Method for separating and purifying active ingredients in composition containing radix puerariae and herba epimedii and radix puerariae and herba epimedii extract

Abstract

The invention discloses a method for separating and purifying active ingredients in a composition containing radix puerariae and herba epimedii and a radix puerariae and herba epimedii extract. The method for separating and purifying the active ingredients in the composition containing the radix puerariae and the herba epimedii comprises the following steps: extracting a radix puerariae medicinal material and a herba epimedii medicinal material by adopting a first alcohol solvent to prepare an extracting solution; enabling the extracting solution to pass through macroporous adsorption resin, sequentially eluting with water and a second alcohol solvent, and collecting an eluent eluted with the second alcohol solvent; wherein the flow rate of the elution with the water is 1.5 BV / h to 3 BV / h, and the flow rate of the elution with the second alcohol solvent is 1 BV / h to 1.5 BV / h. The whole method is simple and convenient to operate, low in cost, stable and reliable in process and suitable for large-scale popularization and production. The content of active ingredients in the prepared radix puerariae and herba epimedii extract is relatively high.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicines, and in particular to a method for separating and purifying active components in a composition containing kudzu root and epimedium, and a kudzu root and epimedium extract. Background Art

[0002] Pueraria root is the dried root of the leguminous plant Pueraria lobata. Its main active ingredients are various isoflavone substances such as soybean flavonoid aglycones, soybean glycosides, and puerarin. It has various pharmacological and health-care effects, such as antibacterial, blood circulation and blood stasis, dilation of coronary arteries and cerebral blood vessels, reduction of myocardial oxygen consumption, improvement of myocardial contractile function, promotion of blood circulation, and enhancement of body immunity. Pueraria root is mostly used in injections, eye drops, capsules and other dosage forms.

[0003] Epimedium is the dried aerial part of the plants Epimedium, Epimedium sagittatum, Epimedium pubescens or Epimedium koreana of the Berberidaceae family. It has the effects of tonifying kidney yang, strengthening tendons and bones, and removing rheumatism. The main active ingredients in Epimedium are flavonoid compounds. Modern pharmacological studies have shown that Epimedium has multiple pharmacological effects in immunity, reproduction, nucleic acid metabolism, cardiovascular and anti-aging. Epimedium is mostly used in tablets, granules and other dosage forms.

[0004] Separating and purifying the active ingredients in Pueraria root and Epimedium can better exert their medicinal value. However, the current method for separating and purifying the active ingredients of the composition containing Pueraria root and Epimedium has poor effect. Summary of the invention

[0005] Based on this, it is necessary to provide a method for separating and purifying active ingredients in a composition containing kudzu root and epimedium, and a kudzu root and epimedium extract.

[0006] In some embodiments, a method for separating and purifying 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;

[0007] Passing the extract through a macroporous adsorption resin, eluting with water and a second alcohol solvent in sequence, and collecting the eluate eluted with the second alcohol solvent;

[0008] The flow rate of elution with water is 1.5 BV / h to 3 BV / h, and the flow rate of elution with the second alcohol solvent is 1 BV / h to 1.5 BV / h.

[0009] In some embodiments, in the provided method for separating and purifying active ingredients in a composition containing Pueraria lobata and Epimedium brevicornum, the pore size of the macroporous adsorption resin is 0.3 mm to 1.2 mm;

[0010] Optionally, the macroporous adsorption resin comprises AB-8 macroporous adsorption resin.

[0011] In some embodiments, in the provided method for separating and purifying active ingredients in a composition containing Pueraria lobata and Epimedium brevicornum, the flow rate of the extract passing through the macroporous adsorption resin is 0.8 BV / h~1.5 BV / h.

[0012] In some embodiments, in the provided method for separating and purifying active ingredients in a composition containing Pueraria lobata and Epimedium, the second alcohol solvent is an ethanol aqueous solution with a concentration of 60% (v / v) to 90% (v / v).

[0013] In some embodiments, in the provided method for separating and purifying active ingredients in a composition containing Pueraria lobata and Epimedium brevicornum, in the step of eluting with water, the mass ratio of the water to the macroporous adsorption resin is (3-5):1.

