阅读说明：本技术 一种基于杜仲叶活性物提取与分离于一体的三相分配方法 (Three-phase distribution method based on integration of extraction and separation of active substances of eucommia ulmoides leaves ) 是由 王润平 杨再波 吕文志 于 2019-11-12 设计创作，主要内容包括：本发明公开了一种基于杜仲叶活性物提取与分离于一体的三相分配方法,包括如下步骤：1)取杜仲叶粉末,加入胆碱色氨酸离子液体水溶液,浸提,然后离心去除不溶性物质；2)将K<Sub>3</Sub>PO<Sub>4</Sub>水溶液加入步骤1)的溶液中,搅拌均匀后,静置至形成三相；3)将步骤2)分相的溶液进行分层分离,其中下层为无机盐K<Sub>3</Sub>PO<Sub>4</Sub>富集相,通过向其中加入乙醇沉淀多糖,再通过旋蒸将乙醇回收后即得多糖；中层为蛋白富集相,直接分离回收；上层为胆碱色氨酸离子液体富集相,通过加入抗溶剂后将其中的绿原酸和黄铜进行沉淀,然后旋蒸回收抗溶剂,即得绿原酸和黄铜。本发明能够对杜仲叶中的蛋白、黄铜和绿原酸一体提取和分离,工艺简单,条件温和,分离时间短,效率高。(The invention discloses a three-phase distribution method based on the integration of extraction and separation of active substances from eucommia leaves, which comprises the following steps: 1) adding the eucommia ulmoides leaf powder into a choline tryptophan ionic liquid aqueous solution, leaching, and centrifuging to remove insoluble substances; 2) will K 3 PO 4 Adding the aqueous solution into the solution obtained in the step 1), uniformly stirring, and standing until three phases are formed; 3) carrying out layered separation on the solution subjected to phase separation in the step 2), wherein the lower layer is inorganic salt K 3 PO 4 Enriching the phase, precipitating polysaccharide by adding ethanol, and recovering ethanol by rotary evaporation to obtain polysaccharide; the middle layer is a protein enrichment phase and is directly separated and recovered; the upper layer is choline tryptophan ionic liquid enrichment phase, chlorogenic acid and brass in the choline tryptophan ionic liquid enrichment phase are precipitated after the anti-solvent is added, and then the anti-solvent is recovered through rotary evaporation, so that the chlorogenic acid and the brass are obtained. The invention can treat protein, brass and chlorogenic acid in eucommia leavesThe method has the advantages of integrated extraction and separation, simple process, mild conditions, short separation time and high efficiency.)

1. A three-phase distribution method based on the integration of extraction and separation of active substances of eucommia ulmoides leaves is characterized by comprising the following steps of:

1) taking choline tryptophan ionic liquid and inorganic salt K₃PO₄And is ready for use;

2) determination of choline tryptophan ionic liquid and inorganic salt K by cloud point method and weight method respectively₃PO₄The phase diagram and the line of the formed ionic liquid aqueous two-phase at 25 ℃;

3) adding the eucommia ulmoides leaf powder into a choline tryptophan ionic liquid aqueous solution, leaching for 40-80min, and then centrifuging to remove insoluble substances;

4) according to the phase diagram and the series line data of the step 2), adding the inorganic salt K₃PO₄Adding the aqueous solution into the solution obtained in the step 3), uniformly stirring, and standing until the solution phase is separated and balanced to form three phases;

5) carrying out layered separation on the solution subjected to phase separation in the step 4), wherein the lower layer is inorganic salt K₃PO₄Enriching the phase by adding ethanol to itPrecipitating polysaccharide, and recovering ethanol by rotary evaporation to obtain polysaccharide; the middle layer is a protein enrichment phase and is directly separated and recovered; the upper layer is choline tryptophan ionic liquid enrichment phase, chlorogenic acid and brass in the choline tryptophan ionic liquid enrichment phase are precipitated after the anti-solvent is added, and then the anti-solvent is recovered through rotary evaporation, so that the chlorogenic acid and the brass are obtained.

2. The eucommia ulmoides leaf active matter extraction and separation integrated three-phase distribution method according to claim 1, wherein: the folium Eucommiae powder is prepared by cleaning with tap water, air drying, and pulverizing into 0.1-1cm powder²Drying the small blocks in an oven at the temperature of 30-50 ℃ for 1-3h to obtain the food.

