Aescin high-purity extraction method based on molecular imprinting technology and application in cosmetics

Abstract

The application provides a high-purity extraction method of escin based on a molecular imprinting technique and application of the escin in cosmetics, and belongs to the technical field of plant extraction. The method comprises the following steps: taking horse chestnut seeds as raw materials, crushing and screening after husking, heating and reflux extraction, vacuum concentration to obtain escin extract, loading into a solid-phase extraction column, gradient elution after elution and gradient elution to obtain escin products; wherein, the solid-phase extraction column is filled with a ternary synergistic molecular imprinting polymer taking itaconic acid, 4-vinylpyridine and acrylamide as functional monomers, hydrogen bonds, ionic bonds and synergistic networks of hydrophobic effects are formed by utilizing the advantages of different monomers, effective adsorption of escin is realized, when the molecular imprinting polymer is applied to solid-phase extraction of the escin extract, escin products with a purity of more than 97% and a yield of more than 98% can be efficiently prepared, and the escin products have high application value in the manufacturing and research of cosmetics, biological medicines and the like.

Description

Technical Field

[0001] This invention relates to the field of plant extraction technology, and in particular to a method for high-purity extraction of aescin based on molecular imprinting technology and its application in cosmetics. Background Technology

[0002] Escin is a triterpenoid saponin active ingredient extracted from the seeds and bark of plants in the genus *Aesculus*. It possesses pharmacological effects such as anti-inflammatory, anti-exudative, venous tone-increasing, and blood circulation-improving properties, and is widely used in the pharmaceutical and cosmetic fields. The main active components of escin are aescin A, B, C, and D, where A and B are β-configurations, and C and D are α-configurations. In addition to the above components, more than 100 saponin compounds have been found in *Aesculus* plants. These compounds have similar chemical skeletons, minimal differences in substituents, and numerous isomers, making it difficult to obtain high-purity products using traditional extraction methods.

[0003] Traditional methods for extracting aescin from aescin mainly include solvent extraction and macroporous adsorption resin column chromatography. These methods suffer from poor selectivity, low purity, complex processes, and high consumption of organic solvents. Molecular imprinting technology (MIT) can achieve highly selective separation of target molecules by preparing polymers with specific recognition capabilities for template molecules. However, molecularly imprinted polymers based on single functional monomers often have limited binding site types and adsorption capacities, making it difficult to effectively distinguish structurally similar substances.

[0004] Therefore, it is of great significance to develop a method for the efficient extraction and purification of aescin based on molecular imprinting technology. Summary of the Invention

[0005] The purpose of this invention is to provide a high-purity extraction method for aescin based on molecular imprinting technology. By systematically optimizing the types and ratios of functional monomers, template-monomer ratios, and polymerization conditions, this method solves the problems of insufficient selectivity and low adsorption capacity of existing molecular imprinted polymers for aescin, achieving efficient separation and purification of aescin with a product purity of over 95% and a yield of over 98%.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for high-purity extraction of aescin based on molecular imprinting technology, comprising the following steps: (1) Using horse chestnut seeds as raw material, remove the shell, crush, pass through a 50-100 mesh sieve, add organic solvent for heating and reflux extraction, and concentrate under reduced pressure to obtain horse chestnut saponin extract; (2) The aescin extract was loaded onto a solid phase extraction column, washed, and then eluted with an ethanol solution in a gradient. The eluent was collected, concentrated, and dried to obtain the aescin product. The molecular polymer packed in the solid-phase extraction column is obtained by polymerization of itaconic acid, 4-vinylpyridine and acrylamide as functional monomers.

[0007] Furthermore, the solid-liquid ratio of horse chestnut seeds to organic solvent is 1:7~15 (g / mL). The organic solvent is a 65-75% ethanol solution.

[0008] Furthermore, the conditions for the heating and reflux extraction are: water bath extraction at 65~75℃, extraction time of 2~3 hours, and extraction times of 2~3 times.

[0009] Furthermore, in step (2), the loading flow rate is 0.5 to 1.5 times the column volume; the eluent is a 5% to 10% ethanol solution with an eluent flow rate of 1 to 2 times the column volume; and the elution solution is a 30% to 70% ethanol solution with an elution flow rate of 0.5 to 1.5 times the column volume.

[0010] The present invention also provides a method for preparing the aforementioned molecular polymer, specifically: 1) The aescin template and functional monomers are dissolved and mixed to obtain a pre-assembled complex; 2) Add crosslinking agent and initiator to the pre-assembled composite, introduce inert gas, carry out polymerization reaction, filter, and obtain crude polymer product; 3) Using a mixture of methanol and glacial acetic acid as the eluent, the crude polymer product is subjected to Soxhlet extraction and dried to obtain the molecular polymer.