[0014] In some embodiments, in the provided method for separating and purifying active ingredients in a composition containing Pueraria lobata and Epimedium, in the step of eluting with the second alcohol solvent, the mass ratio of the second alcohol solvent to the macroporous adsorption resin is (6-10):1.

[0015] In some embodiments, the provided method for separating and purifying active ingredients in a composition containing Puerariae Radix and Epimedium Herba satisfies one or more of the following characteristics:

[0016] In the step of extracting the kudzu root and epimedium medicinal materials using the first alcohol solvent, the first alcohol solvent is an ethanol aqueous solution with a concentration of 60% (v / v) to 90% (v / v);

[0017] Extracting a mixture containing Pueraria root medicinal material and Epimedium medicinal material by using a first alcohol solvent;

[0018] In the step of extracting the kudzu root medicinal material and the epimedium medicinal material using the first alcohol solvent, the mass ratio of the kudzu root medicinal material to the epimedium medicinal material is (1-3): (1-3).

[0019] In some embodiments, the provided method for separating and purifying active ingredients in a composition containing Puerariae Radix and Epimedium Herba satisfies one or more of the following characteristics:

[0020] The extract prepared by concentrating and drying the eluate has a total flavonoid content of 48 wt% to 65 wt%, wherein icariin is 1 wt% to 4.8 wt%, and puerarin is 16.4 wt% to 26.6 wt%;

[0021] The extract prepared by concentrating and drying the eluate has a water-soluble component content of 64 wt % to 67 wt %.

[0022] In some embodiments, a kudzu root and epimedium extract is provided, which is prepared by the method described.

[0023] In some embodiments, the provided Pueraria lobata and Epimedium extract satisfies one or more of the following characteristics:

[0024] The total flavonoid content is 48wt%~65wt%, of which icariin is 1wt%~4.8wt% and puerarin is 16.4wt%~26.6wt%;

[0025] The water-soluble component content is 64wt%~67wt%.

[0026] The above-mentioned method for separating and purifying active ingredients in a composition containing kudzu root and epimedium provides a specific method for using an optimized macroporous adsorption resin to optimize the process for separating and purifying active ingredients in kudzu root and epimedium. The whole method is simple to operate, low in cost, and has a stable and reliable process, which is suitable for large-scale promotion and production. The prepared kudzu root and epimedium extract has a high content of active ingredients. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the implementation methods and examples of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for use in the description of the implementation methods or examples. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0028] Figure 1 The present invention is a flow chart of a method for separating and purifying active ingredients in a composition containing Puerariae Radix and Epimedium Herba in one embodiment;

[0029] Figure 2 It is a trend diagram of the total flavonoid content obtained in Examples 1 to 4 and Comparative Examples 1 to 2;

[0030] Figure 3 It is a trend diagram of puerarin content obtained in Examples 1 to 4 and Comparative Examples 1 to 2;

[0031] Figure 4 The icariin content trend diagram obtained in Examples 1 to 4 and Comparative Examples 1 to 2;

[0032] Figure 5 The following is a trend chart of the water-soluble component content obtained in Examples 1 to 4 and Comparative Examples 1 to 2. DETAILED DESCRIPTION

[0033] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0035] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0036] In the present application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0037] The terms "combination thereof", "any combination thereof", "any combination thereof" and the like used in this application include all suitable combinations of any two or more of the listed items.

[0038] In this application, the "suitable" mentioned in "suitable combination", "suitable method", "any suitable method", etc. is based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0039] In the present application, "preferred", "better", "more preferred" and "suitable" are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of the present application.

[0040] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0041] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0042] In the present invention, in the "first aspect", "second aspect", "third aspect", "fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0043] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0044] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0045] The temperature parameters in this application, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0046] In the present application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0047] The "room temperature" in this application generally refers to 5°C to 30°C, preferably 25±5°C.

[0048] Adsorption capacity: refers to the ability of filter material or ion exchanger to adsorb certain substances or ions.

[0049] Dynamic adsorption: An equal amount of pretreated resin is loaded into the resin adsorption column. The drug solution passes through the resin bed at a certain flow rate, and the drug concentration of the effluent is measured until the adsorption equilibrium is reached.

