Heterocyclic alkaloid compound in purslane as well as extraction and separation method and application thereof

Abstract

The invention belongs to the field of traditional Chinese medicine extraction and separation, and particularly relates to a heterocyclic alkaloid compound in purslane as well as an extraction and separation method and application thereof. The molecular formula of the compound is C9H12N4O5, and the chemical name of the compound is 5-(4-hydroxy-3-((hydroxymethyl) amine) isoxazolidin-5-yl)-5H-pyrrolo [3, 4-d] isoxazolidin-4-ol. The structural formula of the compound is shown in the specification, and the structural formula of the compound is shown in the specification. The invention also provides an extraction and separation method of the compound. Ethanol extraction, ODS column chromatography separation, sephadex gel chromatographic column separation and ultra-high performance liquid chromatography are adopted for separation, purification and preparation. The structure of the compound is determined as a new compound by mass spectrum, hydrogen spectrum, carbon spectrum and two-dimensional nuclear magnetic spectrum analysis methods, and the compound has anti-inflammatory, anti-cholinesterase and antioxidant activity. The heterocyclic alkaloid compound and the salt or derivative thereof can be used as raw materials for drug development and pharmacological activity research.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of traditional Chinese medicine extraction and separation, and relates to a heterocyclic alkaloid compound in Portulaca oleracea L., an extraction and separation method thereof and application, in particular to a compound 5-(4-hydroxy-3-((hydroxymethyl)amino)isoxazolidin-5-yl)-5H-pyrrolo[3,4-d]isoxazol-4-ol extracted, separated and identified from Portulaca oleracea L. and an extraction and separation method thereof. BACKGROUND

[0002] Portulaca oleracea L. is an annual succulent herbaceous plant of Portulacaceae and Portulaca L., and is one of wild plants of Chinese medicine and food, also known as Longniancai, Wuxingcao, Mazhaocai, etc. Portulaca oleracea L. has been used as traditional Chinese medicine for thousands of years, is widely distributed, and is rich in resources, and is commonly found in farmland, vegetable garden, vegetable garden, roadside, etc. below an altitude of 1300 meters. Portulaca oleracea L. likes warm and humid environment, is drought and salt tolerant, and is not low temperature resistant, has phototropism, and has very strong adaptability and tenacious vitality. The dry aboveground part of Portulaca oleracea L. is recorded in the 2020 edition of the People's Republic of China Pharmacopoeia for medicine, tastes sour and cold, belongs to the liver and large intestine channels, has the effects of clearing heat and resolving toxicity, cooling blood and stopping bleeding, and stopping dysentery, and is used for treating heat and toxic dysentery, carbuncle, eczema, erysipelas, snake and insect bites, hematochezia, hemorrhoids, and metrorrhagia.

[0003] Portulaca oleracea L. contains various chemical components, mainly including alkaloids, flavonoids, organic acids, terpenes, coumarins, polysaccharides, amino acids and mineral substances, etc. Among them, alkaloids are the main components in Portulaca oleracea L. The chemical components contained in Portulaca oleracea L. are closely related to its diverse pharmacological effects. Modern pharmacological studies have shown that Portulaca oleracea L. has anti-inflammatory, antioxidant, neuroprotective, antibacterial, antitumor, hypoglycemic, hepatoprotective and immunopotentiating effects. In view of the rich chemical components in Portulaca oleracea L. and its diverse pharmacological activities, it is of great significance to develop and separate the compounds in Portulaca oleracea L. SUMMARY

[0004] In view of the above problems, the present application provides a heterocyclic alkaloid compound in Portulaca oleracea L. and an extraction and separation method and application thereof. It is found through research that the heterocyclic alkaloid compound of the present application has anti-inflammatory activity, anticholinesterase activity and antioxidant activity, and a simple, rapid, environmentally friendly and high-purity extraction and separation method for the compound of the present application is provided.

