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

Abstract

The present invention belongs to the field of traditional Chinese medicine extraction and separation, and particularly relates to a new pyrimidine alkaloid compound in purslane, as well as an extraction, separation method and application thereof. The new pyrimidine alkaloid compound has a molecular formula of C9H 15 N3O5 and is named Olerapyrimidine. An extraction and separation method for the above new compound is also provided, and ethanol reflux extraction, ODS column chromatography, sephadex gel chromatography and high performance liquid chromatography are sequentially used for separation, purification and preparation. Its structure is determined to be a new compound by methods such as mass spectrometry, hydrogen nuclear magnetic resonance spectroscopy, carbon nuclear magnetic resonance spectroscopy and two-dimensional nuclear magnetic spectroscopy, and it has anti-inflammatory activity. The new 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 invention belongs to the technical field of traditional Chinese medicine extraction and separation, and in particular relates to a new pyrimidine alkaloid compound in purslane and an extraction and separation method and application thereof. Background Art

[0002] Portulaca oleracea L., also known as horse amaranth, five-element grass, longevity vegetable, five-sided grass, melon seed vegetable, hemp rope vegetable, horse tooth vegetable, grasshopper vegetable, is an annual herbaceous plant of the genus Portulacaceae, the whole plant is hairless; the stem is lying or leaning, spreading on the ground, multi-branched, cylindrical, 10-15 cm long, light green or dark red, with small seeds, most of which are obliquely spherical, dark brown. It is cultivated in both the north and south of my country, growing in vegetable gardens, farmlands, and roadsides, and is a common weed in the field. The 2020 edition of the "Pharmacopoeia of the People's Republic of China" records that it has the effects of clearing away heat and detoxifying, cooling blood and stopping bleeding, and stopping dysentery. It is commonly used clinically to treat heat-toxic bloody dysentery, carbuncle furuncle, eczema, erysipelas, snake and insect bites, blood in the stool, hemorrhoids, metrorrhagia, etc.

[0003] There are many kinds of chemical components in Purslane, including flavonoids, terpenes and sterols, alkaloids, organic acids and other active ingredients. Among them, alkaloids are the main chemical components of Purslane. The alkaloid components reported so far include oleraisoindole A, oleraindole A, oleraindole B, oleraciamideE, oleraciamideF, oleraciamideG, etc. Because Purslane contains rich chemical substances, it provides a material basis for the study of the pharmacological effects of Purslane. Modern pharmacological studies have found that Purslane has the effects of lowering blood sugar, lowering blood lipids, anti-tumor, anti-oxidation, anti-aging, anti-browning, anti-inflammatory and analgesic, kidney protection activity, anti-virus, neuroprotection, and regulating immune function.

[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 is needed. Summary of the invention

[0005] In view of the above problems, the present invention provides a new pyrimidine alkaloid compound extracted and separated from Portulaca oleracea. Studies have found that the new compound of the present invention has anti-inflammatory 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] In order to achieve the above-mentioned object of the present invention, the present invention provides a new pyrimidine alkaloid compound, the molecular formula of which is C9H 15 N3O5, named Olerapyrimidine, has the chemical formula:

[0007]

[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 pyrimidine alkaloid compound from Portulaca oleracea, which specifically comprises the following steps:

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

[0010] Step 2, after the Chinese medicine solution in step 1 is evaporated to dryness, it is extracted with different proportions of ethyl acetate: ethanol, the extract is filtered, and the extract is concentrated under reduced pressure to an extract for standby use;

[0011] Step 3, dissolve the ethyl acetate:ethanol (1:5) part of the extract in step 2 with ethyl acetate:ethanol in the same ratio, and dissolve the insoluble part with 70% methanol to obtain a medicinal solution for standby use;

[0012] Step 4, separating the Chinese medicine solution in step 3 through an ODS (octadecylsilane bonded silica gel filler) column, using a methanol-water gradient elution to obtain several elution fractions, detecting them through thin layer chromatography, developing the color, combining the same elution fractions, and concentrating the combined fractions to dryness under reduced pressure for standby use;

[0013] Step 5. The fifth fraction obtained in step 4 is further separated by chromatography on a pretreated Sephadex LH-20 column, and isocratically eluted with 70% methanol to obtain several elution fractions, which are detected by thin layer chromatography and color developed. The same elution fractions are combined, and the combined elution fractions are concentrated to dryness under reduced pressure for later use.

[0014] Step 6: The fifth fraction concentrate obtained in step 5 is separated and prepared by HPLC (high performance liquid chromatography), and methanol: 0.1% formic acid is used as a mobile phase for isocratic elution to prepare the novel pyrimidine alkaloid compound Olerapyrimidine of the present invention.

[0015] Furthermore, in step 1, ethanol reflux extraction is performed twice, each time for 2 hours, and the amount used is 8-16 times that of the medicinal material.

