A new pyridine alkaloid compound from Portulaca oleracea and its extraction and separation method and use

Abstract

The present invention belongs to the technical field of traditional Chinese medicine extraction and separation, and particularly relates to a new pyridine alkaloid compound extracted, separated and identified from Portulaca oleracea L. and its extraction and separation method. The molecular formula of the new pyridine alkaloid compound is C 13 H 23 N5O3, and it is named as (1-(1,3,4,5,7,8-hexahydropyrido[3,4-e][1,2,4]triazin-6(2H)-yl)-3-methyl-1-oxopentan-2-yl)carbamic acid according to its structure. The present invention also provides an extraction and separation method for the above alkaloid compound, which sequentially adopts water decoction extraction, macroporous resin column chromatography, ODS column chromatography, Sephadex column chromatography and high performance liquid chromatograph for separation, purification and preparation. Its structure is determined to be the alkaloid compound (1-(1,3,4,5,7,8-hexahydropyrido[3,4-e][1,2,4]triazin-6(2H)-yl)-3-methyl-1-oxopentan-2-yl)carbamic acid by means of mass spectrometry, hydrogen nuclear magnetic resonance spectroscopy, carbon nuclear magnetic resonance spectroscopy and two-dimensional nuclear magnetic resonance spectroscopy. The compound has anti-inflammatory activity, anti-cholinesterase activity and antioxidant activity. The alkaloid compound and its salts or derivatives 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 new pyridine alkaloid compound in purslane and an extraction and separation method and application thereof. Background Art

[0002] Purslane comes from the dried aerial parts of Portulacaoleracea L., a plant of the Portulacaceae family. It is one of the wild plants with both medicinal and edible properties designated by the Ministry of Health of my country. It is also known as long-life vegetable and five-element grass. As a traditional Chinese medicine in my country, Purslane has a history of thousands of years of use. It is widely distributed, rich in resources, and has very strong adaptability and tenacious vitality. The 2020 edition of the "Pharmacopoeia of the People's Republic of China" contains 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 the stool, hemorrhoids, and metrorrhagia.

[0003] Modern research shows that purslane has anti-inflammatory, antioxidant, anticholinesterase, antitumor, hypolipidemic and antibacterial effects, which are mainly attributed to its bioactive components, such as alkaloids, flavonoids, lignans, organic acids, terpenes, furans, polysaccharides and phenolic acids, especially alkaloids, as one of the active substances, which have been found to have significant anti-inflammatory 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 new pyridine alkaloid compound extracted from Portulaca oleracea. Studies have shown that the pyridine alkaloid compound 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] In order to achieve the above-mentioned object of the present invention, the present invention provides a new pyridine alkaloid compound, the molecular formula of which is C 13 H 23 N5O3, named according to its structure (1-(1,3,4,5,7,8-hexahydropyrido[3,4-e][1,2,4]triazin-6(2H)-yl)-3-methyl-1-oxopentan-2-yl)carbamic acid, has the following chemical formula:

[0006]

[0007] (1-(1,3,4,5,7,8-hexahydropyrido[3,4-e][1,2,4]triazin-6(2H)-yl)-3-methyl-1-oxopentan-2-yl)carbamic acid

[0008] In order to achieve the above-mentioned object of the present invention, the present invention also provides a method for extracting and separating a new pyridine alkaloid compound from Portulaca oleracea, which specifically comprises the following steps:

[0009] Step 1: Take dried purslane, decoct in water to extract, concentrate the extract, cool to room temperature, and set aside the liquid medicine.

[0010] Step 2: The concentrated solution in step 1 is subjected to a macroporous resin, and eluted with water and ethanol of different concentrations, and the 30% ethanol portion is recovered under reduced pressure to obtain an extract, and a concentrate is obtained for use.

[0011] 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 30% methanol elution portion and evaporate to dryness to obtain a concentrate for use.

