Method for isolating and purifying active ingredients in composition containing puerariae radix and epimedii folium, and puerariae radix-epimedii folium extract

Abstract

The present application provides a method for isolating and purifying active ingredients in a composition containing Puerariae radix and Epimedii folium, comprising the following steps: treating a Puerariae radix medicinal material and an Epimedii folium medicinal material with a first alcohol solvent to prepare a liquid extract, passing the liquid extract through a macroporous adsorption resin, performing elution with water and elution with a second alcohol solvent in sequence, and collecting the eluate after the elution with the second alcohol solvent, wherein the flow rate for the elution with water is 1.5 BV / h to 3 BV / h, and the flow rate for the elution with the second alcohol solvent is 1 BV / h to 1.5 BV / h.

Description

Method for isolation and purification of active ingredients in compositions containing kudzu root and epimedium, and kudzu root and epimedium extracts

[0001] Related applications

[0002] This application claims priority to Chinese Patent Application No. 202411837196X, filed on December 13, 2024, entitled “Method for separating and purifying active ingredients in a composition containing kudzu root and epimedium and kudzu root and epimedium extract”, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application relates to the field of traditional Chinese medicine technology, and to a method for separating and purifying active ingredients in a composition containing kudzu root and epimedium, as well as kudzu root and epimedium extract. Background Technology

[0004] Kudzu root is the dried root of the legume *Pueraria lobata*. Its main active ingredients are daidzein, daidzein, puerarin, and other isoflavones, which possess various pharmacological and health-promoting effects, including antibacterial properties, promoting blood circulation and removing blood stasis, dilating coronary and cerebral arteries, reducing myocardial oxygen consumption, improving myocardial contractility, promoting blood circulation, and enhancing immunity. Kudzu root is commonly used in injections, eye drops, and capsules.

[0005] Epimedium, the dried aerial parts of *Epimedium brevicornu*, *Epimedium sagittatum*, *Epimedium pubescens*, or *Epimedium koreanum* (betel berry family), is used in traditional Chinese medicine. It is believed to tonify kidney yang, strengthen tendons and bones, and dispel wind and dampness. The main active components of Epimedium are flavonoids. Modern pharmacological studies have shown that Epimedium has various medicinal effects in areas such as immunity, reproduction, nucleic acid metabolism, cardiovascular health, and anti-aging. Epimedium is commonly used in tablet and granule formulations.

[0006] Separating and purifying the active ingredients in kudzu root and epimedium can better enhance their medicinal value. However, current methods for separating and purifying the active ingredients in compositions containing kudzu root and epimedium are not very effective. Summary of the Invention

[0007] According to various embodiments of this application, a method for separating and purifying active ingredients in a composition containing kudzu root and epimedium, and a kudzu root and epimedium extract are provided.

[0008] In some embodiments, a method for separating and purifying the active ingredients in a composition containing kudzu root and epimedium is provided, comprising the following steps: extracting kudzu root and epimedium with a first alcohol solvent to prepare an extract;

[0009] The extract was passed through a macroporous adsorption resin and eluted sequentially with water and a second alcohol solvent. The eluent after elution with the second alcohol solvent was collected.

[0010] The flow rate for elution with water is 1.5 BV / h to 3 BV / h, and the flow rate for elution with a second alcohol solvent is 1 BV / h to 1.5 BV / h.

[0011] In some embodiments, in the method for separating and purifying the active ingredients in a composition containing kudzu root and epimedium, the pore size of the macroporous adsorption resin is 0.3 mm to 1.2 mm.

[0012] In some embodiments, in the provided method for separating and purifying the active ingredients in a composition containing kudzu root and epimedium, the macroporous adsorption resin comprises AB-8 macroporous adsorption resin.

[0013] In some embodiments, in the provided method for separating and purifying the active ingredients in a composition containing kudzu root and epimedium, the flow rate of the extract through the macroporous adsorption resin is 0.8 BV / h to 1.5 BV / h.

[0014] In some embodiments, in the method for separating and purifying the active ingredients in a composition containing kudzu root and epimedium, the second alcohol solvent is an aqueous ethanol solution with a concentration of 60% (v / v) to 90% (v / v).

[0015] In some embodiments, the method for separating and purifying the active ingredients in a composition containing kudzu root and epimedium, wherein in the elution step with water, the mass ratio of water to the macroporous adsorption resin is (3-5):1.