[0005] To achieve the above-mentioned purpose of the present application, the present application provides a heterocyclic alkaloid compound, the molecular formula of which is C9H 12N4O5, chemical name 5-(4-hydroxy-3-((hydroxymethyl)amino)isoxazolidin-5-yl)-5H-pyrrolo[3,4-d]isoxazol-4-ol, Chinese name 5-(4-hydroxy-3-((hydroxymethyl)amino)isoxazolidin-5-yl)-5H-pyrrolo[3,4-d]isoxazol-4-ol, chemical structural formula:

[0006]

[0007] To achieve the above-mentioned purposes of the present application, the present application further provides an extraction and separation method of 5-(4-hydroxy-3-((hydroxymethyl)amino)isoxazolidin-5-yl)-5H-pyrrolo[3,4-d]isoxazol-4-ol, comprising the following specific steps:

[0008] Step 1: Take the dried medicinal material of Portulaca oleracea, and extract it with ethanol. Filter the ethanol extract, combine the filtrate, and concentrate it under reduced pressure. Let it cool to room temperature to obtain a medicinal liquid for standby use.

[0009] Step 2: After the medicinal liquid in step 1 is evaporated, it is extracted with different proportions of ethyl acetate and ethanol. The extract is filtered, and each is concentrated under reduced pressure to obtain an extract for standby use.

[0010] Step 3: After the extract in step 2 is dissolved in water in the ethyl acetate:ethanol (1:5) part, it is separated by ODS column chromatography, eluted with methanol-water gradient, detected by thin layer chromatography, colored, combined and evaporated, and divided into six parts.

[0011] Step 4: The concentrated material obtained in step 3 is further separated by Sephadex gel column (Sephadex LH-20), eluted with methanol-water isocratic elution, detected by thin layer chromatography, colored, combined and evaporated to obtain eight parts.

[0012] Step 5: The concentrated material obtained in step 4 is further separated by Sephadex gel column (Sephadex LH-20), eluted with methanol-water isocratic elution, detected by thin layer chromatography, colored, combined and evaporated to obtain eight parts.

[0013] Step 6: The evaporated material obtained in step 5 is further separated by ODS column, eluted with different methanol-water gradient, and the obtained gradient eluted material is further purified by UHPLC preparation, eluted with methanol-0.1% formic acid (volume percent) isocratic elution to obtain the heterocyclic alkaloid compound.

[0014] Further, in step 1, the ethanol reflux extraction is performed twice, each time for 2 hours, and the amount of the medicinal material is 8-16 times.

[0015] Further, in step 2, the mobile phase elution program is as follows: ethyl acetate: ethanol (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 recovery ethanol gradient elution.

[0016] Further, in step 3, the volume ratio of methanol and water is 10:0, 30:70, 50:50, 70:30, 90:10 and 100:0; and the ODS particle size is 40-70 μm.

[0017] Further, in step 4, the volume ratio of methanol and water is 30:70.

[0018] Further, in step 5, the volume ratio of methanol and water is 10:0.

[0019] Further, in step 6, the volume ratio of methanol and water used is 10:0, 50:50, 30:70, 20:80, 15:85; and the ODS particle size is 40-70 μm.

[0020] Further, in steps 3 and 6, the ODS pretreatment process is as follows: methanol is soaked for 24 hours, after column loading, methanol is washed until no turbidity is generated when water is added dropwise, and then the initial mobile phase is balanced.

[0021] Further, in step 6, the volume ratio of methanol-0.1% formic acid used is 5:95, and the compound retention time is 10.562 min.

[0022] The application also provides a use of 5-(4-hydroxy-3-((hydroxymethyl)amino)isoxazolidin-5-yl)-5H-pyrrolo[3,4-d]isoxazol-4-ol separated from the Portulaca oleracea medicinal material in preparation of anti-inflammatory drugs, anti-cholinesterase drugs and antioxidant drugs.

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

[0024] The separation and pharmacological activity research of the Portulaca oleracea 5-(4-hydroxy-3-((hydroxymethyl)amino)isoxazolidin-5-yl)-5H-pyrrolo[3,4-d]isoxazol-4-ol in the plant are not reported in the prior art; the present application provides a heterocyclic alkaloid compound derived from Portulaca oleracea and an extraction and separation method and application thereof, and the heterocyclic alkaloid compound is successfully extracted and separated through steps such as ethanol extraction, ODS column chromatography separation, dextran gel column chromatography separation and ultra-high performance liquid chromatography separation and purification, the method is simple, rapid and environmentally friendly in process; in addition, the above compound has anti-inflammatory activity, anticholinesterase activity and antioxidant activity, and therefore the 5-(4-hydroxy-3-((hydroxymethyl)amino)isoxazolidin-5-yl)-5H-pyrrolo[3,4-d]isoxazol-4-ol and salts and derivatives thereof can be used as a raw material for drug development and pharmacological activity research. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 for the 5-(4-hydroxy-3-((hydroxymethyl)amino)isoxazolidin-5-yl)-5H-pyrrolo[3,4-d]isoxazol-4-ol of the present application 1 H-NMR spectrum.

