Aqueous two-phase system taking SAB disodium salt as salting-out agent and research method of aqueous two-phase system

Abstract

The invention discloses an aqueous two-phase system taking SAB disodium salt as a salting-out agent and a research method of the aqueous two-phase system, and belongs to the technical field of aqueous two-phase systems. A phase forming reagent of the aqueous two-phase system is ethanol, SAB disodium salt and a carbohydrate, and the aqueous two-phase system is prepared from the following raw materials in percentage by mass: 10%-30% of ethanol, 5%-30% of SAB disodium salt, 10%-40% of carbohydrate and the balance of water. The research method provided by the invention has good practicability and reliability, and can provide an effective means for component distribution prediction of an actual salvia miltiorrhiza water extraction and alcohol precipitation system.

Description

Technical Field

[0001] The present invention belongs to the technical field of aqueous two-phase systems, and particularly relates to an aqueous two-phase system using disodium SAB as a salting-out agent and a research method thereof. Background Art

[0002] An aqueous two-phase system refers to an aqueous system formed by two or more substances that spontaneously form two immiscible aqueous phases with a clear interface under certain conditions. The small molecule alcohol-inorganic salt aqueous two-phase system is one of them, which is composed of small molecule alcohol, inorganic salt and water. The small molecule alcohol is methanol, ethanol, n-propanol, isopropanol, etc., and the inorganic salt is ammonium sulfate, sodium sulfate, potassium phosphate, etc. The extraction and separation principle of this system is based on the selective partitioning of biomass in the aqueous two-phase system. That is, when a biological substance enters the aqueous two-phase system, it will be selectively partitioned between the upper and lower phases. Compared with the conventional extraction partitioning relationship, this partitioning relationship shows a larger or smaller partition coefficient.

[0003] Currently, the small molecule alcohol-inorganic salt aqueous two-phase system has been widely used in the fields of bio-industry, food industry, pharmaceutical industry and analysis and detection. Especially in the separation and purification of biological products, such as the separation and purification of biological macromolecules such as proteins, bioenzymes, bacteria, cells, and biological small molecules such as amino acids and antibiotics, this system shows unique advantages. The small molecule alcohol-inorganic salt aqueous two-phase system has the advantages of low cost and easy recovery. Disodium SAB, that is, the disodium salt form of Salvianolic acid B, was observed to have fluidity in the precipitation during the industrial production of danshen alcohol precipitation, and a liquid-liquid two-phase phenomenon occurred, that is, in addition to the upper layer being liquid and the bottom having solid, there were also some fluids. However, the existing alcohol precipitation mechanism cannot explain this phenomenon. Therefore, the present invention develops an aqueous two-phase system using disodium SAB as a salting-out agent and a research method thereof to solve the technical problems in the prior art of improving the separation selectivity of biological products, reducing the influence on the activity of target substances, and providing a theoretical basis for the industrial production of danshen alcohol precipitation from the perspective of a new aqueous two-phase system with the natural components of medicinal materials as salts. Summary of the Invention

[0004] The object of the invention is to provide an aqueous two-phase system using disodium SAB as a salting-out agent and a research method thereof to solve the technical problems in the prior art of improving the separation selectivity of biological products, reducing the influence on the activity of target substances, and providing a theoretical basis for the industrial production of danshen alcohol precipitation from the perspective of a new aqueous two-phase system with the natural components of medicinal materials as salts.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions: An aqueous two-phase system with disodium SAB as the salting-out agent, wherein the phase-forming reagents of the aqueous two-phase system are ethanol, disodium SAB and saccharide compounds, and the aqueous two-phase system is prepared from the following raw materials in mass percentages: 10%-30% ethanol, 5%-30% disodium SAB, 10%-40% saccharide compounds, and the balance is water.

[0006] Further, the saccharide compound is D-fructose or sucrose; The preparation method of the disodium SAB includes the following steps: S1. Extraction of SAB: Add Salvia miltiorrhiza powder to an ethanol solution, reflux and extract, and then filter to obtain an SAB extract; S2. Separation and purification: Concentrate the SAB extract by vacuum distillation, and then separate and purify it by macroporous adsorption resin column chromatography and recrystallization to obtain high-purity SAB; S3. Preparation of disodium salt: Dissolve high-purity SAB in deionized water, adjust the pH value with a sodium hydroxide solution, and then evaporate, concentrate, crystallize and dry to obtain disodium SAB.

