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

Abstract

The present invention belongs to the field of traditional Chinese medicine extraction and separation technology, and relates to a nitrosoindole alkaloid compound in purslane and its extraction and separation method and use. The new alkaloid has the molecular formula of C 11 H 14 N2O5, chemically known as 2-(hydroxymethyl)-5,6-dimethoxy-1-nitrosoindolin-3-ol. A method for extracting and isolating the novel alkaloid is also provided, comprising sequentially employing water decoction, macroporous resin column chromatography, ODS column chromatography, Sephadex LH-20 column chromatography, and high-performance liquid chromatography for separation, purification, and preparation. Its structure was confirmed as a novel alkaloid using mass spectrometry, proton spectrometry, carbon spectrometry, and two-dimensional nuclear magnetic resonance spectroscopy. The novel alkaloid exhibits anti-inflammatory, anticholinesterase, and antioxidant activities. The novel alkaloid and its salts or derivatives can be used as raw materials for drug development and pharmacological activity research.

Description

Technical Field

[0001] The present invention belongs to the technical field of extraction and separation of traditional Chinese medicines, and relates to a nitrosoindole alkaloid compound in purslane, an extraction and separation method and use thereof, and in particular to a new alkaloid compound Oleraindole G extracted, separated and identified from purslane, and an extraction and separation method thereof. Background Art

[0002] Purslane, derived from the dried aerial parts of Portulaca oleracea L., is a wild plant designated by my country's Ministry of Health as both a medicine and a food. Also known as longevity vegetable and five-element grass, purslane has been used as a traditional Chinese medicine for thousands of years. Widely distributed and abundant in resources, it possesses exceptional adaptability and tenacious vitality. The 2020 edition of the Pharmacopoeia of the People's Republic of China lists the dried aerial parts of purslane as a medicinal herb. It has a sour and cold flavor 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, blood in stool, hemorrhoids, and metrorrhagia.

[0003] Modern research indicates that Portulaca oleracea possesses anti-inflammatory, antioxidant, anticholinesterase, antitumor, lipid-lowering, and antimicrobial effects, primarily attributable to its bioactive components, such as alkaloids, flavonoids, lignans, organic acids, terpenes, furans, polysaccharides, and phenolic acids. Alkaloids, in particular, have been shown to exhibit significant anti-inflammatory effects. Portulaca oleracea contains a wide variety of chemical components with diverse pharmacological activities. The discovery and isolation of compounds from Portulaca oleracea provides a foundation for in-depth research on the plant. Summary of the Invention

[0004] To address the above problems, the present invention provides a nitrosoindole alkaloid compound extracted from Portulaca oleracea. Studies have found that the alkaloid of the present invention has anti-inflammatory, anticholinesterase and antioxidant activities. At the same time, a simple, rapid, environmentally friendly and high-purity extraction and separation method for the compound of the present invention is provided.

[0005] In order to achieve the above-mentioned purpose of the present invention, the present invention provides a nitrosoindole alkaloid compound, the molecular formula of which is C 11 H 14 N2O5, named 2-(hydroxymethyl)-5,6-dimethoxy-1-nitrosoindolin-3-ol or Oleraindole G according to its structure, has the following chemical formula:

[0006]

[0007] To achieve the above-mentioned object of the present invention, the present invention also provides a method for extracting and separating the nitrosoindole alkaloid compound Oleraindole G, which specifically comprises the following steps:

[0008] Step 1: Take dried purslane, decoct in water and extract, concentrate the extract, cool to room temperature, and set aside the medicinal solution.

[0009] Step 2: The concentrated solution in step 1 is subjected to macroporous resin, and eluted with water and ethanol of different concentrations, and the 50% ethanol portion is recovered under reduced pressure to obtain an extract to obtain a concentrate for use.

[0010] 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 60% methanol elution fractions and evaporate to dryness to obtain a concentrate for use.

[0011] Step 4: The concentrate obtained in step 3 is further separated by ODS column chromatography, using a methanol-water gradient elution, detected by thin layer chromatography, and color developed. The 70% methanol elution portion is combined and evaporated to dryness to obtain a concentrate for use.