[0016] Furthermore, the mobile phase elution program used in step 2 is ethyl acetate (1:0), ethyl acetate: ethanol (5:1), ethyl acetate: ethanol (2:1), ethyl acetate: ethanol (1:1), ethyl acetate: ethanol (1:2), ethyl acetate: ethanol (1:5), and ethanol is recovered for gradient elution.

[0017] Furthermore, in step 4, the volume ratio of methanol to water is 50:50, 70:30, 90:10, and 100:0 for gradient elution, and the ODS particle size is 40 to 70 μm.

[0018] Furthermore, the pretreatment process of the 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.

[0019] Furthermore, the methanol elution procedure in step 5 is 70% methanol isocratic elution.

[0020] Furthermore, in the step 6, the volume ratio of methanol: 0.1% formic acid-water in the isocratic elution is 5:95, and the retention time of the compound is 5.623 min.

[0021] The novel pyrimidine alkaloid compound of the invention can be used for preparing anti-inflammatory drugs.

[0022] Compared with the prior art, the invention has the beneficial effects.

[0023] The separation and pharmacological activity research of the new pyrimidine alkaloid compound from purslane described in the present invention has not been reported in existing papers and journals; the present invention provides a new pyrimidine alkaloid compound derived from purslane and an extraction and separation method for the new compound of the present invention, which uses ethanol extraction, extraction with different proportions of ethyl acetate and ethanol, ODS medium-pressure column, Sephadex LH-20 and high-performance liquid chromatography for separation, purification and preparation in sequence, and successfully extracts and separates the new compound. The method has only six operating steps, and the operation method is simple and fast. The extraction and separation process mainly uses ethanol extraction and methanol elution. The process method is environmentally friendly, and the purity of the compound separated by the method is greater than 90%. In addition, studies have shown that the compound has an anti-inflammatory effect. Therefore, the new compound of the present invention and its salts and derivatives can be used as synthetic leads for other compounds, as well as raw materials for new drug development and pharmacological activity research, and can also be used to prepare anti-inflammatory drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a high-resolution mass spectrum of the novel pyrimidine alkaloid compound Olerapyrimidine of the present invention.

[0025] Figure 2 The novel pyrimidine alkaloid compound Olerapyrimidine of the present invention 1 H-NMR spectrum.

[0026] Figure 3 The novel pyrimidine alkaloid compound Olerapyrimidine of the present invention 13 C-NMR spectrum.

[0027] Figure 4 The DEPT spectrum of the novel pyrimidine alkaloid compound Olerapyrimidine of the present invention is shown in FIG.

[0028] Figure 5 This is the HMBC spectrum of the novel pyrimidine alkaloid compound Olerapyrimidine of the present invention.

[0029] Figure 6 The HSQC spectrum of the novel pyrimidine alkaloid compound Olerapyrimidine of the present invention is shown in FIG.

[0030] Figure 7 The novel pyrimidine alkaloid compound Olerapyrimidine of the present invention 1 H- 1 HCOSY spectrum.

[0031] Figure 8 The ROESY spectrum of the novel pyrimidine alkaloid compound Olerapyrimidine of the present invention is shown in FIG. DETAILED DESCRIPTION

[0032] 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.

[0033] The present invention provides a new compound, the molecular formula of which is C9H 15 N3O5, named Olerapyrimidine, has the chemical formula:

[0034]

[0035] Table 1 shows the NMR data of the new pyrimidine alkaloid compound: 1 H-NMR and 13 C-NMR in methanol.

[0036] Table 1 NMR data of the new pyrimidine alkaloid Olerapyrimidine.

[0037]

[0038]

[0039] The structural identification of the compounds of the present invention is shown in Figure 1-Figure 8 .

[0040] Olerapyrimidine: Yellow oil, 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: 246.1083 [M+H] + The quasi-molecular ion peak (calculated value [C9H16 N3O5] + =246.1084). Combined 1 H-NMR, 13 C-NMR and DEPT data suggest that the possible molecular formula of the compound is C9H 15 N3O5, degree of unsaturation is 3. 13 The C-NMR spectrum and DEPT spectrum showed 9 carbon signals, including 1 methylene carbon (δ C :63.54)、6 methyl carbons (δ C :65.02;72.74;75.45;88.24;91.29;141.54)、2 quaternary carbons (δ C :123.28;148.02).

