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

Abstract

The present invention belongs to the technical field of traditional Chinese medicine extraction and separation, and particularly relates to a new amine alkaloid compound in purslane, and its extraction, separation method and application. The new compound has a molecular formula of C6H6N2O3 and is named 5-(iminomethyl)-1,3-oxazepine-4,6-diol. The invention also provides a method for extracting and separating the above new compound, which successively adopts water extraction, macroporous resin adsorption and elution, ODS medium-pressure column, Sephadex LH-20 column chromatography and UPLC preparation, and successfully extracts and separates a new amine alkaloid compound. Its structure is determined by methods such as mass spectrometry, hydrogen spectrum, carbon spectrum and two-dimensional nuclear magnetic spectroscopy. The new compound has anti-inflammatory and anti-cholinesterase effects. The new compound and its salts or derivatives of the present invention can be used as lead compounds for the synthesis of other compounds, as well as raw materials for new drug development and pharmacological activity research, and are used for preparing anti-inflammatory and anti-cholinesterase drugs or health products.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine extraction and separation, and in particular relates to a new amine alkaloid compound in purslane and an extraction and separation method and application thereof. Background Art

[0002] Purslane (Portulaca oleracea L.), also known as longevity vegetable, melon seed vegetable, and pig fat vegetable, is an annual herbaceous plant of the Portulacaceae family. Purslane is widely distributed and rich in resources. It is one of the 78 wild plants with medicinal and edible properties specified by the Ministry of Health of my country. Purslane is included in the 2020 edition of the "Pharmacopoeia of the People's Republic of China". 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, metrorrhagia, etc.

[0003] Modern pharmacological studies on purslane have shown that it has anti-inflammatory, antibacterial, antihypertensive, antilipidemic, anti-glycemic, anti-uric acid, antioxidant, anti-tumor, neuroprotective, skeletal muscle and smooth muscle relaxation, immune function regulation and other effects. The main chemical components of purslane include alkaloids, coumarins, terpenes, alkaloids, amino acids, lignans, volatile oils, polysaccharides, various pigments and organic acid salts, which provide the material basis for its diverse pharmacological effects. Among them, alkaloid compounds are a major class of chemical components in purslane. The alkaloid components reported so far include purslanine, tropaeoline, allantoin, β-carline, N-trans-feruloyltyramine and N-trans-feruloyl-3-methoxytyramine.

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

[0005] In view of the above problems, the present invention provides a new amine alkaloid compound extracted and separated from Portulaca oleracea. Studies have found that the new compound of the present invention has anti-inflammatory and anticholinesterase effects. At the same time, a simple, rapid, environmentally friendly and high-purity extraction and separation method for the new compound of the present invention is provided.

[0006] To achieve the above-mentioned purpose of the present invention, the present invention provides a new amine alkaloid compound, the molecular formula of which is C6H6N2O3, named 5-(iminomethyl)-1,3-oxazepine-4,6-diol, and the chemical structural formula is:

[0007]

[0008] The present invention also provides a method for extracting and separating a new amine alkaloid compound from Portulaca oleracea, which specifically comprises the following steps:

[0009] Step 1, take dried purslane medicinal material, extract it with water, filter the water extract, combine the filtrate, concentrate it under reduced pressure, cool it to room temperature, and obtain the medicinal solution for use;

[0010] Step 2, evaporating the Chinese medicine solution in step 1 to dryness, adsorbing it with a macroporous resin, eluting it with a water-ethanol gradient, and concentrating the hot water portion under reduced pressure to obtain an extract, thereby obtaining a hot water extract;

[0011] Step 3, separating the hot water extract in step 2 through an ODS (Octadecylsilyl, octadecylsilane bonded silica gel filler) column, using a methanol-water gradient elution, detecting several elution portions through thin layer chromatography, developing color, combining the elution portions with color, and concentrating the combined elution portions to dryness under reduced pressure for later use;

[0012] Step 4, the obtained product in step 5 is further separated by chromatography on a pretreated Sephadex LH-20 column, isocratically eluted with 20% methanol to obtain several elution fractions, which are detected by thin layer chromatography, developed, and the 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 separated and prepared by HPLC (high performance liquid chromatography) using 5% methanol: 0.1% formic acid as the mobile phase to prepare the compound 5-(iminomethyl)-1,3-oxazepine-4,6-diol of the present invention.

