Pyridine alkaloid in purslane as well as extraction and separation method and application thereof

Abstract

The invention belongs to the technical field of traditional Chinese medicine extraction and separation, and particularly relates to a pyridine alkaloid compound extracted and separated from purslane as well as an extraction and separation method and application of the pyridine alkaloid compound. The molecular formula of the compound is C7H9NO2, and the chemical name of the compound is 5-ethofyridin-2-ol. The invention further provides an extraction and separation method of the compound. The compound is prepared by sequentially adopting ethanol reflux extraction, ethyl acetate and ethanol reflux extraction in a volume ratio of 2: 1, ODS medium-pressure column chromatography, sephadex LH-20 chromatography and high performance liquid chromatography. The structure is determined by mass spectrum, carbon spectrum, hydrogen spectrum and two-dimensional nuclear magnetic spectrum analysis methods. The compound has anti-inflammatory and anti-oxidation effects, and the compound and salts or derivatives thereof can be used as raw materials for drug development and pharmacological activity research.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine extraction and separation, and relates to a pyridine alkaloid in purslane and an extraction and separation method and application thereof, and in particular to a compound 5-ethoxypyridin-2-ol extracted, separated and identified from purslane medicinal materials and an extraction and separation method and application thereof. Background Art

[0002] Portulaca oleracea L. has alternate or nearly opposite leaves and cylindrical stems, which are often light brown-red. It is an annual fleshy herb from the genus Portulaca in the family Portulacaceae. Portulaca oleracea was first recorded in the Annotated Compendium of Materia Medica and is a plant used for both medicine and food. Portulaca oleracea has strong resistance to harsh environments such as drought and high salt. As a traditional Chinese medicine, Portulaca oleracea has a history of thousands of years and is one of the traditional Chinese medicines in my country. Portulaca oleracea is not only tenacious, but also has excellent ecological restoration capabilities and strong adaptability. At present, it is widely distributed in temperate and tropical regions around the world. With the development of modern Chinese medicine, Portulaca oleracea is described in the 2020 edition of the "Chinese Pharmacopoeia" as sour in taste, cold in nature, and belongs to the liver and large intestine meridians. It has the effects of clearing heat and detoxifying, cooling blood and stopping bleeding, and stopping dysentery. It can be used to treat heat-toxic bloody dysentery, carbuncles, furuncles, eczema, erysipelas, snake and insect bites, blood in the stool, hemorrhoids, and metrorrhagia.

[0003] Modern pharmacological studies on purslane have shown that it has anti-inflammatory, antibacterial, antiviral, antihypertensive, antilipidemic, anti-oxidant, anti-tumor, and immune function regulating effects. The main chemical components of purslane include flavonoids, coumarins, terpenes, steroids, alkaloids, amino acids, various pigments and minerals. Among them, alkaloids are a major type of chemical component in purslane. The alkaloid components reported so far include norepinephrine, dopamine, a small amount of dopa, adenosine, uracil, adenine, allantoin, N-trans-feruloyltyramine; there are also cyclic dipeptide alkaloids, etc.

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

[0005] In view of the above problems, the present invention provides pyridine alkaloids extracted and separated from Portulaca oleracea. Studies have shown that the compounds of the present invention have anti-inflammatory and antioxidant effects. At the same time, a simple, rapid, environmentally friendly and high-purity extraction and separation method for the compounds of the present invention is provided.

[0006] To achieve the above-mentioned purpose of the present invention, the present invention provides a pyridine alkaloid, the molecular formula of which is C7H9NO2, named 5-ethoxypyridin-2-ol, and the chemical structural formula is:

[0007]

[0008] To achieve the above-mentioned object of the present invention, the present invention also provides a method for extracting and separating pyridine alkaloids from Portulaca oleracea, the specific steps of which are:

[0009] Step 1, take dried purslane, extract with ethanol reflux, 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, evaporating the Chinese medicine solution in step 1 to dryness, extracting with ethanol and ethyl acetate, and recovering under reduced pressure to obtain an extract, thereby obtaining an extract;

[0011] Step 3, the extract in step 2 is separated by chromatography on a pretreated ODS column (Octadecylsilyl, octadecylsilane bonded silica gel filler), and gradient eluted with methanol-water to obtain several elution parts, which are detected by thin layer chromatography and color developed, and each color developed elution part is respectively concentrated to dryness under reduced pressure to obtain a concentrate for standby use;

[0012] Step 4, separating the concentrate obtained in step 3 by chromatography on a pretreated Sephadex LH-20 column, eluting isocratically with methanol 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;

[0013] Step 5: The concentrate obtained in step 4 is subjected to HPLC (high performance liquid chromatography) separation and preparation, with methanol: 0.1% formic acid as the mobile phase, to prepare the compound of the present invention.

