A method for the extraction and separation of indole alkaloids and its application

Abstract

This invention belongs to the field of extraction and separation of traditional Chinese medicine, specifically relating to a method for the extraction and separation of indole alkaloids and its application. The compound is named oleraindolinpyrro, with the molecular formula C2. 12 H 14 N₂O₃, with the structural formula shown in formula (I). This invention employs ethanol reflux extraction, ODS column chromatography, Sephadex LH-20 gel column chromatography, and high-performance liquid chromatography to isolate this compound from Portulaca oleracea with a purity exceeding 96%. Pharmacological experiments show that this compound significantly inhibits the release of IL-1β and TNF-α from LPS-induced RAW264.7 macrophages, exhibiting anti-inflammatory activity. The method of this invention is simple, environmentally friendly, and produces high purity; the obtained compound can be used to prepare anti-inflammatory drugs or as a lead compound for research.

Description

Technical Field

[0001] This invention relates to the field of extraction and separation of traditional Chinese medicine, specifically to a method for extracting and separating indole alkaloids and its application. More particularly, it relates to a novel compound extracted, separated, and identified from purslane and its extraction and separation method. Background Technology

[0002] Purslane (Portulaca oleracea L.), also known as horse purslane, longevity vegetable, and ant vegetable, belongs to the Portulacaceae family. It is an annual herbaceous succulent plant distributed worldwide. Purslane is renowned for its nutritional and medicinal value and has been used as a traditional Chinese medicine for thousands of years.

[0003] Purslane has the effects of clearing heat and detoxifying, cooling blood and stopping bleeding, and stopping dysentery. Modern research shows that purslane mainly contains flavonoids, alkaloids, polysaccharides, phenolic acids, fatty acids and vitamins, and has anti-inflammatory, antioxidant, lipid-regulating, uric acid-lowering, antifungal and antitumor activities.

[0004] Most of the chemical components isolated from purslane are currently known, and their structural novelty is relatively low. Therefore, the development and isolation of new compounds from purslane is urgently needed. This patent provides a method for the efficient extraction and separation of new alkaloid compounds from purslane, while clarifying the structural characteristics of these new compounds. This provides key technical support for the in-depth development of purslane medicinal resources, the discovery of new drug lead compounds, and the research and development of related products, belonging to the core technology field of traditional Chinese medicine modernization and innovative utilization of natural products. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a method for the extraction and separation of indole alkaloids and its applications. Research has revealed that the novel compounds of this invention possess anti-inflammatory and antioxidant effects, and a simple, rapid, environmentally friendly, and high-purity extraction and separation method is provided for these novel compounds.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solutions.

[0007] This invention discloses an indole alkaloid compound, characterized in that the molecular formula of the compound is C2. 12 H 14 N₂O₃, named oleraindolinpyrro, has the following chemical structural formula: .

[0008] This invention also discloses a method for extracting and separating the above-mentioned indole alkaloid compounds, characterized by comprising the following steps: Step 1: Weigh out dried purslane medicinal material. Extract the dried purslane medicinal material twice by reflux with ethanol, each time for 2 hours, using 10 times the amount of medicinal material. Filter the ethanol extract, combine the filtrates and concentrate under reduced pressure. Let it cool to room temperature to obtain the medicinal liquid for later use. Step 2: After evaporating the herbal liquid from Step 1 to dryness, extract with ethyl acetate: ethanol in different ratios. Filter the extracts and concentrate them under reduced pressure to obtain extracts for later use. Step 3: Separate the ethyl acetate:ethanol fraction from Step 2 using an ODS (octadecylsilane-bonded silica gel) column. Elute the fractions using a methanol-water gradient. Detect the fractions using thin-layer chromatography with potassium bismuth iodide as a colorimetric indicator. Combine the colored fractions and concentrate them to dryness under reduced pressure to obtain A1-A7, which are then set aside. Step 4: The A6 obtained in Step 3 was separated by pretreated dextran gel column chromatography (Sephadex LH-20) with isocratic elution using 70% methanol to obtain several eluted fractions. These fractions were then detected by thin-layer chromatography with potassium bismuth iodide as a colorimetric indicator. Each colored eluted fraction was concentrated to dryness under reduced pressure to obtain B1-B1. 20 spare; Step 5: Obtain B from Step 4 17 Pretreated ODS (octadecylsilane-bonded silica gel packing material) was eluted with a methanol-water gradient, detected by thin-layer chromatography, and colored with potassium bismuth iodide. The colored eluted fractions were concentrated to dryness under reduced pressure to obtain concentrates C1-C8. Step 6 involves HPLC separation and preparation of the indole alkaloid compound obtained in step 5, using methanol:0.1% formic acid as the mobile phase.