[0012] Step 4: The concentrate obtained in step 3 is separated by ODS column chromatography again, using methanol-water gradient elution, detected by thin layer chromatography, color development, and the 30% methanol elution portion is combined and evaporated to dryness to obtain a concentrate for use.

[0013] Step 5: The product obtained in step 4 is further separated by chromatography on a pretreated Sephadex LH-20 column, and isocratically eluted with 10% methanol to obtain several elution fractions, which are detected by thin layer chromatography, developed, and the elution fractions with developed color are combined, and the combined elution fractions are concentrated to dryness under reduced pressure for later use;

[0014] Step 6: The concentrate obtained in step 5 is separated and prepared by HPLC (high performance liquid chromatography), and methanol-0.1% formic acid (volume percentage) is used as a mobile phase for isocratic elution to finally obtain the new pyridine alkaloid compound (1-(1,3,4,5,7,8-hexahydropyrido[3,4-e][1,2,4]triazin-6(2H)-yl)-3-methyl-1-oxopentan-2-yl)carbamic acid of the present invention.

[0015] Furthermore, in step 1, the water decocting and extraction are performed twice, each time for 2 hours, and the amount of water used is 8 to 16 times the amount of the medicinal material.

[0016] 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.

[0017] Furthermore, in step 3, the volume ratio of methanol to water is 10:90, 30:70, 50:50, 70:30 and 100:0 for gradient elution; and the particle size of ODS is 40 to 70 μm.

[0018] Furthermore, in step 4, the volume ratio of methanol to water is 10:90, 30:70, 50:50, 70:30 and 100:0 for gradient elution; and the particle size of ODS is 40 to 70 μm.

[0019] Furthermore, in step 5, the methanol elution procedure is 10% methanol isocratic elution.

[0020] Furthermore, in step 6, the volume ratio of methanol to 0.1% formic acid is 7:93, and the retention time of the compounds is 2.394 min.

[0021] Furthermore, in step 6, 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.

[0022] The present invention also provides a use of the (1-(1,3,4,5,7,8-hexahydropyrido[3,4-e][1,2,4]triazin-6(2H)-yl)-3-methyl-1-oxopentan-2-yl)carbamic acid separated from the purslane medicinal material in the preparation of anti-inflammatory drugs, anticholinesterase drugs and antioxidant drugs.

[0023] Compared with the prior art, the invention has the beneficial effects.

[0024] The separation and pharmacological activity research of the new pyridine alkaloid compound (1-(1,3,4,5,7,8-hexahydropyrido[3,4-e][1,2,4]triazin-6(2H)-yl)-3-methyl-1-oxopentan-2-yl)carbamic acid 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, which sequentially adopts water decoction extraction, macroporous resin column chromatography, ODS column chromatography, dextran gel column (Sephadex LH-20) chromatography and high performance liquid chromatography were used for separation, purification and preparation, and a pyridine alkaloid compound was successfully extracted and separated. The method has only six steps, the operation method is simple and fast, water is used for extraction, the process method is environmentally friendly, 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, anticholinesterase activity and antioxidant activity; therefore, the compound (1-(1,3,4,5,7,8-hexahydropyrido[3,4-e][1,2,4]triazin-6(2H)-yl)-3-methyl-1-oxopentan-2-yl)carbamic acid and its salts and derivatives can be used as raw materials for drug development and pharmacological activity research. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The novel pyridine alkaloid compound (1-(1,3,4,5,7,8-hexahydropyrido[3,4-e][1,2,4]triazin-6(2H)-yl)-3-methyl-1-oxopentan-2-yl)carbamic acid of the present invention is 1 H-NMR spectrum.

[0026] Figure 2 The novel pyridine alkaloid compound (1-(1,3,4,5,7,8-hexahydropyrido[3,4-e][1,2,4]triazin-6(2H)-yl)-3-methyl-1-oxopentan-2-yl)carbamic acid of the present invention is 13 C-NMR spectrum.

