Establishment of mathematical model for macroporous resin static adsorption and desorption combined with HPLC and its application

Abstract

This invention discloses a mathematical model establishment method based on the combination of static adsorption and desorption of macroporous resin and HPLC. The capacity factor is extracted by combining static adsorption and desorption of macroporous resin with HPLC technology, and a mathematical model is established to optimize the conditions of pH and ethanol concentration gradient of the eluent. The efficient separation and purification of seven polyphenolic compounds from Smilax glabra are achieved by using offline two-dimensional macroporous resin column chromatography and preparative high performance liquid chromatography.

Description

Technical Field

[0001] This invention belongs to the field of drug separation and purification technology. Specifically, it relates to a mathematical model for optimizing macroporous resin separation and purification elution methods based on macroporous resin static adsorption and desorption data combined with HPLC technology, and its application. Background Technology

[0002] Smilax glabra As a traditional Chinese medicinal herb, over 200 compounds have been identified in its rhizome, among which polyphenols (such as astilbene, isoflavone, and scutellarin) are the main active substances, possessing pharmacological effects such as anti-inflammatory, antioxidant, immunomodulatory, and hepatoprotective properties. However, the complexity of its chemical composition poses a severe challenge to its separation and purification. High structural similarity: Polyphenolic compounds are mostly chiral isomers (such as astilbene and isoflavone), differing only slightly in hydroxyl substitution positions or glycosyl linkages, making them difficult to distinguish using traditional chromatographic methods; coexistence of low-abundance components: In addition to polyphenols, the alcohol extract also contains a large amount of polysaccharides, tannins, and pigments, interfering with the adsorption selectivity of the target analyte; stability issues: Some polyphenols are easily degraded at extreme pH or high temperatures, such as 5-caffeoylshikimic acid, which readily undergoes ester bond hydrolysis at pH > 7. Existing separation processes often result in the loss of active ingredients due to improper condition control.

[0003] Macroporous resin technology has become a research hotspot in the field of natural product separation due to its high adsorption capacity and reusability. However, in practical applications, the screening of elution separation optimization methods and the determination of elution separation procedures have significant limitations: (1) Relying on experience, it is difficult for operators who are not familiar with the macroporous resin elution separation operation procedures to complete this step quickly; (2) The optimization process relies on a large number of trial and error experiments, which wastes sample and reagent resources; (3) There is a lack of a set of highly operable methods that can be practically applied to actual operations; (4) Single-factor optimization defects: Most separation methods only focus on the ethanol concentration gradient and ignore the synergistic effect of pH on the desorption kinetics of the target component; (5) The optimization parameters for laboratory scale (column volume 50 mL) cannot be directly used for industrialization (column volume > 100 L). Summary of the Invention

[0004] The purpose of this invention is to provide a method for establishing a mathematical model based on the combination of static adsorption and desorption of macroporous resins using HPLC.

[0005] Another objective of this invention is to provide an application of the mathematical model established by the method in the separation of polyphenols from Smilax glabra.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a method for establishing a mathematical model based on the combination of static adsorption and desorption of macroporous resin and HPLC, comprising the following steps:

[0008] Based on the adsorption and desorption characteristics of Smilax glabra polyphenols on macroporous resin, capacity factors were extracted. Fit the capacity factor A mathematical model was developed for the pH and ethanol concentration gradient of the eluent, and the optimal elution conditions were determined.

[0009] The mathematical model is established as follows:

[0010] 1. Theoretical Model of a Plate

[0011] This model envisions the chromatographic column as an arbitrary number of theoretical plates, each consisting of identical repeating portions of the stationary and mobile phases. It assumes the solute splits between the two phases to reach equilibrium; the solute's partition coefficient... The consistency across all plates indicates the nature of the substance. Adsorption-desorption equilibrium:

[0012] (1);

[0013] in and These represent the mobile phase and the stationary phase, respectively.

[0014] Capacity factor for:

[0015] (2);

[0016] in and These are the masses of the stationary phase and the mobile phase, respectively.

[0017] Assuming that the distribution of the mobile phase and the stationary phase reaches equilibrium instantaneously, then the distribution coefficient... Represented as:

[0018] (3);

[0019] In the formula, and Substances in the stationary phase and mobile phase, respectively (solute) The molar concentration of )

[0020] 2. Linear Solvent Strength Model

[0021] Under isobaric elution conditions, capacity factor The logarithm is usually related to the volume fraction of the strong eluting solvent in the mobile phase. A linear relationship exists:

[0022] (4);

[0023] In the formula, This is the extrapolated value of the retention coefficient in water. =0; The strength parameter of a strong elution solvent that affects solute elution;

[0024] 3. pH value of the mobile phase

[0025] (5);

[0026] In the formula, This refers to the solute gradient elution time. The time the column stays is It is a constant; Substituting this into equation (5), we obtain the following relationship between retention rate and pH:

[0027] (6);

[0028] Add a parameter , Formula (6) can be derived as follows:

[0029] (7);

[0030] 4. Compared to

[0031] In this invention, the volume of the flowing phase within the column is used. With the volume of the stationary phase The ratio is expressed and defined as follows: :

[0032] (8);

[0033] Using the capacity factor mentioned above and distribution constant The definition can be written as :

[0034] (9);

[0035] Under constant temperature and pressure Maintain consistency to ensure comparability between consistent chromatographic systems;

[0036] (10);

[0037] Add a parameter and defined as :

[0038] (11);

[0039] Then equation (10) can be written as:

[0040] (12);

[0041] From equation (4), we can obtain that ,but:

[0042] (13);

[0043] From equation (7), we can obtain that ,but:

[0044] (14);

[0045] From equations (13) and (14), it can be seen that, under the comparison conditions, the following can be calculated: , The relationship between pH and pH is achieved by comparing conditions under constant temperature and atmospheric pressure.

