A method for optimizing solvent system of counter-current chromatography for separation of complex samples of natural products and its application

Abstract

The application discloses a method for optimizing a countercurrent chromatography solvent system for separating a natural product complex sample and application thereof. The natural product sample is analyzed by using liquid chromatography, and average values of multiple components are calculated; according to types of chromatographic columns, corresponding solvent system mathematical models are brought in, and solvent systems for countercurrent chromatography are calculated; the calculated n-hexane / ethyl acetate / alcohol / water system is verified, a distribution coefficient K is calculated, solvent systems with relatively large K value deviations are corrected, and multiple solvent systems suitable for sample separation are obtained; a selectivity factor of adjacent chromatographic peaks is calculated α , and solvent system conditions with maximum or α ≥1.5 are selected α , so that an optimal countercurrent chromatography solvent system for separating a natural product complex sample is obtained. The high-efficiency optimization strategy of the countercurrent chromatography solvent system can meet the dual requirements of optimization and applicability of the solvent system when CCC is used to separate a natural product complex sample.

Description

Technical Field

[0001] This invention belongs to the field of natural product separation technology, specifically relating to an optimization method for a countercurrent chromatography solvent system for separating complex natural product samples and its application. Background Technology

[0002] "Multi-component, multi-target, multi-pathway, and multi-effect" is the main form in which traditional Chinese medicine (TCM) and natural drugs exert their biological functions. This dictates that the separation and analysis of mixtures of active ingredients are frequently involved in various stages of the research, development, and production of TCM and natural drugs. However, these mixtures often have different chemical structures, functional groups, and properties. Therefore, the separation and analysis of complex natural product systems often requires a wider variety of chromatographic systems (different stationary and mobile phases). Counter-current chromatography (CCC) is a chromatographic technique that utilizes the countercurrent partitioning of liquid and liquid phases to achieve separation. Compared with reversed-phase high-performance liquid chromatography (RP-HPLC), CCC not only eliminates the use of a solid stationary phase and offers flexible operation, but also provides an unlimited selection of solvent systems, offering a more economical and rational separation method for the preparation of active ingredients in TCM, TCM fingerprinting, and the pretreatment of complex metabolomics samples. However, opportunities and challenges go hand in hand. The liquid-liquid two-phase solvent system is a huge advantage of CCC in performing the separation of traditional Chinese medicine and natural products. At the same time, the selection and optimization of solvent system conditions has always been a time-consuming and laborious trial-and-error process full of uncertainties.

[0003] Rapid selection of various CCC solvent systems is fundamental to optimizing CCC solvent system conditions. Currently, for the most commonly used hexane / ethyl acetate / methanol / water (HEMWat) solvent system, domestic and international experts have proposed many useful solvent system selection strategies: the use of solvent families provides a general direction for CCC users to select solvent systems; theoretical models such as the Universal Functional Group Activity Coefficient (UNIFAC) model, the Cosmo-Real Solvent-Conductor Screening (COSMO-RS) model, and the Non-Random Two-Liquid (NRTL) model are used to calculate the solvent composition and compound solubility of each phase of the solvent system, enabling virtual prediction of the solvent system, but insufficient for predicting unknown compounds in complex systems; polarity is a key parameter affecting the dissolution and partitioning of compounds in CCC solvent systems. The average polarity calculation method to find solvent systems with polarities comparable to the target compound to be separated is widely used in complex samples, but the reliability of this method requires further verification with more examples; the generally useful estimate of solvent systems method (GUESS) based on thin-layer chromatography defines the K value of the optimal countercurrent separation solvent system as corresponding to the R value of the silica gel TLC plate in the organic phase of the solvent system. f The range of values ​​reduces the number of partition experiments required when selecting a countercurrent chromatography solvent system. In the HEMWat 9 × 9 table designed by Liang Junling et al., a clear linear relationship exists between the logarithm of the partition coefficient (log K) of a compound (base 10) and the solvent system composition (the ratio of n-hexane or methanol), and a "four-point screening" method based on the 9 × 9 chart strategy was proposed. Wang Xu et al. used an HPLC-assisted linear mathematical model for rapid selection of Arizona-type solvent systems. As mentioned above, the establishment of various CCC solvent system selection strategies simplifies the CCC application experiment process, but lacks an optimization process for solvent types. Due to the excessive difficulty in selecting solvent systems, higher-level optimization of solvent system conditions is even more challenging to achieve. Summary of the Invention

[0004] To address the lack of effectiveness in optimizing CCC solvent system conditions in existing technologies, this invention provides an optimization method for countercurrent chromatography solvent systems used for the separation of complex natural product samples and its application. By changing the type (methanol, ethanol, isopropanol) and solvent ratio of the alcohol solvent in the hexane / ethyl acetate / alcohol solvent / water system, the properties of the solvent system are altered. A mathematical method is established to describe the separation characteristics and applicable range of different solvent systems, thereby establishing an efficient optimization strategy for the hexane / ethyl acetate / alcohol solvent / water countercurrent chromatography solvent system.

