Method for directional gradient purification of active components of Chinese herbal medicine and application thereof in preparation of antler glue composite health care product

Abstract

The application discloses a kind of Chinese herbal medicine active component directional cascade purification method and its application in preparation antler glue composite health care product, belong to biological active substance separation extraction purification technical field.The Chinese herbal medicine active component cascade purification method, including: the flavone / polyphenol component-containing extract is separated and purified by simulated moving bed continuous chromatography system, and flavone / polyphenol refined active component is obtained;The polysaccharide active component-containing extract is purified by crude extraction, redissolving and ultrafiltration membrane and nanofiltration membrane combined membrane separation system, and polysaccharide refined active component is obtained;Two components are mixed to obtain the purified Chinese herbal medicine active component mixture.The traditional Chinese medicine composition includes antler glue and the purified Chinese herbal medicine active component mixture.The Chinese herbal medicine active component mixture prepared by the application has high purity and is easy to scale production.

Description

Technical Field

[0001] This invention relates to the field of bioactive substance separation, extraction and purification technology, and in particular to a method for targeted stepwise purification of active components of traditional Chinese medicine and its application in the preparation of deer antler glue compound health products. Background Technology

[0002] Deer antler glue is a solid glue made from the antlers of male sika deer (Cervus nippon Temminck) or red deer (Cervus elaphus Linnaeus) through boiling and concentrating in water. Deer antler glue is salty and warm in nature, and enters the liver and kidney meridians. It has the effects of warming and tonifying the liver and kidneys, and nourishing essence and blood. The Compendium of Materia Medica records that deer antler glue, when used hot, "nourishes the kidneys, replenishes deficiency, strengthens essence, and invigorates blood." Modern research shows that deer antler glue is rich in collagen, various amino acids, and trace elements, and has various pharmacological activities such as promoting bone cell proliferation, enhancing immune function, and improving anemia.

[0003] The extraction of active ingredients from traditional Chinese medicine (such as the antioxidant effects of flavonoids and the immunomodulatory effects of polysaccharides) faces the following bottlenecks: (1) There is a lack of systematic procedures for the simultaneous extraction and classification purification of compound Chinese medicinal materials. In existing compound products, various Chinese medicinal herbs are often extracted separately and then simply mixed, or processed using a single, crude process after compounding. Due to the significant differences in the physicochemical properties (polarity, molecular weight, solubility) of flavonoids / polyphenols and polysaccharides, it is difficult to achieve the same extraction rate and purity for both types of components when using the same extraction and purification process after compounding. This results in low yield of the target active ingredient and high impurity content, affecting the efficacy and quality control of the final product.

[0004] (2) The purification process of flavonoids / polyphenols is inefficient and difficult to scale up. In existing technologies, the purification of flavonoids / polyphenols mostly employs batch operations using macroporous adsorption resin columns. While this process achieves some separation efficiency, it suffers from several significant drawbacks: low resin utilization, high solvent consumption, low automation, and poor batch-to-batch reproducibility. Although studies have reported the use of simulated moving bed (SMB) technology to separate flavonoids from Ginkgo biloba leaves and resveratrol from Polygonum cuspidatum, current technologies lack standardized continuous SMB processes and a systematic approach from process parameter optimization to large-scale production. SMB essentially represents a technique for continuousizing traditional macroporous adsorption resin column chromatography. While the stationary phase remains macroporous adsorption resin, continuous operation is achieved through a simulated moving bed principle, overcoming the inefficiencies of batch operations. However, current technologies lack in-depth research on the systematic application of macroporous adsorption resin SMB in the purification of flavonoids from traditional Chinese medicine compound preparations.

[0005] (3) The technology for separating and purifying polysaccharide components is outdated. Traditional polysaccharide purification methods involve repeated alcohol precipitation and Sevag deproteinization, which are cumbersome, time-consuming, and consume large amounts of organic solvents. Furthermore, prolonged high-temperature treatments can easily lead to polysaccharide degradation and loss of activity. Although membrane separation technology has been applied in the field of oligosaccharides, a systematic membrane separation process for polysaccharide components in compound medicinal materials has not yet been developed in the field of traditional Chinese medicine polysaccharide purification. Therefore, it is necessary to improve the methods for the stepwise purification of active components from traditional Chinese medicine. Summary of the Invention

[0006] The purpose of this invention is to provide a method for targeted stepwise purification of active components of traditional Chinese medicine and its application in the preparation of deer antler gelatin compound health products, so as to solve the problems of low purification efficiency and difficulty in scaling up existing technologies.

