Use of [C 12 Method for selective extraction of theobromine using a [C mim]Br / STC[4] aqueous two-phase system

Abstract

The present invention discloses a method for utilizing [C 12 mim]Br / STC[4] aqueous two-phase system for selective extraction of theobromine. 12 The solution containing theobromine to be extracted was added to the aqueous biphasic system of mim]Br / 4 wt% STC[4]. After mixing, the mixture was allowed to stand and centrifuged to completely separate the two phases. The distribution coefficients of Van, Syr, MA, Trp, and Cep were less than 1, namely 0.23, 0.24, 0.43, 0.44, and 0.56, respectively. The distribution coefficients of Tyr, Phe, Theo, and TB were greater than 1, namely 2.78, 2.07, and 12.45, respectively. The selectivity of TB for Syr, Van, MA, Trp, Cep, Tyr, Theo, and Phe were 51.88, 54.13, 28.95, 28.30, 22.23, 4.48, 6.01, and 5.00, respectively, achieving efficient and selective extraction of theobromine.

Description

Technical Field

[0001] The present invention belongs to the technical field of separation and purification, and relates to a method for separating and purifying a carbonyl ion by using ionic liquid / 4-sulfonic acid thia[4]arene sodium salt ([C 12 A method for selective extraction of theobromine using a two-phase aqueous system (mim]Br / STC[4]). Background Art

[0002] Alkaloids are an important class of nitrogenous alkaline organic compounds found in nature. They possess diverse structures, a variety of physiological activities, and significant pharmacological effects. Theobromine (3,7-dimethylxanthine, TB) is an alkaloid and a key bioactive component in cocoa. It not only stimulates the human central nervous system and enhances cognitive function, but also captures free radicals, exerting a strong antioxidant effect. Furthermore, theobromine can unclog blood vessels, promote vasodilation, reduce cholesterol accumulation within blood vessels, reduce inflammation, and decrease platelet aggregation in the blood, potentially preventing cardiovascular disease and inhibiting atherosclerosis. Therefore, the extraction of theobromine is of great value and practical significance.

[0003] Currently, the main extraction methods for alkaloids include solvent extraction, compression extraction, supercritical fluid extraction, microwave extraction, and aqueous two-phase extraction (ATPS). The aqueous two-phase system, in which several substances are dissolved in water under certain conditions to form two macroscopic liquid phases, offers advantages such as low interfacial tension, simple operation, low cost, rapid phase separation, and easy scalability. Due to its biocompatibility and environmental friendliness, it is considered a novel green separation system.

[0004] There are few reports on the extraction of theobromine using an aqueous two-phase system. et al. (A. A.PM.Tavares,et al.Aqueous biphasic systems comprisingcopolymers and cholinium-based salts or ionic liquids:Insights on the mechanisms responsible for their creation.Separation and PurificationTechnology 2020,248,117050)When theobromine (TB), theophylline (Theo), caffeine (Caf), and nicotine (Nic) were extracted using 30wt% PL6200 / 25wt% [Ch][DHP], the partition coefficient (K TB )=2.43,K Theo =4.96, K Caf =4.30,KNic =0.31. Ren et al. (J. Ren, Z. Li, et al. Choline derivative ionic liquids-based aqueous two-phase systems: Phase diagrams and partition of purine alkaloids. The Journal of Chemical Thermodynamics 2018, 118, 51-57) used [C6MDEA]Br / K3PO4 ATPS to extract Caf, TB and 2,6-aminopurine and found that the partition coefficients of these three substances were 42, 15 and 4.5, respectively. Dinis et al. (Ionic Liquids) used 25.7wt% (PEG 400+PEG 2000) / 35.7wt% [Ch][DHp] to extract TB, Theo, Caf and Nic and found that Caf, Theo and TB migrated to the PEG-rich phase, while Nic was distributed to the IL-rich phase. Caf =15.8,K Theo =10,K TB =7.9, K Nic =0.3. All of the above methods demonstrate that when using ATPS to extract theobromine, its partition coefficient is similar to that of theophylline, and selective extraction of theobromine by ATPS is not achieved. In fact, currently used ATPS rarely effectively separates biomolecules with similar structures or properties, and its selectivity for target molecules is far from satisfactory. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for utilizing [C 12 A method for selective extraction of theobromine using a mim]Br / STC[4] aqueous two-phase system.

