A new pyran compound from purslane and its extraction and separation method and use

Abstract

The present invention belongs to the field of extraction and separation of traditional Chinese medicine, and relates to a new pyran compound in purslane and its extraction and separation method and use. The new compound has a molecular formula of C 20 H 36 O8, based on its structural chemical name, is 2‑(1‑(3‑hydroxy‑2,2,6‑trimethyl‑6‑vinyltetrahydro‑2 H ‑pyran‑3‑yl)‑2‑methylpropoxy)‑6‑(hydroxymethyl)tetrahydro‑2 H ‑pyran‑3,4,5‑triol. A method for extracting and separating the above new compound is also provided, which sequentially uses water decoction extraction, macroporous resin column chromatography, ODS column chromatography, dextran gel column chromatography and high performance liquid chromatography for separation, purification and preparation. Its structure is determined to be a new compound by mass spectrometry, hydrogen spectrum, carbon spectrum and two-dimensional nuclear magnetic spectrum analysis. The new compound has anti-inflammatory activity, anticholinesterase activity and antioxidant activity. The new compound of the present invention and its salt or derivative can be used as a raw material for drug development and pharmacological activity research.

Description

Technical Field

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

[0002] Purslane comes from the dried aerial parts of Portulaca oleracea L., a plant of the Portulacaceae family. It is one of the wild plants that are both medicinal and edible as designated by the Ministry of Health of my country. It is also known as long-life vegetable and five-element grass. Purslane was first recorded in the "Compendium of Materia Medica" in the Northern and Southern Dynasties. As a traditional Chinese medicine, it 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 efficacy and indications of Purslane recorded in the herbal books of various dynasties all emphasize its function of "dispersing blood and reducing swelling". It is commonly used to treat "carbuncle" diseases. Many ancient books also record that Purslane has the function of treating dysentery and bloody dysentery. The 2020 edition of the "Pharmacopoeia of the People's Republic of China" lists the dried aerial parts of Portulaca oleracea as a medicine. It tastes sour and cold, and enters the liver and large intestine meridians. It has the effects of clearing heat and detoxifying, cooling blood and stopping bleeding, and stopping dysentery. It is used for heat-toxic bloody dysentery, carbuncles, furuncles, eczema, erysipelas, snake and insect bites, bloody stools, hemorrhoidal bleeding, and metrorrhagia.

[0003] Purslane contains a variety of chemical components, mainly including: alkaloids, flavonoids, organic acids, terpenes, coumarins, polysaccharides, amino acids and minerals. The chemical components contained in Purslane are closely related to its diverse pharmacological effects. Modern pharmacological studies have shown that Purslane has antioxidant, neuroprotective, anti-inflammatory, antibacterial, anti-tumor, hypoglycemic, liver-protective and immune-enhancing effects. Purslane has a wide variety of chemical components and diverse pharmacological activities. 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 pyran compound in purslane and its extraction and separation method and use. Studies have found that the new pyran compound of the present invention has anti-inflammatory, anticholinesterase and antioxidant activities, and at the same time provides a simple, rapid, environmentally friendly and high-purity extraction and separation method for the compound of the present invention.

[0005] In order to achieve the above-mentioned object of the present invention, the present invention provides a new pyran compound, the molecular formula of which is C 20 H 36 O 8, named according to the structure 2-(1-(3-hydroxy-2,2,6-trimethyl-6-vinyltetrahydro-2H-pyran-3-yl)-2-methylpropoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol, the chemical formula is:

[0006]

[0007] 2-(1-(3-hydroxy-2,2,6-trimethyl-6-vinyltetrahydro-2H-pyran-3-yl)-2-methylpropoxy)-6-(hydroxymethyl)tetrahydr

[0008] o-2H-pyran-3,4,5-triol

[0009] To achieve the above-mentioned object of the present invention, the present invention also provides a method for extracting and separating pyran compounds from Portulaca oleracea, which specifically comprises the following steps:

[0010] Step 1: Take dried purslane, decoct in water to extract, concentrate the extract, cool to room temperature, and set aside the liquid medicine.

[0011] 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.

[0012] 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 50% methanol elution portion and evaporate to dryness to obtain a concentrate for use.

[0013] 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.

[0014] Step 5: The concentrate obtained in step 4 is further separated by chromatography on a pretreated Sephadex LH-20 column, eluted with 30% methanol to obtain several elution fractions, 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 for use.

