Method for efficiently dissolving heme

Abstract

The invention relates to the technical field of heme processing and utilization, and particularly discloses a method for efficiently dissolving heme. The preparation method comprises the following steps: mixing a hydrogen bond donor and a hydrogen bond acceptor according to a molar ratio of 1: 0.5-1: 8, heating in a water bath to prepare a deep-eutectic solvent, adding heme into the prepared deep-eutectic solvent, and fully and uniformly mixing the heme and the deep-eutectic solvent according to a volume ratio of 1-3.5 mg / ml. The method is simple, the steps are easy to operate, by adopting the method, the solubility of the heme is improved, the stability of the heme can be maintained, and a foundation is laid for further development and utilization of the heme.

Description

Technical Field

[0001] The present invention relates to the technical field of heme processing and utilization, and particularly to a method for efficiently dissolving heme. Background Art

[0002] Heme is an important metal porphyrin compound in the body and widely exists as an important prosthetic group in proteins such as myoglobin, hemoglobin, cytochrome, and catalase, participating in the transportation, exchange, and electron transfer of oxygen in the body and promoting the formation and maturation of red blood cells. In addition to its physiological functions, heme can also be used as a nutritional supplement and natural pigment in the fields of feed processing and food processing, and participate in organic synthesis fields such as catalysis and preparation of metal framework compounds. In addition, heme is widely used in the pharmaceutical field, as an excellent iron agent for the treatment of iron deficiency anemia, and can also be used as a precursor substance for the preparation of expensive drugs such as hematoporphyrin and bilirubin.

[0003] The molecular formula of heme is C 34 H 32 FeN4O4, with a molecular weight of 616.50. The ferrous ion is embedded in the center of the porphyrin ring and forms coordination with the nitrogen atoms of the four pyrrole rings respectively. Heme is mainly obtained by extracting from livestock and poultry blood. During the extraction process, the ferrous ion is prone to oxidation and exists in a more stable form of hemin chloride.

[0004] The structure of heme determines its special solubility. Heme is insoluble in solvents such as water, dilute acid, chloroform, and ether, slightly soluble in hot pyridine, and soluble in reagents such as hot alcohol, ammonia water, sodium hydroxide solution, and highly toxic trimethylamine. It can be seen that the solubility of heme overly depends on temperature and pH, increasing the difficulty of heme in analysis, extraction, and application. In terms of analysis and detection, heme is almost insoluble in conventional chromatographic analysis reagents such as water, methanol, ethanol, acetonitrile, and n-hexane, which is not conducive to the analysis of heme in precision equipment such as high-performance liquid chromatography and liquid chromatography-mass spectrometry. In terms of extraction, the matrix components where heme is located are complex and the heme content is low. The selectivity and specificity of existing extraction solvents are not high, resulting in low extraction efficiency of heme. In the application field, the poor solubility of heme affects the conversion efficiency of preparing high-value-added drugs such as porphyrins and bile pigments, restricting the application field of heme. Although heme can be dissolved in alkaline solutions, under alkaline conditions, heme often converts to hydroxyheme for quantification, and when stored in alkaline conditions for a long time, heme is also prone to degradation or formation of polymers. Summary of the Invention

[0005] The present invention provides a method for efficiently dissolving heme, which solves the problem that the solubility of heme is affected by the type of solvent, as well as the pH and temperature of the solvent, resulting in poor solubility and dissolution stability.

[0006] The present invention adopts the following technical solution: A method for highly efficient dissolution of heme, comprising the following steps: (1) Preparation of deep eutectic solvent: Take a hydrogen bond donor and a hydrogen bond acceptor and mix them in a molar ratio of 1:0.5 to 1:8, and prepare the deep eutectic solvent by heating in a water bath. (2) Dissolution: Add heme to the prepared deep eutectic solvent, and fully mix at a mass-to-volume ratio of heme to deep eutectic solvent of 1 to 3.5 mg / ml, heat in a water bath at 30 to 90 °C, with a stirring rate of 50 to 500 rpm, and a heating and dissolution time of 5 to 30 min. The heme is enriched in the deep eutectic solvent. Further, the hydrogen bond donor is any one of urea, oxalic acid, levulinic acid, glycine, L-proline, ethylene glycol, 1,2-propanediol, glycerol, 1,4-butanediol, triethylene glycol, or polyethylene glycol 200.