[0050] Purification and separation technology is widely used in traditional Chinese medicine. By means of centrifugation, filtration, extraction, evaporation and other means, impurities and harmful substances in the mixture can be effectively removed, thereby significantly improving the purity of the substance. In the process of refining, the purification step can be achieved through adsorption, immunoaffinity, electrophoresis and other advanced technologies.

[0051] Most flavonoids are crystalline solids, and a few are amorphous powders. Flavonoid glycosides are generally soluble in water, methanol, ethanol, ethyl acetate, pyridine and other solvents, but are difficult to dissolve in organic solvents such as ether, chloroform, and benzene. Flavonoids have antibacterial, antiviral, immunomodulatory and neuroprotective pharmacological effects.

[0052] Methods for purifying flavonoid compounds include macroporous adsorption resin method, recrystallization method, extraction method, membrane separation technology, metal complexation technology, activated carbon adsorption method, preparative liquid chromatography and other technologies. Among them, macroporous adsorption resin technology has the characteristics of simple operation, no pollution, low energy consumption and good separation.

[0053] Macroporous resin adsorption separation technology uses a class of organic polymer adsorbents to achieve separation through adsorption and molecular sieve principles, based on the different adsorption forces of different components and the size of their molecular weights, by eluting them with a certain solvent on the macroporous adsorption resin. Macroporous adsorption resins are particularly suitable for the separation, purification and large-scale production of flavonoids and saponins because of their large adsorption capacity, simple regeneration and reliable effect.

[0054] The rigidity of macroporous adsorption resin is not strong, and a high flow rate will cause the resin particles to break, and the effective ingredients will flow out without being adsorbed. In addition, different types of resins have different regeneration capabilities and inconsistent regeneration methods. Generally, macroporous adsorption resins need to be regenerated after being reused more than twice, and the adsorption capacity of regenerated macroporous adsorption resins for effective ingredients such as flavonoids decreases. The pretreatment of macroporous resins mostly uses acid and alkali washing, and organic solvents and harmful residues cannot be completely removed. Macroporous resins have poor adsorption capacity for sugars, and macroporous resins such as HPD100, HPD300, HPD500, D101, D201, D301, NKA9, and S-8 have poor adsorption and desorption capacity for total flavonoids of Epimedium and Pueraria, and the separation effect is poor. It cannot ensure that other components are separated, which affects the study of the physiological activity of flavonoid components.

[0055] In order to improve the purity of total flavonoids, etc., it is necessary to extract and separate the composition containing Pueraria lobata and Epimedium. This patent uses alcohol extraction and macroporous adsorption resin separation technology to separate and purify the active ingredients in the composition containing Pueraria lobata and Epimedium.

[0056] In some embodiments, a method for separating and purifying active ingredients in a composition containing Pueraria lobata and Epimedium is provided, comprising the following steps:

[0057] Extracting the kudzu root and epimedium medicinal materials with a first alcohol solvent to prepare an extract;

[0058] Passing the extract through a macroporous adsorption resin, eluting with water and a second alcohol solvent in sequence, and collecting the eluate eluted with the second alcohol solvent;

[0059] The flow rate of elution with water is 1.5 BV / h to 3 BV / h, and the flow rate of elution with the second alcohol solvent is 1 BV / h to 1.5 BV / h.

[0060] Figure 1 The present invention is a flow chart of a method for separating and purifying active ingredients in a composition containing Pueraria lobata and Epimedium brevicornum in one embodiment.

[0061] Without limitation, the medicinal materials used include one or more of crude drugs, decoction pieces and processing intermediates, decoction pieces prepared by different processing techniques, etc.

[0062] In some embodiments, a method for separating and purifying 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;

[0063] Passing the extract through a macroporous adsorption resin, eluting with water and a second alcohol solvent in sequence, and collecting the eluate eluted with the second alcohol solvent;

[0064] Among them, the flow rate of elution with water is 1.5BV / h~3BV / h, for example, the flow rate of elution with water is 1.5BV / h, 2BV / h, 2.5BV / h, 3BV / h or a range selected from any two of the aforementioned values, and the flow rate of elution with the second alcohol solvent is 1BV / h~1.5BV / h, for example, the flow rate of elution with the second alcohol solvent can be 1BV / h, 1.1BV / h, 1.2BV / h, 1.3BV / h, 1.4BV / h, 1.5BV / h or a range selected from any two of the aforementioned values. In some embodiments, in the provided method for separating and purifying active ingredients in a composition containing Pueraria lobata 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 foregoing values.

