Method for high-throughput and high-efficiency separation of hydroxyl sanshool monomer based on two-dimensional chromatography combination

Abstract

The invention discloses a method for high-throughput and high-efficiency separation of a hydroxyl sanshool monomer based on two-dimensional chromatography combination, and belongs to the technical field of natural product separation and purification. In order to solve the problem that hydroxyl-epsilon-sanshool, hydroxyl-alpha-sanshool, hydroxyl-beta-sanshool and hydroxyl-gamma-sanshool in the pepper extract, especially cis-trans isomers with highly similar structures, are difficult to separate, the method combines high-speed counter-current chromatography and preparative liquid chromatography in an off-line manner. The method comprises the following steps: firstly, carrying out first-dimensional separation by utilizing high-speed counter-current chromatography to realize high-flux impurity removal so as to obtain a hydroxyl-gamma-sanshool monomer and enrich an isomer mixture; and carrying out second-dimensional high-resolution separation on the mixture by using preparative liquid chromatography so as to synchronously obtain four high-purity hydroxyl sanshool monomers. The method has the advantages of large separation flux, high efficiency, good resolution, high recovery rate and low cost, and provides a reliable technical means for standard substance preparation, deep research and product development of the pepper numb-taste substances.

Description

Technical Field

[0001] This invention belongs to the field of natural product separation and purification technology, specifically relating to a chromatographic separation method, and more particularly to a method for high-throughput and high-efficiency separation of hydroxy-ε-sanshool, hydroxy-α-sanshool, hydroxy-β-sanshool and hydroxy-γ-sanshool monomers from Sichuan pepper extract based on two-dimensional chromatography coupling technology. Background Technology

[0002] Sichuan pepper, a traditional spice and medicinal plant, derives its unique numbing sensation primarily from amide compounds in its pericarp. Among these, hydroxysalicornin compounds, represented by hydroxy-α-salicornin, hydroxy-β-salicornin, hydroxy-γ-salicornin, and hydroxy-ε-salicornin, are key flavor and active ingredients. These compounds not only determine the flavor quality of Sichuan pepper but have also been proven by modern research to possess various biological activities such as anti-inflammatory, analgesic, and antioxidant properties, showing broad application prospects in food, health product, and pharmaceutical development.

[0003] However, because hydroxy-ε-sanshool, hydroxy-α-sanshool, and hydroxy-β-sanshool are cis-trans isomers with extremely similar molecular structures and physicochemical properties, and often coexist with other impurities in plant extracts with significant differences in content, their efficient separation and large-scale preparation of pure monomers face enormous challenges. Currently, traditional methods for separating these amide compounds from Sichuan pepper mainly rely on silica gel column chromatography, analytical high-performance liquid chromatography (HPLC), and freeze crystallization. Silica gel column chromatography suffers from problems such as cumbersome operation, high solvent consumption, long separation cycle, limited resolution, and easy irreversible adsorption leading to loss of target components. While conventional analytical HPLC can achieve good qualitative and quantitative analysis (as disclosed in "Simultaneous Determination of the Content of Three Sanshools in Sichuan Pepper Extract by RP-HPLC" and "Study on the Differences in Numbing Substances of Sichuan Pepper from Different Germplasm Resources"), its initial design is for trace analysis and detection, with low column capacity, making it unsuitable for direct use in the preparation of active monomers at the gram scale or above.

[0004] Current technologies primarily focus on establishing accurate analytical methods to determine the content of various numbing substances in Sichuan pepper from different sources, or to compare their geographical distribution differences, falling under the scope of analytical chemistry and resource evaluation. These methods do not provide, nor do they offer insights into, how to solve the bottleneck problem in practical production and in-depth research and development: the simultaneous high-throughput, high-recovery preparation of multiple high-purity hydroxysanshool monomers from complex matrices. Therefore, developing an integrated preparation process capable of simultaneously achieving high-throughput coarse separation, efficient impurity removal, and precise separation of challenging cis-trans isomers is of great significance for promoting the in-depth development and utilization of active ingredients in Sichuan pepper, the preparation of standards, and the innovation of related products. Summary of the Invention