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

[0015] Furthermore, the mobile phase elution procedure used in step 2 is a gradient elution of cold water, hot water, 30% ethanol, 50% ethanol, and 70% ethanol.

[0016] Furthermore, in step 3, the volume ratio of water to methanol is 100:0, 95:5, 90:10, 80:20 and 50:50 for gradient elution.

[0017] Furthermore, the elution procedure with 20% methanol in step 4 is isocratic elution.

[0018] Furthermore, the pretreatment process of ODS and dextran gel in step 4 is: soaking in methanol for 24 hours, loading on the column, washing with methanol until there is no turbidity when dripped into water, and then balancing with the initial mobile phase.

[0019] Furthermore, in step 5, the volume ratio of methanol to water in the isocratic elution of methanol:0.1% formic acid is 5:95.

[0020] The new amine alkaloid compound 5-(iminomethyl)-1,3-oxazepine-4,6-diol can be used to prepare anti-inflammatory and anti-cholinesterase drugs.

[0021] Compared with the prior art, the invention has the beneficial effects.

[0022] The separation and pharmacological activity research of the new amine alkaloid compound from purslane described in the present invention has not been reported in existing paper journals; the present invention provides a new amine alkaloid compound derived from purslane and an extraction and separation method for the new compound of the present invention, which sequentially adopts water extraction, macroporous resin adsorption elution, ODS medium-pressure column, Sephadex LH-20 and high-performance liquid chromatography for separation, purification and preparation, and successfully extracts and separates the new compound. The method has only five operating steps, and the operation method is simple and fast. The extraction and separation process mainly adopts water extraction and methanol elution, and the process method is environmentally friendly. The purity of the compound separated by the method is relatively high, all greater than 90%. In addition, studies have shown that the compound has anti-inflammatory and anti-cholinesterase effects. Therefore, the new compound of the present invention and its salts and derivatives can be used as synthetic leads of 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 anti-cholinesterase drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a high-resolution mass spectrum of the new compound 5-(iminomethyl)-1,3-oxazepine-4,6-diol of the present invention.

[0024] Figure 2 The novel compound 5-(iminomethyl)-1,3-oxazepine-4,6-diol of the present invention 1 H-NMR spectrum.

[0025] Figure 3 The novel compound 5-(iminomethyl)-1,3-oxazepine-4,6-diol of the present invention 13 C-NMR spectrum.

[0026] Figure 4 This is the HMBC spectrum of the new compound 5-(iminomethyl)-1,3-oxazepine-4,6-diol of the present invention.

[0027] Figure 5 The novel compound 5-(iminomethyl)-1,3-oxazepine-4,6-diol of the present invention 1 H- 1 H COSY spectrum.

[0028] Figure 6 This is the HSQC spectrum of the new compound 5-(iminomethyl)-1,3-oxazepine-4,6-diol of the present invention.

[0029] Figure 7 The ROESY spectrum of the new compound 5-(iminomethyl)-1,3-oxazepine-4,6-diol of the present invention is shown in FIG. DETAILED DESCRIPTION

[0030] The present invention provides a new compound with a molecular formula of C6H6N2O3 and named as 5-(iminomethyl)-1,3-oxazepine-4,6-diol. The chemical formula is:

[0031]

[0032] The new compound is named 5-(iminomethyl)-1,3-oxazepine-4,6-diol according to its structure. Table 1 shows the NMR data of the new compound: 1 H-NMR and 13 C-NMR in DMSO.

[0033] Table 1 NMR data of the new compound 5-(iminomethyl)-1,3-oxazepine-4,6-diol.

