CN114632098B - A method for preparing refined flavone product from caulis Spatholobi - Google Patents
A method for preparing refined flavone product from caulis Spatholobi Download PDFInfo
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- CN114632098B CN114632098B CN202210541177.7A CN202210541177A CN114632098B CN 114632098 B CN114632098 B CN 114632098B CN 202210541177 A CN202210541177 A CN 202210541177A CN 114632098 B CN114632098 B CN 114632098B
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- A61K36/18—Magnoliophyta (angiosperms)
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- A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
- A61K36/486—Millettia
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation or decoction
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- A61K2236/30—Extraction of the material
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Abstract
The invention provides a method for preparing a flavone refined product by utilizing suberect spatholobus stem. S1, preparing a eutectic solvent by heating choline chloride and phenoxyethanol at constant temperature in a water bath kettle; step S2, placing the dried caulis Spatholobi powder in a eutectic solvent, adding compound glycosidase, stirring to obtain caulis Spatholobi whole powder homogenate, and cooling to room temperature; step S3, performing centrifugal treatment on the caulis Spatholobi whole powder homogenate cooled to room temperature, and collecting the supernatant after the centrifugal treatment as a flavone crude extract; step S4, standing the crude flavone extracting solution, and after the crude flavone extracting solution separates out a flavone precipitate, performing centrifugal treatment on the flavone precipitate to obtain a solid phase of the flavone precipitate; step S5, washing the solid phase of the flavone precipitate with ethanol solution, and centrifuging after washing to remove the eutectic solvent to obtain total flavone extract; and step S6, sterilizing and drying the total flavone extract to obtain the refined total flavone.
Description
Technical Field
The invention relates to the field of flavone preparation, in particular to a method for preparing a refined flavone product by utilizing suberect spatholobus stem.
Background
Caulis Spatholobi is a common Chinese medicinal material collected from Chinese pharmacopoeia, and is derived from dried rattan of Spinaceus went of Leguminosae. Spatholobus stem, bitter in taste, sweet in flavor and warm in nature, is mostly ascribed to liver and kidney meridians. Has the functions of promoting blood circulation, enriching blood, regulating menstruation, relieving pain, relaxing muscles and tendons and activating collaterals. It is used clinically in treating rheumatic arthralgia, menoxenia, etc. The caulis Spatholobi extract is mainly distributed in Guangxi, Guangdong, Yunnan and Fujian places, and pharmacological research shows that the caulis Spatholobi extract has multiple activities of resisting mutation, virus, tumor, inflammation, oxidation and the like. Wherein the flavonoids are the main components of caulis Spatholobi, and mainly comprise flavonoids, flavonols, flavanones, isoflavones, flavans, etc.
Clinical experiments show that the caulis spatholobi has low toxicity, high safety and wide range, and is suitable for being widely applied to clinic. So the medicien thinks that the scientific application of caulis Spatholobi can well protect the body and can be used in daily life. At present, the administration mode of the caulis spatholobi is mainly the modes of traditional Chinese medicine decoction, wine soaking, water-soaked tea drinking, cooked medicated diet and the like. At present, the medicinal materials of the caulis spatholobi in the market are mostly from wild picking, and with the increase of the medicine demand in recent years and the driving of a benefit chain, wild resources are also about to be exhausted. At present, wild resources of Guangxi province and Guangdong province in China are nearly exhausted, only Yunnan has certain wild resources, but the storage amount is very low.
However, only 5% of the flavonoids of spatholobus stem are released in the conventional eating mode, which causes serious resource waste, unobvious functional effect and the like. Secondly, the extraction method of the caulis spatholobi total flavonoids is mostly an alkali-dissolving acid-precipitating method, and has the defects of high organic reagent concentration, large dosage, high extraction temperature, complex separation and purification, severe reaction conditions, low yield and the like.
The content of flavone in caulis Spatholobi is up to more than 10%. Therefore, the research on how to further improve the utilization rate of the flavone in the caulis spatholobi has certain practical significance, and the method is also a new direction for processing and applying the caulis spatholobi.
Disclosure of Invention
The invention aims to provide a method for preparing a refined flavone product by utilizing caulis spatholobi, which aims to solve the technical problems in the prior art, has low cost, simple process, high efficiency and strong activity, and belongs to a green and environment-friendly production process of the refined flavone product of caulis spatholobi for improving the acne function.