[0046] 5. Capacity factor Calculation

[0047] first, :

[0048] (15);

[0049] In the formula, and These represent the concentration and volume of the solute in the stationary phase, respectively. and These represent the concentration and volume of the solute in the mobile phase, respectively; and the concentration of the substance before adsorption during MR adsorption. and the concentration of the adsorbed substance It is readily available; the volume of the liquid in the system remains essentially unchanged before and after adsorption, i.e. This leads to the equation ;substance The peak area is positively correlated with its concentration C; therefore, it is calculated as follows:

[0050] (16);

[0051] It is the peak area of ​​the substance in the sample before adsorption; It is the peak area of ​​the substance in the sample solution after adsorption is complete;

[0052] Since the total mass of compounds adsorbed by the resin is equal to the mass of substances eluted by the 90% ethanol eluent, ;

[0053] (17);

[0054] 6. Calculation and Similarity Analysis

[0055] From equations (8) and (11), it can be seen that when the fixed phases are the same, ;in and The MR quality of the experimental group and the prediction group are respectively; and These represent the elution buffer volumes for the experimental group and the prediction group, respectively.

[0056] 7. Optimization of elution pH

[0057] Relative retention rate :

[0058] (18);

[0059] 8. Elution gradient optimization

[0060] R is the resolution, and n is the number of plates; relative resolution is used. and capacity factor The value is used as an indicator to select the gradient of strong elution solvent. It is the separation degree after simplification; equation This indicates that selectivity and capacity factors determine resin column separation.

[0061] The polyphenols in Smilax glabra are selected from 5-caffeoylshikimic acid, neoastilbestrol, neoastilbestrol, neoisaltilbestrol, isaltilbestrol, scutellarin, and isofragicin.

[0062] The adsorption and desorption characteristics of Smilax glabra polyphenols on macroporous resin are as follows:

[0063] The pretreatment and static adsorption of macroporous resins are shown below:

[0064] (1) Resin activation: Soak the macroporous resin in 5-95% (preferably 95%) ethanol for 2-28 hours (preferably 24 hours). After it has fully swelled, pack it into a column and rinse it with deionized water until the eluent has no alcohol odor. The pretreated macroporous resin is then used for subsequent separation experiments.

[0065] (2) Static adsorption equilibrium: Take the activated macroporous resin and add it to the Smilax glabra extract solution. The mass ratio of macroporous resin to Smilax glabra extract is 1:1~10 (preferably 1:1.5). Shake (condition: shake for 12 hours at 25℃ and 200 rpm). After the system reaches adsorption equilibrium, seal the resin, wash the resin with deionized water, and dry the surface moisture of the resin.

[0066] (3) Static desorption experiment: The macroporous resin obtained in step (2) was added to an ethanol solution with a concentration of 0%~90% and a pH of 2.0~7.0. The pH of the solution was adjusted by hydrochloric acid (preferably 1 mol / L) and sodium hydroxide solution. Shaking (condition: shaking for 24 hours at 25℃ and 200 rpm) was performed. After the desorption reached equilibrium, the desorption solution sample was collected and the peak area of ​​Smilax glabra polyphenols was determined by high performance liquid chromatography (HPLC).

[0067] Static adsorption and desorption experiments were conducted to determine the capacity factor of Smilax glabra polyphenols on macroporous resin. Based on this data, a capacity factor is constructed. pH of the eluent and volume fraction of ethanol A mathematical model of the relationship between them was developed, and the optimal pH of the eluent and the ethanol concentration gradient elution procedure were determined using this model.

[0068] Preparation of the Smilax glabra extract:

[0069] After crushing the rhizome of Smilax glabra, pass it through a 60-mesh sieve to obtain powder. Add the powder to an ethanol solution with a concentration of 5-95% (preferably 70%) and soak at room temperature (preferably for 8 hours). Extract with ultrasonic assistance (power 300 W, frequency 40 kHz). Combine the filtrates, concentrate the filtrate, and test to find no ethanol residue. Dilute with deionized water to obtain an Smilax glabra extract solution.

[0070] The macroporous resin is selected from D101 macroporous resin, and the particle size of the D101 macroporous resin is 0.3~1.2 mm.

[0071] Dynamic elution and separation of Smilax glabra polyphenols using macroporous resin:

[0072] The extract solution of Smilax glabra was loaded onto a separation column packed with activated macroporous resin and subjected to gradient elution. The eluents from each stage were collected and concentrated to obtain a crude purified product. The crude product was purified by preparative high performance liquid chromatography. The eluents of each target component were collected and freeze-dried to obtain the Smilax glabra polyphenols.

[0073] The activated macroporous resin-packed separation column has the following specifications: glass column, 60 cm × 3.9 cm ID; filled with 100 mL of activated macroporous resin, with a column bed height of 30 cm.

[0074] The sample loading parameters for the activated macroporous resin-packed separation column are as follows: Smilax glabra extract solution (preferably with a concentration of 150 mg / mL), loading volume of 0.2~0.6 BV (preferably 0.3 BV), flow rate of 0.5~2 BV / h (preferably 1.0 BV / h), and breakthrough point monitored by HPLC.

[0075] The gradient elution process consists of pure water (preferably 3 BV) at pH 2.0, 15% ethanol solution (preferably 12 BV) at pH 5.5, 35% ethanol solution (preferably 13 BV) at pH 7.0, and 45% ethanol solution (preferably 7 BV) at pH 2.0. The pH of all solutions is adjusted using 1 mol / L hydrochloric acid.