[0034]

[0035]

[0036] The structural identification of the compounds of the present invention is shown in Figure 1-Figure 7 .

[0037] 5-(iminomethyl)-1,3-oxazepine-4,6-diol: gray powder, insoluble in methanol, soluble in water. HPLC-ESI-Q-TOF-MS gives m / z: 153.0305 [MH] - The quasi-molecular ion peak of 1 H-NMR and 13 Based on the C-NMR spectrum data, it is speculated that the possible molecular formula of the compound is C6H6N2O3, with an unsaturation degree of 5. 13 The C-NMR spectrum shows six carbon signals, namely three tertiary carbons (δ C :139.42;152.44;163.40), 3 quaternary carbons (δ C:117.51;151.23;155.38). 1 The H-NMR spectrum shows three methine signals as δ H 8.10(1H, s), δ H 8.11(1H, s), δ H 8.15 (1H, s), 1 active hydrogen signal is δ H 7.13.

[0038] The HSQC spectrum showed that C-7(δ C :139.42) and H-7(δ H 8.10) connected, C-2(δ C :152.44) and H-2(δ H 8.11) connected, C-1′(δ C :163.40) and H-1′(δ H 8.15)) are connected. The HMBC spectrum shows that H-7 is related to C-2, and H-2 is related to C-7; 13 C-NMR spectrum, C-2 and C-7 are both in low field, indicating that C-2 and C-7 are connected by oxygen atoms. HMBC spectrum also shows that H-2 and C-4 (δ C 155.38) related, H-2 and C-6 (δ C 151.23) related; combined 13 In the C-NMR spectrum, C-4 and C-6 are both in the low field, indicating that C-4 and C-6 are connected to the hydroxyl group respectively; and because C-2 is a methine, it indicates that C-2 and C-4 are connected through the nitrogen atom. According to the HMBC spectrum, H-1′ and C-4, C-5 (δ C :117.51), C-6 are all related, H-7 is related to C-5, combined 13 C-NMR spectrum shows that C-5 is in the low field and is a quaternary carbon, indicating the presence of a seven-membered ring containing heteroatoms (N, O). 1 Active hydrogen signal in H-NMR spectrum and 13 In the C-NMR spectrum, C-1' is in the low field, and it is inferred that C-1' is connected to the imino group. Therefore, based on the above information, it can be determined that this new compound has the above structure.

[0039] The present invention also provides a method for extracting and separating the above-mentioned compound, the specific steps of which are:

[0040] Step 1: Weigh 250 kg of dried purslane medicinal material, extract it with water, the amount of water is 10 times the amount of the medicinal material, extract it twice, each time for 2 hours, filter the water extract, combine the filtrate, concentrate it under reduced pressure to 100 L, cool it to room temperature, and obtain the medicinal solution for use.

[0041] Step 2: evaporate the drug solution obtained in step 1 to dryness, then adsorb and elute with AB-8 resin, and gradiently elute with cold water, hot water, 30% ethanol, 50% ethanol, and 70% ethanol. Recover the drug solution to an extract under reduced pressure below 80°C to obtain a hot water extract.

[0042] Step 3: The hot water extract in step 2 is separated by medium- and low-pressure ODS columns, wherein the filler particle size is 40 to 70 μm, and gradient elution is performed using methanol-water (0 / 100, 5 / 95, 10 / 90, 20 / 80, 50 / 50, 100 / 0, v / v) to obtain 35 fractions in total (i.e., 35 bottles in total, each with 100 mL), which are detected by thin layer chromatography and color developed. The colored 10 to 19 eluted fractions are combined, and the combined 10 to 19 fractions are concentrated to dryness under reduced pressure below 75° C. for later use.

[0043] Step 4: The obtained product in step 3 is separated by pre-treated Sephadex gel column chromatography (Sephadex LH-20), and isocratically eluted with 20% methanol to obtain 20 elution fractions (i.e., a total of 20 bottles, each with 40 mL), which are detected by thin layer chromatography, color is developed, and the 2 to 6 colored fractions are retained, and concentrated to dryness under reduced pressure below 70°C for standby use. The pre-treatment process of the ODS and the Sephadex gel is to soak in methanol for 24 hours, load the column, wash with methanol until there is no turbidity when dripped into water, and then balance with the initial mobile phase.

