CN110638837A

CN110638837A - Propolis extract and preparation method and application thereof

Info

Publication number: CN110638837A
Application number: CN201911056586.2A
Authority: CN
Inventors: 谢雁鸣; 邵旭; 张凯; 郭彧
Original assignee: Guangdong Guoyuan Guoyao Pharmaceutical Co ltd
Current assignee: Guoyuan Guoyao Guangdong Pharmaceutical Group Co ltd
Priority date: 2019-10-31
Filing date: 2019-10-31
Publication date: 2020-01-03
Anticipated expiration: 2039-10-31
Also published as: CN110638837B

Abstract

The invention belongs to the technical field of biological medicines, and provides a preparation method of a propolis extract, which comprises the following steps: extracting the crushed propolis raw material with ethanol water solution with concentration of more than 70% to obtain crude propolis extract; freezing the crude extract, and dispersing in 70% ethanol water solution to obtain propolis concentrated solution; mixing the propolis concentrated solution with silica gel to make active ingredients in the propolis concentrated solution adsorbed onto silica gel powder to prepare silica gel adsorption powder; sequentially carrying out solid-liquid extraction on the silica gel adsorption powder by adopting a first extractant, a second extractant and a third extractant, collecting the part of the third extractant, and preparing a propolis extract; wherein the first extractant is petroleum ether, the second extractant is a mixed solvent formed by mixing acetone and petroleum ether according to the volume ratio of less than 2:1, and the third extractant is a mixed solvent formed by mixing acetone and petroleum ether according to the volume ratio of (2-4) to 1; decolorizing the propolis extract.

Description

Propolis extract and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a propolis extract and a preparation method and application thereof.

Background

Propolis is a natural adhesive substance prepared by mixing the resin secreted by young buds and callus of poplar, willow, pine and other plants with the secretion of jaw gland, wax gland and other plants. The Chinese and foreign research results show that the propolis has pharmacological effects of resisting oxidation, bacteria, inflammation, viruses, tumors and radiation, removing free radicals, promoting tissue regeneration, assisting in reducing blood sugar and blood fat and the like as a natural medicine and a health-care product with long application history, and the flavonoid compound with wide biological activity is a large class of compound with highest content and most abundant types in the propolis, and the content and the types of the flavonoid compound are incomparable with other natural products.

The biological activity of propolis is the result of the interaction of its various chemical components. However, the chemical components of propolis are extremely complex, and more than 300 components are known at present, including resin, beeswax, flavone, polyphenol, terpene, esters, volatile oil and the like, and are affected by plant sources, bee species and other factors, which brings great difficulty to separation and purification and research and application of active ingredients in propolis, and causes a problem that the content of active ingredients such as flavonoid compounds in propolis extract is relatively low, for example: the content of poplar can be as low as 2%, galangin can be as low as 1%, caffeic acid phenethyl ester can be as low as 0.5%, etc. Meanwhile, the preparation process reported in the prior literature has the advantages that the total flavonoids in the propolis with high content are only counted in grams and basically stay in a laboratory stage, and in the silica gel column chromatography process, more toxic solvents such as chloroform, benzene, ethyl acetate and the like are required to be consumed, so that the industrial mass production condition is not met.

Disclosure of Invention

The invention mainly aims to provide a preparation method of a propolis extract, and aims to solve the problems that the existing preparation process of the propolis extract is low in active ingredient content and is not suitable for industrial mass production and the like.

The invention also aims to provide the propolis extract prepared by the preparation method and application.

In order to achieve the purpose, the invention adopts the following technical scheme:

in one aspect, the present invention provides a method for preparing a propolis extract, comprising the steps of:

providing crude propolis, and crushing the crude propolis to obtain crushed propolis;

extracting the propolis raw crushed materials with ethanol water solution with the weight percentage concentration of ethanol of more than 70%, collecting extract, and concentrating to obtain a propolis crude extract;

freezing the crude propolis extract, dispersing in ethanol water solution with ethanol concentration of above 70%, filtering, and concentrating the filtrate to obtain propolis concentrated solution;

providing silica gel, mixing the propolis concentrated solution with the silica gel to enable active ingredients in the propolis concentrated solution to be adsorbed on the silica gel powder, and drying to obtain silica gel adsorption powder;

sequentially carrying out solid-liquid extraction on the silica gel adsorption powder by adopting a first extractant, a second extractant and a third extractant, collecting the part of the third extractant, and concentrating to obtain a propolis extract; the first extracting agent is petroleum ether, the second extracting agent is a mixed solvent formed by mixing acetone and petroleum ether according to the volume ratio of less than 2:1, and the third extracting agent is a mixed solvent formed by mixing acetone and petroleum ether according to the volume ratio of (2-4) to 1;

decolorizing the propolis extract, filtering, and concentrating the filtrate to obtain refined propolis extract.

In the preparation method of the propolis extract, the propolis raw crushed materials are extracted by ethanol water solution with the weight percentage concentration of ethanol of more than 70 percent so as to enrich active ingredients such as flavonoid compounds in the propolis; then, freezing the crude propolis extract to separate out residual beeswax in the crude propolis extract, and separating and removing the beeswax by dispersing the beeswax in an ethanol water solution with the weight percentage concentration of ethanol being more than 70%, so that the influence of the residual beeswax on the subsequent separation and purification of the active ingredients of the total flavonoids is avoided, and the further enrichment of the active ingredients of the total flavonoids in the extract is promoted; then, mixing and adsorbing the propolis concentrated solution and silica gel to enable active ingredients in the propolis concentrated solution to be adsorbed on the silica gel powder, performing solid-liquid extraction on the silica gel adsorbed powder by adopting petroleum ether-acetone systems with different volume ratios, collecting acetone-petroleum ether extraction parts with the volume ratio of (2-4):1, and concentrating to obtain a propolis extract, wherein the method is simple and can realize large-scale production of the propolis extract at the level of hundred kilograms; and then, decoloring the propolis extract. The detection shows that the content of total brass active ingredients in the propolis refined extract prepared by the method is as high as 98.1%, and the active ingredients are clear, so that the propolis refined extract can be well applied to preparing health-care food, medicines and beauty raw materials and is safe to use.

