Preparation process of willow flower extract and application thereof

Abstract

This invention discloses a preparation process and application of *Willow Tree* extract in the field of plant extract technology. The process includes washing, drying, and pulverizing the *Willow Tree* raw material, defatting with petroleum ether, ultrasonic extraction with ethanol-water solution, concentration under reduced pressure to obtain the extract, purification via zirconium aminophosphonic acid hybrid layered zeolite chromatography column, gradient elution to collect the active components, concentration, addition of cyclodextrin, freeze-drying, and pulverization to obtain the final product. The zirconium aminophosphonic acid hybrid layered zeolite is activated and expanded by high-temperature calcination with caustic soda using molecular sieves, then grafted with a silane coupling agent under anhydrous conditions to introduce amino groups, followed by amidation with carboxyethylphosphonic acid to introduce phosphonic acid groups, subsequently chelating with zirconium ions, and finally forming a hybrid layered structure through in-situ growth of layered double hydroxides using a hydrothermal method. The process conditions of this invention are mild, resulting in extracts with high purity and good stability. It can be widely used for the extraction of active ingredients from *Willow Tree* in health foods, cosmetics, and pharmaceutical excipients, exhibiting significant antioxidant and health-promoting effects.

Description

Technical Field

[0001] This invention relates to the field of plant extract technology, specifically to a preparation process and application of willowherb extract. Background Technology

[0002] Willowherb (Salvia splendens) is a perennial herb belonging to the genus Willowherb in the family Willowherbaceae. It is widely distributed in northern my country and has a long history of medicinal use. Traditional medicine believes that willowherb has the effects of clearing heat and detoxifying, reducing swelling and relieving pain, and promoting blood circulation. Modern pharmacological studies have shown that it is rich in flavonoids, phenolic acids, polysaccharides, and other bioactive components, exhibiting significant antioxidant, anti-inflammatory, antibacterial, and immunomodulatory effects. With the increasing demand for natural plant extracts, willowherb extract has broad application prospects in health foods, cosmetics, and pharmaceutical excipients. However, the active ingredients in willowherb are numerous, structurally complex, and present in low concentrations. How to efficiently extract and purify these active ingredients while maintaining their bioactivity stability has become a key technological bottleneck restricting the in-depth development and utilization of willowherb resources.

[0003] Currently, the preparation processes for willowherb flower extract mainly employ traditional techniques such as solvent extraction, ultrasonic-assisted extraction, or microwave-assisted extraction. While these methods are simple to operate, they suffer from problems such as poor extraction selectivity, incomplete impurity removal, and significant loss of active ingredients. In particular, coexisting components such as chlorophyll, lipid-soluble impurities, and macromolecular polysaccharides are difficult to separate effectively, resulting in low purity, dark color, and poor stability of the extract, severely impacting the quality and application effects of subsequent products. In the separation and purification stage, conventional macroporous resins, silica gel, or polyamide chromatography materials, although offering some separation effect, suffer from drawbacks such as low adsorption capacity, insufficient selectivity, and difficulty in regeneration, making it difficult to achieve efficient enrichment of the target active ingredients in willowherb flower. Furthermore, the existing process parameters are not precisely controlled, and the extraction and purification processes lack systematic optimization, leading to large batch-to-batch quality fluctuations and failing to meet the stability requirements of industrial production.

[0004] In the field of chromatographic separation materials, traditional zeolite molecular sieves, while possessing regular pore structures and large specific surface areas, exhibit limited surface chemical properties and lack selective recognition capabilities for specific active ingredients. In recent years, research on organic-inorganic hybrid functional materials has provided new avenues for separation and purification technologies; however, existing modified zeolite materials generally suffer from low functional group loading, poor structural stability, and complex preparation processes. Materials involving multi-step chemical modifications, in particular, often require the introduction of non-commercially available intermediates or stringent reaction conditions, increasing production costs and technical difficulty. Furthermore, the coordination chemistry of zirconium-based materials with phosphonic acid groups demonstrates unique advantages in separation science, but research reports on their combination with zeolite supports and layered double hydroxides to construct hybrid layered structures are scarce, and related preparation processes are still immature. Therefore, developing a readily available, controllable, and highly efficient preparation process for *Willow Tree* extract, along with corresponding functionalized chromatographic materials, is of significant theoretical and practical value for enhancing the utilization value of *Willow Tree* resources and promoting advancements in natural product extraction technologies. Summary of the Invention

[0005] The purpose of this invention is to provide a preparation process and application of willowherb flower extract, which solves the technical problems of low purity, large loss of active ingredients, poor separation selectivity and insufficient product stability in existing willowherb flower extract preparation processes.

[0006] The present invention achieves the above objectives through the following technical solutions: A process for preparing an extract of willowherb flowers includes the following steps: S1. By weight, collect 80-120 parts of willowherb flowers, rinse with deionized water, and air dry to obtain clean willowherb flowers; dry the clean willowherb flowers and crush them to obtain dried raw materials; pulverize the dried raw materials and sieve them to obtain willowherb flower coarse powder; add 500-600 parts of petroleum ether to the willowherb flower coarse powder and stir at room temperature to defatted it; filter to obtain filter cake; place the filter cake in a fume hood to obtain defatted willowherb flower powder; S2, add defatted willowherb flower powder to a mixed solution of 1200-1300 parts of ethanol and water to obtain a mixture; extract the mixture by ultrasonication; after extraction, filter to obtain a filtrate; concentrate the filtrate under reduced pressure to obtain a concentrated willowherb flower extract. S3. Dilute the concentrated extract of willowherb flowers with deionized water and adjust the pH value to obtain a diluted solution. Pass the diluted solution through a chromatography column pre-packed with 5-10 parts of zirconium-aminophosphonic acid hybrid layered zeolite. Rinse the chromatography column with deionized water. Then elute with an ethanol aqueous solution and discard the eluent. Elute again with an ethanol aqueous solution and collect the eluent. S4. Concentrate the eluent collected in step S3 under reduced pressure to obtain a concentrated extract; add β-cyclodextrin to the concentrated extract and mix to obtain a mixture; freeze-dry the mixture to obtain a dried product; pulverize the dried product and sieve it.

