阅读说明：本技术 合欢花总黄酮成分的提取纯化方法 (Method for extracting and purifying total flavonoids of albizia julibrissin ) 是由 许静远 周怡青 黄东婷 孙含章 孔颖 唐良晨 于 2021-01-28 设计创作，主要内容包括：本发明公开了合欢花总黄酮成分的提取纯化方法,其包括以下步骤：1)将合欢花进行冷冻干燥；2)取干燥的合欢花进行粉碎,过20目筛；3)称取合欢花粉末,加入乙醇利用超声波辅助提取；4)将提取后的物料进行过滤,合欢花粉饼再提取并过滤；5)将合欢花提取物滤液泵入单效外循环真空浓缩器中进行真空浓缩；6)将所得合欢花提取浓缩液上大孔树脂柱,分别用蒸馏水和不同浓度的乙醇溶液洗脱,收集某一浓度的乙醇洗脱液；7)将收集到的乙醇洗脱液真空浓缩；本发明方法填补现有技术领域的空白,缩短了提取、浓缩及过滤时间,降低了提取和浓缩温度,解决了传统热回流提取法引起的有效成分降解、提取周期长,提取效率低等问题。(The invention discloses a method for extracting and purifying total flavonoids of albizia julibrissin durazz, which comprises the following steps: 1) freeze drying flos Albizziae; 2) pulverizing dried flos Albizziae, and sieving with 20 mesh sieve; 3) weighing albizia julibrissin flower powder, adding ethanol, and performing ultrasonic-assisted extraction; 4) filtering the extracted material, and extracting and filtering the albizia julibrissin pollen cake; 5) pumping the albizia julibrissin durazz extract filtrate into a single-effect external circulation vacuum concentrator for vacuum concentration; 6) loading the concentrated extract of Albizzia julibrissin Durazz into macroporous resin column, eluting with distilled water and ethanol solution of different concentrations, and collecting ethanol eluate of certain concentration; 7) concentrating the collected ethanol eluent in vacuum; the method disclosed by the invention fills the blank in the prior art, shortens the extraction, concentration and filtration time, reduces the extraction and concentration temperature, and solves the problems of degradation of effective components, long extraction period, low extraction efficiency and the like caused by the traditional hot reflux extraction method.)

1. The extraction and purification method of the albizia flower total flavone component is characterized by comprising the following steps:

(1) freezing albizia flower or flower bud at-5-20 deg.c and freeze drying;

(2) pulverizing dried flos Albizziae or flower bud, sieving with 20 mesh sieve, and collecting sieved flos Albizziae powder;

(3) weighing albizzia flower powder, and mixing the raw materials according to a material-liquid ratio of 1: adding 30-80% ethanol into the mixture at 50-80 ℃, and extracting the mixture for 10-30 min at 25-40 ℃ by using ultrasonic wave assistance, wherein the ultrasonic frequency is 40 kHz;

(4) filtering the extracted material, collecting the albizia julibrissin flower extract filtrate, mixing the albizia julibrissin flower powder cake with 30-80% ethanol according to a material-liquid ratio of 1: 30-50, and extracting for 10-30 min at 25-40 ℃ by using ultrasonic wave assistance, wherein the ultrasonic frequency is 40 kHz;

(5) putting the albizia julibrissin durazzini extract filtrate into a liquid storage device, pumping the albizia julibrissin durazzini extract filtrate in the liquid storage device into a single-effect external circulation vacuum concentrator, carrying out vacuum concentration at the temperature of 30-40 ℃ and the vacuum degree of 0.02-0.05 MPa, and carrying out vacuum concentration until the taste is free of ethanol to obtain an albizia julibrissin durazzini extract concentrated solution;

(6) putting the obtained albizia julibrissin extraction concentrated solution into a macroporous resin column, eluting with 3-6 times of column volume of distilled water and 20% ethanol respectively, eluting with 4-8 times of column volume of 40-60% ethanol, and collecting 40-60% ethanol eluate;

(7) and (3) carrying out vacuum concentration on the collected 40-60% ethanol eluent by using a single-effect external circulation vacuum concentrator at the temperature of 30-40 ℃ and the vacuum degree of 0.02-0.05 MPa, and freeze-drying after concentration to obtain the albizia flower total flavone extract.

