CN112010832B - High-efficiency preparation method and application of high-purity amide alkaloid component - Google Patents

High-efficiency preparation method and application of high-purity amide alkaloid component Download PDF

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CN112010832B
CN112010832B CN202010864744.3A CN202010864744A CN112010832B CN 112010832 B CN112010832 B CN 112010832B CN 202010864744 A CN202010864744 A CN 202010864744A CN 112010832 B CN112010832 B CN 112010832B
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methanol
ethyl acetate
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CN112010832A (en
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孙蓉
王岱杰
李晓骄阳
施树云
刘闰平
崔莉
吕其刚
黄娜娜
吴东进
邝江莹
鹿庆华
叶兰
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Shandong University
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    • C07D317/00—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms
    • C07D317/08—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3
    • C07D317/44—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D317/46—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems condensed with one six-membered ring
    • C07D317/48—Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring
    • C07D317/50—Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to atoms of the carbocyclic ring
    • C07D317/60—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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Abstract

The invention provides a high-efficiency preparation method and application of high-purity amide alkaloid components, belonging to the technical field of extraction and preparation of effective components of traditional Chinese medicines. According to the invention, the total amide alkaloids in the piper longum are enriched through the pH value, so that a method for separating and purifying the piperlongum amide alkaloids by high-speed countercurrent chromatography is established, ten amide alkaloids such as piperlonguminine and the like are finally separated from crude extracts of the piper longum, a foundation is laid for further researching the pharmacological properties of the piperlongum amide alkaloids, research and development of related medicines and the like, meanwhile, the separation and extraction method disclosed by the invention is simple, good in separation degree, high in purity and universal, and a new solution is provided for extraction and analysis of related compounds such as alkaloids in the piper longum and piper plants.

Description

High-efficiency preparation method and application of high-purity amide alkaloid component
Technical Field
The invention belongs to the technical field of extraction and preparation of effective components of traditional Chinese medicines, and particularly relates to a high-efficiency preparation method and application of high-purity amide alkaloid components.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Piper Longum L is a representative plant of the genus Piper, has specific fragrance, pungent and spicy taste and flavoring effect, is widely used in food processing processes of burning, roasting, stewed and the like, and is also one of important pot-stewed spices. The piper longum has wide pharmacological action, can be used for treating various diseases such as dyspepsia, sleep problems, asthma, nausea and the like, has the effects of warming spleen and stomach for dispelling cold, relieving pain and killing insects, and is widely used as an auxiliary medicine for treating malaria, stomachache and cholera. The piperitamide alkaloids are the main chemical components of the plants of the genus Piper, have various structural types, have biological activities such as insect resistance, antibiosis and depression resistance and the like, and are one of the hot points of research in the fields of pharmacy and pharmacology.
High-speed Counter-current Chromatography (HSCCC) is a continuous High-efficiency and rapid liquid-liquid distribution Chromatography separation technology which is developed in 30 years, does not need any solid support, avoids various problems of easy dead adsorption, loss, denaturation and the like of a sample caused by a solid support or a carrier, and obviously reduces the distribution efficiency and the solvent consumption when other liquid Chromatography is used for preparative separation, ensures higher peak resolution of HSCCC, has large separation amount, no sample loss, High recovery rate, mild separation environment and saves the solvent. The high-speed counter-current chromatography can directly carry out a large amount of crude extraction samples or synthesis mixtures, the separation result can reach quite high purity, and the method is widely applied to the preparation, separation and purification of chemical substances in the fields of biology, medicine, environmental protection and the like. However, the inventor finds that the separation and purification of a plurality of amide organisms from piper longum by using HSCCC has not been reported.
Disclosure of Invention
Based on the defects of the prior art, the invention provides a high-efficiency preparation method and application of high-purity amide alkaloid components, the invention enriches the total amide alkaloid in the piper longum through the pH value, thereby establishing a method for separating and purifying the piper longum amide alkaloid based on high-speed countercurrent chromatography, and based on the method, the invention separates and obtains ten amide alkaloid components such as the piperlongum longum alkaloid and the like from the crude extract of the piper longum, and provides a material basis for further researching the pharmacology and the research and development of related medicaments of the piper longum amide alkaloid, so the invention has good value of practical application.
In a first aspect of the present invention, there is provided a process for efficiently producing a high-purity amide alkaloid component, the process at least comprising:
dissolving the total extract of the piperlongumine alkaloids in a solvent system, and performing one-dimensional countercurrent chromatography separation by using high-speed countercurrent chromatography to obtain two piperlongumine alkaloids; carrying out off-line two-dimensional gradient countercurrent chromatography separation on the components which are remained in the separation column and are not eluted, namely the tail blowing part by utilizing high-speed countercurrent chromatography to obtain eight types of piperlongumine alkaloids; the solvent system in the separation and extraction method is n-hexane/ethyl acetate/methanol/water.