[0009] Further, the volume ratio of ethyl acetate to ethanol in step 2 is 5:1, 2:1, 1:1, 1:2, and 1:5, respectively.

[0010] Furthermore, in step 3, the volume ratios of methanol and water are 50:50, 70:30, 90:10, and 100:0, respectively, for gradient elution.

[0011] Furthermore, in step 5, the volume ratios of methanol and water are 50:50, 70:30, 90:10, and 100:0, respectively, for gradient elution.

[0012] Furthermore, in step 4, the pretreatment method for Sephadex LH-20 gel is to soak it in methanol for 24 hours and then equilibrate it with 70% methanol as the initial mobile phase after loading it onto the column.

[0013] Furthermore, the detection wavelengths of the high-performance liquid chromatography in step 6 are 210 nm and 254 nm; the methanol:0.1% formic acid ratio in step 6 is 20:80, v / v.

[0014] This invention also discloses the application of the above-described oleraindolinpyrro compound in the preparation of anti-inflammatory drugs.

[0015] The present invention also discloses an anti-inflammatory pharmaceutical composition, characterized in that it comprises an effective amount of the indole alkaloid compound described above or the indole alkaloid compound prepared by the method described above, and pharmaceutically acceptable excipients.

[0016] Furthermore, the dosage form of the pharmaceutical composition is tablets, capsules, injections, or granules.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0018] The isolation and pharmacological activity studies of the novel alkaloid compounds from purslane described in this invention have not been reported in existing academic papers. This invention provides novel alkaloid compounds derived from purslane and an extraction and separation method for these novel compounds. The method successfully extracts and separates the novel compounds using ethanol reflux extraction, ODS column, Sephadex LH-20, and high-performance liquid chromatography. This method is simple and rapid, and the extraction and separation process mainly employs an environmentally friendly water extraction process. Furthermore, the compounds obtained by this method have high purity, all exceeding 90%. In addition, studies have shown that these compounds possess anti-inflammatory effects. Therefore, the novel compounds of this invention, along with their salts and derivatives, can serve as lead compounds for the synthesis of other compounds, as well as raw materials for new drug development and pharmacological activity studies. They can also be used to prepare anti-inflammatory drugs. Attached Figure Description

[0019] Figure 1 This is a high-resolution mass spectrum of the novel alkaloid compound oleraindolinpyrro from this invention.

[0020] Figure 2 The novel compound oleraindolinpyrro of this invention 1 H-NMR spectrum.

[0021] Figure 3 The novel alkaloid compound oleraindolinpyrro of this invention 13 C-NMR spectrum.

[0022] Figure 4 The DEPT 135 spectrum of the novel alkaloid compound oleraindolinpyrro of this invention is shown.

[0023] Figure 5 The HSQC spectrum of the novel alkaloid compound oleraindolinpyrro of this invention is shown.

[0024] Figure 6 The HMBC spectrum of the novel alkaloid compound oleraindolinpyrro of this invention is shown.

[0025] Figure 7 The novel alkaloid compound oleraindolinpyrro of this invention 1 H- 1 H COSY spectrum.

[0026] Figure 8 The ROESY spectrum of the novel alkaloid compound oleraindolinpyrro of this invention is shown. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to specific embodiments. However, this should not be construed as limiting the scope of the above-described subject matter of the present invention to the following embodiments; all technologies implemented based on the content of the present invention fall within the scope of the present invention.

[0028] Unless otherwise specified, all reagents and materials used in this invention are commercially available.

[0029] This invention provides a new compound with the molecular formula C 12 H 14 N₂O₃, named oleraindolinpyrro, has the following chemical structural formula: oleraindolinpyrro Table 1 shows the NMR data of this new compound: 1 H-NMR and 13 C-NMR in deuterated methanol.