[0016] In some embodiments, in the method for separating and purifying the active ingredients in a composition containing kudzu root and epimedium, in the elution step using the second alcohol solvent, the mass ratio of the second alcohol solvent to the macroporous adsorption resin is (6-10):1.

[0017] In some embodiments, the method for separating and purifying the active ingredients in a composition containing kudzu root and epimedium,

[0018] In the step of extracting kudzu root and epimedium with a first alcohol solvent, the first alcohol solvent is an aqueous ethanol solution with a concentration of 60% (v / v) to 90% (v / v).

[0019] In some embodiments, the method for separating and purifying the active ingredients in a composition containing kudzu root and epimedium involves using a first alcohol solvent to extract the mixture containing kudzu root and epimedium.

[0020] In some embodiments, in the method for separating and purifying the active ingredients in a composition containing kudzu root and epimedium, in the step of extracting kudzu root and epimedium with a first alcohol solvent, the mass ratio of kudzu root and epimedium is (1-3):(1-3).

[0021] In some embodiments, the method for separating and purifying the active ingredients in a composition containing kudzu root and epimedium,

[0022] The extract prepared by concentrating and drying the eluent has a total flavonoid content of 48wt% to 65wt%, of which icariin is 1wt% to 4.8wt% and puerarin is 16.4wt% to 26.6wt%.

[0023] In some embodiments, in the method for separating and purifying the active ingredients in the provided composition containing kudzu root and epimedium, the extract prepared by concentrating and drying the eluent has a water-soluble component content of 64wt% to 67wt%.

[0024] In some embodiments, a kudzu and epimedium extract is provided, which is prepared using the method described above.

[0025] In some embodiments, the provided kudzu and epimedium extract contains,

[0026] The total flavonoid content is 48wt% to 65wt%, of which icariin is 1wt% to 4.8wt% and puerarin is 16.4wt% to 26.6wt%.

[0027] In some embodiments, the water-soluble component content of the provided kudzu and epimedium extract is 64wt% to 67wt%.

[0028] Details of one or more embodiments of the present invention are set forth in the following drawings and description. Other features, objects, and advantages of the invention will become apparent from the specification, drawings, and claims. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the accompanying drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0030] Figure 1 is a flowchart of a method for separating and purifying active ingredients in a composition containing kudzu root and epimedium in one embodiment.

[0031] Figure 2 shows the trend of total flavonoid content obtained in Examples 1-4 and Comparative Examples 1-2;

[0032] Figure 3 shows the trend of puerarin content obtained in Examples 1-4 and Comparative Examples 1-2;

[0033] Figure 4 shows the trend of icariin content obtained in Examples 1-4 and Comparative Examples 1-2;

[0034] Figure 5 shows the trend of water-soluble component content obtained in Examples 1-4 and Comparative Examples 1-2. Detailed Implementation

[0035] To facilitate understanding of this application, a more complete description will be provided below with reference to the accompanying drawings. Preferred embodiments of this application are shown in the drawings. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of this application.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0037] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0038] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0039] The terms “combinations thereof,” “any combination thereof,” and “any combination thereof” as used in this application include all suitable combinations of any two or more of the listed items.

[0040] In this application, the term "suitable" as used in "suitable combination", "suitable method", "any suitable method", etc., refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0041] In this application, terms such as "preferred," "better," "more suitable," and "ideal" are merely used to describe implementation methods or embodiments that achieve better results, and should be understood not to limit the scope of protection of this application.

[0042] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0043] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0044] In this application, the terms "first aspect," "second aspect," "third aspect," and "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," and "fourth," etc., serve only a non-exhaustive enumeration purpose and should be understood not to constitute a closed limitation on quantity.

[0045] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0046] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0047] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0048] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0049] In this application, "room temperature" generally refers to 5℃ to 30℃, and more preferably 25±5℃.

[0050] Adsorption capacity: refers to the ability of filter media or ion exchangers to adsorb a certain substance or ion.

[0051] Dynamic adsorption: An appropriate amount of pretreated resin is loaded into a resin adsorption column, and the drug solution is passed through the resin bed at a certain flow rate. The drug concentration of the effluent is measured until adsorption equilibrium is reached.

[0052] Purification and separation technologies are widely used in traditional Chinese medicine manufacturing. Methods such as centrifugation, filtration, extraction, and evaporation are employed to effectively remove impurities and harmful substances from mixtures, thereby significantly improving the purity of the substances. During the refining process, purification steps can be achieved using advanced technologies such as adsorption, immunoaffinity, and electrophoresis.