The invention provides a method for preparing a flavone refined product by utilizing suberect spatholobus stem. The refined flavone extract prepared from caulis Spatholobi is used for improving skin acne, and the method comprises:
s1, preparing a eutectic solvent by heating choline chloride and phenoxyethanol at constant temperature in a water bath kettle;
step S2, placing the dried caulis Spatholobi powder in the eutectic solvent, adding compound glycosidase, stirring to obtain caulis Spatholobi whole powder homogenate, and cooling to room temperature, wherein the compound glycosidase comprises rhamnosidase, alpha-glucosidase and beta-glucosidase;
step S3, performing centrifugal treatment on the caulis Spatholobi whole powder homogenate cooled to the room temperature, and collecting the supernatant after the centrifugal treatment as a flavone crude extract;
step S4, standing the crude flavone extracting solution, and after a flavone precipitate is separated from the crude flavone extracting solution, performing centrifugal treatment on the flavone precipitate to obtain a solid phase of the flavone precipitate;
step S5, washing the solid phase of the flavone precipitate with an ethanol solution, and after washing, carrying out centrifugal treatment to remove the eutectic solvent to obtain a total flavone extract;
and step S6, sterilizing and drying the total flavone extract to obtain a refined total flavone.
According to the method provided by the first aspect of the invention, in the step S1, the choline chloride and the phenoxyethanol are weighed and mixed, 1 g of the choline chloride is mixed with 1.5 g of the phenoxyethanol, and the mixture of the choline chloride and the phenoxyethanol is placed in the water bath kettle for constant-temperature heating, wherein the temperature of the water bath kettle is 50-80 ℃, and the constant-temperature heating time of the water bath kettle is 45-90 minutes, so that the eutectic solvent is prepared.
According to the method provided by the first aspect of the invention, in the step S2, the dried caulis spatholobi powder is sieved by a 20-60 mesh sieve and then placed in the eutectic solvent, 1 g of the sieved dried caulis spatholobi powder is mixed with 8-20 ml of the eutectic solvent, stirring is carried out to obtain the whole caulis spatholobi powder homogenate, the whole caulis spatholobi powder homogenate is placed in a beaker, the beaker is sealed by a sealing film, the sealed beaker is subjected to water bath ultrasonic treatment, so that the whole caulis spatholobi powder is uniformly cooled to the room temperature, the treatment temperature of the water bath ultrasonic treatment is 30-80 ℃, the treatment time is 30-120 minutes, and the treatment power is 200-600 watts.
According to the method provided by the first aspect of the invention, in the step S3, the caulis spatholobi whole powder homogenate cooled to the room temperature is centrifuged, the supernatant and the solid precipitate after the centrifugation are collected, the eutectic solvent is added into the solid precipitate again to perform water bath ultrasonic treatment, the treatment temperature is 30-80 ℃, the treatment time is 30-120 minutes, the treatment power is 200-600 watts, the centrifugation treatment is performed again, the supernatant collection treatment is performed, the solid precipitate collection treatment is performed, and after repeating for 2-5 times, all the supernatants are collected and combined.
According to the method provided by the first aspect of the present invention, the rotation speed of the centrifugation treatment in the step S3 is 3000-5000 rpm, the time is 5-15 minutes, and the amount of the eutectic solvent added in each solid precipitate is half of the amount of the eutectic solvent added in the dried caulis Spatholobi powder.
According to the method provided by the first aspect of the present invention, in the step S4, the manner of standing the crude flavone extract includes:
placing the crude flavone extracting solution in a low-temperature water bath for standing, wherein the temperature of the low-temperature water bath is 0-20 ℃; or
Placing the crude flavone extracting solution in an ice water mixture for standing; or
Adding deionized water into the crude flavone extracting solution for standing, wherein 1 ml of the crude flavone extracting solution is mixed with 0.3-0.6 ml of the ionized water; or
And adding purified water into the crude flavone extracting solution, and standing, wherein 1 ml of the crude flavone extracting solution is mixed with 0.3-0.6 ml of the purified water.
According to the method provided by the first aspect of the invention, in the step S4, the rotation speed of the centrifugation treatment of the flavone precipitate is 3000-.
According to the method provided by the first aspect of the present invention, in the step S5, the eutectic solvent in the solid phase of the flavone precipitate is removed by three times of ethanol washing and centrifugation treatment to obtain the total flavone extract;
wherein the concentration of the ethanol solution is 10-60%, 1 ml of the solid phase of the flavone precipitate is matched with 3-10 ml of the ethanol solution;
wherein the rotation speed of the centrifugal treatment in the step S5 is 3000-5000 r/min, and the time is 5-15 min;
wherein the hydrolysis degree of flavonoid glycoside in the total flavone extract is more than or equal to 90%.
According to the method provided by the first aspect of the present invention, in the step S6, the drying process includes freeze drying or vacuum low-temperature drying, the temperature of the vacuum low-temperature drying is 40 ℃, the purity of the refined total flavonoids is greater than or equal to 85%, the extraction rate of flavonoids is 70% compared with the total flavonoids in the dried caulis spatholobi powder, and the extraction rate of flavonoids is 7.5% compared with the dried caulis spatholobi powder.