In another aspect, the present invention provides a propolis extract prepared by the above preparation method, wherein the active ingredients of the propolis extract include chrysin, pinocembrin, galangin, caffeic acid phenethyl ester, kaempferol, pinobanksin and acacetin.

The propolis extract provided by the invention is prepared by the preparation method, has the purity of 98.1%, mainly comprises chrysin, pinocembrin, galangin, caffeic acid phenethyl ester, kaempferol, pinobanksin, farnesol and other flavonoid compounds as active ingredients, has definite active ingredients, can be widely applied to preparation of health-care food, medicines and beauty raw materials, and is safe to use.

In another aspect, the present invention provides the use of the aforementioned method of preparation or the aforementioned propolis extract in the preparation of health foods, pharmaceuticals and cosmetic raw materials.

The propolis extract prepared by the preparation method has the purity as high as 98.1 percent, the active ingredients of the propolis extract mainly comprise flavonoids compounds such as chrysin, pinocembrin, galangin, caffeic acid phenethyl ester, kaempferol, pinobanksin and farnesol, the propolis extract has definite active ingredients and wide biological activity, and can be applied to preparing functional products such as health-care food, medicines, beauty raw materials and the like.

Drawings

FIG. 1 shows the HPLC test results of the propolis extract active ingredient in the test examples.

Detailed Description

In order to make the technical problems, technical solutions and advantageous effects to be solved by the present invention more clearly apparent, the present invention is further described in detail below 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.

In the description of the present invention, it is to be understood that the terms "first", "second", "third", etc. are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implying any number of technical features indicated. Thus, features defined as "first", "second", "third" may explicitly or implicitly include one or more of the features.

A method for preparing propolis extract comprises the following steps:

s01, providing crude propolis, and crushing the crude propolis to obtain crushed propolis;

s02, extracting the propolis raw crushed materials by adopting ethanol water solution with the weight percentage concentration of ethanol being more than 70%, collecting extract, and concentrating to obtain a propolis crude extract;

s03, freezing the crude propolis extract, dispersing the frozen crude propolis extract in an ethanol water solution with the weight percentage concentration of ethanol being more than 70%, filtering, and concentrating the filtrate to obtain a propolis concentrated solution;

s04, providing silica gel, mixing the propolis concentrated solution with the silica gel to enable active ingredients in the propolis concentrated solution to be adsorbed on the silica gel powder, and drying to obtain silica gel adsorption powder;

s05, sequentially carrying out solid-liquid extraction on the silica gel adsorption powder by adopting a first extracting agent, a second extracting agent and a third extracting agent, collecting the part of the third extracting agent, and concentrating to obtain a propolis extract; the first extracting agent is petroleum ether, the second extracting agent is a mixed solvent formed by mixing acetone and petroleum ether according to the volume ratio of less than 2:1, and the third extracting agent is a mixed solvent formed by mixing acetone and petroleum ether according to the volume ratio of (2-4) to 1;

s06, decoloring the propolis extract, filtering, and concentrating the filtrate to obtain the refined propolis extract.

Specifically, in step S01, the propolis feather glue is a propolis feather material that has not been extracted with any chemical reagent, and may be selected from propolis feather glues originated from various origins, such as huangshangaoxi, atlantic golden village, henna flat top mountain, shanxi jin city, etc.

And crushing the crude propolis to obtain crushed propolis materials. The step of pulverizing the propolis raw gum can refer to the conventional operation in the field, for example, in some embodiments, the propolis raw gum is subjected to freezing pulverization to solve the problem of propolis stickiness when the propolis raw gum is pulverized at normal temperature. In one embodiment, the step of pulverizing the propolis raw gum comprises freezing the propolis raw gum at-10 to-20 ℃ for 10 to 20 hours, and then pulverizing the propolis raw gum. In some embodiments, the temperature of the freezing treatment is-10 ℃, -11 ℃, -12 ℃, -13 ℃, -14 ℃, -15 ℃, -16 ℃, -17 ℃, -18 ℃, -19 ℃, -20 ℃, and the time of the freezing treatment is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 hours. Thus, the rapid pulverization of the propolis crude gum to a proper size can be promoted. In some embodiments, the propolis raw gum is subjected to frozen pulverization to a 40-100 mesh size to improve extraction efficiency of the propolis extract.

Specifically, in step S02, the crushed propolis material is extracted with an ethanol aqueous solution with the weight percentage concentration of ethanol being more than 70%, and the extract is collected and concentrated to obtain a crude propolis extract.

The step of extracting the propolis raw crushed materials by using an ethanol water solution with the weight percentage concentration of ethanol being more than 70% can refer to the conventional operation in the field, for example, the extraction is carried out by using methods such as stirring extraction, percolation extraction, ultrasonic extraction, microwave extraction, soaking extraction and the like. The propolis raw crushed materials adopt ethanol water solution with the weight percentage concentration of ethanol of more than 70 percent as an extraction solvent, and active ingredient flavonoid compounds in the propolis can be extracted to the maximum extent. In specific embodiments, the concentration of ethanol in the aqueous ethanol solution is 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% by weight.

In one embodiment, in the step of extracting the crude propolis crushed materials with an ethanol aqueous solution with a concentration of ethanol of 70% by weight or more, the crude propolis crushed materials and the ethanol aqueous solution are extracted for 2-3 hours each time for 1-4 times with stirring according to a feed-to-solution ratio of 1g (3-10) mL. In a specific embodiment, the material-liquid ratio of the propolis raw crushed aggregates to the ethanol aqueous solution is 1g:3mL, 1g:4mL, 1g:5mL, 1g:6mL, 1g:7mL, 1g:8mL, 1g:9mL, 1g:10mL, and by controlling the material-liquid ratio of the propolis raw crushed aggregates to the ethanol aqueous solution to be within the above range, flavonoid compounds in the propolis raw gel can be effectively enriched and extracted, and the extraction efficiency of flavonoid compounds in the propolis extract is remarkably improved. In a further embodiment, the weight percentage concentration of ethanol in the ethanol aqueous solution is preferably 90% or more, and when the propolis raw material particles and the ethanol aqueous solution are stirred and extracted according to a material-liquid ratio of 1g (3-10) mL, and the weight percentage concentration of ethanol in the ethanol aqueous solution is preferably 90% or more, the content of total flavone active ingredients in the obtained propolis crude extract is increased from 6.86% to 32.8% -56.2% of propolis.

