Preparation method of concentrated solution capable of effectively retaining active ingredients of folium cortex eucommiae

Abstract

The invention discloses a preparation method of a concentrated solution capable of effectively retaining active ingredients of folium cortex eucommiae, and belongs to the technical field of natural product extraction.The preparation method comprises the steps that after raw materials are pretreated, the folium cortex eucommiae raw materials are rotationally cleaned with reverse osmosis water in a roller; during extraction, the temperature of the feed liquid is controlled to be 60-70 DEG C, and rotary circulation is performed for 10-20 min; after filtration and centrifugation, the finally obtained filtrate is filtered and concentrated by adopting multi-stage membrane combined concentration equipment, the membrane aperture of a first-stage ultrafiltration membrane is 5-20 nm, the membrane aperture of a second-stage ultrafiltration membrane is 1-5 nm, and the temperature is 20-30 DEG C; and sterilizing and filling to obtain a folium cortex eucommiae concentrated solution product. According to the method, labor and time are saved, the product quality and yield can be improved, the temperature in the extraction process is low, damage of high-temperature heating to effective components is avoided, and gutta-percha can be removed. The folium cortex eucommiae concentrated solution extracted by the method can be used as a medical health-care product and a plant beverage to produce prime pulp, is safe and non-toxic, and is suitable for the fields of health-care products, foods and the like.

Description

TECHNICAL FIELD

[0001] The present application mainly relates to the field of natural product extraction technology, and particularly relates to a preparation method of a concentrated solution capable of effectively preserving active components of Eucommia ulmoides leaves. BACKGROUND

[0002] Modern pharmacological research has confirmed that the efficacy of Eucommia ulmoides leaves is closely related to various active components contained therein, mainly including flavonoids (such as quercetin and kaempferol derivatives), phenylpropanoids (such as chlorogenic acid and caffeic acid), iridoid glycosides (such as onjisaponin and geniposide), and polysaccharides. These active components endow Eucommia ulmoides leaves with various pharmacological activities such as anti-fatigue, immune enhancement, antioxidant, hypoglycemic, hypolipidemic, and hypotensive effects, making them important raw materials for the development of natural health products.

[0003] Currently, common concentration methods include evaporation concentration, freeze concentration, and membrane concentration. ①Evaporation concentration is to evaporate water by heating, so as to achieve the purpose of concentration. This concentration method has defects such as long heating time and high temperature in operation, and is prone to cause problems such as high dissolution of Eucommia ulmoides gum and large loss of active components when applied to the processing of Eucommia ulmoides leaf concentrated solution; ②Freeze concentration is to lower the temperature of water molecules in the extract to the freezing point to form ice crystals, and remove the ice crystals by mechanical means to achieve the purpose of concentration. Although this concentration method can effectively preserve the active components in the Eucommia ulmoides leaf concentrated solution, the dissolution of Eucommia ulmoides gum increases the concentration of the extract, hinders the diffusion of water molecules, and reduces the precipitation rate of ice crystals, which affects the freeze concentration efficiency and increases energy consumption, greatly limiting the industrial application of this method; ③Membrane concentration is to use a selective permeation membrane as a separation medium, and selectively permeate the membrane according to the molecular weight of the active components or impurities in the extract, so as to achieve the purpose of separation and concentration. However, the Eucommia ulmoides leaf extract contains macromolecular impurities such as Eucommia ulmoides gum, which is prone to cause problems such as smaller membrane pore size or clogging in the existing membrane concentration technology operation, affecting the concentration rate.

[0004] The development and utilization of active components of Eucommia ulmoides leaves still face two core technical bottlenecks: First, heat-sensitive active components are prone to degradation and loss. Phenylpropanoids (especially chlorogenic acid) and iridoid glycosides in Eucommia ulmoides leaves are chemically unstable and prone to hydrolysis, oxidation or structural transformation under heat, resulting in a significant decrease or even loss of pharmacological activity.

[0005] If the temperature is simply lowered (such as freeze concentration), the viscosity of the system will increase, which will cause a sharp decrease in the concentration efficiency, and cannot meet the needs of industrial production, forming a technical contradiction that "retention rate and efficiency cannot be achieved simultaneously".

[0006] Secondly, there are product stability issues caused by the inactive ingredient, Eucommia ulmoides gum. Eucommia ulmoides gum, found in Eucommia ulmoides leaves, is a natural macromolecular polymer with a molecular weight of 100-500 kDa. If the extraction or concentration process is not properly controlled, a large amount of Eucommia ulmoides gum can dissolve and remain in the product. Because Eucommia ulmoides gum easily forms colloidal particles in aqueous solutions, it can cause flocculation, turbidity, and stratification in the concentrated Eucommia ulmoides leaf solution and subsequent processed products, severely affecting the sensory quality of the product. At the same time, the dissolved Eucommia ulmoides gum can also interfere with the efficiency of subsequent separation and purification, increasing process costs.

[0007] Furthermore, the cleaning, extraction, and concentration processes in Eucommia ulmoides leaf processing are interconnected, but existing processes mostly optimize only one or a few steps (such as improving only the extraction temperature or concentration method), without forming a synergistic design of parameters throughout the entire process. For example, when extracting active ingredients from Chinese medicinal materials or plant raw materials, the raw materials are usually pulverized. However, due to the characteristics of Eucommia ulmoides leaf raw materials, extraction after pulverization will not only cause a large amount of impurities such as Eucommia gum to dissolve, but also lead to problems such as difficulty in separating the residue and liquid and difficulty in filtration. Improper extraction parameters will exacerbate the dissolution of Eucommia gum. Fluctuations in concentration conditions will affect the retention rate of active ingredients, ultimately resulting in significant differences in the content of active ingredients and clarity of different batches of products, making it difficult to meet the quality stability requirements of the food industry. Summary of the Invention

[0008] This invention addresses the problem of overly simplistic solutions in existing technologies by providing a significantly different approach. It offers a method for preparing a concentrated solution that effectively preserves the active ingredients of Eucommia ulmoides leaves. This method solves the technical problems mentioned in the background section regarding the easy degradation and loss of heat-sensitive active ingredients and the impact of inactive Eucommia ulmoides gum on product quality and production efficiency. It effectively enriches the active ingredients of Eucommia ulmoides leaves and features a short process flow, low production cost, and no pollution.