[0015] Step 6: The concentrate obtained in step 5 is separated and prepared by HPLC (high performance liquid chromatography), and isocratic elution is performed using methanol-0.1% formic acid (volume percentage) as the mobile phase to finally obtain the new pyran compound 2-(1-(3-hydroxy-2,2,6-trimethyl-6-vinyltetrahydro-2H-pyran-3-yl)-2-methylpropoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol of the present invention.

[0016] Furthermore, in step 1, the extraction is performed by decoction in water twice, each time for 2 hours, and the amount of water used is 8 to 16 times the amount of the medicinal material.

[0017] 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.

[0018] Furthermore, in step 3, the volume ratio of methanol to water is 10:0, 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 4, the volume ratio of methanol to water is 10:0, 30:70, 50:50, 70:30 and 100:0 for gradient elution; and the particle size of ODS is 40 to 70 μm.

[0020] Furthermore, in step 5, the methanol elution procedure is 30% methanol isocratic elution.

[0021] Furthermore, the pretreatment process of ODS and Sephadex LH-20 in step 3, step 4 and step 5 is to soak in methanol for 24 hours; after loading, wash with methanol until there is no turbidity when dripped into water, and then balance with the initial mobile phase.

[0022] Furthermore, in step 6, the volume ratio of methanol to 0.1% formic acid is 30:70, and the retention time of the compound is 14.204 min.

[0023] The present invention also provides a use of 2-(1-(3-hydroxy-2,2,6-trimethyl-6-vinyltetrahydro-2H-pyran-3-yl)-2-methylpropoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol separated from the purslane medicinal material in the preparation of anti-inflammatory drugs, anticholinesterase drugs and antioxidant drugs.

[0024] Compared with the prior art, the invention has the beneficial effects.

[0025] The separation and pharmacological activity research of the new pyran compound 2-(1-(3-hydroxy-2,2,6-trimethyl-6-vinyltetrahydro-2H-pyran-3-yl)-2-methylpropoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol in purslane described in the present invention has not been reported in plants; the present invention provides a new pyran 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 new pyran compound was successfully extracted and separated. The method has only six operating steps, the operation method is simple and fast, water is used for extraction, and the process method is environmentally friendly. In addition, studies have shown that the above compounds have anti-inflammatory activity, anti-cholinesterase activity and antioxidant activity. Therefore, the new compound 2-(1-(3-hydroxy-2,2,6-trimethyl-6-vinyltetrahydro-2H-pyran-3-yl)-2-methylpropoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol and its salts and derivatives can be used as raw materials for drug development and pharmacological activity research. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The novel compound 2-(1-(3-hydroxy-2,2,6-trimethyl-6-vinyltetrahydro-2H-pyran-3-yl)-2-methylpropoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol of the present invention is 1 H-NMR spectrum.

[0027] Figure 2 The novel compound 2-(1-(3-hydroxy-2,2,6-trimethyl-6-vinyltetrahydro-2H-pyran-3-yl)-2-methylpropoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol of the present invention is 13 C-NMR spectrum.

[0028] Figure 3 The DEPT spectrum of the novel compound 2-(1-(3-hydroxy-2,2,6-trimethyl-6-vinyltetrahydro-2H-pyran-3-yl)-2-methylpropoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol of the present invention is shown in FIG.

[0029] Figure 4 The HSQC spectrum of the novel compound 2-(1-(3-hydroxy-2,2,6-trimethyl-6-vinyltetrahydro-2H-pyran-3-yl)-2-methylpropoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol of the present invention is shown.

[0030] Figure 5 The figure is the HMBC spectrum of the novel compound 2-(1-(3-hydroxy-2,2,6-trimethyl-6-vinyltetrahydro-2H-pyran-3-yl)-2-methylpropoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol of the present invention.

[0031] Figure 6 The figure is the COESY spectrum of the novel compound 2-(1-(3-hydroxy-2,2,6-trimethyl-6-vinyltetrahydro-2H-pyran-3-yl)-2-methylpropoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol of the present invention.

[0032] Figure 7 The figure is the ROESY spectrum of the novel compound 2-(1-(3-hydroxy-2,2,6-trimethyl-6-vinyltetrahydro-2H-pyran-3-yl)-2-methylpropoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol of the present invention.

[0033] Figure 8The high-resolution mass spectrum of the novel compound 2-(1-(3-hydroxy-2,2,6-trimethyl-6-vinyltetrahydro-2H-pyran-3-yl)-2-methylpropoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol of the present invention. DETAILED DESCRIPTION

[0034] 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.

