阅读说明：本技术 一种高含量葛根总黄酮及其制备方法 (High-content pueraria flavonid and preparation method thereof ) 是由 于华忠 宋科 郭婕 吴海顺 李伟业 于 2020-12-15 设计创作，主要内容包括：本申请提供一种高含量葛根总黄酮及其制备方法,依次通过超声波逆流提取、树脂纯化、结晶、干燥工艺组合应用,提取得到黄酮含量高的葛根提取物,与常见的葛根提取物提取工艺相比,本申请制得的葛根总黄酮含量达到95％以上,且工艺简单、生产周期短,产品颜色浅,生产成本低,易实现大规模生产,适用于工业化大生产的高含量的葛根总黄酮,可广泛用于保健食品(用于解酒、丰胸、延缓女性更年期、糖尿病引起的并发症等)等领域。(The application provides a high-content pueraria flavonid and a preparation method thereof, the pueraria flavonid extract with high flavone content is obtained by extracting and combining ultrasonic countercurrent extraction, resin purification, crystallization and drying processes in sequence, and compared with the common pueraria flavonid extraction process, the prepared pueraria flavonid has the advantages of high content of more than 95 percent, simple process, short production period, light product color, low production cost and easy realization of large-scale production, is suitable for industrial mass production, is high-content pueraria flavonid, and can be widely used in the fields of health-care food (for relieving alcoholism, enlarging breast, delaying female climacteric, complications caused by diabetes and the like) and the like.)

1. A preparation method of high-content pueraria flavonid is characterized by comprising the following steps: the method comprises the following steps:

s101, ultrasonic continuous countercurrent extraction: crushing a kudzu root medicinal material, adding a 3-5 times volume fraction of 30-60% ethanol solution, performing ultrasonic continuous countercurrent extraction at 50-80 ℃, and discharging for 1-2 hours from feeding;

s102, concentration: concentrating the extracting solution until the liquid medicine has no alcohol smell, and adding 1-3 times of hot water with volume of 60-80 ℃;

s103, resin separation: loading the concentrated solution onto macroporous resin, washing with water by 1-3 times of column volume to remove impurities, eluting with ethanol with the volume fraction of 10-45% at the temperature of 30-50 ℃, and collecting an eluent containing flavone through ultraviolet online monitoring;

s104, crystallization: concentrating the eluent containing flavone until the specific gravity of the liquid medicine is 1.1-1.2, cooling to 2-5 ℃, standing and crystallizing;

s105, drying: and drying the crystal to obtain the crystal.

2. The method for preparing high content of pueraria flavonid according to claim 1, which is characterized in that: in the step S101, the radix puerariae is wild radix puerariae and is crushed to 1-2 mm.

3. The method for preparing high content of pueraria flavonid according to claim 1, which is characterized in that: in step S101, the countercurrent extraction is leaching twice, and the ultrasonic frequency is 20 KHz-40 KHz.

4. The method for preparing high content of pueraria flavonid according to claim 1, which is characterized in that: in the step S102 and the step S104, the concentration is performed under reduced pressure, and the reduced pressure concentration condition is 60-80 ℃ and 0.07-0.08 MPa.

5. The method for preparing high content of pueraria flavonid according to claim 1, which is characterized in that: in step S102, the concentration is performed until the liquid medicine has no alcohol smell, specifically until the alcohol concentration is less than 5%.

6. The method for preparing high content of pueraria flavonid according to claim 1, which is characterized in that: in step S103, a deuterium lamp ultraviolet detector with the wavelength of 200 nm-400 nm is adopted for ultraviolet online monitoring.

7. The method for preparing high content of pueraria flavonid according to claim 1, which is characterized in that: in step S103, the macroporous resin is selected from any one of LSA-21 macroporous resin, D101 macroporous resin, HPD-300 macroporous resin and DM-301 macroporous resin.

8. The method for preparing high content of pueraria flavonid according to claim 1, which is characterized in that: in step S105, the crystal is dried by spray drying under the following conditions: the air inlet temperature is 110-120 ℃, the air outlet temperature is 60-85 ℃, the feeding temperature is 60 ℃, and the feeding speed is 10 mL/min.

9. A high-content pueraria flavonid is characterized in that: the preparation method of high-content pueraria flavonid according to any one of claims 1 to 8.

10. The high content pueraria flavonid as claimed in claim 1, wherein: the content of radix Puerariae total flavone is greater than 95%.

Technical Field

The invention belongs to the technical field of pueraria flavonid, and particularly relates to high-content pueraria flavonid and a preparation method thereof.

