WO2019241947A1 - Method of separating and purifying icariin from epimedium extract - Google Patents

Abstract

Disclosed is a method of separating and purifying icariin from Epimedium extract, relating to the technical field of separating and purifying a single component from a plant extract, and is intended to address the issues of complicated processing, high preparation costs, and environmental pollution in existing methods. The method comprises: 1) blending an Epimedium extract with an aqueous acetone solution having a volume fraction of 88%-96%; 2) filtering and collecting a filter cake, and then dissolving the filter cake in an aqueous ethanol solution having a volume fraction of 50%-75%; 3) after the dissolution is completed, filtering and collecting the filtrate, and concentrating the filtrate to remove the ethanol, thereby obtaining a concentrated liquid; 4) at a first temperature, adding acetone to the concentrated liquid, mixing uniformly, and lowering the temperature to a second temperature after sufficient dissolution to perform crystallization; 5) after the crystallization is completed, filtering and collecting a filter cake, and letting the filter cake dry to obtain crude icariin. The method can provide a single component having a icariin content of more than 98% that satisfies the high purity requirement of injection.

Description

Method for separating and purifying icariin from extract of epimedium Technical field

The invention relates to the technical field of isolation and purification of monomers in plant extracts, and in particular to a method for separating and purifying icariin from an extract of epimedium.

Background technique

Epimedium extract refers to the product obtained by extracting the main active ingredient flavonoids from the herb material of Epimedium, using modern Chinese medicine extraction technology, including icariin, Haemidine A, Haemidine B, Haem There are more than 200 ingredients such as Ding C, Fructus A, Fructus B, and Breviin, also known as Epimedium Total Flavones.

Icariin is the component with the largest content in the extract of Epimedium, and it is also a very important medicinal ingredient in Epimedium medicinal materials. It has the function of increasing cardio-cerebral blood flow, promoting hematopoietic function, immune function and bone metabolism, Its structural formula is shown in the figure below.

Icariin monomers are mostly prepared by using the method of separating and purifying the extract from icariin. At present, the separation and purification methods disclosed in the prior art include macroporous resin adsorption, column chromatography, and chromatography. Method and solvent extraction method, but these methods have the disadvantages of complicated process, high preparation cost and environmental pollution. For example, Chinese patent application CN103288902A discloses a preparation method for extracting icariin from epimedium. In this method, macroporous resin is used to separate and purify icariin extract. In the process, different concentrations of ethanol solutions are used for gradients. Elution, cumbersome operation, and the production of a large amount of eluent, the concentration process is time-consuming and energy-consuming, while a large amount of wastewater will be generated. Another example is Chinese patent application CN101607976A, which discloses a method for preparing icariin. The method firstly repeatedly extracts the icariin extract with petroleum ether, chloroform, ethyl acetate and n-butanol until it is almost colorless, and then recovers the extraction solvent. Separation of silica gel column chromatography and polyamide column chromatography in sequence has extremely complicated processes and uses a variety of toxic organic solvents, which is not conducive to environmental protection.

technical problem

The purpose of the present invention is to provide a new method for separating and purifying icariin from the extract of Epimedium, which aims at solving the technical problems of complicated process, high preparation cost and environmental pollution existing in the existing method.

Technical solutions

In order to achieve the above purpose, the inventors conducted a lot of experimental explorations. The research found that although the structure and polarity of icariin and many other components in the extract of icariin are very similar and difficult to separate, these components are in different solvents. However, the solubility is quite different. For example, the solubility of Chaoyangding A, B, C, etc. in water is better than that of icariin. The solubility of brevisin and acetone is better in acetone, while icariin is in 70% acetone aqueous solution and 70% ethanol. Solubility is greatest in aqueous solutions, etc. So using this discovery, the inventors have developed a new method for isolating and purifying icariin from the extract of epimedium, including the following steps: 1) using a volume fraction of 88% -96% acetone in water to extract the epimedium Beating; 2) filtering, filtering to remove impurities such as Breviin Ⅰ, collecting the filter cake, dissolving the filter cake with a volume fraction of 50% -75% ethanol aqueous solution; 3) after dissolving, filtering, filtering off insoluble matters, collecting the filtrate, The filtrate is concentrated to remove ethanol to obtain a concentrated solution; 4) At the first temperature, acetone is added to the concentrated solution and mixed uniformly. After being fully dissolved, the temperature is lowered to the second temperature for crystallization; 5) After the crystallization is completed, Filter and collect the filter cake. After the filter cake is dried, the crude icariin is obtained.

The separation and purification method provided by the present invention is suitable for separating and purifying icariin from the icariin extract whose content range is greater than or equal to 10%.