[0009] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows: A method for preparing a concentrated extract that effectively retains the active components of Eucommia ulmoides leaves includes the following steps: (1) Raw material pretreatment: Select Eucommia leaves that meet the requirements of physicochemical properties, dry them, and set them aside for later use; (2) Raw material cleaning: In the drum, reverse osmosis water is used to rotate and clean the raw material of Eucommia ulmoides leaves; (3) Extraction: Add the raw material after cleaning in step (2) to reverse osmosis water at a material-to-liquid ratio of 1:15-25, rotate and circulate for 10-20 min, control the material temperature at 60-70℃, and collect the extract; (4) Filtration and centrifugation: Filter the extract obtained in step (3) through a 120-160 mesh filter bag to obtain the filtrate; then centrifuge it and put the centrifuged liquid into a 2-10 μm filter bag for safe filtration and collect the filtrate. (5) Membrane concentration: The filtrate obtained in step (4) is concentrated using a multi-stage membrane combination concentration device. The pore size of the first-stage ultrafiltration membrane is 5-20 nm, the pore size of the second-stage ultrafiltration membrane is 1-5 nm, and the temperature is 20-30℃. When the soluble solids in the concentrate are 11.0-13.0%, the concentrate is collected. (6) After sterilization, fill the product to obtain the Eucommia ulmoides leaf concentrate.

[0010] The above-mentioned method for preparing concentrated solution has the following advantages: ① This invention uses a drum rotation cleaning technology. By using the mutual friction between the blades and the drum wall, and between the blades themselves, the surface of the Eucommia ulmoides leaves is damaged, which helps the active ingredients to dissolve better. At the same time, it can also prevent the dissolution of large amounts of Eucommia ulmoides gum and other impurities, making it easier for subsequent separation and concentration operations.

[0011] ② This invention strictly controls the extraction time. Since eucommia gum is an impurity that affects the quality of eucommia leaf concentrate, by strictly controlling the extraction time, the amount of eucommia gum dissolved in the eucommia leaf concentrate can be minimized, while the active ingredients in eucommia leaves can be fully dissolved.

[0012] ③ This invention strictly controls the concentration temperature, keeping it below 40℃ to avoid thermal decomposition of thermally unstable components in the Eucommia ulmoides leaf concentrate due to high-temperature heating.

[0013] ④ This invention employs a multi-stage membrane series combination technology. Since Eucommia gum is a macromolecular compound with large molecules, a filter membrane with a larger pore size can retain it. Therefore, the first-stage membrane filtration uses a large-pore ultrafiltration membrane, allowing the small amount of Eucommia gum dissolved during extraction to be retained by the ultrafiltration membrane, while other active components of Eucommia leaves, even polysaccharides, can permeate, thus achieving the separation of Eucommia gum impurities from other active components. The second-stage membrane uses a smaller-pore ultrafiltration membrane to retain soluble macromolecular substances such as polysaccharides (5-10 kDa) and small-molecule active components such as chlorogenic acid, flavonoids, and iridoids, while allowing water molecules to permeate, resulting in a concentrated solution of a predetermined concentration. The series use of two ultrafiltration membranes effectively solves the problems of large molecules causing membrane pore size reduction or blockage in existing membrane concentration technologies.

[0014] ⑤ This invention uses ultra-high temperature instantaneous sterilization technology, which can not only prevent the loss of the unique herbal aroma of Eucommia ulmoides leaves and retain the nutrients and flavor of the concentrate to a great extent, but also achieve sterilization and extend its shelf life to more than one year, thus improving the service life of the concentrate.

[0015] The Eucommia ulmoides leaf concentrate obtained by this invention can provide a qualified concentrated base material for the industrial production of pharmaceuticals, health products, and plant-based beverages.

[0016] Furthermore, in step (1), Eucommia ulmoides leaves grown from July to October are picked; And / or, the drying temperature is 45-55℃.

[0017] Furthermore, in step (2), the material-to-liquid ratio is 1:8-12; And / or, the cleaning time is 2-4 minutes.

[0018] Further, in step (4), the centrifugation process is as follows: the filtrate is cooled to 15-20 ℃ by plate heat exchange, allowed to stand for 25-35 min, and then centrifuged using a centrifuge with a speed of 5000-7000 rmp / min.

[0019] Furthermore, in step (6), the sterilization operation is as follows: ultra-high temperature instantaneous sterilization is used, and sterilization is performed at a temperature of 136-140℃ for 29-34 seconds.

[0020] Further, in step (6), the filling operation is aseptically filling the sterilized concentrate at a temperature below 40 °C.

[0021] In the above scheme, the separation mechanism of membrane concentration is only "molecular weight screening". If the membrane flux decreases, it can be solved by increasing the operating pressure or strengthening the cleaning, ignoring the influence of membrane surface properties on impurity adsorption. As an improvement to the above scheme, in step (5), the primary ultrafiltration membrane is modified with a polydopamine-chitosan composite layer. Polydopamine acts as an adhesion layer, providing good interfacial bonding force; chitosan acts as a functional layer, providing positively charged groups (amino) and hydrophilic groups (hydroxyl).

[0022] Eucommia gum is a natural polymer containing carboxyl groups (-COOH) on its molecular chain. In aqueous solutions with pH > 4.5, it can dissociate into -COO. -Chitosan carries a negative charge under acidic conditions (amino group) and retains some positive charge even at neutral pH. The positive charge of chitosan and its electrostatic adsorption with Eucommia ulmoides gum enhance the retention rate of Eucommia ulmoides gum. Other extracts from Eucommia ulmoides leaves, such as flavonoids, phenylpropanoids (e.g., chlorogenic acid), and iridoids, have relatively small molecular weights (mostly 300-800 Da), much smaller than the pore size of the primary ultrafiltration membrane, allowing them to easily pass through. Furthermore, flavonoids and iridoids are almost unaffected by the membrane surface charge. Although chlorogenic acid also contains carboxyl groups and theoretically carries a weak negative charge, its extremely small molecular weight means its movement is primarily driven by diffusion, and its electrostatic interaction with the membrane surface is far weaker than that of the large Eucommia ulmoides gum molecules, thus preventing significant retention. Therefore, this invention achieves dual screening of membrane pore size and surface charge through membrane modification, further removing Eucommia ulmoides gum from Eucommia ulmoides leaf extract. Simultaneously, the hydrophilic groups (hydroxyl and amino groups) in the composite layer reduce adsorption fouling on the membrane surface and extend membrane lifespan.

