CN111249340A - Method for systematic separation and purification of corydalis tuber alkaloid - Google Patents

Method for systematic separation and purification of corydalis tuber alkaloid Download PDF

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CN111249340A
CN111249340A CN201811464112.7A CN201811464112A CN111249340A CN 111249340 A CN111249340 A CN 111249340A CN 201811464112 A CN201811464112 A CN 201811464112A CN 111249340 A CN111249340 A CN 111249340A
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corydalis
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梁鑫淼
郭秀洁
王超然
郭志谋
浦辰辉
张剑
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Taizhou Medical City Guoke Huawu Biomedical Technology Co ltd
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Abstract

The invention provides a systematic separation and purification method of corydalis alkaloid. The preparation liquid chromatogram is adopted to efficiently separate and purify alkaloid monomers from alkaloid components, and the separation modes are reversed phase chromatogram and ion exchange chromatogram. The mobile phase consists of acetonitrile, methanol or ethanol with water (with the addition of a certain amount of acid or buffer salt). Linear gradient, step gradient or isocratic elution mode is adopted. Preparing the rhizoma corydalis micro-alkaloid component, the constant tertiary amine alkali component and the quaternary ammonium alkali component by two-dimensional reversed phase/reversed phase, reversed phase and ion exchange chromatography modes respectively. The method can realize the target preparation of alkaloid, can obtain batch of known active alkaloid, and simultaneously enrich and separate trace alkaloid, thereby continuously enriching alkaloid library and providing material basis for the activity research of alkaloid compounds and the development of new drugs with single component.

Description

Method for systematic separation and purification of corydalis tuber alkaloid
Technical Field
The invention relates to separation and purification of alkaloid compounds, in particular to a systematic separation and purification method of corydalis alkaloid.
Background
Rhizoma Corydalis (Rhizoma Corydalis) is dried tuber of Corydalis yanhusuo (Corydalis yanhusuo W.T. Wang) belonging to Corydalis genus of Papaveraceae family. Also named Yanhu, corydalis tuber and corydalis tuber. Has the effects of promoting blood circulation, removing blood stasis, promoting qi circulation and relieving pain, and is a pain killer with a long application history in traditional Chinese medicine and pharmacy. Its main production place is Zhejiang east yang, which is one of the famous Zhejiang eight flavors. It is pungent, bitter and warm in flavor, enters liver and spleen meridians, and is mainly used for various pains and cardiovascular diseases such as chest rib, epigastric abdomen, postpartum stasis, dysmenorrheal and the like clinically. In traditional Chinese medicine, corydalis tuber is used in compatibility, and in 2005 edition, corydalis tuber is used in nearly 30% of compound preparations. Modern researches have shown that corydalis tuber has significant analgesic, sedative and hypnotic effects, and has better clinical effects on coronary heart disease, arrhythmia, gastric ulcer and other diseases (Heqiao, Gaojiali, Zhao Guang Shuang corydalis tuber, chemical components, pharmacological action and quality control research progress, Chinese herbal medicine 2007, 38 (12): 1909-. In view of the remarkable curative effect and wide clinical application of corydalis tuber, the research reports on the chemical components and pharmacological action of corydalis tuber are increased year by year in recent years.
Modern medicinal chemistry and pharmacological research shows that the main active ingredients in the corydalis tuber are tertiary amine and quaternary ammonium type alkaloids, wherein the content of tertiary amine base is about 0.65%, and the content of quaternary ammonium base is about 0.3%. More than 40 kinds of alkaloids have been separated from the genuine corydalis tuber and other corydalis tubers. These alkaloids are isoquinoline alkaloids, and are classified into berberine, protoberberine, apophenanthrene and protopine according to skeleton type.
In the discovery of Corydalis alkaloids, the traditional normal pressure column chromatography, including silica gel and alumina, plays an important role, and is still widely used at present (Zhangli, Quyang, Houjiaming, etc., Corydalis yanhusuo Chemical composition, Shenyang university of pharmacy, 2008, 25(7): 534-95540; T.T. Hu, X. Zhang, S.Z. Ma, et al, A new protoberberine alkali from Corydalis yanhusuo W.T. Wang. Chinese Chemical Letters, 2009, 20(8): 957.). Although these methods are simple and inexpensive, they have disadvantages such as complicated steps, low separation efficiency, and poor reproducibility. Especially, the ion of alkaloid in the corydalis tuber is generally strong, the alkaloid is easy to be dead-absorbed on a silica gel column, and the loss of a sample is large in multiple separation processes. Although the number of the corydalis tuber alkaloids known at present exceeds 40, the corydalis tuber alkaloids are obtained in a 'zero-knock type', and the number of the alkaloids obtained by each researcher independently is limited, so that the basic research on the substances with the corydalis tuber activity is not systematic and comprehensive. Therefore, introduction or development of a more efficient separation and purification method in the separation of corydalis alkaloids is still a more active topic.