[0007] Further, the hydrogen bond acceptor is any one of tetraethylammonium bromide, tetrabutylammonium bromide, tetrabutylammonium chloride, betaine, or choline chloride.

[0008] Further, in the step (2), the stirring rate is 50 to 500 rpm.

[0009] Further, in the step (1), the water bath heating temperature is 25 to 80 °C, and the heating time is 10 to 240 min.

[0010] Further, a method for recycling the deep eutectic solvent: Add 2 to 20 times the volume of water or ethanol of the heme deep eutectic solvent enrichment solution, let it stand at room temperature, and centrifuge to separate and collect the supernatant and precipitate respectively. The precipitate is washed successively with water and 30% to 70% ethanol, and then vacuum dried at 40 to 60 °C to obtain heme. The supernatant is rotary evaporated at a temperature of 40 to 50 °C to remove ethanol or water, and the deep eutectic solvent is recovered after concentration.

[0011] The temperature control and pH dependence of heme dissolution. The pH of the prepared deep eutectic solvent is generally between 4.5 and 7.5, and this pH range is the common precipitation area of heme, which is not conducive to the dissolution of heme. The dissolution of heme can be temporarily provided by increasing the temperature, but the solubility of heme decreases as the temperature decreases, and it cannot fundamentally solve the high efficiency of the heme solvent. The deep eutectic solvent prepared in this study can not only improve the solubility of heme, but also maintain the stability of heme well, and the heme is precipitated in the form of a precipitate through the back-extraction reagent, realizing the lossless dissolution-recovery of heme.

[0012] The method of the present invention is simple and the steps are easy to operate. The prepared deep eutectic solvent has the characteristics of high polarity, low viscosity, and closer to pH neutrality, which is used for the dissolution and enrichment of heme solvent, breaking through the dependence of heme on pH and temperature regulation, realizing the efficient dissolution of heme, improving the dissolution stability of heme, and laying a good foundation for the development and utilization of heme. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 It is the high performance liquid chromatography diagram of the deep eutectic solvent and heme in the deep eutectic solvent in the embodiment of the present invention; Figure 2 It is the diagram of the change of peak area of 1 μg / mL heme stored in different solutions for 4 weeks in the present invention; Figure 3 It is the diagram of the change of peak area of 5 μg / mL heme stored in different solutions for 4 weeks in the present invention; Figure 4 It is the diagram of the change of peak area of 10 μg / mL heme stored in different solutions for 4 weeks in the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0014] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the specific embodiments of the present invention.

[0015] Preparation method of deep eutectic solvent: (1) Take a hydrogen bond donor and a hydrogen bond acceptor and mix them in a molar ratio of 1:0.5 to 1:8; The hydrogen bond donor is any one of urea, oxalic acid, levulinic acid, glycine, L-proline, ethylene glycol, 1,2-propanediol, glycerol, 1,4-butanediol, triethylene glycol or polyethylene glycol 200; The hydrogen bond acceptor is tetraethylammonium bromide, tetrabutylammonium bromide, tetrabutylammonium chloride, betaine, choline chloride; (2) The water bath heating temperature is 30 to 90 °C, and the heating time is 5 min to 30 min; the stirring rate is 50 rpm to 500 rpm to form a uniform transparent liquid, and it is cooled to room temperature without solidification to obtain the deep eutectic solvent.

[0016] Example 1: Deep eutectic solvent 1 (choline chloride-urea) The preparation method is as follows: including the following steps (1) Take the hydrogen bond donor urea and the hydrogen bond acceptor choline chloride and mix them in a molar ratio of 1:2; (2) The water bath heating temperature is 25 °C, and the heating time is 30 min; the stirring rate is 50 rpm to form a uniform transparent liquid, and the deep eutectic solvent: choline chloride-urea is prepared.