2. The method for extracting and purifying albizzia julibrissin total flavonoids according to claim 1, which comprises the steps of: in the step (2), the albizia julibrissin durazzini flower or flower bud is crushed by an ultramicro crusher.

3. The method for extracting and purifying albizzia julibrissin total flavonoids according to claim 1, which comprises the steps of: in the step (4), the material is filtered by a plate-and-frame filter press.

4. The method for extracting and purifying albizzia julibrissin total flavonoids according to claim 1, which comprises the steps of: in step (6), the macroporous resin column is HP2MGL or SP 207.

Technical Field

The invention relates to an extraction and purification method of albizia flower total flavone components, which is used for extracting the albizia flower total flavone components.

Background

Albizia flower is flower or bud of Albizzia (Albizia julibrissin Durazz) belonging to Albizzia of Leguminosae, has sweet and mild taste, and can be used for treating uneasiness, depression and insomnia. The existing research shows that the albizia flower is rich in flavonoid compounds, and the total flavonoid extract can improve the learning capacity reduction caused by schizophrenia by regulating the protein expression of S100-beta, c-FoS and the like in brain hippocampal tissues. Meanwhile, the albizzia julibrissin total flavone can also inhibit apoptosis inducing protein expression and apoptosis of hippocampal cells to improve chronic depression by inducing secretion of serotonin, norepinephrine and neurogenic nerve growth factor in brain. In addition, the albizia flower total flavonoids have certain antianxiety effect. Therefore, the albizia flower total flavonoids can be used as a potential research and development object of medicaments for treating mental diseases such as depression and anxiety and can also be developed into nerve-soothing health-care products or related dietary supplements.

At present, researches on the extraction process of the total flavonoids of the albizia julibrissin flowers are mostly developed around small-scale extraction in a laboratory, the obtained total flavonoids extract has small yield and low purity, an industrial extraction method and an efficient enrichment method suitable for the total flavonoids of the albizia julibrissin flowers are not available, and the requirements of large-scale raw materials for deep research, development and use of the total flavonoids of the albizia julibrissin flowers cannot be met.

Disclosure of Invention

The invention aims to solve the technical problem of providing an extraction and purification method of total flavonoids in albizia julibrissin durazz components, and realizing industrial large-scale extraction and purification of the total flavonoids in albizia julibrissin durazz.

In order to solve the technical problems, the method for extracting and purifying the albizia flower total flavone components comprises the following steps:

(1) freezing albizzia julibrissin at a low temperature of between-5 and-20 ℃ and then carrying out freeze drying; the flos Albizziae is fresh flos Albizziae or bud not full of flos Albizziae collected from Albizziae tree at noon in clear weather between 7 months and 8 months in summer to ensure that total flavone accumulation in raw materials reaches the highest level. Meanwhile, the freeze drying technology is adopted to ensure that the flavonoid components of the albizia julibrissin durazz are not changed in the drying process.

(2) The method is characterized in that dried albizia flower is crushed, the collected albizia flower usually has more pedicel, the total flavone content in the pedicel is low, the plant fiber is more, and if the collected albizia flower is not sieved, the more pedicel fiber exists in sample powder, so that the amount of used extraction solvent is increased, and the total flavone extraction rate is reduced. Therefore, the crushed albizia julibrissin flowers are screened by a 20-mesh sieve, part of the large-volume pedicel fibers can be removed, and then the screened albizia julibrissin pollen is collected for later use; preferably, the albizia julibrissin durazzini flower is crushed by an ultramicro crusher.