[0053] Flavonoids are mostly crystalline solids, with a few being amorphous powders. Flavonoid glycosides are generally readily soluble in solvents such as water, methanol, ethanol, ethyl acetate, and pyridine, but poorly soluble in organic solvents such as ether, chloroform, and benzene. Flavonoids possess pharmacological effects including antibacterial, antiviral, immunomodulatory, and neuroprotective properties.

[0054] Methods for purifying flavonoids include macroporous adsorption resin method, recrystallization method, extraction method, membrane separation technology, metal complexation technology, activated carbon adsorption method, preparative liquid chromatography and other techniques. Among them, macroporous adsorption resin technology has the characteristics of simple operation, no pollution, low energy consumption and good separation.

[0055] Macroporous resin adsorption separation technology utilizes a class of organic polymer adsorbents. Through adsorption and molecular sieving principles, and based on the differences in adsorption capacity and molecular weight of different components, separation is achieved by elution with a specific solvent on a macroporous adsorption resin. Macroporous adsorption resins are particularly suitable for the separation, purification, and large-scale production of flavonoids and saponins due to their large adsorption capacity, simple regeneration, and reliable performance.

[0056] Macroporous adsorption resins lack rigidity, and high flow rates can cause resin particles to break, resulting in the loss of unadsorbed active ingredients. Furthermore, different resin types have varying regeneration capabilities and require different regeneration methods. Generally, macroporous adsorption resins need regeneration after being reused more than twice, but the adsorption capacity for flavonoids and other active ingredients decreases after regeneration. Pretreatment of macroporous resins often involves acid and alkali washing, which cannot completely remove organic solvents and harmful residues. Macroporous resins have poor adsorption capacity for sugars, and resins such as HPD100, HPD300, HPD500, D101, D201, D301, NKA9, and S-8 exhibit poor adsorption and desorption capabilities for total flavonoids from Epimedium and Pueraria lobata, resulting in unsatisfactory separation and failing to ensure the separation of other components, thus affecting the study of the physiological activity of flavonoids.

[0057] To improve the purity of total flavonoids, it is necessary to extract and separate the composition containing kudzu root and epimedium. This application uses alcohol extraction and macroporous adsorption resin separation technology to separate and purify the active ingredients in the composition containing kudzu root and epimedium.

[0058] In some embodiments, a method for separating and purifying the active ingredients in a composition containing kudzu root and epimedium is provided, comprising the following steps:

[0059] Extracts were prepared by using a primary alcohol solvent to extract kudzu root and epimedium.

[0060] The extract was passed through a macroporous adsorption resin and eluted sequentially with water and a second alcohol solvent. The eluent after elution with the second alcohol solvent was collected.

[0061] The flow rate for elution with water is 1.5 BV / h to 3 BV / h, and the flow rate for elution with a second alcohol solvent is 1 BV / h to 1.5 BV / h.

[0062] Figure 1 is a flowchart of a method for separating and purifying the active ingredients in a composition containing kudzu root and epimedium, according to one embodiment. It should be understood that although the steps in the flowchart shown in Figure 1 are displayed sequentially according to the arrows, these steps are not necessarily performed in the order indicated by the arrows. Unless explicitly stated herein, there is no strict order restriction on the execution of these steps, and they can be performed in other orders. Moreover, at least some of the steps in Figure 1 may include multiple sub-steps or multiple stages. These sub-steps or stages are not necessarily completed at the same time, but can be performed at different times, and their execution order is not necessarily sequential, but can be performed alternately or in turn with other steps or at least a portion of the sub-steps or stages of other steps.

[0063] The provided method for separating and purifying the active ingredients in a composition containing kudzu root and epimedium optimizes the process by providing a specific method for using an optimized macroporous adsorption resin. The entire method is simple to operate, low in cost, and the process is stable and reliable, making it suitable for large-scale production. The prepared kudzu root and epimedium extract has a high content of active ingredients.

[0064] Without limitation, the medicinal materials used include one or more of the following: raw medicinal materials, processed medicinal materials and intermediate processed products, and processed medicinal materials obtained by different processing techniques.

[0065] In some embodiments, a method for separating and purifying the active ingredients in a composition containing kudzu root and epimedium is provided, comprising the following steps: extracting kudzu root and epimedium with a first alcohol solvent to prepare an extract;

[0066] The extract was passed through a macroporous adsorption resin and eluted sequentially with water and a second alcohol solvent. The eluent after elution with the second alcohol solvent was collected.