In a second aspect of the present invention, there is provided an amide alkaloid obtained by the above-mentioned production process. The total ten amide alkaloids are piperlonguminine, piperine, piperlongumine A, piperlongumine C, Dehydropipernonaline, pipernonadiene pyridine, piperundecaladine, guineenside, Brachyamide B, (2E,4E,12Z) -N-isobutyl-2,4, 12-octadecatrienamide.
In a third aspect of the invention, there is provided a process for the preparation and/or use of an amide alkaloid as hereinbefore described in any one or more of:
1) researching the pharmacology and medicinal properties of the amide alkaloid;
2) screening and/or developing related drugs of amide alkaloids.
The beneficial technical effects of one or more technical schemes are as follows:
according to the technical scheme, the ten high-purity piperlongumine alkaloid compounds are successfully extracted and prepared from the piper longumine through the high-speed countercurrent chromatography for secondary separation technology, the separation and purification effects are remarkable, and a foundation is laid for further researching the pharmacological and medicinal properties of the piperlongumine alkaloid, research and development of related medicines and the like.
Meanwhile, the preparation method is simple, good in separation degree, high in purity and universal, provides a new solution for extraction and analysis of related compounds such as alkaloids in piper longum and piper plants, and has good practical application value.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the application and, together with the description, serve to explain the application and are not intended to limit the application.
FIG. 1 is a process flow diagram of the present invention;
FIG. 2 is a chemical structural formula of a Piper longum separation monomer according to an embodiment of the present invention;
FIG. 3 is a schematic diagram of one-dimensional high-speed countercurrent chromatography cycle separation of Piper longum total extract according to an embodiment of the present invention;
FIG. 4 is a high-speed countercurrent chromatogram of Piper longum two-dimensional gradient countercurrent chromatographic separation in the embodiment of the present invention;
FIG. 5 is a high performance liquid chromatogram of a total Piper longum extract and a monomer separated by countercurrent chromatography in an embodiment of the invention.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
In one exemplary embodiment of the present invention, there is provided a method for efficiently producing a high-purity amide alkaloid component, the method at least comprising:
dissolving the total extract of the piperlongumine alkaloids in a solvent system, and performing one-dimensional countercurrent chromatography separation by using high-speed countercurrent chromatography to obtain two piperlongumine alkaloids; carrying out off-line two-dimensional gradient countercurrent chromatography separation on the components which are remained in the separation column and are not eluted, namely the tail blowing part by utilizing high-speed countercurrent chromatography to obtain eight types of piperlongumine alkaloids; the solvent system in the separation and extraction method is n-hexane/ethyl acetate/methanol/water.
In another embodiment of the present invention, the preparation method of the total piperlongumine alkaloid extract comprises: pulverizing fructus Piperis Longi, extracting with ethanol under reflux, concentrating, adding water, adjusting pH to strong acidity, and extracting with petroleum ether; adjusting pH of the residual water phase to alkaline, extracting with chloroform, mixing filtrates, and concentrating to obtain chloroform phase. Because the piperlongumine alkaloid is a weak polar compound and has poor stability, the crude extraction method can effectively enrich and extract the piperlongumine alkaloid compound and reduce the introduction of other compound components.
In another embodiment of the invention, the ethanol is 95% ethanol, and the feed-liquid ratio is 1: 5-15 (g/mL), preferably 1: 10; the reflux extraction method comprises the following steps: reflux extraction is carried out for 2-4 h (preferably 3h) at 80-90 ℃ (preferably 85 ℃); and (4) refluxing and extracting for 2-3 times.
In another embodiment of the present invention, the strong acidity is 2-3 (preferably 2); the alkalinity is 9-10 (preferably 9).
In another embodiment of the present invention, in the one-dimensional countercurrent chromatography, the solvent system is n-hexane/ethyl acetate/methanol/water, the volume ratio is 4-6: 3-5: 4-6: 3-5, v/v, and more preferably 5:4:5:4, and the upper phase solution is selected as the stationary phase and the lower phase solution is selected as the mobile phase.
In another embodiment of the invention, the stationary phase is pumped into a separation column of the countercurrent chromatography at a flow rate of 10-30 mL/min (preferably 20 mL/min); the rotating speed is adjusted to 600-800 rpm clockwise, the temperature is controlled to be 20-25 ℃, and the mobile phase starts to balance at the flow rate of 1.0-3.0 mL/min (preferably 2.0mL/min) in a head-to-tail mode.