[0011] Furthermore, in step 1), the molar ratio of the functional monomer itaconic acid, 4-vinylpyridine, acrylamide, and template is 1~2:1~2:2~4:1.

[0012] Furthermore, in step 2), the crosslinking agent is ethylene glycol dimethacrylate, the initiator is azobisisobutyronitrile, and the inert gas is nitrogen or argon.

[0013] Furthermore, in step 2), the polymerization reaction temperature is 55~65℃, the stirring speed is 100~200 rpm, and the reaction time is 20~25h.

[0014] Furthermore, in step 3), the volume ratio of methanol to glacial acetic acid is 8~10:1, the Soxhlet extraction temperature is 65~75℃, and the time is 24~48h.

[0015] In summary, this invention employs an extraction-molecular imprinted column chromatography-elution method to prepare high-purity aescin from horse chestnut seeds: First, 70% ethanol solution is used as the extraction solvent, with a material-to-liquid ratio of 1:8, and extraction is performed twice under reflux at 70°C to obtain an aescin extract; then, molecular imprinted column chromatography is used for elution to obtain high-purity aescin. This method can significantly improve the separation effect of aescin and increase its recovery rate.

[0016] This invention leverages the advantages of different monomers to form a synergistic network of hydrogen bonds, ionic bonds, and hydrophobic interactions, firmly binding aescin. Specifically, IA (itaconic acid) utilizes its two carboxyl groups to form strong ionic bonds with the carboxyl groups on the aglycone of aescin, or to form a dense network of hydrogen bonds with multiple hydroxyl groups. A single IA molecule can provide "two-point anchoring," significantly enhancing the binding force. 4-VP (4-vinylpyridine), with its pyridine nitrogen, is a strong hydrogen bond acceptor and basic site, targeting the carboxyl and hydroxyl groups of aescin. Its aromatic ring can also generate hydrophobic interactions and π-π effects with the steroidal skeleton. AM (acrylamide), as an excellent hydrogen bond "bridge," forms strong hydrogen bonds with the amide group on the glycosyl group and numerous hydroxyl groups on the aglycone of aescin, filling the gap between IA and 4-VP, forming a three-dimensional encapsulation. The advantages of this combination are: comprehensive interaction forces, strong synergistic effect, theoretically one of the strongest binding forces that can be generated in non-covalent imprinting, and the pre-assembled complex is very stable.

[0017] The high-purity extraction method of aescin based on molecular imprinting technology provided by this invention has low raw material costs, can efficiently separate target molecules from complex mixtures, and efficiently prepare aescin products with a purity of over 97% and a yield of over 98%. The process is simple, reproducible, and suitable for industrial production, and has high application value in the manufacturing and research and development of cosmetics, biomedicine and other fields. Detailed Implementation

[0018] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0019] Example 1: Preparation of Molecularly Imprinted Polymers

[0020] Step 1: Preparation of pre-assembly solution

[0021] a. Accurate weighing: Weigh aescin (≥95%, Merck, CAS No.: 6805-41-0): 1 mmol, itaconic acid (IA): 2 mmol, 4-vinylpyridine (4-VP): 2 mmol, acrylamide (AM): 4 mmol; b. Dissolution and mixing: Add all the template and functional monomer to a three-necked flask containing 100 mL of acetonitrile, seal, and sonicate in an ultrasonic (37 kHz) water bath for 15 min until the solid is completely dissolved and the solution becomes clear or homogeneous. c. Pre-assembly: Transfer the flask to a magnetic stirrer and stir slowly (200 rpm) for 4 hours at room temperature (25°C) in the dark to allow the functional monomer and template molecule to fully interact and form a pre-assembled complex.

[0022] Step 2: Polymerization reaction

[0023] d. Add crosslinking agent and initiator: Add the following to the pre-assembled complex: EGDMA (crosslinking agent: ethylene glycol dimethacrylate): 4.0 mmol (ensure crosslinking degree >80%), AIBN (initiator: azobisisobutyronitrile): 0.1 mmol, and sonicate again for 5 min to ensure that all components are completely dissolved and mixed; e. Introduce inert gas: Introduce high-purity nitrogen or argon into the reaction system for 30 minutes to remove oxygen dissolved in the solution (oxygen is an inhibitor of free radical polymerization). f. Thermally initiated polymerization: Connect a three-necked flask to a condenser and place it in a constant temperature water bath at 60°C. Under the conditions of continuous introduction of a small amount of nitrogen and slow stirring (about 150 rpm to prevent bumping), the reaction is carried out for 24 hours. g. Reaction termination and collection: After 24 hours of reaction, the reaction solution will turn into a milky white suspension with white fine particles as precipitate. Stop heating and stirring, and allow it to cool naturally to room temperature (25°C). Filter the reaction product through a microporous membrane (0.45 μm), wash the precipitate three times with a small amount of acetonitrile, and collect the white crude polymer product.