The step of collecting the extract for concentration may be performed by referring to a conventional procedure in the art, for example, vacuum concentration of the extract under reduced pressure. In some embodiments, in the step of collecting the extract for concentration, the extract is concentrated under reduced pressure in vacuum using a rotary evaporator. In some embodiments, the step of collecting and concentrating the extract comprises concentrating the extract to a fluid extract with a relative density of 1.15-1.25mg/mL, and in some embodiments, the obtained crude propolis extract has a relative density of 1.15, 1.20, 1.25, 1.24, 1.22 mg/mL. Concentrating the extractive solution to obtain fluid extract of propolis crude extract with relative density in the above range, and promoting better freezing treatment in the subsequent step S03 to separate out beeswax.

Specifically, in step S03, the crude propolis extract is subjected to freezing treatment, then dispersed in an ethanol aqueous solution with a concentration of ethanol of 70% by weight or more, filtered, and the filtrate is concentrated to obtain a propolis concentrated solution.

Freezing the crude propolis extract to separate out residual beeswax at low temperature, wherein the beeswax is insoluble in water, dispersing the propolis extract separated out with beeswax in ethanol water solution with the weight percentage concentration of more than 70%, separating and removing the beeswax, avoiding the influence of residual beeswax on the subsequent separation and purification of the total flavone active ingredients, and promoting the further enrichment of the total flavone active ingredients in the extract.

As an embodiment, in the step of freezing the crude propolis extract, the crude propolis extract is frozen at-10 to-20 ℃ for 20 to 24 hours. In some embodiments, the temperature of the freezing treatment is-10 ℃, -11 ℃, -12 ℃, -13 ℃, -14 ℃, -15 ℃, -16 ℃, -17 ℃, -18 ℃, -19 ℃, -20 ℃, and the time of the freezing treatment is 20, 21, 22, 23 and 24 hours, so that the beeswax remained in the crude propolis extract can be ensured to be separated out to the maximum extent.

The step of dispersing the crude propolis extract in an ethanol aqueous solution with a concentration of 70% by weight or more of ethanol may be performed by a conventional method in the art, for example, by uniformly dispersing the crude propolis extract in the ethanol aqueous solution by using a mechanical stirring method or an ultrasonic treatment method. In some embodiments, the propolis crude extract is dispersed in an ethanol aqueous solution with a concentration of 70% by weight or more of ethanol by using an ultrasonic treatment method. In some embodiments, the weight percent concentration of ethanol in the aqueous ethanol solution is 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 95%, 100%.

In one embodiment, in the step of dispersing the crude propolis extract in an ethanol aqueous solution with a concentration of 70% by weight or more, the crude propolis extract and the ethanol aqueous solution are mixed and dispersed according to a material-to-liquid ratio of 1g (5-10) mL. In some embodiments, the feed-to-liquid ratio of the crude propolis extract to the aqueous ethanol solution is 1g:5mL, 1g:6mL, 1g:7mL, 1g:8mL, 1g:9mL, 1g:10 mL. In some embodiments, the crude propolis extract is sonicated with the aqueous ethanol solution at a feed to liquid ratio of 1g (5-10) mL. In some embodiments, the crude propolis extract is ultrasonically dispersed in an aqueous ethanol solution having a concentration of 70% to 80% by weight of ethanol.

Specifically, in step S04, providing silica gel, mixing the propolis concentrated solution with the silica gel to adsorb active ingredients in the propolis concentrated solution onto the silica gel powder, and drying to obtain silica gel adsorption powder.

The silica gel is used as an adsorbent for adsorbing chemical components in the propolis concentrated solution, and may be selected from silica gel materials conventional in the art, for example, in some embodiments, the silica gel is selected from column chromatography silica gel.

The step of mixing the propolis concentrated solution with the silica gel may refer to a conventional procedure in the art, for example, adding silica gel to the propolis concentrated solution one by one while stirring, so that the active ingredient in the propolis concentrated solution is adsorbed onto the silica gel powder. In addition, the drying can refer to the conventional drying method in the art, for example, the solvent in the mixture is naturally volatilized at room temperature to be dried or the solvent in the mixture is directly recovered by a concentration method to obtain the silica gel adsorption powder.

As an embodiment, in the step of mixing the propolis concentrated solution with the silica gel, the silica gel in an amount of 20-100% by weight of the propolis raw gel is added into the propolis concentrated solution to be uniformly mixed, so that chemical components in the propolis concentrated solution can be sufficiently adsorbed onto the silica gel to improve the extraction efficiency of flavonoid compounds in the propolis raw gel. In some embodiments, the amount of silica gel added to the propolis concentrate is 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% by weight of the propolis. In some embodiments, the solid-to-liquid ratio of the propolis concentrated solution is 1g:2mL to 1g:3mL, specifically 1g:3mL, 1g:2mL, 1g:2.5mL, and when the solid-to-liquid ratio of the propolis concentrated solution is less than 1g:2mL, a part of propolis active ingredients are easily separated out, the binding degree with silica gel is reduced, and purification is not facilitated; when the solid-to-liquid ratio of the propolis concentrated solution is lower than 1g:3mL, the concentration is too dilute, time and labor are wasted, a large amount of ethanol is wasted, and in addition, the ethanol carries away propolis active ingredients to climb the wall when evaporating, so that the loss is large.