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

[0015] Furthermore, in step 2, the volume ratio of ethyl acetate to ethanol is 2:1, reflux extraction is performed twice, each time for 2 hours, and the amount of ethyl acetate and ethanol used is 10 times that of the medicinal material.

[0016] Furthermore, in step 3, the volume ratio of methanol to water in gradient elution is 40:60, 50:50, 70:30, and 100:0.

[0017] Furthermore, in step 5, the volume ratio of methanol to 0.1% formic acid is 30:70, and the retention time of the compound is 14.693 min.

[0018] Furthermore, the pretreatment process of the ODS and dextran gel is to soak them in methanol for 24 hours, load them onto the column, wash them with methanol until there is no turbidity when dripped into water, and then balance them with the initial mobile phase.

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

[0020] The separation and pharmacological activity research of 5-ethoxypyridin-2-ol in purslane of the present invention has not been reported in existing paper journals; the present invention provides pyridine alkaloids derived from purslane and an extraction and separation method for the compound of the present invention, which sequentially adopts ethanol reflux extraction, ethanol and ethyl acetate extraction with a volume ratio of 1:2, ODS medium-pressure column, Sephadex LH-20 and high-performance liquid chromatography for separation, purification and preparation, and successfully extracts and separates the compound. The method has only five operating steps, and the operation method is simple and fast. The extraction and separation process mainly adopts ethanol and ethyl acetate extraction and methanol elution, and the process method is environmentally friendly. The purity of the compound separated by the method is relatively high and is greater than 90%. In addition, studies have shown that the compound has anti-inflammatory and antioxidant effects. Therefore, the 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 antioxidant drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The compound 5-ethoxypyridin-2-ol of the present invention 1 H-NMR spectrum.

[0022] Figure 2 The compound 5-ethoxypyridin-2-ol of the present invention 13 C-NMR spectrum.

[0023] Figure 3 The DEPT spectrum of the compound 5-ethoxypyridin-2-ol of the present invention is shown in FIG.

[0024] Figure 4 It is the HSQC spectrum of the compound 5-ethoxypyridin-2-ol of the present invention.

[0025] Figure 5 It is the HMBC spectrum of the compound 5-ethoxypyridin-2-ol of the present invention.

[0026] Figure 6 The compound 5-ethoxypyridin-2-ol of the present invention 1 H- 1 H COSY spectrum.

[0027] Figure 7 It is the ROESY spectrum of the compound 5-ethoxypyridin-2-ol of the present invention.

[0028] Figure 8 This is a high-resolution mass spectrum of the compound 5-ethoxypyridin-2-ol of the present invention. DETAILED DESCRIPTION

[0029] The following examples will help to understand the present invention, but these examples are only for illustrating the present invention, and the present invention is not limited to these contents. The operating methods in the examples are all conventional operating methods in the art.

[0030] Example 1.

[0031] The present invention provides a pyridine alkaloid, the molecular formula of which is C7H9NO2, named as 5-ethoxypyridin-2-ol, and the chemical structural formula is:

[0032]

[0033] Table 1 shows the NMR data of 5-ethoxypyridin-2-ol. The solvent used in NMR is deuterated methanol.