[0044] Step 5: The product obtained in step 4 was separated and prepared by HPLC, with methanol:0.1% formic acid (5:95, v / v) as the mobile phase for isocratic elution, and the detection wavelength was 210nm and 254nm, to separate and prepare the new compound 5-(iminomethyl)-1,3-oxazepine-4,6-diol of the present invention, and the purity was 90-99% as determined by normalization method.

[0045] The anti-inflammatory effect of the novel amine alkaloid compound of the present invention.

[0046] 1Main materials.

[0047] 1.1 Drugs and reagents: The new amine alkaloid compounds used in the experiment were prepared by the above method with a purity of 90-99%. They were accurately weighed and diluted with DMSO to the required solutions for each dose group as follows: DMEM high-glucose medium, fetal bovine serum (Hyclone, USA); penicillin and streptomycin (Hangzhou Sijiqing Company); LPS (Sigma, USA); ELISA kits for IL-1β and TNF-α (Cayman, USA); cell lysate, Griess reagent (Biyuntian 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, one group each.

[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°C, CO2 incubator.

[0052] 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 5-(iminomethyl)-1,3-oxazepine-4,6-diol (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 cells in the above groups were cultured for 24h, 10μL of CCK-8 was added to the cells in each well, and after further incubation for 2h at 37℃, 5% CO2, the absorbance value of each well was measured at a wavelength of 450nm by an enzyme marker.

[0053] 2.3ELISA method for determination of inflammatory factors IL-1β and TNF-α: 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 and 5% CO2, the experimental group added the new amine alkaloid compound 5-(iminomethyl)-1,3-oxazepine-4,6-diol (1-20μM) of the present invention, cultured for 1h, added LPS (final concentration of 1μg / mL) to each well, incubated for 24h, and each group was treated with 3 wells. The ELISA method was used to determine the content of IL-1β and TNF-α secreted by RAW264.7 macrophages after being treated with the new alkaloid compound from Purslane.

[0054] 3 Experimental results.

[0055] The experimental results show that the new amine alkaloid compound 5-(iminomethyl)-1,3-oxazepine-4,6-diol of the present invention has no effect on the proliferation of LPS-induced macrophages RAW264.7 at 25 μM, and is safe and non-toxic; and can effectively inhibit the excessive inflammatory cytokines IL-1β and TNF-α produced by LPS-induced macrophages RAW264.7, and is concentration-dependent.

[0056] The results of the relative cell survival rate experiment are shown in Table 2.

[0057] Table 2 Effect of compound 5-(iminomethyl)-1,3-oxazepine-4,6-diol on the relative survival rate of RAW264.7 macrophages.

[0058]

[0059] Note: * P<0.05 compared with the control group. # P<0.05 compared with LPS group.

[0060] The results of ELISA determination of inflammatory factors IL-1β and TNF-α are shown in Table 3.

[0061] Table 3 Effects of compound 5-(iminomethyl)-1,3-oxazepine-4,6-diol on the levels of IL-1β and TNF-α secreted by RAW264.7 cells induced by LPS (mean ± standard deviation, n = 3).

[0062]

[0063]

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

[0065] The anticholinesterase effect of the novel amine alkaloid compounds of the present invention.

[0066] 1Main materials.

[0067] 1.1 Drugs and reagents: The new compounds used in the experiment were prepared by the above method with a purity of 96%. 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.).

[0068] 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.).

[0069] 2 Experimental methods.

[0070] This experiment was conducted to determine the anticholinesterase activity of the new amine alkaloid compound 5-(iminomethyl)-1,3-oxazepine-4,6-diol according to the modified Ellman method. 5-(iminomethyl)-1,3-oxazepine-4,6-diol and physostigmine were accurately weighed and prepared into five series of sample solutions with concentrations of 3.125μM, 6.25μM, 12.5μM, 25.0μM and 50.0μM 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):

[0071] Inhibition rate (%) = (A 空白 -A 样品 ) / A 空白 ×100%

[0072] 3 Experimental results.