The beating in step 1) above is specifically directed to adding a certain solvent having a lower solubility to the target substance and a higher solubility to other substances to the solid substance, and dissolving other unwanted substances in the solvent by stirring, and the target substance and The process in which the solvent is suspended.

Filtration in the present invention refers to a process in which solids and liquids in a solution are separated by a physical method. Common filtration methods are applicable to the present invention, including atmospheric pressure filtration, reduced pressure filtration, centrifugal filtration, and the like.

The concentration in the present invention refers to a process in which a solvent is used to evaporate a solvent to increase the concentration of a solution, and includes a vacuum distillation method, an ultrafiltration method, a dialysis method, an adsorption method, and a freeze-drying method.

Preferably, the concentration of the acetone aqueous solution in the step 1) is 89% -92% by volume.

More preferably, the concentration of the acetone aqueous solution in step 1) is 90% by volume.

Preferably, the amount of the acetone aqueous solution in step 1) is 2-10 ml / g epimedium extract.

More preferably, the amount of the acetone aqueous solution in step 1) is 5 ml / g epimedium extract.

Preferably, the dissolution process of controlling step 2) is performed at a temperature of 50-70 ° C.

More preferably, the dissolution process of controlling step 2) is performed at a temperature of 55-65 ° C.

More preferably, the dissolution process of controlling step 2) is performed at a temperature of 60 ° C.

In order to accelerate the dissolution, the dissolution process in step 2) is preferably performed under stirring.

Preferably, the concentration of the ethanol aqueous solution in the step 2) is 60% -75% by volume.

More preferably, the concentration of the ethanol aqueous solution in step 2) is 70% by volume.

Preferably, the amount of the ethanol aqueous solution in step 2) is 3-10 ml / g filter cake weight.

More preferably, the amount of the ethanol aqueous solution in step 2) is 5 ml / g filter cake weight.

Preferably, in step 3), after the dissolution is completed, the solution is filtered while hot.

Preferably, in the step 3), the volume of the concentrated liquid is 1/5 of the volume of the filtrate.

Preferably, the concentration process of step 3) is concentrated using a reduced pressure distillation method.

Preferably, the concentration process of controlling step 3) is performed at a temperature below 80 ° C.

In order to improve the concentration efficiency, more preferably, the concentration process of step 3) is controlled at a temperature of 60-80 ° C.

Preferably, the added amount of acetone in the step 4) is calculated by volume ratio, and acetone: concentrate = 3: 7 ~ 3: 2.

More preferably, the added amount of acetone in step 4) is calculated by volume ratio, and acetone: concentrated liquid = 1: 1 ~ 3: 2.

Preferably, the first temperature is 50-70 ° C.

More preferably, the first temperature is 55-65 ° C.

More preferably, the first temperature is 60 ° C.

Preferably, the second temperature is room temperature.

More preferably, the second temperature is 20-25 ° C.

More preferably, the crystallization process in step 4) is performed under stirring, and the time for which the crystallization is maintained at the second temperature is at least 1 h.

Further, the above method further includes the following steps: 6) at a third temperature, dissolving the crude icariin obtained in step 5) in a 30% -100% acetone aqueous solution by volume; 7) after the solution is fully dissolved, the temperature is Reduce the temperature to the fourth temperature for crystallization; 8) After the crystallization is completed, filter and collect the filter cake. After the filter cake is dried, a pure icariin is obtained.

Preferably, the concentration of the acetone aqueous solution in step 6) is 30% -60% by volume.

More preferably, the concentration of the acetone aqueous solution in the step 6) is 50% -60% by volume.

Preferably, the amount of the acetone aqueous solution in the step 6) is 25-35 ml / g crude icariin dry weight.

More preferably, the amount of the acetone aqueous solution in step 6) is 29-32 ml / g crude icariin dry weight.

More preferably, the amount of the acetone aqueous solution in step 6) is 30 ml / g crude icariin dry weight.

In order to accelerate the dissolution, the dissolution process of step 6) is preferably performed under stirring.

Preferably, the third temperature is 50-70 ° C.

More preferably, the third temperature is 55-65 ° C.

More preferably, the third temperature is 60 ° C.

Preferably, the fourth temperature is room temperature.

More preferably, the fourth temperature is 20-25 ° C.

More preferably, the crystallization process in step 7) is performed with stirring, and the time for which the crystallization is kept at the fourth temperature is at least 1 h.

The room temperature referred to in the present invention is the room temperature described in the examples of the 2015 Chinese Pharmacopoeia, that is, normal temperature, and refers to 10-30 ° C.