In another embodiment of the invention, in the off-line two-dimensional gradient countercurrent chromatography, the stationary phase is an upper phase of n-hexane/ethyl acetate/methanol/water, the volume ratio is 5-7: 3-5: 5-7: 3-5, v/v, and more preferably 6:4:6:4, and the mobile phase is configured as follows: taking a lower phase of n-hexane/ethyl acetate/methanol/water (5-7: 3-5: 5-7: 3-5, v/v, more preferably 6:4:6:4) as a mobile phase within 320-380 min (preferably 360min) of the initial peak; then taking a lower phase of n-hexane/ethyl acetate/methanol/water (6-8: 2-5: 6-8: 2-5, v/v, preferably 7:3:7:3) as a mobile phase at 320-380 min (preferably 360 min); then, taking a lower phase of n-hexane/ethyl acetate/methanol/water (7-10: 1-3: 7-10: 1-3, v/v, preferably 8:2:8:2) as a mobile phase at 320-380 min (preferably 360 min). The invention elutes the components one by one through the polarity change of the mobile phase.
In another embodiment of the present invention, the preparation method further comprises performing analytical detection on the eluted sample by using HPLC, wherein the specific conditions of HPLC include: the mobile phase is acetonitrile (a) -water; the method is carried out by adopting a gradient elution mode, and the gradient elution method is optimized by prolonging the elution time and adjusting the solvent ratio, so that the liquid phase condition is determined as follows: 0-20min (50% A), 20-45min (50% -100% A), 45-55min (100% A), 55-60min (100% -50% A);
detection wavelength: 250-330nm, preferably 254nm, under the wavelength condition, the chromatographic peak is more, and the condition of the sample to be detected can be more comprehensively reflected.
Flow rate: 0.5 to 2mL/min (preferably 1.0 mL/min).
In another specific embodiment of the present invention, the separation and extraction method further separates and purifies ten alkaloids, which are piperlonguminine, piperine, pseudopiperlongumine a, pseudopiperlongumine C, Dehydropipernonaline, piperonyldipidine, piperundecaladine, guaifenesin, Brachyamide B, (2E,4E,12Z) -N-isobutryl-2, 4, 12-octadecatrienamide.
In another embodiment of the present invention, there is provided an amide alkaloid obtained by the above-mentioned production method. The total ten amide alkaloids are piperlonguminine, piperine, piperlongumine A, piperlongumine C, Dehydropipernonaline, pipernonadiene pyridine, piperundecaladine, guineenside, Brachyamide B, (2E,4E,12Z) -N-isobutyl-2,4, 12-octadecatrienamide.
In another embodiment of the present invention, there is provided the above preparation method and/or use of the amide alkaloid in any one or more of the following:
1) researching the pharmacology and medicinal properties of the amide alkaloid;
2) screening and/or developing related drugs of amide alkaloids.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Examples
1. Sample extraction
Taking 1.8kg of dried long pepper, grinding and screening by using a grinder, carrying out reflux extraction for 3h at 85 ℃ by using 95% ethanol according to a material-liquid ratio of 1:10, and repeating the operation for three times. Mixing the extractive solutions, vacuum filtering, concentrating, adding distilled water to obtain crude extract 250mL, adding hydrochloric acid to adjust pH to 2, and extracting with isovolumetric petroleum ether for 3 times. Adjusting pH of the rest water phase to 9 with ammonium hydroxide, extracting with 250mL chloroform for 3 times, mixing filtrates, concentrating under reduced pressure to obtain chloroform phase 113g, with sample extraction rate of 6.2%, and storing the sample in refrigerator at 4 deg.C.
2. One-dimensional counter-current chromatographic separation of total extract
In the high-speed countercurrent chromatography, the solvent system is n-hexane-ethyl acetate-methanol-water (5: 4:5:4 (v/v)), the upper phase solution is selected as a stationary phase, the lower phase solution is selected as a mobile phase, and ultrasonic degassing is carried out. The stationary phase was pumped into a separation column of countercurrent chromatography at a flow rate of 20 mL/min. The rotation speed is adjusted to 800rpm clockwise, the temperature is controlled to 25 ℃, and the mobile phase starts to balance at the flow rate of 2.0mL/min in a head-to-tail mode. And after the fluid dynamic balance is achieved, sample solution is injected along with a manual sample injection ring, the absorbance of the ultraviolet detector is set to 254nm, the recorder is started, separated components are collected and a spectrogram is collected after sample injection, and the separated components are collected in a marked 10mL glass bottle every 6min so as to be detected. And after the separation process is finished, closing the instrument, ejecting the components remained in the countercurrent chromatographic separation column by using a vacuum pump, and obtaining the retention rate of the stationary phase according to the proportion of the volume of the stationary phase in the tail blowing to the total volume. And carrying out subsequent liquid phase detection by reduced pressure concentration. And the tail blowing part after the separation is carried out for multiple times of enrichment by adopting repeated sample introduction operation. And concentrating the tail-blown part under reduced pressure, and recovering to obtain a tail-blown sample (an enriched mixture of trace amide components). Finally separating to obtain the compound 1 piperlonguminine (62.89mg) and the compound 2 piperine (101.93mg), wherein the purity of liquid phase detection is higher than 95%.