[0007] Further, in S1, the mass-volume ratio of Salvia miltiorrhiza powder to the ethanol solution is 1:5-20 g / mL, the volume concentration of the ethanol solution is 70-95 vt%, the reflux extraction temperature is 60-80 °C, and the reflux extraction time is 2-4 hours; in S2, the vacuum distillation temperature is 40-60 °C, the vacuum degree of vacuum distillation is -0.08 to -0.1 MPa, the model of the macroporous adsorption resin column chromatography is HPD-100, the flow rate is 0.5-2 mL / min, the sample loading amount is 5%-20% of the resin volume, elute with 10-80 vt% ethanol solution, the crystallization temperature is 0-10 °C, the crystallization time is 8-24 hours, and the purity of SAB is greater than 90%; in S3, the pH value is adjusted to 8-10 with the sodium hydroxide solution, the evaporation and concentration temperature is 40-60 °C, the drying temperature is 50-80 °C, and the drying time is 10-24 hours.

[0008] The research method of the aqueous two-phase system with disodium SAB as the salting-out agent includes the following steps: (1) Determination of binodal data by cloud point method: Using ethanol as the organic phase and disodium SAB as the inorganic salt, draw a phase diagram by cloud point titration: dropwise add ethanol and water until it becomes turbid, record the mass of ethanol and the total mass of the system; then dropwise add ultrapure water until it becomes clear, record the mass of water and the total mass of the system; repeat the operation, calculate the mass fractions of ethanol, saccharide and salting-out agent, and draw a binodal diagram; (2) Determination of liquid-liquid equilibrium of the aqueous two-phase system: According to the binodal data, dropwise add water and ethanol until it becomes turbid, centrifuge to separate the upper and lower phases, respectively measure the components of the upper and lower phases, and calculate the distribution coefficient; (3)Actual system prediction research: Based on the binodal data of the aqueous two-phase system, predict the mass fraction of ethanol and the distribution of saccharide components in the actual water extract concentrate of Salvia miltiorrhiza for Samples 1 and 2, and analyze the possibility of falling within the aqueous two-phase region; (4)Experimental results and analysis: Correlate the binodal data using the Merchuk equation, and correlate the liquid-liquid equilibrium data using the Bachman equation, the Othmer-Tobias equation, and the Bancroft equation. Then, in the systems of disodium salt of SAB, ethanol, water and disodium salt of SAB, sucrose, ethanol, water, explore the effect of the mass fraction of disodium salt of SAB on partitioning; in the systems of disodium salt of SAB, ethanol, water and disodium salt of SAB, sucrose, ethanol, water, explore the effect of the mass fraction of ethanol on partitioning; in the systems of disodium salt of SAB, ethanol, water and disodium salt of SAB, sucrose, ethanol, water, explore the effect of the mass fraction of water on partitioning; compare the aqueous two-phase region of the system of disodium salt of SAB, D-fructose, ethanol, water with the system of disodium salt of SAB, ethanol, water to explore the effect of D-fructose on partitioning; finally, predict the component distribution of the actual water extract and alcohol precipitation system of Salvia miltiorrhiza based on the binodal data of the system of disodium salt of SAB, sucrose, ethanol, water.