[0073] The experimental results show that the new compound 5-(iminomethyl)-1,3-oxazepine-4,6-diol 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.

[0074] The anticholinesterase activity of the novel amine alkaloid 5-(iminomethyl)-1,3-oxazepine-4,6-diol of the present invention is shown in Table 4.

[0075] Table 4 Anticholinesterase activity of compound 5-(iminomethyl)-1,3-oxazepine-4,6-diol.

[0076]

[0077] In summary, the present invention provides a new amine alkaloid compound and an extraction and separation method thereof, which uses water extraction, macroporous resin adsorption elution, ODS medium-pressure column, Sephadex LH-20 and high-performance liquid chromatography for separation, purification and preparation, and successfully separates and obtains the new compound. The method is simple, rapid and environmentally friendly, and the compound separated by the method has a high purity. Since the obtained compound has a unique chemical structure and is extracted from the commonly used traditional Chinese medicine Portulaca oleracea, it has anti-inflammatory and anticholinesterase effects; therefore, the new amine alkaloid compound and its salts and derivatives of the present invention can be used as natural products to develop new traditional Chinese medicines, and have broad prospects.

Claims

1. A new amine alkaloid compound isolated from Portulaca oleracea, characterized in that: The molecular formula is: C6H6N2O3, and it is named 5-(iminomethyl)-1,3-oxazepine-4,6-diol according to the structure. Its chemical structure is as follows: 。 2. A method for extracting and separating new amine alkaloid compounds isolated from Portulaca oleracea, characterized in that: The specific steps of the extraction and separation method include: Step 1, take dried purslane medicinal material, extract it with water, filter the water extract, combine the filtrate, concentrate it under reduced pressure, cool it to room temperature, and obtain the medicinal solution for use; Step 2, evaporating the Chinese medicine solution in step 1 to dryness, adsorbing it with a macroporous resin, eluting it with a water-ethanol gradient, and concentrating the hot water portion under reduced pressure to obtain an extract, thereby obtaining a hot water extract; Step 3, separating the hot water extract in step 2 through an ODS column, using a methanol-water gradient elution, partially evaporating the 20% methanol and applying it to an ODS column, sequentially eluting with a methanol-water gradient elution to obtain several elution fractions, detecting by thin layer chromatography, developing color, combining the elution fractions with color, and concentrating the combined elution fractions to dryness under reduced pressure for later use; Step 4, the obtained product in step 5 is further separated by pre-treated dextran gel column chromatography, isocratically eluted with 20% methanol to obtain several elution fractions, detected by thin layer chromatography, color developed, combined the color developed elution fractions, and concentrated the combined elution fractions to dryness under reduced pressure for standby use; Step 5: The concentrate obtained in step 4 is separated and prepared by HPLC using 5% methanol:0.1% formic acid v / v as the mobile phase to prepare compound 5-(iminomethyl)-1,3-oxazepine-4,6-diol.

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

4. The extraction and separation method according to claim 2, characterized in that: The mobile phase elution program used in step 2 is cold water, hot water, 30% ethanol, 50% ethanol, 70% ethanol gradient elution.

5. The extraction and separation method according to claim 2, characterized in that: In step 3, the volume ratio of water to methanol is 100:0, 95:5, 90:10, 80:20 and 50:50 for gradient elution.

6. The extraction and separation method according to claim 2, characterized in that: In step 3, the pretreatment process of ODS and dextran gel is: soaking in methanol for 24 hours, loading on the column, washing with methanol until there is no turbidity when dripped into water, and then balancing with the initial mobile phase.

7. Use of the new amine alkaloid compound isolated from Portulaca oleracea as claimed in claim 1 in the preparation of anti-inflammatory and anticholinesterase drugs.

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