[0067] The elution rate with water is 1.5 BV / h to 3 BV / h, for example, the elution rate with water is 1.5 BV / h, 2 BV / h, 2.5 BV / h, 3 BV / h, or a range selected from any two of the aforementioned values. The elution rate with the second alcohol solvent is 1 BV / h to 1.5 BV / h, for example, the elution rate with the second alcohol solvent can be 1 BV / h, 1.1 BV / h, 1.2 BV / h, 1.3 BV / h, 1.4 BV / h, 1.5 BV / h, or a range selected from any two of the aforementioned values. In some embodiments, in the provided method for separating and purifying the active ingredients in a composition containing kudzu root and epimedium, the pore size of the macroporous adsorption resin is 0.3 mm to 1.2 mm. For example, the pore size of the macroporous adsorption resin can be 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1.0 mm, 1.1 mm, 1.2 mm, or a range selected from any two of the aforementioned values.

[0068] In some embodiments, the method for separating and purifying the active ingredients in a composition containing kudzu root and epimedium includes AB-8 macroporous adsorption resin as the macroporous adsorption resin.

[0069] In some embodiments, in the provided method for separating and purifying the active ingredients in a composition containing kudzu root and epimedium, the flow rate of the extract through the macroporous adsorption resin is 0.8 BV / h to 1.5 BV / h. For example, the flow rate of the extract through the macroporous adsorption resin can be 0.8 BV / h, 0.9 BV / h, 1 BV / h, 1.1 BV / h, 1.2 BV / h, 1.3 BV / h, 1.4 BV / h, 1.5 BV / h, or a range selected from any two of the aforementioned values.

[0070] In some embodiments, in the method for separating and purifying the active ingredients in the composition containing kudzu root and epimedium, the second alcohol solvent is an aqueous ethanol solution with a concentration of 60% (v / v) to 90% (v / v). For example, the concentration of the aqueous ethanol solution can be 60% (v / v), 70% (v / v), 80% (v / v), 90% (v / v), or a range selected from any two of the aforementioned values.

[0071] In some embodiments, in the method for separating and purifying the active ingredients in a composition containing kudzu root and epimedium, in the elution step with water, the mass ratio of water to macroporous adsorption resin is (3-5):1. For example, the mass ratio of water to macroporous adsorption resin can be 3:1, 4:1, 5:1 or a range selected from any two of the aforementioned ratios.

[0072] In some embodiments, in the method for separating and purifying the active ingredients in a composition containing kudzu root and epimedium, in the elution step with a second alcohol solvent, the mass ratio of the second alcohol solvent to the macroporous adsorption resin is (6-10):1. For example, the mass ratio of the second alcohol solvent to the macroporous adsorption resin can be 6:1, 7:1, 8:1, 9:1, 10:1 or selected from any of the aforementioned two ratios.

[0073] In some embodiments, in the method for separating and purifying the active ingredients in a composition containing kudzu root and epimedium, in the step of extracting kudzu root and epimedium with a first alcohol solvent, the first alcohol solvent is an aqueous ethanol solution with a concentration of 60% (v / v) to 90% (v / v). For example, the concentration of the aqueous ethanol solution can be 60% (v / v), 70% (v / v), 80% (v / v), 90% (v / v), or a range selected from any two of the aforementioned values.

[0074] In some embodiments, the method for separating and purifying the active ingredients in a composition containing kudzu root and epimedium involves using a first alcohol solvent to extract the mixture containing kudzu root and epimedium.

[0075] In some embodiments, in the method for separating and purifying the active ingredients in a composition containing kudzu root and epimedium, in the step of extracting kudzu root and epimedium with a first alcohol solvent, the mass ratio of kudzu root and epimedium is (1-3):(1-3), for example, 1:1, 1:2, 1:3, 2:1, 2:3, 3:1, 3:2 or a range selected from any two of the aforementioned ratios.

[0076] In some embodiments, in the method for separating and purifying the active ingredients in the provided composition containing kudzu root and epimedium, the extract prepared by concentrating and drying the eluent has a total flavonoid content of 48wt% to 65wt%, of which icariin is 1wt% to 4.8wt% and puerarin is 16.4wt% to 26.6wt%.

[0077] In some embodiments, in the method for separating and purifying the active ingredients in the provided composition containing kudzu root and epimedium, the extract prepared by concentrating and drying the eluent has a water-soluble component content of 64wt% to 67wt%.