[0009] Further, the pressure for measuring the binodal data in step (1) is one standard atmosphere, and the temperature is 298.2 K; the preparation method of sample 1 in step (3) is to weigh 20.0555 g of the concentrated aqueous extract of Salvia miltiorrhiza in a 50 mL conical flask, and then slowly add 10.0388 g of absolute ethanol drop by drop while magnetically stirring and mixing, and let it stand to obtain sample 1; the preparation method of sample 2 is to weigh 100.0 g of the concentrated aqueous extract of Salvia miltiorrhiza in a 500 mL beaker, and then slowly add 48.7 g of absolute ethanol drop by drop while magnetically stirring and mixing, and let it stand to obtain sample 2; in step (4), the binodal data is correlated by the Merchuk equation, and both the fitting parameters and the determination coefficient R2 are greater than 0.992, indicating that the equation can well describe the equilibrium state of the aqueous two-phase system; the liquid-liquid equilibrium data is correlated by the Bachman equation, the Othmer-Tobias equation and the Bancroft equation. In the system of SAB disodium salt, sucrose, ethanol and water, the R2 values of the fitting parameters are all greater than 0.89, and the data is reliable; in the system of SAB disodium salt, ethanol and water and the system of SAB disodium salt, sucrose, ethanol and water, the SAB distribution coefficient increases with the increase of the mass fraction, and appropriately increasing SAB helps to improve the phase separation ability; in the system of SAB disodium salt, ethanol and water and the system of SAB disodium salt, sucrose, ethanol and water, the increase of the ethanol mass fraction leads to the decrease of the SAB distribution coefficient, and appropriately reducing ethanol helps to improve the phase separation ability; in the system of SAB disodium salt, ethanol and water and the system of SAB disodium salt, sucrose, ethanol and water, the increase of the water mass fraction leads to the increase of the SAB distribution coefficient, and appropriately increasing water helps to improve the phase separation ability; the aqueous two-phase region of the system of SAB disodium salt, D-fructose, ethanol and water is larger than that of the system of SAB disodium salt, ethanol and water, indicating that D-fructose has a stronger phase separation ability for the aqueous two-phase system; according to the binodal data of the system of SAB disodium salt, sucrose, ethanol and water, the data points of sample 1 and sample 2 are relatively close to the binodal data, and the prediction results are close to the actual situation.

[0010] In summary, due to the adoption of the above technical solutions, the beneficial effects of the present invention are as follows: 1. The aqueous two-phase system of the present invention is composed of ethanol, SAB disodium salt, saccharide compounds and water. Due to the salting-out effect, hydrogen bond effect and hydrophobic interaction between solutes, the system will form two immiscible aqueous phases, namely the aqueous two-phase, which provides a mild environment for the separation and purification of biological substances and avoids the damage of traditional organic solvents to biological active substances. In addition, there is less research on the new aqueous two-phase system with the natural components of medicinal materials as salts. This system has lower cost, simpler operation and easier recovery.

[0011] 2. The phase behavior of the aqueous two-phase system can be visually reflected by the binodal curve diagram of the present invention, providing basic data for subsequent research. The partition coefficient is an important parameter to measure the distribution ability of a substance between two phases. By measuring the partition coefficient, the distribution law of the substance in the aqueous two-phase system can be understood, providing a basis for the separation and purification of the substance. According to the binodal curve data of the SAB disodium salt, sucrose, ethanol, and water system, the data points of Sample 1 and Sample 2 are relatively close to the binodal curve data, and the prediction results are close to the actual situation, indicating that this research method has good practicability and reliability and can provide an effective means for predicting the component distribution in the actual water extraction and ethanol precipitation system of Salvia miltiorrhiza. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0013] Figure 1 It is the binodal curve diagram of the aqueous two-phase system measured by the cloud point method in Example 4 of the present invention at 298.2K; Figure 2 It is the triangular phase diagram of different aqueous two-phase systems in Example 4 of the present invention at 298.2K, (a) SAB disodium salt, ethanol, water system, (b) SAB disodium salt, D-fructose, ethanol, water system, (c) SAB disodium salt, sucrose, ethanol, water system; Figure 3 It is the triangular phase diagram of different aqueous two-phase systems in Example 4 of the present invention at 298.2K (■, SAB disodium salt, sucrose, water, ethanol system; ○, Sample 1; △, Sample 2); Figure 4 It is the trend diagram of the influence of the mass fraction of SAB disodium salt on the SAB partition coefficient in Example 4 of the present invention (■, SAB disodium salt, ethanol, water system; ●, SAB disodium salt, D-fructose, ethanol, water system; ▲, SAB disodium salt, sucrose, ethanol, water system); Figure 5 It is the trend diagram of the influence of the mass fraction of ethanol on the SAB partition coefficient in Example 4 of the present invention (■, SAB disodium salt, ethanol, water system; ●, SAB disodium salt, D-fructose, ethanol, water system; ▲, SAB disodium salt, sucrose, ethanol, water system); Figure 6Influence trend diagram of water mass fraction on SAB partition coefficient in Example 4 of the present invention (■, SAB disodium salt, ethanol, water system; ●, SAB disodium salt, D-fructose, ethanol, water system; ▲, SAB disodium salt, sucrose, ethanol, water system); Figure 7 Phase diagram of the aqueous two-phase system in Example 4 of the present invention in a binary plane rectangular coordinate system (■, SAB disodium salt, ethanol, water system; ●, SAB disodium salt, D-fructose, ethanol, water system; ▲, SAB disodium salt, sucrose, ethanol, water system). Detailed implementation manners