[0012] Step 5: The product obtained in step 4 is further chromatographed on a pretreated Sephadex LH-20 column and isocratically eluted with 50% methanol to obtain several elution fractions, which are detected by thin layer chromatography and color developed. The color developed elution fractions are combined and the combined elution fractions are concentrated to dryness under reduced pressure for later use;

[0013] Step 6: The concentrate obtained in step 5 is separated and prepared by HPLC (high performance liquid chromatography) using methanol-0.1% formic acid (volume percentage) as the mobile phase for isocratic elution to finally obtain the new nitrosoindole alkaloid compound Oleraindole G of the present invention.

[0014] Furthermore, in step 1, the extraction is performed twice with water decoction, each time for 2 hours, and the amount of water used is 8 to 16 times the amount of the medicinal material.

[0015] Furthermore, in step 2, the macroporous resin used is AB-8 macroporous resin, and the volume ratios of ethanol and water used are 30:70, 50:50, 70:30 and 100:0.

[0016] Furthermore, in step 3, the volume ratio of methanol to water is 5:95, 20:80, 40:60, 60:40 and 80:20; and the particle size of ODS is 40-70 μm.

[0017] Furthermore, the volume ratio of methanol to water used in step 4 is 30:70, 50:50, 70:30 and 100:0; and the particle size of ODS is 40-70 μm.

[0018] Furthermore, in step 5, the methanol elution procedure is 50% methanol isocratic elution.

[0019] Furthermore, the volume ratio of methanol to 0.1% formic acid used in step 6 is 45:55, and the retention time of the compound is 8.913 min.

[0020] 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 dripping into water, and then balance them with the initial mobile phase.

[0021] The present invention also provides a use of the Oleraindole G isolated from the purslane medicinal material as described above in the preparation of anti-inflammatory drugs, anticholinesterase drugs and antioxidant drugs.

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

[0023] The isolation and pharmacological activity research of Oleraindole G from Portulaca oleracea described in the present invention have not been reported in plants. The present invention provides a new nitrosoindole alkaloid compound derived from Portulaca oleracea and a method for extracting and isolating the compound of the present invention. The method sequentially uses water decoction extraction, macroporous resin column chromatography, ODS column chromatography, Sephadex LH-20 column chromatography, and high-performance liquid chromatography for separation, purification, and preparation. The new nitrosoindole alkaloid compound is successfully extracted and isolated. The method has only six steps and is simple and rapid. The extraction is carried out using water, which is environmentally friendly. The compound isolated by the method has a high purity of greater than 90%. In addition, studies have shown that the above compound has anti-inflammatory, anticholinesterase, and antioxidant activities. Therefore, the new compound Oleraindole G and its salts and derivatives can be used as raw materials for drug development and pharmacological activity research. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The novel alkaloid Oleraindole G of the present invention 1 H-NMR spectrum.

[0025] Figure 2 The novel alkaloid Oleraindole G of the present invention 13 C-NMR spectrum.

[0026] Figure 3This is the DEPT spectrum of the new alkaloid Oleraindole G of the present invention.

[0027] Figure 4 This is the HSQC spectrum of the new alkaloid Oleraindole G of the present invention.

[0028] Figure 5 This is the HMBC spectrum of the new alkaloid Oleraindole G of the present invention.

[0029] Figure 6 The COESY spectrum of the new alkaloid Oleraindole G of the present invention is shown in FIG.

[0030] Figure 7 This is the ROESY spectrum of the new alkaloid Oleraindole G of the present invention.

[0031] Figure 8 This is a high-resolution mass spectrum of the new alkaloid Oleraindole G of the present invention. DETAILED DESCRIPTION

[0032] The following examples will help to understand the present invention, but these examples are only for illustration of 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] Example 1 Oleraindole G.

[0034] The present invention provides a nitrosoindole alkaloid compound, the molecular formula of which is C 11 H 14 N2O5, named 2-(hydroxymethyl)-5,6-dimethoxy-1-nitrosoindolin-3-ol or OleraindoleG according to its structure, has the following chemical formula:

[0035]

[0036] Table 1 NMR data of Oleraindole G.