3. The eucommia ulmoides leaf active matter extraction and separation integrated three-phase distribution method according to claim 1, wherein: and 3) the concentration of the choline tryptophan ionic liquid aqueous solution in the step 3) is 35-50%.

4. The eucommia ulmoides leaf active matter extraction and separation integrated three-phase distribution method according to claim 1, wherein: in the step 3), the solid-to-liquid ratio of the eucommia ulmoides leaf powder to the choline tryptophan ionic liquid aqueous solution is 1: 5-25.

5. The eucommia ulmoides leaf active matter extraction and separation integrated three-phase distribution method according to claim 1, wherein: in the step 3), the temperature for leaching is 20-70 ℃.

6. The eucommia ulmoides leaf active matter extraction and separation integrated three-phase distribution method according to claim 1, wherein: in the step 4), the inorganic salt K is added₃PO₄The mass ratio of the water solution to the folium Eucommiae powder is 10-32:1-3, and inorganic salt K₃PO₄The concentration of the aqueous solution was 45%.

Technical Field

The invention relates to an extraction and separation method of eucommia ulmoides leaf active substances, in particular to a three-phase distribution method based on the integration of extraction and separation of eucommia ulmoides leaf active substances.

Background

Eucommia bark is a traditional Chinese medicinal material, is used as a medicine from old times, has the effects of tonifying liver and kidney, strengthening bones and muscles, reducing blood pressure, preventing miscarriage and the like, has unique curative effect on hypertension, and is considered to be one of the most ideal natural blood pressure reducing medicines. Modern researches also show that eucommia bark has the effects of reducing blood pressure, regulating immunity, resisting viruses, resisting tumors and the like. The eucommia ulmoides leaves have high yield, and the collection of the eucommia ulmoides leaves does not influence the growth of eucommia ulmoides trees, so the research and application of the eucommia ulmoides leaves have great potential value. Meanwhile, the modern analysis means researches prove that the medicinal effective components of the eucommia ulmoides leaves are basically the same as the bark, and the medicinal functions are basically consistent. At present, more than 70 organic substances are separated and identified in folium cortex eucommiae, besides, various mineral substances also exist, and the folium cortex eucommiae can be roughly divided into lignans, iridoids, phenylpropanoids, flavonoids, polysaccharides, gutta percha, amino acids, volatile oil, trace elements, vitamins and the like according to the structural characteristics of the folium cortex eucommiae. Therefore, the method has important application significance in the research of the extraction and separation of the active substances of the eucommia ulmoides leaves.

The ionic liquid aqueous two-phase (ILATPS) has the advantages of low energy consumption, short process time, reliable scalability, and biocompatible environment, and is therefore considered as an economical and efficient downstream processing method. Moreover, ILATPS exhibits lower viscosity, does not form opaque aqueous solutions, and solutions have a wider range of polarity than other aqueous two-phase systems (based on polymer-polymer, polymer-salt, or salt-salt systems); this novel technique has therefore been applied to the extraction of different types of biomolecules, such as antibiotics, amino acids, proteins, hormones and alkaloids. Nevertheless, few studies have been directed to the stripping step that may be required for the isolation of target biomolecules, which may be a major challenge that must be overcome later.

In response to the limitations presented by ionic liquid aqueous two-phase (ILATPS), ionic liquid-based three-phase partitioning (ILTPP) began to emerge as a promising technology for protein recovery. In the ILTPP technique, protein in the solid phase accumulates between the liquid-liquid interfaces of the ionic liquid rich phase and the salt rich phase. The technology combines the advantages of ionic liquid aqueous two-phase systems (ILATPS) and three-phase distribution (TPP), and can obtain the target compound at the two-phase interface of the corresponding ionic liquid/salt aqueous two-phase systems, which is the characteristic of TPP. ILTPP has been successfully applied in the recovery of bovine whey proteins with important nutritional properties, such as lactoferrin, where an ionic liquid/salt system consisting of 1-butyl-3-methylimidazolium trifluoromethanesulfonic acid and phosphate is considered to be the most suitable for three-phase partitioning.