[0006] The technical solutions for achieving the purpose of the present invention are as follows:

[0007] Using [C 12 mim]Br / STC[4] aqueous two-phase system for selective extraction of theobromine, specifically: in [C 12 The solution containing theobromine to be extracted is added into the aqueous two-phase system of mim]Br / STC[4], mixed, allowed to stand, and centrifuged to completely separate the two phases. The upper phase is collected as the extracted theobromine. 12 In the aqueous two-phase system of mim]Br / STC[4], [C 12 The concentration of mim]Br was 7 wt % and the concentration of STC[4] was 4 wt %.

[0008] The structural formula of STC[4] described in the present invention is:

[0009] [C 12 The structural formula of mim]Br is:

[0010] Preferably, the [C 12 In the mim]Br / STC[4] aqueous two-phase system, the solvent is phosphate buffer solution.

[0011] Preferably, the concentration of the phosphate buffer solution is 20 mM, and the pH is 7.0.

[0012] Preferably, the [C 12 The temperature of the mim]Br / STC[4] aqueous two-phase system was 25 °C.

[0013] Compared with the prior art, the present invention has the following advantages:

[0014] (1) The present invention first constructed [C 12 mim]Br / STC[4] aqueous two-phase system has the advantages of small amount of phase-forming components and simple system, and has been successfully applied to the selective extraction of theobromine.

[0015] (2) The present invention can simply and effectively change the distribution behavior of the extracted substances by changing the concentration of the two-phase aqueous components, thereby quickly achieving selective extraction of theobromine. 12 mim]Br / STC[4]ATPS[C 12 When the mim]Br concentration was 7 wt% and the STC[4] concentration was 4 wt%, the partition coefficients of vanillin (Van), syringic acid (Syr), mandelic acid (MA), tryptophan (Trp), and cephalexin (Cep) were less than 1, namely, 0.23, 0.24, 0.43, 0.44, and 0.56, respectively. The partition coefficients of tyrosine (Tyr), phenylalanine (Phe), theophylline (Theo), and theobromine (TB) were greater than 1, namely, 2.78, 2.07, and 12.45, respectively. The selectivity of TB for Syr, Van, MA, Trp, Cep, Tyr, Theo, and Phe were 51.88, 54.13, 28.95, 28.30, 22.23, 4.48, 6.01, and 5.00, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 For [C 12 Phase diagram of mim]Br / STC[4]ATPS at room temperature.

[0017] Figure 2 For the extraction of substances at 12wt% [C12 mim]Br / 5wt% STC[4]ATPS partition coefficient.

[0018] Figure 3 For the extraction of substances at 7wt% [C 12 mim]Br / 4wt% STC[4]ATPS partition coefficient.

[0019] Figure 4 For the extraction of substances at 7wt% [C 12 mim]Br / 5wt% STC[4]ATPS partition coefficient. DETAILED DESCRIPTION

[0020] The present invention will be further described below in conjunction with the embodiments and accompanying drawings.

[0021] To detect different compositions of [C 12 The selectivity of the mim]Br / STC[4] aqueous two-phase system for theobromine. In the following examples, in addition to determining the partition coefficient of TB, the selective extraction effect of the aqueous two-phase system for TB was analyzed by determining the partition coefficients of eight substances with similar structures to TB, including vanillin (Van), syringic acid (Syr), mandelic acid (S-MA), tryptophan (L-Trp), tyrosine (Tyr), phenylalanine (Phe), cephalexin (Cep), and theophylline (Theo). The structural formula of Van is: The structural formula of Syr is: The structural formula of S-MA is: The structural formula of L-Trp is: The structural formula of Tyr is: The structural formula of Phe is: The structural formula of Cep is: Theo's structural formula is: The structural formula of TB is:

[0022] In the following examples, [C 12 mim]Br solution, STC[4] solution and extraction substance (Van, Syr, MA, Trp, Phe, Tyr, Cep, Theo, TB) solution were all prepared using phosphate buffer solution as solvent.

