Preparation method and application of mixed mode monolithic column for separation and screening of effective chemical components in dendrobium candidum

Abstract

The application provides a preparation method of a mixed mode monolithic column for separation and screening of effective chemical components in Dendrobium fimbriatum and application thereof. The mixed mode monolithic column material is prepared by uniformly dispersing a mixture of azobisisobutyronitrile, metal organic framework material MIP-202, methacrylic acid, myristyl methacrylate, n-dodecanol, n-propanol and ethylene glycol dimethacrylate in a water bath. The mixed mode monolithic column material has good mechanical strength and permeability, and by adjusting the proportion of the organic phase in the mobile phase, the separation mode can be changed between hydrophobic and hydrophilic, and the mixed mode monolithic column material can be used for separation and screening of antitumor active components in Dendrobium fimbriatum.

Description

Technical Field

[0001] This invention relates to the field of separation material preparation, specifically to a method for preparing a mixed-mode monolithic column for separating and screening effective chemical components in Dendrobium nobile and its application. Background Technology

[0002] High-performance liquid chromatography (HPLC) uses liquid as the mobile phase and employs a high-pressure delivery system to pump solvents of different polarities into a chromatographic column containing stationary phase packing. Different components are separated in the chromatographic column and flow into a detector for analysis. However, common chromatographic columns have drawbacks such as a single separation mode, high column pressure, and high cost. Furthermore, using them to analyze traditional Chinese medicine can easily damage the chromatographic column packing.

[0003] Dendrobium was first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica) and listed as a superior herb, described as having a sweet and neutral taste and being non-toxic. It is used to treat internal injuries, relieve numbness, lower qi, replenish the five internal organs, alleviate weakness, and strengthen yin. Its medicinal value is abundant, with a history of use spanning over two thousand years. It can strengthen the body and prolong life, thus being listed as a superior tonic in traditional Chinese medicine. *Dendrobium fringeii* (also known as Dendrobium nobile) benefits the stomach and promotes the production of body fluids, quenching thirst. It is used for yin deficiency and fluid depletion, dry mouth and thirst, poor appetite and dry retching, post-illness weakness and fever, and blurred vision. The chemical components of *Dendrobium fringeii*... The compounds include bibenzyl and its derivatives, phenanthrene and its glycosides, phenylpropanoids and their derivatives, fluorenones, small molecule phenolic acids, quinones, polysaccharides, and steroids, with glycosides being the most abundant and exhibiting significant antioxidant activity. Some studies have shown that its extracts have antitumor effects, but research on the specific antitumor components is limited. Therefore, a method is needed to enrich, purify, and screen the effective antitumor components. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing a mixed-mode monolithic column for separating and screening effective chemical components in Dendrobium nobile and its application, so as to overcome the shortcomings of traditional chromatographic stationary phase separation modes, long operation time, and large amount of solvent required for mobile phase.

[0005] The objective of this invention is achieved through the following technical solution: A method for preparing a mixed-mode monolithic column for separating and screening effective chemical components in Dendrobium nobile includes the following steps: (1) Azobisisobutyronitrile, metal-organic framework material MIP-202, methacrylic acid, tetradecyl methacrylate, n-dodecyl alcohol, n-propanol, and ethylene glycol dimethacrylate are mixed sequentially and ultrasonically oscillated to obtain a monolithic column prepolymer solution. (2) Pour the monolithic column prepolymer solution into a stainless steel empty column tube that is sealed at one end and open at the other end, seal the open end, and bathe it in a water bath at 55-65℃ for 10-12 hours. After taking it out and rinsing it, a mixed-mode monolithic column for separating and screening effective chemical components in Dendrobium nobile is obtained.

[0006] Furthermore, the ratio of the amounts of azobisisobutyronitrile, metal-organic framework material MIP-202, methacrylic acid, tetradecyl methacrylate, n-dodecyl alcohol, n-propanol, and ethylene glycol dimethacrylate is 0.010-0.030g: 0.005-0.006g: 0.1-0.3mL: 0.1-0.3mL: 1.2-2.4mL: 1.2-2.4mL: 0.5-0.8mL.

[0007] Furthermore, the rinsing involves connecting the stainless steel hollow column tube after water bath to a high-performance liquid chromatography system, using 100% methanol as the mobile phase, and rinsing at a flow rate of 1 mL / min for 1.5 h.