[0017] During use, add 20 wt% water and mix evenly for the heme dissolution experiment.

[0018] Example 2: Deep eutectic solvent 2 (tetrabutylammonium bromide - ethylene glycol) The preparation method is as follows: including the following steps (1) Mix the hydrogen bond donor ethylene glycol and the hydrogen bond acceptor tetrabutylammonium bromide in a molar ratio of 1:2; (2) The water bath heating temperature is 60 °C, and the heating time is 90 min; the stirring rate is 200 rpm to form a uniform transparent liquid, obtaining the deep eutectic solvent: tetrabutylammonium bromide - ethylene glycol.

[0019] During use, add 15 wt% water and mix evenly for the heme dissolution experiment.

[0020] Example 3: Deep eutectic solvent 3 (betaine - polyethylene glycol 200) The preparation method is as follows: including the following steps (1) Mix the hydrogen bond donor polyethylene glycol 200 and the hydrogen bond acceptor betaine in a molar ratio of 1:5; (2) The water bath heating temperature is 80 °C, and the heating time is 60 min; the stirring rate is 200 rpm to form a uniform transparent liquid, obtaining the deep eutectic solvent: betaine - polyethylene glycol 200.

[0021] During use, add 30 wt% water and mix evenly for the heme dissolution experiment.

[0022] Example 4: Deep eutectic solvent 4 (tetrabutylammonium chloride - 1,2 - propanediol) The preparation method is as follows: including the following steps: (1) Mix the hydrogen bond donor 1,2 - propanediol and the hydrogen bond acceptor tetrabutylammonium chloride in a molar ratio of 1:3; (2) The water bath heating temperature is 80 °C, and the heating time is 60 min; the stirring rate is 500 rpm to form a uniform transparent liquid, obtaining the deep eutectic solvent.

[0023] During use, add 5 wt% water and mix evenly for the heme dissolution experiment.

[0024] Example 5: Deep eutectic solvent 5 (choline chloride - polyethylene glycol 200) The preparation method is as follows: including the following steps (1) Mix the hydrogen bond donor polyethylene glycol 200 and the hydrogen bond acceptor choline chloride in a molar ratio of 1:5; (2)The water bath heating temperature is 40 °C, and the heating time is 240 min; the stirring rate is 50 rpm to form a uniform and transparent liquid, and a deep eutectic solvent: choline chloride - polyethylene glycol 200 is prepared.

[0025] The viscosity and polarity results of the deep eutectic solvents in Examples 1 to 5 are shown in Table 1.

[0026] Table 1 Viscosity and polarity results of several deep eutectic solvents Viscosity (mPa•s) Hydrogen bond basicity Molar transfer energy (kJ / mol) Deep eutectic solvent 1 11.61 0.93 90.24 Deep eutectic solvent 2 25.42 1.14 89.50 Deep eutectic solvent 3 25.42 1.08 97.32 Deep eutectic solvent 4 45.76 0.84 90.58 Deep eutectic solvent 5 61.4 1.18 118.41 It can be seen from Table 1 that the viscosity of the deep eutectic solvent prepared by the present invention is between 11.61 - 61.4 mPa•s, and several deep eutectic solvents have relatively high hydrogen bond basicity and molar transfer energy. Example

[0027] A method for highly efficient dissolution of heme: Add heme to the prepared deep eutectic solvent, and mix well at a mass - to - volume ratio of heme to the volume of the deep eutectic solvent of 1 - 3.5 mg / ml. Heat in a water bath at a temperature of 80 °C for a dissolution time of 20 min, and the stirring rate is 300 rpm, so that heme is fully dissolved in the deep eutectic solvent; Recovery method of deep eutectic solvent: (1)Add an anti - extractant to the deep eutectic solvent enrichment liquid of heme, add water or ethanol with a volume 10 times that of the enrichment liquid, let it stand at room temperature for 20 min, centrifuge at 5000 rpm for 10 min, collect the supernatant and precipitate respectively. The precipitate is heme, and after washing with water and then washing with 70% ethanol in sequence, it is dried in vacuo at 50 °C to obtain heme; (2)Rotate - evaporate the above - obtained supernatant at a temperature of 45 °C to remove ethanol or water, heat on a hot plate to remove water, and recover the deep eutectic solvent after concentration.

