Separation method of alkaloid compounds in senecio trifurcatus

Abstract

The application discloses a method for separating alkaloid compounds in Senecio scandens Buchen. The method comprises the following steps: (1) subjecting dried medicinal materials of Senecio scandens Buchen to alcohol extraction, and then concentrating to obtain an ethanol extract; (2) dispersing the ethanol extract with water, and then sequentially extracting with petroleum ether and ethyl acetate to obtain a petroleum ether part, an ethyl acetate part and a water part; (3) subjecting the water part to D101 macroporous resin separation and purification to obtain an alkaloid crude product; (4) subjecting the alkaloid crude product to medium-pressure preparative liquid chromatography separation to obtain a concentrate; and (5) subjecting the concentrate to preparative high-performance liquid chromatography purification to obtain a product. The alkaloid compounds are successfully obtained by adopting the ethanol extraction, D101 macroporous resin column chromatography, medium-pressure preparative liquid chromatography and preparative HPLC separation and purification, the operation method is simple and rapid, and the compounds obtained by the method have high purity.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of alkaloid extraction, in particular to a separation method of senecionine alkaloid compound in Emilia sonchifolia. BACKGROUND

[0002] Emilia sonchifolia is a plant of the genus Emilia in the family Asteraceae, mainly distributed in East China, Central-South to Southwest. Emilia sonchifolia is used as a whole herb or root for medicine, with the functions of clearing heat and resolving toxins, reducing inflammation and promoting diuresis, relieving pain, cooling blood and removing dampness, etc. It is mainly used for treating upper respiratory tract infection, oral ulcer, pneumonia, mastitis, furuncle, sore, urinary tract infection, skin eczema, cooling blood, removing dampness and relieving itching, etc. Emilia sonchifolia contains a variety of chemical components, mainly including alkaloids, flavonoids, phenolic acids, terpenes and a small amount of amino acids, organic acids, polysaccharides, etc. Among them, the main alkaloids are pyrrolizidine alkaloids, such as senecionine, which have various pharmacological effects such as anti-tumor and anti-inflammatory.

[0003] Since alkaloids have many important physiological activities, it is particularly important to extract and separate alkaloids with high purity from natural plants. Traditional methods for separating alkaloids include solvent extraction, precipitation and extraction, but these methods have many shortcomings, such as low separation efficiency, inability to obtain single components, low product purity, high cost, and large environmental impact.

[0004] Therefore, it is very important to develop an efficient, low-cost and environmentally friendly alkaloid extraction and separation technology from Emilia sonchifolia. SUMMARY

[0005] The purpose of the present application is to provide a separation method of senecionine alkaloid compound in Emilia sonchifolia, 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-mentioned purpose, the first aspect of the present application provides a separation method of senecionine alkaloid compound in Emilia sonchifolia, comprising the following steps:

[0007] (1) After the dried medicinal material of Emilia sonchifolia is alcohol-extracted, the ethanol extract is obtained by concentration;

[0008] (2) The ethanol extract is dispersed with water, and then extracted with petroleum ether and ethyl acetate in sequence to obtain petroleum ether fraction, ethyl acetate fraction and water fraction;

[0009] (3) The water fraction is separated and purified by D101 macroporous resin to obtain a crude alkaloid product extracted and separated from Emilia sonchifolia;

[0010] (4) The crude alkaloid product is separated by medium pressure preparative liquid chromatography to obtain a concentrate;

[0011] (5) The concentrate is purified by preparative high performance liquid chromatography to obtain a Senecionine compound.

[0012] Preferably, in step (1), the dried medicinal material of Euphorbia humifusa is extracted with 85% ethanol, the filtrate is combined and concentrated under reduced pressure to obtain an ethanol extract.

[0013] Preferably, in step (2), the ethanol extract is dispersed in 8-12 times water, and then extracted with equal volumes of petroleum ether and ethyl acetate in sequence to obtain a petroleum ether fraction, an ethyl acetate fraction and a water fraction.