[0007] To achieve the above objectives, the present invention provides the following solution: This invention provides a method for targeted stepwise purification of active components from traditional Chinese medicine, comprising: S1, after compounding Chinese medicinal materials rich in flavonoid / polyphenolic active ingredients and Chinese medicinal materials rich in polysaccharide active ingredients, extraction is performed. The Chinese medicinal materials rich in flavonoid / polyphenolic active ingredients are subjected to alcohol extraction to obtain an extract containing flavonoid / polyphenolic components; the Chinese medicinal materials rich in polysaccharide active ingredients are subjected to water extraction to obtain an extract containing polysaccharide active components. S2, the flavonoid / polyphenol mixed extract is separated and purified by a simulated moving bed continuous chromatography system to obtain the purified active flavonoid / polyphenol components; The extract containing polysaccharide active components was purified by crude extraction, resolution, and a membrane separation system combining ultrafiltration and nanofiltration to obtain refined polysaccharide active components. S3, after mixing the refined active components of flavonoids / polyphenols and polysaccharides, a purified mixture of active components of traditional Chinese medicine is obtained.

[0008] Optionally, in S1, the Chinese medicinal materials rich in flavonoid / polyphenolic active ingredients are selected from one or more of the following: Astragalus membranaceus, Lycium barbarum, Crataegus pinnatifida, Carthamus tinctorius, Rosa rugosa, Chrysanthemum morifolium, Morus alba, Ginkgo biloba, Pueraria lobata, Scutellaria baicalensis, Polygonum cuspidatum, Hippophae rhamnoides, Mentha haplocalyx, Lilium brownii, and Citrus reticulata peel. The Chinese medicinal materials rich in polysaccharide active ingredients are selected from one or more of the following: Astragalus membranaceus, Lycium barbarum, Codonopsis pilosula, Polygonatum sibiricum, Dioscorea opposita, Ziziphus jujuba, Poria cocos, Tremella fuciformis, Lentinus edodes, Ganoderma lucidum, Ophiopogon japonicus, Polygonatum odoratum, Trichosanthes kirilowii, Anemarrhena asphodeloides, and Rehmannia glutinosa. The ingredients are compounded according to the target efficacy and the properties of the medicinal materials. The mass ratio of flavonoid / polyphenolic medicinal materials to polysaccharide medicinal materials in the compounding process is 1:(0.5-3).

[0009] This invention, based on the principles of traditional Chinese medicine's "principal, assistant, adjuvant, and guide" formula composition and modern pharmacological research, combines Chinese medicinal herbs rich in flavonoids / polyphenols with those rich in polysaccharides. For tonifying qi and nourishing blood, Astragalus membranaceus (rich in both flavonoids and polysaccharides), Lycium barbarum (rich in both flavonoids and polysaccharides), Crataegus pinnatifida (rich in flavonoids), and Poria cocos (rich in polysaccharides) are combined in appropriate proportions. For immune regulation, Ganoderma lucidum (rich in polysaccharides), Ginkgo biloba (rich in flavonoids), and Hippophae rhamnoides (rich in flavonoids) are combined in appropriate proportions. The resulting compounded herbal active components exhibit good efficacy.

[0010] Optionally, in S1, the alcohol extraction method involves adding 30%-80% ethanol by volume and refluxing at 60-90°C for 1-3 times, each time for 1-3 hours, more preferably for 2 hours each time; the volume ratio of the ethanol to the total mass of the medicinal materials is (6-15):1.

[0011] The water extraction method is suitable for polysaccharides as the main target components: add 8-20 times the amount of purified water, decoct and extract 1-3 times at 80-100℃, each time for 1.5-3 hours, more preferably 2 hours each time; combine the extracts, filter, and concentrate to an appropriate amount to obtain a mixed extract. The volume ratio of the purified water to the total mass of the medicinal materials is (8-20):1.

[0012] The extraction method of this invention is simultaneous extraction. This simultaneous extraction involves extracting different types of compound Chinese medicinal herbs. The advantage of simultaneous extraction is that it preserves the original "co-dissolved and co-precipitated" state of the herbs to the greatest extent during the extraction process, ensuring that the various active ingredients maintain their natural proportions and complex forms in the extract, which is closer to the traditional Chinese medicine theory of "synergistic effect through decoction." At the same time, it simplifies the production process, improves the dissolution rate and stability of the effective components, and enhances the comprehensive utilization rate of resources.

[0013] Optionally, in S2, the simulated moving bed continuous chromatography system includes 8-24 chromatographic columns, the stationary phase is a macroporous adsorption resin selected from ADS-7, AB-8, HPD-100, NKA-9 and D101 types; the mobile phase is an ethanol-water system with an ethanol volume fraction of 10%-60%, the operating temperature is 25-40℃, and the system pressure is 0.5-3.0 MPa.

[0014] SMB continuous chromatography is essentially a continuous operation of traditional macroporous adsorption resin column chromatography. Its stationary phase is still macroporous adsorption resin, and the separation mechanism remains unchanged. However, by simulating the moving bed principle, it achieves continuous feeding and continuous discharging, overcoming the efficiency bottleneck of traditional batch operation.