3. Off-line two-dimensional gradient countercurrent chromatographic separation of trace amide alkaloids in long pepper
And carrying out secondary separation on the tail-blown part of the sample by adopting a single-pump gradient countercurrent elution mode. Preparing three solvent systems with different proportions, namely n-hexane-ethyl acetate-methanol-water (6: 4:6:4(v/v), n-hexane-ethyl acetate-methanol-water (7: 3:7:3(v/v) and n-hexane-ethyl acetate-methanol-water (8: 2:8:2 (v/v)), balancing a separation column by using a n-hexane-ethyl acetate-methanol-water (6: 4:6:4(v/v) system, injecting 80mg of the sample, continuously eluting for 360min after a peak value is formed by starting a recorder connected with a high-speed counter-current chromatograph, and continuously eluting for 360min under the condition that the lower phase is changed into a n-hexane-ethyl acetate-methanol-water (7: 3(v/v) system, and continuously eluting for 360min under the condition that the lower phase is changed into a n-hexane-ethyl acetate-methanol-water (8: 2:8:2(v/v) system . The components are eluted one by the polarity change of the mobile phase. Finally, compound 3 pseudopiperlongumine A (1.08mg), compound 4 pseudopiperlongumine C (5.83mg), compound 5 Dehydropiperonoline (1.84mg), compound 6 piperonylenediene pyridine (5.25mg), compound 7 piperonylidine (4.33mg), compound 8 piperyleneamide (6.26mg), compound 9 Brachynamide B (3.14mg), compound 10(2E,4E,12Z) -N-isobutyl-2,4,12-octadecatrienamide (2.02mg) are obtained by separation, and the purity of liquid phase detection is higher than 95%.
Using a high performance liquid phaseChromatography of the isolate, liquid phase conditions: waters Symmetry C18column (5 μm,4.6mm × 250mm, i.d.), uv detection wavelength 254nm, flow rate: 1.0mL/min, sample size: 10 μ L, mobile phase eluted with acetonitrile/water gradient: 0-20 minutes, 50% a; 20-45 minutes, 50-100% A; 45-55 minutes, 100% A; 55-60 minutes, 100-50%.
4. Structural identification
And (3) structural identification: respectively measuring MS and NMR spectrums of the separated flavonoid component monomers by an Agilent 5973N mass spectrometer and a Varian 600MHz nuclear magnetic resonance spectrometer, wherein the obtained data are as follows:
compound 1: c16H19NO3,ESI-MS(positive ion mode)m/z:274.17.1H-NMR(CDCl3,400MHz)δ:5.92(1H,overlapped,H-2),7.19(1H,dd,J＝14.8,10.4Hz,H-3),6.68(1H,m,H-4),6.74(1H,overlapped,H-5),6.78(1H,s,H-2'),6.70(1H,overlapped,H-5'),6.88(1H,dd,J＝8.0,1.2Hz,H-6'),5.92(2H,overlapped,H-7'),5.78(1H,brs,H-N),3.18(2H,m,H-1"),1.90(1H,m,H-2"),0.93(overlapped,6H,H-3",4").13C-NMR(CDCl3,100MHz)δ:166.2(C＝O),123.2(CH-2),140.9(CH-3),124.6(CH-4),138.8(CH-5),130.9(C-1'),105.7(CH-2'),148.2(C-3'),148.2(C-4'),108.9(CH-5'),122.6(CH-6'),101.3(CH2-7'),(NH),47.0(CH2-1"),28.6(CH-2"),20.1(Me-3", 4'), identified as piperlonguminine base by literature comparison.