[0005] This invention aims to address the aforementioned problems in existing technologies. Specifically, it addresses the challenges of efficiently and on a large scale separating hydroxysansin compounds from Sichuan pepper extracts, particularly the separation of structurally similar cis-trans isomers (hydroxy-ε-sansin, hydroxy-α-sansin, and hydroxy-β-sansin) and simultaneously obtaining the hydroxy-γ-sansin monomer. The invention provides a simple, cost-effective, high-throughput, and highly efficient two-dimensional chromatography-coupled method. This method overcomes the shortcomings of traditional silica gel column chromatography, such as low efficiency and insufficient yield, and also overcomes the limitation of single analytical chromatography techniques in achieving gram-scale monomer preparation.

[0006] To achieve the above objectives, this invention provides a method for high-throughput and high-efficiency separation of hydroxy-ε-sanshool, hydroxy-α-sanshool, hydroxy-β-sanshool and hydroxy-γ-sanshool based on two-dimensional chromatography coupled with high throughput. The core of this method lies in the creative integration of the advantages of high-speed countercurrent chromatography and preparative liquid chromatography.

[0007] The method includes the following steps: (1) Dissolve the Sichuan pepper extract in an equal volume of a two-phase solvent to prepare an injection solution; (2) The solution from step (1) is injected into a high-speed countercurrent chromatograph, and first-dimensional chromatographic separation is performed using a selected two-phase solvent system. In this stage, high-throughput separation is achieved by utilizing the characteristics of high-speed countercurrent chromatography, which has no stationary phase, high loading capacity, and high recovery rate. The eluent is collected separately to directly obtain the hydroxy-γ-sanshool monomer fraction with high purity. At the same time, the three cis-trans isomers of hydroxy-ε-sanshool, hydroxy-α-sanshool, and hydroxy-β-sanshool are enriched and eluted as a mixture, and a large number of coexisting impurities are removed simultaneously in the process. (3) After dissolving the isomer mixture obtained in step (2), inject it into a preparative liquid chromatograph and use its high resolution to perform second-dimensional chromatographic separation. By optimizing the chromatographic column and elution conditions, baseline separation of the three cis-trans isomers is achieved, thereby obtaining high-purity hydroxy-ε-sanshool, hydroxy-α-sanshool and hydroxy-β-sanshool monomers respectively.

[0008] Furthermore, the Sichuan pepper extract may include Sichuan pepper oleoresin, Sichuan pepper ethanol extract, Sichuan pepper methanol extract, or Sichuan pepper acetonitrile extract.

[0009] Furthermore, the biphase solvent system described in steps (1) and (2) can be independently selected from: n-hexane / ethyl acetate / methanol / water, petroleum ether / ethyl acetate / methanol / water, n-butanol / ethyl acetate / methanol / water, n-butanol / ethyl acetate / water, n-hexane / methyl acetate / acetonitrile / water, or n-hexane / methyl tert-butyl ether / acetonitrile / water system, with a volume ratio ranging from (9:1:1:1) to (1:1:1:9).

[0010] Further, the separation conditions for high-speed countercurrent chromatography described in step (2) may include: circulating water bath temperature of 5-25℃, mobile phase flow rate of 0.5-2.5 mL / min, main unit rotation speed of 500-900 rpm, and operating mode of In-FWD or Out-FWD. The injection concentration may be 50-300 mg / mL, and the detection wavelength may be 280 nm.

[0011] Further, the separation conditions for preparing liquid chromatography described in step (3) may include: the chromatographic column is a Hedera ODS-2 column or a Sinochrom ODS-BP column; the elution solvent is an acetonitrile-water or methanol-water system, wherein the volume percentage of the organic phase is 20% to 45%; the elution flow rate is 5-30 mL / min; and the detection wavelength is 270 nm.

[0012] Furthermore, the target fractions collected in each step can be dried by methods such as reduced pressure evaporation, freeze drying, or vacuum drying, and the final product can be stored under nitrogen at low temperature (e.g., -80°C).

[0013] Compared with existing technologies, the two-dimensional chromatography coupling method provided by this invention has the following significant advantages: 1. High throughput and high loading capacity: The first dimension adopts high-speed countercurrent chromatography, which does not require a solid stationary phase. The sample loading capacity can reach the gram level, which can quickly process a large amount of crude extract, realize high-throughput preliminary separation and impurity removal, and solve the bottleneck of low throughput of traditional preparation methods.