[0041] The 1H-NMR spectrum shows a methylene signal as δ H 3.65, 3.78 (2H, dd, J = 2.25, 12.54 Hz, H-6′), 6 methine signals are δH8.17 (1H, d, H-2, J = 8.22 Hz), δ H 5.87 (1H, d, H-6, J = 6.54Hz), δH4.66 (1H, d, J = 1.32Hz, H-2′), δ H 4.23 (1H, m, H-3′), δ H 3.90 (1H, m, H-4′), δ H 4.08 (1H, m, H-5'). According to 1H-NMR spectrum and 13 The δ H 3.65, 3.78, δH4.66, δ H 4.23, δ H 3.90, δ H 4.08 and δ C 63.70, δ C 65.05, δ C 72.89, δ C 75.57, δ C The signal of 88.28 indicates that the structure contains furanose. According to the HMBC spectrum, H-2 and C-4 (δ C :148.02)、C-6(δ C :91.29) related; H-6 and C-2 (δ C :141.54)、C-4(δ C :148.02) related; H-2′ and C-4 (δ C :148.02)、C-6(δ C:91.29) related; H-3′ and C-5 (δ C :123.28) and C-2(δ C :141.54)、C-4(δ C :148.02)、C-6(δ C :91.29) is located in the low field, it can be inferred that C-2 (δ C :141.54) connected N-1-N3, C-4 (δ C :148.02) is substituted with amino group, C-6 (δ C :91.29) is connected to a hydroxyl substitution, and it can be shown that the furanose part is connected to the pyrimidine ring. Thus, based on the above information, it can be determined that this new compound is the above structure.

[0042] The present invention also provides a method for extracting and separating the above-mentioned pyrimidine alkaloid compound, which specifically comprises the following steps:

[0043] Step 1: Weigh 250 kg of dried purslane medicinal materials, extract with ethanol, the amount of ethanol is 10 times that of the medicinal materials, extract twice, each time for 2 hours, filter the ethanol extract, combine the filtrate, concentrate under reduced pressure to 250 L, cool to room temperature, and obtain the medicinal solution for use.

[0044] Step 2: After the Chinese medicine solution in step 1 is evaporated to dryness, it is extracted with ethyl acetate: ethanol in different proportions, and the extract is filtered to obtain 7 parts in total, which are concentrated under reduced pressure to extracts.

[0045] Step 3: Dissolve the ethyl acetate:ethanol (1:5) portion of the extract in step 2 with ethyl acetate:ethanol in the same ratio, dissolve the insoluble portion with 70% methanol and separate it with a medium- and low-pressure ODS column, wherein the filler particle size is 40-70 μm, and use methanol-water (50:50, 70:3, 90:10, 100:0, v / v) gradient elution to obtain a total of 16 parts, detect by thin layer chromatography, develop color, combine the same parts, and concentrate the combined 5th part to dryness under reduced pressure below 75°C for later use.

[0046] Step 4: The 5th part obtained in step 3 is separated by pre-treated dextran gel column chromatography (Sephadex LH-20), eluted with methanol, and 7 elution parts are obtained (i.e., 7 bottles are obtained, each bottle is 50 mL), detected by thin layer chromatography, color is developed, and the 5th part is retained, concentrated to dryness under reduced pressure below 50°C, and used for standby. The pretreatment process of ODS and dextran 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. .

[0047] Step 5: The product obtained in step 4 was separated and prepared by HPLC, and methanol: 0.1% formic acid water (5:95, v / v) was used as the mobile phase for isocratic elution, and the detection wavelength was 210nm and 254nm, and the new pyrimidine alkaloid compound Olerapyrimidine of the present invention was separated and prepared. The purity was 90-99% as determined by normalization method.

[0048] Example 2.

[0049] The present invention discloses the anti-inflammatory effect of the novel pyrimidine alkaloid compound Olerapyrimidine.

[0050] 1. Main materials.

[0051] 1.1 Drugs and reagents: The new pyrimidine 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 solution 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.).

[0052] 1.2 Cell line: RAW264.7 macrophages (ATCC cell bank, USA).

[0053] 1.3 Grouping: Divided into control group, LPS group and experimental group, one group each.

[0054] 2. Experimental method.

[0055] 2.1 Cell culture: DMEM high glucose medium, with 10% fetal bovine serum, 1% antibiotics (100 U / mL penicillin and 1

[0056] 00 μg / mL streptomycin) and cultured in a 37.5°C, CO2 incubator.

[0057] 2.2 CCK-8 method to determine cell viability: The three groups of RAW264.7 macrophages in the logarithmic growth phase were inoculated in 96-well culture plates, with a cell density of 1×104 / mL, 100μL per well, and cultured overnight at 37°C and 5% CO2. Different concentrations of the compound Olerapyrimidine (5μM~50μM) of the present invention were added to the experimental group. After incubation for 1h, LPS with a concentration of 1μg / mL was added to the LPS group and the experimental group, respectively. A zero adjustment group (culture solution containing DMSO solvent) was 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 2h at 37°C and 5% CO2. The absorbance value of each well was measured at a wavelength of 450nm by an ELISA reader.