According to the method provided by the first aspect of the invention, the total flavone refined product is used for improving skin acne, and the oral administration dosage is as follows: 4.8 mg of said total flavonoids extract corresponds to 1 kg of body weight; the application amount is as follows: 100 micrograms of the total flavone extract corresponds to daily usage by an adult.
The second aspect of the invention provides a refined flavone product prepared from caulis spatholobi, which is used for improving skin acne, and the refined flavone product is obtained based on the method for preparing the refined flavone product from caulis spatholobi provided by the first aspect of the invention.
In conclusion, the technical scheme provided by the invention adopts the eutectic solvent to extract the suberect spatholobus stem flavone and the water-precipitated suberect spatholobus stem flavone refined product, has the advantages of low cost, high efficiency, environmental protection and suitability for industrial production. In addition, the refined caulis spatholobi flavone product which is natural and efficient and can improve the acne functional activity is a product urgently needed in the current market, and has very important social and economic benefits for realizing the high-value utilization of caulis spatholobi.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the description in the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a flow chart of a method for preparing a refined flavone extract from caulis Spatholobi according to an embodiment of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present 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.
The invention provides a method for preparing a flavone refined product by utilizing suberect spatholobus stem, and the flavone refined product prepared by utilizing the suberect spatholobus stem is used for improving skin acne. FIG. 1 is a flow chart of a method for preparing a refined flavone extract from caulis Spatholobi according to an embodiment of the present invention; as shown in fig. 1, the method includes:
s1, preparing a eutectic solvent by heating choline chloride and phenoxyethanol at constant temperature in a water bath kettle;
step S2, placing the dried caulis Spatholobi powder in the eutectic solvent, adding compound glycosidase, stirring to obtain caulis Spatholobi whole powder homogenate, and cooling to room temperature, wherein the compound glycosidase comprises rhamnosidase, alpha-glucosidase and beta-glucosidase;
step S3, performing centrifugal treatment on the caulis Spatholobi whole powder homogenate cooled to the room temperature, and collecting the supernatant after the centrifugal treatment as a flavone crude extract;
step S4, standing the crude flavone extracting solution, and after a flavone precipitate is separated from the crude flavone extracting solution, performing centrifugal treatment on the flavone precipitate to obtain a solid phase of the flavone precipitate;
step S5, washing the solid phase of the flavone precipitate with an ethanol solution, and after washing, carrying out centrifugal treatment to remove the eutectic solvent to obtain a total flavone extract;
and step S6, sterilizing and drying the total flavone extract to obtain a refined total flavone.
In some embodiments, in step S1, the choline chloride and the phenoxyethanol are weighed and mixed, 1 g of the choline chloride is mixed with 1.5 g of the phenoxyethanol, and the mixture of the choline chloride and the phenoxyethanol is placed in the water bath to be heated at a constant temperature, wherein the temperature of the water bath is 50 to 80 ℃, and the constant temperature of the water bath is 45 to 90 minutes, so as to prepare the eutectic solvent.
Specifically, the mass ratio of the choline chloride to the phenoxyethanol is selected from the range of 1: (0.7-2.5), preferably, the mass ratio of the choline chloride to the phenoxyethanol is selected from the range of 1: (0.9-2.0), preferably, the mass ratio of the choline chloride to the phenoxyethanol is selected from the range of 1: (1-1.5). The temperature of the water bath can be selected within the range of 50-90 ℃, preferably 70-80 ℃, and the eutectic solvent is prepared by heating.
In some embodiments, in step S2, the dried caulis spatholobi powder is sieved by a 20-60 mesh sieve and then placed in the eutectic solvent, 1 g of the sieved dried caulis spatholobi powder is mixed with 8-20 ml of the eutectic solvent, stirring is performed to obtain the whole caulis spatholobi powder homogenate, the whole caulis spatholobi powder homogenate is placed in a beaker, the beaker is sealed by a sealing film, the sealed beaker is subjected to water bath ultrasonic treatment, so that the whole caulis spatholobi powder is uniformly cooled to the room temperature, the treatment temperature of the water bath ultrasonic treatment is 30-80 ℃, the treatment time is 30-120 minutes, and the treatment power is 200 + 600 watts.
