Separation method of climbing groundsel herb alkaloid compound in emilia sonchifolia
Through alcohol extraction, D101 macroporous resin column chromatography and high-performance liquid chromatography separation and purification, a problem of low separation efficiency and low purity in red was solved, and a high-purity Keli photoacid was obtained, which was used to prepare anti-tumor drugs.
Patent Information
- Application Number
- CN202511006113.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-22
- Publication Date
- 2025-08-19
- Estimated Expiration
- 2045-07-22
AI Technical Summary
In the prior art, the separation efficiency of alkaloids in Little Red is low, the product purity is low and the cost is high, and the traditional methods have a great impact on the environment.
The separation and purification were carried out using 85% ethanol extraction, D101 macroporous resin column chromatography, silica gel medium pressure column and preparative high performance liquid chromatography, including alcohol extraction, extraction, resin separation and gradient elution, and separation and purification were carried out in combination with medium pressure preparation liquid chromatography and preparative HPLC.
It has achieved efficient separation of alkaloid compounds, with a purity of more than 95%, is easy to operate, and the compounds have potential anti-tumor activity, which can be used to prepare anti-tumor drugs.
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Figure CN120504675A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of alkaloid extraction, in particular to a method for separating senecio alkaloid compounds from Emilia-Romagna. Background Art
[0002] Emilia radiata is a plant of the genus Emilia in the Asteraceae family, primarily distributed in East China, South-Central, and Southwest China. It is cool and bitter in nature, and the whole plant or root is used as medicine. Its efficacy is heat-clearing and detoxifying, anti-inflammatory and diuretic, anti-inflammatory and analgesic, cooling blood and dispersing blood, removing dampness and relieving itching, and it is primarily used to treat upper respiratory tract infections, oral ulcers, pneumonia, mastitis, boils, urinary tract infections, skin eczema, cooling blood and dispersing blood, removing dampness and relieving itching, and other symptoms. Emilia radiata contains a variety of chemical components, primarily including alkaloids, flavonoids, phenolic acids, terpenes, and small amounts of amino acids, organic acids, and polysaccharides. Among these, the main alkaloids are pyrrolizidine alkaloids, such as senecioide, which have multiple pharmacological effects, including anti-tumor and anti-inflammatory effects.
[0003] Since alkaloids have many important physiological activities, it is particularly important to extract and separate high-purity alkaloids from natural plants. Traditional alkaloid separation methods include solvent extraction, precipitation, extraction, etc. However, these methods have many disadvantages, such as low separation efficiency, inability to obtain single components, low product purity, high cost, and great impact on the environment.
[0004] Therefore, it is very important to develop an efficient, low-cost and environmentally friendly technology for extracting and separating alkaloids from Emilia-Romagna. Summary of the Invention
[0005] The purpose of the present invention is to provide a method for separating alkaloid compounds of Senecio graminis from Emilia-Romagna, so as to solve the problems of low separation efficiency, low product purity and high cost in the above-mentioned natural alkaloid extraction process.
[0006] To achieve the above object, the present invention provides a first aspect of a method for separating a Senecio alkaloid compound from Emilia-Romagna, comprising the following steps: (1) Extracting the dried medicinal materials of Radix Eupatorii with alcohol and concentrating the extracted materials to obtain an ethanol extract; (2) dispersing the ethanol extract with water, and extracting with petroleum ether and ethyl acetate in sequence to obtain a petroleum ether fraction, an ethyl acetate fraction, and a water fraction; (3) The water fraction was separated and purified by D101 macroporous resin to obtain a crude product of alkaloids extracted and separated from Emilia-Romagna; (4) separating the crude alkaloid product by medium-pressure preparative liquid chromatography to obtain a concentrate; (5) The concentrate is purified by preparative high performance liquid chromatography to obtain a gram-scale Senecio alkaloid compound.
[0007] Preferably, in step (1), the dried medicinal material of Emilia-Romagna is extracted with 85% ethanol, filtered, the filtrate is combined, and then concentrated and dried under reduced pressure to obtain an ethanol extract.
[0008] Preferably, in step (2), 8 to 12 times of water is added to the ethanol extract for dispersion, and then extracted with equal volumes of petroleum ether and ethyl acetate in sequence to finally obtain a petroleum ether fraction, an ethyl acetate fraction and a water fraction.