[0078] Non-limitingly, the prepared extract includes both water-soluble and water-insoluble components. The water-soluble components include flavonoid glycosides, saponins, and other components, among which flavonoid glycosides include icariin and puerarin. The water-insoluble components include flavonoid aglycones, etc. Total flavonoids include flavonoid aglycones and flavonoid glycosides.

[0079] In some embodiments, a kudzu and epimedium extract is provided, which is prepared using the aforementioned method.

[0080] In some embodiments, the total flavonoid content of the provided kudzu and epimedium extract is 48wt% to 65wt%, of which icariin is 1wt% to 4.8wt% and puerarin is 16.4wt% to 26.6wt%.

[0081] In some embodiments, the water-soluble component content of the provided kudzu and epimedium extract is 64wt% to 67wt%.

[0082] In some embodiments, a dynamic adsorption method was used to compare the adsorption and desorption capabilities of five different macroporous adsorption resins (AB-8, D-101, HPD100, X-5, and D140) for total flavonoids in Epimedium puerarin. The AB-8 macroporous adsorption resin showed better extraction performance and is suitable for the separation and purification of active ingredients in Epimedium puerarin.

[0083] In a non-limiting sense, AB-8 macroporous resin is a polystyrene-type, weakly polar polymer adsorbent that exhibits good adsorption and desorption performance for total flavonoids. Compared with other resin columns, such as HPD-100, NKA9, S-8, D-101, HPD100, X-5, and D140, it has a fast adsorption rate, large adsorption capacity, and is easy to desorb, making it suitable for the purification and adsorption of flavonoids.

[0084] In some embodiments, upon initial use, macroporous adsorption resin is packed into a macroporous adsorption resin column, and the resin column is eluted with approximately twice the column volume of 95% ethanol at a flow rate of 2 BV / h. After elution, the column is soaked for at least 12 hours before use. Before loading the sample, purified water is turned on, and the flow rate is controlled at 2 BV / h to balance the effluent with the added effluent. The purified water is eluted until the effluent has no ethanol odor and no ethanol is detected by an alcohol meter.

[0085] In some embodiments, the total flavonoid content of the provided kudzu and epimedium extract is 48wt% to 61wt%, of which icariin is 1wt% to 1.1wt% and puerarin is 22wt% to 24.5wt%.

[0086] It should be noted that the total flavonoid content involved is measured using a standard curve prepared with puerarin as the reference standard.

[0087] The provided method for separating and purifying the active ingredients in a composition containing kudzu root and epimedium involves loading the extract of the composition containing kudzu root and epimedium onto a column, eluting, collecting the eluent, concentrating, and drying to obtain purified total flavonoids. This method reduces the use of organic solvents throughout the purification process, preventing their influence on the final product. It achieves high total flavonoid transfer rate and high total flavonoid content in the purified product, exhibits good reproducibility, and allows for resin reuse. Furthermore, gas-phase residual solvent detection of the epimedium and kudzu root extracts shows that the contents of toluene and 1,2-diethylbenzene are both below the limits specified in national standards, indicating good safety.

[0088] In the examples below, the qualified particle size (0.3 mm to 1.2 mm) of the AB-8 macroporous adsorption resin used is not less than 90%.

[0089] Example 1: Isolation and purification of active ingredients from a composition containing kudzu root and epimedium.

[0090] 1. Preparation of Pueraria lobata and Epimedium extract

[0091] Take 60g of kudzu root and 30g of epimedium, add 12 times the amount of 80% ethanol each time, heat and reflux to extract 3 times, 1 hour each time. Filter the extract, combine the kudzu root and epimedium ethanol extracts after the three extractions, control the vacuum degree at -0.05 to -0.09 MPa and the temperature at 60 to 80℃, concentrate under reduced pressure to a relative density of 1.03 to 1.08 (70℃), and let the concentrated solution stand at below 10℃ for more than 40 hours to precipitate, and take the supernatant.

[0092] 2. Separation and purification

[0093] (1) Pretreatment of macroporous adsorption resin

[0094] The AB-8 macroporous adsorption resin used in this product is pre-treated. For initial use, load 125g of AB-8 macroporous adsorption resin into a macroporous adsorption resin column. Elute the resin column with approximately twice the column volume of 95% ethanol at a flow rate of 2 BV / h. After elution, continue soaking for at least 12 hours. Before loading the sample, turn on the purified water and control the flow rate to 2 BV / h to balance the effluent with the added effluent. Elute with purified water until the effluent has no ethanol odor and an alcohol meter detects no ethanol.