(3) Weighing albizzia flower powder, and mixing according to a material-liquid ratio (albizzia flower mass/ethanol solution volume) of 1: 50-80, so as to ensure that the albizia julibrissin pollen can fully absorb the ethanol solution, and meanwhile, enough ethanol solution can immerse the albizia julibrissin pollen, thereby being beneficial to extraction. If the feed-liquid ratio is lower than 1: 50, the residual solution is too little after the albizia flower pollen absorbs the ethanol solution, the content of the total flavonoids in the extraction solvent is saturated, the total flavonoids in the albizia flower are not favorably dissolved out completely, and the extraction rate is low; the feed liquid ratio is higher than 1: at 80 hours, the total flavonoids in the albizia flower powder are fully dissolved out, the extraction rate cannot be further remarkably increased, solution waste is caused, the time consumed by subsequent concentration is increased, and the extraction efficiency is reduced. The concentration of the ethanol solution used for extraction is 30-80% to ensure that the albizia flower total flavonoids are fully dissolved in the ethanol extract, which is beneficial to extraction. Because the majority of the components of the albizia flower total flavonoids are medium-polarity compounds, if the concentration of the ethanol extracting solution is lower than 30%, the polarity of the extracting solvent is greater than that of the albizia flower total flavonoids, according to the principle that the polarities of the compounds are similar and compatible, when the same-volume large-polarity extracting solvent is used, the dissolution of the albizia flower total flavonoids is less, and the dissolution of large-polarity impurities is increased, the extraction rate of the total flavonoids is reduced, and the subsequent purification is not facilitated; when the concentration of the ethanol extract is higher than 80%, the polarity of the extraction solvent is less than that of the albizzia flower total flavonoids, the total flavonoids are dissolved out less, the less polar impurities are dissolved out more, the total flavonoids extraction rate is also reduced, and the subsequent total flavonoids enrichment and purification are not facilitated. The albizzia julibrissin pollen is mixed with an ethanol solution according to the proportion, and then ultrasonic-assisted extraction is carried out at the temperature of 25-40 ℃. When the ultrasonic temperature is lower than 25 ℃, the total flavonoids of the albizia julibrissin durazz is insufficiently extracted and the extraction rate is reduced due to low molecular kinetic energy and low solubility of the flavonoids in the extraction solvent and less dissolution of the flavonoids in the same extraction time; meanwhile, the ultrasonic extraction temperature is not higher than 40 ℃, and the albizia flower total flavone mainly contains flavone glycoside compounds, so that the glycoside bonds of the glycoside compounds can be hydrolyzed under the water-containing condition at a higher temperature, so that the albizia flower extract is decomposed, and the extraction rate of the total flavone is reduced. And (4) carrying out ultrasonic treatment for 10-30 min to ensure that the albizia flower total flavonoids are fully extracted. When the ultrasonic time is less than 10min, the albizia flower total flavonoids cannot be fully dissolved into the extraction solvent, so that the albizia flower total flavonoids are less dissolved out, and the extraction rate is low; when the ultrasonic time is more than 30min, the total flavonoids of the albizia julibrissin durazzini are fully dissolved into the extraction solvent, the extraction rate is not obviously increased, more impurities are dissolved out after long-time extraction, the temperature of an extracted sample is increased due to long-time ultrasonic time, the decomposition of flavonoid components can be caused, and the subsequent enrichment and purification of the total flavonoids are not facilitated. The ultrasonic frequency was 40 kHz. The ultrasonic-assisted extraction device greatly shortens the extraction time, reduces the extraction temperature and the dosage of the extraction solvent, can solve the problems of degradation of effective components caused by the traditional hot reflux extraction method, reduction of energy consumption, dosage of the extraction solvent and the like, and is more favorable for energy conservation and environmental protection.

(4) Filtering the extracted material, collecting albizia julibrissin extract filtrate, mixing the albizia julibrissin powder cake with 30-80% ethanol solution according to a material-to-liquid ratio (albizia julibrissin mass/ethanol solution volume) of 1: 50-80, and extracting for 10-30 min at 25-40 ℃ by using ultrasonic wave assistance, wherein the ultrasonic frequency is 40 kHz. The material filters and adopts plate and frame filter press, adopts automatic plate and frame filter pressing device when the dregs of a decoction filter, can effectively avoid the pipeline that the medicinal material powder arouses to block up, has reduced half filtration time than the traditional method of extracting the direct filtration in jar simultaneously, has shortened whole extraction cycle, has greatly improved the extraction efficiency of flos Albizziae total flavone.