[0023] During concentration, the solution pH is controlled at 6.5±0.5. This pH can be adjusted by adding trace amounts of phosphate buffer or sodium citrate to maintain system stability. This ensures that the eucommia gum carries a sufficiently negative charge, ultimately guaranteeing the electrostatic adsorption between chitosan and the eucommia gum.

[0024] Furthermore, the modification treatment of the above-mentioned primary ultrafiltration membrane includes the following steps: a. Select the membrane module and prepare the dopamine solution and chitosan solution; b. New membrane pretreatment: Rinse the membrane module with pure water at low speed to remove protective agents and impurities such as glycerol from the membrane pores; c. Polydopamine undercoating: Inject the pre-prepared dopamine solution into the modification tank. At 25-30 ℃, circulate the solution through the membrane module at a low flow rate of 6-10 L / min for 4-6 hours. Observe the solution color gradually change from colorless to light brown and then to dark brown, which indicates that polydopamine (PDA) has been successfully deposited on the membrane surface. After coating, drain the waste liquid and rinse the membrane module and system with pure water at a medium flow rate until the effluent is colorless to ensure the removal of all physically adsorbed polydopamine particles. d. Chitosan functional layer assembly: The pre-prepared chitosan solution is injected into the modification tank and circulated through the membrane module at 25-30 ℃ for 2-4 hours. During this time, the positively charged chitosan (CS) will be firmly adsorbed onto the negatively charged polydopamine layer through electrostatic and hydrogen bonding. After coating, the waste liquid is drained and rinsed with pure water until neutral to wash away excess acetic acid and unbound chitosan. e. Functional layer fixation: Prepare a 0.1% (w / v) glutaraldehyde aqueous solution as a crosslinking agent, and adjust the pH to 7.0-8.0 with sodium hydroxide; circulate this solution through the membrane module for 1-2 hours. The aldehyde groups of glutaraldehyde will react with the amino groups of chitosan to form a Schiff base reaction, forming a stable crosslinking network; after completion, drain the crosslinking agent waste liquid and rinse the membrane module with a large amount of pure water for at least 1 hour to thoroughly remove residual glutaraldehyde; this step can prevent the weakening of the positive charge of chitosan and dissolution under neutral conditions.

[0025] f. Post-treatment and storage: First, rinse with 0.1 M sodium hydroxide solution for 15-20 minutes, then rinse with pure water until neutral. Fill the membrane module with 1% sodium bisulfate protective solution or pure water, seal and store for later use.

[0026] Rinsing with sodium hydroxide solution serves two purposes: ① to neutralize any residual acid; ② to ensure that the amino groups of chitosan remain partially deprotonated in a near-neutral storage environment, but their positive charge is locked by the cross-linking network, thus enabling them to continue to exert electrostatic adsorption in subsequent use.

[0027] Further, in step a, the preparation steps for the dopamine solution are as follows: weigh dopamine hydrochloride and dissolve it in Tris-HCl buffer solution with pH=8.5 (adjusted with a precision pH meter), wherein the ratio of dopamine hydrochloride to Tris-HCl buffer solution is 1.0 g: 500 mL; stir well; prepare and use immediately to prevent oxidation.

[0028] The steps for preparing the chitosan solution are as follows: Weigh chitosan (degree of deacetylation ≥ 90%) and dissolve it in a 1% (v / v) dilute acetic acid aqueous solution, wherein the ratio of chitosan to dilute acetic acid aqueous solution is 1.5 g: 500 mL; stir until completely dissolved to obtain a clear and transparent chitosan acetic acid solution with a pH of 4.0 ± 0.5.

[0029] Furthermore, in steps (2) and (3), the conductivity of the reverse osmosis water is ≤10 μS / cm. This avoids introducing additional salts and keeps the extract as low in ionic strength as possible.

[0030] Under low ionic strength, on the one hand, salt ions are avoided from interfering with the electrostatic adsorption of chitosan and eucommia gum, ensuring the retention efficiency of the modified membrane, while the already weak electrostatic attraction between chitosan and chlorogenic acid is almost completely suppressed; on the other hand, the impact of salt on the stability of the concentrate is reduced, such as avoiding salt precipitation during subsequent storage.

[0031] Furthermore, in step (5), the filtrate obtained in step (4) is first desalted through a nanofiltration membrane with a molecular weight cutoff of 200-300 Da, and then filtered through a primary ultrafiltration membrane. Nanofiltration membranes can retain substances with molecular weights of several hundred or more, while allowing monovalent salt ions (Na+) to pass through. + , K + , Cl - The nanofiltration retentate, after desalination, then enters the ultrafiltration system for concentration. This actively and efficiently removes a large number of ions, significantly reducing ionic strength and further ensuring quality.

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The present invention uses a drum rotation cleaning method for cleaning. The friction of the blades in the drum achieves gentle surface micro-damage, which removes surface impurities and opens the dissolution channels of active ingredients through slight damage. At the same time, compared with the raw material crushing treatment method, it can avoid excessive compression that leads to a large amount of dissolution of Eucommia ulmoides gum. Compared with the existing raw material cleaning steps (such as the use of basket cleaning) which only focus on removing surface contaminants such as dust and mud, the cleaning method provided by the present invention focuses on the impact on the cell structure of Eucommia ulmoides leaves and the subsequent dissolution of components. It controls the material-liquid ratio and cleaning time to avoid excessive cleaning that would cause leaf damage and aggravate the dissolution of Eucommia ulmoides gum, and also avoids insufficient cleaning that would affect the dissolution efficiency of active ingredients.

[0033] (2) This invention strictly controls the extraction temperature to 60-70℃ and the concentration temperature to 20-30℃ to avoid the decomposition of heat-sensitive components; the physical sieving method of membrane filtration replaces thermal evaporation, without thermal damage, and can effectively separate active ingredients such as chlorogenic acid and flavonoids and eucommia gum, avoiding a sharp drop in concentration efficiency due to increased system viscosity, which is beneficial to balance retention rate and efficiency; the use of high temperature to kill microorganisms instantly can shorten the heat exposure time of active ingredients to the maximum extent compared with traditional long-term heating sterilization, while ensuring sterilization effect and effectively retaining heat-sensitive components. Data shows that the chlorogenic acid content of this application reaches 728mg / 100mL, and the total flavonoids reach 3630mg / 100mL, which is 2-3 times higher than the retention rate of evaporation concentration.