[0005] This invention is achieved through the following technical solution: An optimization method for a countercurrent chromatography solvent system for separating complex natural product samples includes the following steps: (1) High performance liquid chromatography (HPLC) was used to analyze the natural product samples, and the B% of the target compound and the average values ​​of various components were calculated. % In the formula, t R For retention time; t D 1 represents the delay time of the high performance liquid chromatograph itself; ƙ represents the gradient slope; B0% represents the initial proportion of methanol in the gradient; and B% represents the actual proportion of methanol at the column tip when the sample elutes. (2) Based on the type of chromatographic column used, the average values ​​of multiple components in the natural product sample are calculated. The solvent system for countercurrent chromatography is calculated by substituting the corresponding mathematical model of the solvent system into the solvent system; the solvent system is n-hexane / ethyl acetate / alcohol solvent / water system. (3) The calculated n-hexane / ethyl acetate / alcohol solvent / water system was verified by shaking flask experiment. The peak areas of the samples in the upper and lower phases of different solvent systems were analyzed by HPLC, and the partition coefficient (K) was calculated. Where K is the allocation coefficient, A U A represents the peak area of ​​the sample in the upper phase. L The peak area of ​​the sample in the lower phase; (4) Correction was performed on solvent systems with large K-value deviations to obtain various suitable solvent systems for sample separation, 0.25 <K<2.5; (5) Calculate the selectivity factor of adjacent chromatographic peaks in natural product samples. α Select adjacent chromatographic peaks. α Maximum value or α Solvent system conditions ≥ 1.5 indicate that the solvent system conditions have been optimized, resulting in the optimal countercurrent chromatography solvent system for the separation of complex natural product samples. in, K1 is the selectivity factor of adjacent chromatographic peaks, K2 is the partition coefficient of the second adjacent chromatographic peak, and K1 is the partition coefficient of the first adjacent chromatographic peak.

[0006] Further, the chromatographic column mentioned in step (1) is an octadecyl bonded silica gel column; the n-hexane / ethyl acetate / alcohol solvent / water system is a n-hexane / ethyl acetate / methanol / water system, a n-hexane / ethyl acetate / ethanol / water system, and a n-hexane / ethyl acetate / isopropanol / water system.

[0007] Furthermore, the octadecyl-bonded silica gel column is an Agilent ZORBAX SB-C10. 18 , 5 μm, 4.6×250 mm, Agilent Extend-C 18 , 5 μm, 4.6×250 mm, Aglient TC-C 18 , 5 μm, 4.6×150 mm, Diamonsil C 18 , 5 μm, 4.6×250 mm or Thermo Hypersil GOLD, 5 μm, 4.6×250mm. Furthermore, when different n-hexane / ethyl acetate / alcohol solvent / water systems are presented in 9×9 chart form, for the vertical solvent system family (1:9:4:6 → 8:2:4:6), the horizontal solvent system family (5:5:2:8 → 5:5:8:2), and the diagonal solvent system family (3:7:1:9 → 9:1:7:3), the mathematical models for optimizing the n-hexane / ethyl acetate / alcohol solvent / water solvent system conditions based on different chromatographic columns are as follows (1)-(5): The solvent composition of the vertical solvent system family of n-hexane / ethyl acetate / methanol / water is: X:(10-X):4:6 (X is the proportion of n-hexane, 1 ≤ X ≤ 9), the solvent composition of the horizontal solvent system family is: 5:5:Y:10-Y (Y is the proportion of methanol, 1 ≤ Y ≤ 8), and the solvent composition of the diagonal solvent system family is: X:(10-X):(X-2):(12-X) (3 ≤ X ≤ 9). 9); Solvent composition of the vertical solvent system family in the n-hexane / ethyl acetate / ethanol / water system: M:(10-M):4:6 (M is the proportion of n-hexane, 1 ≤ M ≤ 9), solvent composition of the horizontal solvent system family: 5:5:N:(10-N) (N is the proportion of ethanol, 1 ≤ N ≤ 8), solvent composition of the diagonal solvent system family: M:(10-M):(M-2):(12-M) (3 ≤ M ≤ 9); Solvent composition of the vertical solvent system family in the n-hexane / ethyl acetate / isopropanol / water system: E:(10-E):4:6 (E is the proportion of n-hexane, 1 ≤ E ≤ 9), solvent composition of the diagonal solvent system family: E:(10-E):(E-2):(12-E) (3 ≤ E ≤ 9); (1) The chromatographic column was an Agilent ZORBAX SB-C. 18 The mathematical model for the n-hexane / ethyl acetate / alcohol solvent / water system is shown in the table below: (2) The chromatographic column is an Agilent Extend-C1000-2000-2000-2000-2000-2000-2000-3000-2000-3000-2000-3000-2000-3000-4 ... 18When the conditions are n-hexane / ethyl acetate / alcohol solvent / water system, the mathematical model is shown in the table below: (3) The chromatographic column is Aglient TC-C 18 When the conditions are n-hexane / ethyl acetate / alcohol solvent / water system, the mathematical model is shown in the table below: (4) The chromatographic column is Diamonsil C 18 When the conditions are n-hexane / ethyl acetate / alcohol solvent / water system, the mathematical model is shown in the table below: (5) When the chromatographic column is Thermo Hypersil GOLD, when the conditions are n-hexane / ethyl acetate / alcohol solvent / water system, the mathematical model is shown in the table below: Further, the operating method of the shaking flask experiment described in step (3) is: under the condition of 25 ± 3 ℃, prepare the solvent system for countercurrent chromatography calculated in step (2), shake fully, stand still and equilibrate, then transfer equal volumes of the upper phase solvent and the lower phase solvent into a sample bottle, add the natural product sample, shake fully again, stand still and equilibrate, then transfer equal volumes of the upper phase solvent and the lower phase solvent, blow to dry, dissolve with methanol and filter, which is the sample to be detected by HPLC in step (3).