Thus, high-speed countercurrent chromatography (HSCCC) is applied to the Separation and Purification of alkaloids in Corydalis tuber (wild goose, gold, Xuchao, etc. high-speed countercurrent chromatography technique is applied to separate tetrahydropalmatine from Corydalis tuber. world Science and Technology: traditional Chinese medicine modernization 2006, 8(2): 17-19; Z.L. Liu, Y. Yu, P.N. Shen, et al. Separation and Purification of DL-tetrahydropalmatine of corydalus yanhusuo by high-speed counter-current chromatography [ J ] Separation and Purification Technology 2008, 58(3): Separation 346; Q.yu, S.Q.Q.Tong., J.Z.Yan.J.P.J.P.P.P.J.P.P.P.P.J.P.P.P.P.P.P.J.P.P.P.P.P.P.P.P.P.P.N.C. filtration and Purification of corydalion of corydalus and J.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.C. filtration and Purification of the No. C. C.P.P, 2011, 34(3): 278-; wusanqiao, Feng Zi, Li Xing, etc. high speed countercurrent chromatography is used in separating and purifying tetrahydropalmatine and protopine [ J ]. J. China journal of biochemical medicine, 2009, 30(2): 89-92). From the reported results, the HSCCC can realize the high-efficiency separation of the target alkaloid in the corydalis tuber through condition optimization, and compared with the traditional method, the HSCCC can greatly improve the sample recovery rate and is beneficial to the industrial production of related products. However, the method also has the problems of low separation efficiency, poor separation degree, long separation time, complicated condition optimization and the like, only a few compounds with constant quantities can be obtained at one time, and the separation capability of compounds with trace amounts and similar structures still needs to be improved.
High Performance Liquid Chromatography (HPLC) is widely used for component analysis and content detection of target components such as tetrahydropalmatine and palmatine in corydalis tuber and related formulas and traditional Chinese medicine preparations (Hanjiawei, Zhang Li, Gao Xiao Chun. HPLC method is used for determining tetrahydropalmatine content in compound corydalis tuber analgesic plaster. Chinese pharmacist, 2009, 5: 621-one 623; Weihuizhen, Xiufe, Mirabilite, and the like. HPLC method is used for determining tetrahydropalmatine in corydalis tuber analgesic capsules and uncertainty evaluation thereof. Chinese herbal medicine, 2012, 43(002): 299-one 302) and becomes a standard method for quality control of the medicines. A high performance liquid chromatography method developed on the basis of a small-particle homogeneous separation material has a higher separation capacity than the aforementioned separation method, and thus more compound information can be obtained (B. Ding, T.T. Zhou, G.R. Fan, et al, Qualitative and quantitative determination of content in a biological chip chromatography WT by LC-MS/MS and LC-DAD [ J ] Journal of Pharmaceutical and biological Analysis, 2007, 45(2): 219 and 226; J. Zhang, Y. Jin, J. Dong, et al, Systematic refining and characterization of molecular and characterization of biological and quantitative Analysis [ J ] sample and sample of biological Analysis [ W.522-78 (sample of biological chromatography) is disclosed. All the methods show the advantages of the high performance liquid chromatography method in the aspects of separation capability and on-line characterization, however, due to the tailing of the basic compound on the conventional C18, the sample loading of Corydalis tuber alkaloid on the reversed phase chromatographic column is relatively limited, so the report of liquid phase preparation is less (Shimin, Korea, Yewenyi, etc.. Corydalis tuber chemical component research, natural product research and development, 2011, 23(4): 647-. Therefore, the high performance liquid chromatography preparation of corydalis tuber alkaloid needs to develop a novel separation material to solve the problem of chromatographic peak shape, increase the sample loading amount and improve the separation selectivity so as to ensure that the method has enough value in practical application.
The invention is provided in view of the above.
Disclosure of Invention
The invention provides a method for systematic separation and purification of corydalis alkaloid in order to obtain alkaloid monomers required by corydalis pharmacological research.
The invention relates to a method for systematically separating and purifying rhizoma corydalis alkaloid, which comprises the following specific steps:
(1) the corydalis ambigua methanol extract is segmented by a C18WCX reversed phase/weak cation exchange mixed mode chromatographic column to obtain 3 components, namely a corydalis ambigua trace alkaloid component, a major tertiary amine alkali component and a quaternary ammonium alkali component.
(2) The alkaloid monomers are efficiently separated and purified from the 3 alkaloid components by adopting preparative liquid chromatography: separating and purifying rhizoma corydalis trace alkaloid component by two-dimensional reversed phase/reversed phase chromatography mode; separating and purifying rhizoma corydalis constant tertiary amine alkali component by reverse phase chromatography; separating and purifying the corydalis ambigua quaternary ammonium base component by ion exchange chromatography.