[0065] In some embodiments, in the provided method for separating and purifying active ingredients in a composition containing Pueraria lobata and Epimedium brevicornum, the macroporous adsorption resin comprises AB-8 macroporous adsorption resin.

[0066] In some embodiments, in the provided method for separating and purifying active ingredients in a composition containing Pueraria lobata and Epimedium, the flow rate of the extract through the macroporous adsorption resin is 0.8BV / h~1.5BV / h. For example, the flow rate of the extract through the macroporous adsorption resin can be 0.8BV / h, 0.9BV / h, 1BV / h, 1.1BV / h, 1.2BV / h, 1.3BV / h, 1.4BV / h, 1.5BV / h or a range selected from any two of the foregoing values.

[0067] In some embodiments, in the provided method for separating and purifying active ingredients in a composition containing Pueraria lobata 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 foregoing values.

[0068] In some embodiments, in the provided method for separating and purifying active ingredients in a composition containing Pueraria lobata and Epimedium, in the step of eluting with water, the mass ratio of water to the macroporous adsorption resin is (3~5):1. For example, the mass ratio of water to the macroporous adsorption resin can be 3:1, 4:1, 5:1 or a range selected from any two of the foregoing ratios.

[0069] In some embodiments, in the provided method for separating and purifying active ingredients in a composition containing Pueraria lobata and Epimedium, in the step of eluting 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 a range selected from any two of the foregoing ratios.

[0070] In some embodiments, in the provided method for separating and purifying active ingredients in a composition containing Pueraria lobata and Epimedium brevicornum, in the step of extracting Pueraria lobata and Epimedium brevicornum medicinal materials using a first alcohol solvent, the first alcohol solvent is an ethanol aqueous solution with a concentration of 60% (v / v) to 90% (v / v). For example, the concentration of the ethanol aqueous solution can be 60% (v / v), 70% (v / v), 80% (v / v), 90% (v / v) or a range selected from any two of the foregoing values.

[0071] In some embodiments, in the provided method for separating and purifying active ingredients in a composition containing Pueraria lobata and Epimedium brevicornum, a first alcohol solvent is used to extract a mixture containing Pueraria lobata and Epimedium brevicornum.

[0072] In some embodiments, in the provided method for separating and purifying active ingredients in a composition containing Pueraria lobata and Epimedium brevicornum, in the step of extracting Pueraria lobata and Epimedium brevicornum with a first alcohol solvent, the mass ratio of Pueraria lobata and Epimedium brevicornum 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 foregoing ratios.

[0073] In some embodiments, in the provided method for separating and purifying active ingredients in a composition containing Pueraria lobata and Epimedium, the total flavonoid content in the extract prepared by concentrating and drying the eluate is 48wt%~65wt%, of which icariin is 1wt%~4.8wt%, and puerarin is 16.4wt%~26.6wt%.

[0074] In some embodiments, in the provided method for separating and purifying active ingredients in a composition containing Pueraria lobata and Epimedium, the content of water-soluble components in the extract prepared by concentrating and drying the eluate is 64 wt % to 67 wt %.

[0075] In a non-limiting manner, the extract prepared includes water-soluble components and water-insoluble components. The water-soluble components contain components such as flavonoid glycosides and saponins, wherein the flavonoid glycosides contain icariin and puerarin. The water-insoluble components include flavonoid aglycones, etc. Total flavonoids include flavonoid aglycones and flavonoid glycosides.

[0076] In some embodiments, a kudzu root and epimedium extract is provided, which is prepared by the aforementioned method.

[0077] In some embodiments, the total flavonoids content of the provided Pueraria lobata and Epimedium extract is 48 wt% to 65 wt%, of which icariin is 1 wt% to 4.8 wt%, and puerarin is 16.4 wt% to 26.6 wt%.