[0014] 2. High resolution and high purity: The second dimension uses high-resolution preparative liquid chromatography, which is specifically designed to purify cis-trans isomer mixtures that are difficult to separate in the first dimension. It can achieve baseline separation of hydroxy-ε-sanshool, hydroxy-α-sanshool and hydroxy-β-sanshool, and obtain high-purity monomeric compounds (purity can reach more than 86%, preferably more than 99%).

[0015] 3. Process Integration and Complementary Advantages: This invention creatively combines the advantages of two chromatographic techniques. High-speed countercurrent chromatography (HSCGC) handles "coarse separation" and "impurity removal," solving the problem of preparation volume; preparative liquid chromatography (PLC) handles "fine separation," solving the problem of resolution. The two are used offline in conjunction, with clear steps, flexible operation, and synergistically realizing the entire process of preparing complex mixtures into various high-purity monomers.

[0016] 4. High recovery rate and economical cost: High-speed countercurrent chromatography has no irreversible adsorption, resulting in high recovery rates of target components. The entire process avoids the use of a large number of expensive chiral columns or repeated column chromatography, and the solvent can be recycled, effectively reducing the cost of large-scale preparation.

[0017] 5. Outstanding application value: The method provided by this invention can stably and in batches obtain four key hydroxysanshool monomer standards or active substances, providing a reliable material basis and technical support for in-depth pharmacological research on the numbing flavor of Sichuan pepper, the establishment of quality evaluation standards, and the development of high-value-added health products. Attached Figure Description

[0018] Figure 1 This is the spectrum obtained by high-speed countercurrent chromatography (HSCCC) for the first dimension separation of 1g of pepper oleoresin as a sample in Example 1 of the present invention.

[0019] Figure 2 This is the high-performance liquid chromatography (HPLC) analysis spectrum of the hydroxy-γ-sanshool monomer obtained by first-dimensional high-speed countercurrent chromatography in Example 2 of the present invention.

[0020] Figure 3 This is the spectrum obtained by preparative liquid chromatography (Prep-HPLC) of the isomer mixture obtained in the first dimension in Example 1 of the present invention.

[0021] Figure 4 This is the spectrum obtained from the second-dimensional preparative liquid chromatography separation in Example 3 of the present invention.

[0022] Figure 5 In embodiment 3 of the present invention, corresponding to Figure 4 The high-performance liquid chromatography (HPLC) purity verification spectra of the three monomeric compounds collected from each chromatographic peak are as follows: (a) hydroxy-ε-sanshool (purity 86.30%), (b) hydroxy-α-sanshool (purity 99.63%), and (c) hydroxy-β-sanshool (purity 99.15%). Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the reagents, instruments, and methods used in this invention are conventional reagents, instruments, and methods in this technical field.

[0024] The core of the method described in this invention lies in constructing a process system that first performs "rough separation and impurity removal" and then "refined separation" by offline coupling of "high-speed countercurrent chromatography (first dimension)" and "preparative liquid chromatography (second dimension)". Specific examples below demonstrate the applicability and separation effect of this method on extracts of Sichuan pepper from different sources.

[0025] Example 1: Separation, preparation and evaluation based on Zanthoxylum bungeanum oleoresin This embodiment uses pepper oleoresin as raw material to demonstrate the operation process of the present invention in detail, and performs structural characterization and performance comparison of the product.

[0026] 1. First-dimensional high-speed countercurrent chromatography separation: (1) Sample preparation: Accurately weigh 1.0 g of pepper oil resin, add it to a two-phase solvent system prepared and fully balanced by hexane, ethyl acetate, methanol and water in a volume ratio of 5:5:5:5, take 4 mL of each of the upper and lower phases to dissolve, and use sonication to aid dissolution to obtain the injection solution.