[0024] Step 3: Elution of template molecules (Soxhlet extraction)

[0025] h. Transfer the crude polymer product to the filter paper tube of a Soxhlet extractor. Add 200 mL of eluent in a round-bottom flask with a methanol:glacial acetic acid ratio of 9:1 (v / v) and extract continuously at 70°C for 24–48 h until no aescin is detected in the eluent.

[0026] Step 4: Post-processing and storage

[0027] i. Drying: Transfer the washed molecularly imprinted polymer particles to a petri dish and dry them in a vacuum drying oven at 40°C for 12 hours until constant weight.

[0028] j. Grinding and sieving: Gently grind the dried block polymer and sieve out particles of 150-200 mesh.

[0029] k. Storage: Seal the obtained product in a light-proof, dry container and label it with the name and date.

[0030] Example 2: Extraction and purification of aescin

[0031] (1) Take dried horse chestnut seeds, remove the shells and crush them, pass them through a 100-mesh sieve to obtain horse chestnut seed coarse powder, add 70% ethanol solution at a material-to-liquid ratio of 1:8 (g / mL), heat and reflux in a 70℃ water bath for extraction, filter, the first extraction time is 3h, the second extraction time is 2h, collect and combine the two filtrates, concentrate under reduced pressure at 60℃ until there is no alcohol taste, and obtain concentrated horse chestnut saponin extract.

[0032] (2) The prepared aescin extract concentrate was loaded onto a solid-phase extraction column (filled with the molecular polymer prepared in Example 1) at a loading flow rate of 0.8 column volumes to ensure sufficient contact between aescin and the MIP. After loading, the column was allowed to stand for 15 minutes to enhance the adsorption effect. The column bed was rinsed with 9 column volumes (BV) of 8% ethanol solution at a rinsing flow rate of 1 column volume until the color of the eluent became lighter. Then, gradient elution was performed. First, 30% ethanol was used to elute some weakly retained impurities, and then 70% ethanol solution was used to elute and collect the target component at a elution flow rate of 1 column volume. The collected eluent was concentrated under reduced pressure at 60°C until there was no alcohol odor, and the concentrate was obtained. The concentrate was transferred to an evaporating dish and dried in a vacuum drying oven at 50°C to constant weight to obtain high-purity aescin product.

[0033] Comparative Example 1

[0034] Unlike Example 2, in step (2), the molecular polymer packed in the solid-phase extraction column is obtained by polymerization reaction using 2 mmol of methacrylic acid (MAA), 2 mmol of 4-vinylpyridine (4-VP), and 4 mmol of acrylamide (AM) as functional monomers.

[0035] Comparative Example 2

[0036] Unlike Example 2, in step (2), the molecular polymer packed in the solid-phase extraction column is obtained by polymerization reaction using 2 mmol itaconic acid (IA), 2 mmol 4-vinylpyridine (4-VP), and 4 mmol 2-hydroxyethyl methacrylate (HEMA) as functional monomers.

[0037] Comparative Example 3

[0038] Unlike Example 2, in step (2), the molecular polymer packed in the solid-phase extraction column is obtained by polymerization reaction using 2 mmol itaconic acid (IA) and 2 mmol 4-vinylpyridine (4-VP) as functional monomers.

[0039] Comparative Example 4

[0040] Unlike Example 2, in step (2), the molecular polymer packed in the solid-phase extraction column is obtained by polymerization reaction using 2 mmol of 4-vinylpyridine (4-VP) and 4 mmol of 2-hydroxyethyl methacrylate (HEMA) as functional monomers.

[0041] Comparative Example 5

[0042] Unlike Example 2, the molecular polymer filled in the solid phase extraction column in step (2) is obtained by polymerization reaction using 2 mmol of methacrylic acid (MAA) and 4 mmol of 2-hydroxyethyl methacrylate (HEMA) as functional monomers.

[0043] Comparative Example 6

[0044] Unlike Example 2, the molecular polymer filled in the solid phase extraction column in step (2) is obtained by polymerization reaction using 2 mmol itaconic acid (IA) as the functional monomer.

[0045] Comparative Example 7

[0046] Unlike Example 2, in step (2), the molecular polymer packed in the solid-phase extraction column is obtained by polymerization reaction using 4 mmol itaconic acid (IA), 4 mmol 4-vinylpyridine (4-VP) and 1 mmol acrylamide (AM) as functional monomers.

[0047] Comparative Example 8

[0048] Unlike Example 2, the molecular polymer packed in the solid-phase extraction column used in step (2) is obtained by polymerization reaction using 2 mmol itaconic acid (IA), 2 mmol 4-vinylpyridine (4-VP) and 4 mmol acrylamide (AM) as functional monomers, ethylene glycol dimethacrylate as crosslinking agent and ammonium persulfate as initiator.