[0078] In some embodiments, the provided Pueraria lobata and Epimedium extract has a water-soluble component content of 64 wt % to 67 wt %.

[0079] In some embodiments, a dynamic adsorption method was used to compare the adsorption and desorption capacities of five different macroporous adsorption resins, AB-8, D-101, HPD100, X-5, and D140, on total flavonoids in Pueraria lobata and Epimedium. The AB-8 macroporous adsorption resin performed better in extraction and was suitable for the separation and purification of active ingredients in Pueraria lobata and Epimedium.

[0080] Without limitation, AB-8 macroporous resin is a polystyrene-type, weakly polar polymer adsorbent, which exhibits good adsorption and desorption properties for total flavonoids. Compared with other resin columns, such as HPD-100, NKA9, S-8, D-101, HPD100, X-5, D140, etc., it has a fast adsorption rate for total flavonoids, a large adsorption capacity, and is easy to desorb, making it suitable for the purification and adsorption of flavonoids.

[0081] In some embodiments, when used for the first time, the macroporous adsorption resin is loaded into a macroporous adsorption resin column, and the resin column is eluted with 95% ethanol at a flow rate of 2 BV / h, which is about 2 times the volume of the macroporous adsorption resin column. After the elution is completed, the resin column is soaked for more than 12 hours for standby use. Before loading, the purified water is turned on and the flow rate is controlled to 2 BV / h to balance the outflow and addition. The purified water is eluted until the effluent has no ethanol taste and the alcohol meter has no ethanol.

[0082] In some embodiments, the total flavonoids content of the provided Pueraria lobata and Epimedium extract is 48 wt% to 61 wt%, of which icariin is 1 wt% to 1.1 wt%, and puerarin is 22 wt% to 24.5 wt%.

[0083] It should be noted that the total flavonoid content involved was measured based on a standard curve prepared with puerarin as the reference substance.

[0084] The provided method for separating and purifying active ingredients in a composition containing kudzu root and epimedium comprises applying an extract of the composition containing kudzu root and epimedium to a column, eluting, collecting the eluate, concentrating, and drying to obtain purified total flavonoids. The method reduces the use of organic solvents in the entire purification process, prevents the influence of organic solvents on the final product, and has high total flavonoid transfer rate and total flavonoid content of the purified product, good reproducibility, and the resin can be reused. In addition, the contents of toluene and 1,2-diethylbenzene in the epimedium and kudzu root extracts are less than the limits specified by national standards after gas phase residual solvent detection, and have good safety.

[0085] The qualified particle size (0.3 mm to 1.2 mm) of the AB-8 macroporous adsorption resin used in the following examples is not less than 90%.

[0086] Example 1 Separation and purification of active ingredients from a composition containing Pueraria root and Epimedium

[0087] 1. Preparation of Puerariae Radix and Epimedium Herba Extract

[0088] Take 60g of Pueraria root and 30g of Epimedium, add 12 times the amount of 80% ethanol each time, heat and reflux to extract 3 times, each time for 1 hour, filter the extract, combine the Pueraria root and Epimedium alcohol extracts after three extractions and filtrations, control the vacuum degree at -0.05~-0.09MPa, and the temperature at 60~80℃, and concentrate under reduced pressure to a relative density of 1.03~1.08 (70℃), and let the concentrated solution stand below 10℃ for more than 40 hours for precipitation, and take the supernatant.

[0089] 2. Separation and purification

[0090] (1) Pretreatment of macroporous adsorption resin

[0091] The AB-8 macroporous adsorption resin used in this product has been pre-treated when purchased. When used for the first time, 125g of AB-8 macroporous adsorption resin is loaded into the macroporous adsorption resin column, and the resin column is eluted with 95% ethanol, which is about 2 times the volume of the macroporous adsorption resin column, at a flow rate of 2BV / h. After the elution is completed, continue to soak for more than 12 hours for standby use. Before loading, open the purified water and control the flow rate to 2BV / h to balance the outflow and addition. Elute with purified water until the effluent has no ethanol smell and the alcohol meter has no ethanol.