[0095] (2) Adsorption, elution and collection

[0096] Take 110g of the supernatant and pass it through a 125g AB-8 macroporous adsorption resin column at a flow rate of approximately 1 BV / h. First, elute impurities with 4 times the weight of the resin of water at a flow rate of 1.5 BV / h, and discard the water. Then, desorb the flavonoids with 8 times the weight of the resin of 80% ethanol at a flow rate of 1.0 BV / h, collect the eluent, and control the vacuum degree at -0.05 to -0.09 MPa and the temperature at 60 to 80℃. Concentrate the ethanol eluent under reduced pressure to a relative density of approximately 1.08 to 1.15 (65 to 75℃) to obtain a purified thick extract of Pueraria lobata and Epimedium.

[0097] 3. Drying

[0098] The purified viscous extract of kudzu root and epimedium was placed in a vacuum drying oven and dried at a vacuum level of -0.06 to -0.09 MPa and a temperature below 60°C for approximately 26 hours. The dried extract was then pulverized and passed through a 60-mesh sieve to obtain kudzu root and epimedium extracts. The contents of total flavonoids, puerarin, icariin, and water-soluble components were determined.

[0099] 4. Post-use treatment of macroporous adsorption resin

[0100] Resin treatment during continuous production: Add approximately 1.5 times the column volume of macroporous adsorption resin and soak in 95% ethanol for more than 4 hours. Wash the column with purified water and elute until the effluent has no ethanol odor and no ethanol is detected by an alcohol meter. Then it can be used for the next time.

[0101] Resin treatment during non-continuous production: Soak the resin in 95% ethanol for preservation with approximately 1.5 times the column volume of macroporous adsorption resin to prevent microbial growth and contamination. Before reuse, wash with purified water until the effluent has no ethanol odor and the alcohol meter shows no ethanol.

[0102] Example 2: Isolation and purification of active ingredients from a composition containing kudzu root and epimedium.

[0103] The difference from Example 1 is that, in the adsorption, elution and collection steps 2.(2), the flow rate of water elution is 3 BV / h and the flow rate of 80% ethanol elution is 1.0 BV / h.

[0104] Example 3: Isolation and purification of active ingredients from a composition containing kudzu root and epimedium.

[0105] The difference from Example 1 is that, in the adsorption, elution and collection steps 2.(2), the flow rate of water elution is 4 BV / h and the flow rate of 80% ethanol elution is 1.0 BV / h.

[0106] Example 4: Isolation and purification of active ingredients from a composition containing kudzu root and epimedium.

[0107] The difference from Example 1 is that, in the adsorption, elution and collection steps 2.(2), the flow rate of water elution is 1.5 BV / h, and the flow rate of 80% ethanol elution is 1.5 BV / h.

[0108] Comparative Example 1: Isolation and purification of active ingredients from a composition containing kudzu root and epimedium

[0109] The difference from Example 1 is that, in the adsorption, elution and collection steps 2.(2), the flow rate of water elution is 1.5 BV / h and the flow rate of 80% ethanol elution is 2.0 BV / h.

[0110] Comparative Example 2: Isolation and purification of active ingredients from the composition containing kudzu root and epimedium

[0111] The difference from Example 1 is that, in the adsorption, elution and collection steps 2.(2), the flow rate of water elution is 1.5 BV / h and the flow rate of 80% ethanol elution is 3.0 BV / h.

[0112] Content determination

[0113] 1. Method for determining total flavonoid content

[0114] The total flavonoid content was determined according to the ultraviolet-visible spectrophotometric method (General Chapter 0401, Chinese Pharmacopoeia 2020 Edition).

[0115] Preparation of reference solution: Take an appropriate amount of puerarin reference standard, accurately weigh it, and add ethanol to prepare a solution containing 0.1 mg in 1 ml.

[0116] Preparation of the standard curve: Accurately measure 0.2 ml, 0.4 ml, 0.6 ml, 0.8 ml, and 1.0 ml of the reference solution and place them in separate 10 ml volumetric flasks. Dilute to the mark with 80% ethanol and mix well. Using the corresponding reagents as blanks, measure the absorbance at a wavelength of 250 nm using ultraviolet-visible spectrophotometry (General Chapter 0401 of the Chinese Pharmacopoeia 2020). Plot the standard curve with absorbance as the ordinate and concentration as the abscissa.