The drying treatment in the present invention includes normal pressure drying, reduced pressure drying, freeze drying, spray drying and the like.

Beneficial effect

Compared with the existing method for separating and purifying icariin from the extract of Epimedium, the method provided by the present invention has the following advantages:

1. The process is simple and easy to operate, the cost is low, and the green is pollution-free;

2. The purity of the icariin monomer obtained by applying the method of the present invention is at least 90% or more;

3. A more preferred process using the method of the present invention can obtain an icariin monomer with a content of more than 98%, which is sufficient to meet the high purity requirements of injections, and its yield is not less than 80%.

BRIEF DESCRIPTION OF THE DRAWINGS

1 is an HPLC chart of Epimedium extract used in Examples 1 and 2 of the present invention;

FIG. 2 is an HPLC chart of a pure icariin obtained in Example 2 of the present invention.

Best Mode of the Invention

The preferred process of the method provided by the present invention includes the following steps:

1) Beating Epimedium extract with a volume fraction of 88% -96% acetone aqueous solution, the amount of 88% -96% acetone aqueous solution is 2-10ml / g epimedium extract; 2) filtering, collecting filter cake, Dissolve the filter cake with a volume fraction of 50% -75% ethanol aqueous solution at a temperature of 50-70 ° C, and stir while dissolving. The amount of 50% -75% ethanol aqueous solution is 3-10ml / g filter cake weight; 3) After dissolution, Filtrate while hot, collect the filtrate, and concentrate the filtrate under reduced pressure at 80 ° C to 1/5 of the original volume to obtain a concentrated solution. 4) At 50-70 ° C, use the volume ratio as acetone: concentrate. = 3: 7 ~ 3: 2 Add acetone to the concentrate and stir to mix the two evenly. After fully dissolved, lower the temperature to room temperature and crystallize. Keep the crystals stirred at room temperature for at least 1h; 5 ) After the crystallization is completed, filter and collect the filter cake. The filter cake is dried to obtain the crude icariin.

If an icariin product with a further increased content is to be obtained, the method of the present invention further includes the following steps: 6) dissolving the crude icariin obtained in step 5) in a volume fraction of 30% -100% acetone at a temperature of 50-70 ° C In the aqueous solution, stir while dissolving. The amount of acetone aqueous solution is 25-35ml / g crude icariin dry weight; 7) After it is fully dissolved, reduce the temperature to room temperature and crystallize. It is 1h; 8) After the crystallization is completed, filter, collect the filter cake, and dry the filter cake to obtain the pure icariin.

The filtration in the above process uses suction filtration. The specific operation is as follows: firstly insert not less than 400 mesh filter cloth or filter paper into a Buchner funnel, and then connect the Buchner funnel to a suction filter bottle connected to a vacuum pump. Then pour the solution to be filtered into a Buchner funnel and turn on the vacuum pump for suction filtration until the filtrate and cake are fully separated.

Embodiments of the invention

The present invention will be further described in detail with reference to specific embodiments and drawings. The following embodiments are explanations of the present invention, and the present invention is not limited to the following embodiments.

The raw materials and reagents used in the following examples were purchased from the market unless otherwise specified.

Example 1

Optimization experiment of process conditions of the method of the present invention

1. Determine the concentration of acetone aqueous solution for beating

(1) Prepare an acetone aqueous solution of equal gradient concentration of 0-100% by volume, and test the solubility of each concentration of acetone aqueous solution to icariin and scutellarin Ⅰ at room temperature. The test results are shown in Table 1.

Table 1

Acetone solution concentration	Icariin solubility (mg / ml)	Solubility of Breguet I (mg / ml)
0	0.49527	1.057788
10%	0.75283	0.145435
20%	1.15249	0.681134
30%	1.89396	3.482554
40%	2.68629	12.61701
50%	6.44322	27.04883
60%	9.08015	101.0067
70%	9.29089	167.6433
80%	8.84707	190.8692
90%	2.74241	272.0951
100%	0.93333	64.83548

(2) Beat the Epimedium extract with a volume fraction of 88.6% -96.5% acetone in water. The Epimedium extract is selected from the Epimedium extract produced by Xi'an Huibo Biological Technology Co., Ltd., which contains 20% Icariin has a purity of 40%. Its HPLC chart is shown in Figure 1. After pulping, it is filtered. The content, purity and yield of icariin in the obtained filter cake are shown in Table 2.