[0027] Figure 3The DEPT spectrum of the new pyridine alkaloid compound (1-(1,3,4,5,7,8-hexahydropyrido[3,4-e][1,2,4]triazin-6(2H)-yl)-3-methyl-1-oxopentan-2-yl)carbamic acid of the present invention is shown in FIG.

[0028] Figure 4 The HSQC spectrum of the new pyridine alkaloid compound (1-(1,3,4,5,7,8-hexahydropyrido[3,4-e][1,2,4]triazin-6(2H)-yl)-3-methyl-1-oxopentan-2-yl)carbamic acid of the present invention is shown.

[0029] Figure 5 The figure is the HMBC spectrum of the new pyridine alkaloid compound (1-(1,3,4,5,7,8-hexahydropyrido[3,4-e][1,2,4]triazin-6(2H)-yl)-3-methyl-1-oxopentan-2-yl)carbamic acid of the present invention.

[0030] Figure 6 The COESY spectrum of the new pyridine alkaloid compound (1-(1,3,4,5,7,8-hexahydropyrido[3,4-e][1,2,4]triazin-6(2H)-yl)-3-methyl-1-oxopentan-2-yl)carbamic acid of the present invention is shown.

[0031] Figure 7 The ROESY spectrum of the new pyridine alkaloid compound (1-(1,3,4,5,7,8-hexahydropyrido[3,4-e][1,2,4]triazin-6(2H)-yl)-3-methyl-1-oxopentan-2-yl)carbamic acid of the present invention is shown in FIG.

[0032] Figure 8 The high-resolution mass spectrum of the novel pyridine alkaloid compound (1-(1,3,4,5,7,8-hexahydropyrido[3,4-e][1,2,4]triazin-6(2H)-yl)-3-methyl-1-oxopentan-2-yl)carbamic acid of the present invention is shown. DETAILED DESCRIPTION

[0033] 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.

[0034] Example 1.

[0035] (1-(1,3,4,5,7,8-hexahydropyrido[3,4-e][1,2,4]triazin-6(2H)-yl)-3-methyl-1-oxope ntan-2-yl)carbamic acid

[0036] The present invention provides a new pyridine alkaloid compound, the molecular formula of which is C 13 H 23 N5O3, named according to the structural formula (1-(1,3,4,5,7,8-hexahydropyrido[3,4-e][1,2,4]triazin-6(2H)-yl)-3-methyl-1-oxopentan-2-yl)carbamic acid, has the following chemical structures:

[0037]

[0038] (1-(1,3,4,5,7,8-hexahydropyrido[3,4-e][1,2,4]triazin-6(2H)-yl)-3-methyl-1-oxopentan-2-yl)carbamic acid Table 1 is

[0039] NMR data of (1-(1,3,4,5,7,8-hexahydropyrido[3,4-e][1,2,4]triazin-6(2H)-yl)-3-methyl-1-oxopentan-2-yl)carbamic acid. The solvent used for NMR is DMSO-d6.

[0040] Table 1

[0041] (1-(1,3,4,5,7,8-hexahydropyrido[3,4-e][1,2,4]triazin-6(2H)-yl)-3-methyl-1-oxopentan-

[0042] NMR data of 2-yl)carbamic acid.

[0043]