Specifically, the complex glycosidase is prepared from rhamnosidase, alpha-glucosidase and beta-glucosidase according to a mass ratio of 2: 2: 6, the addition amount accounts for 1-5% of the weight of the dried caulis spatholobi powder. 1 g of dried caulis spatholobi powder after sieving corresponds to 8-40 ml of the eutectic solvent, preferably 1 g of dried caulis spatholobi powder after sieving corresponds to 10-30 ml of the eutectic solvent, preferably 1 g of dried caulis spatholobi powder after sieving corresponds to 15-20 ml of the eutectic solvent, whole caulis spatholobi powder homogenate is obtained by stirring, or whole caulis spatholobi powder homogenate is obtained by oscillating, or whole caulis spatholobi powder homogenate is obtained by vortex, or whole caulis spatholobi powder homogenate is obtained by ultrasonic, the whole caulis spatholobi powder homogenate is put for ultrasonic extraction, the treatment temperature of water bath ultrasonic treatment is 30-80 ℃, the treatment time is 30-120 minutes, and the treatment power is 200-600 watts. Homogenizing the treated caulis Spatholobi powder, and cooling to room temperature.
In some embodiments, in step S3, the caulis spatholobi whole powder homogenate cooled to the room temperature is centrifuged, the supernatant and the solid precipitate after centrifugation are collected, the eutectic solvent is added again to the solid precipitate for water bath ultrasonic treatment at the temperature of 30-80 ℃ for 30-120 minutes at the treatment power of 200-600 watts, and centrifugation, supernatant collection and solid precipitate collection are performed again, and all supernatants are collected and combined after 2-5 times of repetition.
In some embodiments, the rotation speed of the centrifugation in step S3 is 3000-5000 rpm for 5-15 minutes, and the amount of the eutectic solvent added to the solid precipitate is half of the amount of the eutectic solvent added to the dried caulis spatholobi powder.
Specifically, the centrifugation in step S3 is a deslagging process, wherein a clear solution and a solid residue are obtained by an extrusion process or a membrane separation process, and then the solid residue is added with the eutectic solvent again to perform extraction and deslagging, preferably, the number of times of extraction and deslagging is 1-2 times. The amount of the re-added eutectic solvent is 10-100% of the primary extraction amount, preferably the amount of the re-added eutectic solvent is 20-60% of the primary extraction amount, preferably the amount of the re-added eutectic solvent is 30-40% of the primary extraction amount, and the collected clear solution is combined to be used as the crude flavone extracting solution
In some embodiments, in step S4, the step of standing the crude flavone extract includes:
placing the crude flavone extracting solution in a low-temperature water bath for standing, wherein the temperature of the low-temperature water bath is 0-20 ℃; or
Placing the crude flavone extracting solution in an ice water mixture for standing; or
Adding deionized water into the crude flavone extracting solution for standing, wherein 1 ml of the crude flavone extracting solution is mixed with 0.3-0.6 ml of the ionized water; or
And adding purified water into the crude flavone extracting solution, and standing, wherein 1 ml of the crude flavone extracting solution is mixed with 0.3-0.6 ml of the purified water.
Specifically, the temperature of the deionized water is 0 ℃ to room temperature, preferably 0 ℃; the temperature of the purified water is from 0 ℃ to room temperature, preferably 0 ℃.
In some embodiments, in step S4, the rotation speed of the centrifugation process for the flavone precipitate is 3000-.
Specifically, the rotation speed for performing centrifugal treatment on the flavone precipitate can be selected within the range of 3000-10000 rpm, and the time can be selected within the range of 3-15 minutes.
In some embodiments, in the step S5, the eutectic solvent in the solid phase of the flavone precipitate is removed by three times of ethanol washing and centrifugation to obtain the total flavone extract;
wherein the concentration of the ethanol solution is 10-60%, 1 ml of the solid phase of the flavone precipitate is matched with 3-10 ml of the ethanol solution;
wherein the rotation speed of the centrifugal treatment in the step S5 is 3000-5000 r/min, and the time is 5-15 min;
wherein the hydrolysis degree of flavonoid glycoside in the total flavone extract is more than or equal to 90%.
Specifically, the adsorbed eutectic solvent in the solid phase of the flavone precipitate is removed by three times of ethanol washing and centrifugation treatment. The rotation speed of the centrifugal treatment in the step 5 can be selected within the range of 3000-10000 rpm, and the time can be selected within the range of 3-15 minutes;
in some embodiments, in step S6, the drying process includes freeze drying or vacuum low-temperature drying, the temperature of the vacuum low-temperature drying is 40 ℃, the purity of the total flavone refined product is greater than or equal to 85%, the extraction rate of flavone is 70% compared with the total flavone content in the dried caulis Spatholobi powder, and the extraction rate of flavone is 7.5% compared with the dried caulis Spatholobi powder.
In some embodiments, the total flavonoid extract is used for improving acne on the skin by oral administration in an amount of: 4.8 mg of said total flavonoids extract corresponds to 1 kg of body weight; the painting dosage is as follows: 100 micrograms of the total flavone extract corresponds to daily usage by an adult.
Specific example of embodiment 1
S1, preparing a eutectic solvent: accurately weighing choline chloride and phenoxyethanol according to a mass ratio of 1: 1.2, placing the mixture in a water bath kettle at the temperature of 65 ℃ for heating for 60 minutes at constant temperature.