[0015] The SMB system is divided into four zones (Zone I: elution zone, Zone II: purification zone, Zone III: adsorption zone, Zone IV: regeneration zone). By periodically and synchronously switching the feed, discharge, and eluent inlet positions of each column, countercurrent contact between the stationary and mobile phases is simulated to achieve continuous separation of the target flavonoid / polyphenol components from impurities. Operating conditions (switching time, flow rate ratio of each zone) are calculated and optimized based on adsorption parameters obtained from single-column pre-experiments. Optionally, the SMB system is divided into four zones (Zone I: elution zone, Zone II: purification zone, Zone III: adsorption zone, Zone IV: regeneration zone). The switching time for each zone is 900-1200 s, and the flow rate ratios for each zone are: Zone I: 1-5 BV / H; Zone II: 3-10 BV / H; Zone III: 1-5 BV / H; Zone IV: 3-10 BV / H.

[0016] After continuous separation by SMB, the following samples were collected: Extract (rich in flavonoid / polyphenol target components): purity ≥80%, yield ≥85%; Raffinate (rich in highly polar impurities): can be recycled.

[0017] The extract was concentrated under reduced pressure until the solid content was ≥60% to obtain refined active components of flavonoids / polyphenols.

[0018] Traditional macroporous adsorption resin column chromatography is an intermittent operation: sample loading, washing, elution, and regeneration are performed at different times, with the column in a non-productive state for most of the time (e.g., during washing and regeneration). SMB (Synthetic Macroporous Adsorption Resin) achieves continuous operation by connecting multiple columns in series and periodically switching the functional zones (adsorption, purification, elution, and regeneration zones) of each column, allowing each column to play a different role at different times. Feeding, effluent, elution, and regeneration occur simultaneously, significantly improving preparation efficiency and reducing solvent consumption. However, the separation essence of SMB is exactly the same as that of traditional macroporous resin column chromatography; therefore, it can be understood as a "continuous macroporous adsorption resin separation technology."

[0019] Optionally, in S2, the crude extraction method includes: concentrating the extract containing polysaccharide active components under reduced pressure to 1 / 4-1 / 2 of the original volume, adding 95% ethanol to achieve an alcohol content of 60%-80%, and letting it stand overnight at 4-10°C to obtain crude polysaccharide. The reconstitution method includes: reconstitute the crude polysaccharide with deionized water to a concentration of 5-20 mg / mL, and then centrifuge and microfilter to obtain a pretreated crude polysaccharide solution; The centrifugation is performed at 4000-10000 rpm for 15-30 minutes to remove insoluble impurities; The microfiltration process employs a ceramic membrane with a pore size of 0.45-1.0 μm to remove fine particles and colloidal impurities.

[0020] The pretreated crude polysaccharide solution is purified by a membrane separation system combining ultrafiltration and nanofiltration. The membrane separation system includes an ultrafiltration membrane with a molecular weight cutoff of 10-30 kDa and a nanofiltration membrane with a molecular weight cutoff of 150-1000 Da. The operating temperature of both membranes is ≤45℃. The system adopts a cross-flow filtration mode and is supplemented by constant volume washing filtration.

[0021] The ultrafiltration (UF) method described in this invention is used to remove macromolecular impurities. It employs a polyethersulfone (PES) or regenerated cellulose (RC) ultrafiltration membrane with a molecular weight cutoff of 10-30 kDa. The operating mode is cross-flow filtration. The transmembrane pressure is 0.1-0.5 MPa. The operating temperature is ≤45℃. The concentration factor is 3-6 times. Deionized water washing is performed by adding 2-4 times the volume of deionized water for constant volume washing to improve the yield. The permeate (rich in target polysaccharides and small molecule impurities) is collected, while the retentate (macromolecular proteins, colloids, etc.) is discarded.

[0022] The nanofiltration (NF) separation described in this invention is used to remove small molecule impurities. The ultrafiltration permeate is passed through a nanofiltration membrane (such as a polyamide nanofiltration membrane) with a molecular weight cutoff of 150-1000 Da. The operation mode is cross-flow filtration; the transmembrane pressure is 0.5-2.5 MPa; the operating temperature is ≤45℃; the concentration factor is 5-10 times; the deionized water washing filter is performed by adding 2-4 times the volume of deionized water for constant volume washing filter to fully remove small molecule impurities such as monosaccharides, oligosaccharides, and inorganic salts; the retentate (rich in target polysaccharide components) is collected, and the permeate (containing monosaccharides, inorganic salts, etc.) is discarded.

[0023] After being processed by the above-mentioned ultrafiltration-nanofiltration membrane separation system, the obtained nanofiltration retentate is the refined active polysaccharide component. Concentrating it to a solid content of ≥50% yields the refined active polysaccharide component.

[0024] Technical results: Target polysaccharide purity ≥88%, impurity removal rate (based on small molecule sugars) ≥85%, yield ≥85%. The entire membrane separation process is operated at ≤45℃, effectively avoiding polysaccharide degradation and activity loss caused by traditional heating concentration and organic solvent precipitation.