[0008] A method for separating and screening effective chemical components in Dendrobium chrysanthum involves using a hybrid monolithic column (as described above) as a solid-phase extraction adsorbent coupled with high-performance liquid chromatography (HPLC) to separate and screen the effective chemical components in Dendrobium chrysanthum.

[0009] In the preliminary separation of effective chemical components in Dendrobium nobile, firstly, the monolithic column is equilibrated with mobile phase 1, and a certain amount of Dendrobium nobile extract is injected. Mobile phase 1 is used to carry the sample into the monolithic column. At this time, some components are adsorbed in the monolithic column, and the remaining components are distilled out with mobile phase 1 to obtain the distillate of mobile phase 1. Then, mobile phase 2 is used to elute the components adsorbed on the monolithic column, and the eluent of mobile phase 2 is collected. The solvent in the distillate of mobile phase 1 and the eluent of mobile phase 2 is evaporated to obtain the preliminary separation eluent.

[0010] Both mobile phase 1 and mobile phase 2 are mixed solutions of acetonitrile and water; wherein, in mobile phase 1, the volume ratio of acetonitrile to water is 20:80, and in mobile phase 2, the volume ratio of acetonitrile to water is 70:30.

[0011] In the further separation and screening of active components, the preliminary separation eluent was further separated by a reverse-phase semi-preparative column. The eluent with an elution time of 16-18 min was collected, and the effective chemical components in Dendrobium chrysogenum were screened by detecting the cytotoxicity of different fractions of the eluent to HepG-2 human liver cancer cells using the CCK-8 assay.

[0012] The beneficial effects of this invention are as follows: The hybrid monolithic column prepared using the method of this invention exhibits good mechanical strength and permeability. By adjusting the proportion of the organic phase in the mobile phase, the hydrophobic and hydrophilic separation modes can be switched. The hybrid monolithic column can be used for the separation and screening of antitumor active components in Dendrobium nobile. This invention is novel, simple, easy to operate, low-cost, and time-efficient, offering advantages in the analysis of complex samples. Attached Figure Description

[0013] Figure 1 The nitrogen adsorption-desorption isotherm (A) and scanning electron microscope (SEM) image (B) of the monolithic column 1 in the hybrid mode of Example 3 are shown.

[0014] Figure 2 The relationship between column back pressure and flow rate (A) and methanol content (B) for the mixed-mode monolithic column and the single-mode monolithic column in Example 8.

[0015] Figure 3 The logIC50 (B) of different components of traditional Chinese medicine extract separated by a mixed-mode monolithic column 1 under different mobile phase conditions on the growth of HepG-2 cells were obtained (A) and the component with the strongest anti-cancer effect (eluted with 80% water) was obtained.

[0016] Figure 4 The inhibitory effect of each component on the growth of HepG-2 cells (A) and the logIC50 (B) of the component with the strongest anti-cancer effect (component 3) were obtained by using a reversed-phase semi-preparative chromatography column for further separation of the active ingredients.

[0017] Figure 5 The image shows the chromatograms of the extract and the purified antitumor active ingredients obtained from traditional Chinese medicine. Detailed Implementation

[0018] The present invention will be further described below with reference to the embodiments. The embodiments described below are for illustration only and do not limit the scope of protection of the present invention in any way.

[0019] The processes and methods not described in detail in the following examples are conventional methods known in the art. All reagents used in the examples are analytically pure or chemically pure, and can be commercially available or prepared by methods known to those skilled in the art.

[0020] Example 1: Preparation of a monolithic hydrophilic monomer column Accurately weigh 0.018 g of azobisisobutyronitrile and place it in a centrifuge tube. Then add 0.25 mL of methacrylic acid, 1.8 mL of n-dodecyl alcohol, 1.8 mL of n-propanol and 0.665 mL of ethylene glycol dimethacrylate. After dissolving the solid by vortexing, sonicate for 30 min until it is evenly dispersed. Pack it into a stainless steel empty column tube and react in a 60℃ water bath for 11 h to obtain the hydrophilic monomer monolithic column 1.

[0021] The hydrophilic monomer monolithic column 1 was connected to the high-performance liquid chromatography system. Using 100% methanol as the mobile phase, the flow rate was slowly and uniformly increased to 1 mL / min. The monolithic column was flushed for 90 min to remove pore-forming agents and other substances.