[0034] (3) By using a drum to clean the raw material of Eucommia ulmoides leaves and controlling the material-liquid ratio and cleaning time, the present invention can avoid the large-scale dissolution of Eucommia ulmoides gum; by strictly controlling the material-liquid ratio, temperature and time in the extraction operation, the dissolution of Eucommia ulmoides gum is avoided; and in the concentration operation, Eucommia ulmoides gum is intercepted by a first-stage ultrafiltration membrane to avoid the presence of a large amount of Eucommia ulmoides gum in the Eucommia ulmoides leaf concentrate and subsequent processed products, which would lead to flocculation, turbidity and stratification, thus helping to ensure the sensory quality of the product; at the same time, the dissolved Eucommia ulmoides gum is avoided from interfering with the efficiency of subsequent separation and purification.

[0035] (4) This invention avoids excessive dissolution of eucommia gum by using a drum to clean the eucommia leaf raw material and controlling the material-to-liquid ratio and cleaning time; it also avoids aggravating the dissolution of eucommia gum by strictly controlling the material-to-liquid ratio, temperature, and time during the extraction operation; and it avoids affecting the retention rate of active ingredients by strictly controlling parameters such as membrane pore size and temperature during the concentration operation. In other words, this invention, through multiple synergistic effects, can ultimately ensure the consistency of active ingredient content and clarity in different batches of products, thus meeting the requirements for quality stability in the food industry.

[0036] (5) In the optimized scheme of the present invention, the ultrafiltration membrane modified by the polydopamine-chitosan composite layer and pH control achieve a dual separation mechanism of pore size screening and charge adsorption for Eucommia ulmoides gum. At the same time, it does not affect the passage of active ingredients such as chlorogenic acid and flavonoids through the primary ultrafiltration membrane, thus achieving targeted removal of Eucommia ulmoides gum and avoiding the residual risk of organic solvent extraction. The separation efficiency and purity are improved. Meanwhile, the polydopamine bottom layer ensures a strong bond between the composite layer and the membrane substrate, and the hydrophilic groups of the composite layer reduce adsorption pollution on the membrane surface, which helps to solve the membrane clogging problem caused by relying solely on pore size screening. Furthermore, only reverse osmosis water is added in the treatment step before concentration to avoid introducing additional salt. Even before concentration, desalination is performed through a 200-300 Da nanofiltration membrane, so that the ionic strength of the solution is very low when the concentrated membrane is filtered, avoiding salt ions from interfering with the electrostatic adsorption of chitosan and Eucommia ulmoides gum. At the same time, there is no risk of organic solvent residue, which meets food or pharmaceutical standards.

[0037] (6) The process flow of this invention is streamlined around the core steps of cleaning, extraction, purification, concentration, and sterilization. After drying, the product is directly washed by drum washing without additional crushing and soaking. After filtration and centrifugation, the product is directly processed by a multi-stage membrane system to continuously remove eucommia gum and concentrate, eliminating the need for repeated alcohol precipitation and column chromatography. After membrane concentration, the product is directly sterilized at ultra-high temperature and aseptically filled, without the drying and reconstitution steps, which significantly shortens the production cycle. In terms of production cost, the product uses low-temperature extraction at 60-70℃, membrane concentration at 20-30℃, and ultra-high temperature sterilization to shorten the heat exposure time and reduce energy consumption. Drum washing promotes the dissolution of active ingredients, and membrane concentration and physical sieving reduce losses, improving material utilization and resulting in lower production costs. In addition, reverse osmosis water is used as the medium throughout the process. The small amount of food-grade reagents used in membrane modification are thoroughly rinsed away, leaving no organic solvent residue. The production waste liquid does not contain high-concentration pollutants and no solid waste is generated. The protective liquid can be safely discharged after neutralization without secondary pollution.

[0038] The present invention will be explained in detail below with reference to the accompanying drawings and specific embodiments. Attached Figure Description

[0039] Figure 1 This is a process flow diagram of the present invention. Detailed Implementation

[0040] To facilitate understanding of the present invention, a more comprehensive description of the present invention will be given below with reference to the accompanying drawings, which illustrate several embodiments of the present invention. However, the present invention can be implemented in different forms and is not limited to the embodiments described in the text. Rather, these embodiments are provided to make the disclosure of the present invention more thorough and complete.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly associated with those skilled in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments and is not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0042] Example 1: Please refer to the appendix for details. Figure 1 A method for preparing a concentrated extract that effectively retains the active ingredients of Eucommia ulmoides leaves includes the following steps: (1) Raw material pretreatment: Eucommia leaves grown from July to October are picked, and Eucommia leaves that meet the requirements are dried at 45℃ after evaluation of physicochemical indicators. ① Sensory requirements: a. Color: Light green to brownish-black leaves of the current year; b. Odor and taste: Possesses the unique aroma of Eucommia ulmoides leaves, without any off-odors; tastes slightly bitter; c. State: Dried Eucommia ulmoides leaves are brittle and easily broken when rubbed, with a small amount of silvery-white rubbery filaments connected on the broken surface; d. Impurities: Free from damage caused by pests and diseases; free from mold and rot; free from other impurities visible to the naked eye, such as branches, stones, sand, paper scraps, plastics, etc.; free from chemical contamination (engine oil, gasoline, etc.).

[0043] ② Physicochemical indicators: Moisture / (%) ≤12.0; Chlorogenic acid / (g / kg) ≥10.0; Total flavonoids / (g / 100 g) ≥1.0.