[0014] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0015] Instrument model information involved in the present invention: multi-position magnetic stirrer (03-1, Hangzhou Instrument Motor Co., Ltd.), low-temperature constant temperature bath (THD-1008W, Ningbo Tianheng Instrument Factory); electric fan (ZJ01-192, Taizhou Jiaojiang Zhongjie Electric Appliance Factory); vacuum drying oven (DZF-6050, Shanghai Jinghong Experimental Equipment Co., Ltd.); moisture analyzer (870 KF Titrinoplus, Metrohm AG, Switzerland); electronic balance (AB204-N, METTLER TOLEDO); high-performance liquid chromatograph (Agilent 1100 series, Agilent Technologies, USA and LC5090 series, Zhejiang Fuli Analytical Instrument Co., Ltd.); freeze dryer (MICROMODULYO230, Thermo Fisher Scientific, USA); temperature-controlled oscillator (DSHZ-300, Taicang Experimental Equipment Factory); peristaltic pump (2PB20005Ⅱ, Beijing Satellite Manufacturing Factory, China Aerospace Science and Technology Corporation).

[0016] Materials and reagents used in the present invention: D-fructose (57-48-7, purity 99.4%) was purchased from Aladdin Reagent (Shanghai) Co., Ltd.; D-glucose (50-99-7, purity >99.8%) and D-galactose (59-23-4, purity >99%) were purchased from Shanghai Sangon Biotech Co., Ltd.; sucrose (batch number: WXBC1917V, purity ≥99%) was purchased from Sigma Aldrich; volumetric single-component pyridine-free Karl Fischer reagent (3-5 mg of water / mL, Shanghai Macklin Biochemical Co., Ltd.); analytical pure ethanol was purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd.; analytical pure n-butanol was purchased from Jiangsu Qiangsheng Functional Chemical Co., Ltd. Deionized water was prepared by the ultrapure water system (Milli-Q, Millipore Corporation, USA) in the laboratory.

[0017] Example 1:

[0018] This example discloses a preparation method of SAB disodium salt, which includes the following steps: S1. Extraction of SAB: Add 100 g of Salvia miltiorrhiza powder to 500 mL of 70 vt% ethanol solution, carry out reflux extraction, the temperature of reflux extraction is 60 °C, the time of reflux extraction is 2 hours, and then filter to obtain the SAB extract; S2. Separation and purification: Carry out vacuum distillation and concentration on the SAB extract, the temperature of vacuum distillation is 40 °C, the vacuum degree of vacuum distillation is -0.08 MPa, and then carry out separation and purification by HPD-100 macroporous adsorption resin column chromatography and recrystallization, the flow rate is 0.5 mL / min, the sample loading amount is 5% of the resin volume, elute with 10 vt% ethanol solution, the crystallization temperature is 0 °C, and the crystallization time is 8 hours to obtain high-purity SAB; S3. Preparation of disodium salt: Dissolve high-purity SAB in deionized water, add sodium hydroxide solution to adjust the pH value to 8, then carry out evaporation and concentration, crystallization, and drying. The temperature of evaporation and concentration is 40 °C, the temperature of drying is 50 °C, and the time of drying is 10 hours to obtain SAB disodium salt.