[0030] Table 1: NMR data of the alkaloid compound oleraindolinpyrro of this invention.

[0031] This invention relates to the structural identification and derivation of the alkaloid compound oleraindolinpyrro.

[0032] oleraindolinpyrro is a dark green powder, readily soluble in methanol. When spotted onto a silica gel thin-layer plate and sprayed with dilute potassium bismuth iodide solution, the spots turn orange-yellow, indicating that this compound is an alkaloid. UV (MeOH) λmax: 345 nm; UHPLC-ESI-QTOF / MS spectrum gives m / z: 235.1082 [M+H]. + The quasi-molecular ion peak was observed, with a molecular weight of 235.1082. (This is followed by a seemingly unrelated sentence fragment: "combined...") 1 H-NMR,13 Based on C1-NMR and DEPT data, the possible molecular formula of this compound is C10. 12 H 14 N2O3 has an unsaturation degree of 7.

[0033] according to 13 C-NMR and DEPT 135 spectra show that the compound contains 12 carbon signals, including 4 CH2 (δ) atoms. C 38.65, 32.70, 28.81, 28.76), 3 CHs (δ) C 116.24, 112.40, 58.31), 5 quaternary carbons (1 carbonyl carbon δ) C 175.87; 4 olefin carbon δ C 145.62, 145.46, 129.80, 125.55).

[0034] according to 1 H-NMR signal, the two aromatic hydrogens are δ H 6.53 (1H, s, H-4), δ H 6.54 (1H, s, H-7) combined with HMBC correlations shows that H-4 is associated with C-6 and C-8, and H-7 is associated with C-5 and C-9, indicating the presence of a tetrasubstituted benzene ring. Furthermore, HMBC correlations show that H-3 and H-2 are associated with C-8, H-3 with C-4, and H-4 with C-3. Combined with COSY data, H-3 is associated with H-2, indicating an indoline group.

[0035] In addition, C-2' (δ C The presence of a carbonyl group (175.87) in the low-field region indicates the presence of a carbonyl group. Combined with HMBC data showing H-2 is associated with C-3', H-3' with C-8, and C-5' with both C-2' and C-3', and ROSY data showing H-3' is associated with H-4', this indicates that an indoline group is attached to this carbonyl group. Based on this information, the compound can be identified as described above.

[0036] This invention also provides a method for extracting and separating the above-mentioned compounds, the specific steps of which are as follows: Step 1: Weigh 150 kg of dried purslane medicinal material. Extract the dried purslane medicinal material twice by reflux with ethanol, each time for 2 hours, using 10 times the amount of medicinal material. Filter the ethanol extract, combine the filtrates and concentrate under reduced pressure. Let it cool to room temperature to obtain the medicinal liquid for later use. Step 2: After evaporating the herbal liquid from Step 1 to dryness, extract with ethyl acetate:ethanol in different ratios (5:1, 2:1, 1:1, 1:2, 1:5). Filter the extracts and concentrate them under reduced pressure to obtain extracts for later use. Step 3: The ethyl acetate:ethanol (1:5) fraction from Step 2 is separated using an ODS (octadecylsilane-bonded silica gel) column. A gradient elution of methanol-water at volume ratios of 50:50, 70:30, 90:10, and 100:0 is used to obtain several elution fractions. These fractions are then detected by thin-layer chromatography with potassium bismuth iodide as a colorimetric indicator. The colored elution fractions are combined, and the combined elution fractions are concentrated to dryness under reduced pressure to obtain A1-A7, which are then set aside. Step 4: The A6 obtained in Step 3 was separated by pretreated dextran gel column chromatography (Sephadex LH-20) with isocratic elution using 70% methanol to obtain several eluted fractions. These fractions were then detected by thin-layer chromatography with potassium bismuth iodide as a colorimetric indicator. Each colored eluted fraction was concentrated to dryness under reduced pressure to obtain B1-B1. 20 spare; Step 5: Obtain B from Step 4 17 Pretreated ODS (octadecylsilane-bonded silica gel packing material) was eluted with methanol-water gradients of 50:50, 70:30, 90:10 and 100:0, respectively. The eluted material was detected by thin-layer chromatography with potassium bismuth iodide as the colorimetric indicator. The eluted fractions were concentrated to dryness under reduced pressure to obtain concentrates C1-C8. Step 6 involves HPLC separation and preparation of C3 obtained in Step 5, using methanol:0.1% formic acid (20:80, v / v) as the mobile phase and detection wavelengths of 210 nm and 254 nm to obtain the indole alkaloid compound. The purity was determined to be 96% by normalization method.

