阅读说明：本技术 一种莪术中强极性成分的分离制备及分析方法和应用 (Separation preparation and analysis method and application of strong polar components in curcuma zedoary ) 是由 梁鑫淼 相凯婧 周维佳 王纪霞 刘艳芳 于 2019-02-25 设计创作，主要内容包括：本发明提供了一种针对莪术中强极性成分的分离制备及分离分析方法。首先对莪术的极性组分进行制备,以降低样品复杂性。获得极性组分后,使用反相/亲水二维色谱分析方法进行分析：使用极性共聚的反相XAqua C18色谱柱进行第一维分析,酰胺键合的亲水色谱柱XAmide对其进行第二维分析。在发明中,以莪术极性成分为例,开发了二维RPLC×HILIC正交方法,可应用于从植物中分离极性化合物。该方法具有很好的正交性,解决了一般极性化合物在常规色谱填料上的弱保留问题,可以实现莪术中强极性成分的高效制备,为其他天然产物、生物样品以及药物制剂中极性化合物的分离纯化提供了良好的技术方案。(The invention provides a separation preparation and separation analysis method for strong polar components in curcuma zedoary. The polar component of zedoary was prepared first to reduce sample complexity. After obtaining the polar components, analysis was performed using a reverse phase/hydrophilic two-dimensional chromatographic analysis method: the first dimension was performed using a polar copolymerised reversed phase XAqua C18 column, which was subjected to the second dimension by amide-bound hydrophilic column XAmide. In the invention, a two-dimensional RPLC × HILIC orthogonal method is developed by taking the polar component of curcuma zedoary as an example, and the method can be applied to separating polar compounds from plants. The method has good orthogonality, solves the problem of weak retention of common polar compounds on conventional chromatographic packing, can realize high-efficiency preparation of strong polar components in the curcuma zedoary, and provides a good technical scheme for separation and purification of polar compounds in other natural products, biological samples and pharmaceutical preparations.)

1. A method for separating, preparing and analyzing strongly polar components in curcuma zedoary is characterized in that: separating and preparing the strong polar components in the curcuma zedoaria to obtain the strong polar components in the curcuma zedoaria, and then separating and analyzing the prepared strong polar components by a two-dimensional RPLC multiplied by HILIC orthogonal method.

2. The method of claim 1, wherein: the preparation method for separating the strongly polar components in the curcuma zedoary comprises the following specific steps:

weighing 3-10 kg of rhizoma Curcumae tuber, slicing, extracting with 5-20 times of 50-90% ethanol solution under reflux for three times (each time for 1-3 hr), mixing extractive solutions, vacuum filtering, and rotary evaporating at 50-70 deg.C under vacuum to concentrate to 5-10L; sequentially eluting the rotary-distillation concentrated solution with 3-5 times volume of n-heptane and ethyl acetate, and further drying the obtained ethyl acetate eluent under vacuum to obtain a sample; dissolving a sample to a concentration of 180-220mg/mL by adopting a methanol-water mixed solvent with a volume concentration of 50-50%; centrifuging the dissolved sample solution, filtering the supernatant through a 0.45-micrometer membrane to obtain a polar fraction, and preparing the polar fraction by using a Unit C18 column (50 × 265mm, 7 μm, Wasaburra, China); the preparation conditions are as follows: the mobile phase A is 0.05-0.1% formic acid (v/v) water solution, the mobile phase B is methanol, and gradient elution conditions are set as follows: 0 min (25% B) -5 min (30% B) -15 min (45% B) -40 min (70% B) -50 min (95% B) -60 min (95% B), a sample volume of 8-10mL, a flow rate of 70-90mL/min, recording chromatograms at a wavelength of 190-; collecting the peak fraction in the time close to the dead volume, and concentrating for later use to complete the separation and preparation of the strongly polar components in the curcuma zedoary.

3. The method of claim 1, wherein: the two-dimensional RPLC × HILIC orthogonal method is used for separating and analyzing the prepared strong polar components, and the specific method is as follows:

1) separating Curcumae rhizoma strongly polar components by first-dimension reversed-phase high performance liquid chromatography, wherein the chromatographic column is polar copolymerized C-eighteen column, the elution mode is linear gradient elution, and collecting one-dimensional prepared fraction;

2) and (3) separating the one-dimensional preparation fraction of the curcuma zedoary by using a second-dimensional hydrophilic high performance liquid chromatography, wherein a mobile phase consists of acetonitrile (A) and water (B), and 0.05-0.1% of formic acid is added to carry out second-dimensional preparation on the fraction to obtain the two-dimensional fraction of the polar compound.

