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Saponin separation and purification method

A technology for separation and purification of saponins, which is applied in the field of separation and purification of saponins, and can solve problems such as rigid chromatographic separation modes, limited types of hydrophilic chromatographic separation materials, and difficulty in discovering new compounds.

Active Publication Date: 2014-04-16
ZHANGJIAGANG IND TECH RES INST CO LTD DALIAN INST OF CHEM PHYSICS CHINESE ACADEMY OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
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Problems solved by technology

This method mainly has the following problems: one is that the saponin compound has strong polarity and poor solubility in the normal phase eluent, which leads to problems such as tailing and dead adsorption when it is separated on a silica gel column; the other is that , saponins generally only have absorption at the ultraviolet end, which brings inconvenience to the online detection of preparative high-performance liquid chromatography; third, the clustering of a large number of similar structures or isomer saponins exists, which is difficult for conventional C18 preparative chromatograms. The separation ability is a challenge; the fourth is that when the saponins are concentrated in water, a large amount of foam will be generated, which brings inconvenience to the sample treatment after the reversed-phase chromatography column; the fifth is that the controllability of the compound preparation is poor, and the effective target preparation cannot be achieved. , leading to the repeated acquisition of a large number of known compounds, resulting in a great waste of manpower and material resources; Sixth, the chromatographic separation mode is rigid and the separation selectivity is poor, making it more and more difficult to discover new compounds
Due to the limited types of commercially available hydrophilic chromatographic separation materials, there are few reports on their use in the preparation of saponins
In addition, some studies have reported the separation and preparation of ginsenosides by high-speed counter-current chromatography (HSCCC) (Qizhen Du, Gerold Jerzc, Reiner Waibelb, Peter Winterhalterc, Isolation of dammarane saponins from Panax notoginseng by high-speed counter-current chromatography, J. Chromatogr. A 1008 (2003) 173-180.; Young Wan Ha, Soon Sung Lim, In Jin Ha, Yun-Cheol Na, Jung-Ju Seo, Heungsop Shin, Sung Ho Son, Yeong Shik Kim, Preparative isolation of four ginsenosides from Korean red ginseng(steam-treated Panax ginseng C.A.Meyer), by high-speed counter-current chromatography coupled with evaporative light scattering detection, J.Chromatogr.A 1151(2007)37-44.; Yijun Cheng, QionglinLiang, Ping Hu, Yiming Wang, Frank Wu Jun, Guoan Luo, Combination of normal-phase medium-pressure liquid chromatography and high-performance counter-current chromatography for preparation of ginsenoside-Ro from panax ginseng with high recovery and efficiency, Separation and Purification Technology 73(2010) 397-402. , but this method is mainly suitable for the separation and preparation of known and high-content compounds, and the selection of a suitable extraction solvent system is very difficult, and the emulsification phenomenon is easy to occur during the extraction process, which brings inconvenience to the separation and detection

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  • Saponin separation and purification method

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Experimental program
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Effect test

Embodiment 1

[0021] Embodiment 1: Preparation of the first dimension fraction of saponin extract

[0022] The water extract of Notoginseng leaf was separated by preparative HPLC (C18 TDE column; water (A) and acetonitrile (B) mobile phase system; elution gradient was 0-5min, volume concentration 20%→32%B; 5-45min, Volume concentration 32%→68%B; 45-50min, volume concentration 68%→100%B; 50-55min, 100%B); during the separation process, an ultraviolet detector was used for detection, and the detection wavelength was 203nm. 1-55min, one collection per minute, a total of 54 components. Fractions 1-54 respectively, saponins mainly concentrated in Fractions 6-30.