Compound 2: c17H19NO3,ESI-MS(positive ion mode)m/z:286.15.1H-NMR(CDCl3,400MHz)δ:6.42(1H,d,J＝14.6Hz,H-2),7.39(1H,dd,J＝5.1,14.6Hz,H-3),6.71(1H,dd,J＝9.7,14.2Hz,H-4),6.75(1H,d,J＝15.6Hz,H-5),6.96(1H,d,J＝1.7Hz,H-2'),6.78(1H,d,J＝8.0Hz,H-5'),6.87(1H,dd,J＝1.6,8.0Hz,H-6'),5.97(2H,s,-O-CH2-O-,H-7'),3.63(2H,m,H-2"),1.59(2H,overlapped,H-3"),1.66(2H,m,H-4"),1.58(2H,overlapped,H-5"),3.62(2H,m,H-6").13C-NMR(CDCl3,100MHz)δ:165.4(C＝O),119.8(CH-2),143.0(CH-3),125.3(CH-4),138.6(CH-5),131.0(C-1'),108.6(CH-2'),148.1(C-3'),148.2(C-4'),105.7(CH-5'),122.5(CH-6'),101.3(CH2-7'),43.2(CH2-2"),25.6(CH2-3"),24.6(CH2-4"),26.7(CH2-5"),46.6(CH2-6"). Piperine was identified by literature comparison.
Compound 3: c20H25NO3,ESI-MS(positive ion mode)m/z:328.19.1H-NMR(CDCl3,400MHz)δ:5.71(1H,d,J＝10Hz,H-2),7.26(1H,dd,J＝10.0,7.2Hz,H-3),6.17(1H,overlapped,H-4),6.17(1H,overlapped,H-5),2.32(2H,overlapped,H-6),2.32(2H,overlapped,H-7),6.09(1H,m,H-8),6.31(1H,d,J＝10.8Hz,H-9'),6.88(1H,d,J＝0.8Hz,H-2'),6.81(1H,overlapped,H-5'),6.81(1H,overlapped,H-6'),5.93(2H,s,-O-CH2-O-,H-7'),5.46(1H,brs,H-N),3.20(1H,t,J＝6.4Hz,H-1"),1.8(1H,m,H-2"),0.92(6H,d,J＝6.6Hz,H-3",4").13C-NMR(CDCl3,100MHz)δ:166.1(C＝O),123.8(CH-2),144.4(CH-3),123.8(CH-4),144.4(CH-5),31.9(CH2-6),32.7(CH2-7),129.7(CH-8),128.9(CH-9),132.2(C-1'),105.4(CH-2'),147.9(C-3'),146.7(C-4'),108.2(CH-5'),120.3(CH-6'),100.9(CH2-7'),46.9(CH2-1"),28.6(CH-2"),20.1(Me-3", 4'), identified as pseudo-piperlongumine a by literature alignment.
Compound 4: c20H27NO3,ESI-MS(positive ion mode)m/z:330.21.1H-NMR(CDCl3,400MHz)δ:5,76(1H,d,J＝15.0Hz,H-2),7.25(1H,m,H-3),2.19(2H,overlapped,H-4),1.45(2H,overlapped,H-5),1.45(2H,overlapped,H-6),2.19(2H,overlapped,H-7),6.04(1H,m,H-8),6.28(1H,d,J＝15.6Hz,H-9),6.88(1H,s,H-2'),6.74(1H,overlapped,H-5'),6.74(1H,overlapped,H-6'),5.90(2H,s,-O-CH2-O-,H-3'),5.49(1H,brs,H-N),3.16(1H,t,J＝6.4Hz,H-1"),1.79(1H,m,H-2"),0.92(6H,d,J＝6.4Hz,H-3",4").13C-NMR(CDCl3,100MHz)δ:166.0(C＝O),123.8(CH-2),144.4(CH-3),31.9(CH2-4),27.8(CH2-5),28.9(CH2-6),32.7(CH2-7),128.9(CH-8),129.7(CH-9),132.4(C-1'),105.4(CH-2'),148.0(C-3'),146.6(C-4'),108.2(CH-5'),120.2(CH-6'),100.9(CH2-7'),46.9(CH2-1"),28.6(CH-2"),20.1(Me-3", 4'), identified as pseudo-piperlongumine C by literature alignment.