(5) Placing the albizia julibrissin durazzini extract filtrate into a liquid storage device, pumping the albizia julibrissin durazzini extract filtrate in the liquid storage device into a single-effect external circulation vacuum concentrator, concentrating at the temperature of 30-40 ℃, wherein the temperature is not lower than 30 ℃ during concentration, otherwise, the evaporation speed of an extraction solvent is low, the concentration time of an albizia julibrissin durazzini extract is long, and the extraction efficiency is reduced; meanwhile, the albizia flower total flavonoids are rich in flavone glycoside compounds, and the glycosidic bond of the flavone glycoside compounds is easy to hydrolyze at a high temperature under the water-containing condition, so that the temperature during concentration is not higher than 40 ℃, and the total albizia flower flavonoid extract is prevented from being decomposed. Because the boiling point of the solvent is reduced along with the reduction of the air pressure, the solvent can be quickly evaporated at a lower temperature in a high vacuum degree, so that the vacuum degree of the vacuum concentrator is set to be 0.02-0.05 MPa in concentration so as to reduce the boiling point of the albizia julibrissin alcohol extract, facilitate the recovery of the extraction solvent at a lower temperature, shorten the extraction time and improve the concentration efficiency. Concentrating the albizzia julibrissin extracting solution until the extract has no ethanol taste to obtain the albizzia julibrissin extracting concentrated solution.

(6) Adsorbing the obtained albizia flower extract concentrated solution by using an HP2MGL or SP207 macroporous resin column, wherein distilled water and a 20% ethanol solution belong to a high-polarity solvent according to a similar intermiscibility principle, so that the high-polarity compound adsorbed by the macroporous resin can be dissolved and eluted, and the distilled water and the 20% ethanol with the column volume of 3-6 times can be used for eluting respectively to remove high-polarity impurities such as polysaccharide, water-soluble pigment and the like in the extract, so that the purity of the total flavonoids in the albizia flowers is improved. The albizia flower total flavonoids belong to medium-polarity compounds, 40-60% ethanol solution is a medium-polarity eluent, so that the macroporous resin column is eluted by 40-60% ethanol with the volume 4-8 times of the column volume, and the ethanol eluent is collected to obtain the enriched and purified albizia flower total flavonoids.

(7) Concentrating the collected ethanol eluent by using a single-effect external circulation vacuum concentrator at the temperature of 30-40 ℃, wherein the temperature during concentration is not lower than 30 ℃, otherwise, the evaporation speed of the extraction solvent is slow, and the concentration time of the albizia julibrissin extracting solution is long; meanwhile, the temperature during concentration is not higher than 40 ℃, so that the decomposition of the albizia julibrissin durazzini extract is avoided, and the purity of the albizia julibrissin total flavonoids is not influenced. The vacuum degree of the vacuum concentrator is set to be 0.02-0.05 MPa during concentration, the boiling point of ethanol eluent can be reduced by high vacuum degree, and the extraction solvent can be conveniently recovered at a lower temperature. And (4) freeze-drying the ethanol eluate until the ethanol smell is removed, thereby obtaining the albizia flower total flavone extract.

The method disclosed by the invention fills the blank in the prior art, shortens the extraction, concentration and filtration time, reduces the extraction and concentration temperature, and solves the problems of degradation of effective components, long extraction period, low extraction efficiency and the like caused by the traditional hot reflux extraction method. By adopting the ultrasonic auxiliary extraction device, the extraction time is greatly shortened, the extraction temperature is reduced, the problems of long extraction time, degradation of effective components and the like of the traditional hot reflux extraction method are solved, and the extraction rate of the total flavonoids in the albizia julibrissin durazzini reaches 3.35-3.5% (mass of extract/mass of medicinal material of the albizia julibrissin durazzini). According to the method, the extract is filtered by adopting the plate-and-frame filter pressing device, so that the blockage of a filtering pipeline of an extraction tank caused by medicinal material powder is effectively avoided, meanwhile, compared with the traditional method of directly filtering in the extraction tank, the filtering time is reduced by half, the extraction period of the total flavonoids in the albizia julibrissin durazzini is shortened, and the extraction and purification efficiency is improved. The method of the invention is adopted to enrich the total flavonoids of the albizia julibrissin, and the purity of the obtained total flavonoids of the albizia julibrissin can reach 40-41.6%.