Further, in the two-dimensional reversed phase/reversed phase chromatographic separation mode, the first-dimensional reversed phase chromatographic column is a polar modified carbon eighteen column (XCharge C18), the mobile phase composition is one or two of acetonitrile, methanol or ethanol and water containing formic acid or acetic acid, and the elution mode is a linear gradient; the second dimension reversed phase chromatographic column is common carbon eighteen column (XUnion C18, Unit C18, XTerra MS C18 or SunAire C18), the mobile phase composition is one or two of acetonitrile, methanol or ethanol and acid water (containing one of acetic acid or phosphoric acid, adjusting pH to 5.5-6.0 with triethylamine), and linear gradient, step gradient or isocratic elution mode is adopted.
Further, in the reverse phase chromatographic separation mode, the reverse phase chromatographic column is a common carbon eighteen column (XUnion C18, Unit C18, XTerra MS C18 or SunAire C18), the mobile phase is one or two of acetonitrile, methanol or ethanol mixed with acid water (containing one of acetic acid or phosphoric acid, and the pH is adjusted to 5.5-6.0 by triethylamine), and a linear gradient, a step gradient or an isocratic elution mode is adopted.
Further, in the ion exchange separation mode, the ion exchange column is a silica gel-based strong cation exchange column (XCharge SCX), the mobile phase composition is one or two of acetonitrile, methanol or ethanol, and is mixed with buffer salt (containing phosphate salt and chaotropic salt, and adjusting pH to 3.0 with phosphoric acid), and isocratic elution is adopted.
Further, the chromatographic operating parameters are as follows: the inner diameter of the chromatographic column is 4.6-1000 mm; the sample concentration is 1 mg/mL-1 g/mL; the sample amount is 10 mu L-400 mL; the flow rate is 0.5-480 mL/min; the column temperature is 25-45 ℃.
The method can realize the target preparation of the alkaloid, can obtain batch of known active alkaloid, and simultaneously enrich and separate trace alkaloid, thereby continuously enriching alkaloid libraries and providing a material basis for the activity research of alkaloid compounds and the development of new drugs with single components.
Drawings
FIG. 1 is a reanalysis of the trace alkaloid fractions 21-45 on a Unit C18 column (150 mm. times.4.6 mm, 5 μm) with mobile phase: (A) 0.1% phosphoric acid (pH adjusted to 6.0 with triethylamine), (B) acetonitrile; elution gradient: 0-5 min, 10% B, 5-10 min, 10-15% B, 10-35 min, 15-25% B, 35-40 min, 25-55% B, 40-50 min, 55% B; flow rate: 1 mL/min; column temperature: 30 ℃; detection wavelength: 280 nm;
FIG. 2 chromatograms of minor alkaloid components 34, 36 and 40; (A) a component 34; (B) a component 36; (C) and (4) 40. A chromatographic column: a Unitry C18 column (150 mm. times.4.6 mm, 5 μm); mobile phase: (A) 0.1% phosphoric acid (pH adjusted to 6.0 with triethylamine), (B) acetonitrile; elution gradient: 0-5 min, 10% B, 5-10 min, 10-15% B, 10-35 min, 15-25% B, 35-40 min, 25-55% B, 40-50 min, 55% B; flow rate: 1 mL/min; column temperature: 30 ℃; detection wavelength: 203 nm
FIG. 3 is a preparation spectrum of a tertiary amine base component (component III);
FIG. 4 chromatogram of quaternary ammonium base component (component IV) at different loading volumes; (A) 10 mu L of the solution; (B) 50 mu L of the solution; (C) 100 μ L.
Detailed Description
The present invention is further illustrated below with reference to examples, which are by no means intended to limit the scope of the invention. The chromatographic separation columns used in the examples were purchased from Zhejiang spectra New technology, Inc.
Example 1:
separating rhizoma corydalis methanol extract by preparative High Performance Liquid Chromatography (HPLC), wherein the chromatographic conditions are as follows: the chromatographic column is a reversed phase/weak cation exchange mixed mode (C18 WCX) column; a mobile phase system of water (A) and ethanol (B); the elution gradient is 0-12 min, the volume concentration is 0% B, 12-18 min, and the sample solution; 18-30 min, volume concentration of 0% B, 30-36 min, volume concentration of 30% B, 36-44 min, 50% B; 44-52 min, 100% B; 52-64 min, 50% B-1% acetic acid; the flow rate is 375 mL/min; the concentration of the sample solution is 10 mg/mL, and the sample solution is dissolved by adopting methanol with the volume concentration of 2.5 percent to 97.5 percent; the sample injection volume is 2.25L; collecting into one part (rhizoma corydalis micro alkaloid component, component I) at 30-36 min; collecting into one part (component II) at 36-44 min; collecting in one portion (rhizoma corydalis constant tertiary amine base component, component III) at 44-52 min; collecting at 52-64 min to obtain 4 fractions (rhizoma corydalis quaternary ammonium base fraction, fraction IV), and concentrating each fraction to dry for use.