[0008] Further, the method for correction using K value in step (4) is: if K<0.25, subtract 1 from the calculated value of the mathematical model for n-hexane / ethyl acetate / alcohol solvent / water system; if K>2.5, add 1 to the calculated value of the mathematical model for n-hexane / ethyl acetate / alcohol solvent / water system.

[0009] Further, in the liquid chromatography of step (1), mobile phase A is water, mobile phase B is methanol, and gradient elution is performed.

[0010] The present invention provides the high-efficiency optimization method and application of the countercurrent chromatography solvent system that can be used for separating complex samples of traditional Chinese medicines and natural products.

[0011] The beneficial effects obtained by the present invention are: The present invention establishes a high-efficiency optimization mathematical model for multiple solvent systems by changing the composition of the solvent system and the type of alcohol solvent, and 8 solvent systems satisfying 0.25<K<2.5 can be quickly obtained through one HPLC analysis, and select from them α ≥1.5 or αThe solvent system with the highest value is thus achieved, which realizes efficient optimization of solvent system conditions. Compared with the methods in the current literature, the present invention greatly improves the effectiveness of solvent system optimization, and its optimization efficiency is improved by about 50%.

[0012] Based on the HPLC-assisted linear fitting method, this invention systematically changes the solvent ratio and type of alcohol in the hexane / ethyl acetate / alcohol solvent / water system to alter the properties of the solvent system. By establishing a mathematical method to describe the separation characteristics and applicability of the solvent system, this invention further establishes an efficient selection and optimization strategy for the hexane / ethyl acetate / alcohol solvent / water countercurrent chromatography solvent system, meeting the dual requirements of effectiveness and applicability of CCC solvent system optimization for the separation of complex natural product samples. Attached Figure Description

[0013] Figure 1 The table shows the solvent system in 9×9 format and the solvent system family diagram in three representative directions: horizontal solvent system family (5:5:2:8 → 5:5:8:2); vertical solvent system family (1:9:4:6 → 8:2:4:6); and diagonal solvent system family (3:7:1:9 → 9:1:7:3). Figure 2 This is the HPLC chromatogram of the crude extract of the traditional Chinese medicine Scutellaria baicalensis. Figure 3 This is an HPLC chromatogram of the crude extract of Magnolia officinalis. Figure 4 This is a comparison chart of the "two-point method" and the "mathematical model method". Detailed Implementation

[0014] The technical solution of the present invention will be further described below with reference to the embodiments and accompanying drawings, but it is not limited thereto. Any modifications or equivalent substitutions to the technical solution of the present invention that do not depart from the spirit and scope of the technical solution of the present invention should be covered within the protection scope of the present invention.