[0014] Preferably, in step (3), the specific steps of D101 macroporous resin separation and purification are as follows:

[0015] Macroporous resin pretreatment: D101 macroporous resin is soaked in 95% ethanol for 24 hours, then washed with 95% ethanol until the effluent has no white turbidity after adding water, and then washed with pure water until the effluent is clear and has no alcohol smell, and then used.

[0016] Macroporous resin separation and purification: the pretreated D101 macroporous resin is wet-packed into a glass column, then the water fraction is passed through the glass column packed with D101 macroporous resin for adsorption separation, then D101 macroporous resin is used as the stationary phase and an ethanol-water solvent system is used as the eluent to elute the D101 macroporous resin, to obtain an alkaloid crude product.

[0017] Preferably, in step (3), the specific process of using an ethanol-water solvent system to elute D101 macroporous resin is as follows:

[0018] D101 macroporous resin is eluted with 2BV water, 2BV 20% ethanol, 2BV 30% ethanol, 2BV 40% ethanol, 2BV 60% ethanol, 2BV 80% ethanol and 3BV 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 Euphorbia humifusa, the elution components of 30% ethanol, 40% ethanol and 60% ethanol are collected as crude alkaloids of Euphorbia humifusa, and the elution component of 40% ethanol is collected as the crude product of alkaloids for medium-pressure preparative liquid chromatography separation.

[0019] Preferably, in step (4), the medium-pressure preparative liquid chromatography separation uses YMC ODS reversed-phase C18 packing material with a packing particle size of 50μm, the crude product of alkaloids is separated by passing through C18 silica gel, an ethanol-ultra-pure water gradient solvent system is used for elution, ultra-pure water is used as mobile phase A and ethanol is used as mobile phase B for elution and separation, to obtain several elution fractions, which are detected by HPLC to obtain a concentrate for use.

[0020] Preferably, in step (4), the gradient elution conditions are: A:B from 95:5~0:100, 0~600 minutes; B isomeric 100, 600~700 minutes.

[0021] Preferably, in step (5), the concentrate is purified by preparative high performance liquid chromatography, with ultrapure water as mobile phase A, and methanol containing 0.05% ammonia water as mobile phase B, with the conditions of A:B=70:30 isomeric elution for 30 minutes, and the main peak is collected to obtain the griffithiosine alkaloid compound.

[0022] The second aspect of the present application provides a griffithiosine alkaloid compound in Yidianhong, which is extracted by the above separation method.

[0023] Preferably, the chemical structural formula of the griffithiosine alkaloid compound is: .

[0024] Therefore, the separation method of the griffithiosine alkaloid compound in Yidianhong with the above structure has the following beneficial effects:

[0025] The present application uses 85% ethanol extraction, D101 macroporous resin column chromatography, silica gel medium pressure column, semi-preparative HPLC and preparative HPLC for separation and purification, successfully obtains the alkaloid compound, the operation method is simple and fast, and the purity of the compound separated by the method is higher, all more than 95%. In addition, the above compound has potential tumor cell toxicity activity, and can be used for preparing anti-tumor drugs.

[0026] The technical solutions of the present application are further described in detail below through the drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is the mass spectrum of the 40% ethanol eluted component;

[0028] Figure 2 is the mass spectrum of the concentrate;

[0029] Figure 3 is the high performance liquid chromatogram of the concentrate;

[0030] Figure 4 is the high performance liquid chromatogram of the griffithiosine alkaloid compound;

[0031] Figure 5 is the nuclear magnetic hydrogen spectrum of the griffithiosine alkaloid compound;

[0032] Figure 6 is the nuclear magnetic carbon spectrum of the griffithiosine alkaloid compound. DETAILED DESCRIPTION

[0033] The present application will be further described below. It should be noted that the present embodiment is based on the technical solution of the present application, and gives detailed implementation modes and specific operation processes, but the present application is not limited to the present embodiment.

[0034] Example 1

[0035] A separation method of a Senecionine alkaloid compound in Eriocaulon buergerianum includes the following steps:

[0036] (1) 45 kg of dried Eriocaulon buergerianum medicinal materials are cut into pieces, and are extracted by refluxing with 85% ethanol at 85°C for 3 times. The extraction solutions are combined, concentrated and dried under reduced pressure to obtain 4500 g of total extract, which is an ethanol extract.