Specifically, in step S05, performing solid-liquid extraction on the silica gel adsorption powder sequentially by using a first extraction agent, a second extraction agent and a third extraction agent, collecting the third extraction agent part, and concentrating to obtain a propolis extract; the first extracting agent is petroleum ether, the second extracting agent is a mixed solvent formed by mixing acetone and petroleum ether according to the volume ratio of less than 2:1, and the third extracting agent is a mixed solvent formed by mixing acetone and petroleum ether according to the volume ratio of (2-4) to 1.

According to the embodiment of the invention, a solid-liquid extraction method is adopted, a petroleum ether-acetone elution system is adopted, and the petroleum ether-acetone ratio is gradually adjusted, so that the compound adsorbed on the silica gel can be gradually desorbed into the extractant with the corresponding polarity range from small to large according to the polarity, and the target component flavonoid compound is efficiently enriched to the third extractant position. Compared with the conventional silica gel column chromatography method, the method provided by the embodiment of the invention is simple to operate, is not limited by silica gel column chromatography, and can realize large-scale production of the propolis extract at the level of hundreds of kilograms.

The silica gel adsorption powder is subjected to solid-liquid extraction by sequentially adopting a first extractant, a second extractant and a third extractant, and the conventional solid-liquid extraction operation in the field can be referred to, so that the details are not repeated here for saving space.

As an embodiment, the step of performing solid-liquid extraction on the silica gel adsorption powder by sequentially using a first extractant, a second extractant, a third extractant and a fourth extractant includes:

s041, mixing the silica gel adsorption powder and the first extracting agent according to the volume ratio of 1 (3-10), stirring and extracting for 1-3 times, wherein each time lasts for 1-3 hours, filtering, and naturally volatilizing filter residues to obtain a first extract;

s042, mixing the first extract and the second extractant according to the volume ratio of 1 (3-10), stirring and extracting for 1-3 times, wherein each time lasts for 1-3 hours, filtering, and naturally volatilizing filter residues to obtain a second extract;

s043, mixing the second extract and the third extractant according to the volume ratio of 1 (3-10), stirring and extracting for 1-3 hours, each time for 1-3 hours, and combining the stirred and extracted third extractants.

In step S041, the silica gel adsorbent powder and the first extracting agent are mixed according to a volume ratio of 1 (3-10), so that a part of the compound adsorbed in the silica gel adsorbent powder and having a polarity similar to that of the first extracting agent is desorbed into the first extracting agent, and the first purification and separation of impurities on the silica gel adsorbent powder is realized. In some embodiments, the volume ratio of the silica gel adsorbent powder to the first extractant is 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1: 10.

In step S042, the first extract and the second extractant are mixed according to a volume ratio of 1 (3-10), so that a part of compounds with a polarity similar to that of the second extractant adsorbed in the silica gel adsorption powder is desorbed to the second extractant, and the second purification and separation of impurities on the silica gel adsorption powder is realized. In some embodiments, the volume ratio of the first extract to the second extractant is 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1: 10. In some embodiments, the volume ratio of acetone to petroleum ether in the second extractant is 1:1, 1:2, 1:3, 1: 4.

In a further embodiment, the step of mixing the first extract with the second extractant in a volume ratio of 1 (3-10) comprises:

mixing the first extract with a mixed solvent of acetone and petroleum ether in a volume ratio of 1 (2-4) according to a volume ratio of 1 (3-10), stirring and extracting for 1-3 times, each time for 1-3 hours, filtering, and taking filter residue; then, mixing the filter residue with a mixed solvent of acetone and petroleum ether in a volume ratio of 1:1 according to a volume ratio of 1 (3-10), stirring and extracting for 1-3 times, each time for 1-3 hours, filtering, and naturally volatilizing the filter residue to obtain a second extract.

Therefore, the influence of the residual impurities in the extract on the extraction of the active ingredients can be reduced, so that the enrichment amount of the flavonoid compounds in the third extractant part in the subsequent step S043 is further improved, and the extraction efficiency of the flavonoid compounds is improved.

In the step S043, the third extractant is a mixed solvent in which acetone and petroleum ether are mixed according to the volume ratio of (2-4) to 1, the polarity of the mixed solvent is similar to that of the flavonoid compound, the second extract and the third extractant are mixed according to the volume ratio of 1 (3-10), and the flavonoid compound in the silica gel adsorption powder can be enriched in the third extractant. In some embodiments, the volume ratio of the second extract to the third extractant is 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1: 10. In some embodiments, the volume ratio of petroleum ether to acetone in the third extractant is 1:2, 1:3, 1: 4.

Specifically, in step S05, the propolis extract is decolorized, filtered, and the filtrate is concentrated to obtain a propolis refined extract.

The step of decolorizing the propolis extract can refer to the conventional operation in the field, for example, decolorizing by adopting methods such as activated carbon adsorption, macroporous resin adsorption or chemical reagent adsorption.

As an implementation mode, the propolis extract is dispersed in an ethanol water solution with the weight percentage concentration of 60% -95% of ethanol, and ultrasonic treatment is carried out. Compared with other existing decoloring treatment processes, the embodiment of the invention adopts 60-95 wt% ethanol water solution for ultrasonic treatment, has simple and convenient operation and high decoloring efficiency, and can realize decoloring and purification of the propolis extract at the level of hundred kilograms. In specific embodiments, the aqueous ethanol solution has a concentration of 60%, 65%, 70%, 75%, 80%, 85%, 95% ethanol by weight. In a further embodiment, the time of the sonication is 30 min. In a further embodiment, before the step of dispersing the propolis extract in an ethanol water solution with the weight percentage concentration of ethanol of 60-95%, the propolis extract is subjected to crushing treatment to improve the decoloring efficiency.