[0076] The eluents collected at each stage are as follows: the elution fraction eluted by 15% ethanol solution is component 1, containing 5-caffeoylshikimic acid; the elution fraction eluted by 35% ethanol solution is component 2, containing neoastilbestrol, astilbestrol, neoisaltilbestrol, and isaltilbestrol; and the elution fraction eluted by 45% ethanol solution is component 3, containing scutellarin and isofragicin.

[0077] The chromatographic conditions for the preparative high-performance liquid chromatography were as follows: YMC-Pack ODS-A C18 column (20×250mm, 5μm), injection volume 5mL, detection wavelength 291nm, flow rate 19mL / min; mobile phase A was an aqueous solution containing 0.1% acetic acid, and mobile phase B was methanol.

[0078] The elution conditions for collecting each target component are as follows: Component 1 was eluted isocratically with 18% B for 0-20 min, and 5-caffeoylshikimic acid was collected at 4-6 min; Component 2 was eluted isocratically with 20% B for 0-60 min, and neo-astilbene, astilbene, isoflavone, and neo-isoflavone were collected at 25-45 min; Component 3 was eluted with 35% B for 0-60 min. Elution was performed using a linear gradient of 45% B, and scutellarin and isoflavone were collected at 27–40 min.

[0079] In a second aspect, the present invention provides an application of the mathematical model established by the method in the separation of polyphenols from Smilax glabra.

[0080] By adopting the above technical solution, the present invention has the following advantages and beneficial effects:

[0081] This invention discloses a method for extracting capacity factors based on static adsorption and desorption of macroporous resin combined with HPLC technology. The method for separating and purifying polyphenolic compounds from Smilax glabra using macroporous resin was optimized using a dual-mode gradient elution technique. Capacity factors (CFU) were extracted using a combination of macroporous resin static adsorption / desorption and HPLC. A mathematical model was established to optimize the conditions of pH and ethanol concentration gradient of the eluent. Offline two-dimensional macroporous resin column chromatography (MRLC) and preparative high performance liquid chromatography (Prep-HPLC) were used to achieve efficient separation and purification of seven polyphenolic compounds (5-caffeoylshikimic acid, neoastilbestrol, astilbestrol, neoisaltilbestrol, isaltilbestrol, scutellarin, and isofragicin) from Smilax glabra.

[0082] The method of this invention features high separation efficiency and low solvent consumption, making it suitable for large-scale purification of bioactive components in complex matrices. By optimizing dual-mode gradient elution using a mathematical model and combining it with offline two-dimensional chromatography, the method achieves highly efficient separation of seven polyphenols from Smilax glabra: Separation efficiency: Target compound purity ≥90%, total recovery rate ≥85%; Solvent economy: Ethanol consumption is reduced by 30% compared to traditional methods, and wastewater discharge is reduced by 35%. Attached Figure Description

[0083] Figure 1 This is a schematic diagram of the HPLC analysis of the alcohol extract of Smilax glabra.

[0084] Figure 2 The UV absorbance of seven polyphenols eluted at three different elution flow rates (0.5 BV / h, 1.0 BV / h, and 2.0 BV / h) is shown.

[0085] Figure 3 This is a schematic diagram of the dynamic breakthrough curve of the sample loading volume.

[0086] Figure 4 A schematic diagram illustrating the target peak separation effect under an optimized gradient elution procedure.

[0087] Figure 5 A schematic diagram comparing the eluted and collected components 1, 2, and 3 with the original Smilax glabra alcohol extract.

[0088] Figure 6 A schematic diagram of the liquid phase spectrum for the preparation of component 1 (5-caffeoylshikimic acid).

[0089] Figure 7 A schematic diagram of the purity spectrum of the liquid phase for component 1 (5-caffeoylshikimic acid).

[0090] Figure 8 A schematic diagram of the liquid phase chromatogram prepared for component 2 (neoastilbene, astilbene, neoisoastilbene, isoastilbene).

[0091] Figure 9 A schematic diagram of the liquid phase purity chromatogram for component 2 (neomalacin, astilbin, neoisomalacin, isomalacin).

[0092] Figure 10 A schematic diagram of the liquid phase spectrum prepared for component 3 (lycopene and isolycopene).

[0093] Figure 11 A schematic diagram of the liquid phase purity spectrum for component 3 (lycopene and isolycopene).

[0094] Figure 12 The chromatograms are comparisons of the alcohol extract of Smilax glabra with the standards of each target component.

[0095] Figure 13 This is a schematic diagram for characterizing the purity of the prepared product. Detailed Implementation

[0096] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.

[0097] The core of this invention lies in extracting capacity factors through a combination of static adsorption and desorption using macroporous resin and HPLC technology. Furthermore, by employing a bivariate (pH-ethanol concentration) gradient elution model, the limitations of traditional macroporous resin separation techniques for separating complex polyphenol systems were overcome. Specifically, based on the adsorption and desorption characteristics of Smilax glabra polyphenols on D101 macroporous resin, the capacity factor (…) was extracted. ), and establish capacity factor ( The quantitative relationship between elution conditions and elution parameters is as follows. The specific calculation process is as follows:

[0098] Method Modeling and Simulation

[0099] 1. Theoretical Model of Plate

[0100] The plate model, proposed by Martin and Synge, is a basic chromatographic model that envisions the chromatographic column as an arbitrary number of theoretical plates, each consisting of identical repeating portions of the stationary and mobile phases. It is assumed that the solute splits between the two phases to reach equilibrium (Journal of Chromatography A 1218 (2011) 5166-5174). The partition coefficient of the solute (… The consistency across all plates indicates that the substance... The adsorption-desorption equilibrium (Journal of Chromatography A 1216 (2009) 1737-1755):

[0101] (1)

[0102] in and These represent the mobile phase and the stationary phase, respectively.

[0103] Capacity factor ( )for:

[0104] (2)

[0105] in and These represent the masses of the stationary phase and the mobile phase, respectively.