Background

Radix Puerariae is dried root of Pueraria lobata Ohwi of Leguminosae, and has effects of relieving muscles and fever, promoting fluid production to quench thirst, invigorating yang and relieving diarrhea, dredging meridian passage, etc. Research shows that the kudzuvine root contains rich nutrient components such as flavonoid substances, starch, protein, various trace elements, amino acid, choline derivatives and the like, wherein the kudzuvine root total flavone has very complex components and comprises isoflavone compounds such as puerarin, daidzin, daidzein, formononetin, 4, 7-diglucose daidzin, 3-methyl-puerarin, 7-xylose-puerarin and the like, so that the kudzuvine root has very high nutritional value and medicinal value.

The flavonoids are main functional components of radix Puerariae, and have antibacterial, blood circulation promoting, blood stasis removing, coronary artery blood vessel and cerebral vessels dilating, myocardial oxygen consumption reducing, myocardial contraction improving, blood circulation promoting, and immunity enhancing effects. The pueraria flavonid is mainly used for treating cardiovascular and cerebrovascular diseases such as hypertension, hyperlipidemia, migraine, coronary heart disease, myocardial infarction, angina, retinal artery occlusion, retinal vein occlusion, sudden deafness and the like at present, and replaces the traditional Chinese medicine pueraria in Chinese patent medicines. Meanwhile, the health-care food is widely applied to the fields of health-care food (for relieving alcoholism, enlarging breast, delaying female climacteric syndrome, diabetes-induced complications and the like).

In order to facilitate the administration, the high-purity pueraria flavonid product is concerned by the international market. The content of flavone in radix Puerariae is not high but 3-5%, and extraction and separation are required to obtain high-purity radix Puerariae total flavone. The pueraria flavonid is usually separated and purified by adopting methods such as solvent conventional extraction, solvent extraction, macroporous adsorption resin method, polyamide column chromatography adsorption method and the like. Different methods have advantages and disadvantages, some pueraria flavonid have low purity and yield, and some production processes have long production period. Therefore, how to use a simple, feasible and efficient extraction and separation process has important significance for realizing the industrial production of the pueraria flavonid with the content of more than 95 percent.

Disclosure of Invention

The invention aims to solve the technical problems of low content, complex process and long period of the pueraria flavonid prepared from the pueraria in the prior art, and provides a preparation method of the pueraria flavonid with high content, which prepares a pueraria extract with the flavone content of more than 95 percent by combined application of ultrasonic countercurrent extraction, resin purification, crystallization and drying processes, has simple process and short production period, and is suitable for industrial mass production.

Another object of the present application is to provide a high content of pueraria flavonid.

A preparation method of high-content pueraria flavonid comprises the following steps:

s101, ultrasonic continuous countercurrent extraction: crushing a kudzu root medicinal material, adding a 3-5 times volume fraction of 30-60% ethanol solution, performing ultrasonic continuous countercurrent extraction at 50-80 ℃, and discharging for 1-2 hours from feeding;

s102, concentration: concentrating the extracting solution until the liquid medicine has no alcohol smell, and adding 1-3 times of hot water with volume of 60-80 ℃;

s103, resin separation: loading the concentrated solution onto macroporous resin, washing with water by 1-3 times of column volume to remove impurities, eluting with ethanol with the volume fraction of 10-45% at the temperature of 30-50 ℃, and collecting an eluent containing flavone through ultraviolet online monitoring;

s104, crystallization: concentrating the eluent containing flavone until the specific gravity of the liquid medicine is 1.1-1.2, cooling to 2-5 ℃, standing and crystallizing;

s105, drying: and drying the crystal to obtain the crystal.

The application provides a preparation method of high-content pueraria flavonid, which is characterized in that the pueraria flavonid extract with high flavone content is obtained by extracting through the combined application of ultrasonic countercurrent extraction, resin purification, crystallization and drying processes in sequence.

Preferably, in the step S101, the kudzuvine root is wild kudzuvine root, and is crushed to 1-2 mm. The wild kudzu root used in the application contains rich kudzu root total flavonoids, the kudzu root is crushed to 1 to 2mm by adopting a JWFC-16 universal crusher of the mechanical factory of the spring and joy of the Henan province, the kudzu root powder prepared by crushing treatment is fine and smooth, the content of the kudzu root total flavonoids contained in the kudzu root powder is high, the extraction of the high-content kudzu root total flavonoids is facilitated, and the production period is shortened.

Preferably, in step S101, the countercurrent extraction is leaching twice, and the ultrasonic frequency is 20KHz to 40 KHz. The method has the advantages that the ultrasonic countercurrent extraction efficiency is high, the operation is carried out under the whole closed condition, the loss of the pueraria flavonid in the extraction process is favorably reduced, the activity of the pueraria flavonid in the pueraria powder can be furthest kept, and the content of the pueraria flavonid is improved. Preferably, a dynamic continuous countercurrent extractor of pharmaceutical machinery factory of Jiangsu Danyang is adopted.