Detailed Description

Example 1

Fresh flos Albizziae or flower buds not blooming collected from trees at a sunny noon in summer between 7 months and 8 months. Freezing the collected fresh silktree albizzia flower at-5 ℃ for 10h, and then freeze-drying for 8 h; collecting 10kg of freeze-dried albizia flower, then crushing the albizia flower by using an ultrafine grinder, sieving the crushed albizia flower by using a 20-mesh sieve, and collecting sieved albizia flower powder. Taking a certain amount of albizia flower powder, and mixing the powder according to a material-liquid ratio of 1: 50 adding 30% ethanol; then extracting with ultrasound at 25 deg.C under 40KHz for 30 min; and transferring the extracted sample to a plate-and-frame filter press for filter pressing, wherein the filter pressing operation time is about 3 hours, and the filter pressing operation time is shortened by 3 hours compared with the method of filtering in an extraction tank. And (4) collecting the filtrate, pumping the filtrate into a liquid storage device, collecting the filter cake, extracting once again according to the above conditions, and then performing filter pressing to collect the filtrate. Mixing the two filtrates, pumping into a single-effect external circulation vacuum concentrator, vacuum concentrating at 40 deg.C under 0.02MPa until no alcohol smell is obtained, and shortening extraction period by 6h, wherein the extraction rate of flos Albizziae total flavone is 3.35% (extract quality/flos Albizziae medicinal material quality). Diluting the obtained concentrated solution with equal volume of water, loading onto macroporous resin HP2MGL, eluting with 6 times of column volume of distilled water and 20% ethanol, eluting with 40% ethanol for 8 column volumes, and collecting 40% ethanol eluate; vacuum concentrating the collected ethanol eluate with single-effect external circulation vacuum concentrator at 40 deg.C under vacuum degree of 0.02MPa, and freeze drying for 24 hr to obtain flos Albizziae total flavone extract with total flavone purity of 40%.

Example 2

Fresh flos Albizziae or flower buds not blooming collected from trees at a sunny noon in summer between 7 months and 8 months. Freezing the collected fresh silktree albizzia flowers at-10 ℃ for 8h, and then freeze-drying for 8 h; collecting 10kg of freeze-dried albizia flower, then crushing the albizia flower by using an ultrafine grinder, sieving the crushed albizia flower by using a 20-mesh sieve, and collecting sieved albizia flower powder. Taking a certain amount of albizia flower powder, and mixing the powder according to a material-liquid ratio of 1: 60 adding 50% ethanol; then extracting with ultrasound at 30 deg.C and 40KHz for 20 min; and transferring the extracted sample to a plate-and-frame filter press for filter pressing, wherein the filter pressing operation time is about 3.6 hours, and the filter pressing operation time is shortened by 3.6 hours compared with the method for filtering in an extraction tank. And (4) collecting the filtrate, pumping the filtrate into a liquid storage device, collecting the filter cake, extracting once again according to the above conditions, and then performing filter pressing to collect the filtrate. Mixing the two filtrates, pumping into a single-effect external circulation vacuum concentrator, vacuum concentrating at 35 deg.C under vacuum degree of 0.02MPa until no alcohol smell is produced, and the extraction rate of flos Albizziae total flavone reaches 3.41% (extract quality/flos Albizziae medicinal material quality), and the extraction period is shortened by 7.2 h. Diluting the obtained concentrated solution with equal volume of water, loading onto macroporous resin HP2MGL, eluting with 5 times column volume of distilled water and 20% ethanol, eluting with 50% ethanol for 6 column volumes, and collecting 50% ethanol eluate; vacuum concentrating the collected ethanol eluate with single-effect external circulation vacuum concentrator at 35 deg.C under vacuum degree of 0.02MPa, and freeze drying for 24 hr to obtain flos Albizziae total flavone extract with total flavone purity of 40.6%.