Example 2:
selecting a component I, and performing two-dimensional preparative High Performance Liquid Chromatography (HPLC) separation by adopting a two-dimensional reversed phase/reversed phase chromatographic separation mode. First dimension preparative chromatography conditions: the chromatographic column is polar modified carbon eighteen column (XCharge C18); 0.1% formic acid (A) and acetonitrile (B) mobile phase system; the elution gradient is 0-40 min, 2-20% B; 40-55 min, 20% B; the flow rate is 300 mL/min; the detection wavelength is 280 nm; the sample volume was 35 mL. One portion was collected every minute for 4-55 minutes for a total of 51 fractions, each concentrated to dryness for future use. In order to more intuitively reflect the high orthogonality of the 2-D RPLC/RPLC system, the high content of fractions 21 to 45 obtained from the first dimension preparation column of XCharge C18 was reanalyzed on a Unit C18 column, and the results are shown in FIG. 3. The high orthogonality of the 2-D RPLC/RPLC system is well illustrated by the appearance of more chromatographic peaks in FIG. 1. Second dimension preparative chromatographic conditions: the chromatographic column is a common carbon eighteen column (Unitry C18); 0.1% phosphoric acid (pH 6.0 adjusted with triethylamine) (A) and acetonitrile (B) mobile phase system; the elution gradient is 0-20 min, 15-25% B; 20-25 min, 25-55% B; 25-30 min, 55% B; the flow rate is 20 mL/min; the sample injection amount is 2 mL; the detection wavelength was 203 nm. The three components 34, 36 and 40 are subjected to preparative separation, 10 chromatographic peaks are collected, and the solvents are respectively recovered, as shown in fig. 2. 6 of the compounds were identified as N-methyltetrahydrofangchinamine (F34-1, tetrahydrofangchinamine (F34-2), protopine (F36-2), N-methyltetrahydropalmatine (F40-2), glaucine (F40-3) and tetrahydropalmatine (F40-4), and the other 4 peaks were not enough for nuclear magnetic identification.
The specific structural data for the compounds are as follows:
f34-1: light yellow powder, positive ion ESI-MS gives molecular ion peakm/z：356 [M]+；1H NMR (600 MHz, DMSO-d6):δ7.16 (d,J= 8.4 Hz, 1H, H-12), 7.14 (d,J= 8.4 Hz, 1H, H-11), 6.91 (s, 1H, H-1), 6.83 (s, 1H, H-4), 3.85 (s, 3H, OCH3-9), 3.81 (s, 3H, OCH3-3), 3.78 (s, 3H, OCH3-10), 2.82 (s, 3H, CH3-N);13C NMR (125 MHz, DMSO-d6)δ151.1 (C-10), 148.4 (C-3), 147.3 (C-9), 145.3 (C-2), 125.0 (C-12), 123.4 (C-14a), 122.3 (C-12a), 121.1 (C-4a), 120.4 (C-8a), 113.8 (C-11), 113.2 (C-1), 112.4 (C-4), 65.2 (C-14), 61.1 (C-8), 60.9 (C-6), 60.6 (OCH3-9), 56.4 (OCH3-3), 56.0 (OCH3-10), 38.9 (CH3-N), 28.2 (C-13), 23.2 (C-5). The data are consistent with the literature report of N-methyltetrahydroAfrican tetrandrine (N-methytrahydrocolumine).
F34-2: pale yellow powder, positive ion ESI-MS gives the peak of the excimer ionm/z：342 [M+H]+；1H NMR (600 MHz, DMSO-d6):δ8.17 (1H, s, -OH), 6.89 (1H, s, H-12), 6.72 (1H, s, H-11), 6.89 (1H, s, H-4) 6.66 (1H, s, H-1), 4.11 (1H, d,J= 16.2 Hz, Ha-8), 3.78 (3H, s, -OCH3), 3.75 (3H, s, -OCH3), 3.74 (3H, s, -OCH3).13C NMR (150 MHz, DMSO-d6):δ150.3 (C-9), 146.6 (C-10), 145.1 (C-2), 144.8 (C-3), 130.0 (C-14a), 128.4 (C-12a), 127.8 (C-8a), 125.0 (C-4a), 124.2 (C-12), 112.8 (C-4), 112.3 (C-11), 111.7 (C-1), 60.0, 59.0 (C-14, 9-OCH3), 56.2, 56.0 (2×OCH3) 53.7 (C-8), 51.5 (C-6), 36.0 (C-13), 28.7 (C-5). The data are consistent with the reported tetrahydrocolumbamine.