[0037]

[0038] The solvent used for NMR was CD3OD-d4

[0039] Oleraindole G: Yellow oily substance, easily soluble in methanol. After being spotted on a silica gel thin layer plate, the spot will turn orange when sprayed with ferric chloride test solution, indicating that the compound contains phenolic hydroxyl groups; the spot will turn red when sprayed with potassium bismuth iodide test solution, indicating that the compound contains nitrogen. 1 H-NMR,13 C-NMR and HR-ESI-TOF-MS signals suggest that the possible molecular formula of the compound is C 11 H 14 N2O5, unsaturation is 6. HR-ESI-TOF-MS gives m / z [M+H] + The quasi-molecular ion peak is 251.1396, and the calculated value (C 11 H 15 N2O5 + =251.1390). According to 13 C-NMR and DEPT spectra show that there are 11 carbon resonance peaks in the compound, including 2 methyl groups (δ C 56.70,56.70), 1 methylene (δ C 70.64), 4 methines (δ C 55.72, 83.57, 103.92, 104.42), 4 quaternary carbon resonance peaks (δ C 133.09, 136.19, 149.18, 149.30). The NMR data of the compound are listed in Table 1. 1 H-NMR spectrum and HMBC spectrum showed that H-1(δ H 6.60,1H) and C-3(δ C 136.19) and C-5(δ C 139.30) has a strong correlation; H-4 (δ H 6.60) and C-2(δ C 133.09) and C-6(δ C 149.18) showed a strong correlation, suggesting the presence of a tetrasubstituted benzene ring; H-10 (δ H 3.78) has a strong correlation with C-5, H-11 (δ H 3.78) has a strong correlation with C-6, indicating that C-5 and C-6 are connected to methoxy groups. H 2.87,1H) are related to C-2 and C-3, respectively, and H-8 (δ H 4.80,1H) is related to C-2; and the chemical shifts of C-2 and C-7 are located in the low field, which may be connected through the N atom. It is speculated that pyrrolidine is connected to the benzene ring through C-2 and C-3. H 4.80,1H) has a strong correlation with C-9, C-8 (δ C 83.57) is located in the downfield, indicating that the hydroxyl group is located at the C-8 position; H-9 (δ H 3.22 / 3.75,2H) has a strong correlation with C-8, C-9 (δ CThe chemical shift of 70.64 (above field) indicates a hydroxyl group attached to the C-9 position. Since all carbon-hydrogen information is doubled and very close in the NMR spectrum, and the correlations shown in the HMBC spectrum are nearly identical, the presence of a nitrosamine (a type of isomer that exists due to N-N bond rotation) is inferred in the compound's structure. Based on this information, combined with the molecular weight from the HR-ESI-TOF-MS spectrum, the new alkaloid can be confirmed to have the aforementioned structure.

[0040] The present invention also provides a method for extracting and separating Oleraindole G, which comprises the following specific steps:

[0041] Step 1: Weigh 250 kg of dried purslane medicinal materials, use water decoction to extract, the amount of which is 10 times that of the medicinal materials, extract twice, each time for 2 hours, combine the extracts, heat and concentrate, cool to room temperature, and obtain the medicinal solution for use.

[0042] Step 2: The medicinal solution obtained in step 1 is evaporated to dryness and separated by chromatography on an AB-8 macroporous resin column using an ethanol-water (30:70, 50:50, 70:30 and 100:0, v:v) gradient elution. The 50% ethanol portion is collected and recovered under reduced pressure to obtain an extract to obtain a concentrate for later use.

[0043] Step 3: The concentrate in step 2 is separated by a pretreated ODS column with a filler particle size of 40 to 70 μm, and gradient elution is performed using methanol-water (5:95, 20:80, 40:60, 60:40 and 80:20, v:v). The product is detected by thin layer chromatography and color is developed. The 60% methanol elution fractions are combined, concentrated to dryness under reduced pressure, and set aside.