[0044] (2) Raw material cleaning: First, add room temperature RO (reverse osmosis) water to the drum to rinse the drum. After rinsing, put the raw material processed in step (1) into the drum and add RO water at a material-to-liquid ratio of 1:10. After rotating and circulating for 2 minutes, drain the liquid to complete the cleaning of Eucommia ulmoides leaves. (3) Extraction: Add the raw material cleaned in step (2) to RO water at a material-to-liquid ratio of 1:19, rotate and circulate for 15 minutes, drain the liquid when the temperature of the liquid is about 60-70℃, and collect the extract. (4) Filtration and centrifugation: Filter the extract obtained in step (3) through a 140-mesh filter bag to obtain filtrate. Then, cool the filtrate to 15-20 ℃ through a plate heat exchanger, let it stand for 30 min, and then centrifuge it using a centrifuge with a speed of 6000 rmp / min. Continue to put the obtained centrifuged liquid into a 5μm filter bag for safe filtration and collect the filtrate. (5) Membrane concentration: The Eucommia ulmoides leaf extract (the filtrate obtained in step (4)) is concentrated using a multi-stage membrane combination concentration device. The pore size of the first-stage ultrafiltration membrane is 15-20 nm, the pore size of the second-stage ultrafiltration membrane is 1-5 nm, and the temperature is 30℃. When the soluble solids in the concentrate are 11.5%, the concentrate is collected. (6) Sterilization: The concentrated liquid collected in step (5) is sterilized by ultra-high temperature instantaneous sterilization at a temperature of 136℃ for 29s; (7) Filling: The sterilized concentrate is aseptically filled at a temperature below 40°C to obtain the Eucommia ulmoides leaf concentrate product.

[0045] In step (5), the primary ultrafiltration membrane is modified with a polydopamine-chitosan composite layer, specifically including the following steps: a. Preparation: Select a standard ultrafiltration membrane module made of PES or PVDF, with an initial molecular weight cutoff corresponding to a pore size of approximately 5-20 nm. To prepare dopamine solution: Weigh 2.0g of dopamine hydrochloride and dissolve it in 1000 mL of Tris-HCl buffer solution with pH=8.5 (adjust with a precision pH meter). Stir well and use immediately to prevent oxidation.

[0046] Prepare chitosan solution: Weigh 1.5 g chitosan (degree of deacetylation ≥90%), dissolve it in 500 mL of 1% (v / v) dilute acetic acid aqueous solution, stir until completely dissolved, and obtain a clear and transparent chitosan acetic acid solution with a pH of 4.0±0.5.

[0047] b. Pretreatment of new membrane: Rinse the membrane module with pure water at low speed for 30 minutes to remove protective agents and impurities such as glycerol in the membrane pores; c. Polydopamine undercoating: Inject the pre-prepared dopamine solution into the modification tank. At 25-30 ℃, circulate the solution through the membrane module at a low flow rate of 10 L / min for 4-6 hours. Observe the solution color gradually change from colorless to light brown and then to dark brown, which indicates that polydopamine (PDA) has been successfully deposited on the membrane surface. After coating, drain the waste liquid and rinse the membrane module and system with pure water at a medium flow rate until the effluent is colorless to ensure the removal of all physically adsorbed polydopamine particles. d. Chitosan functional layer assembly: The pre-prepared chitosan solution is injected into the modification tank and circulated through the membrane module at 25-30 ℃ for 2 hours. During this time, the positively charged chitosan (CS) will be firmly adsorbed onto the negatively charged polydopamine layer through electrostatic and hydrogen bonding. After coating, the waste liquid is drained and rinsed with pure water until neutral to wash away excess acetic acid and unbound chitosan. e. Functional layer fixation: Prepare a 0.1% (w / v) glutaraldehyde aqueous solution as a crosslinking agent, and adjust the pH to 7.0-8.0 with sodium hydroxide; circulate this solution through the membrane module for 1 hour. The aldehyde groups of glutaraldehyde will react with the amino groups of chitosan to form a Schiff base reaction, forming a stable crosslinking network; after completion, drain the crosslinking agent waste liquid and rinse the membrane module with a large amount of pure water for at least 1 hour to thoroughly remove residual glutaraldehyde; this step can prevent the weakening of the positive charge of chitosan and dissolution under neutral conditions.

[0048] f. Post-treatment and storage: First, rinse with a 0.1 M sodium hydroxide solution for 15-20 minutes. This neutralizes any residual acid and ensures that the amino groups of chitosan remain partially deprotonated in a near-neutral storage environment, while their positive charge is locked by the cross-linking network, allowing them to continue to exert their electrostatic adsorption effect in subsequent use. Then rinse with pure water until neutral. Fill the membrane module with a 1% sodium bisulfate protective solution or pure water, seal, and store for later use.

[0049] In step (5), during the primary ultrafiltration stage, the pH is adjusted to 6.5 ± 0.5 by adding trace amounts of phosphate buffer or sodium citrate to maintain system stability. This is because Eucommia gum can dissociate into -COO in aqueous solutions with pH > 4.5. - This results in a negative charge. By controlling and adjusting the pH to 6.5±0.5, we can ensure that the eucommia gum is fully negatively charged, thus guaranteeing the electrostatic adsorption between chitosan and eucommia gum.

[0050] Chitosan carries a positive charge (amino group) under acidic conditions and retains some positive charge even at neutral pH. Utilizing the electrostatic adsorption between the positive charge of chitosan and the negatively charged eucommia gum, the retention rate of eucommia gum can be enhanced. Therefore, this invention achieves dual screening of membrane pore size and surface charge through membrane modification, further removing eucommia gum from Eucommia leaf extract. Simultaneously, the hydrophilic groups (hydroxyl and amino groups) in the composite layer reduce adsorption fouling on the membrane surface and extend the membrane's lifespan.

[0051] 1. Nutritional composition testing The nutritional components of the obtained Eucommia ulmoides leaf concentrate are shown in Table 1. Table 1 Nutritional composition of Eucommia ulmoides leaf concentrate

[0052] 2. Shelf life test Accelerated stability tests were conducted on samples of Eucommia ulmoides leaf concentrate at a temperature of 37±2℃ without light exposure, and the sensory flavor, physicochemical indicators, microbiological indicators and content of active ingredients were monitored.

[0053] The results showed that the accelerated stability test sample (equivalent to 25 months at room temperature) met the sensory requirements. The product formed natural plant precipitates. After reconstitution in a water bath, the soluble solids content was 11.4%, which met the standard of 12.0±1.0%. The remaining physicochemical and microbiological indicators also met the product standards.