[0044] (1-(1,3,4,5,7,8-hexahydropyrido[3,4-e][1,2,4]triazin-6(2H)-yl)-3-methyl-1-oxop entan-2-yl)carbamic acid: yellow-brown oily substance, easily soluble in water. After being spotted on a silica gel thin layer plate, the spots are sprayed with dilute potassium bismuth iodide test solution and appear orange-yellow, 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 C 13 H 23 N5O3, unsaturation is 5. HR-ESI-TOF-MS gives m / z [M+K] + The quasi-molecular ion peak is 336.1437, and the calculated value (C 13 H 23 N5O3 + =336.1437). 13 C-NMR and DEPT spectra show that there are 13 carbon resonance peaks in the compound, including 2 methyl groups (δ C 10.66,15.23), 5 methylene groups (δ C 24.64, 26.15, 28.95, 42.00, 55.50), 2 methines (δ C 35.96,36.60), 2 quaternary carbon resonance peaks (δ C 138.50,140.55), 2 carbonyl groups (δ C 173.28, 175.05). The NMR data of the compound are listed in Table 1. 1 H-NMR and HMBC spectra showed that H-2'(δ H 2.55) and C-4'(δ C 138.50) and C-6'(δ C 42.00) has a strong correlation; H-6'(δ H 2.48) has a strong correlation with C-4'; H-5' (δ H 1.88) and C-3'(δ C 140.55) has a strong correlation, and according to 13 The chemical shifts of C-2' and C-6' in the C-NMR spectrum are in the low field, proving that they are connected to the N atom, respectively, so there is a hexahydropyridine ring. On the other side of the pyridine ring, the HMBC spectrum shows that H-9' (δ H3.70) has a strong correlation with C-3', but no correlation with C-4', and the chemical shifts of C-3', C-4', and C-9' are all in the downfield, which is inferred to be connected to N, respectively. It is inferred that it is a six-membered ring with 3 N atoms and is fused through C-4' and C-3'. 1 H-NMR spectrum showed that H-5 and H-6 were methyl groups. 13 C-NMR spectrum showed that C-1 and C-8 were carbonyl groups, and 1 H- 1 The H-COSY spectrum shows that H-6 is correlated with H-3, H-3 is correlated with H-4, H-4 is correlated with H-5, and H-5 is correlated with H-6. Based on the correlation between H-5 and C-3, the correlation between H-6 and C-4, the correlation between H-4 and H-6 and C-2, and the correlation between H-3 and C-1 in the HMBC spectrum, based on the correlation between H-2 and C-8, and C-8 is in the low field, combined with the HR-ESI-TOF-MS mass spectrum, it is reasonable to infer that the compound contains hydroxyl groups, so the structure is inferred to be 3-methyl-2-carbamic acid-1-oxopentane. Based on the strong correlation between H-2' and H-6' and C-1 in the HMBC spectrum, it is shown that 3-methyl-2-carbamic acid-1-oxopentane is connected to pyridine through N-1'-C-1. Based on the above information, combined with the molecular weight of the HR-ESI-TOF-MS spectrum, it can be determined that this pyridine alkaloid compound has the above structure.

[0045] The present invention also provides a method for extracting and separating a pyridine alkaloid compound (1-(1,3,4,5,7,8-hexahydropyrido[3,4-e][1,2,4]triazin-6(2H)-yl)-3-methyl-1-oxopentan-2-yl)carbamic acid, which specifically comprises the following steps:

[0046] Step 1: Weigh 250 kg of dried purslane medicinal materials, use water decoction to extract, the amount used is 10 times 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.

[0047] Step 2: evaporate the drug solution obtained in step 1 to dryness, separate it through AB-8 macroporous resin column chromatography, use ethanol-water (30:70, 50:50, 70:30 and 100:0, v:v) gradient elution, collect the 30% ethanol portion, recover it to the extract under reduced pressure, and obtain the concentrate for use.

[0048] Step 3: Separate the concentrate in step 2 through a pretreated ODS column with a filler particle size of 40 to 70 μm, and use methanol-water (10:0, 30:70, 50:50, 70:30 and 100:0, v:v) gradient elution, detect by thin layer chromatography, develop color, combine the 30% methanol elution fractions, concentrate to dryness under reduced pressure, and set aside.

[0049] Step 4: The obtained product in step 3 is separated by pre-treated ODS column chromatography, the filler particle size is 40-70 μm, and gradient elution is performed with methanol-water (10:0, 30:70, 50:50, 70:30 and 100:0, v / v), and detected by thin layer chromatography, color development, and the 30% methanol elution part is combined, concentrated to dryness under reduced pressure, and used for standby. The ODS pretreatment process is to soak in methanol for 24 hours, after loading the column, wash with methanol until there is no turbidity when dripped into water, and then balance with the initial mobile phase.