[0044] Step 4: The obtained product in step 3 is further separated by pretreated ODS column chromatography, the filler particle size is 40-70 μm, and gradient elution is performed with methanol-water (30:70, 50:50, 70:30 and 100:0, v / v). The product is detected by thin layer chromatography and color is developed. The 70% methanol elution fractions are combined, concentrated to dryness under reduced pressure, and set aside. The ODS pretreatment process is to soak in methanol for 24 hours. After loading the column, it is washed with methanol until there is no turbidity when dripped into water, and then balanced with the initial mobile phase.

[0045] Step 5: The product obtained in step 4 is further chromatographed on a pretreated Sephadex LH-20 column, isocratically eluted with 50% methanol to obtain several elution fractions, which are detected by thin layer chromatography and color developed. The colored elution fractions are combined, and the combined elution fractions are concentrated to dryness under reduced pressure for later use. The pretreatment process of the sephadex is as follows: soaking the column in methanol for 24 hours, loading the column, washing with methanol until there is no turbidity when dripping into water, and then equilibrating with the initial mobile phase.

[0046] 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 45:55 as the mobile phase and detection wavelengths of 210 and 254 nm to obtain the new nitrosoindole alkaloid Oleraindole G of the present invention. The purity was determined by normalization method to be 96%.

[0047] Example 2 Anti-inflammatory effect of Oleraindole G of the present invention.

[0048] 1 Main Materials

[0049] 1.1 Drugs and Reagents: The new alkaloid used in this experiment was prepared by the above-mentioned method with a purity of 96%. DMEM high-glucose medium and fetal bovine serum were obtained from Hyclone (USA); penicillin and streptomycin were obtained from Hangzhou Sijiqing Company; LPS was obtained from Sigma (USA); ELISA kits for IL-1β and TNF-α were obtained from Cayman (USA); and cell lysate was used.

[0050] 1.2 Cell line: RAW264.7 macrophages (ATCC cell bank, USA)

[0051] 1.3 Grouping: Divided into control group, LPS group and experimental group.

[0052] 2 Experimental methods

[0053] 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.

[0054] 2.2 CCK-8 assay for cell viability: The three groups of RAW264.7 macrophages in logarithmic growth phase were inoculated into 96-well culture plates at a cell density of 1×10 4 After overnight incubation at 37°C, 5% CO2, 100 μL per well of the present invention compound Oleraindole G (5 μM to 50 μM) was added to the experimental group at varying concentrations. After incubation for 1 hour, LPS at a concentration of 1 μg / mL was added to both the LPS group and the experimental group. A zero adjustment group (culture medium containing DMSO) was also established. Three replicate wells were set up in each group to examine the effects of drug addition on the cells. After 24 hours of incubation, 10 μL of CCK-8 was added to each well. After an additional 4 hours of incubation at 37°C, 5% CO2, the absorbance of each well was measured at 450 nm using a microplate reader.

[0055] 2.3 ELISA assay for inflammatory cytokines IL-1β and TNF-α: RAW264.7 macrophages in the logarithmic growth phase were seeded in 24-well culture plates at a cell density of 1×10 5Cells were incubated overnight at 37°C in 5% CO2 with 1 mL per well. The experimental group was treated with the compound of this invention, Oleraindole G (1 μM to 20 μM), for 1 hour. LPS (final concentration 1 μg / mL) was then added to each well and incubated for 24 hours. Each treatment was repeated in triplicate. IL-1β and TNF-α levels were measured by ELISA.

[0056] 3 Experimental results

[0057] 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 cytokines IL-1β and TNF-α produced by LPS-induced macrophages RAW264.7 in a concentration-dependent manner.

[0058] The results of the relative cell survival rate experiment are shown in Table 2.

[0059] Table 2: Effects of the compounds of the present invention on the relative survival rate of RAW264.7 macrophages.

[0060]

[0061] The results of ELISA determination of inflammatory factors IL-1β and TNF-α are shown in Table 3.

[0062] Table 3: Effects of the compounds of the present invention on the levels of IL-1β and TNF-α secreted by RAW264.7 cells induced by LPS (mean ± standard deviation, n=3).