Preferably, in step S102 and step S104, the concentration is performed under reduced pressure, and the reduced pressure concentration is performed at 60-80 ℃ and 0.07-0.08 MPa. Its advantages are low boiling point of solution, preventing decomposition of effective substance, high evaporating efficiency and short production period.

Preferably, in step S102, the concentration is performed until the liquid medicine has no alcohol smell, specifically until the ethanol concentration is 5% or less.

Preferably, in step S103, the ultraviolet on-line monitoring uses a deuterium lamp ultraviolet detector with a wavelength of 200nm to 400 nm. The method has the advantages of high sensitivity, wide linear range and better selectivity, and is used for extracting target flavone and improving the content of the flavone, preferably with the wavelength of 220 nm.

Preferably, in step S103, the macroporous resin is selected from any one of LSA-21 macroporous resin, D101 macroporous resin, HPD-300 macroporous resin and DM-301 macroporous resin. Used for adsorbing and removing impurities.

Preferably, in step S105, the crystal is dried by spray drying under the following conditions: the air inlet temperature is 110-120 ℃, the air outlet temperature is 60-85 ℃, the feeding temperature is 60 ℃, and the feeding speed is 10 mL/min. The drying process is rapid, and is favorable for obtaining high-content pueraria flavonid.

A high content radix Puerariae total flavone is prepared by the above preparation method.

Preferably, the content of the pueraria flavonid is more than 95 percent. The pueraria flavonid prepared by the method has the content of more than 95 percent measured by an ultraviolet-visible spectrophotometry, and the high-content pueraria flavonid can be widely used in the fields of health-care food (for dispelling the effects of alcohol, enlarging breast, delaying female climacteric syndrome, complicating diseases caused by diabetes and the like) and the like.

Compared with the prior art, the invention has the following advantages:

the application provides a preparation method of high-content pueraria flavonid, which is characterized in that the pueraria flavonid extract with high flavone content is obtained by extracting through the combined application of ultrasonic countercurrent extraction, resin purification, crystallization and drying processes in sequence.

The application provides a high-content pueraria flavonid, the content of the pueraria flavonid is up to more than 95 percent through ultraviolet-visible spectrophotometry detection, and compared with the common pueraria extract extraction process, the method only organically combines the procedures of ultrasonic continuous countercurrent extraction, column chromatography and crystallization to obtain the pueraria flavonid with high content and light color. The production cost is low, the process is simple, the large-scale production is easy to realize, and the health-care food can be widely used in the fields of health-care food (for relieving alcoholism, enlarging breast, delaying female climacteric syndrome, diabetes-induced complications and the like) and the like.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1:

a preparation method of high-content pueraria flavonid comprises the following steps:

s101, ultrasonic continuous countercurrent extraction: taking 100kg of kudzu root medicinal material, crushing the kudzu root medicinal material to 2mm, adding 3 times of 60% ethanol solution in volume fraction, performing ultrasonic continuous countercurrent extraction at 70 ℃, and adjusting the speed from feeding to discharging for 1 hour;

s102, concentration: concentrating the above extractive solution until the medicinal liquid has no alcohol smell, and adding 2 times volume of hot water of 70 deg.C;

s103, resin separation: loading the concentrated solution onto macroporous resin DM-301, washing with water 2 times the column volume to remove impurities, eluting with ethanol with volume fraction of 35% at 40 deg.C, monitoring on-line by ultraviolet, and collecting eluate containing flavone;

s104, crystallization: concentrating the eluate containing flavone until the specific gravity of the medicinal liquid is 1.15, cooling to 3 deg.C, standing, and crystallizing;

s105, drying: drying the crystal to obtain 6.2kg radix Puerariae total flavone extract.

Example 2:

a preparation method of high-content pueraria flavonid comprises the following steps:

s101, ultrasonic continuous countercurrent extraction: taking 100kg of radix puerariae medicinal material, crushing the radix puerariae medicinal material to 1.5mm, adding 3 times of 60% ethanol solution by volume, carrying out ultrasonic continuous countercurrent extraction at the temperature of 80 ℃, and adjusting the speed from feeding to discharging for 1.5 hours;

s102, concentration: concentrating the above extractive solution until the medicinal liquid has no alcohol smell, and adding 2 times volume of hot water of 70 deg.C;

s103, resin separation: loading the concentrated solution onto macroporous resin LSA-21, washing with water 1 times column volume to remove impurities, eluting with 40% ethanol at 30 deg.C, monitoring by ultraviolet on-line, and collecting eluate containing flavone;

s104, crystallization: concentrating the eluate containing flavone until the specific gravity of the medicinal liquid is 1.18, cooling to 4 deg.C, standing, and crystallizing;

s105, drying: drying the crystal to obtain 6.5kg radix Puerariae total flavone extract.