[0009] Preferably, in step (3), the specific steps of separation and purification using D101 macroporous resin are: Pretreatment of macroporous resin: soak D101 macroporous resin in 95% ethanol for 24 hours, then wash with 95% ethanol until the effluent is no longer turbid when water is added, then wash with pure water until the effluent is clear and has no alcohol smell, and set aside; Macroporous resin separation and purification: The pretreated D101 macroporous resin is wet-loaded into a glass column, and then the water portion is passed through a glass column loaded with D101 macroporous resin wet-loaded for adsorption separation. Then, the D101 macroporous resin is used as the stationary phase and the ethanol-water solvent system is used as the eluent to elute the D101 macroporous resin using the ethanol-water solvent system to obtain a crude alkaloid product.
[0010] Preferably, in step (3), the specific process of eluting the D101 macroporous resin using an ethanol-water solvent system is as follows: The D101 macroporous resin was eluted with 2BV of water, 2BV of 20% ethanol, 2BV of 30% ethanol, 2BV of 40% ethanol, 2BV of 60% ethanol, 2BV of 80% ethanol and 3BV of 100% ethanol in sequence, and the elution components of water, 20% ethanol, 30% ethanol, 80% ethanol and 100% ethanol were collected as non-alkaloids of Emilia-Romagna, and the elution components of 30% ethanol, 40% ethanol and 60% ethanol were collected as crude alkaloids of Emilia-Romagna. The elution component of 40% ethanol was used as the crude alkaloid product for medium-pressure preparative liquid chromatography separation.
[0011] Preferably, in step (4), the medium-pressure preparative liquid chromatography separation uses YMC ODS reverse-phase C18 filler with a filler particle size of 50 μm, and the crude alkaloid product is passed through C18 silica gel for separation. An ethanol-ultrapure water gradient solvent system is used for elution, with ultrapure water as mobile phase A and ethanol as mobile phase B. Elution and separation are performed to obtain several elution fractions, which are detected by HPLC to obtain a concentrate for use.
[0012] Preferably, in step (4), the gradient elution conditions are: A:B from 95:5 to 0:100, 0 to 600 minutes; B equal degree 100, 600 to 700 minutes.
[0013] Preferably, in step (5), the concentrate is purified by preparative high performance liquid chromatography, using ultrapure water as mobile phase A and methanol containing 0.05% ammonia water as mobile phase B, and isocratic elution under the condition of A:B=70:30 for 30 minutes, collecting the main peak to obtain the gram-form senecio alkaloid compound.
[0014] The second aspect of the present invention provides a Senecio alkaloid compound from Emilia-Romagna, which is extracted by the above separation method.
[0015] Preferably, the chemical structural formula of the Senecio alkaloid compound is: .
[0016] Therefore, the present invention adopts the method for separating the gram-form Senecio alkaloid compound from Emilia-Romagna with the above structure, which has the following beneficial effects: The present invention successfully isolated and purified alkaloid compounds using 85% ethanol extraction, D101 macroporous resin column chromatography, silica gel medium-pressure column chromatography, semi-preparative HPLC, and preparative HPLC. The method is simple and rapid, and the compounds isolated by this method have a high purity of greater than 95%. Furthermore, the compounds have potential tumor cytotoxic activity and can be used to prepare anti-tumor drugs.
[0017] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 This is the mass spectrum of the component eluted with 40% ethanol; Figure 2 is the mass spectrum of the concentrate; Figure 3 is the HPLC chromatogram of the concentrate; Figure 4 For grams of Senecio alkaloid compounds HPLC ; Figure 5 This is the H NMR spectrum of the gram-type Senecio alkaloid compound; Figure 6 This is the NMR carbon spectrum of the gram-type Senecio alkaloid compound. DETAILED DESCRIPTION
[0019] The present invention will be further described below. It should be noted that this embodiment is based on the technical solution and provides a detailed implementation method and specific operation process, but the present invention is not limited to this embodiment.
[0020] Example 1
[0021] The method for separating the gram-forming Senecio alkaloid compound from Emilia-Romagna comprises the following steps: (1) Chop 45 kg of dried Radix Eupatorii and extract with 85% ethanol at 85°C for 3 times. Combine the extracts, concentrate under reduced pressure and dry to obtain 4500 g of total extract, which is the ethanol extract.
[0022] (2) The total extract was dispersed with 10 times the amount of distilled water, and extracted with equal volumes of petroleum ether and ethyl acetate, respectively, to obtain 445.4 g of petroleum ether fraction, 554.9 g of ethyl acetate fraction, and 130 g of water fraction.