[0007] In this invention, the preparation process of willowherb extract is based on the synergistic effect of multiple physicochemical principles. Petroleum ether defatting utilizes the principle of "like dissolves like," where non-polar petroleum ether effectively dissolves fat-soluble impurities such as chlorophyll, waxes, and oils. Room temperature conditions prevent the degradation of heat-sensitive active ingredients, and the defatted raw material is more conducive to the subsequent extraction of polar active ingredients. Ultrasonic extraction utilizes the cavitation effect generated by ultrasound in a liquid. When bubbles collapse, they create localized high temperatures, high pressures, and strong shock waves, disrupting the plant cell wall structure and accelerating the diffusion of intracellular active ingredients into the solvent. The ethanol-water mixed solvent has moderate polarity, effectively dissolving target active ingredients such as flavonoids and phenolic acids. During the chromatographic separation process, the pH of the extract is adjusted to a weakly acidic state, allowing phenolic compounds to exist in molecular form, which facilitates their interaction with the chromatographic material. Zirconium ions on the surface of the zirconium-aminophosphonic acid hybrid layered zeolite form coordination bonds with phenolic hydroxyl groups, while phosphonic acid groups form hydrogen bonds with the active ingredient, achieving specific adsorption. Water washing removes weakly adsorbed polysaccharides and proteins, low-concentration ethanol elutes away moderately adsorbed impurities, and high-concentration ethanol disrupts the coordination and hydrogen bonds, eluting the target active ingredient. When the concentrated extract is mixed with cyclodextrin, the hydrophobic cavities of the cyclodextrin can encapsulate the active ingredient molecules, forming stable inclusion complexes that protect the active ingredient from oxidation and photodegradation. Freeze-drying, under low-temperature vacuum conditions, allows water to sublimate directly, avoiding the damage of liquid water to the active ingredient. Simultaneously, it forms a porous and loose structure, improving the product's resolubility and stability, ultimately yielding a high-purity, high-activity, and highly stable willowherb extract.

[0008] According to a preferred embodiment of the present invention, in step S1, the stirring and degreasing time is 2-4 hours.

[0009] According to a preferred embodiment of the present invention, in step S2, the ultrasonic extraction time is 30-50 min.

[0010] According to a preferred embodiment of the present invention, in step S3, the pH value is adjusted to 5.4-5.6.

[0011] According to a preferred embodiment of the present invention, in step S4, the freeze-drying time is 36-40 hours.

[0012] According to a preferred embodiment of the present invention, the preparation steps of the zirconium-aminophosphonic acid hybrid layered zeolite include: A1, by weight, 95-105 parts of molecular sieve are heated to 495-505℃ and calcined, then naturally cooled to room temperature to obtain calcined molecular sieve; the calcined molecular sieve is immersed in 20-40 parts of sodium hydroxide aqueous solution and shaken at 50-70℃; after treatment, it is filtered, washed with deionized water, and then dried at 104-106℃ to obtain activated and expanded pore zeolite support; A2. Activated and expanded zeolite support was added to 1400-1600 parts of anhydrous toluene and ultrasonically dispersed. 15-25 parts of 3-aminopropyltriethoxysilane were added dropwise, nitrogen gas was introduced, and the mixture was refluxed and stirred at 108-112℃. After the reaction was completed, the mixture was cooled and filtered to obtain a solid. The solid was washed with toluene, anhydrous ethanol, and deionized water, and dried under vacuum at 58-62℃ to obtain amino-functionalized zeolite. The amino-functionalized zeolite was dispersed in a mixed solvent of 900-1100 parts of anhydrous ethanol and water, and 25-50 parts of 2-carboxyethylphosphonic acid, 40-80 parts of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 25-50 parts of N-hydroxysuccinimide were added. The mixture was reacted at room temperature to obtain a reaction mixture. The reaction mixture was centrifuged to obtain a solid product. The solid product was washed successively with ethanol, deionized water, and anhydrous ethanol, and dried under vacuum to obtain aminophosphonic acid-modified zeolite. A3. Dissolve 45-55 parts of zirconium oxychloride in deionized water, adjust the pH to 3.0-3.5 to obtain a zirconium ion solution; add aminophosphonic acid-modified zeolite to the zirconium ion solution and shake the reaction at room temperature; after the reaction is complete, filter, wash with acidic deionized water, rinse with anhydrous ethanol, and vacuum dry to obtain zirconium ion coordination-modified zeolite. A4. Zirconium ion-coordinated modified zeolite is dispersed in 2400-2600 parts of deionized water to obtain a dispersion; 160-190 parts of hexamethylenetetramine are dissolved in 600-700 parts of deionized water to obtain a hexamethylenetetramine solution; 150-180 parts of aluminum nitrate nonahydrate are dissolved in 700-800 parts of deionized water to obtain an aluminum nitrate solution; the aluminum nitrate solution is added to the dispersion under stirring and mixed to obtain a mixture; the hexamethylenetetramine solution is added to the mixture under stirring; the pH is adjusted to 6.8-7.2, and the mixture is transferred to a high-pressure reactor for hydrothermal reaction at 90-100℃; after the reaction is completed, the mixture is naturally cooled to room temperature, centrifuged, and a solid precipitate is obtained; the solid precipitate is washed alternately with deionized water and anhydrous ethanol, and vacuum dried to obtain a dried product; the dried product is heat-treated at 120-150℃ under a nitrogen atmosphere, and then ground and sieved.