[0054] Example 2: The difference between this example and Example 1 is that: In step (1), the drying temperature is 50℃; In step (2), the material-to-liquid ratio is 1:8, and the liquid is discharged after rotating and circulating for 3 minutes; In step (3), the material-to-liquid ratio is 1:15, and the rotation cycle is 10 minutes. In step (4), filtration was performed using a 160-mesh filter bag and a 2μm filter bag, respectively; after standing for 25 minutes, centrifugation was performed using a centrifuge with a speed of 7000 rmp / min. In step (5), the pore size of the primary ultrafiltration membrane is 10-15 nm; the temperature is 25℃; and the concentrate is collected when the soluble solids content in the concentrate is 11.0%. In step (6), sterilization is performed at a temperature of 138°C for 32 seconds.

[0055] Everything else is the same as in Example 1.

[0056] Example 3: The difference between this example and Example 1 is that: In step (1), the drying temperature is 55℃; In step (2), the material-to-liquid ratio is 1:12, and the liquid is discharged after rotating and circulating for 4 minutes; In step (3), the material-to-liquid ratio is 1:25, and the rotational circulation lasts for 20 minutes; In step (4), filtration was performed using a 120-mesh filter bag and a 10μm filter bag, respectively; after standing for 35 min, centrifugation was performed using a centrifuge with a speed of 5000 rmp / min. In step (5), the pore size of the primary ultrafiltration membrane is 5-10 nm; the temperature is 20℃; and the concentrate is collected when the soluble solids content in the concentrate is 13.0%. In step (6), sterilization is performed at a temperature of 140°C for 34 seconds.

[0057] Everything else is the same as in Example 1.

[0058] Example 4: The difference between this example and Example 1 is that: The reverse osmosis water used has a conductivity of ≤10 μS / cm, which avoids the introduction of additional salts and keeps the extract as low as possible in terms of ionic strength.

[0059] Everything else is the same as in Example 1.

[0060] Example 5: The difference between this example and Example 1 is that: In step (5), the filtrate obtained in step (4) is first desalted through a nanofiltration membrane with a molecular weight cutoff of 200-300 Da, and then filtered through a primary ultrafiltration membrane. The nanofiltration membrane can retain substances with molecular weights of several hundred or more, while allowing monovalent salt ions to pass through; the desalted nanofiltration retentate then enters the ultrafiltration system for concentration. This actively and efficiently removes a large number of ions, significantly reduces ionic strength, and further ensures quality.

[0061] Everything else is the same as in Example 1.

[0062] Comparative Example 1: The difference from Example 1 is that: In step 5, the primary ultrafiltration membrane was not modified with dopamine-chitosan composite.

[0063] Everything else is the same as in Example 1.

[0064] Comparative Example 2: The difference from Example 1 is that: In step 5, the primary ultrafiltration membrane is modified only with dopamine solution, without being modified with chitosan solution.

[0065] Everything else is the same as in Example 1.

[0066] Comparative Example 3: The difference from Example 1 is that: In step 5, the primary ultrafiltration membrane is modified directly with chitosan solution without being modified with dopamine solution.

[0067] Everything else is the same as in Example 1.

[0068] Comparative Example 4: The difference from Example 1 is that: In step 5, the primary ultrafiltration membrane modification process does not involve functional layer fixation.

[0069] Everything else is the same as in Example 1.

[0070] Comparative Example 5: The difference from Example 1 is that: In step 5, the primary ultrafiltration membrane modification process does not involve post-treatment.

[0071] Everything else is the same as in Example 1.

[0072] Comparative Example 6: The difference from Example 1 is that: In step 5, the pH is controlled at 4.5-5; Everything else is the same as in Example 1.

[0073] Comparative Example 7: The difference from Example 1 is that: In step 2, the Eucommia ulmoides leaves are washed using a hanging basket. Everything else is the same as in Example 1.

[0074] Comparative Example 8: The difference from Example 1 is that: The temperature of the liquid material during extraction in step 3 is approximately 90-100℃; Everything else is the same as in Example 1.

[0075] Comparative Example 9: The difference from Example 1 is that: The rotation cycle time in step 3 is 30 minutes.

[0076] Everything else is the same as in Example 1.

[0077] Comparative Example 10: The difference from Example 1 is that: In step 5, the Eucommia ulmoides leaf solution is concentrated by evaporation instead of through a membrane.

[0078] Everything else is the same as in Example 1.

[0079] Comparative Example 11: The difference from Example 1 is that: The concentration temperature in step 5 is 50℃.

[0080] Everything else is the same as in Example 1.

[0081] The contents of total flavonoids and chlorogenic acid in the Eucommia ulmoides leaf concentrates obtained in Examples 1 to 5 and Comparative Examples 1 to 11 were tested, and the results are shown in Table 2.

[0082] Table 2. Content of total flavonoids and chlorogenic acid in Eucommia ulmoides leaf concentrates obtained by different methods

[0083] As can be seen from Table 2: Comparing Example 1 with Examples 2 and 3, the data for Example 1 are all higher than those for Examples 2 and 3. This indicates that the Eucommia ulmoides leaf product can be further optimized through further parameter adjustments in this invention.

[0084] Comparing Example 4 with Example 1, the chlorogenic acid content and total flavonoid content were slightly increased. This indicates that reverse osmosis water with a conductivity ≤10 μS / cm can avoid introducing additional salts, keeping the extract at a low ionic strength, which is beneficial to improving the electrostatic adsorption efficiency of the modified membrane.

[0085] Comparing Example 5 with Example 1, both the chlorogenic acid content and the total flavonoid content were further increased. This indicates that the 200-300 Da nanofiltration membrane removed monovalent salt ions in advance, further reducing the ionic strength of the system and ensuring that the electrostatic adsorption efficiency of the modified membrane was maximized. At the same time, it reduced the impact of salting out on the dissolution of active ingredients, resulting in a further increase of 3.7% in chlorogenic acid content and 3.5% in total flavonoid content. This verifies the synergistic advantages of the combination of desalination and modified membrane.

[0086] Compared with Comparative Example 1, Example 1 showed a significant increase in both chlorogenic acid content and total flavonoid content. This indicates that the polydopamine-chitosan modified membrane enhances the removal of Eucommia ulmoides gum through a dual mechanism of pore size screening and charge adsorption, reducing the co-adsorption loss of active ingredients and impurities. At the same time, the low conductivity reverse osmosis water avoids salt ion interference, resulting in a 7.2% increase in chlorogenic acid content and a 7.7% increase in total flavonoid content, directly demonstrating the synergistic effect of membrane modification technology.