F36-2: white powder, positive ion ESI-MS gives the peak of the excimer ionm/z：354 [M+H]+；1H NMR (600 MHz, DMSO-d6)δ6.97 (1H, s), 6.82 (1H, s), 6.74 (1H, d,J= 7.8 Hz), 6.70 (1H, d,J= 7.8 Hz), 6.01 (2H, s), 5.98 (2H, s), 1.87 (3H, s);13C NMR (125 MHz, DMSO-d6)δ147.7 (C-3), 146.1 (C-2), 145.7 (C-9), 145.7 (C-10), 136.1 (C-4a), 132.8 (C-14a), 129.8 (C-12a), 125.3 (C-12), 118.3 (C-8a), 110.7 (C-4), 107.7 (C-1), 106.7 (C-11), 101.5 (O-CH2-O), 101.1 (O-CH2-O), 57.6 (C-6), 51.2 (C-8), 46.1 (C-13), 41.5 (N-CH3) 30.6 (C-5). The above data are consistent with the literature reports of protopine (protopine).
F40-2: light yellow powder, positive ion ESI-MS gives molecular ion peakm/z：370 [M]+；1H NMR (600 MHz, DMSO-d6)δ7.19 (d,J= 8.4 Hz, 1H, H-12), 7.16 (d,J= 8.4 Hz, 1H, H-11), 7.02 (s, 1H, H-1), 6.92 (s, 1H, H-4), 3.86 (s, 3H, OCH3), 3.83 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), 3.80 (s, 3H, OCH3), 2.84 (s, 3H, CH3-N).13C NMR (125 MHz, DMSO-d6)δ151.2 (C-10), 149.3 (C-3), 148.7 (C-2), 145.3 (C-9), 124.8 (C-12), 123.4 (C-14a), 123.1 (C-12a), 122.2 (C-4a), 121.1 (C-8a), 113.8 (C-11), 112.2 (C-1), 109.8 (C-4), 65.3 (C-14), 61.0 (C-8), 60.9 (C-6), 60.6 (OCH3-9), 56.4 (OCH3-2), 56.4 (OCH3-3), 56.0 (OCH3-10), 39.0 (CH3-N), 28.0 (C-13), 23.2 (C-5). The data are consistent with the reported N-methyltetrahydropalmatine (N-methyltetrahydropalmatine).
F40-3: brown green powder, positive ion ESI-MS gives the peak of the excimer ionm/z：356 [M+H]+；1H NMR (600 MHz, DMSO-d6):δ7.89 (1H, s, 11-H), 6.95 (1H, s, 8-H), 6.72 (1H, s, 3-H), 3.81 (6H, s, 2×OCH3), 3.78 (3H, s, OCH3), 3.60 (3H, s, OCH3)。13C NMR (150 MHz, DMSO-d6):δ152.0 (C-2), 148.4 (C-9), 147.5 (C-10), 144.2 (C-1), 129.9 (C-7a), 129.3 (C-13a), 127.4 (C-3b), 126.4 (C-11b), 124.2 (C-11a), 112.2 (C-11), 112.1 (C-8), 111.4 (C-3), 62.6 (C-6a), 60.0 (OCH3), 56.1 (OCH3), 56.0 (OCH3), 55.8 (OCH3), 53.1 (C-5), 44.1 (NCH3) 34.3 (C-7), 29.2 (C-4). The above data are consistent with literature reports of glaucine (glaucine).
F40-4: pale yellow powder, positive ion ESI-MS gives the peak of the excimer ionm/z：356 [M+H]+；1H NMR (600 MHz, CDCl3):δ[6.88 (1H, d,J= 8.4 Hz, H-12), 6.80 (1H, d,J= 8.4 Hz, H-11), 6.73 (1H, s, H-4), 6.62 (1H, s, H-1), 4.32(1H, d,J= 15.6 Hz, Ha-8), [3.89 (3H, s, OCH3), 3.87 (3H, s, OCH3), 3.86 (3H, s, OCH3), 3.85 (3H, s, OCH3), 3.62 (1H, d,J= 15.6 Hz, Hb-8), 2.90 (1H, m, H-5b), 2.72 (2H, m, H-13).13C NMR (150 MHz, CDCl3):δ150.4 (C-10), 147.7 (C-3), 147.5 (C-2), 145.5 (C-9), 128.9 (C-12a, C-14a), 127.2 (C-8a), 126.4 (C-4a), 123.8 (C-12), 111.3 (C-4), 111.1 (C-11), 108.5 (C-1), 101.3 (2, 3-OCH2O-), 60.2 (C-14), 59.1 (9-OH3), 56.1, 55.8, 55.8 (2, 3, 10-CH3) 53.5 (C-8), 51.1 (C-6), 35.7 (C-13), 28.6 (C-5). The data are consistent with tetrahydropalmatine (tetrahydropalmatine) reported in the literature.