[0013] In this invention, the preparation of zirconium-aminophosphonic acid hybrid layered zeolite involves a multi-step chemical modification and structure building process. First, the molecular sieve is calcined at high temperature to remove the template agent and impurities within the pores, making the framework structure more stable and generating more surface hydroxyl active sites. Subsequently, alkali treatment partially desiliconizes the zeolite framework, forming a mesoporous structure, significantly increasing the pore volume and specific surface area, providing sufficient reaction sites for subsequent functionalization modifications. In an anhydrous toluene system, the ethoxy group of the silane coupling agent undergoes a condensation reaction with the hydroxyl groups on the zeolite surface, forming stable silicon-oxygen bonds, covalently grafting amino groups onto the zeolite surface, with nitrogen protection preventing amino oxidation. Next, through an amidation reaction, the carboxyl group of the phosphonic acid compound forms an amide bond with the amino group on the zeolite surface under the action of a condensing agent, introducing phosphonic acid groups into the zeolite support. The phosphonic acid groups possess excellent metal ion coordination ability. Subsequently, under acidic conditions, zirconium ions dissociated from zirconium oxychloride coordinate with phosphonic acid groups to form stable zirconium-oxyphosphorus coordination bonds. The zirconium ions, acting as Lewis acid centers, enhance the material's specific recognition ability for phenolic hydroxyl compounds. Finally, via a hydrothermal method, aluminum source is hydrolyzed under alkaline conditions to generate aluminum hydroxide, which reacts in situ with the zirconium-modified zeolite surface, growing to form a layered double hydroxide structure. Hexamethylenetetramine acts as a precipitant, slowly releasing hydroxide ions to control the crystal growth rate, forming an organic-inorganic hybrid layered composite structure. The interlayer domains can accommodate target molecules, and heat treatment further stabilizes the structure, ultimately yielding a chromatographic material with high adsorption capacity and high selectivity.

[0014] According to a preferred embodiment of the present invention, the oscillation treatment at 50-70°C in step A1 is carried out for 1-3 hours.

[0015] According to a preferred embodiment of the present invention, in step A2, the reaction time at room temperature is 12-14 hours.

[0016] According to a preferred embodiment of the present invention, in step A3, the shaking reaction time at room temperature is 12-24 hours.

[0017] The present invention also provides a preparation process for the above-mentioned willowherb extract, which is applied to the extraction of active ingredients from willowherb in health foods, cosmetics or pharmaceutical excipients.

[0018] The beneficial effects of this invention are as follows: The preparation process for willowherb extract provided by this invention has significant advantages in the raw material pretreatment and extraction stages. Degreasing with petroleum ether at room temperature by stirring effectively removes chlorophyll, waxes, and fat-soluble impurities from the willowherb raw material, preventing these components from interfering with the separation of the target active ingredient during subsequent extraction and purification. Simultaneously, room temperature conditions prevent the degradation of heat-sensitive active ingredients. Ultrasonic-assisted extraction using an ethanol-water mixture utilizes the cavitation effect and mechanical vibration of ultrasound to disrupt the plant cell wall structure, accelerating the diffusion of active ingredients into the solvent, significantly shortening the extraction time and increasing the extraction rate. The vacuum concentration process is carried out at a lower temperature, maximizing the preservation of the structural integrity and bioactivity of heat-labile active ingredients such as flavonoids and phenolic acids. The entire extraction process is characterized by mild conditions, simple operation, low energy consumption, suitability for large-scale production, and good batch-to-batch quality stability, providing a high-quality raw material solution for subsequent purification processes.

[0019] The core innovation of this invention lies in the independently developed zirconium aminophosphonic acid hybrid layered zeolite chromatography material, which possesses a unique multi-level structure and selective recognition capability. High-temperature calcination and alkali treatment activate and expand the pores, significantly increasing the specific surface area and pore volume of the zeolite support, providing ample reaction sites for subsequent functionalization modifications. Silanization grafting under anhydrous conditions avoids the self-condensation side reaction of the silane coupling agent, ensuring uniform loading of amino groups on the zeolite surface. Phosphonic acid groups are introduced through amidation, followed by coordination chelation with zirconium ions to form a stable metal-organic coordination structure, endowing the material with specific adsorption capacity for phenolic hydroxyl compounds. Finally, layered double hydroxides are grown in situ using a hydrothermal method to construct an organic-inorganic hybrid layered composite structure, further increasing the interlayer space and surface active sites. This chromatography material exhibits high adsorption capacity, high selectivity, and good regeneration performance, effectively enriching the target active ingredients in *Illicium verum* while removing interfering impurities such as polysaccharides and proteins, significantly improving the purity of the extract.

[0020] The resulting willowherb extract is of excellent quality and has wide-ranging applications. A gradient elution purification strategy is employed: first, weakly adsorbed impurities are removed by elution with water and low-concentration ethanol; then, the target active ingredient is collected by elution with high-concentration ethanol, achieving efficient separation. The concentrated extract is mixed with cyclodextrin and freeze-dried. The cavity structure of cyclodextrin can encapsulate active ingredient molecules, forming stable inclusion complexes that effectively prevent oxidative degradation of the active ingredient, improving the product's storage stability and bioavailability. The freeze-drying process is carried out under low-temperature vacuum conditions, avoiding damage to heat-sensitive components. The resulting product is porous, has good resolubility, light color, and a pure odor. The obtained extract has high purity, retains its active ingredients intact, and exhibits good stability. It can be widely used as a functional factor additive in health foods, an antioxidant active ingredient in cosmetics, and a natural source of pharmaceutical excipients, meeting the market demand for high-quality natural plant extracts in the health industry and demonstrating significant economic and social benefits. Detailed Implementation

[0021] The following detailed embodiments are only used to further illustrate this application and should not be construed as limiting the scope of protection of this application. Those skilled in the art can make some non-essential improvements and adjustments to this application based on the above application content.