[0034] Table 1 NMR data of the compound 5-ethoxypyridin-2-ol of the present invention

[0035]

[0036]

[0037] 5-ethoxypyridin-2-ol: light yellow powder, easily soluble in methanol, insoluble or slightly soluble in water. After spotting on a silica gel thin layer plate, the spot sprayed with ferric chloride solution turned cyan, indicating that the compound contains phenolic hydroxyl groups. UHPLC-ESI-QTOF-MS gave m / z: 138.0559 [MH] - The quasi-molecular ion peak of 138.0559. 1 H-NMR, 13 Based on C-NMR and DEPT data, it was inferred that the possible molecular formula of the compound was C7H9NO2 with a degree of unsaturation of 4. 13 C-NMR and DEPT spectra showed 7 carbon signals, including 1 methyl carbon (δ14.59), 1 methylene carbon (δ62.47), 3 methine carbons (δ139.08; 123.76; 128.02), and 2 quaternary carbons (δ165.99; 158.92). 1 H-NMR spectrum shows one CH3 signal as δ H 1.34 (3H, t, J = 7.14); 1 methylene signal is δ H4.34 (2H, q, J = 6.78, 14.22); 3 methine signals are δ H 8.12 (1H, d, J = 2.7), δ H 7.98 (1H, d, J = 8.58) and δ H 7.25 (1H, dd, J = 2.82, 8.64). HMBC spectrum showed that H-3 and C-2 (δ C 158.92), C-5(δ C 165.99); H-4 is related to C-2, C-6 (δ C 139.08); H-6 is related to C-2, C-4 (δ C 128.02) related, 1 H- 1 The H COSY spectrum shows that H-3 and H-4 are related, and it is speculated that the core structure of this structure is a pyridine ring, and there is a para-substituted structure. Since the chemical shift of C-2 is in the low field, it can be judged that C-2 is connected to a hydroxyl group. The HMBC shows that H-2′(δ H 4.34) and C-3′(δ C 14.59), C-5(δ C 165.99), H-3′(δ H 1.34) and C-2′(δ C 62.47). And the chemical shift of C-6 is in the low field, which suggests that C-6 is connected to C-2′ through the oxygen atom. In addition, 1 H- 1 The H COSY spectrum showed that H-2′ and H-3′ were related, which further confirmed the branched structure. Therefore, based on the above information, it can be determined that this compound is 5-ethoxypyridin-2-ol.

[0038] The present invention also provides a method for extracting and separating the above-mentioned compound, the specific steps of which are:

[0039] Step 1: Weigh 150 kg of dried purslane medicinal material, use 50% ethanol reflux extraction, the amount of 50% ethanol (v / v) is 10 times that of the medicinal material, reflux extraction twice, each time for 2 hours, filter the alcohol extract, combine the filtrate, concentrate under reduced pressure to 200 L, cool to room temperature, and obtain the medicinal solution for use.

[0040] Step 2: evaporate the medicinal solution obtained in step 1 to dryness, extract it with ethyl acetate and ethanol in a volume ratio of 2:1, and recover the extract to an extractum under reduced pressure at a temperature below 40° C. to obtain an extract.

[0041] Step 3: The extract in step 2 is separated by pretreated ODS medium-pressure column chromatography, wherein the filler particle size is 20-40 μm, and gradient elution is performed with methanol-water (40 / 60, 50 / 50, 70 / 30, 100 / 0, v / v) (pressurized to a flow rate of 1 mL / min and room temperature) to obtain 14 fractions (i.e., 32 bottles are obtained by gradient elution, each bottle having 80 mL), which are detected by thin layer chromatography and color developed. The colored fractions 12 to 18 are retained and concentrated to dryness under reduced pressure below 50° C. for later use.

[0042] Step 4: The product obtained in step 3 is further separated by pretreated dextran gel column chromatography (Sephadex LH-20), eluted with methanol, and 20 elution fractions are obtained (i.e., a total of 20 bottles, each bottle 40 mL), detected by thin layer chromatography, and color developed. The colored 10 to 15 fractions are retained, and concentrated to dryness under reduced pressure below 50°C for use.

[0043] Step 5: The product obtained in step 4 was separated and prepared by HPLC, using methanol:0.1% formic acid (30:70, v / v) as the mobile phase and detection wavelengths of 210nm and 254nm to separate and prepare the compound of the present invention. The purity determined by normalization method was 90-99%.

[0044] 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.

[0045] Example 2 Anti-inflammatory effect of 5-ethoxypyridin-2-ol.