[0106] Assuming that the distribution of the mobile phase and the stationary phase reaches equilibrium instantaneously, then the distribution coefficient... Represented as:

[0107] (3)

[0108] In the formula, and Substances in the stationary phase and mobile phase, respectively (solute) The molar concentration of ).

[0109] 2. Linear Solvent Strength Model

[0110] The linear solvent strength model (LSSM) established by Snyder et al. is based on empirical observations, namely, under isobaric elution conditions, the capacity factor ( The logarithm of ) is usually related to the volume fraction of the strong eluting solvent in the mobile phase ( The relationship is linear (Anal. Chem. (2015) 8.):

[0111] (4)

[0112] In the formula, extrapolated value of retention coefficient in water ( =0), The strength of the strong elution solvent, which affects solute elution, is a parameter (Journal of Chromatography A 1653 (2021) 462376). This two-parameter model is widely used in isometric and gradient LC due to its simplicity.

[0113] 3. pH value of the mobile phase

[0114] Schoenmakers et al. reviewed retention models of ionizable solutes in liquid chromatography as a function of pH and solvent composition. Studies have extensively discussed how pH affects retention coefficients in liquid chromatography, and Schneider et al.'s equation is based on a key model of statistical correlation (Anal. Chem. (2013).).

[0115] (5)

[0116] In the formula, This refers to the solute gradient elution time. The time the column stays is It is a constant. Substituting this into equation (5), we obtain the following relationship between retention rate and pH:

[0117] (6)

[0118] Add a parameter Formula (6) can be derived as follows:

[0119] (7)

[0120] 4. Compared to

[0121] The ratio is a fundamental parameter in liquid chromatography. In this invention, the volume of the mobile phase within the column ( ) and the volume of the stationary phase ( The ratio of ) is expressed and defined as (Journal of Chromatography A 1634 (2020) 461668.):

[0122] (8)

[0123] Using the capacity factor mentioned above and distribution constant The definition can be written as :

[0124] (9)

[0125] Under constant temperature and pressure Maintain consistency to ensure comparability between consistent chromatographic systems;

[0126] (10)

[0127] Add a parameter and defined as :

[0128] (11)

[0129] Then equation (10) can be written as:

[0130] (12)

[0131] From equation (4), we can obtain that ,but:

[0132] (13)

[0133] From equation (7), we can obtain that ,but:

[0134] (14)

[0135] From equations (13) and (14), it can be seen that, under the comparison conditions, the following can be calculated: , The relationship between pH and pH is determined by comparing conditions under constant temperature and pressure.

[0136] 5. Capacity factor Calculation

[0137] first, It can be calculated as follows:

[0138] (15)

[0139] In the formula, and These represent the concentration and volume of the solute in the stationary phase, respectively. and These represent the concentration and volume of the solute in the mobile phase, respectively. In the MR adsorption process, the concentration of the substance before adsorption is... and the concentration of the adsorbed substance It is readily available. The volume of the liquid in the system remains essentially unchanged before and after adsorption, i.e. This leads to the equation. .substance The peak area is positively correlated with its concentration (C), therefore, it can also be calculated as:

[0140] (16)

[0141] It is the peak area of ​​the substance in the sample before adsorption; It is the peak area of ​​the substance in the sample solution after adsorption is complete.

[0142] Since the total mass of compounds adsorbed by the resin is approximately equal to the mass of substances eluted by the 90% ethanol eluent, ;

[0143] (17)

[0144] 6. Calculation and Similarity Analysis

[0145] From equations (8) and (11), it can be seen that when the fixed phases are the same, .in and The MR quality of the experimental group and the prediction group are respectively. and The values ​​represent the elution volumes for the experimental group and the prediction group, respectively.

[0146] 7. Optimization of elution pH

[0147] Relative retention rate ( The values ​​are correct for variables such as flow rate and column conditions that affect reproducibility. They reflect the effectiveness of the stationary phase in separating components, among which... A higher value indicates better selectivity and higher component resolution (Journal of Chromatography A 1217 (2010) 1557-1566).

[0148] (18)

[0149] 8. Elution gradient optimization

[0150] The equation For understanding chromatographic separation, which is crucial (Anal. Chem. 89(2017) 9926-9933.), R is the resolution and n is the number of plates. The relative resolution is... and capacity factor ( The value is used as an indicator to select the gradient of strong elution solvent. It is the separation degree after the simplified formula. Equation This indicates that selectivity and capacity factors determine resin column separation.

[0151] Example 1

[0152] Key equipment and reagents: ultrasonic extractor, preparative HPLC system (Waters Prep 150 LC), UV detector (Hitachi L-7420), analytical HPLC (UltiMate3000, Thermo Scientific), D101 macroporous resin (Anhui Bengbu Resin Co., Ltd.), HPLC grade methanol, acetic acid, and standards (5-caffeoylshikimic acid, astilbene, etc., National Institutes for Food and Drug Control).

[0153] Preparation of Smilax glabra extract:

[0154] The rhizome of Smilax glabra was pulverized and passed through a 60-mesh sieve to obtain powder. 100 g of powder was added to 2000 mL of 70% ethanol solution and soaked at room temperature for 8 hours. Ultrasonic extraction was performed (300 W power, 40 kHz frequency) for 1 hour each time, and repeated twice. The filtrates were combined and concentrated under reduced pressure at 45℃ to 25% of the original volume (about 500 mL). No ethanol residue was detected. The solution was diluted with deionized water to a concentration of 150 mg / mL (calculated based on the weight of the raw medicinal material) to obtain the Smilax glabra extract solution.