[0015] instrument The high-performance liquid chromatograph (HPLC) system was an Agilent 1260 HPLC system (with a pre-column delay volume of 800 μL, equipped with a G1311C quaternary pump, a G1329B autosampler, a G1316A column oven, a G1315D DAD detector, and an Agilent ChemStation workstation); the column was an Agilent ZORBAX SB-C. 18 (5 μm, 4.6×250 mm), AgilentExtend-C 18 (5 μm, 4.6 × 250 mm) and Aglient TC-C 18 (5 μm, 4.6×150 mm);Diamonsil C18 (5 μm, 4.6×250 mm); Thermo Hypersil GOLD (5 μm, 4.6×250 mm).

[0016] Medicines and reagents Hexane, ethyl acetate, ethanol, and isopropanol were all of analytical grade; water was Wahaha purified water; methanol used for liquid chromatography analysis was of high performance liquid chromatography grade.

[0017] Standard substances: Apigenin (CAS: 520-36-5, purity ≥98%, batch number: T04S8F43072), Curcumin (CAS: 458-37-7, purity ≥98%, batch number: M13GB148328), Diosmetin (CAS: 520-34-3, purity ≥98%, batch number: T22F7X9844), Paclitaxel (CAS: 33069-62-4, purity ≥98%, batch number: J04S11H123510), Magnolin (CAS: 33069-62-4, purity ≥98%, batch number: J04S11H123510), Magnolol (CAS: 33069-62-4, purity ≥98%, batch number: J04S11H123510) lol, CAS: 528-43-8, purity ≥98%, batch number: Y27J10C91584), Androgapholide (CAS: 82209-72-1, purity ≥98%, batch number: X20J12H138184), Isoimperatorin (CAS: 482-45-1, purity ≥98%, batch number: M19GB148925), Resveratrol (CAS: 501-36-0, purity ≥98%, batch number: R26J10S94040), Tanshinone II A (Tanshinone II A, CAS: 568-72-9, purity ≥98%, batch number: J10GB151070), and Celastrol (CAS: 34157-83-0, purity ≥98%, batch number: N01GB166246). The traditional Chinese medicine Scutellaria baicalensis is a plant of the Lamiaceae family, Scutellaria baicalensis (…). Scutellaria baicalensis The dried root of *Magnolia officinalis*, a plant in the Magnoliaceae family. Magnolia officinalis Dried bark of Rehd.et Wils.

[0018] HPLC test method: HPLC conditions: Mobile phase: water (A)-methanol (B), gradient elution: 0-45 min, 10%→100% B; 45-55 min, 100% B. Flow rate: 1.0 mL / min. -1The column temperature was 30 ℃, the injection volume was 10 μL, and the detection wavelengths were apigenin 268 nm, curcumin 452 nm, geraniol 346 nm, paclitaxel 230 nm, magnolol 290 nm, andrographolide 226 nm, isoimperatorin 250 nm, resveratrol 308 nm, tanshinone IIA 270 nm, and triptolide 425 nm. The peak areas (A) of the target chromatographic peaks were recorded.

[0019] The formula for calculating B% is as follows: Formula type, t R For retention time; t D 1 represents the delay time of the high performance liquid chromatograph itself; ƙ represents the gradient slope; B0% represents the initial proportion of methanol in the gradient; and B% represents the actual proportion of methanol at the column tip when the sample elutes. Method for calculating the distribution coefficient (K) (shaking flask experiment) At room temperature (25 ± 3 ℃), prepare a 10 mL HSCCC solvent system. After thorough mixing and equilibration, accurately transfer 2 mL each of the upper and lower phase solvents into a 10 mL sample vial. Add 1 mg of each of the 10 standards to each vial, shake thoroughly, and allow to equilibrate. Accurately transfer 1 mL each of the upper and lower phase solvents into a 10 mL sample vial, dry with nitrogen evaporation, accurately transfer 2 mL of methanol to dissolve the sample, filter through a 0.22 μm organic filter into a liquid chromatography vial, and perform HPLC analysis under the chromatographic conditions described below. The formula for calculating the partition coefficient K is: In the formula, A U A represents the peak area of ​​the sample in the upper phase. L This represents the peak area of ​​the sample in the lower phase.