[0023] Taking the preparation of TB solution, Van solution, and Phe solution as an example, the details are as follows:

[0024] A 1.5 mM TB solution, a 50 mM Van solution, and a 130 mM Phe solution were prepared in a 20 mM phosphate buffer solution at pH 7.0, respectively.

[0025] The characteristic UV absorption peaks of Van, Syr, MA, Trp, Phe, Tyr, TB, Theo and Cep are located at 280nm, 262nm, 257nm, 279nm, 257nm, 274nm, 272nm, 272nm and 261nm respectively. 12 A series of Van, Syr, MA, Trp, Phe, Tyr, TB, Theo and Cep solutions were prepared using mim]Br ATPS upper phase as solvent. Their absorbances at characteristic UV absorption peaks were tested, and standard curves of the above nine substances were drawn based on their concentrations and absorbance values.

[0026] ATPS is composed of STC[4], [C 12 The extract is composed of [mim]Br, with a concentration ranging from 1.5 to 130 mM, and is added to the aforementioned ATPS. The mixture is stirred at 25°C and standard atmospheric pressure for 30 minutes and then allowed to stand to reach equilibrium. The absorbance of the extract at the characteristic absorption peak in the upper phase of the ATPS is measured using a UV-Vis spectrometer. Combined with a standard curve for the extract, the partition coefficient of the extract between the two phases of the ATPS can be determined.

[0027] The calculation formula of the extraction material partition coefficient is as follows:

[0028] K=C 上 V 上 / (C 总 V 总 -C 上 V 上 ),

[0029] Where, K: the distribution coefficient of the substance; C 总 : total concentration of substances in ATPS; V 总 : total volume of ATPS; C 上 : concentration of substances in the ATPS upper phase; V 上 : Volume of ATPS upper phase.

[0030] The selectivity is calculated as follows:

[0031] S1=K TB / K Syr , S2=K TB / K Van , S3=K TB / K MA , S4=K TB / K Trp , S5=K TB / K Cep , S6=K TB / K Tyr , S7=KTB / K Theo , S8=K TB / K Phe ,

[0032] Among them, S: selectivity, K TB : Partition coefficient of TB, K Syr : Syr distribution coefficient, K Van : Van's distribution coefficient, K MA : Partition coefficient of MA, K Trp : Partition coefficient of Trp, K Cep : Partition coefficient of Cep, K Tyr : Tyr distribution coefficient, K Theo : Theo's partition coefficient, K Phe : Partition coefficient of Phe.

[0033] Example 1

[0034] [C 12 Phase diagram of mim]Br / STC[4]ATPS at room temperature:

[0035] (1) Prepare a series of 5-20wt% gradient [C 12 mim]Br solution, prepare 40wt% STC[4] solution, slowly add 40wt% STC[4] solution dropwise to the above [C 12 mim]Br solution, when the system becomes turbid, record the weight of the added STC[4] solution, and the composition of a series of cloud point components can be calculated.

[0036] (2) Change the concentration of STC[4] to 30 wt% and repeat step (1).

[0037] (3) Prepare a series of 2-7 wt% gradient STC[4] solutions, prepare 30 wt% [C 12 mim]Br solution, 30wt% [C 12 mim]Br solution was slowly added dropwise to the above STC[4] solution. When the system became turbid, the amount of [C 12 The composition of a series of cloud point components can be calculated based on the weight of the mim]Br solution.

[0038] Through a series of cloud point components, [C 12 mim]Br / STC[4]ATPS phase diagram, the results are as follows Figure 1 shown.

[0039] Example 2

[0040] The extracted material was 12 wt% [C 12mim]Br / 5wt%STC[4]ATPS distribution coefficient:

[0041] (1) Prepare 12 wt% [C 12 mim]Br / 5wt% STC[4] ATPS, the target molecule Syr or Van or MA or Trp or Tyr or Phe or Cep or Theo or TB (1.5-130mM) is added to the above ATPS, mixed in a mixer at 25°C for 30 minutes, and then allowed to stand to allow the two phases to reach equilibrium. The two phases are separated by centrifugation for 5 minutes and the volume of the upper phase is measured.