[0155] Step 1: Pretreatment and static adsorption experiment of D101 macroporous resin:

[0156] (1) Resin activation: 100 g of D101 macroporous resin with a particle size of 0.3~1.2 mm was soaked in 95% ethanol for 24 hours. After it was fully swollen, it was packed into a column and washed with deionized water at a flow rate of 3 BV / h until the eluent had no alcohol smell. The pretreated D101 macroporous resin was obtained and used for subsequent separation experiments.

[0157] (2) Static adsorption equilibrium: Take 2.0 g of the activated D101 macroporous resin in step (1) and place it in a 100 mL stoppered conical flask. Add 20 mL of Smilax glabra extract solution with a concentration of 150 mg / mL. Shake at 25℃ and 200 rpm for 12 hours. After the system reaches adsorption equilibrium, seal the resin and wash it with 2 times the resin column volume (BV) of deionized water. Then, dry the surface of the resin.

[0158] (3) Static desorption experiment: The D101 macroporous resin obtained in step (2) was placed in a 100 mL stoppered conical flask, and 20 mL of ethanol solution with a concentration of 0%~90% and a pH of 2.0~7.0 was added. The pH of the solution was adjusted by 1 mol / L hydrochloric acid and sodium hydroxide solution. The solution was shaken at 25℃ and 200 rpm for 24 hours. After the desorption reached equilibrium, the desorption solution sample was collected, and the peak area of ​​Smilax glabra polyphenols was determined by high performance liquid chromatography (HPLC). Figure 1 The HPLC chromatogram of the alcohol extract of Smilax glabra is shown.

[0159] Static adsorption and desorption experiments were conducted to determine the capacity factor of Smilax glabra polyphenols on D101 macroporous resin. Based on this data, a capacity factor is constructed ( ) and the pH of the eluent, the volume fraction of ethanol ( A mathematical relationship model between the eluent and the ethanol concentration gradient elution procedure was developed using this model.

[0160] Table 1 shows the volume factors of seven polyphenols (5-caffeoylshikimic acid, neoastilbestrol, astilbestrol, neoisoastilbestrol, isoastilbestrol, scutellarin, and scutellarin) in the extract of Smilax glabra at pH 2.0. Table 2 shows the extraction values ​​of the capacity factor at pH 2.0. ) and organic phase volume fraction The linear relationship is shown in Table 3, which represents the capacity factor of 5-caffeoylshikimic acid. The linear relationship between pH and the volume fraction of the organic phase.

[0161] Table 1

[0162]

[0163] Note: C1: 5-Caffeoylshikimic acid; C2: Neoastilbestrol; C3: Astilbestrol; C4: Neoisaltilbestrol; C5: Isoastilbestrol; C6: Scutiginoside; C7: Isofragiginoside.

[0164] Table 2

[0165]

[0166] Note: C1: 5-Caffeoylshikimic acid; C2: Neoastilbestrol; C3: Astilbestrol; C4: Neoisaltilbestrol; C5: Isoastilbestrol; C6: Scutiginoside; C7: Isofragiginoside.

[0167] Table 3

[0168]

[0169] The second step involved optimizing the elution procedure for separating Smilax glabra using a D101 macroporous resin-packed chromatography column.

[0170] In Tables 2 and 3, the mathematical relationships between the capacity factor and the concentration of the elution solvent ethanol and pH are obtained by fitting under experimental conditions. In practical applications, a ratio correction needs to be introduced to match the actual situation of macroporous resin packed columns; therefore, the ratio needs to be calculated.

[0171] The calculation is based on formula (8), and the results are shown in Table 4. For the quality of fixed macroporous resin, This represents the volume of the mobile phase.

[0172] Table 4: Experimental and Actual Values ​​of D101 Macroporous Resin Compared to value

[0173]

[0174] parameters Substituting the numerical values ​​into formulas (13) and (14), the actual mathematical relationships between the capacity factor and the eluent ethanol concentration and pH in the macroporous resin packed column were obtained, as shown in Tables 5 and 6. Table 5 shows the actual capacity factor in the D101 macroporous resin packed chromatography column at pH 2.0. ) and organic phase volume fraction A linear relationship;

[0175] Table 5

[0176]

[0177] Note: C1: 5-Caffeoylshikimic acid; C2: Neoastilbestrol; C3: Astilbestrol; C4: Neoisaltilbestrol; C5: Isoastilbestrol; C6: Scutiginoside; C7: Isofragiginoside.

[0178] Table 6 shows the capacity factor of 5-caffeoylshikimic acid in actual packed chromatography columns of D101 macroporous resin. The linear relationship between pH and the volume fraction of the organic phase.

[0179] Table 6

[0180]

[0181] Optimization reference formula for elution pH (18), optimization reference for elution gradient:

[0182] The equation For understanding chromatographic separation, which is crucial (Anal. Chem. 89(2017) 9926-9933.), R is the resolution and n is the number of plates. The relative resolution is... and capacity factor ( The value is used as an indicator to select the gradient of strong elution solvent. It is the separation degree after the simplified formula. Equation This indicates that selectivity and capacity factors determine resin column separation.

[0183] The gradient elution procedure determined through steps one and two is as follows:

[0184] (1) First gradient: 15% ethanol (pH 5.5), elution volume 12 BV (1200 mL), collect fraction 1 (C1: 5-caffeoylshikimic acid);

[0185] (2) Second gradient: 35% ethanol (pH 7.0), elution volume 13 BV (1300 mL), collect fraction 2 (C2~C5, C2: neoastilbestrol; C3: astilbestrol; C4: neoisaltilbestrol; C5: isaltilbestrol).

[0186] (3) Third gradient: 45% ethanol (pH 2.0), elution volume 7 BV (700 mL), collect fraction 3 (C6~C7, C6: scutellarin isosorbide; C7: scutellarin);

[0187] (4) The eluent was concentrated by vacuum rotary evaporation (45℃, -0.08 MPa) to obtain the crude purified product.