Compound 5: c21H25NO3,ESI-MS(positive ion mode)m/z:340.23.1H-NMR(CDCl3,400MHz)δ:5.98(1H,d,J＝15.0Hz,H-2),6.80(1H,m,H-3),2.31(1H,m,H-4),1.64(1H,m,H-5),1.55(2H,overlapped,H-6),2.31(2H,overlapped,H-7),6.19(1H,m,H-8),6.29(1H,m,H-9),6.88(1H,overlapped,H-2'),6.77(1H,overlapped,H-5'),6.76(1H,overlapped,H-6'),5.94(2H,s,-O-CH2-O-,H-7'),3.61(2H,m,H-2"),1.66(2H,overlapped,H-3"),1.64(2H,m,H-4"),1.58(2H,overlapped,H-5"),3.49(2H,m,H-6").13C-NMR(CDCl3,100MHz)δ:165.6(C＝O),120.4(CH-2),142.5(CH-3),127.8(CH-4),141.1(CH-5),32.2(CH2-6),32.9(CH2-7),129.4(CH-8),130.1(CH-9),132.1(C-1'),105.5(CH-2'),148.0(C-3'),146.8(C-4'),108.2(CH-5'),119.1(CH-6'),101.0(CH2-7'),43.2(CH2-2"),26.7(CH2-3"),24.7(CH2-4"),25.7(CH2-5"),46.7(CH2-6"). Dehydropiperonolide was identified by literature alignment.
Compound 6: c21H27NO3,ESI-MS(positive ion mode)m/z:342.44.1H-NMR(CDCl3,400MHz)δ:5.9(1H,d,J＝15.0Hz,H-2),6.89(1H,m,H-3),2.2(2H,overlapped,H-4),1.50(2H,overlapped,H-5),1.50(2H,overlapped,H-6),2.20(2H,overlapped,H-7),6.00(1H,m,H-8),6.30(1H,m,H-9),6.88(1H,d,J＝15.4Hz,H-2'),6.74(1H,overlapped,H-5'),6.74(1H,overlapped,H-6'),5.92(2H,s,-O-CH2-O-,H-7'),3.50(2H,m,H-2"),1.64(2H,overlapped,H-3"),1.64(2H,m,H-4"),1.61(2H,overlapped,H-5"),3.47(2H,overlapped,H-6").13C-NMR(CDCl3,100MHz)δ:165.6(C＝O),120.5(CH-2),145.7(CH-3),32.4(CH2-4),27.9(CH2-5),29.0(CH2-6),32.7(CH2-7),128.9(CH-8),129.6(CH-9),132.3(C-1'),105.4(CH-2'),147.9(C-3'),146.5(C-4'),108.0(CH-5'),120.2(CH-6'),100.9(CH2-7'),42.8(CH2-2"),26.3(CH2-3"),24.6(CH2-4"),25.9(CH2-5"),46.7(CH2-6"). Piperlongnonadienylpyridine was identified by literature alignment.
Compound 7:C23H29NO3,ESI-MS(positive ion mode)m/z:368.21.1H-NMR(CDCl3,400MHz)δ:6.27(1H,d,J＝14.8Hz,H-2),7.21(1H,m,H-3),6.20(1H,m,H-4),6.09(1H,m,H-5),2.31(2H,overlapped,H-6),1.36(2H,overlapped,H-7),1.36(2H,overlapped,H-8),2.31(2H,overlapped,H-9),6.02(1H,m,H-10),6.31(1H,d,J＝15.6Hz,H-11),6.89(1H,s,H-1'),6.74(1H,overlapped,H-5'),6.74(1H,overlapped,H-6'),5.92(2H,-O-CH2-O-,H-7'),3.47(2H,m,H-2'),1.57(2H,overlapped,H-3'),1.65(2H,m,H-4'),1.57(2H,overlapped,H-5'),2.04(2H,overlapped,H-6').13C-NMR(CDCl3,100MHz)δ:165.9(C＝O),118.9(CH-2),142.7(CH-3),129.4(CH-4),141.4(CH-5),32.2(CH2-6),28.7(CH2-7),29.7(CH2-8),33.9(CH2-9),127.8(CH-10),130.2(CH-11),132.2(C-1'),105.5(CH-2'),148.0(C-3'),146.6(C-4'),108.3(CH-5'),120.4(CH-6'),101.0(CH2-7'),43.3(CH2-2"),25.6(CH2-3"),24.6(CH2-4"),26.7(CH2-5"),47.0(CH2-6"). Piperundecaladine was identified by literature alignment.