[0117] Assay: Accurately weigh approximately 15 mg of the kudzu root and epimedium extracts prepared in Examples 1-4 and Comparative Examples 1-2, place them in a 50 ml volumetric flask, add an appropriate amount of 80% ethanol, shake to dissolve, add 80% ethanol to the mark, shake well, and filter. Accurately measure 1.0 ml of the filtrate, place it in a 25 ml volumetric flask, add 80% ethanol to the mark, and shake well. Following the method under the preparation of the standard curve, starting from "using the corresponding reagent as a blank", determine the absorbance according to the method, read the amount of puerarin in the test solution from the standard curve, and calculate to obtain the result.

[0118] The calculation formulas are as follows: Sample concentration (mg / ml) = Standard concentration × Sample absorbance / Standard absorbance; Total flavonoid content (wt%) = (Sample concentration × 50 × 25) / [Sample weight × (1 - Moisture content of the test sample)] × 100%.

[0119] 2. Method for determining puerarin content

[0120] The content of puerarin was determined by high performance liquid chromatography (General Chapter 0512, Chinese Pharmacopoeia 2020).

[0121] Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase; acetonitrile-0.5% phosphoric acid solution (10:90) was used as the mobile phase; the detection wavelength was 250 nm; and the column temperature was 45℃. The theoretical plate number, calculated based on the puerarin peak, should not be less than 2500.

[0122] Preparation of reference solution: Take an appropriate amount of puerarin reference standard, accurately weigh it, dissolve it in ethanol, and then dilute it with 50% ethanol to prepare a solution containing 80 μg per ml.

[0123] Preparation of the test solution: Weigh approximately 20 mg of the kudzu root and epimedium extracts prepared in Examples 1-4 and Comparative Examples 1-2 accurately, place them in a 50 ml volumetric flask, add an appropriate amount of 80% ethanol, sonicate for 30 minutes (power 250 W, frequency 40 kHz), cool, dilute to the mark with 80% ethanol, shake well, filter through a microporous membrane (0.45 μm), and collect the filtrate to obtain the test solution.

[0124] Determination method: Accurately pipette 5 μl of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0125] The calculation formulas are as follows: Sample concentration (mg / ml) = Standard concentration × Sample peak area / Standard peak area; Puerarin content (wt%) = (Sample concentration × 50) / [Sample weight × (1 - Moisture content of the test sample)] × 100%.

[0126] 3. Method for determining the content of Epimedium

[0127] The content of Epimedium was determined by high performance liquid chromatography (General Chapter 0512, Chinese Pharmacopoeia 2020 Edition).

[0128] Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase; acetonitrile-water (27:73) was used as the mobile phase; the detection wavelength was 270 nm; and the column temperature was 30 °C. The theoretical plate number, calculated based on the icariin peak, should not be less than 1500.

[0129] Preparation of reference solution: Take an appropriate amount of icariin reference standard, accurately weigh it, and add methanol to prepare a solution with a concentration of 0.154 mg / ml.

[0130] Preparation of the test solution: Accurately weigh 70 mg of the kudzu root and epimedium extracts prepared in Examples 1-4 and Comparative Examples 1-2, place them in a stoppered conical flask, accurately add 10 ml of 80% ethanol, seal tightly, weigh, sonicate for 30 minutes (power 250 W, frequency 40 kHz), remove, cool, weigh again, replenish the lost weight with 80% ethanol, shake well, filter through a microporous membrane (0.45 μm), and collect the filtrate to obtain the test solution.

[0131] Determination method: Accurately pipette 10 μl of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0132] The calculation formulas are as follows: Sample concentration (mg / ml) = Reference concentration × Sample peak area / Reference peak area; Icariin content (wt%) = (Sample concentration × 10) / [Sample weight × (1 - Moisture content of the test sample)] × 100%.

[0133] 4. Methods for determining water-soluble components

[0134] The determination of water-soluble components was performed according to the cold maceration method for water-soluble extracts (General Chapter 2201, Chinese Pharmacopoeia 2020).

[0135] Take 0.1 g of the kudzu root and epimedium extracts prepared in Examples 1-4 and Comparative Examples 1-2, place them in a stoppered graduated test tube, add 5 ml of purified water, and shake thoroughly to obtain a suspension; filter the suspension through filter paper to obtain a clear filtrate; transfer the filtrate to a weighing bottle that has been dried to constant weight, dry at 105℃ to constant weight, and weigh the dried product. Water-soluble component content (wt%) = weight of dried product / weight of extract sample × 100%.