[0188] The third step involved optimizing the elution flow rate and sample loading volume of the D101 macroporous resin-packed chromatography column for the separation of Smilax glabra (Tufuling).

[0189] Three different elution flow rates (0.5 BV / h) were investigated. Figure 2 As shown in Figure A), 1.0 BV / h ( Figure 2 As shown in Figure B), 2.0 BV / h ( Figure 2 The effect of (as shown in C) on the elution effect is as follows: Figure 2 As shown, Figure 2 The UV absorbance spectra of seven polyphenolic substances eluted at three different elution flow rates (0.5 BV / h, 1.0 BV / h, and 2.0 BV / h) are shown. The optimal elution separation of each target substance was observed at an elution flow rate of 1.0 BV / h; therefore, this flow rate was determined to be 1.0 BV / h. The sample loading amount was determined using the breakthrough curve method, and the results are shown below. Figure 3 As shown, Figure 3 This is a schematic diagram of the dynamic breakthrough curve of the sample loading volume. When the sample loading amount is 0.3 BV, after adsorption by D101 macroporous resin, the overall concentration of each target substance in the effluent is less than 10%, and the adsorption effect is ideal. Therefore, the optimal sample loading amount is determined to be 0.3 BV.

[0190] Step 4: Dynamic elution and separation of Smilax glabra samples using D101 macroporous resin:

[0191] Resin column specifications: glass column (60 cm × 3.9 cm ID), packed with 100 mL column volume of activated D101 macroporous resin, column bed height 30 cm; Sample loading parameters: Smilax glabra extract at a concentration of 150 mg / mL, loading volume 0.3 BV (30 mL), flow rate 1.0 BV / h (100 mL / h), breakthrough point monitored by HPLC (target analyte leakage ≤10%).

[0192] A 150 mg / mL concentration of Smilax glabra extract was loaded onto an activated D101 macroporous resin-packed separation column and subjected to gradient elution at a flow rate of 1 BV / h. The elution program consisted of 3 BV of pure water at pH 2.0, 12 BV of 15% ethanol solution at pH 5.5, 13 BV of 35% ethanol solution at pH 7.0, and 7 BV of 45% ethanol solution at pH 2.0. The pH of all solutions was adjusted using 1 mol / L hydrochloric acid.

[0193] The eluents from each stage were collected separately: the elution fraction eluted by 15% ethanol solution was component 1, containing 5-caffeoylshikimic acid; the elution fraction eluted by 35% ethanol solution was component 2, containing neoastilbestrol, astilbestrol, neoisaltilbestrol, and isaltilbestrol; and the elution fraction eluted by 45% ethanol solution was component 3, containing scutellarin and isofragicin.

[0194] Figure 4 A schematic diagram illustrating the target peak separation effect under an optimized gradient elution procedure. Figure 5 This diagram illustrates the comparison between the eluted fractions 1, 2, and 3 and the original ethanol extract of *Smilax glabra*. In the diagram, A represents the HPLC chromatogram of the ethanol extract, B represents the HPLC chromatogram of fraction 1, C represents the HPLC chromatogram of fraction 2, and D represents the HPLC chromatogram of fraction 3. As can be seen from the figure, the optimized macroporous resin gradient elution program enables excellent separation of the target components, with regular peak shapes and clear positioning. Furthermore, compared to the original ethanol extract, fractions 1, 2, and 3 show highly efficient enrichment of the target substances, with each fraction corresponding to a specific target compound group, demonstrating ideal separation selectivity and enrichment effects.

[0195] The three components were concentrated by vacuum rotary evaporation at 45℃ and -0.08 MPa to obtain the corresponding crude products. The crude products were further purified by preparative high-performance liquid chromatography (HPLC) under the following conditions: YMC-Pack ODS-A C18 column (20×250 mm, 5 μm), injection volume 5 mL, detection wavelength 291 nm, flow rate 19 mL / min; mobile phase A was an aqueous solution containing 0.1% acetic acid, and mobile phase B was methanol.

[0196] The preparation, elution, and collection conditions for each component are as follows: Component 1 was eluted isocratically with 18% B for 0-20 min, and 5-caffeoylshikimic acid was collected at 4-6 min; Component 2 was eluted isocratically with 20% B for 0-60 min, and neo-astilbene, astilbene, isoflavone, and neo-isoflavone were collected at 25-45 min; Component 3 was eluted with 35% B for 0-60 min. Elution was performed using a linear gradient of 45% B, and scutellarin and isofragellarin were collected separately over 27–40 min. The eluents of each target component were collected and freeze-dried to obtain 5-caffeoylshikimic acid, neoastilbestrol, astilbestrol, neoisofragestrol, isofragestrol, scutellarin, and isofragellarin with a purity of not less than 90%.

[0197] Figures 6-11 The images show the preparative liquid chromatograms and chromatographic purity diagrams for each target component in components 1, 2, and 3, respectively. Figure 6 A schematic diagram of the liquid phase spectrum for the preparation of component 1 (5-caffeoylshikimic acid). Figure 7 A schematic diagram of the purity spectrum of the liquid phase for component 1 (5-caffeoylshikimic acid). Figure 8 A schematic diagram of the liquid phase chromatogram prepared for component 2 (neoastilbene, astilbene, neoisoastilbene, isoastilbene). Figure 9 A schematic diagram of the liquid phase purity chromatogram for component 2 (neomalacin, astilbin, neoisomalacin, isomalacin). Figure 10 A schematic diagram of the liquid phase spectrum prepared for component 3 (lycopene and isolycopene). Figure 11 A schematic diagram of the liquid phase purity spectrum for component 3 (lycopene and isolycopene). Figure 12 The image shows the chromatograms of the alcohol extract of Smilax glabra and the standards for each target component. Figure 13 The chromatograms used to characterize the purity of the prepared products are shown in the figure. As can be seen from the figure, after preparation and purification, each target component exhibits a single main peak with regular peak shape and no interfering peaks, achieving a purity of over 90% as expected. This fully demonstrates that the separation and purification of each monomer component under these conditions is ideal, and high-purity target compounds can be successfully obtained.