[0025] Optionally, in S3, the flavonoid / polyphenol refined active component has a total flavonoid purity of ≥90% based on dry weight; and the polysaccharide refined active component has a total polysaccharide purity of ≥88% based on dry weight.

[0026] This invention provides a traditional Chinese medicine composition comprising deer antler glue and a purified mixture of active components from traditional Chinese herbs, wherein the mass ratio of the purified mixture of active components from traditional Chinese herbs to the deer antler glue is (1-5):(1-5). The obtained flavonoid / polyphenolic refined active components and the obtained polysaccharide refined active components are remixed uniformly under low temperature conditions according to the original compounding ratio (or the ratio is adjusted according to the target efficacy) to obtain the purified mixture of active components from traditional Chinese herbs.

[0027] Existing deer antler gelatin compound products often involve a simple physical mixing of deer antler gelatin and herbal extracts, failing to achieve synergistic binding at the molecular or active component level. This crude mixing method hinders intermolecular interactions between active ingredients (such as hydrogen bonds and hydrophobic interactions), affecting the synergistic efficacy and bioavailability of the compound products. The deer antler gelatin polypeptide chains of this invention contain abundant amino, carboxyl, and hydroxyl functional groups; the herbal flavonoids / polyphenols contain multiple phenolic hydroxyl groups; and the polysaccharides contain hydroxyl groups and glycosidic bonds. These three types of active components can form intermolecular complexes through non-covalent interactions such as hydrogen bonds, hydrophobic interactions, and electrostatic interactions. This molecular-level binding is expected to produce a synergistic effect.

[0028] The quality indicators of the deer antler glue and its active components described in this invention comply with the relevant provisions of the 2020 edition of the Chinese Pharmacopoeia. It can be used directly as crushed deer antler glue blocks or processed into deer antler glue powder. This invention does not limit the source or specific preparation method of the deer antler glue; commercially available deer antler glue products that meet pharmacopoeia standards can be used, or it can be prepared by conventional methods.

[0029] This invention provides the application of traditional Chinese medicine compositions in the preparation of health products, wherein the dosage form of the health products is a paste, chewable tablet, or soft candy.

[0030] Optionally, the preparation method of the health product includes: mixing and compounding the purified mixture of active ingredients of traditional Chinese medicine with deer antler glue at a low temperature of 4-10℃.

[0031] During low-temperature mixing, the following intermolecular interactions drive the formation of active complexes: Hydrogen bonding: The phenolic hydroxyl groups (-OH) of flavonoids / polyphenols and the hydroxyl groups (-OH) of polysaccharides in the mixture of active components of traditional Chinese medicine form hydrogen bonds with the amino (-NH2) and carboxyl (-COOH) groups of the deer antler glue polypeptide chain; Hydrophobic interactions: Hydrophobic stacking occurs between the aromatic rings of flavonoids and the hydrophobic amino acid residues on the polypeptide chains of antler gum; Electrostatic interaction: Ion-ion or ion-dipole interaction between charged groups.

[0032] The aforementioned non-covalent bond forces enable the mixture of active components of traditional Chinese medicine and deer antler glue to form a uniform and stable active complex at the molecular level, which is different from the traditional simple physical mixing method.

[0033] Optionally, the preparation method of the ointment dosage form includes: heating and stirring the compounded mixture until it reaches the flag-hanging state, filling it into a glass bottle, venting and sealing it, sterilizing it (100℃, 30min), and cooling it to obtain the compound deer antler gelatin ointment.

[0034] The preparation method of chewable tablets includes: mixing the compound mixture with an appropriate amount of filler (such as microcrystalline cellulose, lactose) and lubricant (such as magnesium stearate), granulating, drying, sizing, and compressing to obtain compound deer antler gum chewable tablets.

[0035] The preparation method of the gummy dosage form includes: mixing the compound mixture with gelatin, pectin and other gelling agents, casting into molds and drying to obtain compound antler gum gummy.

[0036] Optionally, the health product may also contain excipients. By weight, 1 part of the traditional Chinese medicine composition contains 0.8-1.2 parts of rice wine, 0.6-1 part of rock sugar, 0.4-0.8 parts of honey, and 0.04-0.08 parts of β-cyclodextrin.

[0037] The present invention discloses the following technical effects: 1. This invention provides a method for the stepwise purification of active components from traditional Chinese medicine, the method comprising two modules, wherein, The first module involves the compounding and simultaneous extraction of traditional Chinese medicine (TCM) herbs: TCM herbs rich in flavonoids / polyphenols and those rich in polysaccharides are compounded according to TCM formulation principles and simultaneously extracted using the same extraction process (alcohol extraction or water extraction) to obtain an extract containing both flavonoids / polyphenols and polysaccharides. The second module involves classified and graded purification: The extract containing flavonoids / polyphenols is purified using SMB continuous chromatography (i.e., continuous macroporous adsorption resin separation technology) to obtain high-purity refined flavonoids / polyphenols; the extract containing polysaccharides is purified using ultrafiltration-nanofiltration membrane separation technology to obtain high-purity refined polysaccharides. After purification, the two purified components are remixed to obtain a purified mixture of active TCM herbs.