[0050] Step 5: The obtained product in step 4 is further chromatographed and separated on a pretreated Sephadex LH-20 column, and isocratically eluted with 10% methanol to obtain several elution parts, which are detected by thin layer chromatography, color developed, and the elution parts with color developed are combined, and the combined elution parts are concentrated to dryness under reduced pressure for standby use. The pretreatment process of the dextran gel is to soak it in methanol for 24 hours, load it onto the column, wash it with methanol until there is no turbidity when it is dropped into water, and then balance it with the initial mobile phase.

[0051] Step 6: The fraction obtained in step 5 was separated and prepared by HPLC, with 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 separate and prepare the pyridine alkaloid compound of the present invention, and the purity determined by normalization method was 96%.

[0052] Example 2.

[0053] Anti-inflammatory effects of (1-(1,3,4,5,7,8-hexahydropyrido[3,4-e][1,2,4]triazin-6(2H)-yl)-3-methyl-1-oxopentan-2-yl)carbamic acid.

[0054] 1Main materials.

[0055] 1.1 Drugs and reagents: The pyridine alkaloid compounds used in the experiment were prepared by the above method with a purity of 96%. 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.

[0056] 1.2 Cell line: RAW264.7 macrophages (ATCC cell bank, USA).

[0057] 1.3 Grouping: Divided into control group, LPS group and experimental group.

[0058] 2 Experimental methods.

[0059] 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.

[0060] 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 (1-(1,3,4,5,7,8-hexahydropyrido[3,4-e][1,2,4]triazin-6(2H)-yl)-3-methyl-1-oxopentan-2-yl)carbamic acid (5μM~50μM) were added to the experimental group, and LPS with a concentration of 1μg / mL was added to the LPS group and the experimental group respectively after incubation for 1h. A zero adjustment group (culture solution containing DMSO solvent) was also set up, and 3 replicates 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 the cells were incubated for another 4h at 37℃ and 5% CO2, and the absorbance of each well was measured at a wavelength of 450nm by an enzyme marker.

[0061] 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 was added with the compound of the present invention (1-(1,3,4,5,7,8-hexahydropyrido[3,4-e][1,2,4]triazin-6(2H)-yl)-3-methyl-1-oxopentan-2-yl)carbamic acid (1μM~20μM) and cultured for 1h, then LPS (final concentration of 1μg / mL) was added to each well and incubated for 24h, with 3 wells in each group. ELISA method was used to determine the content of IL-1β and TNF-α.

[0062] 3 Experimental results.

[0063] The experimental results show that the compound of the present invention (1-(1,3,4,5,7,8-hexahydropyrido[3,4-e][1,2,4]triazin-6(2H)-yl)-3-methyl-1-oxopentan-2-yl)carbamic acid has no effect on the proliferation of LPS-induced macrophages RAW264.7; it can effectively inhibit the secretion of excessive inflammatory cytokines IL-1β and TNF-α produced by LPS-induced macrophages RAW264.7 in a concentration-dependent manner.

[0064] The results of the relative cell survival rate experiment are shown in Table 2.

[0065] Table 2 Effects of the compounds of the present invention on the relative survival rate of RAW264.7 macrophages.

[0066]

[0067]

[0068] The results of ELISA determination of inflammatory factors IL-1β and TNF-α are shown in Table 3.

[0069] Table 3 Effects of the compounds of the present invention on the levels of IL-1β and TNF-α secreted by RAW264.7 cells induced by LPS (mean±standard deviation, n=3).

[0070]

[0071] Note: * P<0.05 compared with the control group. # P<0.05 compared with LPS group.

[0072] Example 3.