Example 3:
selecting a component III, and performing preparative High Performance Liquid Chromatography (HPLC) separation in a reversed phase mode, wherein the chromatographic conditions are as follows: the chromatographic column is a common carbon eighteen column (XUnion C18); 0.1% phosphoric acid (pH 6.0 adjusted with triethylamine) (A) and acetonitrile (B) mobile phase system; the elution gradient is 0-30 min, 30-60% B; the flow rate is 20 mL/min; the sample injection amount is 2 mL; the detection wavelength was 203 nm. The preparation spectrum is shown in FIG. 3. 6 chromatographic peaks are collected, and the solvents are respectively recovered, wherein the P2 and P4 are not enough for nuclear magnetic identification, and the structural data of other 4 compounds are as follows:
p1: brown green powder, positive ion ESI-MS gives the peak of the excimer ionm/z：356 [M+H]+；1H NMR (600 MHz, CDCl3):δ8.06 (1H, s, 11-H), 6.77 (1H, s, 8-H), 6.61 (1H, s, 3-H), 3.91 (3H, s, OCH3), 3.88 (6H, s, 2×OCH3), 3.64 (3H, s, OCH3).13C NMR (150 MHz, CDCl3):δ153.2 (C-2), 148.5 (C-9), 148.0 (C-10), 145.1 (C-1), 127.3 (C-7a), 126.7 (C-13a), 126.3 (C-3b), 123.7 (C-11b), 121.7 (C-11a), 111.6 (C-11), 110.8 (C-8), 110.2 (C-3), 61.4 (C-6a), 60.3 (OCH3), 55.9 (OCH3), 55.9 (OCH3), 55.8 (OCH3), 51.8 (C-5), 40.2 (NCH3) 32.0 (C-7), 25.7 (C-4). The above data are consistent with literature reports of glaucine (glaucine).
P3: pale yellow powder, positive ion ESI-MS gives the peak of the excimer ionm/z：356 [M+H]+；1H NMR (600 MHz, CDCl3):δ[6.88 (1H, d,J= 8.4 Hz, H-12), 6.80 (1H, d,J= 8.4 Hz, H-11), 6.73 (1H, s, H-4), 6.62 (1H, s, H-1), 4.32(1H, d,J= 15.6 Hz, Ha-8), [3.89 (3H, s, OCH3), 3.87 (3H, s, OCH3), 3.86 (3H, s, OCH3), 3.85 (3H, s, OCH3), 3.62 (1H, d,J= 15.6 Hz, Hb-8), 2.90 (1H, m, H-5b), 2.72 (2H, m, H-13).13C NMR (150 MHz, CDCl3):δ150.4 (C-10), 147.7 (C-3), 147.5 (C-2), 145.5 (C-9), 128.9 (C-12a, C-14a), 127.2 (C-8a), 126.4 (C-4a), 123.8 (C-12), 111.3 (C-4), 111.1 (C-11), 108.5 (C-1), 101.3 (2, 3-OCH2O-), 60.2 (C-14), 59.1 (9-OH3), 56.1, 55.8, 55.8 (2, 3, 10-CH3) 53.5 (C-8), 51.1 (C-6), 35.7 (C-13), 28.6 (C-5). The data are consistent with tetrahydropalmatine (tetrahydropalmatine) reported in the literature.
P5: white powder, positive ion ESI-MS gives the peak of the excimer ionm/z：340 [M+H]+；1H NMR (600 MHz, CDCl3):δ[6.89 (1H, d,J= 8.4 Hz, H-12), 6.86 (1H, d,J= 8.4 Hz, H-11), 6.68 (1H, s, H-4), 6.62 (1H, s, H-1), 5.95 (2H, s, -O-CH2-O-), 4.53(1H, d,J= 15.6 Hz, Ha-8), 3.88 (3H, s, OCH3), 3.85 (3H, s, OCH3), 4.01 (1H, d,J= 15.6 Hz, Hb-8), 3.13 (3H, m, H-5a, Hb-8, H-14).13C NMR (150 MHz, CDCl3):δ150.8 (C-10), 147.3 (C-2), 147.0 (C-3), 145.2 (C-9), 123.9 (C-12), 112.4 (C-11), 108.7 (C-4), 105.3 (C-1), 60.4 (C-14), 55.9 (-OCH3) 29.7 (C-5). The data are consistent with the literature report of tetrahydroberberine (canadine).