[0042] (2) The absorbance values ​​of the ATPS upper phase after extraction of Syr, Van, MA, Trp, Tyr, Phe, Cep, Theo, and TB at 262 nm, 280 nm, 257 nm, 279 nm, 274 nm, 257 nm, 261 nm, 272 nm, and 272 nm were detected by ultraviolet spectrometer, and the concentrations of Syr, Van, MA, Trp, Tyr, Phe, Cep, Theo, and TB in the upper phase were calculated based on the standard curve of the above-mentioned extracted substances.

[0043] According to the calculation formula of the distribution coefficient, the distribution coefficients of Syr, Van, MA, Trp, Tyr, Phe, Cep, Theo, and TB were calculated. The results are as follows: Figure 2 According to the selectivity calculation formula, the selectivity of TB for other substances was calculated, and the results are shown in Table 1.

[0044] Example 3

[0045] The extracted material was 7 wt% [C 12 mim]Br / 4wt%STC[4]ATPS distribution coefficient:

[0046] (1) Prepare 7wt% [C 12 mim]Br / 4wt% STC[4] ATPS, the target molecule Syr or Van or MA or Trp or Tyr or Phe or Cep or Theo or TB (1.5-130mM)) is added to the above ATPS, mixed in a mixer at 25°C for 30 minutes, and then allowed to stand to allow the two phases to reach equilibrium. The two phases are separated by centrifugation for 5 minutes and the volume of the upper phase is measured.

[0047] (2) The absorbance values ​​of the ATPS upper phase after extraction of Syr, Van, MA, Trp, Tyr, Phe, Cep, Theo, and TB at 262 nm, 280 nm, 257 nm, 279 nm, 274 nm, 257 nm, 261 nm, 272 nm, and 272 nm were detected by ultraviolet spectrometer, and the concentrations of Syr, Van, MA, Trp, Tyr, Phe, Cep, Theo, and TB in the upper phase were calculated based on the standard curve of the above-mentioned extracted substances.

[0048] According to the calculation formula of the distribution coefficient, the distribution coefficients of Syr, Van, MA, Trp, Tyr, Phe, Cep, Theo, and TB were calculated. The results are as follows: Figure 3 According to the selectivity calculation formula, the selectivity of TB for other substances was calculated, and the results are shown in Table 1.

[0049] Example 4

[0050] The extracted material was 7 wt% [C 12 mim]Br / 5wt%STC[4]ATPS distribution coefficient:

[0051] (1) The concentration of the preparation system is 7 wt% [C 12 mim]Br / 5wt% STC[4] ATPS, the target molecule Syr or Van or MA or Trp or Tyr or Phe or Cep or Theo or TB (1.5-130mM) is added to the above ATPS, mixed in a mixer at 25°C for 30 minutes, and then allowed to stand to allow the two phases to reach equilibrium. The two phases are separated by centrifugation for 5 minutes and the volume of the upper phase is measured.

[0052] (2) The absorbance values ​​of the ATPS upper phase after extraction of Syr, Van, Tyr, and TB at 262 nm, 280 nm, 274 nm, and 272 nm were detected by ultraviolet spectrometer, and the concentrations of Syr, Van, Tyr, and TB in the upper phase were calculated based on the standard curve of the above-mentioned extracted substances.

[0053] According to the calculation formula of the distribution coefficient, the distribution coefficients of Syr, Van, Tyr and TB are calculated. The results are as follows: Figure 4 According to the selectivity calculation formula, the selectivity of TB for other substances was calculated, and the results are shown in Table 1.

[0054] Table 1[C 12 Relationship between the selectivity of mim]Br / STC[4]ATPS for TB and its composition

[0055]

[0056] In [C12 mim]Br, STC[4] ATPS phase diagram ( Figure 1 ), the area between the two curves is the two-phase region. Compared with the phase diagrams of traditional PEG / salt and IL / salt ATPS, the [C 12 The mass concentration of mim]Br and STC[4] is small and the amount used is small.