[0022] Example 1 This embodiment provides a preparation process for willowherb flower extract, including the following steps: Step S1: Collect 100g of willowherb flowers, rinse 3 times with 500g of deionized water, and air dry to obtain clean willowherb flowers. Dry the clean willowherb flowers at 50℃ for 24h and then crush them to obtain the dried raw material. Crush the dried raw material through a 40-mesh sieve to obtain willowherb flower coarse powder. Add 550g of petroleum ether to the willowherb flower coarse powder and stir at 25℃ for 3h to degrease. Filter to obtain a filter cake. Place the filter cake in a fume hood to evaporate the solvent for 24h to obtain degreased willowherb flower powder. Step S2: Add defatted willowherb flower powder to 1250g of a mixed solution of ethanol and water with a volume ratio of 70:30 to obtain a mixture. Extract the mixture by ultrasonication for 40min. After extraction, filter to obtain filtrate. Concentrate the filtrate under reduced pressure at 55℃ to one-tenth of the original volume to obtain willowherb flower extract concentrate. Step S3: Dilute the concentrated extract of willowherb flowers with 200g of deionized water, adjust the pH to 5.5 with dilute hydrochloric acid to obtain a diluted solution, pass the diluted solution through a chromatography column pre-packed with 8g of zirconium-aminophosphonic acid hybrid layered zeolite, control the flow rate at 1mL / min, rinse the chromatography column with 300g of deionized water, then elute with 200g of ethanol aqueous solution with a volume ratio of 20:80 and discard the eluent, then elute with 300g of ethanol aqueous solution with a volume ratio of 60:40 and collect the eluent; Step S4: The eluent collected in step S3 is concentrated under reduced pressure at 55°C to obtain a concentrated extract. 5g of β-cyclodextrin is added to the concentrated extract and mixed evenly to obtain a mixture. The mixture is freeze-dried at -50°C for 38h to obtain a dried product. The dried product is pulverized and passed through an 80-mesh sieve to obtain the finished willowherb extract.

[0023] Preparation of zirconium-aminophosphonic acid hybrid layered zeolites: Step A1: Weigh 100g of molecular sieve and place it in a muffle furnace. Heat the furnace to 500℃ and calcine for 4 hours. Allow it to cool naturally to room temperature to obtain the calcined molecular sieve. Immerse the calcined molecular sieve in 30g of 10% sodium hydroxide aqueous solution and shake it at 60℃ for 2 hours. After the treatment, filter the solution, wash it three times with 500g of deionized water, and then dry it at 105℃ for 12 hours to obtain the activated and expanded zeolite support. Step A2: The activated and expanded zeolite support was added to 1500g of anhydrous toluene and ultrasonically dispersed for 30min. 20g of 3-aminopropyltriethoxysilane was added dropwise, and the mixture was purged with nitrogen and refluxed at 110℃ with stirring for 8h. After the reaction, the mixture was cooled to room temperature and filtered to obtain a solid. The solid was washed three times each with 200g of toluene, 200g of anhydrous ethanol, and 200g of deionized water. It was then vacuum dried at 60℃ for 12h to obtain amino-functionalized zeolite. The amino-functionalized zeolite was dispersed in 10... 35g of 2-carboxyethylphosphonic acid, 60g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 35g of N-hydroxysuccinimide were added to a 1:1 mixture of anhydrous ethanol and water. The mixture was reacted at 25°C for 13 hours. After the reaction was completed, the solid product was obtained by centrifugation. The solid product was washed three times each with 200g of ethanol, 200g of deionized water, and 200g of anhydrous ethanol. The product was then dried under vacuum for 12 hours to obtain aminophosphonic acid-modified zeolite. Step A3: Dissolve 50g of zirconium oxychloride in 200g of deionized water, adjust the pH to 3.2 with dilute hydrochloric acid to obtain a zirconium ion solution, add aminophosphonic acid-modified zeolite to the zirconium ion solution, and shake and react at room temperature of 25℃ for 18h. After the reaction is completed, filter, wash 3 times with 300g of acidic deionized water, rinse 2 times with 200g of anhydrous ethanol, and vacuum dry for 12h to obtain zirconium ion coordination-modified zeolite. Step A4: Disperse the zirconium ion-coordinated zeolite in 2500g of deionized water to obtain a dispersion. Dissolve 175g of hexamethylenetetramine in 650g of deionized water to obtain a hexamethylenetetramine solution. Dissolve 165g of aluminum nitrate nonahydrate in 750g of deionized water to obtain an aluminum nitrate solution. Add the aluminum nitrate solution to the dispersion under stirring and mix for 30min to obtain a mixture. Add the hexamethylenetetramine solution to the mixture under stirring. Adjust the pH to 7.0 with sodium hydroxide solution. Transfer to a high-pressure reactor and hydrothermally react at 95℃ for 12h. After the reaction, allow to cool to room temperature naturally. Centrifuge to obtain a solid precipitate. Wash the solid precipitate alternately with 500g of deionized water and 500g of anhydrous ethanol 5 times each. Vacuum dry for 12h to obtain a dried product. Heat-treat the dried product at 135℃ for 6h under a nitrogen atmosphere. Grind through a 100-mesh sieve to obtain zirconium-aminophosphonic acid hybrid layered zeolite.

[0024] Example 2 The specific implementation method is the same as in Example 1, except that a willowherb extract is prepared: Step S1: Collect 80g of willowherb flowers, rinse 3 times with 500g of deionized water, and air dry to obtain clean willowherb flowers. Dry the clean willowherb flowers at 50℃ for 24h and then crush them to obtain the dried raw material. Crush the dried raw material through a 40-mesh sieve to obtain willowherb flower coarse powder. Add 500g of petroleum ether to the willowherb flower coarse powder and stir at 25℃ for 2h to degrease. Filter to obtain a filter cake. Place the filter cake in a fume hood to evaporate the solvent for 24h to obtain degreased willowherb flower powder. Step S2: Add defatted willowherb flower powder to 1200g of a mixed solution of ethanol and water with a volume ratio of 70:30 to obtain a mixture. Extract the mixture by ultrasonication for 30min. After extraction, filter to obtain filtrate. Concentrate the filtrate under reduced pressure at 55℃ to one-tenth of the original volume to obtain willowherb flower extract concentrate. Step S3: Dilute the concentrated extract of willowherb flowers with 200g of deionized water, adjust the pH to 5.4 with dilute hydrochloric acid to obtain a diluted solution, pass the diluted solution through a chromatography column pre-packed with 5g of zirconium-aminophosphonic acid hybrid layered zeolite, control the flow rate at 1mL / min, rinse the chromatography column with 300g of deionized water, then elute with 200g of ethanol aqueous solution with a volume ratio of 20:80 and discard the eluent, then elute with 300g of ethanol aqueous solution with a volume ratio of 60:40 and collect the eluent; Step S4: The eluent collected in step S3 is concentrated under reduced pressure at 55°C to obtain a concentrated extract. 5g of β-cyclodextrin is added to the concentrated extract and mixed evenly to obtain a mixture. The mixture is freeze-dried at -50°C for 36h to obtain a dried product. The dried product is pulverized and passed through an 80-mesh sieve to obtain the willowherb extract product.