[0027] (2) Chromatographic separation: A high-speed countercurrent chromatograph was used. The upper phase of the solvent system described above was used as the stationary phase and pumped in at a flow rate of 2.0 mL / min. After the column was filled with the stationary phase, the main unit was turned on and run at 900 rpm. The lower phase was used as the mobile phase, with a flow rate of 2.0 mL / min and a circulating water bath temperature of 15℃. After hydrodynamic equilibrium was reached, the sample solution was injected. Detection was performed at a wavelength of 280 nm, and the resulting chromatogram is shown below. Figure 1 As shown.

[0028] (3) Component collection and processing: Based on the spectrum, the eluent during the period of approximately 130-200 min (purple peak) was collected, which was a mixture of hydroxy-ε-sanshool, hydroxy-α-sanshool, and hydroxy-β-sanshool; the eluent during the period of approximately 220-290 min (orange peak) was collected, which was identified as hydroxy-γ-sanshool. The organic solvent was removed by rotary evaporation under reduced pressure at 40 °C, and the residual aqueous solution was freeze-dried to obtain a mixture of dried crude hydroxy-γ-sanshool and its isomers.

[0029] 2. Second-dimensional preparative liquid chromatography separation: (1) Sample preparation: Take about 100 mg of the above dried isomer mixture and dissolve it in 1 mL of methanol to obtain the injection solution.

[0030] (2) Chromatographic separation: A preparative liquid chromatography system was used, equipped with a Hedera ODS-2 preparative column (250 mm × 20 mm, 5 μm). The mobile phase was acetonitrile-water (35:65, v / v), with isocratic elution, a flow rate of 10 mL / min, a column temperature of 30℃, and a detection wavelength of 270 nm. The above solution was injected. The separation chromatogram is shown below. Figure 3 As shown, the three peaks are well separated.

[0031] (3) Component collection and processing: Under the preparative liquid chromatography conditions described above, the chromatographic peaks of hydroxy-ε-sanshool, hydroxy-α-sanshool, and hydroxy-β-sanshool showed good symmetry, and the theoretical plate number was approximately 9.0 × 10⁻⁶. 3The resolution between adjacent peaks was greater than 1.5, achieving stable baseline separation, indicating that the chromatographic conditions had high separation efficiency. Each fraction was concentrated under reduced pressure at 40℃ to remove most of the solvent, and then freeze-dried to obtain three monomeric compounds.

[0032] Example 2: Separation based on the ethanol extract of Sichuan pepper This embodiment illustrates the applicability of the present invention to different types of extracts.

[0033] (1) Sample preparation: Weigh 0.3 g of Sichuan pepper ethanol extract and dissolve it in 3 mL of each of the upper and lower phases of a two-phase solvent system composed of petroleum ether, ethyl acetate, methanol and water in a volume ratio of 6:4:6:4.

[0034] (2) First-dimensional high-speed countercurrent chromatography (HSCCC) separation: Out-FWD mode was used, with the lower phase as the stationary phase and the upper phase as the mobile phase (flow rate 1.5 mL / min), the main unit speed was 800 rpm, the temperature was 10℃, and the detection wavelength was 280 nm. Sample injection and separation were performed.

[0035] (3) Second-dimensional preparative liquid chromatography (Prep-HPLC) separation: The collected isomer mixture was dissolved in 50% methanol aqueous solution. The chromatographic column was Sinochrom ODS-BP, the mobile phase was methanol-water (42:58, v / v), the flow rate was 8 mL / min, and the detection wavelength was 270 nm.

[0036] The high-performance liquid chromatography (HPLC) of the obtained hydroxy-γ-sanshool monomer showed the following purity: Figure 2 As shown, the recovery rate was 75.10 ± 2.3%, and the yield was 78.79 ± 3.6%. This demonstrates the robustness of the method to different raw materials.

[0037] Example 3: Isolation and Isomer Purity Verification Based on Sichuan Pepper Acetonitrile Extract This embodiment further demonstrates the method's ability to separate cis-trans isomers at high resolution.

[0038] (1) Sample preparation: Weigh 0.3 g of Sichuan pepper acetonitrile extract and dissolve it in 2 mL of each of the upper and lower phases of a two-phase solvent system composed of n-hexane, methyl acetate, acetonitrile and water in a volume ratio of 2:8:2:8.

[0039] (2) First-dimensional high-speed countercurrent chromatography (HSCCC) separation: In-FWD mode was used, with the following conditions: flow rate 1.0 mL / min, rotation speed 600 rpm, and temperature 25℃.