Table 2

Acetone solution concentration	purity	content	Yield
88.6%	92.44%	73.11%	74.0%
88.9%	91.99%	78.71%	80.2%
89.9%	91.84%	77.65%	86.5%
90.8%	90.76%	77.13%	88.4%
92.7%	89.90%	65.32%	86.2%
94.6%	87.89%	58.09%	83.8%
96.5%	84.43%	59.55%	77.4%

Discussion: The purpose of beating is to retain icariin and remove impurities as much as possible. Breguet Ⅰ is the material with the highest proportion of impurities and difficult to remove. Therefore, the acetone aqueous solution should be selected to have low solubility and icariin. Concentration with high solubility for Breguet I. At the same time, the purity, content and yield of icariin in the filter cake obtained after beating and filtering should also be considered. Based on the data in Tables 1 and 2, it is finally determined that the concentration range of the acetone aqueous solution for beating is 88% -96% by volume, preferably 89% -92%, and most preferably 90%.

2. Determine the concentration of ethanol solution for dissolving the filter cake

At room temperature, 0% to 100% volume fractions of ethanol solutions with equal gradient concentrations were prepared, and the solubility of each concentration solution in icariin and brevin Ⅰ was tested. The test results are shown in Table 3.

table 3

Ethanol concentration	Icariin solubility (mg / ml)	Solubility of Breguet I (mg / ml)
0	0.36969	——
10%	0.43688	——
20%	0.58151	——
30%	1.13148	——
40%	2.04397	4.474874
50%	3.64252	7.673487
60%	3.77094	10.91854
70%	4.39277	18.03678
80%	3.15803	21.82047
90%	1.69284	62.90661
100%	0.6477	4.474874

Discussion: The purpose of dissolving the filter cake with an aqueous ethanol solution is to further remove impurities and increase the content during subsequent crystallization. Therefore, the ethanol aqueous solution should be selected to have a high solubility for icariin and a low solubility for brevin Ⅰ. At the same time, the yield during subsequent crystallization should also be considered. Based on the data in Table 3, it is finally determined that the concentration range of the ethanol aqueous solution for dissolving the filter cake is 50% -75% by volume, preferably 60% -75%, and most preferably 70%.

3. Determine the crystallization conditions

(1) Through the solubility test of icariin in aqueous solutions of methanol, ethanol and acetone, the aqueous solution of acetone was finally selected as the best organic solvent in the crystallization process.

(2) In an acetone aqueous solution, the temperature of the solution has a greater effect on the solubility of icariin. The idea of the method of the present invention is to dissolve icariin in an acetone aqueous solution in a temperature range with large solubility, and then pass The temperature is adjusted to a range with lower solubility, so that the icariin crystals are precipitated from the acetone aqueous solution, and at the same time, the impurities are not precipitated as much as possible, thereby achieving the effect of separation and purification. According to experimental measurements, the solubility of icariin in acetone aqueous solution increases with increasing temperature and decreases with decreasing temperature. Among them, the solubility starts to increase slightly at 30 ° C, and begins to increase significantly at 50 ° C. increase. In the temperature range below 50 ° C, the solubility is generally relatively small, and the demand for acetone aqueous solution is large during dissolution, which is not conducive to cost control; and when the temperature is too high, the acetone aqueous solution is easy to boil, and reflux equipment is required. Therefore, it was finally determined that the temperature for dissolving icariin is 50-70 ° C, preferably 55-65 ° C, and most preferably 60 ° C; the temperature during crystallization is room temperature, that is, 10-30 ° C, preferably 20-25 ° C .

(3) Determination of the concentration of acetone aqueous solution for crystallization

Prepare an acetone aqueous solution of equal gradient concentration with a volume fraction of 30% -100%, and take an equal amount of dried icariin at 55-65 ° C and stir and dissolve in the prepared acetone aqueous solution of each concentration. After being fully dissolved, the temperature was lowered to 20-25 ° C, and the crystals were precipitated while maintaining the temperature at 20-25 ° C. After 1 hour, the filter cake was collected, and the content and yield of icariin in each concentration of acetone aqueous solution were measured. ， 结果 见表 4。 The results are shown in Table 4.

Table 4

Acetone solution concentration	content	Yield
30%	96.92%	86.4%
40%	97.78%	84.7%
50%	98.27%	82.2%
60%	98.93%	80.8%
70%	99.08%	74.6%
80%	97.96%	76.3%
90%	96.32%	83.8%
100%	94.85%	87.3%

Discussion: The purpose of crystallization is to increase the product content as much as possible while reducing product loss. Based on the data in Table 4, the concentration range of the acetone aqueous solution for crystallization is 30% -100%, preferably 30% -60%. Most preferred is 50% -60%.