[0026] Figure 2 for the 5-(4-hydroxy-3-((hydroxymethyl)amino)isoxazolidin-5-yl)-5H-pyrrolo[3,4-d]isoxazol-4-ol of the present application 13 C-NMR spectrum.

[0027] Figure 3 for the 5-(4-hydroxy-3-((hydroxymethyl)amino)isoxazolidin-5-yl)-5H-pyrrolo[3,4-d]isoxazol-4-ol DEPT spectrum of the present application.

[0028] Figure 4 for the 5-(4-hydroxy-3-((hydroxymethyl)amino)isoxazolidin-5-yl)-5H-pyrrolo[3,4-d]isoxazol-4-ol HSQC spectrum of the present application.

[0029] Figure 5 HMBC spectrum of 5-(4-hydroxy-3-((hydroxymethyl)amino)isoxazolidin-5-yl)-5H- pyrrolo[3,4-d]isoxazol-4-ol of the present application.

[0030] Figure 6 HMBC spectrum of 5-(4-hydroxy-3-((hydroxymethyl)amino)isoxazolidin-5-yl)-5H- pyrrolo[3,4-d]isoxazol-4-ol of the present application. 1 H- 1 H COSY spectrum of 5-(4-hydroxy-3-((hydroxymethyl)amino)isoxazolidin-5-yl)-5H- pyrrolo[3,4-d]isoxazol-4-ol of the present application.

[0031] Figure 7 ROESY spectrum of 5-(4-hydroxy-3-((hydroxymethyl)amino)isoxazolidin-5-yl)-5H- pyrrolo[3,4-d]isoxazol-4-ol of the present application.

[0032] Figure 8 High resolution mass spectrum of 5-(4-hydroxy-3-((hydroxymethyl)amino)isoxazolidin-5-yl)-5H- pyrrolo[3,4-d]isoxazol-4-ol of the present application. DETAILED DESCRIPTION

[0033] The following examples will aid in the understanding of the present application, but the present application is not limited to these examples. The methods of operation in the examples are conventional methods in the art.

[0034] Example 1 5-(4-hydroxy-3-((hydroxymethyl)amino)isoxazolidin-5-yl)-5H-pyrrolo[3,4-d]isoxazol-4-ol.

[0035] The present application provides a heterocyclic alkaloid compound, the molecular formula of which is C9H 12 N4O5, the chemical name of which is 5-(4-hydroxy-3-((hydroxymethyl)amino)isoxazolidin-5-yl)-5H-pyrrolo[3,4-d]isoxazol-4-ol, and the chemical structural formula of which is:

[0036]

[0037] Table 1 shows the NMR data of 5-(4-hydroxy-3-((hydroxymethyl)amino)isoxazolidin-5-yl)-5H-pyrrolo[3,4-d]isoxazol-4-ol, with CD3OD as the solvent used for NMR.

[0038] Table 1. NMR data of 5-(4-hydroxy-3-((hydroxymethyl)amino)isoxazolidin-5-yl)-5H-pyrrolo[3,4-d]isoxazol-4-ol of the present invention.

[0039]

[0040]

[0041] 5-(4-hydroxy-3-((hydroxymethyl)amino)isoxazolidin-5-yl)-5H-pyrrolo[3,4-d]is oxazol-4-ol: White powder, readily soluble in methanol, slightly soluble in water. When spotted onto a silica gel thin-layer plate and sprayed with dilute potassium bismuth iodide solution, the spot turns orange-yellow, indicating that this compound is an alkaloid. UHPLC-ESI-Q-TOF-MS gives m / z: 257.0884 [M+H] + The quasi-molecular ion peak (calculated value [C9H)) 13 N4O5] + (257.0880). Combined 1 H-NMR, 13 Based on C-NMR and DEPT data, the possible molecular formula of this compound is C9H. 12 N4O5, with an unsaturation degree of 5.