In summary, in the preparation method of the propolis extract provided by the embodiment of the present invention, the propolis raw material is extracted with an ethanol aqueous solution with a weight percentage concentration of ethanol of more than 70% to enrich active ingredients such as flavonoids in propolis; then, freezing the crude propolis extract to separate out residual beeswax in the crude propolis extract, and separating and removing the beeswax by dispersing the beeswax in an ethanol water solution with the weight percentage concentration of ethanol being more than 70%, so that the influence of the residual beeswax on the subsequent separation and purification of the active ingredients of the total flavonoids is avoided, and the further enrichment of the active ingredients of the total flavonoids in the extract is promoted; then, mixing and adsorbing the propolis concentrated solution and silica gel to enable active ingredients in the propolis concentrated solution to be adsorbed on the silica gel powder, performing solid-liquid extraction on the silica gel adsorbed powder by adopting petroleum ether-acetone systems with different volume ratios, collecting petroleum ether-acetone extraction parts with volume ratios of 1 (2-4), and concentrating to obtain a propolis extract, wherein the method is simple and can realize large-scale production of the propolis extract at the level of hundred kilograms; and then, decoloring the propolis extract. The detection shows that the content of total brass active ingredients in the propolis refined extract prepared by the method is as high as 98.1%, and the active ingredients are clear, so that the propolis refined extract can be well applied to preparing health-care food, medicines and beauty raw materials and is safe to use.

Based on the technical scheme, the embodiment of the invention also provides a propolis extract and application thereof.

Correspondingly, the propolis extract is prepared by the preparation method, and the active ingredients in the propolis extract comprise chrysin, pinocembrin, galangin, caffeic acid phenethyl ester, kaempferol, pinobanksin and acacetin.

The propolis extract provided by the embodiment of the invention is prepared by the preparation method, the purity is as high as 98.1%, the active ingredients of the propolis extract mainly comprise chrysin, pinocembrin, galangin, caffeic acid phenethyl ester, kaempferol, pinobanksin, farnesene and other flavonoid compounds, the active ingredients are clear, and the propolis extract can be widely applied to preparation of health-care food, medicines and beauty raw materials and is safe to use.

As an embodiment, in the propolis extract, the weight percentage of the chrysin is 18% to 23%, the weight percentage of the pinocembrin is 12% to 17%, the weight percentage of the galangin is 7% to 12%, the weight percentage of the caffeic acid phenethyl ester is 6% to 11%, the weight percentage of the kaempferol is 9% to 14%, the weight percentage of the pinobanksin is 7% to 12%, and the weight percentage of the farnesin is 4% to 9%. Compared with the propolis extract prepared by the prior art, the propolis extract prepared by the method in the embodiment has obviously improved contents of flavonoids such as chrysin, pinocembrin, galangin, caffeic acid phenethyl ester, kaempferol, pinobanksin and acacetin.

Accordingly, the above preparation method or the above propolis extract can be used for preparing health food, medicine and skin caring materials.

Further, based on the above technical solution, the embodiment of the present invention further provides a method for detecting the propolis extract prepared by the above preparation method, which can effectively separate the active ingredients of chrysin, pinocembrin, galangin, caffeic acid phenethyl ester, kaempferol, pinobanksin and farnesol in the propolis extract, so as to achieve baseline separation, and realize qualitative and quantitative detection of the active ingredients in the test solution.

Correspondingly, the detection method of the propolis extract comprises the following active ingredients: chrysin, pinocembrin, galangin, caffeic acid phenethyl ester, kaempferol, pinobanksin and acacetin;

A. the detection method of the propolis extract comprises the following steps:

B. dissolving the propolis extract in a solvent to prepare a test solution;

C. detecting the test solution by high performance liquid chromatography to obtain qualitative and quantitative detection result of the propolis extract active components;

wherein, the conditions of the high performance liquid chromatography are as follows: octadecylsilane chemically bonded silica gel column; using acetonitrile as fluidity A, using phosphoric acid aqueous solution as fluidity B, and carrying out gradient elution, wherein the procedure of the gradient elution is as follows:

in some embodiments, the concentration of the propolis extract in the test solution is 0.02-0.08 mg/L.

In some embodiments, the step of dissolving the propolis extract in a solvent includes: at least one of alcohol, an alcohol aqueous solution, acetonitrile, an acetonitrile aqueous solution, and a phosphoric acid aqueous solution. Wherein the alcohol includes, but is not limited to, methanol, ethanol, and the like.

In some embodiments, the phosphoric acid in the aqueous phosphoric acid solution has a concentration of 0.01% to 0.20% by weight.

In order to make the above implementation details and operations of the present invention clearly understood by those skilled in the art, and to make the progress of the method for preparing propolis extract according to the embodiment of the present invention obvious, the implementation of the present invention will be illustrated by the following examples.

Example 1

The embodiment prepares the propolis extract, and specifically comprises the following steps:

s11, taking propolis crude gum with uniform quality from Achestak Huangshan of Anhui, cleaning to remove physical impurities, and freezing at-15 deg.C for 10 hr to obtain frozen propolis crude gum; pulverizing about 1kg of frozen propolis into coarse powder at 2-10 deg.C to obtain propolis crude crushed material;

s12, as 1g: extracting crushed propolis raw materials with 95% ethanol for 3 times (2 hr each time) at a ratio of 5mL, filtering, mixing filtrates, concentrating the filtrate with rotary evaporator to fluid extract to obtain crude propolis extract (relative density at 25 deg.C: 1.15);

s13, freezing the crude propolis extract prepared in the step S12 at-15 ℃ for 24 hours, adding 75% ethanol at room temperature according to the material-liquid ratio of 1g:5mL for ultrasonic extraction, filtering and separating ethanol extract and beeswax impurity precipitate, concentrating the ethanol extract and recovering ethanol to obtain a concentrated propolis solution (the solid-liquid ratio at 25 ℃ is 1g:3 mL);

s14, adding 600g of silica gel into the propolis concentrated solution prepared in the step S13, uniformly mixing, concentrating by using a rotary evaporator to recover ethanol, pumping to dry until the powder is completely dried, and grinding to uniform fine powder to obtain silica gel adsorption powder;