[0038] 2. This invention provides a traditional Chinese medicine composition that synergistically combines a mixture of active components of traditional Chinese herbs with deer antler glue. The active components of traditional Chinese herbs (such as the antioxidant effects of flavonoids and the immunomodulatory effects of polysaccharides) and the warming and tonifying kidney yang effects of deer antler glue have good synergistic potential, which conforms to the theory of "essence and blood sharing the same origin" and the principle of "principal, assistant, adjuvant, and guide" in traditional Chinese medicine formulation, and has excellent effects.

[0039] 3. This invention provides the application of traditional Chinese medicine composition in the preparation of health products. The purified mixture of active components of traditional Chinese medicine and active components of deer antler glue are synergistically compounded at the molecular level under low temperature conditions through non-covalent bond forces to prepare compound health products.

[0040] This invention establishes a complete technical system of "compounding of traditional Chinese medicine → simultaneous extraction → classified and graded purification → synergistic compounding of mixture of active components of traditional Chinese medicine with deer antler glue", resulting in a mixture of active components of traditional Chinese medicine with high purity. Detailed Implementation

[0041] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0042] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0043] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0044] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0045] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0046] In the following embodiments, (1) The total flavonoid content was determined by ultraviolet-visible spectrophotometry (with rutin as a reference standard), and the total flavonoid content was ≥80% (based on the dry weight of the purified component).

[0047] (2) The total polysaccharide content was determined by the phenol-sulfuric acid method (with glucose as a reference standard), and the total polysaccharide content was ≥90% (based on the dry weight of the purified component).

[0048] (3) Quality control of mixtures of active components of traditional Chinese medicine: The purity of total flavonoids > 80% and the purity of total polysaccharides > 88% should both meet the corresponding standards; The ratio of flavonoids to polysaccharides can be adjusted according to the prescription requirements; The yield of purified active components of flavonoids / polyphenols was determined using conventional methods; The removal rate and yield of small molecule sugars in the refined active components of polysaccharides were determined using conventional methods.

[0049] (4) Finished product quality control: The total flavonoid / polyphenol content should be 3%-5%, the total polysaccharide content should be 10%-15%, and the protein / peptide content should be 18%-25%, etc., all of which should meet the enterprise standards. Feature mapping / fingerprint mapping is used to control batch-to-batch consistency of finished products; Microbial limit tests should comply with the relevant regulations for health food products.

[0050] Example 1: Preparation of Compound Deer Antler Glue Paste (Combination of Astragalus membranaceus, Lycium barbarum, Crataegus pinnatifida, and Poria cocos) 1. Compound preparation and simultaneous extraction of traditional Chinese medicine Take 200g of Astragalus membranaceus, 200g of Lycium barbarum, 100g of Crataegus pinnatifida, and 100g of Poria cocos. Astragalus membranaceus and Lycium barbarum have both flavonoid and polysaccharide effects. Crataegus pinnatifida is rich in flavonoids and Poria cocos is rich in polysaccharides. The mass ratio of flavonoid medicinal materials (crataegus pinnatifida, Astragalus membranaceus, and Lycium barbarum) to polysaccharide medicinal materials (Poria cocos, Astragalus membranaceus, and Lycium barbarum) is about 5:5. Mix and grind them together.

[0051] The Chinese medicinal herbs (hawthorn, astragalus, and wolfberry) from which flavonoids are extracted were added to 60% ethanol at a volume ratio of 15:1 to the total mass of the medicinal materials. The mixture was refluxed twice at 80°C for 2 hours each time. The extracts were combined, filtered, and the ethanol was recovered under reduced pressure until no alcohol odor was detected. The mixture was then concentrated to a relative density of 1.05-1.10 (60°C) to obtain approximately 800 mL of mixed flavonoid extract. The total flavonoid content in the mixed extract was approximately 5 mg / mL.

[0052] Deionized water was added to the residue of Astragalus membranaceus and Lycium barbarum obtained after flavonoid extraction and the medicinal material Poria cocos from which polysaccharides were extracted for water extraction. The liquid-to-solid ratio was 18:1 (g / g). The extraction was carried out twice at 95℃ for 2 hours each time. The extracts were combined, filtered, and concentrated to a relative density of 1.05-1.10 (60℃) to obtain about 800 mL of polysaccharide mixed extract with a total polysaccharide content of about 6 mg / mL.

[0053] 2. Classification and stepwise purification Route A: SMB continuous chromatography purification of flavonoid / polyphenol components SMB Continuous Purification: 800 mL of the flavonoid mixed extract obtained in step 1 was pretreated using a ceramic membrane microfiltration process with a pore size of 0.45-1.0 μm before being introduced into the SMB system. The SMB system was configured with 20 columns packed with AB-8 macroporous adsorption resin. The mobile phase was 50% ethanol-water (v / v), the operating temperature was 30℃, and the system pressure was 0.5 MPa. Based on the adsorption parameters (leakage curve, elution curve) obtained from the single-column pre-experiment, the flow ratio and switching time of each zone of the SMB were calculated. After continuous separation by SMB, the extract was collected and concentrated under reduced pressure until the solid content was ≥60%, yielding purified flavonoid / polyphenol active components (total flavonoid purity 91.6%, yield 88.3%).