[0046] 1 Main Materials

[0047] 1.1 Drugs and reagents: The compounds used in the experiment were prepared by the above method with a purity of 90%. 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-6, TNF-α, and PGE2 (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.

[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% CO2 incubator.

[0052] 2.2 MTT colorimetric assay for cell viability: RAW264.7 macrophages in the logarithmic growth phase were inoculated in 96-well culture plates at a cell density of 1×10 4 / mL, 100μL per well, temperature 37℃, 5% CO2, after overnight culture, the experimental group added different concentrations of the compound 5-ethoxypyridin-2-ol (10-100μM) of the present invention, incubated for 1h, LPS with a final concentration of 1μg / mL was added to the LPS group and the experimental group, and a zero adjustment group (culture solution containing DMSO solvent) was set up, and 3 duplicate wells were set up in each group to investigate the effect of the addition of drugs on the cells. After the above-mentioned groups of cells were cultured for 24h, 5mg / mL MTT20μL was added to the cells in each well, and the temperature was 37℃, 5% CO2 was continued to be incubated for 4h, and the culture was terminated, the liquid in the well was aspirated, and 100μL dimethyl sulfoxide (DMSO) was added to each well, and oscillated for 10min to fully dissolve the crystals in the cells, and the absorbance value of each well was measured at a wavelength of 570nm by an enzyme marker.

[0053] 2.3 The NO content was determined by Griess method to investigate the inhibitory effect of the compounds of the present invention on the NO production of mouse macrophages RAW264.7 induced by LPS. Mouse macrophages RAW264.7 were cultured in a high-glucose cell culture medium DMEM containing 10% fetal bovine serum after passage. The experimental group was added with different concentrations of the compound of the present invention 5-ethoxypyridin-2-ol (5-50 μM), incubated at 37°C, 5% CO2 for 1 hour, and then induced an inflammatory response with LPS (final concentration of 1 μg / mL). The supernatant was collected after 24 hours, and each group was treated with 3 repeated wells. The NO content in the cell supernatant was determined by Griess method, and the NO level was reflected according to the effect of different concentrations of the compounds of the present invention on the NO release of RAW264.7 cells induced by LPS.

[0054] 2.4ELISA method for determination of inflammatory factors IL-6, TNF-α and inflammatory mediator PGE2: RAW264.7 macrophages in 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 compound 5-ethoxypyridin-2-ol (5-50μM) 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-6, TNF-α and PGE2 secreted by RAW264.7 macrophages after being treated with the purslane-derived compound.

[0055] 3 Experimental results

[0056] The experimental results show that the compound of the present invention has no effect on the proliferation of LPS-induced macrophages RAW264.7, is safe and non-toxic; and can effectively inhibit the excessive inflammatory cytokines IL-6, TNF-α and inflammatory mediators NO and PGE2 produced by LPS-induced macrophages RAW264.7 in a concentration-dependent manner.

[0057] The results of the relative cell survival rate experiment are shown in Table 2.

[0058] Table 2 Effect of the present invention on the relative survival rate of RAW264.7 macrophages

[0059]

[0060] Note: * P<0.05 compared with the control group (the high concentration group had a significant difference)

[0061] The experimental results of determining the NO content using the Griess method are shown in Table 3.

[0062] Table 3 Effect of the present invention on NO release from RAW264.7 cells induced by LPS (mean ± standard deviation, n = 3)

[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-6, TNF-α and inflammatory mediator PGE2 are shown in Table 4.

[0066] Table 4 Effects of the present invention on the content of IL-6, TNF-α and PGE2 secreted by RAW264.7 cells induced by LPS (mean ± standard deviation, n = 3)

[0067]

[0068] Note: *P<0.05 compared with the control group. # P<0.05 compared with LPS group.

[0069] Example 3 Antioxidant effect of 5-ethoxypyridin-2-ol.

[0070] 1 Main Materials

[0071] 1.1 Drugs and reagents: The compounds used in the experiment were prepared by the above method with a purity of 90%. They were accurately weighed and diluted with methanol to the required solution for each dose group as follows: DPPH (1,1-diphenyl-2-picrylhydrazyl free radical) (Sigma-Fluka); BHA (tert-butyl hydroxyanisole) (Shanghai Xiangrui Technology Co., Ltd.); methanol, chromatographic grade (Changtai Xingye Co., Ltd.).