[0020] Selection factor ( α Calculation method of ) in, K1 is the selectivity factor for adjacent chromatographic peaks, K2 is the partition coefficient of the second adjacent chromatographic peak, and K1 is the partition coefficient of the first adjacent chromatographic peak. Example 1 Establishment of a mathematical model for solvent system optimization: (1) 9×9 diagrams for the hexane / ethyl acetate / methanol / water (HEMWat) system, the hexane / ethyl acetate / ethanol / water (HEEWat) system, and the hexane / ethyl acetate / isopropanol / water (HEIWat) system (e.g.) Figure 1As shown, representative solvent system families in three directions—horizontal (5:5:2:8 → 5:5:8:2), vertical (1:9:4:6 → 8:2:4:6), and diagonal (3:7:1:9 → 9:1:7:3)—were selected for exploration. The solvent composition of the vertical solvent system family in the HEMWat system can be described as hexane: ethyl acetate: methanol: water = X:(10-X):4:6 (1 ≤ X ≤ 9), where X is the proportion of hexane. The solvent composition of the horizontal solvent system family in the HEMWat system can be described as hexane: ethyl acetate: methanol: water = 5:5:Y:(10-Y) (1 ≤ Y ≤ 8, when X>8, the two-phase solvent system cannot form two phases), where Y is the proportion of methanol. The solvent composition of the diagonal solvent system family in the HEMWat system can be described as X:(10-X):(X-2):(12-X) (3 ≤ X ≤ 9). Similarly, in the 9×9 table of the HEMWat system, the solvent composition of the vertical solvent system family in the HEMWat system can be hexane: ethyl acetate: ethanol: water = M:(10-M):4:6 (1 ≤ M ≤ 9), where M is the proportion of hexane. The solvent composition of the HEEWat system's horizontal solvent system family can be described as hexane: ethyl acetate: ethanol: water = 5:5: N:(10-N) (1≤N≤7, when N>7, the two-phase solvent system cannot form two phases), where N is the proportion of ethanol. The solvent composition of the HEEWat system's diagonal solvent system family can be described as M:(10-M):(M-2):(12-M) (3≤M≤9). Similarly, in the 9×9 table of the HEIWat system, the solvent composition of the solvent system family in the vertical direction of the HEIWat system can be described as hexane: ethyl acetate: ethanol: water = E:(10-E):4:6 (1 ≤ E ≤ 9), where E is the proportion of hexane; the solvent composition of the solvent system family in the horizontal direction of the HEIWat system can be described as hexane: ethyl acetate: ethanol: water = 5:5:F:(10-F) (1 ≤ F ≤ 7, when F>7, the two-phase solvent system cannot form two phases), where F is the proportion of isopropanol. The solvent composition of the solvent system family in the diagonal direction of the HEIWat system can be described as E:(10-E):(E-2):(12-E) (3 ≤ E ≤ 9).

[0021] (2) The composition and distribution relationship of solvent systems (HEMWat, HEEWat, and HEIWat) and the applicable range of solvent systems were described using template molecules (10 natural compounds: apigenin, curcumin, geraniol, paclitaxel, magnolol, andrographolide, isoimperatorin, resveratrol, tanshinone IIA, and tripterygium wilfordii). The polarity of template molecules (methanol content B%) was characterized by HPLC. A highly efficient optimization model for the hexane / ethyl acetate / alcohol solvent / water solvent system was established by analyzing the linear correlation between template molecule polarity and solvent composition. The relevant mathematical models for different chromatographic columns are shown in Tables 1-5 below. Table 1. Mathematical model of the n-hexane / ethyl acetate / alcohol solvent / water system (Chromatographic column: Agilent ZORBAX SB-C) 18 ) Table 2. Mathematical Model of the n-Hexane / Ethyl Acetate / Alcohol Solvent / Water System (Column: Agilent Extend-C) 18 ) Table 3. Mathematical model of the n-hexane / ethyl acetate / alcohol solvent / water system (chromatographic column: Aglient TC-C) 18 ) Table 4. Mathematical model of the n-hexane / ethyl acetate / alcohol solvent / water system (chromatographic column: Diamonsil C10) 18 ) Table 5. Mathematical model of the n-hexane / ethyl acetate / alcohol solvent / water system (Chromatographic column: Thermo HypersilGOLD) Example 2 Optimization of HSCCC solvent system for Scutellaria baicalensis (Agilent ZORBAX SB-C18 column (5 μm, 4.6 × 250 mm)) (1) The crude extract of Scutellaria baicalensis from natural product samples was analyzed by HPLC (chromatogram as shown in Figure 1). Figure 2 (As shown), calculate the target compound B% and the average values ​​of multiple components. %; four main chromatographic peaks in Scutellaria baicalensis: Compound 1: 31.78 min, 71.96% B; Compound 2: 33.51 min, 75.42% B; Compound 3: 34.71 min, 77.82% B; Compound 4: 35.65 min, 79.70% B; the average methanol proportion at the column front when four main compounds in Scutellaria baicalensis elute ( %) is 76.23%; (2) According to the type of chromatographic column used, substitute the average value of multiple components of the natural product sample % into the corresponding solvent system mathematical model of the solvent, and calculate the countercurrent chromatography solvent system (n-hexane / ethyl acetate / alcohol solvent / water system) for the traditional Chinese medicine Scutellaria baicalensis; (3) Perform shake-flask experiment to verify the calculated n-hexane / ethyl acetate / alcohol solvent / water system, analyze the peak area of the sample in the upper and lower phases of different solvent systems by HPLC, and calculate the K value; (4) Correct the solvent system with large K value deviation to obtain a variety of solvent systems suitable for sample separation (0.25<K<2.5); if K<0.25, subtract 1 from the calculated value of the mathematical model for n-hexane / ethyl acetate / alcohol solvent / water system conditions; if K>2.5, add 1 to the calculated value of the mathematical model for n-hexane / ethyl acetate / alcohol solvent / water system conditions; (5) Calculate the selectivity factor of adjacent chromatographic peaks in Scutellaria baicalensis α , select each adjacent chromatographic peak α with the maximum value or α the solvent system condition with ≥ 1.5, that is, the condition optimization of the solvent system is completed, and the optimal countercurrent chromatography solvent system for separating target compounds in Scutellaria baicalensis is obtained; According to the above "mathematical model method", the countercurrent chromatography solvent system, partition coefficient K and selectivity factor screened for four compounds in Scutellaria baicalensis α are shown in Table 6 below. As can be seen from Table 6, the optimal solvent system for Scutellaria baicalensis extract is HEEat = 6.5:3.5:4.0:6.0.