s15, adding 5 times of the first extracting agent into the silica gel adsorption powder prepared in the step S14 by using petroleum ether with the boiling point of 60-90 ℃ as the first extracting agent, stirring for 4 hours, filtering, and naturally volatilizing filter residues to obtain a first extract;

adopting petroleum ether-acetone (3:1, vol/vol) as a second extractant, adding the second extractant with the volume 5 times that of the first extractant, stirring for 2 hours, filtering, and naturally volatilizing filter residues to obtain a second extract;

adopting petroleum ether-acetone (1:1, vol/vol) as a third extractant, adding the third extractant with the volume 5 times that of the second extractant, stirring for 2 hours, filtering, and naturally volatilizing filter residues to obtain a third extract;

adopting petroleum ether-acetone (1:3, vol/vol) as a fourth extractant, adding the fourth extractant with the volume 5 times that of the third extractant, stirring and extracting for 2 times, 2h each time, filtering, combining filtrates, concentrating the filtrate by using a rotary evaporator to dry to obtain a propolis extract;

s16, pulverizing propolis extract into fine powder, adding 65% ethanol 3 times the volume of propolis extract, performing ultrasonic treatment for 30min, decolorizing, filtering, and vacuum drying the filter residue under reduced pressure for 5 hr to obtain refined propolis extract 105g with light yellow to yellow color.

Example 2

s21, taking propolis crude gum which is produced from Dabie mountain Jinzhai in Anhui, washing to remove physical impurities, and freezing at-20 ℃ for 15 hours to obtain frozen propolis crude gum; pulverizing about 1kg of frozen propolis into coarse powder at 2-10 deg.C to obtain propolis crude crushed material;

s22, as 1g: extracting the crushed crude propolis material with ethanol for 2 times at a ratio of 8mL, each time for 3 hours, filtering, mixing filtrates, concentrating the filtrate with rotary evaporator to fluid extract to obtain crude propolis extract (relative density at 25 deg.C: 1.20);

s23, freezing the crude propolis extract prepared in the step S22 at-18 ℃ for 20 hours, adding 80% ethanol at room temperature according to the material-liquid ratio of 1g:10mL for ultrasonic extraction, filtering and separating ethanol extract and beeswax impurity precipitate, concentrating the ethanol extract and recovering ethanol to obtain a concentrated propolis solution (the solid-liquid ratio at 25 ℃ is 1g:2 mL);

s24, adding 500g of silica gel into the propolis concentrated solution prepared in the step S23, uniformly mixing, concentrating by using a rotary evaporator to recover ethanol, pumping to dry until the powder is completely dried, and grinding to uniform fine powder to obtain silica gel adsorption powder;

s25, adding 8 times of the first extracting agent into the silica gel adsorption powder prepared in the step S24 by using petroleum ether with the boiling point of 60-90 ℃ as the first extracting agent, stirring for 2 hours, filtering, and naturally volatilizing filter residues to obtain a first extract;

adopting petroleum ether-acetone (3:1, vol/vol) as a second extractant, adding 8 times of the second extractant into the first extract, stirring for 2 hours, filtering, and naturally volatilizing filter residues to obtain a second extract;

adopting petroleum ether-acetone (1:1, vol/vol) as a third extractant, adding 8 times of the third extractant into the second extract, stirring for 2 hours, filtering, and naturally volatilizing filter residues to obtain a third extract;

adopting petroleum ether-acetone (1:3, vol/vol) as a fourth extractant, adding 8 times of the fourth extractant into the third extract, stirring and extracting for 2 times, 3h each time, filtering, combining filtrates, concentrating the filtrate by using a rotary evaporator to dry to obtain propolis extract;

s26, pulverizing propolis extract into fine powder, adding 75% ethanol 3 times the volume of propolis extract, performing ultrasonic treatment for 30min, decolorizing, filtering, and vacuum drying the filter residue under reduced pressure for 6 hr to obtain refined propolis extract 115g with light yellow to yellow color.

Example 3

s31, taking propolis crude gum which is produced from Henan Taiwan mountain and has uniform quality, cleaning to remove physical impurities, and freezing at-18 ℃ for 15 hours to obtain frozen propolis crude gum; pulverizing about 1kg of frozen propolis into coarse powder at 2-10 deg.C to obtain propolis crude crushed material;

s32, as 1g: extracting the crushed crude propolis material with ethanol at a ratio of 10mL for 3 times with each time of 3 hours under stirring, filtering, mixing filtrates, concentrating the filtrate with rotary evaporator to fluid extract to obtain crude propolis extract (relative density at 25 deg.C: 1.25);

s33, freezing the crude propolis extract prepared in the step S32 at-18 ℃ for 24 hours, adding 80% ethanol at room temperature according to the material-liquid ratio of 1g:6mL for ultrasonic extraction, filtering and separating ethanol extract and beeswax impurity precipitate, concentrating the ethanol extract and recovering ethanol to obtain a concentrated propolis solution (the solid-liquid ratio at 25 ℃ is 1g:2.5 mL);

s34, adding 800g of silica gel into the propolis concentrated solution prepared in the step S33, uniformly mixing, concentrating by using a rotary evaporator to recover ethanol, pumping to dry until the powder is completely dried, and grinding to uniform fine powder to obtain silica gel adsorption powder;

s35, adding 10 times of the first extracting agent into the silica gel adsorption powder prepared in the step S34 by taking petroleum ether with the boiling point of 60-90 ℃ as the first extracting agent, stirring for 1 hour, filtering, and naturally volatilizing filter residues to serve as a first extract;

adopting petroleum ether-acetone (3:1, vol/vol) as a second extractant, adding 10 times of the second extractant into the first extract, stirring for 1h, filtering, and naturally volatilizing filter residues to obtain a second extract;

adopting petroleum ether-acetone (1:1, vol/vol) as a third extractant, adding 10 times of the third extractant into the second extract, stirring for 2 hours, filtering, and naturally volatilizing filter residues to obtain a third extract;

adopting petroleum ether-acetone (1:3, vol/vol) as a fourth extractant, adding 10 times of the fourth extractant into the third extract, stirring and extracting for 3 times, 2h each time, filtering, combining filtrates, concentrating the filtrate by using a rotary evaporator to dry to obtain propolis extract;

s36, pulverizing propolis extract into fine powder, adding 75% ethanol 3 times the volume of propolis extract, performing ultrasonic treatment for 30min, decolorizing, filtering, and vacuum drying the filter residue under reduced pressure for 6 hr to obtain refined propolis extract 125g with light yellow to yellow color.