[0058] 2.3ELISA method to determine the inflammatory factors IL-1β and TNF-α: RAW264.7 macrophages in the logarithmic growth phase were inoculated in a 24-well culture plate, with a cell density of 1×105 / mL, 1 mL per well, and cultured overnight at 37°C and 5% CO2. Olerapyrimidine (1-20 μM), a new pyrimidine alkaloid compound of the present invention, was added to the experimental group. After 1 hour of incubation, LPS (final concentration of 1 μg / mL) was added to each well and incubated for 24 hours. Each group of treatments was repeated 3 wells. The ELISA method was used to determine the content of IL-1β and TNF-α secreted by RAW264.7 macrophages after treatment with the new alkaloid compound from Purslane.

[0059] 3. Experimental results.

[0060] The experimental results show that the new pyrimidine alkaloid compound of the present invention has no effect on the proliferation of LPS-induced macrophages RAW264.7 at 20 μM, 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 in a concentration-dependent manner.

[0061] The relative survival rate experimental results of the novel pyrimidine alkaloid compound Olerapyrimidine of the present invention are shown in Table 2.

[0062] Table 2 Relative survival rates of new pyrimidine alkaloid compounds.

[0063]

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

[0065] The results of ELISA determination of inflammatory factors IL-1β and TNF-α are shown in Table 3.

[0066] Table 3 Effects of the novel pyrimidine alkaloid compound Olerapyrimidine 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).

[0067]

[0068]

[0069] Note: * P<0.05 compared with the control group. # P<0.05 compared with LPS group.

[0070] In summary, the present invention provides a new pyrimidine alkaloid compound and an extraction and separation method thereof, which uses ethanol extraction, ODS medium pressure column, Sephadex LH-20 and high performance liquid chromatography for separation, purification and preparation in sequence, 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 new pyrimidine alkaloid compound has a unique chemical structure and is extracted from the commonly used traditional Chinese medicine Portulaca oleracea, it has an anti-inflammatory effect. Therefore, the new pyrimidine 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 pyrimidine alkaloid compound from Portulaca oleracea, characterized in that: The molecular formula is C9H 15 N3O5, named Olerapyrimidine, has the chemical formula: 。 2. A method for extracting and separating a new pyrimidine alkaloid compound from Portulaca oleracea, characterized in that: The specific steps include: Step 1, take dried purslane medicinal material, extract it with ethanol, filter the ethanol extract, combine the filtrate, concentrate it under reduced pressure, cool it to room temperature, and obtain a medicinal solution for use; Step 2, after the Chinese medicine solution in step 1 is evaporated to dryness, it is extracted with ethyl acetate: ethanol in different proportions, the extract is filtered, and the extract is concentrated under reduced pressure to an extract for standby use; Step 3, dissolve the extract in step 2 in a ratio of ethyl acetate to ethanol of 1:5 with ethyl acetate to ethanol in the same ratio, and dissolve the insoluble part with 70% methanol to obtain a liquid medicine for standby use; Step 4, separating the Chinese medicine solution in step 3 through an ODS column, using a methanol-water gradient elution to obtain several elution parts, detecting them through thin layer chromatography, developing the color, combining the same elution parts, and concentrating the combined parts to dryness under reduced pressure for later use; Step 5, the fifth fraction obtained in step 4 is further separated by pretreated dextran gel column chromatography, isocratically eluted with 70% methanol to obtain several elution fractions, detected by thin layer chromatography, color developed, the same elution fractions are combined, and the combined elution fractions are concentrated to dryness under reduced pressure for standby use; Step 6: The fifth fraction concentrate obtained in step 5 is separated and prepared by HPLC, and isocratic elution is performed using methanol:0.1% formic acid as the mobile phase to prepare the novel pyrimidine alkaloid compound Olerapyrimidine as claimed in claim 1.

3. The extraction and separation method according to claim 2, characterized in that: In the step 1, ethanol reflux extraction is performed twice, each time for 2 hours, and the amount used is 8 to 16 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 ethyl acetate 1:0, ethyl acetate:ethanol 5:1, ethyl acetate:ethanol 2:1, ethyl acetate:ethanol 1:1, ethyl acetate:ethanol 1:2, ethyl acetate:ethanol 1:5, and ethanol recovery gradient elution.

5. The extraction and separation method according to claim 2, characterized in that: The volume ratio of methanol to water used in step 4 is 50:50, 70:3, 90:10 and 100:0 for gradient elution; the particle size of ODS is 40-70 μm.

6. The extraction and separation method according to claim 2, characterized in that: The volume ratio of methanol to 0.1% formic acid water used in step 6 is 5:95, and the retention time of the compound is 5.623 min.

7. Use of a new pyrimidine alkaloid compound from Portulaca oleracea as claimed in claim 1 in the preparation of anti-inflammatory drugs.

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