[0092] (2) Adsorption, elution and collection

[0093] Take 110g of the supernatant and pass it through a 125g AB-8 macroporous adsorption resin column at a flow rate of about 1BV / h. First, use 4 times the weight of the resin to elute the impurities with a flow rate of 1.5BV / h, and discard the water. Then use 80% ethanol 8 times the weight of the resin to desorb the flavonoids at a flow rate of 1.0BV / h, collect the eluate, control the vacuum degree at -0.05~-0.09MPa, and the temperature at 60~80℃, and decompress and concentrate the alcohol eluate to a relative density of about 1.08~1.15 (65~75℃), and obtain the purified thick paste of Pueraria lobata and Epimedium.

[0094] 3. Drying

[0095] Put the purified thick paste of Pueraria root and Epimedium into a vacuum drying oven, set the vacuum degree to -0.06~-0.09MPa, dry it at a temperature below 60℃ for about 26 hours, crush it, and pass it through a 60-mesh sieve to obtain the extract of Pueraria root and Epimedium. Determine the content of total flavonoids, puerarin, icariin, and water-soluble components.

[0096] 4. Treatment of macroporous adsorption resin after use

[0097] Treatment of resin during continuous production: Add about 1.5 times the volume of the macroporous adsorption resin column and soak for more than 4 hours in 95% ethanol, wash the column with purified water, and elute until the effluent has no ethanol smell and the alcohol meter measures no ethanol, then it can be used again.

[0098] Treatment of resin in non-continuous production: Add about 1.5 times the volume of the macroporous adsorption resin column and immerse it in 95% ethanol to prevent microbial growth and contamination. Before reuse, elute with purified water until the effluent has no ethanol smell and the alcohol meter measures no ethanol.

[0099] Example 2 Separation and purification of active ingredients from a composition containing Pueraria root and Epimedium

[0100] The difference from Example 1 is that in 2. (2) the adsorption, elution and collection steps, the flow rate of water elution is 3 BV / h, and the flow rate of 80% ethanol elution is 1.0 BV / h.

[0101] Example 3 Separation and purification of active ingredients from a composition containing Pueraria root and Epimedium

[0102] The difference from Example 1 is that in 2. (2) the adsorption, elution and collection steps, the flow rate of water elution is 4 BV / h, and the flow rate of 80% ethanol elution is 1.0 BV / h.

[0103] Example 4 Separation and purification of active ingredients from a composition containing Pueraria root and Epimedium

[0104] The difference from Example 1 is that in 2. (2) the adsorption, elution and collection steps, the flow rate of water elution is 1.5 BV / h, and the flow rate of 80% ethanol elution is 1.5 BV / h.

[0105] Comparative Example 1 Separation and purification of active ingredients in a composition containing Pueraria root and Epimedium

[0106] The difference from Example 1 is that in 2. (2) the adsorption, elution and collection steps, the flow rate of water elution is 1.5 BV / h, and the flow rate of 80% ethanol elution is 2.0 BV / h.

[0107] Comparative Example 2 Separation and purification of active ingredients in a composition containing Pueraria root and Epimedium

[0108] The difference from Example 1 is that in 2. (2) the adsorption, elution and collection steps, the flow rate of water elution is 1.5 BV / h, and the flow rate of 80% ethanol elution is 3.0 BV / h.

[0109] Content determination

[0110] 1. Determination of total flavonoids content

[0111] The total flavonoids content was determined according to the UV-visible spectrophotometry (General Rule 0401 of the Chinese Pharmacopoeia 2020 Edition).

[0112] Preparation of reference solution: Take an appropriate amount of puerarin reference substance, weigh it accurately, and add ethanol to make a 1 ml solution containing 0.1 mg.

[0113] Preparation of standard curve: accurately measure 0.2ml, 0.4ml, 0.6ml, 0.8ml, and 1.0ml of the reference solution, place them in 10ml volumetric flasks, dilute to the mark with 80% ethanol, and shake well. Use the corresponding reagent as blank, measure the absorbance at a wavelength of 250nm according to the UV-visible spectrophotometry (General Rules 0401 of the 2020 edition of the Chinese Pharmacopoeia), and draw a standard curve with absorbance as the ordinate and concentration as the abscissa.