[0054] The SMB system is divided into four zones (Zone I: elution zone, Zone II: purification zone, Zone III: adsorption zone, Zone IV: regeneration zone). The switching time for each zone is 1080 s, and the flow rate ratios for each zone are: Zone I: 3 BV / H; Zone II: 6 BV / H; Zone III: 2 BV / H; Zone IV: 8 BV / H.

[0055] Route B: Purification of polysaccharide components using a membrane separation system combining ultrafiltration and nanofiltration membranes. Take 800 mL of the polysaccharide mixed extract obtained in step 1, concentrate it under reduced pressure to 1 / 3 of the original volume, add 95% ethanol until the final alcohol content of the extract reaches 70%, let it stand overnight at 4℃, centrifuge to collect the precipitate, wash it with anhydrous ethanol, and dry it under vacuum at 50℃ to obtain crude polysaccharide.

[0056] The crude polysaccharide was reconstituted with deionized water to a concentration of 10 mg / mL, centrifuged at 8000 rpm for 20 min to obtain the supernatant. The supernatant was then microfiltered using a 0.45-1.0 μm ceramic membrane to obtain a pretreated crude polysaccharide solution. This pretreated crude polysaccharide solution was then passed sequentially through an ultrafiltration membrane and a nanofiltration membrane. The ultrafiltration membrane is a 10kDa PES spiral wound membrane. The transmembrane pressure of ultrafiltration is 0.3MPa, the temperature is 40℃, the concentration is 4 times, the constant volume washing filter is added with 3 times the volume of water, and the permeate is collected; the operation mode is cross-flow filtration. The operating mode is cross-flow filtration: the nanofiltration membrane is a 1000Da polyamide nanofiltration membrane. The permeate is passed through the nanofiltration membrane, the transmembrane pressure is set to 1.5MPa, the temperature is 40℃, the concentration is 6 times, the constant volume washing filter is added with 3 times the volume of water, and the retentate is collected.

[0057] The retentate was concentrated to a solids content of ≥50% to obtain polysaccharide-refined active components, with a total polysaccharide purity of 90.2%, a small molecule sugar removal rate of 87.5%, and a yield of 86.7%.

[0058] 3. Synergistic compounding with deer antler glue Recombination of the active components of traditional Chinese medicine: The flavonoid / polyphenolic refined components obtained from route A and the polysaccharide refined components obtained from route B are mixed evenly at 4-8℃ (calculated according to the original medicinal material ratio: the raw drug equivalent ratio of the flavonoid / polyphenolic refined components obtained from route A and the polysaccharide refined components obtained from route B is calculated according to the mass ratio of each medicinal material in the original prescription), to obtain the purified active components of traditional Chinese medicine.

[0059] Take 500g of deer antler gelatin powder that meets the standards of the Chinese Pharmacopoeia, and mix it with the above-mentioned purified mixture of active ingredients of traditional Chinese medicine at a mass ratio of 1:1 under conditions of 4-8℃. Add 400g of rice wine, 500g of rock sugar, 200g of honey, and 40g of β-cyclodextrin (by weight), heat and stir until it reaches the "hanging flag" state, fill into containers, sterilize at 100℃ for 30 minutes, and cool to obtain compound deer antler gelatin ointment.

[0060] The quality test results of the compound deer antler gelatin ointment showed that the total flavonoid content was 4.6%, the total polysaccharide content was 12.3%, the protein content was 18.5%, and the microbial limits met the regulations for health food.

[0061] Example 2: Preparation of a compound product of sea buckthorn, wolfberry and Ganoderma lucidum 1. Compound preparation and simultaneous extraction of traditional Chinese medicine Take 200g of sea buckthorn (rich in flavonoids), 100g of wolfberry (rich in flavonoids and polysaccharides), and 100g of Ganoderma lucidum (rich in polysaccharides), and mix and grind them together according to the mass ratio of flavonoids to polysaccharides of approximately 3:2.

[0062] Flavonoid extraction: Take 200g of sea buckthorn and 100g of wolfberry and add 60% ethanol. The volume ratio of ethanol to the total mass of medicinal materials is 15:1. Reflux extraction is performed twice at 80℃, 2 hours each time. Combine the extracts, filter, and recover the ethanol under reduced pressure until there is no alcohol odor. Concentrate to a relative density of 1.05-1.10 (60℃) to obtain about 800mL of flavonoid mixed extract. The total flavonoid content in the flavonoid mixed extract is about 8.6mg / mL.

