboxamide.

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

[0032]

[0033] Table 1 shows the NMR data of N-(1-aminoethyl)-5-oxopyrrolidine-2-carboxamide. The solvent used for NMR was 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, freely soluble in methanol and slightly soluble in water. Spotted on a silica gel thin layer plate, the spot developed an orange-yellow color after spraying with dilute potassium bismuth iodide solution, indicating that the compound is an alkaloid. UHPLC-ESI-Q-TOF-MS yielded m / z: 172.1082 [M+H] + The quasi-molecular ion peak, (calculated value [C7H 14 N3O2] + is 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 degree 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. In addition, H-3′(δ 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) related, indicating that C-4′(δ C 17.61) is attached to C-3′; based on the mass spectrometry molecular weight, NH2 is inferred to be present in the structure. Based on the above information, the structure is inferred to be 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 steps:

[0038] Step 1: Weigh 250 kg of dried purslane medicinal materials, use ethanol reflux extraction, the amount of ethanol 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.

[0039] Step 2: After evaporating the medicinal 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, reduce the pressure to recover the extract, and obtain the concentrate for use.

[0040] Step 3: The concentrate in step 2 is separated by a silica gel column, eluted with ethyl acetate, and recovered under reduced pressure to obtain a concentrate for use.

[0041] Step 4: The concentrate in step 3 was 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 was developed. The eluted portions were combined and evaporated to dryness to obtain 3 parts.

[0042] 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, and color developed. The eluted fractions are combined and evaporated to dryness to obtain 4 fractions.

[0043] Step 6: 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 obtain the compound of the present invention. The purity was determined to be 90% by normalization method.

[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 compound used in this experiment was prepared by the above-mentioned method with a purity of 90%. 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 were 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 N-(1-aminoethyl)-2-oxopyrrolidine-1-carboxamide (5μM ~ 50μM), incubation for 1 hour, and then added LPS at a concentration of 1μg / mL to the LPS group and experimental group, respectively. A zero adjustment group (culture medium containing DMSO solvent) was also set up. Each group had 3 replicates to investigate the effect of drug addition on the cells. After 24 hours of cell culture in each group, 10μL of CCK-8 was added to each well of cells. After a further 4 hours of incubation at 37 ℃, 5% CO2 conditions, the absorbance of each well was measured at a wavelength of 450nm using 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, N-(1-aminoethyl)-2-oxopyrrolidine-1-carboxamide (1 μM to 20 μM). After incubation for 1 hour, LPS (final concentration of 1 μg / mL) was 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 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 the 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 experiments were prepared by the above-mentioned method with a purity of 90%. 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] The anticholinesterase activity of each compound was determined using a modified Ellman method. Accurately weigh N-(1-aminoethyl)-5-oxopyrrolidine-2-carboxamide and physostigmine and prepare a series of five sample solutions in methanol at concentrations of 31.25 μM, 62.5 μM, 125 μM, 250 μM, and 500 μM. The specific operation is as follows: 140 μL of phosphate buffered saline (0.1 M, pH = 8.0, containing 0.1 mol / L sodium dihydrogen phosphate and sodium dihydrogen phosphate) was added to a 96-well microplate, 20 μL of sample solution, 15 μL of AChE (0.2 U / mL), and 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 group of samples was measured at a wavelength of 405 nm. 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 anticholinesterase 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 activity of the compounds of the present invention.

[0074]

[0075] In summary, the compound N-(1-aminoethyl)-2-oxopyrrolidine-1-carboxamide provided by the present invention, and its extraction and separation method and application, are successfully extracted and isolated by sequentially using ethanol reflux extraction, silica gel column chromatography, dextran gel column chromatography, and high-performance liquid chromatography. The operation method is simple, rapid, and environmentally friendly, and the compound isolated by this method is of high purity. Since the obtained compound is extracted from the commonly used traditional Chinese medicine Portulaca oleracea and has anti-inflammatory and anticholinesterase activities, 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 structure is: 。 2. A method for extracting and separating pyrrolidone compounds isolated from the purslane medicinal material according to claim 1, characterized in that: The specific steps include: Step 1: Take dried purslane medicinal material, use ethanol reflux extraction, concentrate the extract, and cool to room temperature to obtain a medicinal solution for use; Step 2: The extract in step 1 is crushed and loaded into a column. The column is eluted with ethyl acetate and ethanol in a volume ratio of 5:1, 2:1, 1:1, 1:2, and 1:

5. The ethyl acetate:ethanol ratio of 1:5 is recovered under reduced pressure to the extract to obtain a concentrate for later 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 from step 3 was 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 product was detected by thin layer chromatography and color was developed. The eluted fractions were 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, and color developed. The eluted fractions are combined and evaporated to dryness to obtain four fractions; Step 6: Fraction 2 in step 5 was separated and prepared by HPLC, and isocratic elution was performed using methanol-0.1% formic acid in a volume ratio of 5:95 as the mobile phase to finally obtain the compound.

3. The extraction and separation method according to claim 2, wherein 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 isolated from the Portulaca oleracea medicinal material according to claim 1 in the preparation of anti-inflammatory drugs and anticholinesterase drugs.

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