[0063]

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

[0065] Example 3 Anticholinesterase effect of Oleraindole G of the present invention.

[0066] 1 Main Materials

[0067] 1.1 Drugs and Reagents: The new alkaloid used in this experiment was prepared by the above method with a purity of 96%. Physostigmine was obtained from Shanghai Hanxiang Biotechnology Co., Ltd., acetylcholine iodide (ATCI) and acetylcholinesterase (AChE) were obtained from Dalian Meilun Biotechnology Co., Ltd., dithiodinitroformic acid (DTNB) was obtained from Shanghai Jinshui Biotechnology Co., Ltd., and sodium dihydrogen phosphate and sodium dihydrogen phosphate were obtained from Shanghai Sinopharm Reagent Co., Ltd.

[0068] 1.2 Experimental instruments and equipment: HBS-1096A 96-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.).

[0069] 2 Experimental methods

[0070] In this experiment, the anticholinesterase activity of each compound was determined using a modified Ellman method. Oleraindole G and physostigmine were accurately weighed and prepared in methanol to prepare five sample solutions at concentrations of 31.25 μM, 62.5 μM, 125 μM, 250 μM, and 500 μM. The following steps were performed: 140 μL of phosphate buffered saline (0.1 M, pH 8.0, containing 0.1 mol / L sodium dihydrogen phosphate and sodium dihydrogen phosphate), 20 μL of sample solution, and 15 μL of AChE (0.2 U / mL) were added to a 96-well microplate. After incubation at 37°C for 10 minutes, 10 μL of ATCI (4 mmol / L) and 10 μL of DTNB (15 mmol / L) were added. After incubation at 37°C for 20 minutes, the 96-well plate was placed in a microplate reader, and the absorbance of each sample was measured at a wavelength of 405 nm. The blank group was treated with methanol instead of the sample solution, and the positive control group was treated with physostigmine instead of the sample solution. The cholinesterase inhibition rate of each compound was calculated according to the following formula (A represents absorbance):

[0071] Inhibition rate (%) = (A 空白 -A 样品 ) / A 空白 ×100%

[0072] 3 Experimental results

[0073] The experimental results show that the new alkaloid 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.

[0074] The anticholinesterase activities of the novel alkaloids of the present invention are shown in Table 4.

[0075] Table 4: Anticholinesterase activity of the compounds of the present invention.

[0076]

[0077]

[0078] Example 4 Antioxidant effect of Oleraindole G of the present invention.

[0079] 1 Main Materials

[0080] 1.1 Drugs and Reagents: The new alkaloid used in the experiment was prepared by the above method with a purity of 96%. 1,1-Diphenyl-2-picrylhydrazyl free radical (DPPH) was obtained from Sigma-Aldrich, USA; butylated hydroxyanisole (BHA) was obtained from Shanghai Xiangrui Biological Co., Ltd.; and methanol (chromatographically pure, Tianjin Kaixin Chemical Industry Co., Ltd.) was used.

[0081] 1.2 Experimental instruments and equipment: Hitachi UV-3010 ultraviolet-visible spectrophotometer (Hitachi, Japan), 1 / 100,000 balance (METTLER, Switzerland).

[0082] 2 Experimental methods

[0083] This experiment uses the DPPH free radical scavenging method to determine the antioxidant activity of each compound. Oleraindole G and BHA were accurately weighed and prepared with methanol to prepare five series of sample solutions with concentrations of 12.5μM, 25μM, 50μM, 100μM and 200μM. In addition, the DPPH solution was prepared as needed, and an appropriate amount of DPPH was accurately weighed and prepared with methanol to a solution with a concentration of 80μM. The whole process was kept away from light. The specific operation is as follows: 1mL of sample solution was fully mixed with 1mL of DPPH solution, and placed at room temperature in a light-proof environment for 10 minutes. After setting the detection wavelength of the ultraviolet spectrophotometer to 517nm, the absorbance value of the sample was measured. Among them, methanol was used instead of the sample solution as the blank group, BHA was used instead of the sample solution as the positive control group, and a mixed solution of 1mL of methanol and 1mL of sample solution was used as the control group. The DPPH scavenging rate of each compound was calculated according to the following formula (A represents absorbance):

[0084] DPPH clearance rate (%) = (1-(A 样品 -A 对照 ) / A 空白 )×100%

[0085] 3 Experimental results

[0086] The experimental results show that the new alkaloid of the present invention exhibits certain antioxidant activity, and the antioxidant effect is enhanced with the increase of the compound concentration, showing a dose-dependent trend.