[0042] Combination 13 C-NMR, DEPT, and HSQC spectra showed nine carbon signals, including three quaternary carbons (δ¹⁸O₁⁻). C : 157.59, 121.01, 149.95), five methines (δ C : 72.71, 75.45, 91.29, 142.01, 153.67), one methylene (δ C (63.47). According to 1 H- 1H COSY spectrum, H-3 is related to H-4, H-4 is related to H-5, indicating that there are three hydrogen, H-3 and H-5 are connected through H-4. According to the HMBC spectrum, H3, H4 are related to C5, C3, C5 are in the low field, indicating that the ring contains heteroatoms, and C5 chemical shift is high, indicating that O is connected with C5, C3 is connected with N. HMBC spectrum shows that C3 is related to H2'', C2'' is in the low field, it is speculated that there is a hydroxyl group and C3 is connected with C2'' through N. C4 chemical shift also increases, it is speculated that it is connected with the hydroxyl group. According to the above data, it is speculated that the structure contains isoxazolidinol structure, and C3 is connected with a carbinolaminez structure.

[0043] According to the HMBC spectrum, H2' is related to C4', C5', H8' is related to C3', C5', and C2', C3', C5', C8' are in the low field, it is speculated that C2, C5 are connected through N and C5 is connected with a hydroxyl group, C3' is connected with O, and C8' is connected with N. It is speculated that the structure of the compound is 5H-pyrrolo[3,4-d]isoxazol-4-ol, according to the HMBC, H5 is related to C2', C5', indicating that 5H-pyrrolo[3,4-d]isoxazol-4-ol is connected with isoxazolidinol structure through C5. Therefore, it is speculated that the structure of the compound is 5-(4-hydroxy-3-((hydroxymethyl)amino)isoxazolidin-5-yl)-5H-pyrrolo[3,4-d]isoxazol-4-ol.

[0044] The present application also provides an extraction and separation method of 5-(4-hydroxy-3-((hydroxymethyl)amino)isoxazolidin-5-yl)-5H-pyrrolo[3,4-d]isoxazol-4-ol, and the specific steps are as follows:

[0045] Step 1: Take the dried medicinal material of Portulaca oleracea, and extract it with ethanol. Filter the ethanol extract, combine the filtrate, and concentrate under reduced pressure. Let it cool to room temperature, and obtain the medicinal liquid for standby use.

[0046] Step 2: Steam the medicinal liquid in step 1, and then extract it with different proportions of ethyl acetate and ethanol. Filter the extract, and concentrate under reduced pressure to obtain the extract for standby use.

[0047] Step 3: Dissolve the extract in step 2 in water in the ethyl acetate and ethanol (1:5) part, and then separate it by ODS column chromatography. Elute it with methanol-water gradient, and detect it by thin layer chromatography. Color, combine and steam dry, and divide it into six parts.

[0048] Step 4: The concentrate obtained in step 3 was separated by Sephadex LH-20 column, eluted with methanol-water isocratic elution, detected by thin layer chromatography, developed, combined and evaporated to dryness to obtain eight fractions;

[0049] Step 5: The concentrate obtained in step 4 was separated by Sephadex LH-20 column, eluted with methanol-water isocratic elution, detected by thin layer chromatography, developed, combined and evaporated to dryness to obtain the evaporated residue;

[0050] Step 6: The evaporated residue obtained in step 5 was separated by ODS column, eluted with different methanol-water gradient, and the gradient eluted substance was prepared and purified by UHPLC, eluted with methanol-0.1% formic acid water (volume percent) isocratic elution to obtain the heterocyclic alkaloid compound.

[0051] Example 2 Anti-inflammatory effect of 5-(4-hydroxy-3-((hydroxymethyl)amino)isoxazolidin-5-yl)-5H-pyrrolo[3,4-d]isoxazol-4-ol of the present application.