Example 4

s41, taking propolis crude gum with uniform quality from Shanxi Jincheng, washing to remove physical impurities, and freezing at-10 ℃ for 15 hours to obtain frozen propolis crude gum; pulverizing about 1kg of frozen propolis into coarse powder at 2-10 deg.C to obtain propolis crude crushed material;

s42, as 1g: extracting crushed propolis raw materials with 90% ethanol for 2 times (2 hr each time) at a ratio of 10mL, filtering, mixing filtrates, concentrating the filtrate with rotary evaporator to fluid extract to obtain crude propolis extract (relative density at 25 deg.C: 1.24);

s43, freezing the crude propolis extract prepared in the step S42 at-18 ℃ for 20 hours, adding 80% ethanol at room temperature according to the material-liquid ratio of 1g:6mL for ultrasonic extraction, filtering and separating ethanol extract and beeswax impurity precipitate, concentrating the ethanol extract and recovering ethanol to obtain a concentrated propolis solution (the solid-liquid ratio at 25 ℃ is 1g:2 mL);

s44, adding 700g of silica gel into the propolis concentrated solution prepared in the step S43, uniformly mixing, concentrating by using a rotary evaporator to recover ethanol, pumping to dry until the powder is completely dried, and grinding to uniform fine powder to obtain silica gel adsorption powder;

s45, adding 7 times of the first extracting agent into the silica gel adsorption powder prepared in the step S44 by using petroleum ether with the boiling point of 60-90 ℃ as the first extracting agent, stirring for 1 hour, filtering, and naturally volatilizing filter residues to obtain a first extract;

adopting petroleum ether-acetone (3:1, vol/vol) as a second extractant, adding the second extractant with the volume 7 times that of the first extractant, stirring for 1h, filtering, and naturally volatilizing filter residues to obtain a second extract;

adopting petroleum ether-acetone (1:1, vol/vol) as a third extractant, adding 7 times of the third extractant into the second extract, stirring for 2 hours, filtering, and naturally volatilizing filter residues to obtain a third extract;

adopting petroleum ether-acetone (1:3, vol/vol) as a fourth extractant, adding the fourth extractant with the volume 7 times that of the third extractant, stirring and extracting for 2 times, 3 hours each time, filtering, combining filtrates, concentrating the filtrate by using a rotary evaporator until the filtrate is dried to obtain a propolis extract;

s46, pulverizing propolis extract into fine powder, adding 95% ethanol 3 times the volume of propolis extract, performing ultrasonic treatment for 30min, decolorizing, filtering, and vacuum drying the filter residue under reduced pressure for 6 hr to obtain refined extract 112g of propolis with light yellow to yellow color.

Example 5

s51, taking propolis crude gum with uniform quality from Zhongshan of Guangdong, cleaning to remove physical impurities, and freezing at-16 deg.C for 20 hr to obtain frozen propolis crude gum; pulverizing about 1kg of frozen propolis into coarse powder at 2-10 deg.C to obtain propolis crude crushed material;

s52, as 1g: extracting crushed propolis raw materials with 90% ethanol for 2 times (3 hr each time) at a ratio of 5mL, filtering, mixing filtrates, concentrating the filtrate with rotary evaporator to fluid extract to obtain crude propolis extract (relative density at 25 deg.C: 1.22);

s53, freezing the crude propolis extract prepared in the step S52 at-18 ℃ for 24 hours, adding 80% ethanol at room temperature according to a material-liquid ratio of 1g:7mL, carrying out ultrasonic extraction, filtering and separating an ethanol extract and beeswax impurity precipitates, concentrating the ethanol extract, and recovering ethanol to obtain a concentrated propolis solution (the solid-liquid ratio at 25 ℃ is 1g:2 mL);

s54, adding 600g of silica gel into the propolis concentrated solution prepared in the step S53, uniformly mixing, concentrating by using a rotary evaporator to recover ethanol, pumping to dry until the powder is completely dried, and grinding to uniform fine powder to obtain silica gel adsorption powder;

s55, adding 5 times of the first extracting agent into the silica gel adsorption powder prepared in the step S54 by using petroleum ether with the boiling point of 60-90 ℃ as the first extracting agent, stirring for 1 hour, filtering, and naturally volatilizing filter residues to obtain a first extract;

adopting petroleum ether-acetone (3:1, vol/vol) as a second extractant, adding the second extractant with the volume 5 times that of the first extractant, stirring for 1h, filtering, and naturally volatilizing filter residues to obtain a second extract;

s56, pulverizing propolis extract into fine powder, adding 95% ethanol 3 times the volume of propolis extract, performing ultrasonic treatment for 30min, decolorizing, filtering, and vacuum drying the filter residue under reduced pressure for 6 hr to obtain refined propolis extract 122g with light yellow to yellow color.