[0114] Determination method: Take about 15 mg of the Pueraria root and Epimedium extracts prepared in Examples 1 to 4 and Comparative Examples 1 to 2, accurately weigh, place in a 50 ml volumetric flask, add an appropriate amount of 80% ethanol, shake to dissolve, add 80% ethanol to the scale, shake well, and filter. Accurately measure 1.0 ml of the filtrate, place in a 25 ml volumetric flask, add 80% ethanol to the scale, shake well. According to the method under the preparation of the standard curve, from "taking the corresponding reagent as blank", determine the absorbance according to the law, read the amount of puerarin in the test solution from the standard curve, calculate, and obtain.

[0115] The calculation formula is as follows:

[0116] ;

[0117] .

[0118] 2. Puerarin content determination method

[0119] The puerarin content was determined according to the high performance liquid chromatography method (General Rule 0512 of the 2020 edition of the Chinese Pharmacopoeia).

[0120] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel as filler; acetonitrile-0.5% phosphoric acid solution (10:90) as mobile phase; detection wavelength of 250nm; column temperature of 45℃. The theoretical plate number calculated based on the puerarin peak should be no less than 2500.

[0121] Preparation of reference solution: Take an appropriate amount of puerarin reference substance, weigh it accurately, add ethanol to dissolve it, and then add 50% ethanol to dilute it to make a solution containing 80 μg per 1 ml.

[0122] Preparation of the test solution: Take about 20 mg of the Pueraria lobata and Epimedium extracts prepared in Examples 1 to 4 and Comparative Examples 1 to 2, accurately weigh, place in a 50 ml volumetric flask, add an appropriate amount of 80% ethanol, ultrasonically treat for 30 minutes (power 250 W, frequency 40 kHz), cool, add 80% ethanol to dilute to the scale, shake well, filter with a microporous filter membrane (0.45 μm), and take the filtrate to obtain.

[0123] Determination method: Accurately pipette 5 μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0124] The calculation formula is as follows:

[0125] ;

[0126] .

[0127] 3. Determination of Epimedium Content

[0128] The method for determining the content of Epimedium is according to the high performance liquid chromatography method (General Rule 0512 of the 2020 edition of the Chinese Pharmacopoeia).

[0129] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel as filler; acetonitrile-water (27:73) as mobile phase; detection wavelength at 270nm; column temperature at 30°C. The theoretical plate number calculated based on the icariin peak should be no less than 1500.

[0130] Preparation of reference solution: Take an appropriate amount of icariin reference substance, weigh accurately, and add methanol to prepare a solution with a concentration of 0.154 mg / ml.

[0131] Preparation of the test solution: Take 70 mg of the Pueraria lobata and Epimedium extracts prepared in Examples 1 to 4 and Comparative Examples 1 to 2, accurately weigh, place in a stoppered conical flask, accurately add 10 ml of 80% ethanol, seal, weigh, and ultrasonically treat for 30 minutes (power 250 W, frequency 40 kHz), take out, cool, weigh again, make up the lost weight with 80% ethanol, shake well, filter with a microporous filter membrane (0.45 μm), and take the filtrate to obtain.

[0132] Determination method: Accurately pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0133] The calculation formula is as follows:

[0134] ;

[0135] .

[0136] 4. Determination of water-soluble components

[0137] The method for determining water-soluble components is determined by referring to the cold extraction method of water-soluble extracts (General Rules 2201 of the Chinese Pharmacopoeia 2020 Edition).

[0138] Take 0.1 g of the Pueraria lobata and Epimedium extracts prepared in Examples 1 to 4 and Comparative Examples 1 to 2, place 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° C. to constant weight, and weigh the dry weight.

[0139] .

[0140] The measured contents of total flavonoids, puerarin, icariin and water-soluble components are shown in Table 1 below. Figure 2 The total flavonoid content trend diagram obtained in Examples 1 to 4 and Comparative Examples 1 to 2 is shown in FIG. Figure 3 The puerarin content trend diagram obtained in Examples 1 to 4 and Comparative Examples 1 to 2 is shown in FIG. Figure 4 This is a trend chart of icariin content obtained in Examples 1 to 4 and Comparative Examples 1 to 2, Figure 5 The following is a trend chart of the water-soluble component content obtained in Examples 1 to 4 and Comparative Examples 1 to 2.