[0037] (2) The total extract is dispersed with 10 times the amount of distilled water, and is extracted with equal volumes of petroleum ether and ethyl acetate in sequence to obtain 445.4 g of a petroleum ether part, 554.9 g of an ethyl acetate part and 130 g of a water part.

[0038] (3) The water part is subjected to D101 macroporous resin separation and purification, specifically as follows:

[0039] Macroporous resin pretreatment: the D101 macroporous resin is soaked with 95% ethanol for 24 h, and then is washed with 95% ethanol until the outflow liquid does not show white turbidity after adding water, and then is washed with pure water until the outflow liquid is clear and free of alcohol smell, and is ready for use;

[0040] Macroporous resin separation and purification: the pretreated D101 macroporous resin is wet-loaded into a glass column, and then the water part is passed through the glass column loaded with the wet D101 macroporous resin at a flow rate of 150 mL·m -1 -2, to be adsorbed and separated. Then, with the D101 macroporous resin as the stationary phase and an ethanol-water solvent system as the eluent, the D101 macroporous resin is eluted 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. The elution components of water, 20% ethanol, 30% ethanol, 80% ethanol and 100% ethanol are collected as non-alkaloids of Eriocaulon buergerianum, the elution components of 30% ethanol, 40% ethanol and 60% ethanol are collected as crude alkaloids of Eriocaulon buergerianum, and the elution component of 40% ethanol is collected as a crude product of alkaloids for medium-pressure preparative liquid chromatography separation.

[0041] The mass spectrum of the elution component of 40% ethanol is shown in Figure 1 . As can be seen from Figure 1 , the elution component of 40% ethanol mainly contains compounds with mass fractions of 418 and 366.

[0042] (4) The eluent of 40% ethanol was separated by YMC ODS-C18 reversed-phase medium pressure column chromatography (MPLC), eluted with a gradient solvent system of ethanol-ultra-pure water, using ultra-pure water as mobile phase A and ethanol as mobile phase B, and the gradient elution conditions were as follows: A:B from 95:5 to 0:100, 0-600 min; B isocratic 100, 600-700 min. The elution was separated and a plurality of elution fractions were obtained. The elution fractions were detected by HPLC and concentrated for use.

[0043] The mass spectrum of the concentrate is shown in Figure 2 , and the high performance liquid chromatogram is shown in Figure 3 . As can be seen from Figure 2 , the concentrate mainly contains a compound with a mass fraction of 366.

[0044] (5) The obtained concentrate was separated and purified by preparative HPLC (high performance liquid chromatography), using a Waters XBridge BEH C18 25×1 cm chromatographic column, with ultra-pure water as mobile phase A and methanol containing 0.05% ammonia water as mobile phase B, and the conditions were A:B=70:30 isocratic elution for 30 min. The main peak was collected to obtain the Githagine compound.

[0045] The high performance liquid chromatogram of the above Githagine compound is shown in Figure 4 , and the nuclear magnetic hydrogen spectrum and nuclear magnetic carbon spectrum are shown in Figure 5 and Figure 6 . The Githagine compound was successfully separated.

[0046] The Githagine compound is a white powder, MS: [366 M+H]. The molecular formula is C 19 H 27 NO6, and the chemical structural formula is: .

[0047] The nuclear magnetic hydrogen spectrum and nuclear magnetic carbon spectrum data are as follows:

[0048] 1H-NMR (600 MHz, CD3OD): 6.13 (1H, s, 2-H), 5.85 (1H, dd, J = 7.4 Hz, 18-H), 5.40 (1H, d, J = 11.1, 7-H), 4.98 (1H, t, J = 3.3 Hz, 9-HD, 4.34 (1H, d, 9-H), 3.39 (1H, s, 3-H), 3.25-3.22 (1H, tt, J = 3.0 Hz, 3-H), 2.85-2.72 (2H, m, J = 4.2 Hz, 5-D, 2.54 (1H, m, 12-H), 2.36 (1H, m, 13-H), 2.28 (1H, d, J = 13 Hz, 13-H), 2.08 (3H, s, 20-H) 1.90 (3H, dd, J = 7.2 Hz, 19-H), 1.80 (1H, dd, J = 2.4 Hz, 6-H), 1.68 (1H, m, 6-H), 1.33 (3H, s, 16-H), 0.91 (3H, d, J = 6.8 Hz, 17-H).