[0035] Example 1.

[0036] 2-(1-(3-hydroxy-2,2,6-trimethyl-6-vinyltetrahydro-2H-pyran-3-yl)-2-methylprop oxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol

[0037] The present invention provides a new pyran compound, the molecular formula of which is C 20 H 36 O 8 According to the structural formula, it is named 2-(1-(3-hydroxy-2,2,6-trimethyl-6-vinyltetrahydro-2H-pyran-3-yl)-2-methylpropoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol, and its chemical structure is:

[0038]

[0039] 2-(1-(3-hydroxy-2,2,6-trimethyl-6-vinyltetrahydro-2H-pyran-3-yl)-2-methylpropoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol

[0040] Table 1 shows the NMR data of 2-(1-(3-hydroxy-2,2,6-trimethyl-6-vinyltetrahydro-2H-pyran-3-yl)-2-methylpropoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol. The solvent used for NMR is CD 3 OD.

[0041] Table 1 NMR data of 2-(1-(3-hydroxy-2,2,6-trimethyl-6-vinyltetrahydro-2H-pyran-3-yl)-2-methylpropoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol of the present invention.

[0042]

[0043]

[0044] 2-(1-(3-hydroxy-2,2,6-trimethyl-6-vinyltetrahydro-2H-pyran-3-yl)-2-methylprop oxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol: yellow oil, easily soluble in methanol. 1 H-NMR, 13 C-NMR and HR-ESI-TOF-MS signals indicate that the possible molecular formula of the compound is C 20 H 36 O 8 , the degree of unsaturation is 3. HR-ESI-TOF-MS gives m / z [MC 6 H 11 O 5 ] - The quasi-molecular ion peak is 241.1805, and the calculated value (C 14 H 25 O 3 - is 241.1804). 1 In the H NMR spectrum, δ Η 1.21(3H,s,H-7),δ Η 1.12(3H,s,H-11),δ Η 1.08(3H,s,H-10),δ Η0.90 (3H, d, J = 2.76 Hz, H-4') and δ Η 0.89 (3H, d, J = 2.76 Hz, H-3′) indicates that there are five methyl groups in the structure, δ Η 5.88(1H,m,H-8),δ Η 5.14 / 4.97 (2H, d, J = 1.56 Hz, H-9) suggests that there may be an olefin structure in the structure, δ Η 1.81 / 1.45(2H,m,H-4),δ Η 1.64 / 1.31 (2H, m, H-5) suggests that there are two methylene groups in the structure, δ Η 3.56(1H,d,J=5.82Hz,H-1′), δ Η 1.54 (1H, m, H-2′) indicates that there are two methine groups in the structure. 1 HNMR spectrum shows δ Η 4.28(1H,d,J=7.8Hz,H-1″), δ Η 3.29(1H,m,H-2″),δ Η 3.11(1H,m,H-3″),δ Η 3.23 (1H, m, H-4″), δ Η 3.57 (1H, m, H-5″) and δ Η 3.62(2H,m,H-6″) six signals, 13 C NMR spectrum showed that C-1″(δ C 103.39), C-2″(δ C 78.07), C-3″(δ C 77.77), C-4″(δ C 71.75), C-5″(δ C 82.82), C-6″(δ C 62.85) six signals, suggesting the existence of β-D-glucose structure in the structure. 1 H NMR (600 MHz, MeOD-d 4 )and 13 C NMR (150 MHz, MeOD-d 4 ) are listed in Table 1. HMBC shows that H-4 and C-2 (δ C 80.20), C-6(δ C 73.84), H-5 and C-3 (δ C 73.87) is relevant, and 1 H- 1HCOSY shows that H-4 is related to H-5, suggesting that there may be a heterocyclic structure in the structure. Since the chemical shift values ​​of C-2 and C-6 increase, it is speculated that C-2 and C-6 are connected to O, that is, there is a pyran ring structure in the structure. The chemical shift value of C-3 increases, and it is speculated that there is a hydroxyl substitution at the C-3 position. The HMBC spectrum shows that H-10 and H-11 are related to C-3, and it is speculated that there are two methyl substitutions at the C-2 position. H-7 and C-5 (δ C 26.66), it is speculated that there is a methyl substitution at the C-6 position, and H-7 and C-8 (δ C 146.59), H-9 is related to C-6, and it is speculated that there is an olefinic substitution at the C-6 position. In HMBC, H-3′, H-4′ and C-1′ (δ C 82.80), H-2′ is related to C-3, and 1 H- 1 HCOSY showed that H-1′ and H-2′ were related, indicating that the isobutyl group was connected to C-3. HMBC showed that H-1′ and C-1″ were related, indicating that β-D-glucose was connected to C-1′. Based on the above information, it can be determined that this new compound has the above structure.