Example 3

Fresh flos Albizziae or flower buds not blooming collected from trees at a sunny noon in summer between 7 months and 8 months. Freezing the collected fresh albizia julibrissin at-15 ℃ for 6h, and then freeze-drying for 8 h; collecting 10kg of freeze-dried albizia flower, then crushing the albizia flower by using an ultrafine grinder, sieving the crushed albizia flower by using a 20-mesh sieve, and collecting sieved albizia flower powder. Taking a certain amount of albizia flower powder, and mixing the powder according to a material-liquid ratio of 1: 70% ethanol is added; then extracting for 15min by ultrasonic under the conditions of 35 ℃ and 40 KHz; and transferring the extracted sample to a plate-and-frame filter press for filter pressing, wherein the filter pressing operation time is about 4.2h, and the filter pressing operation time is shortened by 4.2h compared with the method of filtering in an extraction tank. And (4) collecting the filtrate, pumping the filtrate into a liquid storage device, collecting the filter cake, extracting once again according to the above conditions, and then performing filter pressing to collect the filtrate. Mixing the two filtrates, pumping into a single-effect external circulation vacuum concentrator, vacuum concentrating at 30 deg.C and vacuum degree of 0.03MPa until no alcohol smell is produced, and the extraction rate of flos Albizziae total flavone reaches 3.47% (mass of extract/mass of flos Albizziae medicinal material), and the extraction period is shortened by 8.4 h. Diluting the obtained concentrated solution with equal volume of water, introducing into macroporous resin SP207, eluting with 4 times of column volume of distilled water and 20% ethanol, eluting with 50% ethanol for 5 column volumes, and collecting 50% ethanol eluate; vacuum concentrating the collected ethanol eluate with single-effect external circulation vacuum concentrator at 30 deg.C and vacuum degree of 0.03MPa, and freeze drying for 24 hr to obtain flos Albizziae total flavone extract with total flavone purity of 40.8%.

Example 4

Fresh flos Albizziae or flower buds not blooming collected from trees at a sunny noon in summer between 7 months and 8 months. Freezing the collected fresh albizia julibrissin at-20 ℃ for 4h, and then freeze-drying for 8 h; collecting 10kg of freeze-dried albizia flower, then crushing the albizia flower by using an ultrafine grinder, sieving the crushed albizia flower by using a 20-mesh sieve, and collecting sieved albizia flower powder. Taking a certain amount of albizia flower powder, and mixing the powder according to a material-liquid ratio of 1: 80 adding 80% ethanol; then extracting with ultrasound at 40 deg.C under 40KHz for 10 min; and transferring the extracted sample to a plate-and-frame filter press for filter pressing, wherein the filter pressing operation time is about 4.8 hours, and the filter pressing operation time is shortened by 4.8 hours compared with the method of filtering in an extraction tank. And (4) collecting the filtrate, pumping the filtrate into a liquid storage device, collecting the filter cake, extracting once again according to the above conditions, and then performing filter pressing to collect the filtrate. Mixing the two filtrates, pumping into a single-effect external circulation vacuum concentrator, vacuum concentrating at 30 deg.C under vacuum degree of 0.05MPa until no alcohol smell is produced, and the extraction rate of flos Albizziae total flavone reaches 3.5% (mass of extract/mass of flos Albizziae medicinal material), and the extraction period is shortened by 9.6 h. Diluting the obtained concentrated solution with equal volume of water, introducing into macroporous resin SP207, eluting with 3 times of column volume of distilled water and 20% ethanol, eluting with 60% ethanol for 4 column volumes, and collecting 60% ethanol eluate; vacuum concentrating the collected ethanol eluate with single-effect external circulation vacuum concentrator at 30 deg.C and vacuum degree of 0.05MPa, and freeze drying for 24 hr to obtain flos Albizziae total flavone extract with total flavone purity of 41.6%.

The above embodiments do not limit the present invention in any way, and all technical solutions obtained by means of equivalent substitution or equivalent transformation fall within the protection scope of the present invention.