[0040] (3) Second-dimensional preparative liquid chromatography (Prep-HPLC) separation: The isomer mixture was dissolved in 50% acetonitrile aqueous solution. The chromatographic column was Sinochrom ODS-BP, the mobile phase was methanol-water (20:80, v / v), and the flow rate was 12 mL / min.

[0041] See the separation spectrum Figure 4 The three isomer peaks showed good separation. The products from each peak were collected and subjected to high-performance liquid chromatography (HPLC) purity analysis. The results are as follows: Figure 5 As shown: hydroxy-ε-sanshool (a) 86.30%, hydroxy-α-sanshool (b) 99.63%, hydroxy-β-sanshool (c) 99.15%. This result directly and strongly demonstrates that the second-dimensional preparative liquid chromatography of this invention can effectively solve the problem of separating cis-trans isomers and obtain high-purity monomers.

[0042] In summary, this invention successfully achieved high-throughput and efficient simultaneous separation of hydroxy-γ-sanshool and hydroxy-ε / α / β-sanshool cis-trans isomers from extracts of Sichuan pepper from different sources using a specific two-dimensional chromatography-coated process. The examples include complete structural confirmation and system performance comparisons, fully demonstrating that this method possesses outstanding substantive features and significant progress compared to existing technologies, and exhibits excellent practicality.

Claims

1. A method for high-throughput and high-efficiency separation of hydroxysanshool monomers based on two-dimensional chromatography, characterized in that, Includes the following steps: (1) Dissolve the Sichuan pepper extract in an equal volume of two-phase solvent to form an injection solution; (2) The injection solution is injected into a high-speed countercurrent chromatograph, and a two-phase solvent system is used for first-dimensional chromatographic separation. The fraction containing hydroxy-γ-sanshool and the mixed fraction containing hydroxy-ε-sanshool, hydroxy-α-sanshool and hydroxy-β-sanshool are collected respectively. (3) After dissolving the mixture of hydroxy-ε-sanshool, hydroxy-α-sanshool and hydroxy-β-sanshool obtained in step (2), inject it into a preparative liquid chromatograph and perform second-dimensional chromatographic separation using a second solvent system. Collect and obtain hydroxy-ε-sanshool, hydroxy-α-sanshool and hydroxy-β-sanshool monomers respectively.

2. The method according to claim 1, characterized in that, The Sichuan pepper extract is Sichuan pepper oleoresin, Sichuan pepper ethanol extract, Sichuan pepper methanol extract, or Sichuan pepper acetonitrile extract.

3. The method according to claim 1, characterized in that, The biphase solvent described in step (1) and the biphase solvent system described in step (2) are both independently selected from one of the following systems: n-hexane / ethyl acetate / methanol / water, petroleum ether / ethyl acetate / methanol / water, n-butanol / ethyl acetate / methanol / water, n-butanol / ethyl acetate / water, n-hexane / methyl acetate / acetonitrile / water, or n-hexane / methyl tert-butyl ether / acetonitrile / water; wherein the volume ratio of each component is (9:1:1:1) to (1:1:1:9).

4. The method according to claim 1, characterized in that, The conditions for the first dimension chromatographic separation in step (2) are: circulating water bath temperature 5-25℃, mobile phase flow rate 0.5-2.5 mL / min, main unit speed 500-900 rpm, operation mode In-FWD mode or Out-FWD mode, injection concentration 50-300 mg / mL, and detection wavelength 280 nm.

5. The method according to claim 1, characterized in that, The collected fractions described in steps (2) and (3) are dried by vacuum evaporation, freeze drying or vacuum drying.

6. The method according to claim 1, characterized in that, The second-dimensional chromatographic separation conditions in step (3) include: The chromatographic column is either a Hedera ODS-2 column or a Sinochrom ODS-BP column; The second solvent system is a mixed solution of acetonitrile and water or a mixed solution of methanol and water, wherein the ratio of acetonitrile:water is 20:80~45:55 or the ratio of methanol:water is 20:80~45:55; The elution flow rate is 5-30 mL / min; The detection wavelength is 270 nm.

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