[0022] Table 6 Countercurrent chromatography solvent systems, partition coefficients K and selectivity factors of four target compounds in Scutellaria baicalensis screened by "mathematical model method" α .

[0023] Example 3 Optimization of HSCCC solvent system for traditional Chinese medicine Magnolia officinalis (chromatographic column: Agilent ZORBAX SB-C18 (5 μm, 4.6×250 mm)) (1) HPLC chromatographic analysis is performed on the crude extract of Magnoliae Officinalis Cortex, a natural product sample (the chromatogram is shown in Figure 3 ), calculate the target compound B% and the average value of various components %; two main chromatographic peaks in Magnoliae Officinalis Cortex: Compound 5: 38.41 min, 85.22% B; Compound 6: 40.05 min, 88.5% B. The average methanol proportion at the column front when the two main compounds in Magnoliae Officinalis Cortex elute ( %) is 86.86%; ; (2) According to the type of chromatographic column used, the average value of various components in the natural product sample % is substituted into the corresponding solvent system mathematical model of the solvent to calculate the countercurrent chromatography solvent system (n-hexane / ethyl acetate / alcohol solvent / water system) for traditional Chinese medicine Magnoliae Officinalis Cortex; (3) Shake flask experiment is used to verify the calculated n-hexane / ethyl acetate / alcohol solvent / water system, HPLC is used to analyze the peak area of the sample in the upper and lower phases of different solvent systems, and the K value is calculated; (4) Correct the solvent system with large K value deviation to obtain a variety of solvent systems suitable for sample separation (0.25<K<2.5); if K<0.25, subtract 1 from the calculated value of the n-hexane / ethyl acetate / alcohol solvent / water system mathematical model; if K>2.5, add 1 to the calculated value of the n-hexane / ethyl acetate / alcohol solvent / water system mathematical model; (5) Calculate the selectivity factor of adjacent chromatographic peaks in Magnoliae Officinalis Cortex α , select for each pair of adjacent chromatographic peaks α with the maximum value or α the solvent system condition with ≥ 1.5, the condition optimization of the solvent system is completed, and the optimal countercurrent chromatography solvent system for separating the target compounds in Magnoliae Officinalis Cortex is obtained; According to the above "mathematical model method", the countercurrent chromatography solvent systems, partition coefficients K and selectivity factors screened for the 2 compounds in Magnoliae Officinalis Cortex α are shown in Table 7 below. It can be seen from Table 7 that the optimal solvent system for Magnoliae Officinalis Cortex extract is HEMWat = 7.7:2.3:5.7:4.3.