[0052] 1 Main materials

[0053] 1.1 Pharmaceutical and reagent: The new natural product used in the experiment was prepared by the above method, and the purity was 96%. DMEM high-sugar medium, fetal bovine serum (Hyclone, USA); penicillin, streptomycin (Hangzhou Sijiqing Company); LPS (Sigma, USA); IL-1β ELISA kit (Cayman, USA); cell lysis solution.

[0054] 1.2 Cell strain: RAW 264.7 macrophage cells (ATCC cell bank, USA)

[0055] 1.3 Grouping: divided into control group, LPS group and experimental group.

[0056] 2 Experimental method

[0057] 2.1 Cell culture: 10% fetal bovine serum and 1% antibiotic (100 U / mL penicillin and 100 μg / mL streptomycin) were added to DMEM high-sugar medium, and the medium was stored at 4°C.

[0058] 2.2 CCK-8 method for determining cell viability: The logarithmic growth phase RAW264.7 macrophage cells of the above three groups were inoculated in a 96-well culture plate, and the cell density was 1×10 4After incubation at 37℃, 5% CO2 for overnight, the experimental group was added with different concentrations of the compound 5-(4-hydroxy-3-((hydroxymethyl)amino)isoxazolidin-5-yl)-5H-pyrrolo[3,4-d]isoxazol-4-ol (5 μM-50 μM) of the application, and incubated for 1 h, and then LPS with a concentration of 1 μg / mL was added to the LPS group and the experimental group, and a zero group (culture solution containing DMSO solvent) was set up, 3 replicate wells were set up for each group, and the effect on the cells after the addition of the drug was investigated. After the culture of the cells in the above groups for 24 h, 10 μL of CCK-8 was added to the cells in each well, and the incubation was continued at 37℃, 5% CO2 for 4 h, and then the absorbance of each well was determined by an enzyme label instrument at a wavelength of 450 nm.

[0059] 2.3 Determination of inflammatory factor IL-1β by ELISA method: logarithmically growing RAW264.7 macrophages were inoculated in a 24-well culture plate, and the cell density was 1×10 5 After incubation at 37℃, 5% CO2 for overnight, the experimental group was added with the compound 5-(4-hydroxy-3-((hydroxymethyl)amino)isoxazolidin-5-yl)-5H-pyrrolo[3,4-d]isoxazol-4-ol (1 μM-20 μM) of the application, and incubated for 1 h, and then LPS with a final concentration of 1 μg / mL was added to each well, and the incubation was continued for 24 h, and 3 replicate wells were set up for each treatment group. The content of IL-1β was determined by the ELISA method.

[0060] 3 Experimental results

[0061] The experimental results show that the compound of the application has no effect on the proliferation of LPS-induced macrophages RAW264.7; and can effectively inhibit the secretion of excessive inflammatory cytokine IL-1β produced by LPS-induced macrophages RAW264.7, and is in a concentration-dependent manner.

[0062] The experimental results of the relative survival rate of the cells are shown in Table 2.

[0063] Table 2 Effect of the compound of the application on the relative survival rate of RAW264.7 macrophages

[0064]

[0065]

[0066] The results of the determination of the inflammatory factor IL-1β by the ELISA method are shown in Table 3.

[0067] Table 3 Effect of the compound of the present application on the content of IL-1β secreted by LPS-induced RAW264.7 cells (mean ± standard deviation, n = 3).

[0068]

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

[0070] Example 3 Anti-cholinesterase effect of 5-(4-hydroxy-3-((hydroxymethyl)amino)isoxazolidin-5-yl)-5H-pyrrolo[3,4-d]isoxazol-4-ol of the present application.

[0071] 1 Main materials

[0072] 1.1 Drugs and reagents: The new natural product used in the experiment was prepared by the above method, and the purity was 96%. Physostigmine (Shanghai Hanxiang Biological Technology Co., Ltd.), iodine acetylcholine sulfate (ATCI) and acetylcholinesterase (AChE) (Dalian Meilun Biological Technology Co., Ltd.), dithiothreitol (DTNB) (Shanghai Jinshui Biological Technology Co., Ltd.), disodium hydrogen phosphate and sodium dihydrogen phosphate (Shanghai Gaoke Reagent Co., Ltd.).