[0022] The back pressure of the monolithic column was measured to be 1.9 MPa, and the theoretical plate number was 6150 plates / m. The specific surface area of ​​the continuous bed was measured to be 67.3816 m². 2 / g. This indicates that the material lacking tetradecyl methacrylate and MIP-202 has lower permeability compared to the mixed-mode material.

[0023] Example 2: Preparation of a monolithic hydrophobic monomer column Accurately weigh 0.018 g of azobisisobutyronitrile (AIBN) and 0.005 g of metal-organic framework material MIP-202 into a centrifuge tube. Then add 0.25 mL of tetradecyl methacrylate, 1.8 mL of n-dodecyl alcohol, 1.8 mL of n-propanol, and 0.665 mL of ethylene glycol dimethacrylate. Vortex to dissolve the solid, then sonicate for 30 min until uniformly dispersed. Transfer the solution to a stainless steel empty column and react in a 60°C water bath for 11 h to obtain the final product. Subsequent rinsing procedures are as described in Example 1.

[0024] The back pressure of the monolithic column was measured to be 1.0 MPa, and the theoretical plate number was 5140 plates / m. The specific surface area of ​​the continuous bed was measured to be 113.8626 m². 2 / g. This indicates that the material lacking methacrylic acid has reduced separation ability compared to the mixed-mode material.

[0025] Example 3: Preparation of Hybrid Mode Monolithic Column 1 Accurately weigh 0.018 g of azobisisobutyronitrile (AIBN) and 0.005 g of MIP-202 into a centrifuge tube. Then add 0.135 mL of methacrylic acid, 0.112 mL of tetradecyl methacrylate, 1.8 mL of n-dodecyl alcohol, 1.8 mL of n-propanol, and 0.665 mL of ethylene glycol. Vortex to dissolve the solids, then sonicate for 30 min until uniformly dispersed. Transfer the solution to a stainless steel empty column and react in a 60°C water bath for 11 h to obtain the final product. Subsequent rinsing procedures are as described in Example 1.

[0026] The back pressure of the monolithic column was measured to be 1.1 MPa, and the theoretical plate number was 8545 plates / m. The specific surface area of ​​the continuous bed was measured to be 207.1361 m². 2 / g.

[0027] The nitrogen adsorption-desorption isotherm of the monolithic column 1 in the hybrid mode is shown in the figure below. Figure 1 As shown in (A), the isotherm of the monolithic column 1 in the hybrid mode is a type IV isotherm, indicating that the monolithic material contains a mesoporous structure. The microstructure of the continuous bed prepared above was observed using a scanning electron microscope, as shown... Figure 1 As shown in (B), it can be seen that it exhibits a porous structure with stacked particles.

[0028] Example 4: Preparation of the monolithic column 2 in the hybrid mode Accurately weigh 0.030 g of azobisisobutyronitrile (AIBN) and 0.006 g of MIP-202 into a centrifuge tube. Then add 0.3 mL of methacrylic acid, 0.3 mL of tetradecyl methacrylate, 1.9 mL of n-dodecyl alcohol, 1.9 mL of n-propanol, and 0.7 mL of ethylene glycol. Vortex to dissolve the solid, then sonicate for 30 min until uniformly dispersed. Transfer the solution to a stainless steel empty column and react in a 60°C water bath for 11 h to obtain the final product. Subsequent rinsing procedures are as described in Example 1.

[0029] The back pressure of the monolithic column was measured to be 1.3 MPa, and the theoretical plate number was 7545 plates / m. The specific surface area of ​​the continuous bed was measured to be 167.2627 m². 2 / g.

[0030] Example 5: Preparation of the monolithic column 3 in the hybrid mode Accurately weigh 0.018 g of azobisisobutyronitrile (AIBN) and 0.005 g of MIP-202 into a centrifuge tube. Then add 0.135 mL of methacrylic acid, 0.112 mL of tetradecyl methacrylate, 2 mL of n-dodecyl alcohol, 2 mL of n-propanol, and 0.665 mL of ethylene glycol. Vortex to dissolve the solid, then sonicate for 30 min until uniformly dispersed. Transfer the solution to a stainless steel empty column and react in a 60°C water bath for 11 h to obtain the final product. Subsequent rinsing procedures are as described in Example 1.

[0031] The measured column back pressure of the monolithic column was 0.9 MPa, the theoretical plate number was 1645 plates / m, and a peak leading edge was observed. This indicates that the increase of pore-forming agent reduced the separation capacity of the monolithic column, and the presence of gaps between the monolithic material and the steel column edge led to the peak leading edge.