[0025] Preparation of zirconium-aminophosphonic acid hybrid layered zeolites: Step A1: Weigh 95g of molecular sieve and place it in a muffle furnace. Heat the furnace to 495℃ and calcine for 5 hours. Allow it to cool naturally to room temperature to obtain the calcined molecular sieve. Immerse the calcined molecular sieve in 20g of 10% sodium hydroxide aqueous solution and shake it at 50℃ for 1 hour. After the treatment, filter the solution, wash it three times with 500g of deionized water, and then dry it at 104℃ for 12 hours to obtain the activated and expanded zeolite support. Step A2: The activated and expanded zeolite support was added to 1400g of anhydrous toluene and ultrasonically dispersed for 30min. 15g of 3-aminopropyltriethoxysilane was added dropwise, and the mixture was purged with nitrogen for protection. The mixture was refluxed and stirred at 108℃ for 8h. After the reaction, the mixture was cooled to room temperature and filtered to obtain a solid. The solid was washed three times each with 200g of toluene, 200g of anhydrous ethanol, and 200g of deionized water. The solid was then vacuum dried at 58℃ for 12h to obtain amino-functionalized zeolite. The amino-functionalized zeolite was dispersed in 9... 25g of 2-carboxyethylphosphonic acid, 40g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 25g of N-hydroxysuccinimide were added to a 1:1 mixture of anhydrous ethanol and water. The mixture was reacted at 25°C for 12 hours. After the reaction was completed, the solid product was obtained by centrifugation. The solid product was washed three times each with 200g of ethanol, 200g of deionized water, and 200g of anhydrous ethanol. The product was then dried under vacuum for 12 hours to obtain aminophosphonic acid-modified zeolite. Step A3: Dissolve 45g of zirconium oxychloride in 200g of deionized water, adjust the pH to 3.0 with dilute hydrochloric acid to obtain a zirconium ion solution, add aminophosphonic acid-modified zeolite to the zirconium ion solution, and shake the reaction at room temperature of 25℃ for 12h. After the reaction is completed, filter, wash 3 times with 300g of acidic deionized water, rinse 2 times with 200g of anhydrous ethanol, and vacuum dry for 12h to obtain zirconium ion coordination-modified zeolite. Step A4: Disperse the zirconium ion-coordinated zeolite in 2400g of deionized water to obtain a dispersion. Dissolve 160g of hexamethylenetetramine in 600g of deionized water to obtain a hexamethylenetetramine solution. Dissolve 150g of aluminum nitrate nonahydrate in 700g of deionized water to obtain an aluminum nitrate solution. Add the aluminum nitrate solution to the dispersion under stirring and mix for 30min to obtain a mixture. Add the hexamethylenetetramine solution to the mixture under stirring. Adjust the pH to 6.8 with sodium hydroxide solution. Transfer to a high-pressure reactor and hydrothermally react at 90℃ for 12h. After the reaction, allow to cool naturally to room temperature. Centrifuge to obtain a solid precipitate. Wash the solid precipitate alternately with 500g of deionized water and 500g of anhydrous ethanol 5 times each. Vacuum dry for 12h to obtain a dried product. Heat-treat the dried product at 120℃ for 6h under a nitrogen atmosphere. Grind through a 100-mesh sieve to obtain zirconium-aminophosphonic acid hybrid layered zeolite.

[0026] Example 3 The specific implementation method is the same as in Example 1, except that a willowherb extract is prepared: Step S1: Collect 120g of willowherb flowers, rinse 3 times with 500g of deionized water, and air dry to obtain clean willowherb flowers. Dry the clean willowherb flowers at 50℃ for 24h and then crush them to obtain the dried raw material. Crush the dried raw material through a 40-mesh sieve to obtain willowherb flower coarse powder. Add 600g of petroleum ether to the willowherb flower coarse powder and stir at 25℃ for 4h to degrease. Filter to obtain a filter cake. Place the filter cake in a fume hood to evaporate the solvent for 24h to obtain degreased willowherb flower powder. Step S2: Add defatted willowherb flower powder to 1300g of a mixed solution of ethanol and water with a volume ratio of 70:30 to obtain a mixture. Extract the mixture by ultrasonication for 50min. After extraction, filter to obtain a filtrate. Concentrate the filtrate under reduced pressure at 55℃ to one-tenth of the original volume to obtain willowherb flower extract concentrate. Step S3: Dilute the concentrated extract of willowherb flowers with 200g of deionized water, adjust the pH to 5.6 with dilute hydrochloric acid to obtain a diluted solution, pass the diluted solution through a chromatography column pre-packed with 10g of zirconium-aminophosphonic acid hybrid layered zeolite, control the flow rate at 1mL / min, rinse the chromatography column with 300g of deionized water, then elute with 200g of ethanol aqueous solution with a volume ratio of 20:80 and discard the eluent, then elute with 300g of ethanol aqueous solution with a volume ratio of 60:40 and collect the eluent; Step S4: The eluent collected in step S3 is concentrated under reduced pressure at 55°C to obtain a concentrated extract. 5g of β-cyclodextrin is added to the concentrated extract and mixed evenly to obtain a mixture. The mixture is freeze-dried at -50°C for 40h to obtain a dried product. The dried product is pulverized and passed through an 80-mesh sieve to obtain the willowherb extract product.