[0198] Initial impurity removal in this step: 3 BV of deionized water at pH 2.0 can effectively elute highly polar impurities (such as polysaccharides and tannins) while maintaining the molecular state of the target polyphenols to enhance resin adsorption.

[0199] In this step, gradient elution is performed: the first gradient is a 15% ethanol solution with a pH of 5.5 at 12 BV. This solution utilizes the fact that the pH is lower than the isoelectric point of substances such as astilbene to increase their electrostatic adsorption with the resin and selectively desorb low hydrophobic components (such as 5-caffeoylshikimic acid).

[0200] This step involves gradient elution: the second gradient is a 13 BV solution of 35% ethanol at pH 7.0, which induces the deprotonation of flavonoid glycosides (such as isoflavone) by raising the pH to neutral, and then binds to a moderate concentration of ethanol. =35%) to achieve efficient, high-resolution separation;

[0201] In this step, gradient elution is performed: the third gradient is a 45% ethanol solution at pH 2.0 (7 BV), which provides a strong solvation effect to thoroughly desorb highly hydrophobic components (such as isoflavones). =0.074), while acidic conditions inhibit the oxidative degradation of the target compound.

[0202] The compounds collected in this invention were verified by HPLC-DAD: the retention time was ≥99% matched with the standard; mass spectrometry identification: using UPLC-Q-TOF-MS (negative ion mode), the molecular weight of the target compound deviated from the literature value by ≤5 ppm.

[0203] Resin regeneration efficiency: After four adsorption-desorption cycles, the adsorption capacity of D101 resin for astilbin decreased by only 4.3%.

[0204] Solvent consumption comparison: Compared with the traditional single gradient method, dual-mode gradient elution reduces ethanol consumption by 42% (from 38 BV to 22 BV) and wastewater discharge by 35%.

[0205] Purity and recovery: The HPLC purity of the seven target polyphenols was ≥90%, and the total recovery rate was ≥85%, which was significantly higher than that of the silica gel column method (60%~70%) and the HSCCC method (75%~80%).

[0206] This invention optimizes dual-mode gradient elution conditions using a mathematical model, combining D101 macroporous resin with preparative HPLC to achieve highly efficient separation of Smilax glabra polyphenols, with target compound purity ≥90%. This provides a new method for the large-scale preparation of active ingredients from natural products. Through mathematically model-driven dual-mode gradient optimization combined with offline two-dimensional chromatography, this invention solves three major challenges in the separation of Smilax glabra polyphenols: low selectivity, high solvent consumption, and poor scale-up stability. It provides an efficient, green, and scalable solution for the industrial separation of natural products.

[0207] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for establishing a mathematical model based on HPLC combining static adsorption and desorption of macroporous resin, characterized in that, Includes the following steps: Based on the adsorption and desorption characteristics of Smilax glabra polyphenols on macroporous resin, capacity factors were extracted. Fit the capacity factor A mathematical model was developed for the pH and ethanol concentration gradient of the eluent, and the optimal elution conditions were determined. The mathematical model is established as follows: (1). Theoretical model of the plate This model envisions the chromatographic column as an arbitrary number of theoretical plates, each consisting of identical repeating portions of the stationary and mobile phases. It assumes the solute splits between the two phases to reach equilibrium; the solute's partition coefficient... The consistency across all plates indicates the nature of the substance. Adsorption-desorption equilibrium: （1）； in and These represent the mobile phase and the stationary phase, respectively. Capacity factor for: （2）； in and These are the masses of the stationary phase and the mobile phase, respectively. Assuming that the distribution of the mobile phase and the stationary phase reaches equilibrium instantaneously, then the distribution coefficient... Represented as: （3）； In the formula, and Substances in the stationary phase and mobile phase, respectively molar concentration; (2). Linear solvent intensity model Under isobaric elution conditions, capacity factor The logarithm is usually related to the volume fraction of the strong eluting solvent in the mobile phase. A linear relationship exists: （4）； In the formula, This is the extrapolated value of the retention coefficient in water. =0; The strength parameter of a strong elution solvent that affects solute elution; (3). pH value of the mobile phase （5）； In the formula, This refers to the solute gradient elution time. The time the column stays is It is a constant; Substituting this into equation (5), we obtain the following relationship between retention rate and pH: （6）； Add a parameter , Formula (6) can be derived as follows: （7）； (4). Compared to In this invention, the volume of the flowing phase within the column is used. With the volume of the stationary phase The ratio is expressed and defined as follows: : （8）； Using the capacity factor mentioned above and distribution constant The definition can be written as : （9）； Under constant temperature and pressure Maintain consistency to ensure comparability between consistent chromatographic systems; （10）； Add a parameter and defined as : （11）； Then equation (10) can be written as: （12）； From equation (4), we can obtain that ,but: （13）； From equation (7), we can obtain that ,but: （14）； From equations (13) and (14), it can be seen that, under the comparison conditions, the following can be calculated: , The relationship between pH and pH is achieved by comparing conditions under constant temperature and atmospheric pressure. (5). Capacity factor Calculation first, : （15）； In the formula, and These represent the concentration and volume of the solute in the stationary phase, respectively. and These represent the concentration and volume of the solute in the mobile phase, respectively; and the concentration of the substance before adsorption during MR adsorption. and the concentration of the adsorbed substance It is readily available; the volume of the liquid in the system remains essentially unchanged before and after adsorption, i.e. This leads to the equation ;substance The peak area is positively correlated with its concentration C; therefore, it is calculated as follows: （16）； It is the peak area of ​​the substance in the sample before adsorption; It is the peak area of ​​the substance in the sample solution after adsorption is complete; Since the total mass of compounds adsorbed by the resin is equal to the mass of substances eluted by the 90% ethanol eluent, ; （17）； (6). Calculation and similarity analysis From equations (8) and (11), it can be seen that when the fixed phases are the same, ;in and The MR quality of the experimental group and the prediction group are respectively; and These represent the elution buffer volumes for the experimental group and the prediction group, respectively. (7). Optimization of elution pH Relative retention rate : （18）； (8). Elution gradient optimization R is the resolution, and n is the number of trays; With relative resolution and capacity factor The value is used as an indicator to select the gradient of strong elution solvent. It is the separation degree after simplified public declaration; equation This indicates that selectivity and capacity factors determine resin column separation.