[0063] After extracting flavonoids, the wolfberry residue and Ganoderma lucidum (a traditional Chinese medicine used for polysaccharide extraction) were subjected to water extraction with deionized water at a liquid-to-solid ratio of 20:1 (g / g). The extraction was carried out twice at 95℃ for 2 hours each time. The extracts were combined, filtered, and concentrated to a relative density of 1.05-1.10 (60℃) to obtain approximately 800 mL of polysaccharide mixed extract. The total polysaccharide content in the polysaccharide mixed extract was approximately 16.5 mg / mL.

[0064] 2. Classification and stepwise purification Route A (SMB method): SMB continuous chromatography purification of flavonoid / polyphenol components: AB-8 type macroporous adsorption resin was used as the stationary phase, with 8 columns in the SMB system. The mobile phase was 40% ethanol-water (v / v). The rest was the same as in Example 1. The purified flavonoid components of sea buckthorn and wolfberry were obtained (total flavonoid purity 92.3%, yield 87.1%). Route B (Membrane Separation Method): Purification of polysaccharide components using a membrane separation system combining ultrafiltration and nanofiltration: filtration was performed using a 10kDa ultrafiltration membrane and a 500Da nanofiltration membrane, with the rest as in Example 1, to obtain refined polysaccharide components of Ganoderma lucidum and Lycium barbarum (total polysaccharide purity 90.8%, small molecule sugar removal rate 86.3%, yield 88.0%).

[0065] 3. The mixture of active ingredients from traditional Chinese medicine was recombined and compounded with deer antler glue. The purified active components of the two types of traditional Chinese medicine were remixed to obtain a purified mixture of active components. The purified mixture of active components was then mixed with deer antler glue powder at a mass ratio of 1:1 at low temperature. Excipients were added to prepare compound deer antler glue chewable tablets, and the rest was the same as in Example 1.

[0066] The quality test results of the finished compound deer antler gelatin chewable tablets showed that the total flavonoid content was 3.5%, the total polysaccharide content was 12.7%, the protein content was 22.6%, and the microbial limits met the regulations for health food.

[0067] Example 3: Preparation of a compound product of ginkgo leaves, Ganoderma lucidum, and Poria cocos 1. Compound preparation and simultaneous extraction of traditional Chinese medicine Take 150g of ginkgo leaves (rich in flavonoids), 200g of Ganoderma lucidum (rich in polysaccharides), and 150g of Poria cocos (rich in polysaccharides), and mix them according to a mass ratio of flavonoids to polysaccharides of approximately 3:7. The rest is the same as in Example 1.

[0068] The total flavonoid content in the flavonoid mixed extract was 4.2 mg / mL. The total polysaccharide content in the polysaccharide mixed extract was 5.8 mg / mL.

[0069] 2. Classification and stepwise purification Route A (SMB method): AB-8 type macroporous adsorption resin was used as the stationary phase, 12 columns of SMB system were used, and the mobile phase was 30% ethanol-water. The rest was the same as in Example 1. The purified Ginkgo leaf flavonoid fraction was obtained (total flavonoid purity ≥90.5%, yield 86.4%).

[0070] Route B (membrane separation method): using a 30kDa ultrafiltration membrane and a 300Da nanofiltration membrane in combination, the rest is the same as in Example 1, to obtain the refined components of Ganoderma lucidum and Poria cocos polysaccharides (total polysaccharide purity 89.7%, small molecule sugar removal rate 85.3%, yield 87.2%).

[0071] 3. Synergistic compounding with deer antler glue The flavonoid / polyphenolic refined components obtained from route A and the polysaccharide refined components obtained from route B are mixed evenly at 4-8℃ (calculated according to the proportion of the original medicinal materials) to obtain a purified mixture of active components of traditional Chinese medicine.

[0072] The purified mixture of active components of traditional Chinese medicine was mixed with deer antler gelatin powder at a ratio of 3:2 at low temperature, and excipients were added to prepare compound deer antler gelatin ointment. The rest was the same as in Example 1.

[0073] The quality test results of the compound deer antler gelatin ointment showed that the total flavonoid content was 4.8%, the total polysaccharide content was 14.2%, the protein content was 19.7%, and the microbial limits met the regulations for health food.

[0074] This invention, through a RAW264.7 macrophage immune activation experiment, confirms that the deer antler gelatin ointment prepared in Example 1 of this invention can significantly promote the secretion of TNF-α (235±1.3 pg / mL) and IL-6 (351±4.7 pg / mL) by macrophages. p <0.01), promoting NO release of 11.8±0.6 μmol / L ( p <0.01), enhancing macrophage phagocytic function ( p <0.05), exerting an immune-enhancing effect from three levels: cytokine secretion, effector molecule release, and phagocytic function, indicating that the health product of this invention has significant immunomodulatory activity.