[0037] The anti-inflammatory effects of the alkaloid compounds of this invention.

[0038] 1. Main materials.

[0039] 1.1 Reagents and Chemicals: The novel compounds used in the experiments were prepared by the above method, with a purity of 90%–99%. Accurately weighed, they were diluted with DMSO to the required solutions for each of the following dosage groups. DMEM high-glucose culture medium, fetal bovine serum (Hyclone, USA); penicillin, streptomycin (Hangzhou Sijiqing Pharmaceutical Group Co., Ltd.); LPS (Sigma-Aldrich, USA); IL-1 β TNF-α ELISA kit (Cayman, USA); cell lysis buffer.

[0040] 1.2 Cell line: RAW264.7 macrophages (ATCC Cell Bank, USA) 1.3 Grouping: control group, LPS group and experimental group, one group each.

[0041] 2. Experimental methods.

[0042] 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°C, 5% CO2 incubator.

[0043] 2.2 Cell viability was determined using the CCK8 assay. Logarithmically growing RAW264.7 macrophages from the three groups were seeded in 96-well culture plates at a cell density of 1 × 10⁻⁶ cells / well. 4 Cells were cultured overnight at 37°C with 100 μL per well and 5% CO2 at a concentration of 1 μg / mL. Different concentrations of the alkaloid compound oleraindolinpyrro (1 μM–20 μM) were added to the experimental group. After incubation for 1 h, 1 μg / mL LPS was added to both the LPS group and the experimental group. A zeroing group (culture medium containing DMSO) was also set up. Each group had three replicates to investigate the effect of drug addition on cells. After 24 h of cell culture, 10 μL of CCK8 was added to each well, and the cells were incubated for another 4 h at 37°C with 5% CO2. The absorbance of each well was measured at 450 nm using a microplate reader.

[0044] 2.3 ELISA method for measuring inflammatory factor IL-1 β And TNF-α: Log-phase RAW264.7 macrophages were seeded in 24-well culture plates at a cell density of 1×10⁻⁶ cells / well. 5 The cells were incubated at 37°C with 5% CO2 overnight, 1 mL per well. The experimental group was treated with the alkaloid compound oleraindolinpyrro (1 μM–20 μM). After 1 h of incubation, LPS (final concentration 1 μg / mL) was added to each well, and the cells were incubated for a total of 24 h. Each treatment was repeated in triplicate. IL-1 secreted by RAW264.7 macrophages treated with the novel compound derived from purslane was measured by ELISA. β And the content of TNF-α.

[0045] 3. Experimental results.

[0046] Experimental results show that the novel alkaloid compound of this invention has no effect on the proliferation of LPS-induced macrophages RAW264.7, and is safe and non-toxic; and can effectively inhibit the excessive production of inflammatory cytokines IL-1β and TNF-α inflammatory mediators by LPS-induced macrophages RAW264.7 in a concentration-dependent manner.

[0047] The results of the relative cell viability experiment are shown in Table 2.

[0048] Table 2: Effect of this invention on the relative survival rate of RAW264.7 macrophages.

[0049] Note: *P<0.05 compared with the control group (the high concentration group showed a significant difference).

[0050] ELISA method for measuring inflammatory factor IL-1 β The results for TNF-α inflammatory mediators are shown in Table 3.

[0051] Table 3: The effect of this invention on LPS-induced IL-1 secretion in RAW264.7 cells β Effects on TNF-α levels Note: # P<0.05 compared with the control group, * P<0.05 compared with the LPS group, mean ± SD, n =3.