[0073] 1.2 Experimental instruments and equipment: HBS-1096A 96-well enzyme label instrument (Nanjing Detong Experimental Equipment Co., Ltd.), hundred-thousandth balance (Switzerland METTLER), HH-4 type digital constant-temperature water bath (Jintan Ronghua Instrument Manufacturing Co., Ltd. in Jiangsu Province).

[0074] 2 Experimental method

[0075] The experiment determines the anti-cholinesterase activity of each compound according to a modified Ellman method. 5-(4-hydroxy-3-((hydroxymethyl)amino)isoxazolidin-5-yl)-5H-pyrrolo[3,4-d]isoxazol-4-ol and physostigmine are precisely weighed, and sample solutions with five series of concentrations of 31.25 μM, 62.5 μM, 125 μM, 250 μM and 500 μM are prepared by using methanol. The specific operation is as follows: 140 μL of phosphate buffered saline solution (0.1 M, pH = 8.0, containing 0.1 mol / L disodium hydrogen phosphate and sodium dihydrogen phosphate), 20 μL of sample solution and 15 μL of AChE (0.2 U / mL) are added to a 96-well enzyme-labeled plate, and then incubated at 37°C for 10 minutes, 10 μL of ATCI (4 mmol / L) and 10 μL of DTNB (15 mmol / L) are added, and then incubated at 37°C for 20 minutes, and then the 96-well plate is placed in an enzyme-labeled instrument, and the absorbance values of samples in each group are measured at a wavelength of 405 nm. Among them, methanol is used instead of the sample solution as a blank group, and physostigmine is used instead of the sample solution as a positive control group. The cholinesterase inhibition rate of each compound is calculated according to the following formula (A represents absorbance):

[0076] Inhibition rate (%) = (A 空白 -A 样品 ) / A 空白 × 100%

[0077] 3 Experimental results

[0078] The experimental results show that the new natural product of the application exhibits certain anti-cholinesterase activity, and the inhibition of cholinesterase is enhanced with the increase of the concentration of the compound, showing a dose-dependent trend.

[0079] The anti-cholinesterase activity of the new natural product of the application is shown in Table 4.

[0080] Table 4 Anti-cholinesterase activity of the compound of the application.

[0081]

[0082] Example 4 Antioxidation of 5-(4-hydroxy-3-((hydroxymethyl)amino)isoxazolidin-5-yl)-5H-pyrrolo[3,4-d]isoxazol-4-ol of the application.

[0083] 1 Main materials

[0084] 1.1 Drugs and reagents: The new natural product used in the experiment was prepared by the above method, and the purity was 96%. 1,1-diphenyl-2-pyrrole hydrazine radical (DPPH) (Sigma, USA), butylated hydroxyanisole (BHA) (Shanghai Xiangrui Biological Co., Ltd.), methanol (chromatographic pure, Tianjin Kaixin Chemical Industry Co., Ltd.)

[0085] 1.2 Experimental instruments and equipment: Hitachi UV-3010 ultraviolet visible spectrophotometer (Japan Hitachi), hundredth scale (Switzerland METTLER).

[0086] 2 Experimental method

[0087] The experiment uses DPPH radical scavenging method to determine the antioxidant activity of each compound. 5-(4-hydroxy-3-((hydroxymethyl)amino)isoxazolidin-5-yl)-5H-pyrrolo[3,4-d]isoxazol-4-ol and BHA are precisely weighed, and five series of concentration 12.5 μM, 25 μM, 50 μM, 100 μM and 200 μM sample solutions are prepared with methanol. In addition, the DPPH solution is prepared on site, and an appropriate amount of DPPH is precisely weighed and prepared into a solution with a concentration of 80 μM with methanol. The whole process is operated in the dark. The specific operation is as follows: 1 mL of sample solution is mixed with 1 mL of DPPH solution, and placed in a dark environment at room temperature for 10 min. After setting the detection wavelength of the ultraviolet spectrophotometer to 517 nm, the absorbance value of the sample is measured. Among them, methanol instead of sample solution as blank group, BHA instead of sample solution as positive control group, 1 mL methanol and 1 mL sample solution as control group. The DPPH scavenging rate of each compound is calculated according to the following formula (A represents absorbance):

[0088] DPPH scavenging rate (%) = (1-(A 样品 -A 对照 ) / A 空白 )×100% 3 Experimental results

[0089] The experimental results show that the new natural product of the application shows certain antioxidant activity, and the antioxidant effect is enhanced with the increase of the concentration of the compound, showing a dose-dependent trend.