4. A method according to claim 3, characterized by: the first dimension chromatographic column is one of reversed phase XAquaC18 column (10 × 250mm, 5 μm), XAqua C18 column (4.6 × 150mm,5 μm), XCharge C18PN column (4.6 × 150mm,5 μm) and Xbridge column (4.6 × 150mm,5 μm), and the second dimension chromatographic column is one of amide bonded hydrophilic chromatographic column XAmide column (2.1 × 250mm, 5 μm, Hua Shi, China), XAmide column (4.6 × 150mm,5 μm), Inertsil Diol column (4.6 × 150mm,5 μm) and BEH Silica column (4.6 × 150mm,5 μm);

the chromatographic operating parameters were as follows: the inner diameter of the chromatographic column is 2.1-50 mm; the sample injection concentration is 1mg/mL-140 mg/mL; the sample injection amount is 5 mu L-9 mL; the flow rate is 0.2-80 mL/min; the column temperature was 30 ℃.

5. A method according to claim 3, characterized by: the two-dimensional RPLC × HILIC orthogonal method is used for separating and analyzing the prepared strong polar components, and comprises the following specific steps:

separating the curcuma zedoary strongly polar component prepared in the step 1) by a first dimension polar copolymerization XAqua C18 column (10 multiplied by 250mm), selecting the wavelength of an ultraviolet detector as 190-400nm, injecting 50-200 mu L of sample, wherein a mobile phase consists of acetonitrile (A) and water (B), 0.05-0.1 percent of formic acid is added, and gradient elution parameters are as follows: 0 min (0% a) -6 min (0% a) -13 min (2% a) -16 min (8% a) -17 min (14% a) -20 min (24% a) -30 min (40% a) -35 min (70% a) -40 min (95% a) -45 min (95% a) at a flow rate of 3-3.5mL/min, collecting fractions according to chromatographic peak time, taking fractions once per minute from 3 min, taking 23 one-dimensional fractions in total, and nitrogen-blown concentration to 1mL for a second-dimensional hydrophilic chromatographic separation;

step 2) a second dimension analysis of 23 one-dimensional fractions was performed on a combined HPLC system consisting of 2998 photodiode matrix detector (Waters, USA) using XAmide column (2.1 × 250mm, 5 μm, hua spectral, china), mobile phase consisting of acetonitrile (a) and water (B), each with addition of 0.05% -0.1% formic acid, gradient elution conditions set as: 0 min (98% A) -12 min (98% A) -13 min (92% A) -30 min (75% A) -40 min (50% A) -50 min (5% A) -55 min (5% A) at a flow rate of 0.2mL/min, and chromatograms were recorded at 190-400 nm.

6. The use of the method according to any one of claims 1 to 5 for the separation and analysis of highly polar components in zedoary.

Technical Field

The invention relates to a separation analysis or separation preparation method and application of a strong polar compound in curcuma zedoary, in particular to an off-line two-dimensional chromatographic separation method, which utilizes the orthogonality of two-dimensional chromatography to improve the separation degree and analyze and separate the components of the polar compound.

The method can be used for separating and preparing the polar components of the curcuma zedoary so as to carry out subsequent activity tracing.

Technical Field

Traditional Chinese medicines are generally taken by decocting with water, a plurality of polar compounds exist in solution, and the compounds with different polarities act on different positions to finally achieve the effect of treating diseases. Experimenters have focused on the isolation and analysis of the natural components of the drug to find the active ingredients therein and to determine the mechanism of action. The search for pharmacologically active molecules in traditional Chinese medicine based on therapeutic effects will accelerate the discovery of new drugs. The active action of the polar components should not be ignored, so it is very important to separate the polar natural products in the Chinese medicine. (Y.M, Liu et al. journal of separation science,32(2009)2871)

High Performance Liquid Chromatography (HPLC) is one of the most efficient separation methods. The stationary phase of Reverse Phase Liquid Chromatography (RPLC) is typically a technique where particles are covalently bonded with alkyl chains to create hydrophobicity, with stronger affinity for compounds that are hydrophobic or less polar. When polar compounds are separated, the polar compounds are easily eluted directly from pores, and finally problems such as no retention or poor retention effect are caused. When the reversed phase chromatography is surface bonded with polar groups, retention of polar compounds can be improved. Hydrophilic interaction liquid chromatography (HILIC) is realized by bonding polar groups such as amino groups, amido bonds and the like on the surface of silica gel, in the HILIC, a mobile phase forms a water-rich layer rather than a water-deficient mobile phase on the surface of a polar stationary phase, and compared with a compound with lower polarity, the interaction between a compound with higher polarity and a fixed water layer is stronger. Are commonly used to isolate polar compounds such as sugars, polypeptides, saponins, and the like. Although High Performance Liquid Chromatography (HPLC) is one of the most effective separation methods, the separation effect in analyzing complex samples is limited by factors such as column performance, selectivity and peak capacity. HILIC separation of very polar compounds reveals a large amount of chemical information that is overlooked in the normal RPLC analysis. However, the complexity of polar compounds poses a huge challenge to their isolation beyond the capabilities of single-dimensional Chromatography (Z, Liang et al journal of Chromatography a,1224(2012) 61).