[0024] Table 7 Countercurrent chromatography solvent systems, partition coefficients K and selectivity factors of two compounds in Magnoliae Officinalis Cortex screened by "mathematical model method" α Comparative Example 1 The "two-point method" is used to screen and calculate the solvent system, partition coefficient K and selectivity factor of 4 compounds in Scutellariae Radix α , and the results are shown in Table 8 below: Table 8. Solvent systems, partition coefficients K, and selectivity factors of four compounds from Scutellaria baicalensis screened using the "two-point method". α Comparative Example 2 The solvent system, partition coefficient K, and selectivity factor of four compounds in Magnolia officinalis were determined using a two-point method. α The results of the screening and calculation are shown in Table 9 below: Table 9. Solvent systems, partition coefficients K, and selectivity factors α of two compounds from Magnolia officinalis screened using the "two-point method". Comparison of the "two-point method" and the "mathematical model method" A comparison between the "two-point method" and the "mathematical model method" reveals (e.g.) Figure 4 As shown, when screening the optimal solvent system from the same family of solvent systems, the screening results of the two methods are similar, but the number of shake-flask experiments for the Chinese herb Scutellaria baicalensis can be reduced from 18 in the two-point method to 12 in the mathematical model method, while the number of shake-flask experiments for the Chinese herb Magnolia officinalis can be reduced from 15 to 5. This greatly improves the experimental efficiency and achieves efficient optimization of the solvent system.

Claims

1. An optimization method for a countercurrent chromatography solvent system for the separation of complex natural product samples, characterized in that, Includes the following steps: (1) High performance liquid chromatography was used to analyze the natural product samples, and the percentage of the target compound B and the average value of multiple components were calculated. % In the formula, t R For retention period; t D 1 represents the delay time of the high performance liquid chromatograph itself; ƙ represents the gradient slope; B0% represents the initial proportion of methanol in the gradient; and B% represents the actual proportion of methanol at the column tip when the sample elutes. (2) Based on the type of chromatographic column used, the average values ​​of multiple components in the natural product sample are calculated. The solvent system for countercurrent chromatography is calculated by substituting the corresponding mathematical model of the solvent system; the solvent system is n-hexane / ethyl acetate / alcohol solvent / water system. (3) The calculated n-hexane / ethyl acetate / alcohol solvent / water system was verified by shaking flask experiment. The peak areas of the samples in the upper and lower phases of different solvent systems were analyzed by HPLC, and the partition coefficient K was calculated. Where K is the allocation coefficient, A U A represents the peak area of ​​the sample in the upper phase. L The peak area of ​​the sample in the lower phase; (4) Correct the solvent system with large K value deviation to obtain a variety of suitable sample separation solvent systems, 0.25 < K < 2.5; (5) Calculate the selectivity factor of adjacent chromatographic peaks in natural product samples. α Select adjacent chromatographic peaks α Maximum value or α Solvent system conditions ≥ 1.5 indicate that the solvent system conditions have been optimized, resulting in the optimal countercurrent chromatography solvent system for the separation of complex natural product samples. in, K1 is the selectivity factor for adjacent chromatographic peaks, K2 is the partition coefficient of the second adjacent chromatographic peak, and K1 is the partition coefficient of the first adjacent chromatographic peak. The chromatographic column used was an Agilent ZORBAX SB-C. 18 The mathematical model for the hexane / ethyl acetate / alcohol solvent / water system is shown in the table below: (2) The chromatographic column is an Agilent Extend-C. 18 The mathematical model for the hexane / ethyl acetate / alcohol solvent / water system is shown in the table below: (3) The chromatographic column is an Aglient TC-C. 18 The mathematical model for the hexane / ethyl acetate / alcohol solvent / water system is shown in the table below: (4) The chromatographic column was Diamonsil C10. 18 The mathematical model for the hexane / ethyl acetate / alcohol solvent / water system is shown in the table below: (5) When the chromatographic column is Thermo Hypersil GOLD, the mathematical model of the n-hexane / ethyl acetate / alcohol solvent / water system is shown in the table below: ； Where X represents the percentage of n-hexane, Y represents the percentage of methanol, M represents the percentage of n-hexane, N represents the percentage of ethanol, and E represents the percentage of n-hexane.

2. The method for optimizing a countercurrent chromatography solvent system for separating complex natural product samples according to claim 1, characterized in that, The chromatographic column mentioned in step (1) is an octadecyl bonded silica gel column; the n-hexane / ethyl acetate / alcohol solvent / water system is a n-hexane / ethyl acetate / methanol / water system, a n-hexane / ethyl acetate / ethanol / water system, and a n-hexane / ethyl acetate / isopropanol / water system.

3. The method for optimizing a countercurrent chromatography solvent system for separating complex natural product samples according to claim 2, characterized in that, The octadecyl-bonded silica gel column is an Agilent ZORBAX SB-C10. 18 , 5 μm, 4.6×250 mm, Agilent Extend-C 18 , 5 μm, 4.6×250 mm, Aglient TC-C 18 , 5 μm, 4.6×150 mm, Diamonsil C 18 , 5 μm, 4.6×250 mm or Thermo Hypersil GOLD, 5 μm, 4.6×250 mm.