P6: pale yellow powder, positive ion ESI-MS gives the peak of the excimer ionm/z：370 [M+H]+；1H NMR (600 MHz, CDCl3):δ6.90 (1H, d,J= 8.4 Hz, H-12), 6.82 (1H, d,J= 8.4 Hz, H-11), 6.68 (1H, s, H-1), 6.61 (1H, s, H-4), 4.20 (1H, d,J= 15.6 Hz, 8-Hb), 3.88 (6H, s, 2×OCH3), 3.86 (3H, s, OCH3), 3.85 (3H, s, OCH3), 3.71 (1H, brs, H-14), 3.50 (1H, d,J= 15.6 Hz, 8-Ha), 3.23 (1H, m, H-13), 3.10 (2H, m, H-6), 2.60 (2H, m, H-5), 0.95 (3H, d,J= 7.2 Hz, OCH3).13C NMR (150 MHz, CDCl3):δ150.0 (C-10), 147.6 (C-3), 147.2 (C-2), 144.8 (C-9), 134.8 (C-12a), 128.3 (C-8a, C-14a, C-4a), 124.0 (C-12), 111.1 (C-4), 110.9 (C-11), 108.6 (C-1), 63.0 (C-14), 60.1 (9-OH3), 56.1,55.8, 55.8 (2, 3, 10-CH3) 54.4 (C-8), 51.4 (C-6), 38.2 (C-13), 29.2 (C-5). The data are consistent with the corydaline (coreydaline) reported in the literature.
Example 4:
selecting a component IV, and separating by ion exchange chromatography separation mode preparative High Performance Liquid Chromatography (HPLC), wherein the chromatographic conditions are as follows: the chromatographic column is a silica gel-based strong cation exchange column (XCharge SCX); volume fraction 45% acetonitrile + volume fraction 10% 100 mM sodium dihydrogen phosphate (pH adjusted to 2.8 with phosphoric acid) + volume fraction 30% 100 mM sodium trifluoroacetate + volume fraction 15% water isocratically; the flow rate is 3 mL/min; the detection wavelength is 320 nm; the sample size was 100. mu.L. The spectrum is shown in FIG. 4. Collecting each chromatographic peak, and respectively recovering the solvent, wherein the P7 content is not enough for nuclear magnetic identification, and the structural data of other 4 compounds are as follows:
the specific structural data is as follows:
p8: yellow powder, positive ion ESI-MS gives molecular ion peakm/z：352 [M]+；1H NMR (400 MHz, MeOD)δ9.64 (s, 1H, H-8), 8.06 (d,J= 9.2 Hz, 1H, H-12), 7.85 (d,J= 9.2 Hz, 1H, H-11), 7.40 (s, 1H, H-1), 7.14 (s, 1H, H-4), 4.79 (t, 2H, H-6), 4.18 (s, 3H, OCH3-9), 3.98 (s, 3H, OCH3-3), 3.94 (s, 3H, OCH3-2), 3.18 (t, 2H, H-5), 3.03 (s, 3H, CH3-13);13C NMR (100 MHz, MeOD) δ 151.3 (C-3), 150.3 (C-10), 147.9 (C-2), 142.3 (C-9), 142.1 (C-8), 135.8 (C-14), 133.2 (C-12a), 131.6 (C-4a), 130.9 (C-11), 130.2 (C-13), 121.9 (C-8a), 120.7 (C-12), 119.5 (C-14a), 114.3 (C-1), 110.3 (C-4), 60.7 (OCH3-9), 57.2 (C-6), 55.6 (OCH3-2), 55.2 (OCH3-3), 27.3 (C-5), 16.7 (CH3-13). The above data are consistent with the literature reports of 13-methyl dehydrocorydalmine (13-methyl hydrocorydalmine).
P9: yellow powder, positive ion ESI-MS gives molecular ion peakm/z：352 [M]+；1H NMR (400 MHz, DMSO-d6)δ9.86 (s, 1H, H-8), 8.20 (d,J= 9.4 Hz, 1H, H-12), 8.17 (d,J= 9.4 Hz, 1H, H-11), 7.37 (s, 1H, H-1), 6.92 (s, 1H, H-4), 4.93-4.71 (m, 2H, H-6), 4.11 (s, 3H, OCH3-9), 4.09 (s, 3H, OCH3-10), 3.87 (s, 3H, OCH3-2), 3.11-3.03 (m, 2H, H-5), 2.98 (s, 3H, CH3-13);13C NMR (100 MHz, DMSO-d6)δ150.4 (C-10), 149.7 (C-14a), 146.8 (C-2), 144.4 (C-9), 144.2 (C-8), 136.9 (C-14), 133.6 (C-12a), 132.4 (C-4a), 129.7 (C-13), 126.3 (C-12), 121.6 (C-11), 121.0 (C-8a), 118.1 (C-3), 115.6 (C-1), 115.1 (C-4), 62.4 (OCH3-9), 57.3 (C-6), 57.3 (OCH3-10), 56.7 (OCH3-2), 27.1 (C-5), 18.1 (CH3-13). The data are consistent with the literature reports of dehydrocorydaline (dehydrocorybulbine).