Example 3:

a preparation method of high-content pueraria flavonid comprises the following steps:

s101, ultrasonic continuous countercurrent extraction: taking 100kg of radix puerariae medicinal material, crushing the radix puerariae medicinal material to 2mm, adding 5 times of 30% ethanol solution by volume, performing ultrasonic continuous countercurrent extraction at the temperature of 60 ℃, and adjusting the speed from feeding to discharging for 1 hour;

s102, concentration: concentrating the above extractive solution until the medicinal liquid has no alcohol smell, and adding 1 volume of 80 deg.C hot water;

s103, resin separation: loading the concentrated solution onto macroporous resin HPD-300, washing with water 1 times column volume to remove impurities, eluting with ethanol with volume fraction of 45% at 50 deg.C, monitoring on-line by ultraviolet, and collecting eluate containing flavone;

s104, crystallization: concentrating the eluate containing flavone until the specific gravity of the medicinal liquid is 1.1, cooling to 2 deg.C, standing, and crystallizing;

s105, drying: drying the crystal to obtain 6.1kg radix Puerariae total flavone extract.

Example 4:

a preparation method of high-content pueraria flavonid comprises the following steps:

s101, ultrasonic continuous countercurrent extraction: taking 100kg of kudzu root medicinal material, crushing to 1mm, adding 4 times of 40% ethanol solution by volume, performing ultrasonic continuous countercurrent extraction at 50 ℃, and adjusting the speed from feeding to discharging for 2 hours;

s102, concentration: concentrating the above extractive solution until the medicinal liquid has no alcohol smell, and adding 3 times volume of 60 deg.C hot water;

s103, resin separation: loading the concentrated solution onto macroporous resin DM-301, washing with water 1 times column volume to remove impurities, eluting with ethanol with volume fraction of 10% at 30 deg.C, monitoring on-line by ultraviolet, and collecting eluate containing flavone;

s104, crystallization: concentrating the eluate containing flavone until the specific gravity of the medicinal liquid is 1.2, cooling to 5 deg.C, standing, and crystallizing;

s105, drying: drying the crystal to obtain 6.0kg radix Puerariae total flavone extract.

The product prepared by the implementation of 1-4 is subjected to the content determination of the pueraria flavonid, the determination method adopts an ultraviolet-visible spectrophotometry, and the determination steps are as follows:

1. accurately weighing 10mg of puerarin standard substance which is dried to constant mass, adding a proper amount of 50% ethanol with volume fraction for full dissolution, transferring to a 50m L volumetric flask, adding 50% ethanol with volume fraction for constant volume, and fully shaking up to obtain puerarin standard solution with certain concentration. And sequentially taking 5 parts of standard solutions 2, 4, 6, 8 and 10m L with equal interval volumes from the standard solutions, respectively placing the standard solutions into 5 identical 10m L volumetric flasks, and adding ethanol with the volume fraction of 50% to dilute the standard solutions to a constant volume. At the same time, the volume fraction of 50% ethanol, 1.0m L, was diluted to 10m L with distilled water as a blank, and the absorbance value was measured at 250nm with an ultraviolet-visible spectrophotometer. And drawing a standard curve by taking the concentration of the puerarin as an abscissa and the absorbance as an ordinate.

2. Weighing 50mg radix Puerariae product sample in 10m L small beaker, dissolving with 50% ethanol solution, filtering, transferring filtrate into 50ml volumetric flask, diluting with 50% ethanol to 50ml, measuring absorbance value with ultraviolet-visible spectrophotometer at 250nm, and calculating radix Puerariae total flavone content according to standard curve.

The test results are shown in table 1:

TABLE 1 Total flavonoid content test results of radix Puerariae

Examples	Example 1	Example 2	Example 3	Example 4
					Radix Puerariae Total Flavonoids content	96.3％	95.8％	96.1％	95.9％

According to the test results, the content of the pueraria flavonid prepared by the preparation method is more than 95%, so that the pueraria flavonid with high content is obtained, the simple, feasible and efficient extraction and separation process is provided, the industrial production of the pueraria flavonid with 95% content is realized, the pueraria extract with high content of the flavonid is obtained by the combined application of ultrasonic countercurrent extraction, resin purification, crystallization and drying processes in sequence, and compared with the common extraction process of the pueraria extract, the content of the pueraria flavonid prepared by the preparation method reaches more than 95%, and the preparation method is simple in process, short in production period, light in product color, low in production cost, easy to realize large-scale production, and suitable for the industrial large-scale production of the pueraria flavonid with high content.

The above description is only exemplary of the present application and should not be taken as limiting the present application, as any modification, equivalent replacement, or improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.