2. The method for establishing a mathematical model based on the combination of static adsorption and desorption of macroporous resin and HPLC according to claim 1, characterized in that, The polyphenols in Smilax glabra are selected from 5-caffeoylshikimic acid, neoastilbestrol, neoastilbestrol, neoisaltilbestrol, isaltilbestrol, scutellarin, and isofragicin.

3. The method for establishing a mathematical model based on the combination of static adsorption and desorption of macroporous resin and HPLC according to claim 1, characterized in that, The adsorption and desorption characteristics of Smilax glabra polyphenols on macroporous resin are as follows: The pretreatment and static adsorption of macroporous resins are shown below: (1) Resin activation: Soak the macroporous resin in 5-95% ethanol for 2-28 hours. After it has fully swelled, pack it into a column and rinse it with deionized water until the eluent has no alcohol smell. The pretreated macroporous resin is then used for subsequent separation experiments. (2) Static adsorption equilibrium: Take the activated macroporous resin and add it to the Smilax glabra extract solution. The mass ratio of macroporous resin to Smilax glabra extract is 1:1~10. Shake and wait for the system to reach adsorption equilibrium. Then, seal the resin, wash the resin with deionized water, and dry the surface of the resin. (3) Static desorption experiment: The macroporous resin obtained in step (2) is added to an ethanol solution with a concentration of 0%~90% and a pH of 2.0~7.

0. The pH of the solution is adjusted by hydrochloric acid and sodium hydroxide solution. After oscillation and reaching equilibrium, the eluent sample was collected, and the peak area of ​​Smilax glabra polyphenols was determined by high performance liquid chromatography. Static adsorption and desorption experiments were conducted to determine the capacity factor of Smilax glabra polyphenols on macroporous resin. Based on this data, a capacity factor is constructed. pH of the eluent and volume fraction of ethanol A mathematical model of the relationship between them was developed, and the optimal pH of the eluent and the ethanol concentration gradient elution procedure were determined using this model.

4. The method for establishing a mathematical model based on the combination of static adsorption and desorption of macroporous resin and HPLC according to claim 1, characterized in that, Dynamic elution and separation of Smilax glabra polyphenols using macroporous resin: The extract solution of Smilax glabra was loaded onto a separation column packed with activated macroporous resin and subjected to gradient elution. The eluents from each stage were collected and concentrated to obtain a crude purified product. The crude product was purified by preparative high performance liquid chromatography. The eluents of each target component were collected and freeze-dried to obtain the Smilax glabra polyphenols.

5. The method for establishing a mathematical model based on the combination of static adsorption and desorption of macroporous resin and HPLC according to claim 4, characterized in that, The activated macroporous resin-filled separation column: Resin column specification: glass column; filled with activated macroporous resin.

6. The method for establishing a mathematical model based on the combination of static adsorption and desorption of macroporous resin and HPLC according to claim 4, characterized in that, The sample loading parameters for the activated macroporous resin-packed separation column are: Smilax glabra extract solution, loading volume 0.2~0.6 BV, flow rate 0.5~2 BV / h.

7. The method for establishing a mathematical model based on the combination of static adsorption and desorption of macroporous resin and HPLC according to claim 4, characterized in that, The gradient elution process consists of pure water at pH 2.0, 15% ethanol solution at pH 5.5, 35% ethanol solution at pH 7.0, and 45% ethanol solution at pH 2.0, with the pH of all solutions adjusted using hydrochloric acid.

8. The method for establishing a mathematical model based on the combination of static adsorption and desorption of macroporous resin and HPLC according to claim 7, characterized in that, The eluents collected at each stage are as follows: the elution fraction eluted by 15% ethanol solution is component 1, containing 5-caffeoylshikimic acid; the elution fraction eluted by 35% ethanol solution is component 2, containing neoastilbestrol, astilbestrol, neoisaltilbestrol, and isaltilbestrol; and the elution fraction eluted by 45% ethanol solution is component 3, containing scutellarin and isofragicin.

9. The method for establishing a mathematical model based on the combination of static adsorption and desorption of macroporous resin and HPLC according to claim 7, characterized in that, The elution conditions for collecting each target component are as follows: Component 1 was eluted isocratically with 18% B for 0-20 min, and 5-caffeoylshikimic acid was collected at 4-6 min; Component 2 was eluted isocratically with 20% B for 0-60 min, and neo-astilbene, astilbene, isoflavone, and neo-isoflavone were collected at 25-45 min; Component 3 was eluted with 35% B for 0-60 min. Elution was performed using a linear gradient of 45% B, and scutellarin and isoflavone were collected at 27–40 min.

10. The application of a mathematical model established by the method according to any one of claims 1 to 9 in the separation of Smilax glabra polyphenols.

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