[0052] In summary, this invention provides a special compound and its extraction and separation method. The method employs ethanol reflux extraction, ODS column, Sephadex LH-20, and high-performance liquid chromatography for separation, purification, and preparation. A new compound was successfully isolated. This method is simple, rapid, and environmentally friendly, and the compound obtained by this method has high purity. Due to the unique chemical structure of the obtained compound, which is extracted from the commonly used traditional Chinese medicine purslane, it possesses anti-inflammatory properties. Therefore, the special compound of this invention, along with its salts and derivatives, can be used as natural products to develop new traditional Chinese medicine drugs, showing broad prospects.

[0053] The above description is merely a preferred embodiment of the present invention and is not intended to limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An indole alkaloid compound, characterized in that, The molecular formula of the compound is C 12 H 14 N₂O₃, named oleraindolinpyrro, has the following chemical structural formula: 。 2. A method for extracting and separating indole alkaloids as described in claim 1, characterized in that, Includes the following steps: Step 1: Weigh out dried purslane medicinal material. Extract the dried purslane medicinal material twice by reflux with ethanol, each time for 2 hours, using 10 times the amount of medicinal material. Filter the ethanol extract, combine the filtrates and concentrate under reduced pressure. Let it cool to room temperature to obtain the medicinal liquid for later use. Step 2: After evaporating the herbal liquid from Step 1 to dryness, extract with ethyl acetate: ethanol in different ratios. Filter the extracts and concentrate them under reduced pressure to obtain extracts for later use. Step 3: Separate the ethyl acetate:ethanol fraction from Step 2 using an ODS (octadecylsilane-bonded silica gel) column. Elute the fractions using a methanol-water gradient. Detect the fractions using thin-layer chromatography with potassium bismuth iodide as a colorimetric indicator. Combine the colored fractions and concentrate them to dryness under reduced pressure to obtain A1-A7, which are then set aside. Step 4: The A6 obtained in Step 3 was separated by pretreated dextran gel column chromatography (Sephadex LH-20) with isocratic elution using 70% methanol to obtain several eluted fractions. These fractions were then detected by thin-layer chromatography with potassium bismuth iodide as a colorimetric indicator. Each colored eluted fraction was concentrated to dryness under reduced pressure to obtain B1-B1. 20 spare; Step 5: Obtain B from Step 4 17 Pretreated ODS (octadecylsilane-bonded silica gel packing material) was eluted with a methanol-water gradient, detected by thin-layer chromatography, and colored with potassium bismuth iodide. The colored eluted fractions were concentrated to dryness under reduced pressure to obtain concentrates C1-C8. Step 6 involves HPLC separation and preparation of the indole alkaloid compound obtained in step 5, using methanol:0.1% formic acid as the mobile phase.

3. The extraction and separation method as described in claim 2, characterized in that, The volume ratios of ethyl acetate to ethanol in step 2 are 5:1, 2:1, 1:1, 1:2, and 1:5, respectively.

4. The extraction and separation method as described in claim 2, characterized in that, In step 3, the volume ratios of methanol and water are 50:50, 70:30, 90:10, and 100:0, respectively, for gradient elution.

5. The extraction and separation method as described in claim 2, characterized in that, In step 5, the volume ratios of methanol and water are 50:50, 70:30, 90:10, and 100:0, respectively, for gradient elution.

6. The extraction and separation method as described in claim 2, characterized in that, In step 4, the pretreatment method for Sephadex LH-20 gel is to soak it in methanol for 24 hours and then equilibrate it with 70% methanol as the initial mobile phase after loading it onto the column.

7. The extraction and separation method as described in claim 2, characterized in that, In step 6, the detection wavelengths of high performance liquid chromatography are 210 nm and 254 nm; the methanol:0.1% formic acid ratio in step 6 is 20:80, v / v.

8. The use of a compound of oleraindolinpyrro as described in claim 1 in the preparation of an anti-inflammatory drug.

9. An anti-inflammatory pharmaceutical composition, characterized in that, It contains an effective amount of the indole alkaloid compound of claim 1 or 2, and pharmaceutically acceptable excipients.

10. The anti-inflammatory pharmaceutical composition according to claim 9, characterized in that, The dosage form of the pharmaceutical composition is tablets, capsules, injections, or granules.

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