[0045] The present invention also provides a method for extracting and separating 2-(1-(3-hydroxy-2,2,6-trimethyl-6-vinyltetrahydro-2H-pyran-3-yl)-2-methylpropoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol, 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 under reduced pressure to extract, 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, 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 50% 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 the gradient elution is carried out with methanol-water (10:0, 30:70, 50:50, 70:30 and 100:0, v:v), and the detection is carried out 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 30% 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, using methanol and 0.1% formic acid in a volume ratio of 30:70 as the mobile phase and detection wavelengths of 210 and 254 nm to separate and prepare the new compound of the present invention.

[0052] Example 2.

[0053] Anti-inflammatory effects of 2-(1-(3-hydroxy-2,2,6-trimethyl-6-vinyltetrahydro-2H-pyran-3-yl)-2-methylprop oxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol.

[0054] 1 Main Materials

[0055] 1.1 Drugs and reagents: The new compound used in the experiment was prepared by the above method, and after purification, the purity was 95%. DMEM high glucose medium, fetal bovine serum (Hyclone, USA); penicillin, streptomycin (Hangzhou Sijiqing Company); LPS (Sigma, USA); IL-1β ELISA kit (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 method to determine 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 pcs / mL, 100 μL per well, 37°C, 5% CO 2 After culturing overnight under the conditions, different concentrations of the compound of the present invention (5μM-50μM) were added to the experimental group. After incubation for 1 hour, 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. Each group had 3 replicate wells to investigate the effect of the addition of drugs on the cells. After the cells in the above groups were cultured for 24 hours, 10μL of CCK-8 was added to the cells in each well at 37°C and 5% CO 2 After incubation for 4 h under the same conditions, the absorbance of each well was measured at a wavelength of 450 nm using an ELISA reader.

[0061] 2.3ELISA method for determination of inflammatory factor IL-1β: RAW264.7 macrophages in the logarithmic growth phase were inoculated in 24-well culture plates at a cell density of 1×10 5 pcs / mL, 1 mL per well, temperature 37°C, 5% CO 2 The cells were cultured overnight under the same conditions. The experimental group was added with the compound of the present invention (1 μM to 20 μM) for 1 hour, and then LPS (final concentration of 1 μg / mL) was added to each well for 24 hours. Each group was treated with 3 wells. The IL-1β content was determined by ELISA.

[0062] 3 Experimental results.

[0063] The experimental results show that the compound of the present invention has no effect on the proliferation of LPS-induced macrophages RAW264.7; it can effectively inhibit the secretion of excessive inflammatory cytokine IL-1β produced by LPS-induced macrophages RAW264.7 in a concentration-dependent manner.

[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] The results of ELISA determination of inflammatory factor IL-1β are shown in Table 4.

[0068] Table 3 Effects of the compounds of the present invention on the IL-1β content secreted by RAW264.7 cells induced by LPS (mean±standard deviation, n=3).

[0069]

[0070] Note: * P<0.05 compared with the control group. # P<0.05 compared with LPS group.

[0071] Example 3.

[0072] Anticholinesterase effects of 2-(1-(3-hydroxy-2,2,6-trimethyl-6-vinyltetrahydro-2H-pyran-3-yl)-2-methylprop oxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol.

[0073] 1 Main Materials

[0074] 1.1 Drugs and reagents: The new compounds used in the experiment were prepared by the above method and purified to a purity of 95%. 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.).

[0075] 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.).

[0076] 2 Experimental methods.