[0019] Example 2:

[0020] This example discloses a preparation method of SAB disodium salt, which includes the following steps: S1. Extraction of SAB: Add 100 g of Salvia miltiorrhiza powder to 500 mL of 85 vt% ethanol solution, carry out reflux extraction, the temperature of reflux extraction is 70 °C, the time of reflux extraction is 3 hours, and then filter to obtain the SAB extract; S2. Separation and purification: The SAB extract is concentrated by vacuum distillation. The temperature of vacuum distillation is 40 - 60 °C, and the vacuum degree is -0.09 MPa. Then, it is separated and purified by HPD - 100 macroporous adsorption resin column chromatography and recrystallization. The flow rate is 1 mL / min, the sample loading amount is 10% of the resin volume, eluted with 50 vt% ethanol solution, the crystallization temperature is 5 °C, and the crystallization time is 20 hours to obtain high - purity SAB; S3. Preparation of disodium salt: Dissolve high - purity SAB in deionized water, add sodium hydroxide solution to adjust the pH value to 9, then evaporate and concentrate, crystallize, and dry. The temperature of evaporation and concentration is 50 °C, the drying temperature is 70 °C, and the drying time is 16 hours to obtain SAB disodium salt.

[0021] Example 3:

[0022] This example discloses a method for preparing SAB disodium salt, which includes the following steps: S1. Extraction of SAB: Add 100 g of Salvia miltiorrhiza powder to 2000 mL of 95 vt% ethanol solution, reflux and extract. The reflux extraction temperature is 80 °C, and the reflux extraction time is 4 hours. Then filter to obtain the SAB extract; S2. Separation and purification: The SAB extract is concentrated by vacuum distillation. The temperature of vacuum distillation is 60 °C, and the vacuum degree is -0.1 MPa. Then, it is separated and purified by HPD - 100 macroporous adsorption resin column chromatography and recrystallization. The flow rate is 2 mL / min, the sample loading amount is 20% of the resin volume, eluted with 80 vt% ethanol solution, the crystallization temperature is 10 °C, and the crystallization time is 24 hours to obtain high - purity SAB; S3. Preparation of disodium salt: Dissolve high - purity SAB in deionized water, add sodium hydroxide solution to adjust the pH value to 10, then evaporate and concentrate, crystallize, and dry. The temperature of evaporation and concentration is 60 °C, the drying temperature is 80 °C, and the drying time is 24 hours to obtain SAB disodium salt.

[0023] Example 4:

[0024] This example discloses a research method for an aqueous two - phase system using SAB disodium salt as a salting - out agent, including the following steps: (1)Cloud point method for determining binodal data: Using ethanol as the organic phase and the disodium salt of SAB as the inorganic salt, the phase diagram was plotted by cloud point titration: First, use a pipette to dropwise add ethanol and water into a glass jacketed container. Then, accurately weigh the sugar (D-fructose or sucrose) and the disodium salt of SAB into the glass jacketed container respectively, and stir magnetically while adding to completely dissolve them. Then, add ethanol dropwise until the system becomes turbid. Record the mass of ethanol added and the total mass of the system. The container was temperature-controlled by a low-temperature constant temperature bath. Then, slowly add ultrapure water and stir magnetically until the system just becomes clear. Record the mass of ultrapure water added and the total mass of the system; continue to slowly add ethanol to make the system turbid again, and then add water until the system becomes clear. Record the mass of ultrapure water added and the total mass of the system; repeat the above operations. Use the gravimetric method to calculate the mass fractions of ethanol, D-fructose / sucrose, and the disodium salt of SAB in the total system at each turbidity, and plot the phase diagram. The curve in the phase diagram is the binodal. The results are shown in Figure 1 as shown. The Merchuk equation was used to correlate the binodal data, and the calculation formula is as follows:

[0025] where W1 and W2 are the mass fractions of ethanol and the salting-out agent (disodium salt of SAB or disodium salt of SAB and sugar) in the system respectively, A 0, B 0 and C 0 are the fitting parameters of the equation respectively. The correlation parameters and the determination coefficient R 2 are shown in Table 1: Table 1 Fitting results of binodal data (298.2K)

[0026] SE α is the standard deviation of the parameter. It can be seen from Table 1 that R 2 are all greater than 0.992, indicating that the above equations can all well correlate the binodal data.