[0072] 1.2 Grouping: Divided into control group, experimental group and blank group.

[0073] 2 Experimental methods

[0074] The ability to eliminate DPPH free radicals was determined by colorimetry: the experimental group took 1 mL of DPPH solution (80 μM) and added it to a cuvette, and then added 1 mL of 5-ethoxypyridin-2-ol (6.25-100 μM) of different concentrations; the control group took 1 mL of methanol solution and added it to a cuvette, and then added 1 mL of sample solution of different concentrations; the blank group took 1 mL of DPPH solution and added it to a cuvette, and then added 1 mL of methanol solution. All three groups were thoroughly mixed, allowed to stand at room temperature and away from light for 10 minutes, and the absorbance was measured at 517 nm. Each sample was measured three times on average and the average value was taken. The positive control was a BHA solution of different concentrations (6.25-100 μM). The scavenging rate of the sample for DPPH free radicals was calculated according to the following formula, and its free radical scavenging rate IC was further calculated. 50 value.

[0075] DPPH removal rate (%) = 1-(A1-A2) / A0×100%

[0076] Among them, A0 is the absorbance value of the blank group; A1 is the absorbance value of the sample group; A2 is the absorbance value of the control group.

[0077] 3 Experimental results

[0078] The experimental results show that the compounds of the present invention have a scavenging effect on DPPH free radicals, and as the drug concentration increases, the scavenging rate also increases significantly.

[0079] The compounds of the present invention have an IC value of DPPH free radical. 50 See Table 5 for values.

[0080] Table 5 The scavenging effect of the compounds of the present invention on DPPH free radicals

[0081] Group <![CDATA[IC 50 (μM)]]> BHA 58.68 5-ethoxypyridin-2-ol 52.45

[0082] In summary, the present invention provides a compound 5-ethoxypyridin-2-ol and an extraction and separation method and application thereof, wherein ethanol reflux extraction, ethyl acetate and ethanol volume ratio 2:1 extraction, ODS medium pressure column chromatography, dextran gel column chromatography and high performance liquid chromatography are sequentially used for separation, purification and preparation, and the compound is successfully separated and obtained. The 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 purslane and has anti-inflammatory and antioxidant effects, the compound and its salt and derivatives of the present invention can provide an experimental basis for drug development and have broad prospects.

Claims

1. A new pyridine alkaloid isolated from Portulaca oleracea, characterized in that: The molecular formula is: C7H9NO2, the chemical name is 5-ethoxypyridin-2-ol, and the chemical structure is: 。 2. A method for extracting and separating pyridine alkaloids separated from the purslane medicinal material according to claim 1, characterized in that: The specific steps include: Step 1: Take dried purslane medicinal material, extract it with ethanol reflux, filter the ethanol extract, combine the filtrate and concentrate it under reduced pressure, cool it to room temperature, and obtain the medicinal solution for use; Step 2: After the Chinese medicine solution in step 1 is evaporated to dryness, it is extracted with ethyl acetate and ethanol, and the extract is recovered under reduced pressure to obtain an extract; Step 3: evaporate the drug solution in step 2 to dryness and apply it to an ODS column, and use a gradient elution of methanol and water in a volume ratio of 40:60, 50:50, 70:30, and 100:0, detect by thin layer chromatography, develop color, and concentrate each color-developed elution portion to dryness under reduced pressure to obtain a concentrate for use; Step 4: The extract obtained in step 3 is separated by pre-treated dextran gel column chromatography, isocratically eluted with methanol, detected by thin layer chromatography, color developed, and the colored elution fractions are combined, and the combined elution fractions are concentrated to dryness under reduced pressure; Step 5: The concentrate obtained in step 4 is subjected to HPLC separation and preparation, using methanol:0.1% formic acid in a volume ratio of 30:70 as the mobile phase to prepare the compound of the present invention.

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

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

5. Use of the pyridine alkaloids separated from the Portulaca oleracea medicinal material according to claim 1 in the preparation of anti-inflammatory drugs and antioxidant drugs.

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