S2, sieving the dried caulis spatholobi powder with a 30-mesh sieve, placing the dried caulis spatholobi powder into a eutectic solvent according to the material-liquid ratio of 1: 20 (mass/volume), adding 3% (mass ratio) of rhamnosidase, alpha-glucosidase and beta-glucosidase, and mixing the components in a mass ratio of 2: 2: 6, stirring to obtain caulis spatholobi whole powder homogenate, sealing a beaker by using a sealing film, carrying out water bath at 60 ℃, carrying out ultrasonic treatment at 400W for 80 minutes, and cooling to room temperature.
And S3, centrifuging the extracting solution in the S2 at 4000 rpm for 5 minutes, collecting the supernatant, adding a eutectic solvent which is 10 times of the original material into the solid, extracting for 2 times again, centrifuging, and combining the supernatants to obtain the crude flavone extracting solution.
S4, placing the crude flavone extracting solution in a low-temperature water bath at 4 ℃, standing, centrifuging at 4000 rpm for 5 minutes after the solution precipitates, separating a solid phase and a liquid phase, combining and collecting the solid phase.
S5, mixing the solid phase of S4 according to the ratio of material to liquid of 1: 4 (volume/volume), adding 30 percent ethanol, stirring and washing, centrifuging for 5 minutes at 4000 rpm, and repeating the washing and centrifuging operation for three times to obtain the caulis spatholobi total flavone extract without the eutectic solvent.
S6, sterilizing the caulis Spatholobi total flavone extract, and freeze drying to obtain refined powder of caulis Spatholobi total flavone.
Test analysis shows that the total flavone content in the obtained refined powder of the total flavone of the caulis spatholobi is 85.3 mass percent, the extraction rate is more than or equal to 71.9 mass percent (compared with the total flavone content in the dried caulis spatholobi powder), the extraction rate is more than or equal to 7.6 mass percent (compared with the dried caulis spatholobi powder), and the hydrolysis degree of the flavonoid glycoside is more than or equal to 92.1 percent.
Specific example two of the embodiment
S1, preparing a eutectic solvent: accurately weighing choline chloride and phenoxyethanol according to a mass ratio of 1:1, and placing the mixture in a water bath kettle at the temperature of 70 ℃ for heating for 90 minutes at constant temperature.
S2, sieving the dried caulis Spatholobi powder with a 50-mesh sieve, placing the dried caulis Spatholobi powder into a eutectic solvent according to the material-liquid ratio of 1:15 (mass/volume), adding 4% (mass ratio) of rhamnosidase, alpha-glucosidase and beta-glucosidase, and mixing the components in a mass ratio of 2: 2: 6, mixing to obtain caulis spatholobi whole powder homogenate, sealing a beaker by using a sealing film, carrying out water bath at 50 ℃, carrying out ultrasonic treatment at 450W for 100 minutes, and cooling to room temperature.
And centrifuging the extracting solution in the S2 at the speed of S3 and 4500 rpm for 10 minutes, collecting supernatant, adding 8 times of eutectic solvent of the original material into the solid, extracting for 3 times again, centrifuging, and combining the supernatants to obtain the crude flavone extracting solution.
S4, placing the crude flavone extract in a low-temperature water bath at 2 ℃, standing, centrifuging at 4500 rpm for 10 minutes after the solution precipitates, separating a solid phase and a liquid phase, combining and collecting the solid phase.
S5, mixing the solid phase of S4 according to the ratio of material to liquid of 1: 6 (volume/volume) adding 40% ethanol, stirring, washing, centrifuging at 4500 rpm for 10 min, and repeating washing and centrifuging for three times to obtain caulis Spatholobi total flavone extract without eutectic solvent.
S6, sterilizing the caulis Spatholobi total flavone extract, and vacuum drying at 40 deg.C to obtain refined powder of caulis Spatholobi total flavone.
Test analysis shows that the total flavone content in the obtained refined powder of the total flavone of the caulis spatholobi is 88.2 percent by mass, the extraction rate is more than or equal to 73.6 percent by mass (compared with the total flavone content in the dried caulis spatholobi powder), the extraction rate is more than or equal to 7.8 percent by mass (compared with the dried caulis spatholobi powder), and the hydrolysis degree of the flavonoid glycoside is more than or equal to 92.9 percent.
Specific example of embodiment three
S1, preparing a eutectic solvent: accurately weighing choline chloride and phenoxyethanol according to a mass ratio of 1.2: 1, and placing the mixture in a water bath kettle at the temperature of 50 ℃ for constant temperature heating for 55 minutes.