[0075] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for targeted stepwise purification of active components from traditional Chinese medicine, characterized in that, include: S1, after compounding Chinese medicinal materials rich in flavonoid / polyphenolic active ingredients and Chinese medicinal materials rich in polysaccharide active ingredients, extraction is performed. The Chinese medicinal materials rich in flavonoid / polyphenolic active ingredients are subjected to alcohol extraction to obtain an extract containing flavonoid / polyphenolic components; the Chinese medicinal materials rich in polysaccharide active ingredients are subjected to water extraction to obtain an extract containing polysaccharide active components. S2, the flavonoid / polyphenol mixed extract is separated and purified by a simulated moving bed continuous chromatography system to obtain the purified active flavonoid / polyphenol components; The extract containing polysaccharide active components was purified by crude extraction, resolution, and a membrane separation system combining ultrafiltration and nanofiltration to obtain refined polysaccharide active components. S3, after mixing the refined active components of flavonoids / polyphenols and the refined active components of polysaccharides, a purified mixture of active components of traditional Chinese medicine is obtained.

2. The method according to claim 1, characterized in that, In S1, the Chinese medicinal materials rich in flavonoid / polyphenolic active ingredients are selected from one or more of the following: Astragalus membranaceus, wolfberry, hawthorn, safflower, rose, chrysanthemum, mulberry, ginkgo leaf, kudzu root, scutellaria baicalensis, polygonum cuspidatum, sea buckthorn, mint, lily and orange peel. The Chinese medicinal materials rich in polysaccharide active ingredients are selected from one or more of the following: Astragalus membranaceus, Lycium barbarum, Codonopsis pilosula, Polygonatum sibiricum, Dioscorea opposita, Ziziphus jujuba, Poria cocos, Tremella fuciformis, Lentinus edodes, Ganoderma lucidum, Ophiopogon japonicus, Polygonatum odoratum, Trichosanthes kirilowii, Anemarrhena asphodeloides, and Rehmannia glutinosa. The mass ratio of flavonoid / polyphenolic medicinal materials to polysaccharide medicinal materials in the compound preparation is 1:(0.5-3).

3. The method according to claim 1, characterized in that, In S1, the alcohol extraction method involves adding 30%-80% ethanol by volume and refluxing at 60-90℃ for 1-3 times, each time for 1-3 hours, wherein the volume of ethanol is 6-15 times the total mass of the medicinal materials; the water extraction method involves adding 8-20 times the total mass of the medicinal materials and decocting at 80-100℃ for 1-3 times, each time for 1-3 hours.

4. The method according to claim 1, characterized in that, In S2, the simulated moving bed continuous chromatography system comprises 8-24 chromatographic columns, with a stationary phase of macroporous adsorption resin selected from ADS-7, AB-8, HPD-100, NKA-9, and D101 types; the mobile phase is an ethanol-water system with an ethanol volume fraction of 10%-60%, an operating temperature of 25-40℃, and a system pressure of 0.5-3.0 MPa.

5. The method according to claim 1, characterized in that, In S2, the coarse extraction method includes: The extract containing polysaccharide active components was concentrated under reduced pressure to 1 / 4-1 / 2 of its original volume, and 95% ethanol was added until the alcohol content reached 60%-80%. The mixture was then allowed to stand overnight at 4-10°C to obtain crude polysaccharide. The reconstitution method includes: reconstitute the crude polysaccharide with deionized water to a concentration of 5-20 mg / mL, and then centrifuge and microfilter to obtain a pretreated crude polysaccharide solution; The pretreated crude polysaccharide solution is purified by a membrane separation system combining ultrafiltration and nanofiltration membranes. The membrane separation system includes an ultrafiltration membrane with a molecular weight cutoff of 10-30 kDa and a nanofiltration membrane with a molecular weight cutoff of 150-1000 Da. The operating temperature of both membranes is ≤45℃. The system adopts a cross-flow filtration mode and is supplemented by constant volume washing filtration.

6. The method according to claim 1, characterized in that, In S3, the total flavonoid purity of the refined flavonoid / polyphenol active components is ≥90% by dry weight; and the total polysaccharide purity of the refined polysaccharide active components is ≥88% by dry weight.

7. A traditional Chinese medicine composition, characterized in that, The mixture includes deer antler glue and a purified mixture of active components of traditional Chinese medicine obtained by the method of any one of claims 1-6, wherein the mass ratio of the purified mixture of active components of traditional Chinese medicine to the deer antler glue is (1-5):(1-5).

8. The application of the traditional Chinese medicine composition according to claim 7 in the preparation of health products, characterized in that, The health supplement is available in the form of a paste, chewable tablet, or soft candy.

9. The application according to claim 8, characterized in that, The preparation method of the health product includes: mixing and compounding the purified mixture of active ingredients of traditional Chinese medicine with deer antler glue at a low temperature of 4-10℃.

10. The application according to claim 8, characterized in that, The health product also requires the addition of excipients. By mass, 1 part of the traditional Chinese medicine composition includes 0.8-1.2 parts of rice wine, 0.6-1 part of rock sugar, 0.4-0.8 parts of honey, and 0.04-0.08 parts of β-cyclodextrin.