[0027] (2)Liquid-liquid equilibrium determination of aqueous two-phase system: According to the binodal data, first use a pipette to dropwise add water and ethanol into a glass jacketed container. Then, accurately weigh the sugar (D-fructose or sucrose) and the disodium salt of SAB into the glass jacketed container respectively, and stir magnetically while adding to completely dissolve them. Then, add a certain amount of ethanol until the system becomes turbid, and stir magnetically evenly. The container was temperature-controlled by a low-temperature constant temperature bath. After centrifuging at 3000 rpm for 10 min to complete phase separation (i.e., it is divided into clear upper and lower phases with an obvious phase interface), use a pipette to take out the upper and lower phases in the centrifuge tube respectively, weigh and analyze. The liquid-liquid equilibrium data of the aqueous two-phase system are shown in Table 2. Where , and are the mass fractions of ethanol, salting-out agent (disodium SAB or disodium SAB and saccharides), and water in the organic phase, respectively, , and are the mass fractions of ethanol, salting-out agent (disodium SAB or disodium SAB and saccharides), and water in the aqueous phase, respectively, and the subscripts 1, 2, and 3 represent ethanol, salting-out agent, and water, respectively.

[0028] Table 2 Liquid-liquid phase equilibrium composition and distribution coefficient of aqueous two-phase system (298.2K)

[0029] It can be seen from Table 2 that the mass fraction of the salting-out agent in the aqueous phase is significantly higher than that in the organic phase; in the disodium SAB, ethanol, water system and the disodium SAB, sucrose, ethanol, water system, the total mass fraction of disodium SAB and saccharides in the organic phase increases with the decrease of the total mass fraction of disodium SAB and saccharides in the aqueous phase. The distribution coefficients are shown in Table 2. In most cases, the distribution coefficients of SAB and saccharides increase with the decrease of the mass fraction of the salting-out agent in the aqueous phase.

[0030] The liquid-liquid equilibrium triangular phase diagrams of 3 different aqueous two-phase systems at atmospheric pressure and a temperature of 298.2K are shown in Figure 2 . It can be seen from Figure 2 that in the disodium SAB, ethanol, water system and the disodium SAB, sucrose, ethanol, water system, the tie-line length is basically unchanged; in the disodium SAB, D-fructose, ethanol, water system, the tie-line length is also basically unchanged.

[0031] (3) Prediction research on the actual system: Weigh 20.0555 g of the concentrated aqueous extract of Salvia miltiorrhiza into a 50 mL conical flask, and then slowly add 10.0388 g of absolute ethanol dropwise while magnetically stirring and mixing. Let it stand to obtain Sample 1; similarly, weigh 100.0 g of the concentrated aqueous extract of Salvia miltiorrhiza into a 500 mL beaker, and then slowly add 48.7 g of absolute ethanol dropwise while magnetically stirring and mixing. Let it stand to obtain Sample 2. According to the binodal data of the disodium SAB, sucrose, ethanol, water aqueous two-phase system, predict whether the above Samples 1 and 2 fall into the two-phase region.

[0032] Predict the actual system according to the binodal data of the disodium SAB, sucrose, ethanol, water system. As Figure 3 shown, the data points of Samples 1 and 2 are far from the binodal. The possible reason is that the sample is mainly composed of stachyose and also contains other types of phenolic acid salts.

[0033] (4)Experimental results and analysis: The mass fractions of the active ingredients (including danshensu, protocatechuic aldehyde, rosmarinic acid, SAB, coumaric acid, and hydroxysafflor yellow A) in danshen and safflower in the upper and lower phases were determined and calculated by HPLC. The mass fractions of saccharides (D-fructose or sucrose or stachyose) were determined and calculated by HPLC-ELSD. The mass fraction of water was determined and calculated by a moisture analyzer. The mass fraction of ethanol was calculated by the law of conservation of mass.

[0034] The Bachman equation, Othmer-Tobias equation, and Bancroft equation were used for the correlation equations of the liquid-liquid equilibrium tie lines. The calculation formulas are as follows.

[0035]

[0036] where A1, B1, A2, B2, k2, and r are the parameters of the equations respectively.

[0037] The fitting parameters and R of the Bachman equation, Othmer-Tobias equation, and Bancroft equation in the SAB disodium salt, sucrose, ethanol, water system 2 values are shown in Table 3.

[0038] Table 3 Fitting parameters and R of the Bachman equation, Bancroft equation, and Other-Tobias equation 2 values (SAB disodium salt, sucrose, ethanol, water aqueous two-phase system)

[0039] SE α is the standard deviation of the parameter.

[0040] According to Table 3, for the SAB disodium salt, sucrose, ethanol, water system, the R 2 values fitted by the three equations are all greater than 0.89.