[0073] Anticholinesterase effects of (1-(1,3,4,5,7,8-hexahydropyrido[3,4-e][1,2,4]triazin-6(2H)-yl)-3-methyl-1-oxopentan-2-yl)carbamic acid.

[0074] 1Main materials.

[0075] 1.1 Drugs and reagents: The pyridine alkaloid 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.).

[0076] 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.).

[0077] 2 Experimental methods.

[0078] In this experiment, the anticholinesterase activity of each compound was determined according to the modified Ellman method. (1-(1,3,4,5,7,8-hexahydropyrido[3,4-e][1,2,4]triazin-6(2H)-yl)-3-methyl-1-oxopentan-2-yl)carbamic acid and physostigmine were accurately weighed and prepared into five series of sample solutions with concentrations of 31.25μM, 62.5μM, 125μM, 250μM and 500μM 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):

[0079] Inhibition rate (%) = (A 空白 -A 样品 ) / A 空白 ×100%

[0080] 3 Experimental results.

[0081] The experimental results show that the pyridine alkaloid compound 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.

[0082] The anticholinesterase activities of the pyridine alkaloid compounds of the present invention are shown in Table 4.

[0083] Table 4 Anticholinesterase activities of the compounds of the present invention.

[0084]

[0085] Example 4.

[0086] Antioxidant effect of (1-(1,3,4,5,7,8-hexahydropyrido[3,4-e][1,2,4]triazin-6(2H)-yl)-3-methyl-1-oxopentan-2-yl)carbamic acid.

[0087] 1Main materials.

[0088] 1.1 Drugs and reagents: The pyridine alkaloid compounds used in the experiment were prepared by the above method with a purity of 96%. 1,1-Diphenyl-2-picrylhydrazyl free radical (DPPH) (Sigma, USA), butylated hydroxyanisole (BHA) (Shanghai Xiangrui Biological Co., Ltd.), methanol (chromatographic grade, Tianjin Kaixin Chemical Industry Co., Ltd.).

[0089] 1.2 Experimental instruments and equipment: Hitachi UV-3010 ultraviolet-visible spectrophotometer (Hitachi, Japan), 1 / 100,000 balance (METTLER, Switzerland).

[0090] 2 Experimental methods.

[0091] In this experiment, the DPPH free radical scavenging method was used to determine the antioxidant activity of each compound. (1-(1,3,4,5,7,8-hexahydropyrido[3,4-e][1,2,4]triazin-6(2H)-yl)-3-methyl-1-oxopentan-2-yl)carbamic acid and BHA were accurately weighed and prepared into five series of sample solutions with concentrations of 12.5μM, 25μM, 50μM, 100μM and 200μM with methanol. In addition, the DPPH solution was prepared as needed. An appropriate amount of DPPH was accurately weighed and prepared into a solution with a concentration of 80μM with methanol. The whole process was protected from light. The specific operation is as follows: 1mL of the sample solution was fully mixed with 1mL of the DPPH solution, and placed at room temperature in a light-proof environment for 10min. After setting the detection wavelength of the UV spectrophotometer to 517nm, the absorbance value of the sample was measured. Among them, methanol was used instead of sample solution as blank group, BHA was used instead of sample solution as positive control group, and a mixed solution of 1 mL methanol and 1 mL sample solution was used as control group. The DPPH scavenging rate of each compound was calculated according to the following formula (A represents absorbance):

[0092] DPPH clearance rate (%) = (1-(A 样品 -A 对照 ) / A 空白 )×100%

[0093] 3 Experimental results.

[0094] The experimental results show that the pyridine alkaloid compound of the present invention exhibits certain antioxidant activity, and the antioxidant effect is enhanced with the increase of the concentration of the compound, showing a dose-dependent trend.

[0095] The antioxidant activities of the pyridine alkaloid compounds of the present invention are shown in Table 5.

[0096] Table 5 Antioxidant activity of the compounds of the present invention.