Compound 8: c24H33NO3,ESI-MS(positive ion mode)m/z:384.26.1H-NMR(CDCl3,400MHz)δ:5.72(1H,d,J＝14.8Hz,H-2),6.07(1H,m,H-3),6.10(1H,m,H-4),7.17(1H,dd,J＝14.8,10Hz,H-5),2.16(2H,overlapped,H-6),1.26-1.46(8H,overlapped,H-7),2.16(2H,overlapped,H-8),1.26-1.46(2H,overlapped,H-9),1.26-1.46(2H,overlapped,H-10),1.26-1.46(2H,overlapped,H-11),6.06(1H,overlapped,H-12),6.28(1H,d,J＝15.6Hz,H-13),6.89(1H,s,H-2'),6.74(1H,overlapped,H-5'),6.74(1H,overlapped,H-6'),5.92(2H,-O-CH2-O-,H-7'),5.45(1H,brs,H-N),3.14(1H,t,J＝6.4Hz,H-1"),1.80(1H,m,H-2"),0.92(6H,d,J＝6.4Hz,H-3",4").13C-NMR(CDCl3,100MHz)δ:166.4(C＝O),121.8(CH-2),143.1(CH-3),128.3(CH-4),141.3(CH-5),32.9(CH2-6),29.0(CH2-7),32.9(CH2-8),29.3(CH2-9),29.0(CH2-10),32.9(CH2-11),129.4(CH-12),130.3(CH-13),132.5(C-1'),105.4(CH-2'),147.9(C-3'),146.6(C-4'),108.2(CH-5'),120.2(CH-6'),100.9(CH2-7'),46.9(CH2-1"),28.6(CH-2"),20.1(Me-3", 4'), identified as guaifenesin by literature alignment.
Compound 9: c26H37NO3,ESI-MS(positive ion mode)m/z:412.28.1H-NMR(CDCl3,400MHz)δ:6.01(1H,d,J＝14.8Hz,H-2),6.06(1H,overlapped,H-3),6.04(1H,overlapped,H-4),7.16(2H,dd,J＝14.8,10.0Hz,H-5),2.15(2H,overlapped,H-6),1.80(2H,overlapped,H-7),1.76(2H,overlapped,H-8),1.61(2H,overlapped,H-9),1.42(2H,overlapped,H-10),1.29(2H,overlapped,H-11),1.25(2H,overlapped,H-12),2.17(1H,overlapped,H-13),6.05(1H,overlapped,H-14),6.30(1H,d,J＝15.6Hz,H-15),6.89(1H,s,H-2'),6.72(1H,overlapped,H-5'),6.74(1H,overlapped,H-6'),5.93(2H,-O-CH2-O-,H-7'),5.45(1H,brs,H-N),3.16(1H,t,J＝6.8Hz,H-1"),1.80(1H,m,H-2"),0.93(6H,d,J＝6.8Hz,H-3",4").13C-NMR(CDCl3,100MHz)δ:166.4(C＝O),121.3(CH-2),143.0(CH-3),128.2(CH-4),140.8(CH-5),32.9(CH2-6),29.1(CH2-7),29.4(CH2-8),29.4(CH2-9),28.8(CH2-10),29.4(CH2-11),29.1(CH2-12),32.9(CH2-13),129.5(CH-14),129.3(CH-15),130.8(C-1'),105.4(CH-2'),145.8(C-3'),143.1(C-4'),108.3(CH-5'),121.3(CH-6'),100.9(CH2-7'),46.9(CH2-1"),8.6(CH-2"),20.1(Me-3", 4'), identified by literature alignment as Brachyamide B.
Compound 10: c22H39NO,ESI-MS(positive ion mode)m/z:334.12.1H-NMR(CDCl3,400MHz)δ:5.69(1H,d,J＝15.0Hz,H-2),7.21(1H,overlapped,H-3),6,18(1H,overlapped,H-4),6.05(1H,m,H-5),2.12(2H,m,H-6),1.53(2H,-O-CH2-O-,H-7),1.29(2H,m,H-8),1.29(2H,overlapped,H-9),1,29(2H,overlapped,H-10),1.31(2H,overlapped,H-11),1.3(1H,m,H-12),2,01(1H,m,H-13),5.39(2H,m,H-14),5.39(2H,overlapped,H-15),2.02(2H,overlapped,H-16),1.28(2H,overlapped,H-17),1.40(2H,m,H-18),5.47(1H,brs,H-N),3.20(2H,t,J＝4.29Hz,H-1"),1.75(1H,m,H-2"),0.92(6H,d,J＝6.8Hz,H-3",4").13C-NMR(CDCl3,100MHz)δ:166.2(C＝O),121.9(CH-2),143.3(CH-3),122.3(CH-4),141.1(CH-5),33.2(CH2-6),28.9(CH2-7),30.4(CH2-8),29.1(CH2-9),29.2(CH2-10),29.3(CH2-11),128.1(CH-12),129.8(CH-13),29.7(CH2-14),29.6(CH2-15),27.0(CH2-16),29.2(CH2-17),31.8(CH2-18),47.0(CH2-1"),28.6(CH-2"),20.1(Me-3", 4'), identified by literature alignment as (2E,4E,12Z) -N-isobutyl-2,4, 12-octadecacatrienamide.