[0041] The distribution coefficients of SAB in different aqueous two-phase systems are as Figure 4 shown. In the SAB disodium salt, ethanol, water system and the SAB disodium salt, sucrose, ethanol, water system, the distribution coefficient of SAB increases with the increase of the mass fraction of SAB disodium salt, which indicates that SAB tends to transfer to the upper phase (organic phase) with the increase of the mass fraction of SAB disodium salt. Therefore, appropriately increasing the SAB disodium salt helps to reduce the loss of SAB during the alcohol precipitation process; while in the SAB disodium salt, D-fructose, ethanol, water system, the distribution coefficient of SAB first increases and then slightly decreases with the increase of the mass fraction of SAB disodium salt, and the overall change is not significant. Therefore, appropriately increasing the SAB disodium salt helps to improve the phase separation ability.

[0042] The distribution coefficients of SAB in different aqueous two-phase systems are as Figure 5 shown. In the systems of SAB disodium salt, ethanol, water and SAB disodium salt, sucrose, ethanol, water, the distribution coefficient of SAB decreases with the increase of the mass fraction of ethanol; while in the system of SAB disodium salt, D-fructose, ethanol, water, the distribution coefficient of SAB first increases and then slightly decreases with the increase of the mass fraction of ethanol, and the overall change is not significant. Therefore, appropriately reducing the addition of ethanol helps to improve the phase separation ability, and SAB tends to transfer to the upper phase (organic phase).

[0043] The distribution coefficients of SAB in different aqueous two-phase systems are as Figure 6 shown. In the systems of SAB disodium salt, ethanol, water and SAB disodium salt, sucrose, ethanol, water, the distribution coefficient of SAB increases with the increase of the mass fraction of water; while in the system of SAB disodium salt, D-fructose, ethanol, water, the distribution coefficient of SAB first increases and then slightly decreases with the increase of the mass fraction of water, and the overall change is not significant. Therefore, appropriately increasing the water helps to improve the phase separation ability, and SAB tends to transfer to the upper phase (organic phase).

[0044] To compare the effects of different sugar components on the extraction and separation in aqueous two-phase systems, the phase equilibrium data of each phase can be plotted in a binary right-angle coordinate system to obtain a phase diagram, as Figure 7 shown. The upper right region of the binodal curve is the two-phase region, while the lower left region is the homogeneous region. It can be seen from the figure that when sucrose is added to the system of SAB disodium salt, ethanol, water, the two-phase region remains basically unchanged; while when D-fructose is added, the binodal is closer to the coordinate axes and the two-phase region increases significantly, indicating that it has a stronger phase separation ability in the construction of aqueous two-phase systems. This may be due to the much larger water solubility of D-fructose than sucrose. Therefore, compared with the system of SAB disodium salt, ethanol, water, the new aqueous two-phase system formed after adding D-fructose can form an aqueous two-phase with less SAB, which is of great significance for practical applications.

[0045] Experimental example: Under the condition of 25 °C, determination of the active ingredient-sucrose-ethanol-water aqueous two-phase simulation system of (Salvia miltiorrhiza, Carthamus tinctorius): According to the SAB disodium salt-sucrose-ethanol-water aqueous two-phase system, some active ingredients in Salvia miltiorrhiza and Carthamus tinctorius were added to study the advantages of this aqueous two-phase system. The results are shown in Table 4. It can be seen from Table 4 that in the SAB disodium salt-sucrose-ethanol-water aqueous two-phase system, when the mass fraction of the initially added SAB disodium salt is 0.075, the distribution coefficient of SAB is 0.3357; when some active ingredients of Salvia miltiorrhiza and Carthamus tinctorius are added to form an aqueous two-phase system closer to the actual system, the distribution coefficient of SAB remains basically unchanged, which indicates that the SAB disodium salt-sucrose-ethanol-water aqueous two-phase system can better simulate the actual system; while the distribution coefficient of protocatechuic aldehyde can reach about 9.9, which indicates that protocatechuic aldehyde can be effectively separated and purified through this aqueous two-phase system; while the distribution coefficients of rosmarinic acid, coumaric acid and hydroxysafflor yellow A are not high, and the possible reason is that rosmarinic acid, coumaric acid and hydroxysafflor yellow A mainly exist in ionic form in the upper phase; while protocatechuic aldehyde is an aldehyde substance and mainly exists in molecular form.