[0136] The measured contents of total flavonoids, puerarin, icariin, and water-soluble components are shown in Table 1 below. Figure 2 shows the trend of total flavonoid content obtained in Examples 1-4 and Comparative Examples 1-2; Figure 3 shows the trend of puerarin content obtained in Examples 1-4 and Comparative Examples 1-2; Figure 4 shows the trend of icariin content obtained in Examples 1-4 and Comparative Examples 1-2; and Figure 5 shows the trend of water-soluble component content obtained in Examples 1-4 and Comparative Examples 1-2.

[0137] Table 1. Content changes at different elution rates

[0138] As shown in Table 1 and Figures 2-5, the provided separation and purification method can effectively separate and purify the active ingredients in Epimedium puerarin. With a fixed ethanol elution flow rate, different water elution flow rates had no significant effect on total flavonoids, icariin, and puerarin, but significantly affected the content of water-soluble components. The best results were achieved with water elution rates of 1.5 BV / h to 3 BV / h, while the effect was poor at 4 BV / h. Similarly, with a fixed water elution flow rate, the best results were achieved with ethanol elution rates of 1 BV / h to 1.5 BV / h, while the effect was poor at 2 BV / h to 3 BV / h.

[0139] The entire method is simple to operate, low in cost, and the process is stable and reliable, making it suitable for large-scale production.

[0140] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0141] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A method for separating and purifying the active ingredients in a composition containing kudzu root and epimedium, characterized in that, The process includes the following steps: extracting kudzu root and epimedium with a primary alcohol solvent to prepare an extract; The extract is passed through a macroporous adsorption resin and eluted sequentially with water and a second alcohol solvent. The eluent after elution with the second alcohol solvent is collected. The flow rate for elution with water is 1.5 BV / h to 3 BV / h, and the flow rate for elution with the second alcohol solvent is 1 BV / h to 1.5 BV / h.

2. The method according to claim 1, characterized in that, The macroporous adsorption resin has a pore size of 0.3 mm to 1.2 mm.

3. The method according to claim 1 or 2, characterized in that, The macroporous adsorption resin includes AB-8 macroporous adsorption resin.

4. The method according to any one of claims 1 to 3, characterized in that, The flow rate of the extract through the macroporous adsorption resin is 0.8 BV / h to 1.5 BV / h.

5. The method according to any one of claims 1 to 4, characterized in that, The second alcohol solvent is an aqueous ethanol solution with a concentration of 60% (v / v) to 90% (v / v).

6. The method according to any one of claims 1 to 5, characterized in that, In the elution step using water, the mass ratio of water to the macroporous adsorption resin is (3-5):

1.

7. The method according to any one of claims 1 to 6, characterized in that, In the elution step using the second alcohol solvent, the mass ratio of the second alcohol solvent to the macroporous adsorption resin is (6-10):

1.

8. The method according to any one of claims 1 to 7, characterized in that, In the step of extracting kudzu root and epimedium with a first alcohol solvent, the first alcohol solvent is an aqueous ethanol solution with a concentration of 60% (v / v) to 90% (v / v).

9. The method according to any one of claims 1 to 8, characterized in that, The mixture containing kudzu root and epimedium was extracted using a first alcohol solvent.

10. The method according to any one of claims 1 to 9, characterized in that, In the step of extracting kudzu root and epimedium with a first alcohol solvent, the mass ratio of kudzu root and epimedium is (1-3):(1-3).

11. The method according to any one of claims 1 to 10, characterized in that, The extract prepared by concentrating and drying the eluent has a total flavonoid content of 48wt% to 65wt%, of which icariin is 1wt% to 4.8wt% and puerarin is 16.4wt% to 26.6wt%.

12. The method according to any one of claims 1 to 11, characterized in that, The extract prepared by concentrating and drying the eluent has a water-soluble component content of 64wt% to 67wt%.

13. A kudzu root and epimedium extract, characterized in that, It is prepared by the method described in any one of claims 1 to 12.

14. The kudzu root and epimedium extract according to claim 13, characterized in that, The total flavonoid content is 48wt% to 65wt%, of which icariin is 1wt% to 4.8wt% and puerarin is 16.4wt% to 26.6wt%.

15. The kudzu root and epimedium extract according to claim 13 or 14, characterized in that, The water-soluble component content is 64wt% to 67wt%.

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