Embodiment 2

[0023] Embodiment 2: the preparation of compound P1 and P2

[0024] Fraction 9 was selected for second-dimensional preparative HPLC separation using hydrophilic chromatography mode (the chromatographic column is an amide column; mobile phase system of water (A) and acetonitrile (B); volume concentration 76% B isocratic elution) The flow rate is 20mL / min; the detection wavelength is 203nm; the injection volume is 1mL. Each chromatographic peak was collected, the solvent was recovered separately, and two compounds, P1 and P2, were obtained through NMR experiments. The purity detected by HPLC is greater than 95%, and the data are as follows: P1, white powder, HR-ESI-MS: [M+H] + (m / z):1211.6409. 1 H NMR(600MHz,pyridine-d5)δ:0.77(1H,s,H-19),0.92(2H,s,H-18,30),1.08(1H,s,H-29),1.25(1H, s,H-28),1.60(1H,s,H-21),1.62(1H,s,H-27),1.64(1H,s,H-26),3.28(1H,dd,J=4.2, 11.4,H-3),5.29(1H,t,J=7.2,H-24),4.92(1H,d,J=7.8),5.50(1H,d,J=7.8),5.40(1H,d, J=6.6),5.13(1H,d,J=7.8),4.85(1H,brs). 13 C ...

Embodiment 3

[0025] Embodiment 3: the preparation of compound P3-P6

[0026] Fraction 11 was selected for second-dimensional preparative HPLC separation using hydrophilic chromatography mode (the chromatographic column is an amide column; mobile phase system of water (A) and acetonitrile (B); volume concentration 80% B isocratic elution) The flow rate is 20mL / min; the detection wavelength is 203nm; the injection volume is 1mL. Each chromatographic peak was collected, the solvent was recovered separately, and four compounds P3-P6 were obtained through NMR experiments. The purity detected by HPLC is greater than 95%, and the data are as follows: P3, white powder, HR-ESI-MS: [M+H] + (m / z):1079.5989. 1 H NMR(600MHz,pyridine-d5)δ:0.78(1H,s,H-19),0.92(1H,s,H-30),0.93(1H,s,H-18),1.08(1H,s, H-29),1.26(1H,s,H-28),1.60(1H,s,H-21),1.62(1H,s,H-27),1.64(1H,s,H-26),3.24 (1H,dd,J=4.2,11.4,H-3),5.29(1H,t,J=6.6,H-24),4.90(1H,d,J=7.8),5.35(1H,d,J= 7.8),5.12(1H,d,J=7.8),4.85(1H,brs). 13 C NMR: see Tab...

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Abstract

The invention provides a saponin separation and purification method. The method adopts two dimensions to prepare liquid chromatogram to high-efficiently separate and purify saponin monomers from a saponin extract, a first dimension adopts a reversed-phase chromatographic column, and a second dimension adopts a hydrophilic chromatography column. A mobile phase is prepared from acetonitrile, methyl alcohol, ethyl alcohol or water, does not add buffer salt, and is convenient for post-processing of sample preparation. A linear gradient, stagewise gradient or isocratic elution mode is adopted. A sanchi leaf aqueous extract is selected to be prepared into three representative fractions through the first dimension and is prepared into eight saponin monomers through the second dimension, namely the hydrophilic chromatography column, wherein the eight saponin monomers contain two pairs of isomerides and a new saponin. The method can realize the objective preparation of saponin, can obtain batch of known active saponins and enrich separated trace saponins at the same time, therefore constantly enriches a saponin library, and provides a material base for the activity research of saponin compounds and the development of new drugs with single component.

Description

technical field [0001] The present invention relates to the separation and purification of saponin compounds, in particular to a method of separating and preparing saponin monomers from saponin extracts by two-dimensional preparative chromatographic separation technology, including constant known saponins, saponin isomers and trace new saponins, etc. method. Background technique [0002] Saponins are a class of glycosides in which Sapogenins are Triterpenoids or Spirostane compounds. Saponins can be divided into monosaccharide chain saponins, disaccharide chain saponins and trisaccharide chain saponins according to the number of sugar chains linked by aglycone. In the chemical structure of saponins, because aglycones have different degrees of lipophilicity and sugar chains have strong hydrophilicity, saponin becomes a kind of surfactant, and its aqueous solution can produce persistent foam when shaken vigorously. [0003] Saponins not only have properties such as surface a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07J17/00C07J75/00B01D15/18
Inventor 梁鑫淼郭秀洁张秀莉郭志谋丰加涛肖远胜
Owner ZHANGJIAGANG IND TECH RES INST CO LTD DALIAN INST OF CHEM PHYSICS CHINESE ACADEMY OF SCI
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