[0087] The antioxidant activities of the new alkaloids of the present invention are shown in Table 5.

[0088] Table 5: Antioxidant activity of compounds of the present invention.

[0089]

[0090] In summary, the present invention provides the compound Oleraindole G and its extraction and isolation method. Separation, purification, and preparation are performed sequentially using water decoction extraction, macroporous resin column chromatography, ODS column chromatography, Sephadex LH-20, and high-performance liquid chromatography. A new alkaloid compound is successfully extracted and isolated. This method is simple, rapid, and environmentally friendly, and the compound isolated by this method is of high purity. Because the resulting compound is extracted from the commonly used traditional Chinese medicine Portulaca oleracea and exhibits anti-inflammatory, anticholinesterase, and antioxidant activities, the new compound Oleraindole G and its salts and derivatives of the present invention may provide insights into drug development and have broad development prospects.

Claims

1. A nitrosoindole alkaloid compound isolated from Portulaca oleracea, characterized in that: The molecular formula is: C 11 H 14 N2O5, chemical structure is: 。 2. The method for extracting and separating nitrosoindole alkaloid compounds from Portulaca oleracea according to claim 1, wherein: The specific steps include: Step 1: Take dried purslane, decoct in water, concentrate the extract, and cool to room temperature to obtain a 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. The 50% ethanol portion is recovered under reduced pressure to obtain an extract, and the concentrate is used for standby use; the macroporous resin used is AB-8 macroporous resin, and the volume ratios of ethanol and water used are 30:70, 50:50, 70:30 and 100:0; Step 3: The concentrate in step 2 was separated by ODS column, eluted with methanol-water gradient, detected by thin layer chromatography, and developed. The 60% methanol elution fraction was combined and evaporated to dryness to obtain a concentrate for later use; the volume ratio of methanol to water used was 5:95, 20:80, 40:60, 60:40 and 80:20; the ODS particle size was 40-70 μm; Step 4: The concentrate obtained in step 3 was further separated by ODS column chromatography using a methanol-water gradient elution, detected by thin layer chromatography, and color was developed. The 70% methanol elution fractions were combined and evaporated to dryness to obtain a concentrate for later use; the volume ratios of methanol and water used were 30:70, 50:50, 70:30, and 100:0; The particle size of ODS is 40-70 μm; Step 5: The product obtained in step 4 is further separated by pre-treated dextran gel column chromatography, and isocratically eluted with 50% methanol to obtain several elution fractions, which are detected by thin layer chromatography and developed. The elution fractions with color development are combined, and the combined elution fractions are concentrated to dryness under reduced pressure for later use; Step 6: The concentrate obtained in step 5 was separated and prepared by HPLC, with methanol-0.1% by volume formic acid as the mobile phase for isocratic elution, the volume ratio of methanol-0.1% formic acid used was 45:55, and the retention time of the compound was 8.913 minutes; the nitrosoindole alkaloid compound was obtained.

3. The extraction and separation method according to claim 2, wherein In the step 1, the extraction is performed by decocting in water twice, each time for 2 hours, and the dosage is 8 to 16 times of the medicinal material.

4. The extraction and separation method according to claim 2, wherein The pretreatment process of the ODS and dextran gel is as follows: soaking in methanol for 24 hours, loading onto the column, washing with methanol until no turbidity is observed when dripped into water, and then balancing with the initial mobile phase.

5. The nitrosoindole alkaloid compound according to claim 1 is used for preparing anti-inflammatory drugs, anticholinesterase drugs and antioxidant drugs.

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