P10: yellow powder, positive ion ESI-MS gives molecular ion peakm/z：366 [M]+；1H NMR (400 MHz, DMSO-d6)δ10.36 (1H, s, H-8), 7.93 (1H, d,J= 9.20, H-12), 7.87 (1H, d,J= 9.20, H-11), 7.27(1H, s, H-1), 6.93 (1H, s, H-4), 5.16 (2H, br. s, H-6), 3.24 (2H, br. s, H-5), 3.95 (3H, s, OCH3-2), 4.30 (3H, s, OCH3-3), 4.08 (3H, s, OCH3-9), 4.00 (3H, s, OCH3-10), 2.98(3H, s, CH3-13). The above data are consistent with literature reports of dehydrocorydaline (dehydrocorydaline).
P11: yellow powder, positive ion ESI-MS gives molecular ion peakm/z：352 [M]+；1H NMR (400 MHz, DMSO-d6)δ10.16 (1H, s, H-8), 8.86(1H, s, H-13), 7.98 (1H, d,J= 9.20, H-12), 7.71 (1H, d,J= 8.40, H-11), 7.45(1H, s, H-1), 6.74 (1H, s, H-4), 5.17 (2H, br. s, H-6), 3.29 (2H, br. s, H-5), 3.97 (3H, s, OCH3-2), 4.24 (3H, s, OCH3-3), 4.08 (3H, s, OCH3-9), 4.03 (3H, s, OCH3-10). The above data are consistent with the literature reports of palmatine (palmatine).
The method can realize the target preparation of alkaloid, can obtain batch of known active alkaloid, and simultaneously enrich and separate trace alkaloid, thereby continuously enriching alkaloid library and providing material basis for the activity research of alkaloid compounds and the development of new drugs with single component
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 changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. 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.

Claims (8)

1. A method for systematic separation and purification of corydalis tuber alkaloid is characterized in that: the method comprises the following steps:
(1) the corydalis ambigua methanol extract is segmented by a C18WCX reversed phase/weak cation exchange mixed mode chromatographic column to obtain 3 components, namely a corydalis ambigua trace alkaloid component, a major tertiary amine alkali component and a quaternary ammonium alkali component.
2, (2) separating and purifying alkaloid monomers from the 3 alkaloid components by using preparative liquid chromatography: separating and purifying rhizoma corydalis trace alkaloid component by two-dimensional reversed phase/reversed phase chromatography mode; separating and purifying rhizoma corydalis constant tertiary amine alkali component by reverse phase chromatography; separating and purifying the corydalis ambigua quaternary ammonium base component by ion exchange chromatography.
3. The method for systematic separation and purification of corydalis alkaloids according to claim 1, wherein: in the two-dimensional reversed phase/reversed phase chromatographic separation mode, a first-dimensional reversed phase chromatographic column is a polarity modified carbon eighteen column (XCharge C18), a mobile phase component is one or two of acetonitrile, methanol or ethanol and water containing formic acid or acetic acid, and the elution mode is a linear gradient; the second dimension reversed phase chromatographic column is common carbon eighteen column (XUnion C18, Unit C18, XTerra MS C18 or SunAire C18), the mobile phase composition is one or two of acetonitrile, methanol or ethanol mixed with acid water, and linear gradient, step gradient or isocratic elution mode is adopted.
4. The method for systematic separation and purification of corydalis alkaloids according to claim 1, wherein: in the reverse phase chromatographic separation mode, the reverse phase chromatographic column is a common carbon eighteen column (XUnion C18, Unit C18, XTerra MS C18 or SunAire C18), the mobile phase composition is one or two of acetonitrile, methanol or ethanol mixed with acid water, and a linear gradient, step gradient or isocratic elution mode is adopted.
5. The method for systematic separation and purification of corydalis alkaloids according to claim 3 or 4, wherein: the acid water contains one of acetic acid or phosphoric acid, and the pH is adjusted to 5.0-6.5 by triethylamine.
6. The method for systematic separation and purification of corydalis alkaloids according to claim 1, wherein: in the ion exchange separation mode, the ion exchange column is a silica gel-based strong cation exchange column (XCharge SCX), the mobile phase composition is one or two of acetonitrile, methanol or ethanol mixed with buffer salt, and isocratic elution mode is adopted.
7. The method for systematic separation and purification of corydalis alkaloids according to claim 6, wherein: the buffer salt contains phosphate and chaotropic salt, and the pH is adjusted to 1.5-5.0 by phosphoric acid.
8. The method for systematic separation and purification of corydalis alkaloids according to claim 1, wherein: the chromatographic operating parameters were as follows: the chromatographic operating parameters were as follows: the inner diameter of the chromatographic column is 4.6-1000 mm; the sample concentration is 1 mg/mL-1 g/mL; the sample amount is 10 mu L-400 mL; the flow rate is 0.5-480 mL/min; the column temperature is 25-45 ℃.
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