[0027] Preparation of zirconium-aminophosphonic acid hybrid layered zeolites: Step A1: Weigh 105g of molecular sieve and place it in a muffle furnace. Heat the furnace to 505℃ and calcine for 4 hours. Allow it to cool naturally to room temperature to obtain the calcined molecular sieve. Immerse the calcined molecular sieve in 40g of 10% sodium hydroxide aqueous solution and shake it at 70℃ for 3 hours. After the treatment, filter the solution, wash it three times with 500g of deionized water, and then dry it at 106℃ for 12 hours to obtain the activated and expanded zeolite support. Step A2: The activated and expanded zeolite support was added to 1600g of anhydrous toluene and ultrasonically dispersed for 30min. 25g of 3-aminopropyltriethoxysilane was added dropwise, and the mixture was purged with nitrogen for protection. The mixture was refluxed and stirred at 112℃ for 8h. After the reaction, the mixture was cooled to room temperature and filtered to obtain a solid. The solid was washed three times each with 200g of toluene, 200g of anhydrous ethanol, and 200g of deionized water. The solid was then vacuum dried at 62℃ for 12h to obtain amino-functionalized zeolite. The amino-functionalized zeolite was dispersed in 11... 50g of 2-carboxyethylphosphonic acid, 80g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 50g of N-hydroxysuccinimide were added to a 1:1 mixture of anhydrous ethanol and water. The mixture was reacted at 25°C for 14 hours. After the reaction was completed, the solid product was obtained by centrifugation. The solid product was washed three times each with 200g of ethanol, 200g of deionized water, and 200g of anhydrous ethanol. The product was then vacuum dried for 12 hours to obtain aminophosphonic acid-modified zeolite. Step A3: Dissolve 55g of zirconium oxychloride in 200g of deionized water, adjust the pH to 3.5 with dilute hydrochloric acid to obtain a zirconium ion solution, add aminophosphonic acid-modified zeolite to the zirconium ion solution, and shake and react at room temperature of 25℃ for 24h. After the reaction is completed, filter, wash 3 times with 300g of acidic deionized water, rinse 2 times with 200g of anhydrous ethanol, and vacuum dry for 12h to obtain zirconium ion coordination-modified zeolite. Step A4: Disperse the zirconium ion-coordinated zeolite in 2600g of deionized water to obtain a dispersion. Dissolve 190g of hexamethylenetetramine in 700g of deionized water to obtain a hexamethylenetetramine solution. Dissolve 180g of aluminum nitrate nonahydrate in 800g of deionized water to obtain an aluminum nitrate solution. Add the aluminum nitrate solution to the dispersion under stirring and mix for 30min to obtain a mixture. Add the hexamethylenetetramine solution to the mixture under stirring. Adjust the pH to 7.2 with sodium hydroxide solution. Transfer to a high-pressure reactor and hydrothermally react at 100℃ for 12h. After the reaction, allow to cool naturally to room temperature. Centrifuge to obtain a solid precipitate. Wash the solid precipitate alternately with 500g of deionized water and 500g of anhydrous ethanol 5 times each. Vacuum dry for 12h to obtain a dried product. Heat-treat the dried product at 150℃ for 6h under a nitrogen atmosphere. Grind through a 100-mesh sieve to obtain zirconium-aminophosphonic acid hybrid layered zeolite.

[0028] Comparative Example 1 The specific implementation method is the same as in Example 1, except that zirconium oxychloride is not added in step A3 for zirconium ion coordination modification, and the aminophosphonic acid modified zeolite is directly subjected to the hydrothermal reaction in step A4. The remaining steps and the preparation process steps S1 to S4 of the willowherb extract are the same as in Example 1.

[0029] Comparative Example 2 The specific implementation method is the same as in Example 1, except that in step A2, 2-carboxyethylphosphonic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide are not added for aminophosphonic acid modification. Instead, the amino-functionalized zeolite is directly modified by zirconium ion coordination in step A3. The remaining steps and the preparation process steps S1 to S4 of the willowherb extract are the same as in Example 1.

[0030] Comparative Example 3 The specific implementation method is the same as in Example 1, except that amino functionalization modification is not performed in step A2, zirconium ion coordination modification is not performed in step A3, and the activated and expanded zeolite support is directly subjected to the hydrothermal reaction in step A4. The remaining steps and the preparation process steps S1 to S4 of the willowherb extract are the same as in Example 1.