Two-dimensional liquid chromatography (2D-LC) is considered to be a very effective analytical method. The use of two chromatographic columns with different separation mechanisms can significantly improve the resolution and peak capacity (2D-LC peak capacity is the product of each dimension), and is widely used for the research of natural products due to its good orthogonality. Since the elution order in HILIC is opposite to that in RPLC separation, this means that HILIC is suitable for analyzing solutes that are problematic in that they are not retained or are poorly retained in RPLC. The increased retention of hydrophilic solutes in HILIC avoids the use of very low organic solvent concentrations and ion pair additives in RPLC. And the high organic modifier content provides two additional advantages to HILIC: ESI-MS has high sensitivity, lower viscosity of HILIC eluate and faster separation speed compared to standard RPLC eluate (Y.M, Liu et al. journal of separation science,32(2009) 2871). Thus, the different separation mechanisms of the two dimensions will result in good orthogonality. We will develop this two-dimensional chromatographic method to analyze the polar components in zedoary.

Zedoary is a traditional Chinese medicine, is widely distributed in various places of China and is widely used in folks, the chemical components of the zedoary mainly comprise volatile oil, curcumin, polysaccharides, sterols, phenolic acids, alkaloids and the like, and modern pharmacological studies show that the zedoary has various pharmacological effects of resisting tumors, resisting platelet aggregation, resisting thrombus, regulating blood fat, resisting atherosclerosis, resisting tissue fibrosis, resisting inflammation and easing pain, resisting bacteria and viruses, reducing blood sugar, resisting oxidation and the like (X.J, Chen et al. Few reports have focused on the polar compounds of zedoary, which may be closely related to the difficulty in separating and purifying the polar components of zedoary. Therefore, the invention develops a separation analysis method of 2D-LC by means of RPLC and HILIC aiming at the polar components of the curcuma zedoary.

Disclosure of Invention

The invention aims to provide a method for separating and analyzing strongly polar components in curcuma zedoary or a development method for preparing the strongly polar components.

The method is used for separating and separating the strong polar compounds in the curcuma zedoary by respectively carrying out reversed phase separation and hydrophilic separation on the polar components in the curcuma zedoary by utilizing the characteristics of good orthogonality of two-dimensional chromatography and obvious increase of peak capacity.

In order to achieve the purpose, the invention adopts the technical scheme that: separating and preparing the strong polar components in the curcuma zedoaria to obtain the strong polar components in the curcuma zedoaria, and then separating and analyzing the prepared strong polar components by a two-dimensional RPLC multiplied by HILIC orthogonal method.

The first dimension adopts a two-dimensional chromatographic analysis method combining a polarity reverse phase chromatographic column with a second dimension hydrophilic chromatographic column to separate and analyze the strong polar compounds in the curcuma zedoary, the mobile phases are acetonitrile (A) and water (B), 0.05 to 0.1 percent of formic acid is added, and the ultraviolet detector detects the wavelength of 190-. Wherein the polarity inversion column is XAqua C18 column, and the hydrophilic column is XAmid column. The chromatographic operating parameters were as follows: the inner diameter of the chromatographic column is 2.1-50 mm; the sample concentration is 1mg/mL-140 mg/mL; the sample injection amount is 5 mu L-9 mL; the flow rate is 0.2-80 mL/min; the column temperature was 30 ℃.

The separation and preparation operation steps of the strongly polar components in the zedoary are as follows:

the preparation method of the curcuma zedoary polar component comprises the following steps:

weighing 3-10 kg of rhizoma Curcumae tuber, slicing, extracting with 5-20 times of 50-90% ethanol solution under reflux for three times (each time for 1-3 hr), mixing extractive solutions, vacuum filtering, and rotary evaporating at 50-70 deg.C under vacuum to concentrate to 5-10L; sequentially eluting the rotary-distillation concentrated solution with 3-5 times volume of n-heptane and ethyl acetate, and further drying the obtained ethyl acetate eluent under vacuum to obtain a sample; dissolving a sample to a concentration of 180-220mg/mL by adopting a methanol-water mixed solvent with a volume concentration of 50-50%; centrifuging the dissolved sample solution, filtering the supernatant through a 0.45-micrometer membrane to obtain a polar fraction, and preparing the polar fraction by using a Unit C18 column (50 × 265mm, 7 μm, Wasaburra, China); the preparation conditions are as follows: the mobile phase A is 0.05-0.1% formic acid (v/v) water solution, the mobile phase B is methanol, and gradient elution conditions are set as follows: 0 min (25% B) -5 min (30% B) -15 min (45% B) -40 min (70% B) -50 min (95% B) -60 min (95% B), a sample volume of 8-10mL, a flow rate of 70-90mL/min, recording chromatograms at a wavelength of 190-; collecting the peak fraction in the time close to the dead volume, concentrating for later use, and completing the separation and preparation of the strongly polar components in the curcuma zedoary.