[0023] (3) The water fraction was separated and purified by D101 macroporous resin, specifically: Pretreatment of macroporous resin: soak D101 macroporous resin in 95% ethanol for 24 hours, then wash with 95% ethanol until the effluent is no longer turbid when water is added, then wash with pure water until the effluent is clear and has no alcohol smell, and set aside; Macroporous resin separation and purification: The pretreated D101 macroporous resin was wet loaded into a glass column, and then the water portion was -1 The method comprises the following steps: passing the liquid through a glass column equipped with a D101 macroporous resin wet method at a flow rate of 100 μg / min for adsorption separation, and then using the D101 macroporous resin as the stationary phase and an ethanol-water solvent system as the eluent, eluting the D101 macroporous resin with 2 BV of water, 2 BV of 20% ethanol, 2 BV of 30% ethanol, 2 BV of 40% ethanol, 2 BV of 60% ethanol, 2 BV of 80% ethanol and 3 BV of 100% ethanol in sequence, collecting the elution components of water, 20% ethanol, 30% ethanol, 80% ethanol and 100% ethanol as non-alkaloids of Emilia-Romagna, collecting the elution components of 30% ethanol, 40% ethanol and 60% ethanol as crude alkaloids of Emilia-Romagna, and using the elution component of 40% ethanol as the crude alkaloid product for medium-pressure preparative liquid chromatography separation.
[0024] The mass spectrum of the elution component with 40% ethanol is shown in Figure 1 .from Figure 1 It can be seen that the 40% ethanol elution component mainly contains compounds with mass fractions of 418 and 366.
[0025] (4) The eluate with 40% ethanol was separated by YMC ODS-C18 reverse phase medium pressure column chromatography (MPLC). A gradient solvent system of ethanol-ultrapure water was used for elution. Ultrapure water was used as mobile phase A and ethanol was used as mobile phase B. The gradient elution conditions were: A:B from 95:5 to 0:100, 0 to 600 minutes; B was 100, elution and separation were performed from 600 to 700 minutes, and several elution parts were obtained. The elution parts were detected by HPLC and the concentrate was prepared for use.
[0026] The mass spectrum of the concentrate is shown in Figure 2 , HPLC see Figure 3 .from Figure 2It can be seen that the concentrate mainly contains 366 mass fractions of compounds.
[0027] (5) The obtained concentrate was separated and purified by preparative HPLC (high performance liquid chromatography) using a Waters XBridge BEH C18 25×1 cm column, ultrapure water as mobile phase A, methanol containing 0.05% ammonia water as mobile phase B, and isocratic elution at the ratio of A:B=70:30 for 30 minutes. The main peak was collected to obtain the gram-type Senecio alkaloid compound.
[0028] The HPLC results of the above mentioned Senecio alkaloid compounds are shown in Figure 4 , H NMR and C NMR spectra are shown in Figure 5 and Figure 6 , the alkaloid compounds of Senecio gram-formis were successfully isolated.
[0029] The compound of Senecio alkaloids is a white powder, MS: [366M+H]. The molecular formula is C 19 H 27 NO6, chemical structure is: .
[0030] The H-NMR and C-NMR data are as follows: 1 H-NMR(600MHz,CD3OD):6.13(1H,s,2-H),5.85(1H,dd,J=7.4Hz,18-H),5.40(1H,d,J=11.1,7-H ),4.98(1H,t,J=3.3Hz,9-HD,4.34(1H,d,9-H),3.39(1H,s,3-H),3.25-3.22(1H,tt,J=3.0Hz,3 -H),2.85-2.72(2H,m,J=4.2Hz,5-D,2.54(1H,m,12-H),2.36(1H,m,13-H),2.28(1H,d,J=13Hz, 13-H),2.08(3H,s,20-H)1.90(3H,dd,J=7.2Hz,19-H),1.80(1H,dd,J=2.4Hz,6-H),1.68(1H,m, 6-H),1.33(3H,s,16-H),0.91(3H,d,J=6.8Hz,17-H). 13C-NMR(150MHz,CD3OD):134.3(C-1),137.4(C-2),58.5(C-3),53.2(C-5),36.4(C-6),78.0(C-7),192.1(C-8),64.4(C-9),178.0(C-11) ,76.6(C-12),38.6(C-13),37.7(C-14),131.8(C-15),166.4(C-16),24.5(C-18),10.9(C-19),137.1(C-20),15.3(C-21),40.3(NCH3).
[0031] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.