The economic and environmental aspects involved in ionic liquid based three-phase dispensing are highly dependent on the consumption of the reagents used, especially ionic liquids, which may sometimes be more effective than some volatile organic solvents due to their relatively high price and environmental impact. From previous studies it can be concluded that 0.8-5% of the ionic liquid cannot be reused during the use of the ILTPP, depending on the experimental conditions at the time. Although these lost ionic liquids are of little value, they can result in significant economic and environmental impact, since ionic liquids are one of the major constituents, accounting for over 20% of the ILTPP system; and the biodegradability and the biocompatibility of the commonly used traditional imidazole ionic liquid are poor. Therefore, corresponding measures should be taken in the process to enhance the recovery of the ionic liquid.

The related documents of ionic liquid three-phase partitioning (ILTPP) reported at present are all based on traditional imidazole ionic liquids, and generally relate to the separation of some model protein molecules, and the separation of choline amino acid ionic liquids with better biocompatibility and biodegradability and proteins in complex plant matrixes is not related at present.

Disclosure of Invention

The invention aims to provide a three-phase distribution method based on the integration of extraction and separation of active substances of eucommia ulmoides leaves. The method can be used for integrally extracting and separating the protein, the brass and the chlorogenic acid in the eucommia leaves, and has the advantages of simple process, mild condition, short separation time and high efficiency.

The technical scheme of the invention is as follows: a three-phase distribution method based on the integration of extraction and separation of active substances of eucommia ulmoides leaves comprises the following steps:

1) taking choline tryptophan ionic liquid ([ Ch)][Trp]) And inorganic salt K₃PO₄And is ready for use;

2) determination of choline tryptophan ionic liquid and inorganic salt K by cloud point method and weight method respectively₃PO₄The phase diagram and the line of the formed ionic liquid aqueous two-phase at 25 ℃;

3) adding the eucommia ulmoides leaf powder into a choline tryptophan ionic liquid aqueous solution, leaching for 40-80min, and then centrifuging to remove insoluble substances;

4) according to the phase diagram and the series line data of the step 2), adding the inorganic salt K₃PO₄Adding the aqueous solution into the solution obtained in the step 3), uniformly stirring, and standing until the solution phase is separated and balanced to form three phases;

5) carrying out layered separation on the solution subjected to phase separation in the step 4), wherein the lower layer is inorganic salt K₃PO₄Enriching the phase, precipitating polysaccharide by adding ethanol, and recovering ethanol by rotary evaporation to obtain polysaccharide; the middle layer is a protein enrichment phase and is directly separated and recovered; the upper layer is choline tryptophan ionic liquid enrichment phase, chlorogenic acid and brass in the choline tryptophan ionic liquid enrichment phase are precipitated after the anti-solvent is added, and then the anti-solvent is recovered through rotary evaporation, so that the chlorogenic acid and the brass are obtained.

In the above three-phase distribution method based on the integration of extraction and separation of active ingredients from folium Eucommiae, the folium Eucommiae powder is prepared by washing folium Eucommiae with tap water, air drying, and pulverizing into 0.1-1cm²Drying the small blocks in an oven at the temperature of 30-50 ℃ for 1-3h to obtain the food.

In the three-phase distribution method based on the integration of extraction and separation of active substances from eucommia ulmoides leaves, the concentration of the aqueous solution of the choline tryptophan ionic liquid in the step 3) is 35-50%.

In the aforementioned three-phase distribution method based on the integration of extraction and separation of active substances from folium cortex eucommiae, in step 3), the solid-to-liquid ratio of the folium cortex eucommiae powder to the choline tryptophan ionic liquid aqueous solution is 1: 5-25.

In the aforementioned three-phase distribution method based on the integration of extraction and separation of active ingredients from folium cortex eucommiae, in the step 3), the temperature for extraction is 20-70 ℃.

In the aforementioned three-phase distribution method based on the integration of extraction and separation of active ingredients from folium cortex eucommiae, in step 4), inorganic salt K is added₃PO₄The mass ratio of the water solution to the folium Eucommiae powder is 10-32:1-3, and inorganic salt K₃PO₄The concentration of the aqueous solution was 45%.

The invention has the advantages of

The invention can carry out integrated extraction and separation on the protein, the brass and the chlorogenic acid in the eucommia leaves by the process, and the whole process has the advantages of simple process, mild condition, short separation time and high efficiency.

Drawings

FIG. 1 is [ Ch][Trp]+K₃PO₄+ H₂Phase diagram and line of O ionic liquid aqueous two-phase system;

fig. 2 is a flow chart of the present invention.

Detailed Description

The present invention is further illustrated by the following examples, which are not to be construed as limiting the invention.

Examples of the invention