[0049] Test case

[0050] Adsorption capacity: Accurately weigh 20.0 mg of MIP particles (150-200 mesh) prepared in each example or comparative example, place them in a 25 mL stoppered conical flask, and add 10 mL of a 1.0 mg / mL aescin acetonitrile solution. Incubate at 150 rpm for 12 h in a constant-temperature shaker at 25 °C. After adsorption is complete, filter the supernatant through a 0.22 μm microporous membrane and determine the concentration of aescin in the solution before and after adsorption using a UV-Vis spectrophotometer at a wavelength of 220 nm. Adsorption capacity Q (mg / g) = (C0 - C e )×V / m; where C0 is the initial concentration (mg / mL), Ce The equilibrium concentration is given by (mg / mL), V is the solution volume (mL), and m is the mass of MIP (g). The purity of each product group was determined by high-performance liquid chromatography (HPLC) and external standard method. Yield = (final product mass × product purity) / (aescin content in raw material) × 100%. Recovery rate determination: Before sample loading, the total mass of aescin (m1) was measured. All eluents after gradient elution were collected, concentrated, dried, weighed, and their purity determined. The mass of aescin obtained from elution (m2) was calculated. Recovery rate = m2 / m1 × 100%.

[0051] Table 1

[0052] As shown in Table 1, the synergy between functional monomers affects the imprinting effect. In the absence of IA or AM (Comparative Examples 3-6), all indicators are significantly lower than in Example 2, indicating that all three monomers used in this invention are indispensable. Furthermore, in Comparative Example 1 (where MAA replaces IA) and Comparative Example 2 (where HEMA replaces AM), the adsorption capacity of the resulting molecular polymer is significantly lower than in Example 2, indicating that the synergistic effect requires the participation of all three monomers. Even with substitutions using similar materials, without the functional monomer combination of this invention, it is difficult to form a dense ion-hydrogen bond synergistic network, failing to provide sufficient binding strength and recognition accuracy for aescin, thus hindering the achievement of excellent imprinting and extraction effects. Comparative Example 7 suggests that the ratio of the three functional monomers also affects the extraction effect; only at a specific ratio can the best results be obtained. Comparative Example 8 shows that after changing the initiator to ammonium persulfate, the adsorption capacity is significantly lower than in Example 2, indicating that the initiation system of Comparative Example 8 is not compatible with the functional monomer combination designed in this invention, and not any single initiator can achieve the imprinting and extraction effects of this application.

[0053] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for high-purity extraction of aescin based on molecular imprinting technology, characterized in that, Includes the following steps: (1) Using horse chestnut seeds as raw material, remove the shell, crush, pass through a 50-100 mesh sieve, add organic solvent for heating and reflux extraction, and concentrate under reduced pressure to obtain horse chestnut saponin extract; (2) The aescin extract was loaded onto a solid phase extraction column, washed, and then eluted with an ethanol solution in a gradient. The eluent was collected, concentrated, and dried to obtain the aescin product. The molecular polymer packed in the solid-phase extraction column is obtained by polymerization of itaconic acid, 4-vinylpyridine and acrylamide as functional monomers.

2. The extraction method as described in claim 1, characterized in that, In step (2), the sample loading rate is 0.5 to 1.5 times the column volume; The eluent is a 5%~10% ethanol solution, and the eluent flow rate is 1~3 times the column volume; The elution solvent is 30-70% ethanol, and the elution flow rate is 0.5-1.5 times the column volume.

3. The extraction method as described in claim 1, characterized in that, The preparation method of the molecular polymer is as follows: 1) The aescin template and functional monomers are dissolved and mixed to obtain a pre-assembled complex; 2) Add crosslinking agent and initiator to the pre-assembled composite, introduce inert gas, carry out polymerization reaction, filter, and obtain crude polymer product; 3) Using a mixture of methanol and glacial acetic acid as the eluent, the crude polymer product is subjected to Soxhlet extraction and dried to obtain the molecular polymer.

4. The extraction method as described in claim 3, characterized in that, The molar ratio of itaconic acid, 4-vinylpyridine, acrylamide, and template in step 1) is 1~2:1~2:2~4:

1.

5. The extraction method as described in claim 4, characterized in that, The crosslinking agent mentioned in step 2) is ethylene glycol dimethacrylate; the initiator is azobisisobutyronitrile; and the inert gas is nitrogen or argon.

6. The extraction method as described in claim 5, characterized in that, In step 2), the polymerization reaction temperature is 55~65℃, the stirring speed is 100~200 rpm, and the reaction time is 20~25h.

7. The extraction method as described in claim 6, characterized in that, In step 3), the volume ratio of methanol to glacial acetic acid is 8~10:1, the Soxhlet extraction temperature is 65~75℃, and the time is 24~48h.

8. The use of aescin obtained by the extraction method according to any one of claims 1 to 7 in cosmetics.

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