[0031] Performance testing The preparation processes of the willowherb extracts prepared in Examples 1-3 and Comparative Examples 1-3 were subjected to performance testing according to the following methods, which included the following steps: The yield of the extract was determined by weighing. The initial mass m0 of the willowherb flower raw material was accurately weighed, and the mass m1 of the final willowherb flower extract product was obtained by weighing. The yield Y was calculated as m1 divided by m0 multiplied by 100%, i.e., Y = (m1 / m0) × 100%. The total flavonoid content was determined using the aluminum nitrate colorimetric method. 10 mg of willowherb extract sample was accurately weighed, dissolved in methanol, and diluted to 10 mL to obtain a sample stock solution. 1 mL of the sample solution was added to 0.1 mL of 5% sodium nitrite solution, shaken well, and allowed to stand for 6 min. Then, 0.1 mL of 10% aluminum nitrate solution was added, shaken well, and allowed to stand for 6 min. Finally, 1 mL of 4% sodium hydroxide solution was added, and the solution was diluted to 5 mL with methanol, shaken well, and allowed to stand for 15 min. The absorbance was measured at 510 nm using a UV-Vis spectrophotometer. Standard solutions with mass concentrations of 0.02 mg / mL, 0.04 mg / mL, 0.06 mg / mL, 0.08 mg / mL, and 0.10 mg / mL were prepared using rutin as a standard. The absorbance was measured using the same method, and a standard curve was plotted. The regression equation was A = 0.0125C + 0.002, and R² = 0.9995. The total flavonoid content in the sample was calculated based on the standard curve, in mg / g. The total phenol content was determined using the Folin-Ciocalteu colorimetric method. 10 mg of willowherb extract sample was accurately weighed, dissolved in distilled water, and diluted to 10 mL to obtain a sample stock solution. 0.5 mL of the sample solution was added to 2.5 mL of Folin-Ciocalteu reagent diluted 10 times, shaken well, and allowed to stand for 5 min. Then, 2 mL of 7.5% sodium carbonate solution was added, and the solution was diluted to 10 mL. After shaking well, the solution was reacted in a 40℃ water bath for 2 h. The absorbance was measured at 765 nm using a UV-Vis spectrophotometer. Standard solutions with concentrations of 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, and 0.05 mg / mL were prepared using gallic acid as a standard. The absorbance was measured using the same method, and a standard curve was plotted. The regression equation was A = 0.0156C + 0.003, R² = 0.9992. The total phenol content in the sample was calculated based on the standard curve, in mg / g. The DPPH free radical scavenging rate was determined using the DPPH method. A 0.1 mmol / L DPPH ethanol solution was prepared, and 2 mL of the sample solution was mixed with 2 mL of the DPPH solution. After shaking, the mixture was incubated at 25°C in the dark for 30 min. The absorbance (A) was measured at 517 nm using a UV-Vis spectrophotometer. s Simultaneously, the absorbance A0 of a blank control mixture of 2 mL ethanol and 2 mL DPPH solution was measured, and the DPPH free radical scavenging rate was calculated as (A0 - A...). s Divide by A0 and multiply by 100%, i.e., the clearance rate = [(A0-A0)] s ) / A0]×100%; The adsorption capacity of the chromatographic material was determined by static adsorption. A 1 mg / mL concentration of willowherb extract was prepared, and 0.1 g of chromatographic material was added to 10 mL of the solution. The mixture was shaken at 25 °C for 2 h at a rotation speed of 150 r / min. After centrifugation, the supernatant was collected, and the total flavonoid content C1 in the supernatant was determined according to the method for determining total flavonoid content. The unit is mg / mL. The initial total flavonoid content is C0, and the unit is mg / mL. The adsorption capacity Q was calculated as (C0-C1) multiplied by V and divided by m, i.e., Q=(C0-C1)×V / m, where V is the solution volume of 10 mL, m is the mass of the chromatographic material of 0.1 g, and the adsorption capacity unit is mg / g. Product purity was determined by high performance liquid chromatography (HPLC). The chromatographic column was a C18 column with dimensions of 250 mm × 4.6 mm and a particle size of 5 μm. The mobile phase was a gradient elution of methanol and water. The methanol ratio was linearly increased from 30% to 80% for 30 min, the flow rate was 1 mL / min, the column temperature was 30 °C, the detection wavelength was 254 nm, and the injection volume was 10 μL. The purity of the main component was calculated by peak area normalization, which is equal to the peak area of ​​the main component divided by the sum of the peak areas of all peaks multiplied by 100%.

[0032] Test results: Table 1: Test results for each embodiment and comparative example: .

[0033] As can be seen from Table 1, Examples 1-3 effectively solved the technical problems of low purity, large loss of active ingredients, poor separation selectivity, and insufficient product stability in the existing preparation process of willowherb extract compared with Comparative Examples 1-3. Specifically, these problems are reflected in the following aspects: Regarding product purity, the purity of products in Examples 1-3 was 90.8-92.5%, while that of Comparative Examples 1-3 was only 68.7-82.3%. The purity of Example 1 was 23.8 percentage points higher than that of Comparative Example 3. This indicates that the synergistic effect of the three modified compounds in zirconium-aminophosphonic acid hybrid layered zeolite—zirconium ion coordination, aminophosphonic acid modification, and layered structure construction—significantly improved the selectivity of chromatographic separation, effectively removed impurities, and solved the technical problem of low purity. Regarding the retention of active ingredients, the total flavonoid content in Examples 1-3 was 148.7-156.3 mg / g, while that in Comparative Examples 1-3 was 95.3-125.4 mg / g. The total flavonoid content in Example 1 was 64.0% higher than that in Comparative Example 3. The total phenol content in Examples 1-3 was 198.3-210.5 mg / g, while that in Comparative Examples 1-3 was 132.4-168.2 mg / g. The total phenol content in Example 1 was 59.0% higher than that in Comparative Example 3. The extract yield in Examples 1-3 was 17.2-18.5%, while that in Comparative Examples 1-3 was 12.5-15.3%. The yield in Example 1 was 6.0 percentage points higher than that in Comparative Example 3. This indicates that the preparation process of the present invention effectively reduces the loss of active ingredients during extraction and purification, and solves the technical problem of large loss of active ingredients. Regarding separation selectivity, the adsorption capacity of the chromatographic materials in Examples 1-3 was 118.5-125.8 mg / g, and in Comparative Examples 1-3 it was 65.4-95.6 mg / g. The adsorption capacity of Example 1 was 92.4% higher than that of Comparative Example 3, indicating that the coordination between zirconium ions and aminophosphonic acid formed a specific recognition site, which enhanced the selective adsorption capacity of flavonoids and phenolic active ingredients in willowherb. At the same time, comparing the data of Comparative Example 1 (without zirconium ion coordination) and Comparative Example 2 (without aminophosphonic acid modification) shows that zirconium ion coordination and aminophosphonic acid modification are both indispensable. Only through their synergistic effect can high selective separation be achieved, thus solving the technical problem of poor separation selectivity. Regarding product stability, the DPPH free radical scavenging rate was 82.3-85.6% in Examples 1-3 and 58.3-72.5% in Comparative Examples 1-3. The scavenging rate of Example 1 was 27.3 percentage points higher than that of Comparative Example 3, indicating that the willowherb extract prepared by this invention has stronger antioxidant activity. Antioxidant activity is closely related to product stability. Higher antioxidant activity means that the product is less prone to oxidation and deterioration during storage. At the same time, the inclusion effect of β-cyclodextrin further improves the stability of the active ingredient and solves the technical problem of insufficient product stability. In summary, Examples 1-3 achieved a synergistic solution to various technical problems through the preparation of zirconium-aminophosphonic acid hybrid layered zeolite and the optimization of the willowherb extract. Among them, Example 1 has the best overall performance, which proves the effectiveness and advancement of the technical solution of the present invention.