[0077] This experiment determines the anticholinesterase activity of each compound according to the modified Ellman method. The compound of the present invention and physostigmine were accurately weighed and prepared into five series of sample solutions with concentrations of 31.25μM, 62.5μM, 125μM, 250μM and 500μM with methanol. The specific operation is as follows: 140μL of phosphate buffered saline (0.1M, pH=8.0, containing 0.1mol / L disodium hydrogen phosphate and sodium dihydrogen phosphate), 20μL of sample solution, 15μL of AChE (0.2U / mL) were added to a 96-well ELISA plate, and after incubation at 37°C for 10 minutes, 10μL of ATCI (4mmol / L) and 10μL of DTNB (15mmol / L) were added, and after incubation at 37°C for 20 minutes, the 96-well plate was placed in an ELISA instrument, and the absorbance value of each group of samples was measured 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):

[0078] Inhibition rate (%) = (A 空白 -A 样品 ) / A 空白 ×100%

[0079] 3 Experimental results.

[0080] The experimental results show that the new 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.

[0081] The anticholinesterase activities of the novel compounds of the present invention are shown in Table 4.

[0082] Table 4 Anticholinesterase activities of the compounds of the present invention.

[0083]

[0084]

[0085] Example 4.

[0086] Antioxidant effect of 2-(1-(3-hydroxy-2,2,6-trimethyl-6-vinyltetrahydro-2H-pyran-3-yl)-2-methylprop oxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol.

[0087] 1Main materials.

[0088] 1.1 Drugs and reagents: The new compounds used in the experiment were prepared by the above method, and after purification, the purity was 95%. 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] This experiment uses the DPPH free radical scavenging method to determine the antioxidant activity of each compound. Accurately weigh the compound of the present invention and BHA, and use methanol to prepare five series of sample solutions with concentrations of 12.5μM, 25μM, 50μM, 100μM and 200μM. In addition, the DPPH solution is prepared as needed, and an appropriate amount of DPPH is accurately weighed and used to prepare a solution with a concentration of 80μM with methanol. The whole process is protected from light. The specific operation is as follows: 1mL of the sample solution is fully mixed with 1mL of the DPPH solution, and placed at room temperature and in a light-proof environment for 10min. After setting the detection wavelength of the ultraviolet spectrophotometer to 517nm, the absorbance value of the sample is measured. Among them, methanol is used instead of the sample solution as the blank group, BHA is used instead of the sample solution as the positive control group, and a mixed solution of 1mL of methanol and 1mL of the sample solution is used as the control group. The DPPH scavenging rate of each compound is 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 new compound of the present invention exhibits certain antioxidant activity, and the antioxidant effect increases with the increase of the compound concentration, showing a dose-dependent trend.

[0095] The antioxidant activities of the novel 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 2-(1-(3-hydroxy-2,2,6-trimethyl-6-vinyltetrahy dro-2H-pyran-3-yl)-2-methylpropoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol 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 new pyran 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 and has anti-inflammatory activity, anticholinesterase activity and antioxidant activity, the new compound 2-(1-(3-hydroxy-2,2,6-trimethyl-6-vinyltetrahydro-2H-pyran-3-yl)-2-methylpropoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol and its salts and derivatives can provide ideas for drug development and have broad development prospects.

Claims

1. A new pyran compound isolated from Portulaca oleracea, characterized in that: The molecular formula is: C 20 H 36 O8, named according to the structure, is 2-(1-(3-hydroxy-2,2,6-trimethyl-6-vinyltetrahydro-2H-pyran-3-yl)-2-methylpropoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol, and its chemical formula is: 。 2. A method for extracting and separating new pyran compounds isolated 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 50% 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 concentrate obtained in step 4 is further separated by pre-treated dextran gel column chromatography, eluted with 30% methanol to obtain several elution fractions, 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 for standby use; Step 6: The concentrate obtained in step 5 was separated and prepared by HPLC, and methanol-0.1% formic acid was used as the mobile phase for isocratic elution to finally obtain a new pyran compound 2-(1-(3-hydroxy-2,2,6-trimethyl-6-vinyltetrahydro-2H-pyran-3-yl)-2-methylpropoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol.

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: The macroporous resin used in step 2 is AB-8 macroporous resin, and the volume ratio of ethanol to water used in gradient elution is 30:70, 50:50, 70:30 and 100:

0.

5. The extraction and separation method according to claim 2, characterized in that: The volume ratio of methanol to water used in step 3 is 10:0, 30:70, 50:50, 70:30 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 water used in step 4 is 10:0, 30:70, 50:50, 70:30 and 100:0 for gradient elution; the particle size of ODS is 40-70 μm.

7. The extraction and separation method according to claim 2, characterized in that: The volume ratio of methanol to 0.1% formic acid used in step 6 is 30:70, and the retention time of the compound is 14.204 min.

8. Use of the new pyran 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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