Claims
1. A method for separating alkaloid compounds of Senecio graminis from Radix Emilia-Romagna, characterized in that: The following steps are involved: (1) Extracting the dried medicinal materials of Radix Eupatorii with alcohol and concentrating the extracted materials to obtain an ethanol extract; (2) dispersing the ethanol extract with water, and extracting with petroleum ether and ethyl acetate in sequence to obtain a petroleum ether fraction, an ethyl acetate fraction, and a water fraction; (3) The water fraction was separated and purified by D101 macroporous resin to obtain a crude product of alkaloids extracted and separated from Emilia-Romagna; (4) separating the crude alkaloid product by medium-pressure preparative liquid chromatography to obtain a concentrate; (5) The concentrate is purified by preparative high performance liquid chromatography to obtain a gram-scale Senecio alkaloid compound.
2. The method for separating the alkaloid compounds of Radix Emilia-Romagna from Radix Emilia-Romagna according to claim 1, wherein: In step (1), the dried medicinal material of Emilia-Romagna is extracted with 85% ethanol, filtered, the filtrate is combined, and then concentrated and dried under reduced pressure to obtain an ethanol extract.
3. The method for separating the alkaloid compounds of Radix Emilia-Romagna from Radix Emilia-Romagna according to claim 2, wherein: In step (2), 8 to 12 times of water is added to the ethanol extract for dispersion, and then extracted with equal volumes of petroleum ether and ethyl acetate in sequence to finally obtain a petroleum ether fraction, an ethyl acetate fraction and a water fraction.
4. The method for separating the alkaloid compounds of Radix Emilia-Romagna from Radix Emilia-Romagna according to claim 3, wherein: In step (3), the specific steps of separation and purification using D101 macroporous resin are as follows: Pretreatment of macroporous resin: soak D101 macroporous resin in 95% ethanol for 24 hours, then wash with 95% ethanol until the effluent is no longer turbid when water is added, then wash with pure water until the effluent is clear and has no alcohol smell, and set aside; Macroporous resin separation and purification: The pretreated D101 macroporous resin is wet-loaded into a glass column, and then the water portion is passed through a glass column loaded with D101 macroporous resin wet-loaded for adsorption separation. Then, the D101 macroporous resin is used as the stationary phase and the ethanol-water solvent system is used as the eluent to elute the D101 macroporous resin using the ethanol-water solvent system to obtain a crude alkaloid product.
5. The method for separating the alkaloid compounds of Radix Emilia-Romagna from Radix Emilia-Romagna according to claim 4, wherein: In step (3), the specific process of eluting the D101 macroporous resin using an ethanol-water solvent system is as follows: The D101 macroporous resin is eluted with 2BV of water, 2BV of 20% ethanol, 2BV of 30% ethanol, 2BV of 40% ethanol, 2BV of 60% ethanol, 2BV of 80% ethanol and 3BV of 100% ethanol in sequence, and the elution components of water, 20% ethanol, 30% ethanol, 80% ethanol and 100% ethanol are collected as non-alkaloids of Emilia-Romagna, the elution components of 30% ethanol, 40% ethanol and 60% ethanol are collected as crude alkaloids of Emilia-Romagna, and the elution component of 40% ethanol is used as the crude alkaloid product for medium-pressure preparative liquid chromatography separation.
6. The method for separating the alkaloid compounds of Radix Emilia-Romagna from Radix Emilia-Romagna according to claim 1, wherein: In step (4), medium-pressure preparative liquid chromatography separation uses YMC ODS reverse-phase C18 filler with a filler particle size of 50 μm. The crude alkaloid product is passed through C18 silica gel for separation and eluted using an ethanol-ultrapure water gradient solvent system. Ultrapure water is used as mobile phase A and ethanol is used as mobile phase B. Elution and separation are performed to obtain several elution fractions, which are detected by HPLC to obtain a concentrate for use.
7. The method for separating the alkaloid compounds of Senecio graminis from Emilia-Romagna according to claim 6, wherein: In step (4), the gradient elution conditions are: A:B from 95:5 to 0:100, 0 to 600 min; B equal degree 100, 600 to 700 min, and the flow rate is 15 ml / min.
8. The method for separating the alkaloid compounds of Senecio graminis from Emilia-Romagna according to claim 1, wherein: In step (5), the concentrate is purified by preparative high performance liquid chromatography using ultrapure water as mobile phase A and methanol containing 0.05% ammonia water as mobile phase B, and isocratic elution is performed under the condition of A:B=70:30 for 30 minutes, and the main peak is collected to obtain the gram-type Senecio alkaloid compound.
9. A Senecio alkaloid compound from Emilia-Romagna, characterized in that: The product is extracted by the separation method according to any one of claims 1 to 8.
10. The Emilia-Romagna alkaloid compound according to claim 9, characterized in that: The chemical structural formula of the gram-formula Senecio alkaloid compound is: .