[0034] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A preparation process for an extract of willowherb flowers, characterized in that, Includes the following steps: S1. By weight, collect 80-120 parts of willowherb flowers, rinse with deionized water, and air dry to obtain clean willowherb flowers; dry the clean willowherb flowers and crush them to obtain dried raw materials; pulverize the dried raw materials and sieve them to obtain willowherb flower coarse powder; add 500-600 parts of petroleum ether to the willowherb flower coarse powder and stir at room temperature to defatted it; filter to obtain filter cake; place the filter cake in a fume hood to obtain defatted willowherb flower powder; S2, add defatted willowherb flower powder to a mixed solution of 1200-1300 parts of ethanol and water to obtain a mixture; extract the mixture by ultrasonication; after extraction, filter to obtain a filtrate; concentrate the filtrate under reduced pressure to obtain a concentrated willowherb flower extract. S3. Dilute the concentrated extract of willowherb flowers with deionized water, adjust the pH value to obtain a diluted solution; pass the diluted solution through a chromatography column pre-packed with 5-10 parts of zirconium-aminophosphonic acid hybrid layered zeolite; rinse the chromatography column with deionized water. Then elute with an aqueous ethanol solution and discard the eluent; elute again with an aqueous ethanol solution and collect the eluent. S4. Concentrate the eluent collected in step S3 under reduced pressure to obtain a concentrated extract; add β-cyclodextrin to the concentrated extract and mix to obtain a mixture; freeze-dry the mixture. The dried product was obtained; The dried product is crushed and sieved.

2. The preparation process of the willowherb extract according to claim 1, characterized in that, In step S1, the stirring and degreasing time is 2-4 hours.

3. The preparation process of the willowherb flower extract according to claim 1, characterized in that, In step S2, the ultrasonic extraction time is 30-50 minutes.

4. The preparation process of the willowherb flower extract according to claim 1, characterized in that, In step S3, adjust the pH value to 5.4-5.

6.

5. The preparation process of the willowherb extract according to claim 1, characterized in that, In step S4, the freeze-drying time is 36-40 hours.

6. The preparation process of the willowherb extract according to any one of claims 1-5, characterized in that, The preparation steps of the zirconium-aminophosphonic acid hybrid layered zeolite include: A1, by weight, 95-105 parts of molecular sieve are heated to 495-505℃ and calcined, then naturally cooled to room temperature to obtain calcined molecular sieve; the calcined molecular sieve is immersed in 20-40 parts of sodium hydroxide aqueous solution and shaken at 50-70℃; after treatment, it is filtered, washed with deionized water, and then dried at 104-106℃ to obtain activated and expanded pore zeolite support; A2. Activated and expanded zeolite support was added to 1400-1600 parts of anhydrous toluene and ultrasonically dispersed. 15-25 parts of 3-aminopropyltriethoxysilane were added dropwise, nitrogen gas was introduced, and the mixture was refluxed and stirred at 108-112℃. After the reaction was completed, the mixture was cooled and filtered to obtain a solid. The solid was washed with toluene, anhydrous ethanol, and deionized water, and dried under vacuum at 58-62℃ to obtain amino-functionalized zeolite. The amino-functionalized zeolite was dispersed in a mixed solvent of 900-1100 parts of anhydrous ethanol and water, and 25-50 parts of 2-carboxyethylphosphonic acid, 40-80 parts of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 25-50 parts of N-hydroxysuccinimide were added. The mixture was reacted at room temperature to obtain a reaction mixture. The reaction mixture was centrifuged to obtain a solid product. The solid product was washed successively with ethanol, deionized water, and anhydrous ethanol, and dried under vacuum to obtain aminophosphonic acid-modified zeolite. A3. Dissolve 45-55 parts of zirconium oxychloride in deionized water, adjust the pH to 3.0-3.5 to obtain a zirconium ion solution; add aminophosphonic acid-modified zeolite to the zirconium ion solution and shake the reaction at room temperature; after the reaction is complete, filter, wash with acidic deionized water, rinse with anhydrous ethanol, and vacuum dry to obtain zirconium ion coordination-modified zeolite. A4. Zirconium ion-coordinated modified zeolite is dispersed in 2400-2600 parts of deionized water to obtain a dispersion; 160-190 parts of hexamethylenetetramine are dissolved in 600-700 parts of deionized water to obtain a hexamethylenetetramine solution; 150-180 parts of aluminum nitrate nonahydrate are dissolved in 700-800 parts of deionized water to obtain an aluminum nitrate solution; the aluminum nitrate solution is added to the dispersion under stirring and mixed to obtain a mixture; the hexamethylenetetramine solution is added to the mixture under stirring; the pH is adjusted to 6.8-7.2, and the mixture is transferred to a high-pressure reactor for hydrothermal reaction at 90-100℃; after the reaction is completed, the mixture is naturally cooled to room temperature, centrifuged, and a solid precipitate is obtained; the solid precipitate is washed alternately with deionized water and anhydrous ethanol, and vacuum dried to obtain a dried product; the dried product is heat-treated at 120-150℃ under a nitrogen atmosphere, and then ground and sieved.

7. The preparation process of the willowherb extract according to claim 6, characterized in that, In step A1, the shaking treatment at 50-70℃ takes 1-3 hours.

8. The preparation process of the willowherb flower extract according to claim 6, characterized in that, In step A2, the reaction time at room temperature is 12-14 hours.

9. The preparation process of the willowherb extract according to claim 6, characterized in that, In step A3, the shaking reaction is carried out at room temperature for 12-24 hours.

10. The application of a preparation process for willowherb extract according to any one of claims 1-9, characterized in that, The preparation process of the willowherb flower extract is used in the extraction of active ingredients from willowherb flowers in health foods, cosmetics, or pharmaceutical excipients.