[0057] Figure 2 It shows that the use of 12wt% [C 12 When Syr, Van, MA, Trp, Tyr, Phe, Cep, Theo and TB were extracted by mim]Br / 5wt%STC[4]ATPS, Syr, Van, MA, Trp and Cep were all distributed to the lower phase of ATPS, and their distribution coefficients were 0.14, 0.12, 0.18, 0.23 and 0.32, respectively; Tyr, Theo and Phe were almost evenly distributed between the two phases, and their distribution coefficients were 1.01, 1.30 and 1.51, respectively, and the distribution coefficient of TB was 1.76; at this time, Theo had a high selectivity for Syr, Van, MA, Trp and Cep, and their S were 12.57, 14.67, 9.78, 7.65 and 5.5, respectively, while its selectivity for Phe, Tyr and Theo was poor, and their S were only 1.17, 1.35 and 1.74.

[0058] Figure 3 It shows that at 7wt% [C 12 When nine substances with similar structures were extracted from mim]Br / 4wt%STC[4]ATPS, the distribution coefficients of Van were 0.23, Syr, MA and Trp, and Cep, respectively, were 0.44 and 0.56, respectively. All five substances were concentrated in the lower phase of ATPS. The distribution coefficients of Phe and Tyr were 2.49 and 2.78, respectively, and the distribution coefficient of Theo was 2.07. The highest distribution coefficient of TB was 12.45. At this time, about 92% of TB was concentrated in the upper phase of ATPS. 12 mim]Br / 4wt%STC[4]ATPS can successfully achieve the selective extraction of TB, and its S value is the highest (K TB / K Van ) is 54.13. More importantly, this system can effectively increase the difference between the distribution behaviors of Theo and TB, resulting in a larger difference in their distribution coefficients, which is currently unattainable by other ATPS.

[0059] Figure 4 It shows that using 7wt% [C 12When the concentration of [C Figure 2 , the concentration of [C 12 mim]Br was decreased from 12wt% to 7wt%, the distribution behavior of Syr and Van was slightly affected, the distribution coefficient of Tyr increased from 1.01 to 1.57, and the distribution coefficient of TB increased from 1.76 to 8.77. Compared with the Figure 3 , when the concentration of STC[4] was increased from 4wt% to 5wt%, the distribution coefficients of Syr and Van changed little (0.24-0.17, 0.23-0.27), the distribution coefficient of Tyr was 1.49, and the distribution coefficient of TB changed significantly, decreasing from 12.45 to 8.77.

[0060] In summary, the concentration of phase-forming components determines the distribution behavior of extract substances to a great extent. By adjusting the concentration of [C 12 mim]Br / STC[4] phase-forming components, when the ATPS composition was 7wt% [C 12 mim]Br / 4wt% STC[4], TB could be efficiently and selectively extracted from 9 structurally similar extract substances, and the Smax (K TB / K Van ) was 54.13.

Claims

1. Using [C 12 mim]Br / STC[4] aqueous two-phase system selectively extracting theobromine, characterized in that: Specifically: In [C 12 The solution containing theobromine to be extracted is added into the aqueous two-phase system of mim]Br / STC[4], mixed, allowed to stand, and centrifuged to completely separate the two phases. The upper phase is collected as the extracted theobromine. 12 In the aqueous two-phase system of mim]Br / STC[4], [C 12 The concentration of mim]Br is 7 wt %, the concentration of STC[4] is 4 wt %, and the structural formula of STC[4] is: ,[C 12 The structural formula of mim]Br is: , the [C 12 In the mim]Br / STC[4] aqueous two-phase system, the solvent is phosphate buffer solution.

2. The method according to claim 1, wherein The concentration of phosphate buffer solution is 20 mM, pH = 7.

0.

3. The method according to claim 1, characterized in that The [C 12 The temperature of the mim]Br / STC[4] aqueous two-phase system was 25 °C.

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