[0087] Comparing Example 1 with Comparative Examples 2 to 5, the data in Example 1 were all higher than those in Comparative Examples 2 to 5. Comparative Example 2, which only underwent dopamine modification and lacked the electrostatic adsorption effect of the chitosan positively charged layer, resulted in insufficient retention of impurities such as Eucommia ulmoides gum, leading to easy loss of active ingredients. Comparative Example 3, directly modified with chitosan, lacked a polydopamine adhesion layer, resulting in weak chitosan bonding and easy detachment. Comparative Example 4, without functional layer fixation, and Comparative Example 5, without post-treatment, both led to decreased membrane modification stability and reduced retention of active ingredients. This indicates that dopamine modification, chitosan modification, functional layer fixation, and post-treatment all contribute to increasing the content of active ingredients in Eucommia ulmoides leaf concentrate.

[0088] Compared with Comparative Example 6, the chlorogenic acid content and total flavonoid content of the Eucommia ulmoides leaf concentrate obtained in Example 1 were approximately 1.04 times that of Comparative Example 7. This indicates that improper pH control could not ensure that the Eucommia ulmoides gum was sufficiently negatively charged, thus affecting the electrostatic adsorption of the Eucommia ulmoides gum by the modified primary ultrafiltration membrane. This invention, through pH control, can improve the retention rate of active ingredients.

[0089] Compared with Comparative Example 7, the chlorogenic acid content and total flavonoid content of the Eucommia ulmoides leaf concentrate obtained in Example 1 were approximately 1.03 times that of Comparative Example 7. This indicates that the basket cleaning method used in Comparative Example 7 may be inefficient and uneven, while the present invention uses a rotating drum cleaning method, which achieves gentle surface damage through friction between the leaves. This is beneficial for the efficient dissolution of subsequent active ingredients and avoids excessive pulverization or compression that would lead to the dissolution of large amounts of Eucommia ulmoides gum, thus laying a good foundation for subsequent purification and concentration.

[0090] Compared with Comparative Example 8, the chlorogenic acid content of the Eucommia ulmoides leaf concentrate obtained in Example 1 was approximately 1.62 times that of Comparative Example 8, and the total flavonoid content was approximately 1.61 times that of Comparative Example 8. This indicates that although extraction at 90-100℃ in Comparative Example 8 could increase the extraction rate, it destroyed heat-sensitive components. In contrast, this invention strictly controls the extraction temperature at 60-70℃ and the extraction time to a precise 15 minutes. This condition represents the optimal balance between extraction efficiency and component retention, ensuring sufficient dissolution of active ingredients while maximally inhibiting the dissolution of Eucommia ulmoides gum and the decomposition of heat-sensitive components.

[0091] Comparing Example 1 with Comparative Example 9, the data for Comparative Example 9 are close to those of Example 1, but still slightly lower. This is because extending the extraction time did not increase the content of active ingredients; in fact, it decreased. This indicates that at the optimized temperature of 60-70°C and a material-to-liquid ratio of 1:19, most of the active ingredients were efficiently dissolved within 15 minutes. Further extending the extraction time not only increases energy consumption and time costs but may also cause a small amount of components to slowly decompose or undergo other adverse reactions due to prolonged heat contact. Therefore, the extraction time determined in this invention is an efficient and economical optimized value.

[0092] Compared with Comparative Example 10, the chlorogenic acid content of the Eucommia ulmoides leaf concentrate obtained in Example 1 was approximately 3.08 times that of Comparative Example 10, and the total flavonoid content was approximately 3.53 times that of Comparative Example 10. This is because evaporation concentration requires continuous heating and boiling to remove water, which generates high-temperature thermal stress, leading to the significant degradation, oxidation, and inactivation of key heat-sensitive active ingredients in Eucommia ulmoides leaves (such as chlorogenic acid and flavonoids). In contrast, the membrane concentration method used in this invention is carried out entirely at low temperatures, a purely physical sieving process that perfectly avoids thermal damage, thereby maximizing the preservation of the product's bioactivity. Furthermore, evaporation concentration indiscriminately concentrates all soluble substances, including the target active ingredient and large molecular weight impurities such as Eucommia ulmoides gum. Increased impurity concentration may exacerbate the instability of subsequent products. The multi-stage membrane system of this invention has molecular sieving capabilities, effectively retaining and removing large molecular weight Eucommia ulmoides gum while concentrating the target small molecule active ingredient, fundamentally solving the quality problems of easy turbidity and precipitation in traditional processes.

[0093] Compared to Comparative Example 11, the chlorogenic acid content of the Eucommia ulmoides leaf concentrate obtained in Example 1 was approximately 1.09 times that of Comparative Example 11, and the total flavonoid content was approximately 1.1 times that of Comparative Example 10. Although 50°C is far below the evaporation temperature, it is still sufficient to accelerate chemical reactions (such as oxidation) compared to 30°C. During longer equipment cycle concentration processes, this 20°C temperature difference can lead to a slow but not negligible loss of active ingredients. This invention strictly controls the membrane concentration temperature at 20-30°C, further ensuring a low-temperature environment throughout the entire process from extraction to concentration, achieving comprehensive and end-to-end protection of the active ingredients.

[0094] In summary, the contents of total flavonoids and chlorogenic acid in the examples were higher than those in comparative examples one to five. This demonstrates that the present invention is not a simple application of a single technology, but rather a sophisticated system with interconnected and synergistic effects. Specifically, drum washing and low-temperature extraction aim to efficiently dissolve the target components and inhibit the dissolution of impurities; multi-stage membrane concentration focuses on the efficient enrichment of active ingredients at low temperatures and the simultaneous removal of macromolecular impurities; ultra-high temperature instantaneous sterilization and low-temperature filling aim to maximize the preservation of product flavor, quality, and stability. Through low-temperature control, directional separation, and precise processes in extraction and concentration, the present invention successfully solves the key technical bottlenecks of traditional methods, such as significant loss of active ingredients, easy product turbidity, and unstable quality. Ultimately, it yields a high-quality Eucommia ulmoides leaf concentrate with high active ingredient content, high clarity, and good stability, providing a reliable high-quality raw material for its industrial application in the pharmaceutical, health product, and food industries.