It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited thereto, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications and equivalents can be made in the technical solutions described in the foregoing embodiments, or equivalents thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. Although the present invention has been described with reference to the specific embodiments, it should be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.

Claims (7)

1. A preparation method of long pepper amide alkaloid components comprises ten long pepper amide alkaloid components, wherein the structural formulas of the ten long pepper amide alkaloid components are shown as follows:
Figure 959912DEST_PATH_IMAGE001
，
characterized in that the preparation method at least comprises the following steps:
dissolving the total extract of the piperlongumine alkaloids in a solvent system, and performing one-dimensional countercurrent chromatography separation by using high-speed countercurrent chromatography to obtain two piperlongumine alkaloids; carrying out off-line two-dimensional gradient countercurrent chromatography separation on the components which are remained in the separation column and are not eluted, namely the tail blowing part by utilizing high-speed countercurrent chromatography to obtain eight types of piperlongumine alkaloids; the solvent system in the high-speed counter-current chromatography is n-hexane/ethyl acetate/methanol/water;
in the one-dimensional countercurrent chromatographic separation, the solvent system is n-hexane/ethyl acetate/methanol/water with the volume ratio of 5:4:5:4,v/vselecting the upper phase solution as a stationary phase and the lower phase solution as a mobile phase;
in the off-line two-dimensional gradient countercurrent chromatography separation, the stationary phase is an upper phase of n-hexane/ethyl acetate/methanol/water with the volume ratio of 6:4:6:4,v/v；
pumping the stationary phase into a separation column of the counter-current chromatography at a flow rate of 20 mL/min; clockwise regulating the rotating speed to 600-800 rpm, starting to control the temperature to 20-25 ℃, and beginning to balance the mobile phase at the flow rate of 2.0mL/min in a head-to-tail mode;
the flow phases were set as follows: within 320-380 min of the initial peak, taking n-hexane/ethyl acetate/methanol/water phase as a mobile phase with the volume ratio of 6:4:6:4,v/v(ii) a Then taking n-hexane/ethyl acetate/methanol/aqueous phase as a mobile phase at a volume ratio of 7:3:7:3 within 320-380 min,v/v(ii) a Then taking n-hexane/ethyl acetate/methanol/aqueous phase as a mobile phase at a volume ratio of 8:2:8:2 within 320-380 min,v/v；
the preparation method of the piperlongumine alkaloid total extract comprises the following steps: pulverizing fructus Piperis Longi, extracting with ethanol under reflux, concentrating, adding water, adjusting pH to strong acidity, and extracting with petroleum ether; adjusting pH of the residual water phase to alkaline, extracting with chloroform, mixing filtrates, and concentrating to obtain chloroform phase.
2. The preparation method according to claim 1, wherein the ethanol is 95% ethanol, and the feed-liquid ratio is 1: 5-15 g/mL; the reflux extraction method comprises the following steps: reflux extraction is carried out for 2-4 h at the temperature of 80-90 ℃; reflux extraction is carried out for 2-3 times;
the strong acid pH is 2-3; the alkaline pH is 9-10.
3. The preparation method according to claim 2, wherein the feed-to-liquid ratio is 1:10 g/mL; the reflux extraction method comprises the following steps: reflux extracting at 85 deg.C for 3 hr;
the strong acid pH is 2; the alkaline pH was 9.
4. The method of claim 1, wherein in an off-line two-dimensional gradient countercurrent chromatographic separation,
the flow phases were set as follows: within 360min of the initial peak, taking n-hexane/ethyl acetate/methanol/water phase as a mobile phase with the volume ratio of 6:4:6:4,v/v(ii) a Then taking n-hexane/ethyl acetate/methanol/water phase as a mobile phase within 360min, wherein the volume ratio is 7:3:7:3,v/v(ii) a Then taking n-hexane/ethyl acetate/methanol/water phase as a mobile phase within 360min, wherein the volume ratio is 8:2:8:2,v/v。
5. the method of claim 1, further comprising performing analytical testing on the eluted sample using HPLC, wherein the HPLC specific conditions comprise: the mobile phase is acetonitrile-water; the method is carried out by adopting a gradient elution mode, and the gradient elution method is optimized by prolonging the elution time and adjusting the solvent ratio, so that the liquid phase condition is determined as follows: 0-20min, 50% acetonitrile; 20-45min, 50% -100% acetonitrile; 45-55min, 100% acetonitrile; 55-60min, 100% -50% acetonitrile.
6. The method of claim 5, wherein the detection wavelength: 250-330 nm; flow rate: 0.5-2 mL/min.
7. The method of claim 6, wherein the detection wavelength: 254 nm; flow rate: 1.0 mL/min.
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