[0032] Example 6: Preparation of the monolithic column 4 in the hybrid mode Accurately weigh 0.018 g of azobisisobutyronitrile (AIBN) and 0.005 g of MIP-202 into a centrifuge tube. Then add 0.135 mL of methacrylic acid, 0.112 mL of tetradecyl methacrylate, 1.8 mL of n-dodecyl alcohol, 1.8 mL of n-propanol, and 0.8 mL of ethylene glycol. Vortex to dissolve the solid, then sonicate for 30 min until uniformly dispersed. Transfer the solution to a stainless steel empty column and react in a 60°C water bath for 11 h to obtain the final product. Subsequent rinsing procedures are as described in Example 1.

[0033] The measured back pressure of the monolithic column was 1.7 MPa, and the theoretical plate number was 4565 plates / m. The increased crosslinking agent led to a higher back pressure, which has adverse effects on the entire liquid phase system. The monolithic column also faces the risk of collapse; at a flow rate of 1 mL / min, it is easily washed away, making subsequent experiments impossible.

[0034] Example 7: Preparation of the Hybrid Mode Monolithic Column 5 Accurately weigh 0.018 g of azobisisobutyronitrile (AIBN) and 0.007 g of MIP-202 into a centrifuge tube. Then add 0.135 mL of methacrylic acid, 0.112 mL of tetradecyl methacrylate, 1.8 mL of n-dodecyl alcohol, 1.8 mL of n-propanol, and 0.665 mL of ethylene glycol. Vortex to dissolve the solids, then sonicate for 30 min until uniformly dispersed. Transfer the solution to a stainless steel empty column and react in a 60°C water bath for 11 h to obtain the final product. Subsequent rinsing procedures are as described in Example 1.

[0035] Due to an excessive amount of MIP-202, the overall material distribution was uneven, and the excess material was washed out during the rinsing process, making it impossible to conduct subsequent experiments.

[0036] Example 8: Investigation of the mechanical strength and permeability of a monolithic column The prepared monolithic column was connected to a high-performance liquid chromatography (HPLC) system. Using 100% methanol as the mobile phase, the flow rate was slowly increased from 0.1 mL / min to 1 mL / min. The back pressure and flow rate of the mixed-mode monolithic column 1, the hydrophilic single-function monolithic column, and the hydrophobic single-function monolithic column were recorded respectively. The results are as follows: Figure 2 As shown in (A).

[0037] At a flow rate of 1 mL / min, the proportion of methanol in the mobile phase was adjusted from 0% to 100%, and the relationship between the column back pressure and methanol content was recorded for both mixed-mode and single-mode monolithic columns. The results are as follows: Figure 2 As shown in (B).

[0038] Depend on Figure 2 It can be seen that the back pressure of the hybrid monolithic column is between that of hydrophilic and hydrophobic monolithic columns, exhibiting good permeability and mechanical strength.

[0039] Based on the results of Examples 1, 2 and 3, the hybrid mode column has the highest theoretical plate number and the largest specific surface area, indicating that it has stronger separation ability and higher column efficiency.

[0040] Example 9: Hybrid mode monolithic column used for the separation of chemical components and screening of antitumor active ingredients in Dendrobium nobile. The prepared monolithic column 1 in mixed mode was connected to a high-performance liquid chromatography (HPLC) system. A certain amount of *Dendrobium nobile* extract was injected into the column using acetonitrile:water (5:95, 10:90, 20:80, 30:70, v / v) as the mobile phase, and the eluents were collected and labeled as 95% water eluent, 90% water eluent, 80% water eluent, and 70% water eluent, respectively. Then, acetonitrile / water (70 / 30, v / v) solution was used as the eluting mobile phase to elute the components enriched on the monolithic column, and the distillates were collected and labeled as 95% water enriched, 90% water enriched, 80% water enriched, and 70% water enriched, respectively. The solvent was evaporated, and cells without added drugs were used as a control group to test the antitumor effects of the above components. The results were compared with the antitumor effects of *Dendrobium nobile* extract. The comparison results are as follows: Figure 3 As shown.

[0041] Depend on Figure 3 It can be seen that the cell survival rate of the 80% water-eluted portion was low (Figure A), but the anti-tumor effect was significant, with an IC50 value of [missing information]. 50 The concentration was 173.5 μg / mL (Figure B). Compared with Dendrobium nobile extract, its antitumor effect was significantly improved. Therefore, the 80% water-eluting fraction was used for subsequent experiments.