[0095] The present invention has been described by way of example in conjunction with the accompanying drawings. Obviously, the specific implementation of the present invention is not limited to the above-described manner. Any non-substantial improvement made by adopting the inventive concept and technical solution of the present invention, or the direct application of the inventive concept and technical solution of the present invention to other occasions without modification, shall be within the protection scope of the present invention.

Claims

1. A method for preparing a concentrated extract that effectively retains the active ingredients of Eucommia ulmoides leaves, characterized in that: Includes the following steps: (1) Raw material pretreatment: Select Eucommia leaves that meet the requirements of physicochemical properties, dry them, and set them aside for later use; (2) Raw material cleaning: In the drum, reverse osmosis water is used to rotate and clean the raw material of Eucommia ulmoides leaves; (3) Extraction: Add the raw material cleaned in step (2) to reverse osmosis water at a material-to-liquid ratio of 1:15-25, rotate and circulate for 10-20 minutes, control the material temperature at 60-70℃, and collect the extract; (4) Filtration and centrifugation: Filter the extract obtained in step (3) through a 120-160 mesh filter bag to obtain the filtrate; then centrifuge it and put the centrifuged liquid into a 2-10 μm filter bag for safe filtration and collect the filtrate. (5) Membrane concentration: The filtrate obtained in step (4) is concentrated using a multi-stage membrane combination concentration device. The pore size of the first-stage ultrafiltration membrane is 5-20 nm, the pore size of the second-stage ultrafiltration membrane is 1-5 nm, and the temperature is 20-30℃. When the soluble solids in the concentrate are 11.0-13.0%, the concentrate is collected. (6) After sterilization, fill the product to obtain the Eucommia ulmoides leaf concentrate.

2. The method for preparing a concentrated extract that effectively retains the active ingredients of Eucommia ulmoides leaves according to claim 1, characterized in that: In step (1), Eucommia ulmoides leaves that grow from July to October are picked; And / or, the drying temperature is 45-55℃.

3. The method for preparing a concentrated extract that effectively retains the active components of Eucommia ulmoides leaves according to claim 1, characterized in that: In step (2), the material-to-liquid ratio is 1:8-12; And / or, the cleaning time is 2-4 minutes.

4. The method for preparing a concentrated extract that effectively retains the active components of Eucommia ulmoides leaves according to claim 1, characterized in that: In step (2), the conductivity of the reverse osmosis water is ≤10μS / cm.

5. The method for preparing a concentrated extract that effectively retains the active components of Eucommia ulmoides leaves according to claim 4, characterized in that: In step (4), the centrifugation process is as follows: the filtrate is cooled to 15-20 ℃ by plate heat exchange, allowed to stand for 25-35 min, and then centrifuged using a centrifuge with a speed of 5000-7000 rmp / min.

6. The method for preparing a concentrated extract that effectively retains the active components of Eucommia ulmoides leaves according to claim 1, characterized in that: In step (5), the primary ultrafiltration membrane is modified with a polydopamine-chitosan composite layer, and the solution pH is controlled at 6.5±0.

5.

7. The method for preparing a concentrated extract that effectively retains the active ingredients of Eucommia ulmoides leaves according to claim 6, characterized in that: In step (5), the modification treatment of the primary ultrafiltration membrane includes the following steps: a. Select the membrane module and prepare the dopamine solution and chitosan solution; b. New membrane pretreatment: Rinse the membrane module with pure water at low speed to remove the protective agent and impurities in the membrane pores; c. Polydopamine undercoating: Inject the pre-prepared dopamine solution into the modification tank, and circulate the solution through the membrane module at a low flow rate of 6-10 L / min at 25-30 ℃ for 4-6 hours; After coating, drain the waste liquid and rinse the membrane module and system with pure water until the effluent is colorless to ensure that all physically adsorbed polydopamine particles are removed. d. Chitosan functional layer assembly: Inject the pre-prepared chitosan solution into the modification tank and circulate it through the membrane module at 25-30 ℃ for 2-4 hours; After coating, drain the waste liquid and rinse with pure water until neutral to wash away excess acetic acid and unbound chitosan. e. Functional layer fixation: Prepare a 0.1% (w / v) glutaraldehyde aqueous solution as a crosslinking agent, and adjust the pH to 7.0-8.0 with sodium hydroxide; circulate the solution through the membrane module for 1-2 hours; after completion, drain the crosslinking agent waste liquid, and rinse the membrane module with a large amount of pure water for at least 1 hour to thoroughly remove residual glutaraldehyde; f. Post-treatment and storage: First, rinse with 0.1 M sodium hydroxide solution for 15-20 minutes, then rinse with pure water until neutral. Fill the membrane module with 1% sodium bisulfate protective solution or pure water, seal and store for later use.

8. The method for preparing a concentrated extract that effectively retains the active ingredients of Eucommia ulmoides leaves according to claim 7, characterized in that: In step a, the preparation steps for the dopamine solution are as follows: Weigh dopamine hydrochloride and dissolve it in Tris-HCl buffer at pH=8.5, wherein the ratio of dopamine hydrochloride to Tris-HCl buffer is 1.0 g: 500 mL; stir until homogeneous. And / or, the preparation steps of the chitosan solution are as follows: weigh chitosan and dissolve it in a 1% (v / v) dilute acetic acid aqueous solution, wherein the ratio of chitosan to dilute acetic acid aqueous solution is 1.5 g: 500 mL; stir until completely dissolved to obtain a clear and transparent chitosan acetic acid solution with a pH of 4.0±0.

5.

9. The method for preparing a concentrated extract that effectively retains the active ingredients of Eucommia ulmoides leaves according to claim 1, characterized in that: In step (5), the filtrate obtained in step (4) is first desalted through a nanofiltration membrane with a molecular weight cutoff of 200-300 Da, and then filtered through a first-stage ultrafiltration membrane.

10. The method for preparing a concentrated extract that effectively retains the active components of Eucommia ulmoides leaves according to claim 1, characterized in that: In step (6), the sterilization operation is as follows: ultra-high temperature instantaneous sterilization is used, and sterilization is performed at a temperature of 136-140℃ for 29-34 seconds; And / or, the filling operation is aseptically filling the sterilized concentrate at a temperature below 40 °C.

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