[0049] 13 C-NMR (150 MHz, 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).

[0050] Finally, it should be noted that the above examples are merely intended to illustrate the technical solutions of the present application but not to limit the present application. Even though the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still make modifications or equivalent replacements to the technical solutions of the present application without departing from the spirit and scope of the present application.

Claims

1. A method for separating gram-type alkaloids from *Senecio scandens*, characterized in that: Includes the following steps: (1) After ethanol extraction of dried *Rhodiola rosea*, the ethanol extract was concentrated to obtain an ethanol extract; (2) The ethanol extract was dispersed in water and extracted sequentially with petroleum ether and ethyl acetate to obtain the petroleum ether fraction, the ethyl acetate fraction and the water fraction; (3) The water fraction was separated and purified by D101 macroporous resin to obtain the crude alkaloid product extracted from *Ipomoea aquatica*. The specific steps of D101 macroporous resin separation and purification are as follows: macroporous resin pretreatment: D101 macroporous resin was soaked in 95% ethanol for 24 hours, then washed with 95% ethanol until the effluent was clear and free of white turbidity when water was added, and then washed with pure water until the effluent was clear and free of alcohol odor. Macroporous resin separation and purification: the pretreated D101 macroporous resin was wet-packed into a glass column, and then the water fraction was passed through the glass column packed with D101 macroporous resin for adsorption and separation. Then, D101 macroporous resin was used as the stationary phase and ethanol-water solvent system was used as the eluent to elute D101 macroporous resin to obtain the crude alkaloid product. The specific process of eluting D101 macroporous resin using an ethanol-water solvent system is as follows: D101 macroporous resin is eluted sequentially 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. The eluents of water, 20% ethanol, 30% ethanol, 80% ethanol, and 100% ethanol are collected as non-alkaloids of *Ipomoea aquamarine*, and the eluents of 40% ethanol and 60% ethanol are collected as crude alkaloids of *Ipomoea aquamarine*. The crude alkaloid product, obtained by elution using the eluent of 40% ethanol, is then subjected to medium-pressure preparative liquid chromatography. (4) The crude alkaloid product was separated by medium-pressure preparative liquid chromatography. YMCODS reversed-phase C18 packing was used for medium-pressure preparative liquid chromatography to obtain a concentrate. The particle size of the medium-pressure preparative liquid chromatography packing was 50 μm. The crude alkaloid product was passed into C18 silica gel for separation. An ethanol-ultrapure water gradient solvent system was used for elution. Ultrapure water was used as mobile phase A and ethanol was used as mobile phase B for elution separation. Several eluted fractions were obtained. The concentrate was obtained by HPLC detection. The gradient elution conditions were: A:B from 95:5 to 0:100, 0 to 600 minutes; B phase isocratic ratio 100, 600 to 700 minutes, flow rate 15 ml / min. (5) The concentrate was purified by preparative high performance liquid chromatography. Ultrapure water was used as mobile phase A, and methanol containing 0.05% ammonia was used as mobile phase B. Isocratic elution was performed for 30 minutes under the condition that A:B = 70:

30. The main peak was collected to obtain the alkaloid compound styrax. The chemical structural formula of the alkaloid compound clade-Senecio scandens is: 。 2. The method for separating gram-type alkaloids from *Senecio scandens* according to claim 1, characterized in that: In step (1), the dried medicinal material of *Ipomoea aquatica* is extracted with 85% ethanol, filtered, the filtrates are combined, and then concentrated and dried under reduced pressure to obtain an ethanol extract.

3. The method for separating gram-type alkaloids from *Senecio scandens* according to claim 2, characterized in that: In step (2), 8 to 12 times the amount of water is added to the ethanol extract for dispersion, and then the extract is carried out sequentially with equal volumes of petroleum ether and ethyl acetate to finally obtain the petroleum ether fraction, the ethyl acetate fraction and the water fraction.

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