[0046] Table 4 Distribution coefficients of the active ingredient-sucrose-ethanol-water aqueous two-phase system of (Salvia miltiorrhiza, Carthamus tinctorius)

[0047] Since the distribution coefficient of SAB is between 0.28 and 0.48, this indicates that SAB has a large loss during the actual water extraction and alcohol precipitation process of Salvia miltiorrhiza. A method for extracting total phenolic acids of Salvia miltiorrhiza from the alcohol precipitation precipitate of Salvia miltiorrhiza can be adopted, and the percolation process is used to recover the total phenolic acids of Salvia miltiorrhiza including SAB from the dried alcohol precipitation precipitate of Salvia miltiorrhiza, so as to minimize the loss of SAB and improve the utilization rate of Salvia miltiorrhiza medicinal materials.

[0048] As mentioned above, it is only the preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, makes equivalent substitutions or changes, and all should be covered within the protection scope of the present invention.

[0049] The preferred embodiments of the present invention disclosed above are only used to help explain the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the present invention to only the specific embodiments. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principle and practical application of the present invention, so that those skilled in the art in the technical field can well understand and utilize the present invention. The present invention is only limited by the claims and their full scope and equivalents.

Claims

1. An aqueous two-phase system with disodium SAB as the salting-out agent, characterized in that, The phase-forming reagents of the aqueous two-phase system are ethanol, disodium SAB, and saccharide compounds. The aqueous two-phase system is prepared from the following raw materials by mass percentage: 10%-30% ethanol, 5%-30% disodium SAB, 10%-40% saccharide compounds, and the balance is water.

2. The aqueous two-phase system using disodium SAB as the salting-out agent according to claim 1, wherein The saccharide compound is D-fructose or sucrose.

3. The aqueous two-phase system using disodium SAB as a salting-out agent according to claim 1, characterized in that, The preparation method of disodium SAB includes the following steps: S1. Extraction of SAB: Add Salvia miltiorrhiza powder to an ethanol solution, reflux and extract, and then filter to obtain an SAB extract. S2. Separation and purification: Concentrate the SAB extract by vacuum distillation, and then separate and purify it by macroporous adsorption resin column chromatography and recrystallization to obtain high-purity SAB. S3. Preparation of disodium salt: Dissolve high-purity SAB in deionized water, add a sodium hydroxide solution to adjust the pH value, and then evaporate, concentrate, crystallize, and dry to obtain disodium SAB.

4. The aqueous two-phase system using disodium SAB as the salting-out agent according to claim 3, characterized in that, In S1, the mass-volume ratio of Salvia miltiorrhiza powder to the ethanol solution is 1:5-20 g / mL, and the volume concentration of the ethanol solution is 70-95 vt%.

5. The aqueous two-phase system using disodium SAB as a salting-out agent according to claim 3, characterized in that, In S2, the sample loading amount is 5%-20% of the resin volume, and it is eluted with an ethanol solution of 10-80 vt%. The purity of SAB is greater than 90%. In S3, the pH value is adjusted to 8-10 with a sodium hydroxide solution.

6. The research method of the aqueous two-phase system with SAB disodium salt as the salting-out agent, characterized in that, It includes the following steps: (1) Using ethanol as the organic phase and disodium SAB as the inorganic salt, the binodal data is measured by the cloud point method. (2) According to the binodal data, the liquid-liquid equilibrium of the aqueous two-phase system is measured. (3) According to the binodal data of the aqueous two-phase system, the prediction research of the actual system is carried out. (4) The binodal data is correlated by the Merchuk equation, and the liquid-liquid equilibrium data is correlated by the Bachman equation, the Othmer-Tobias equation, and the Bancroft equation, and the experimental results are analyzed.

7. The research method of the aqueous two-phase system using disodium SAB as the salting-out agent according to claim 6, characterized in that, In step (1), the pressure for measuring the binodal data is one standard atmosphere, and the temperature is 298.2K.

8. The research method of the aqueous two-phase system using disodium SAB as a salting-out agent according to claim 6, characterized in that, In step (4), the binodal data is correlated by the Merchuk equation, and the fitting parameters and the coefficient of determination R 2 are both greater than 0.992.

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