[0090] The antioxidant activity of the new natural product of the application is shown in Table 5.

[0091] Table 5 Antioxidant activity of the compound of the application.

[0092]

[0093]

[0094] In summary, the present application provides a compound 5-(4-hydroxy-3-((hydroxymethyl)amino)isoxazolidin-5-yl)-5H-pyrrolo[3,4-d]isoxazol-4-ol and its extraction and separation method, which adopts steps of ethanol extraction, ODS column chromatography separation, dextran gel column separation and ultra-high performance liquid chromatography separation and purification, successfully extracts and separates a heterocyclic alkaloid compound, the operation method is simple, rapid, environmental protection, and the compound obtained by the method has high purity. Since the obtained compound is extracted from the commonly used traditional Chinese medicine Portulaca oleracea, has anti-inflammatory activity, anticholinesterase activity and antioxidant activity, therefore the 5-(4-hydroxy-3-((hydroxymethyl)amino)isoxazolidin-5-yl)-5H-pyrrolo[3,4-d]isoxazol-4-ol and its salts and derivatives of the present application can provide ideas for drug development, and have broad development prospects.

Claims

1. A heterocyclic alkaloid compound from purslane, characterized in that, The molecular formula is C9H 12 N4O5, chemical structural formula is: 。 2. A method for extracting and separating a heterocyclic alkaloid compound from purslane, characterized in that, Specifically, the steps include the following: Step 1: Take dried purslane, extract it with ethanol, filter the ethanol extract, combine the filtrates and concentrate under reduced pressure, cool to room temperature, and obtain the medicinal liquid for later use; Step 2: After evaporating the herbal liquid from Step 1 to dryness, extract it with ethyl acetate: ethanol in different ratios. Filter the extracts and concentrate them under reduced pressure to obtain an extract for later use. Step 3: The portion of the extract in Step 2 with an ethyl acetate:ethanol ratio of 1:5 was dissolved in water, separated by ODS column chromatography, using methanol-water gradient elution, detected by thin-layer chromatography, colorimetric analysis, combined and evaporated to dryness, and separated into six fractions; Step 4: The concentrate obtained from the third fraction in Step 3 is further separated by dextran gel chromatography column, eluted with methanol-water isocratic, detected by thin-layer chromatography, colorimetrically developed, combined and evaporated to dryness to obtain eight fractions; Step 5: The second fraction concentrate obtained in Step 4 is further separated by dextran gel chromatography column, eluted with methanol-water isocratic, detected by thin-layer chromatography, colorimetric, combined and evaporated to dryness to obtain the evaporated product; Step 6: The evaporated product obtained in Step 5 is separated by an ODS column and eluted with different methanol-water gradients. The resulting gradient-eluted substances are purified by UHPLC and isocratic elution is performed using methanol-0.1% formic acid as the mobile phase to finally obtain the heterocyclic alkaloid compound.

3. The extraction and separation method according to claim 2, characterized in that, In step 1, ethanol is refluxed 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 as described in claim 2, characterized in that, In step 2, the mobile phase elution program used is ethyl acetate and ethanol with volume ratios of 1:0, 5:1, 2:1, 1:1, 1:2, and 1:5, with ethanol recovery gradient elution.

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

70.

7. The extraction and separation method as described in claim 2, characterized in that, In step 5, the volume ratio of methanol to water used is 10:

0.

8. The extraction and separation method as described in claim 2, characterized in that, In step 6, the volume ratio of methanol to water used is 10:0, 50:50, 30:70, 20:80, or 15:85; the ODS particle size is 40–70 μm; the volume ratio of methanol to 0.1% formic acid used is 5:95; and the compound retention time is 10.562 min.

9. An application of the heterocyclic alkaloid compound as described in claim 1, characterized in that, The compound is used to prepare anti-inflammatory drugs, anticholinesterase drugs, and antioxidant drugs.

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