S2, sieving the dried caulis spatholobi powder with a 40-mesh sieve, placing the dried caulis spatholobi powder into a eutectic solvent according to the material-liquid ratio of 1: 10 (mass/volume), adding 2% (mass ratio) of rhamnosidase, alpha-glucosidase and beta-glucosidase, and mixing the components in a mass ratio of 2: 2: 6, stirring to obtain caulis spatholobi whole powder homogenate, sealing a beaker by using a sealing film, carrying out water bath at 30 ℃, carrying out ultrasonic treatment at 500W for 100 minutes, and cooling to room temperature.
And S3, centrifuging the extracting solution in the S2 at 3000 r/min for 8 min, collecting the supernatant, adding a eutectic solvent which is 5 times of the original material into the solid, extracting for 4 times again, centrifuging, and combining the supernatants to obtain the crude flavone extracting solution.
S4, mixing the crude flavone extract according to the material-liquid ratio of 1: 0.5 (volume/volume) of deionized water is added, the mixture is kept stand, after the solution is precipitated, the mixture is centrifuged at 3000 rpm for 8 minutes to separate a solid phase and a liquid phase, and the solid phase is combined and collected.
S5, mixing the solid phase of S4 according to the ratio of material to liquid of 1: 8 (volume/volume) adding 20% ethanol, stirring, washing, centrifuging at 3000 r/min for 8 min, and repeating washing and centrifuging for three times to obtain caulis Spatholobi total flavone extract without eutectic solvent.
S6, sterilizing the caulis Spatholobi total flavone extract, and vacuum drying at 40 deg.C to obtain refined powder of caulis Spatholobi total flavone.
Test analysis shows that the total flavone content in the obtained refined powder of the total flavone of the caulis spatholobi is 86.4 mass percent, the extraction rate is more than or equal to 70.2 mass percent (compared with the total flavone content in the dried caulis spatholobi powder), the extraction rate is more than or equal to 7.5 mass percent (compared with the dried caulis spatholobi powder), and the hydrolysis degree of the flavonoid glycoside is more than or equal to 90.3%.
The refined total flavone extract has acne improving effect, and can be administered orally or applied topically. The method verifies the functional activity of improving the acne.
And (3) testing a sample: specific examples of the embodiment include refined powder of caulis Spatholobi total flavonoids, caulis Spatholobi powder, and tanshinone (positive control).
Experimental animals and breeding environment: SPF male golden hamster, 100 + -10 g, 6-8 weeks old, purchased from Beijing Wittingli laboratory animal technology, Inc. The basic feed comprises the following components: 64% of carbohydrate, 21% of protein, 4% of fat, 5% of fiber and 6% of water. The feed is purchased from Beijing Weitonglihua laboratory animal technology Co. The feeding environment has good ventilation, and the day and night change rule (8: 00-20: 00) is 23 + -2 deg.C at room temperature and 55 + -5% of relative humidity.
Grouping experiments: the SPF male golden yellow hamster is bred adaptively for one week and fed freely. One week later, the animals were randomly divided into 2 groups (oral group and smear group) including a normal group, a positive control group (tanshinone/vitamin a cream), and a sample group (examples 1 to 3, and divided into two low and high dose groups), and 5 animals were tested per group for a total of 16 groups. Intragastric administration dosage: the low dose is 30 mg/kg, the high dose is 60 mg/kg, and the dose of tanshinone control group is 20 mg/kg. Coating dosage: the low dose was 10 mg/mouse, the high dose was 20 mg/mouse, and the dose of the vitamin A cream control group was 10 mg/mouse. The experimental period was 4 weeks.
Measuring the TG content of sebaceous gland speckle tissue: after the experiment is finished, the hair on the sebaceous gland plaques on the two sides of the golden hamster is cleaned by a shaver, the sebaceous gland plaque areas on the two sides of 0d and 28d are measured, and the sebaceous gland plaques on the two sides of the abdomen of the golden hamster are cut off. Weighing about 0.1 g of golden hamster sebaceous gland plaques, crushing the golden hamster sebaceous gland plaques into small blocks by using scissors, adding physiological saline according to the proportion of 1:9, grinding the golden hamster sebaceous gland plaques into tissue homogenate by using a glass homogenizer, carrying out the whole process in an ice bath, centrifuging 3000 g of the tissue homogenate for 10 min after grinding, and measuring the TG content and the Caspase-3 content in the supernate of the golden hamster sebaceous gland plaques.