Example 6

s61, taking propolis crude gum with uniform quality from Taishan mountain, cleaning to remove physical impurities, and freezing at-15 deg.C for 20 hr to obtain frozen propolis crude gum; pulverizing about 1kg of frozen propolis into coarse powder at 2-10 deg.C to obtain propolis crude crushed material;

s62, as 1g: extracting crushed propolis raw materials with 90% ethanol for 2 times (each time for 3 hr) at a material-to-liquid ratio of 10mL, filtering, mixing filtrates, concentrating the filtrate with rotary evaporator to fluid extract to obtain crude propolis extract (relative density at 25 deg.C: 1.22);

s63, freezing the crude propolis extract prepared in the step S62 at-18 ℃ for 24 hours, adding 80% ethanol at room temperature according to a material-liquid ratio of 1g:8mL for ultrasonic extraction, filtering and separating an ethanol extract and beeswax impurity precipitates, concentrating the ethanol extract and recovering ethanol to obtain a concentrated propolis solution (the solid-liquid ratio at 25 ℃ is 1g:2 mL);

s64, adding 600g of silica gel into the propolis concentrated solution prepared in the step S63, uniformly mixing, concentrating by using a rotary evaporator to recover ethanol, pumping to dry until the powder is completely dried, and grinding to uniform fine powder to obtain silica gel adsorption powder;

s65, adding 5 times of the first extracting agent into the silica gel adsorption powder prepared in the step S64 by using petroleum ether with the boiling point of 60-90 ℃ as the first extracting agent, stirring for 1 hour, filtering, and naturally volatilizing filter residues to obtain a first extract;

s66, pulverizing propolis extract into fine powder, adding 95% ethanol 3 times the volume of propolis extract, performing ultrasonic treatment for 30min, decolorizing, filtering, and vacuum drying the filter residue under reduced pressure for 6 hr to obtain 128g of propolis refined extract with light yellow to yellow color.

Test example

Dissolving the refined extracts of propolis obtained in examples 1-6 in methanol respectively to obtain test solution with concentration of 0.02-0.08 mg/mL; detecting the test solution by HPLC, qualitatively and quantitatively detecting the active ingredients in the test solution, wherein the conditions of the high performance liquid chromatography are as follows:

a chromatographic column: octadecylsilane chemically bonded silica gel column;

column temperature: 30 ℃;

the detection wavelength is 289 nm;

the theoretical plate number is not less than 3000 calculated according to the pinocembrin peak;

using acetonitrile as fluidity A, and phosphoric acid aqueous solution with the weight percentage concentration of 0.1% as fluidity B, and performing gradient elution, wherein the procedure of the gradient elution is shown in Table 1:

TABLE 1

Time of day	Mobile phase A	Mobile phase B
			0-55 minutes	34％	66％
55-60 minutes	100％	0
			60-72 minutes	66％	34％

The qualitative and quantitative detection results of the propolis extract active ingredients shown in table 2 and fig. 1 were obtained by HPLC detection. As shown by the results, the active ingredients such as chrysin, pinocembrin, galangin, caffeic acid phenethyl ester, kaempferol, pinobanksin and acacetin in the propolis refined extract are separated from the baseline, and the total content of the active ingredients accounts for 61.45-98.1% of the weight of the propolis refined extract. Wherein, the chrysin accounts for 18 to 23 percent, the pinocembrin accounts for 12 to 17 percent, the galangin accounts for 7 to 12 percent, the caffeic acid phenethyl ester accounts for 6 to 11 percent, the kaempferol accounts for 9 to 14 percent, the pinobanksin accounts for 7 to 12 percent, and the farnesin accounts for 4 to 9 percent.

TABLE 2

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims

1. The preparation method of the propolis extract is characterized by comprising the following steps:

2. The preparation method according to claim 1, wherein in the step of extracting the propolis raw crushed materials with an ethanol aqueous solution with a weight percentage concentration of ethanol of more than 70%, the propolis raw crushed materials and the ethanol aqueous solution are extracted for 2-3 hours for 1-4 times with stirring according to a feed-liquid ratio of 1g (3-10) mL.

3. The method according to claim 1, wherein in the step of freezing the crude propolis extract, the crude propolis extract is frozen at-10 to-20 ℃ for 20 to 24 hours.

4. The preparation method of claim 1, wherein in the step of dispersing the crude propolis extract in an ethanol aqueous solution with a concentration of ethanol of 70% by weight or more, the crude propolis extract and the ethanol aqueous solution are mixed according to a feed-to-liquid ratio of 1g (5-10) mL.

5. The method for preparing the propolis liquid concentrate according to any one of claims 1 to 4, wherein in the step of mixing the propolis liquid concentrate with the silica gel, the silica gel is added to the propolis liquid concentrate to be uniformly mixed, the silica gel being 20 to 100% by weight of the propolis feather gel.

6. The production method according to any one of claims 1 to 4, wherein the step of subjecting the silica gel-adsorbed powder to solid-liquid extraction using a first extractant, a second extractant, and a third extractant in this order comprises:

mixing the silica gel adsorption powder and the first extracting agent according to the volume ratio of 1 (3-10), stirring and extracting for 1-3 times, each time for 1-3 hours, filtering, and naturally volatilizing filter residues to obtain a first extract;

mixing the first extract and the second extractant according to the volume ratio of 1 (3-10), stirring and extracting for 1-3 times, each time for 1-3 hours, filtering, and naturally volatilizing filter residues to obtain a second extract;

and mixing the second extract and the third extractant according to the volume ratio of 1 (3-10), stirring and extracting for 1-3 hours, each time for 1-3 hours, and combining the stirred and extracted third extractants.

7. The preparation method according to any one of claims 1 to 4, wherein in the step of decoloring the propolis extract, the propolis extract is dispersed in an ethanol aqueous solution with a concentration of 60 to 95 percent by weight of ethanol, and subjected to ultrasonic treatment.

8. A propolis extract obtained by the method according to any one of claims 1 to 7, wherein the active ingredients of the propolis extract include chrysin, pinocembrin, galangin, caffeic acid phenethyl ester, kaempferol, pinobanksin and acacetin.

9. The propolis extract according to claim 8, wherein the propolis extract contains 18 to 23 weight percent of chrysin, 12 to 17 weight percent of pinocembrin, 7 to 12 weight percent of galangin, 6 to 11 weight percent of caffeic acid phenethyl ester, 9 to 14 weight percent of kaempferol, 7 to 12 weight percent of pinobanksin and 4 to 9 weight percent of acacetin.

10. Use of the preparation method of any one of claims 1 to 7 or the propolis extract of claim 8 or 9 for the preparation of health foods, pharmaceuticals and cosmetic materials.