[0141] Table 1 Content changes at different elution speeds

[0142]

[0143] From Table 1 and Figure 2~Figure 5 It can be seen that the separation and purification method provided can effectively separate and purify the active ingredients in Pueraria Epimedium. When the ethanol elution flow rate is fixed, different water elution flow rates have no significant effect on total flavonoids, icariin, and puerarin, but have a significant effect on the content of water-soluble components. When the water elution flow rate is 1.5BV / h~3BV / h, the effect is best, and when it is 4BV / h, the effect is poor; when the water elution flow rate is fixed, the ethanol elution rate is 1BV / h~1.5BV / h, the effect is best, and when it is 2BV / h~3BV / h, the effect is poor.

[0144] The whole method is easy to operate, low in cost, and has a stable and reliable process, and is suitable for large-scale production.

[0145] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described 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.

[0146] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims, and the description and drawings may be used to interpret the content of the claims.

Claims

1. A method for separating and purifying active ingredients in a composition containing Pueraria lobata and Epimedium, characterized in that: The method comprises the following steps: extracting kudzu root medicinal material and epimedium medicinal material by using a first alcohol solvent to prepare an extract; The extract is passed through a macroporous adsorption resin, and eluted with water and a second alcohol solvent in sequence, and the eluate after elution with the second alcohol solvent is collected; wherein, the flow rate of elution with water is 1.5BV / h~3BV / h, and the flow rate of elution with the second alcohol solvent is 1BV / h~1.5BV / h.

2. The method according to claim 1, characterized in that The pore size of the macroporous adsorption resin is 0.3 mm to 1.2 mm; Optionally, the macroporous adsorption resin comprises AB-8 macroporous adsorption resin.

3. The method according to claim 1 or 2, characterized in that: The flow rate of the extract through the macroporous adsorption resin is 0.8 BV / h~1.5 BV / h.

4. The method according to any one of claims 1 to 3, characterized in that The second alcohol solvent is an ethanol aqueous solution with a concentration of 60% (v / v) to 90% (v / v).

5. The method according to any one of claims 1 to 4, characterized in that: In the step of eluting with water, the mass ratio of the water to the macroporous adsorption resin is (3-5):

1.

6. The method according to any one of claims 1 to 5, characterized in that In the step of eluting with the second alcohol solvent, the mass ratio of the second alcohol solvent to the macroporous adsorption resin is (6-10):

1.

7. The method according to any one of claims 1 to 6, characterized in that: Meet one or more of the following characteristics: In the step of extracting the kudzu root and epimedium medicinal materials using the first alcohol solvent, the first alcohol solvent is an ethanol aqueous solution with a concentration of 60% (v / v) to 90% (v / v); Extracting a mixture containing Pueraria root medicinal material and Epimedium medicinal material by using a first alcohol solvent; In the step of extracting the kudzu root medicinal material and the epimedium medicinal material using the first alcohol solvent, the mass ratio of the kudzu root medicinal material to the epimedium medicinal material is (1-3): (1-3).

8. The method according to any one of claims 1 to 7, characterized in that: Meet one or more of the following characteristics: The extract prepared by concentrating and drying the eluate has a total flavonoid content of 48 wt% to 65 wt%, wherein icariin is 1 wt% to 4.8 wt%, and puerarin is 16.4 wt% to 26.6 wt%; The extract prepared by concentrating and drying the eluate has a water-soluble component content of 64 wt % to 67 wt %.

9. A kudzu root and epimedium extract, characterized in that: The method is prepared by any one of claims 1 to 8.

10. The Pueraria lobata and Epimedium extract according to claim 9, characterized in that Meet one or more of the following characteristics: The total flavonoid content is 48wt%~65wt%, of which icariin is 1wt%~4.8wt% and puerarin is 16.4wt%~26.6wt%; The water-soluble component content is 64wt%~67wt%.

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