TABLE 1 evaluation of the activity of refined extract of Spatholobus suberectus Dunn for improving acne function by oral administration
TABLE 2 evaluation of the activity of applying refined extract of Spatholobus suberectus Dunn for improving acne function
As can be seen from the results in Table 1, the refined extract of spatholobus stem flavone prepared by the invention has obviously better activity for improving the acne function by mouth than tanshinone. From the content of TG, compared with a normal group (0.83 mmol/g prot), the tanshinone control group is reduced to 0.49 mmol/g prot, but the content of TG is weaker than that of the low-dose group (0.35-0.45 mmol/g prot) in examples 1-3, and the capacity of the refined product of the sargentodoxa cuneata flavone prepared by the method for inhibiting triglyceride in sebaceous gland plaques is better than that of the tanshinone control group. From the aspect of the area increase of the sebaceous gland plaques, after tanshinone is taken, the activity of an apoptotic protein Caspase-3 in the sebaceous gland plaques of golden hamster can be improved to promote the apoptosis of the sebaceous gland plaques so as to control the increase of the sebaceous gland plaques. However, when golden hamster is refined by eating the spatholobus stem flavone prepared by the invention, the activity of Caspase-3 and the increase rate of sebaceous gland plaques in the low and high dose groups are superior to those of a tanshinone control group, and the area of the sebaceous gland plaques has no significant difference with that of the tanshinone control group.
As can be seen from the results in Table 2, the activity of improving the acne function by applying the refined caulis Spatholobi flavone extract prepared by the method is obviously superior to that of tanshinone. From the TG content, compared with a normal group (1.19 mmol/g prot), the tanshinone control group is reduced to 0.68 mmol/g prot, but the tanshinone control group is weaker than the high-dose group (0.56-0.68 mmol/g prot) in examples 1-3, and the capacity of the refined sargentodoxa cuneata flavone product prepared by the method for inhibiting triglyceride in sebaceous gland plaques is better than that of the tanshinone control group. From the aspect of the area increase of the sebaceous gland plaques, after tanshinone is taken, the activity of an apoptotic protein Caspase-3 in the sebaceous gland plaques of golden hamster can be improved to promote the apoptosis of the sebaceous gland plaques so as to control the increase of the sebaceous gland plaques. However, when golden hamster is refined by eating the spatholobus stem flavone prepared by the invention, the activity of Caspase-3 and the increase rate of sebaceous gland plaques in a high-dose group are superior to those in a tanshinone control group.
The results show that the refined caulis spatholobi flavone product prepared by the invention has obvious effect of improving the acne functional activity after being taken orally or smeared, and fully illustrates the effectiveness and superiority of the technology of the invention. In view of the advantages of high efficiency and low cost of the preparation method of the invention, the obtained caulis spatholobi flavone refined product has good effect of improving acne, therefore, the technology can be used for industrially developing caulis spatholobi flavone refined product for improving acne functional activity.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made in the above embodiments by those of ordinary skill in the art without departing from the principle and spirit of the present invention.
The second aspect of the invention provides a refined flavone extract prepared from caulis spatholobi, which is used for improving skin acne and is obtained based on the method for preparing the refined flavone extract from caulis spatholobi provided by the first aspect of the invention.
In summary, the solutions of the first and second aspects provided by the present invention have the following beneficial effects: (1) according to the invention, the eutectic solvent is adopted to extract the flavone, the eutectic solvent suitable for the system is selected, the high distribution coefficient of the flavone in the eutectic solvent is fully utilized, and the stable hydrogen bond effect formed between the flavone and the eutectic solvent promotes the transfer of the flavone to a solvent phase, so that the solubility and the extraction rate are greatly improved, the traditional high-temperature heating time and the solvent consumption are reduced, the flavone can be recycled as safe and green ionic liquid, and the safety of the product is fully ensured. (2) The cold water precipitation promotion purification technology adopted by the invention avoids the steps of adding high-concentration organic reagent, treating at high temperature and removing solvent, so that the product is safer and more stable, thereby improving the function effect of improving acne of the product. (3) The method adopts the eutectic solvent extraction-cold water precipitation promotion purification-freeze drying technology, not only can avoid a large amount of ash components brought by the traditional process, but also can simplify the separation and purification steps and shorten the operation time, and greatly improves the purity of the product (more than or equal to 85 mass percent). The technology has the advantages of good separation effect, high safety, environmental protection and high production efficiency, effectively reduces the production cost and is suitable for industrial production.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A method for preparing a refined flavone product from caulis Spatholobi is characterized in that the refined flavone product prepared from caulis Spatholobi is used for improving skin acne, and the method comprises the following steps:
s1, preparing a eutectic solvent by heating choline chloride and phenoxyethanol at a constant temperature in a water bath kettle, wherein the mass ratio of the choline chloride to the phenoxyethanol is 1: 0.7-2.5;
step S2, placing the dried caulis Spatholobi powder in the eutectic solvent, adding compound glycosidase, stirring to obtain caulis Spatholobi whole powder homogenate, and cooling to room temperature, wherein the compound glycosidase comprises rhamnosidase, alpha-glucosidase and beta-glucosidase;
step S3, performing centrifugal treatment on the caulis Spatholobi whole powder homogenate cooled to the room temperature, and collecting the supernatant after the centrifugal treatment as a flavone crude extract;
step S4, standing the crude flavone extracting solution, and after a flavone precipitate is separated from the crude flavone extracting solution, performing centrifugal treatment on the flavone precipitate to obtain a solid phase of the flavone precipitate;
step S5, washing the solid phase of the flavone precipitate with an ethanol solution, and after washing, carrying out centrifugal treatment to remove the eutectic solvent to obtain a total flavone extract;
and step S6, sterilizing and drying the total flavone extract to obtain a refined total flavone.