[0097]

[0098]

[0099] In summary, the present invention provides a compound (1-(1,3,4,5,7,8-hexahydropyrido[3,4-e][1,2,4]triazin-6(2H)-yl)-3-methyl-1-oxopentan-2-yl)carbamic acid and an extraction and separation method thereof, which sequentially adopts water decoction extraction, macroporous resin column chromatography, ODS column chromatography, Sephadex LH-20 and high performance liquid chromatography for separation, purification and preparation, and successfully extracts and separates a pyridine alkaloid compound. 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, it has anti-inflammatory activity, anticholinesterase activity and antioxidant activity; therefore, the pyridine alkaloid compound (1-(1,3,4,5,7,8-hexahydropyrido[3,4-e][1,2,4]triazin-6(2H)-yl)-3-methyl-1-oxopentan-2-yl)carbamic acid and its salts and derivatives can provide ideas for drug development and have broad development prospects.

Claims

1. An alkaloid compound isolated from Portulaca oleracea, characterized in that: The molecular formula is: C 13 H 23 N5O3, named according to its structure (1-(1,3,4,5,7,8-hexahydropyrido[3,4-e][1,2,4]triazine-6(2H)-yl)-3-methyl-1-oxopentan-2-yl)carbamic acid, has the chemical formula: 。 2. A method for extracting and separating an alkaloid compound from Portulaca oleracea, characterized in that: The specific steps include: Step 1: Take dried purslane medicinal material, decoct it in water, concentrate the extract, cool it to room temperature, and obtain the 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, and the 30% ethanol portion is recovered under reduced pressure to obtain an extract, and a concentrate is obtained for standby use; Step 3: Separate the concentrate in step 2 through an ODS column, use a methanol-water gradient elution, detect by thin layer chromatography, develop color, combine the 30% methanol elution portion and evaporate to dryness to obtain a concentrate for use; Step 4: The concentrate obtained in step 3 is separated by ODS column chromatography, eluted by methanol-water gradient, detected by thin layer chromatography, color developed, and the 30% methanol elution portion is combined and evaporated to dryness to obtain a concentrate for use; Step 5: The product obtained in step 4 is further separated by pre-treated dextran gel column chromatography, isocratically eluted with 10% 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; Step 6: The concentrate obtained in step 5 is separated and prepared by HPLC, and isocratic elution is performed using volume percentage methanol-0.1% formic acid as the mobile phase to finally obtain the alkaloid compound (1-(1,3,4,5,7,8-hexahydropyrido[3,4-e][1,2,4]triazine-6(2H)-yl)-3-methyl-1-oxopentan-2-yl)carbamic acid.

3. The extraction and separation method according to claim 2, characterized in that: In the step 1, the extraction is performed by decoction twice, each time for 2 hours, and the amount of water used is 8 to 16 times the amount of the medicinal material.

4. The extraction and separation method according to claim 2, characterized in that: In the 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.

5. The extraction and separation method according to claim 2, characterized in that: In the step 3, the volume ratio of methanol to water is 10:90, 30:70, 50:50, 70:30 and 100:0 for gradient elution; and the particle size of ODS is 40-70 μm.

6. The extraction and separation method according to claim 2, characterized in that: In step 4, the volume ratio of methanol to water is 10:90, 30:70, 50:50, 70:30 and 100:0 for gradient elution; and the particle size of ODS is 40-70 μm.

7. The extraction and separation method according to claim 2, characterized in that: The pretreatment process of the ODS and dextran gel is to soak in methanol for 24 hours, load onto the column, wash with methanol until there is no turbidity when dripped into water, and then balance with the initial mobile phase.

8. The extraction and separation method according to claim 2, characterized in that: In step 6, the volume ratio of methanol to 0.1% formic acid is 7:93, and the retention time of the compound is 2.394 min.

9. Use of the alkaloid compound isolated from Portulaca oleracea as claimed in claim 1 in the preparation of anti-inflammatory drugs, anticholinesterase drugs and antioxidant drugs.

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