4. The method for optimizing a countercurrent chromatography solvent system for separating complex natural product samples according to claim 3, characterized in that, When different n-hexane / ethyl acetate / alcohol solvent / water systems are presented in 9×9 graph form, for the vertical solvent system family 1:9:4:6→8:2:4:6, the horizontal solvent system family 5:5:2:8→5:5:8:2, and the diagonal solvent system family 3:7:1:9→9:1:7:3, the mathematical models for optimizing the conditions of the n-hexane / ethyl acetate / alcohol solvent / water solvent system based on different chromatographic columns are as follows (1)~(5). The solvent composition of the vertical solvent system family of n-hexane / ethyl acetate / methanol / water is: X:(10-X):4:6, where X is the proportion of n-hexane, 1 ≤ X ≤ 9; the solvent composition of the horizontal solvent system family is: 5:5:Y:10-Y, where Y is the proportion of methanol, 1 ≤ Y ≤ 8; the solvent composition of the diagonal solvent system family is: X:(10-X):(X-2):(12-X), where 3 ≤ X ≤ 9; Solvent composition of the vertical solvent system family in the n-hexane / ethyl acetate / ethanol / water system: M:(10-M):4:6, where M is the proportion of n-hexane, 1 ≤ M ≤ 9; Solvent composition of the horizontal solvent system family: 5:5:N:(10-N), where N is the proportion of ethanol, 1 ≤ N ≤ 8; Solvent composition of the diagonal solvent system family: M:(10-M):(M-2):(12-M), where 3 ≤ M ≤ 9; Solvent composition of the vertical solvent system family in the n-hexane / ethyl acetate / isopropanol / water system: E:(10-E):4:6, where E is the proportion of n-hexane, 1 ≤ E ≤ 9; Solvent composition of the diagonal solvent system family: E:(10-E):(E-2):(12-E), where 3 ≤ E ≤ 9; (1) The chromatographic column was an Agilent ZORBAX SB-C. 18 The mathematical model for the hexane / ethyl acetate / alcohol solvent / water system is shown in the table below: (2) The chromatographic column is an Agilent Extend-C1000-2000-2000-2000-2000-2000-2000-3000-2000-3000-2000-3000-2000-3000-4 ... 18 The mathematical model for the hexane / ethyl acetate / alcohol solvent / water system is shown in the table below: (3) The chromatographic column is an Aglient TC-C. 18 The mathematical model for the hexane / ethyl acetate / alcohol solvent / water system is shown in the table below: (4) The chromatographic column was Diamonsil C10. 18 The mathematical model for the hexane / ethyl acetate / alcohol solvent / water system is shown in the table below: (5) When the chromatographic column is Thermo Hypersil GOLD, the mathematical model of the n-hexane / ethyl acetate / alcohol solvent / water system is shown in the table below: 。 5. The method for optimizing a countercurrent chromatography solvent system for separating complex natural product samples according to claim 1, characterized in that, The operation method of the shake flask experiment described in step (3) is as follows: under the condition of 25±3℃, prepare the solvent system for countercurrent chromatography calculated in step (2), shake it thoroughly and let it stand for equilibrium, transfer equal volumes of the upper phase solvent and the lower phase solvent into the sample bottle, add the natural product sample, shake it thoroughly again and let it stand for equilibrium, transfer equal volumes of the upper phase solvent and the lower phase solvent, blow it dry, dissolve it with methanol and filter it, which is the HPLC detection sample in step (3).

6. The method for optimizing a countercurrent chromatography solvent system for separating complex natural product samples according to claim 1, characterized in that, The method for correction using the K value in step (4) is as follows: If K < 0.25, the calculated value of the mathematical model for the n-hexane / ethyl acetate / alcohol solvent / water system is reduced by 1; if K > 2.5, the calculated value of the mathematical model for the n-hexane / ethyl acetate / alcohol solvent / water system is increased by 1.

7. The method for optimizing a countercurrent chromatography solvent system for separating complex natural product samples according to claim 1, characterized in that, Step (1) In liquid chromatography, mobile phase A is water and mobile phase B is methanol, with gradient elution.

8. The method for optimizing a countercurrent chromatography solvent system for separating complex natural product samples according to any one of claims 1 to 7 is applied to the optimization of a countercurrent chromatography solvent system for complex sample systems of traditional Chinese medicine and natural products.

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