2. The method of claim 1, wherein in step S1, the choline chloride and the phenoxyethanol are weighed and mixed, 1 g of the choline chloride is mixed with 1.5 g of the phenoxyethanol, and the mixture of the choline chloride and the phenoxyethanol is placed in the water bath kettle for heating at a constant temperature, wherein the temperature of the water bath kettle is 50-80 ℃, and the heating time at the constant temperature of the water bath kettle is 45-90 minutes, so as to prepare the eutectic solvent.
3. The method as claimed in claim 2, wherein in step S2, the dried caulis Spatholobi powder is sieved with 20-60 mesh sieve and then placed in the eutectic solvent, 1 g of the sieved dried caulis Spatholobi powder is mixed with 8-20 ml of the eutectic solvent, stirring is performed to obtain the caulis Spatholobi whole powder homogenate, the caulis Spatholobi whole powder homogenate is placed in a beaker, the beaker is sealed by a sealing film, the sealed beaker is subjected to water bath ultrasonic treatment to uniformly cool the caulis Spatholobi whole powder to the room temperature, the treatment temperature of the water bath ultrasonic treatment is 30-80 ℃, the treatment time is 30-120 minutes, and the treatment power is 200-600 watts.
4. The method as claimed in claim 3, wherein the step S3 comprises centrifuging the whole caulis Spatholobi powder homogenate cooled to room temperature, collecting supernatant and solid precipitate after the centrifugation, adding the eutectic solvent into the solid precipitate again to perform ultrasonic treatment in water bath at 30-80 deg.C for 30-120 min at 200W, centrifuging again, collecting supernatant, collecting solid precipitate, repeating for 2-5 times, and collecting and combining all supernatants.
5. The method as claimed in claim 4, wherein the rotation speed of the centrifugation step S3 is 3000-5000 rpm for 5-15 min, and the amount of the eutectic solvent added to the solid precipitate is half of the amount of the eutectic solvent added to the dried caulis Spatholobi powder.
6. The method of claim 5, wherein the step S4 of leaving the crude flavone extractive solution to stand comprises:
placing the crude flavone extracting solution in a low-temperature water bath for standing, wherein the temperature of the low-temperature water bath is 0-20 ℃; or
Placing the crude flavone extracting solution in an ice water mixture for standing; or
Adding deionized water into the crude flavone extracting solution for standing, wherein 1 ml of the crude flavone extracting solution is mixed with 0.3-0.6 ml of the ionized water; or
And adding purified water into the crude flavone extracting solution, and standing, wherein 1 ml of the crude flavone extracting solution is mixed with 0.3-0.6 ml of the purified water.
7. The method as claimed in claim 6, wherein the rotation speed of the step S4 is 3000-5000 rpm for 5-15 min, so as to obtain the solid phase and the liquid phase of the flavone precipitate, and the solid phase is collected.
8. The method of claim 7, wherein the eutectic solvent in the solid phase of the flavone precipitate is removed by three times of ethanol washing and centrifugation in step S5 to obtain the total flavone extract;
wherein the concentration of the ethanol solution is 10-60%, 1 ml of the solid phase of the flavone precipitate is matched with 3-10 ml of the ethanol solution;
wherein the rotation speed of the centrifugal treatment in the step S5 is 3000-5000 r/min, and the time is 5-15 min;
wherein the hydrolysis degree of flavonoid glycoside in the total flavone extract is more than or equal to 90%.
9. The method of claim 8, wherein the drying process comprises freeze drying or vacuum low-temperature drying at 40 ℃, the purity of the total flavone refined product is greater than or equal to 85%, the extraction rate of flavone is 70% compared with the total flavone content in the dried caulis Spatholobi powder, and the extraction rate of flavone is 7.5% compared with the dried caulis Spatholobi powder in step S6.
10. The method for preparing refined flavone extract from spatholobus stem according to any of claims 1-9, wherein the refined total flavone extract is administered orally in an amount of: 4.8 mg of said total flavonoids extract corresponds to 1 kg of body weight; the application amount is as follows: 100 micrograms of the total flavone extract corresponds to daily usage by an adult.
Priority Applications (1)
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