Example 2

Take 1000g of Epimedium extract produced by Xi'an Huibo Biotechnology Co., Ltd. (Among them, the content of Icariin is 20%, the purity is 40%, and the HPLC chart is shown in Figure 1), and 5000mL 90% acetone aqueous solution at room temperature Beat for 30min, filter, and dissolve the filter cake with 1000mL of 70% ethanol aqueous solution at 60 ° C for 1h. The insolubles are filtered off. The filtrate is concentrated under reduced pressure to remove the ethanol. After concentration, the solution is 200mL, 200mL of acetone is added, and the temperature is maintained at 60 ° C for 1h. Crystallize at 25 ° C, stir for 1h, filter, dry at 70 ° C for 8h, weigh 178.91g; filter cake again dissolve with 5400mL of 50% acetone aqueous solution at 60 ° C, stir for 1h, cool to 20-25 ° C for crystallization, stir and hold for 1h, filter, The filter cake was dried under vacuum at 50 ° C to obtain 164.72 g of icariin product. The purity was 98.22%, the content was 98.08%, and the molar yield was 82.36%.

Example 3

Take 1000g of Epimedium extract produced by Xi'an Huibo Biotechnology Co., Ltd. (Among them, the content of Icariin is 10% and the purity is 40%), beaten with 5000mL 90% acetone aqueous solution at room temperature for 30min, filter, and use 500mL filter cake Dissolve 70% ethanol in water at 60 ℃ for 1h, filter off the insoluble matter, and concentrate the filtrate under reduced pressure to remove ethanol. After concentration, the solution is 100mL, 150mL of acetone is added, the temperature is maintained at 60 ℃ for 1h, the temperature is reduced to 20-25 ℃, and the crystal is stirred for 1h, filtered Dry at 70 ° C for 8h and weigh 88.7g; filter cake was dissolved again with 2600ml 60% acetone aqueous solution at 60 ° C for 1h, cooled to 20-25 ° C for crystallization, stirred and maintained for 1h, filtered, and filtered cake was dried at 50 ° C under vacuum to obtain 80.12g Icariin product has a purity of 98.67%, a content of 98.49%, and a molar yield of 80.12%.

Claims (10)

1. A method for isolating and purifying icariin from an extract of epimedium, which comprises the following steps: 1) beating the epimedium extract with a volume fraction of 88% -96% acetone aqueous solution; 2) filtering, collecting and filtering Cake, dissolve the filter cake with 50% -75% ethanol solution by volume; 3) After dissolution, filter, collect the filtrate, concentrate the filtrate to remove the ethanol to obtain a concentrated solution; 4) At the first temperature, add to the concentrated solution Acetone was mixed uniformly, and after being fully dissolved, the temperature was lowered to the second temperature for crystallization; 5) After the crystallization was completed, filtration was performed, and the filter cake was collected. The filter cake was dried to obtain the crude icariin.
2. The method for separating and purifying icariin from an extract of epimedium according to claim 1, wherein the amount of the acetone aqueous solution in step 1) is 2-10 ml / g of epimedium extract.
3. The method for separating and purifying icariin from an extract of epimedium according to claim 1, wherein the amount of the ethanol aqueous solution in step 2) is 3-10 ml / g of filter cake weight.
4. The method for separating and purifying icariin from the extract of epimedium according to claim 1, characterized in that: controlling the dissolution process of step 2) is performed at a temperature of 50-70 ° C, and said step 3) After the dissolution is complete, filter while still hot.
5. The method for separating and purifying icariin from the extract of epimedium according to claim 1, characterized in that the concentration process of step 3) is controlled at a temperature below 80 ° C.
6. The method for separating and purifying icariin from an extract of epimedium according to claim 1, characterized in that: the added amount of acetone in step 4) is calculated by volume ratio, and acetone: concentrated liquid = 3: 7 ~ 3: 2.
7. The method for separating and purifying icariin from an extract of epimedium according to claim 1, wherein in the step 4), the first temperature is 50-70 ° C, and the second temperature It's room temperature.
8. The method for separating and purifying icariin from an extract of icariin according to claim 1, further comprising the step of: 6) dissolving the crude icariin obtained in step 5) at a third temperature. In a volume fraction of 30% -100% acetone aqueous solution; 7) after being fully dissolved, reduce the temperature to a fourth temperature for crystallization; 8) after crystallization is completed, filter, collect the filter cake, and filter cake is dried Get pure icariin.
9. The method for separating and purifying icariin from an extract of icariin according to claim 8, characterized in that the amount of the acetone aqueous solution in step 6) is 25-35ml / g crude icariin dry weight.
10. The method for separating and purifying icariin from an extract of epimedium according to claim 8, wherein the third temperature is 50-70 ° C, and the fourth temperature is room temperature.