The method for separating and analyzing the prepared strong polar components by the two-dimensional RPLC multiplied HILIC orthogonal method comprises the following steps:

1) separating Curcumae rhizoma strongly polar components by first-dimension reversed-phase high performance liquid chromatography, wherein the chromatographic column is polar copolymerized C-eighteen column, the elution mode is linear gradient elution, and collecting one-dimensional prepared fraction;

the method comprises the following specific steps: the strongly polar component of Curcumae rhizoma is separated by one of first dimension polar copolymerization XAqua C18 column (10 × 250mm), XAqua C18 column (4.6 × 150mm,5 μm), XCharge C18PN column (4.6 × 150mm,5 μm), and Xbridge column (4.6 × 150mm,5 μm). The wavelength of the ultraviolet detector is selected to be 190-400nm, and the sample injection is 50-200 mu L. The mobile phase consists of acetonitrile (A) and water (B), 0.05% -0.1% formic acid is added, and gradient elution parameters are as follows: 0 min (0% A) -6 min (0% A) -13 min (2% A) -16 min (8% A) -17 min (14% A) -20 min (24% A) -30 min (40% A) -35 min (70% A) -40 min (95% A) -45 min (95% A) with a flow rate of 3-3.5 mL/min. Fractions were collected according to chromatographic peak time, taken once per minute from 3 min, 23 one-dimensional fractions in total, and concentrated to 1mL by nitrogen-blown chromatography for second-dimensional hydrophilic chromatographic separation.

2) And (3) separating the one-dimensional preparation fraction of the curcuma zedoary by using a second-dimensional hydrophilic high performance liquid chromatography, wherein a mobile phase consists of acetonitrile (A) and water (B), and 0.05-0.1% of formic acid is added to carry out second-dimensional preparation on the fraction to obtain the two-dimensional fraction of the polar compound.

The method comprises the following specific steps:

1) the second dimension analysis of the 23 one-dimensional fractions was performed on a combined HPLC system consisting of 2998 photodiode matrix detector (Waters, USA) using one of XAmide column (2.1X 250mm, 5 μm, Waals, China), XAmide column (4.6X 150mm,5 μm), Inertsil Diol column (4.6X 150mm,5 μm), BEH Silica column (4.6X 150mm,5 μm). The mobile phase consists of acetonitrile (A) and water (B), and 0.05-0.1% of formic acid is added. The gradient elution conditions were set as: 0 min (98% a) -12 min (98% a) -13 min (92% a) -30 min (75% a) -40 min (50% a) -50 min (5% a) -55 min (5% a). The flow rate was 0.2 mL/min. Chromatograms were recorded at 190-400 nm.

The method can be applied to the separation preparation and the separation analysis of the strong polar components in the curcuma zedoary.

The advantages of the invention

Provides an off-line reversed phase/hydrophilic two-dimensional chromatographic analysis method of the strong polar compounds in the curcuma zedoary, and provides good separation effect for the compounds which are relatively weak or not retained on the reversed phase. The method solves the problem of weak retention of polar compounds on conventional chromatographic packing, can realize high-efficiency preparation of the strong polar compounds in the curcuma zedoary, and provides a good technical scheme for separation and purification of the polar compounds in other natural products, biological samples and pharmaceutical preparations.

Drawings

FIG. 1 is a polar fraction collection chromatogram of the present invention (λ 254 nm);

fig. 2 shows a reverse phase chromatography column screening chromatogram (λ ═ 254nm) (a) XAqua C18(b) XCharge C18PN (C) XBridge C18;

fig. 3 is a reversed-phase mobile phase investigation chromatogram (λ 254nm) of the present invention (a) acetonitrile (b) methanol in mobile phase;

FIG. 4 is a one-dimensional reverse phase preparative chromatogram of the present invention (λ 254 nm);

FIG. 5 shows a screening chromatogram of a hydrophilic column (λ ═ 254nm) (a) XAmide (b) Inertsil Diol (c) BEH Silica according to the present invention;

fig. 6 shows the chromatogram for investigation of hydrophilic mobile phase (λ ═ 254nm) mobile phase: (a) no salt added (b)5mM ammonium formate (c)10mM ammonium formate;

FIG. 7 shows three-dimensional chromatograms (λ 254nm) of 23 polar fractions of Curcumae rhizoma.

Detailed Description

The present invention will now be further described with reference to examples. The examples are given solely for the purpose of illustration and are not intended to be limiting.