[0042] Example 10: Further tracking and purification of antitumor active ingredients in Dendrobium nobile A reversed-phase semi-preparative chromatographic column was connected to a high-performance liquid chromatography (HPLC) system. Acetonitrile:water (10:90, v / v) was used as the mobile phase. The solution reconstituted from the 80% water elution fraction in Example 5 was injected into the HPLC system. The fractions were collected at 10-12 min (1), 12-14 min (2), and 16-18 min (3), respectively. The solvent was evaporated. Using cells without added drugs as a control group, the antitumor effect was tested. The results are as follows: Figure 4 As shown in Figure A, cell viability is displayed, and Figure B shows the logIC50 of the component with the strongest anti-cancer effect (component 3). It can be seen that the anti-tumor effect is most pronounced in the 16-18 min fraction (3), with its IC50... 50 The concentration was 90.14 μg / mL, which significantly improved the antitumor effect compared to the extract of Dendrobium nobile and the fraction elute with 80% water. The chromatograms of the components after two-step purification are shown below. Figure 5 As shown.

Claims

1. A method for preparing a mixed-mode monolithic column for separating and screening effective chemical components in Dendrobium nobile, characterized in that, Includes the following steps: (1) Azobisisobutyronitrile, metal-organic framework material MIP-202, methacrylic acid, tetradecyl methacrylate, n-dodecyl alcohol, n-propanol and ethylene glycol dimethacrylate are mixed sequentially and ultrasonically oscillated to obtain a monolithic column prepolymer solution. (2) Pour the prepolymer solution of the monolithic column into a stainless steel empty column tube that is sealed at one end and open at the other end, seal the open end, and bathe it in a water bath at 55-65℃ for 10-12 hours. After taking it out and rinsing it, a mixed-mode monolithic column for the separation and screening of effective chemical components in Dendrobium nobile is obtained. The ratio of azobisisobutyronitrile, metal-organic framework material MIP-202, methacrylic acid, tetradecyl methacrylate, n-dodecyl alcohol, n-propanol, and ethylene glycol dimethacrylate is 0.010-0.030g: 0.005-0.006g: 0.1-0.3mL: 0.1-0.3mL: 1.2-1.9mL: 1.2-1.9mL: 0.5-0.7mL.

2. The method for preparing a mixed-mode monolithic column for separating and screening effective chemical components in Dendrobium nobile according to claim 1, characterized in that, The rinsing process involves connecting a stainless steel hollow column tube after a water bath to a high-performance liquid chromatography (HPLC) system, using 100% methanol as the mobile phase, and rinsing at a flow rate of 1 mL / min for 1.5 h.

3. A method for separating and screening effective chemical components in Dendrobium nobile, characterized in that, The mixed-mode monolithic column prepared by the preparation method described in claim 1 for the separation and screening of effective chemical components in Dendrobium chrysogenum is used as a solid-phase extraction adsorbent and coupled with high performance liquid chromatography to separate and screen the effective chemical components in Dendrobium chrysogenum.

4. The method for separating and screening effective chemical components in Dendrobium nobile according to claim 3, characterized in that, First, mobile phase 1 and Dendrobium officinale extract are injected into the mixed-mode monolithic column used for the separation and screening of effective chemical components in Dendrobium officinale, and the distillate of mobile phase 1 is collected; then, mobile phase 2 is injected, and the eluent of mobile phase 2 is collected. The solvent in the distillate of mobile phase 1 and the eluent of mobile phase 2 is evaporated to obtain a preliminary separated eluent.

5. The method for separating and screening effective chemical components in Dendrobium nobile according to claim 4, characterized in that, Both mobile phase 1 and mobile phase 2 are mixed solutions of acetonitrile and water; wherein, in mobile phase 1, the volume ratio of acetonitrile to water is 20:80, and in mobile phase 2, the volume ratio of acetonitrile to water is 70:

30.

6. A method for separating and screening effective chemical components in Dendrobium nobile according to claim 4, characterized in that, The initial eluent was separated by passing it through a reverse-phase semi-preparative column. The eluent with an elution time of 16-18 min was collected and then separated by passing it through a normal-phase semi-preparative column to obtain different fractions of the eluent. The effective chemical components in Dendrobium chrysogenum were screened by detecting the cytotoxicity of different fractions of the eluent to HepG-2 human liver cancer cells using the CCK-8 assay.

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