[0028] Example 7: Solubility of heme in deep eutectic solvent The saturated solubility of heme in the deep eutectic solvent is detected by high - performance liquid chromatography analysis. The parameter settings of the high - performance liquid chromatograph are as follows: Detector: Diode array detector or ultraviolet detector; Chromatographic column: C18 chromatographic column (4.6 mm×250 mm, 5 μm); Detection wavelength: 399 nm; Mobile phase: A is acetonitrile, B is 0.05% trifluoroacetic acid aqueous solution, gradient elution; 0 min A 40%, 12 min A 80%, 18 min A 40%, 20 min A 40% Flow rate: 0.6 mL / min; Column temperature: 30 °C; Injection volume: 10 μL.

[0029] Inject the deep eutectic solvent and the deep eutectic solvent after dissolving heme for analysis. The results are as Figure 1 shown. As Figure 1 shown in (a) of Figure 1 is the chromatogram of the deep eutectic solvent at 398 nm, and (b) in

[0030] is the chromatogram of heme dissolved in DES at this wavelength. It can be seen from the figure that the deep eutectic solvent can fully dissolve heme and has no interference on the peak emergence of the heme chromatogram. Therefore, this method can accurately determine the saturation solubility of heme.

[0031] The results of the saturation solubility of heme in several deep eutectic solvents are shown in Table 2. Solvent pH Heme saturation solubility (μg / mL) 0.1 M sodium hydroxide solution 13 1972.0 Acidic acetone 1.5 71.0 Deep eutectic solvent 1 7.18 1047.4 Deep eutectic solvent 2 7.54 2279.9 Deep eutectic solvent 3 6.16 2254.3 Deep eutectic solvent 4 6.37 1164.2 Deep eutectic solvent 5 7.86 3557.4 As can be seen from Table 2, the solubility of heme in acidic acetone is only 71.0 μg / mL, and its solubility in sodium hydroxide solution can reach 1972.0 μg / mL. The solubility of heme shows strong pH dependence.

[0032] Compared with these two common solvents, the pH of the prepared deep eutectic solvent is between 6.16 and 7.86, close to neutral. The saturation solubility of heme is 1047.4 - 3557.4 μg / mL. Heme has good solubility in the deep eutectic solvents prepared in the present invention. Especially in deep eutectic solvent 5, the saturation solubility of heme reaches 3557.4 μg / mL, which can prove that the deep eutectic solvent can effectively improve the pH dependence of heme.

[0033] Example VIII: Dissolution stability of heme The dissolution stability of heme is an important aspect concerned in the processes of heme analysis, extraction, and storage. Under alkaline conditions, although the solubility of heme is good, heme is easily converted into hydroxyheme under alkaline conditions, and with the prolongation of storage time, heme is prone to degradation or form dimers. When analyzing heme, it is often necessary to prepare it freshly, which not only increases the experimental operation steps but also increases the consumption of reagents.

[0034] Dissolve heme in the deep eutectic solvent (choline chloride - polyethylene glycol 200) prepared in Example 5 and sodium hydroxide solution respectively, prepare a concentration of 1 - 20 μg / mL, store at 4°C for 4 weeks, and analyze the corresponding heme content. The results are as Figure 2 、 Figure 3 and Figure 4 shown.

[0035] From Figure 2It can be seen that in the sodium hydroxide solution, with the extension of storage time, the peak area of low-concentration heme decreased significantly. When 1 μg / mL heme was stored for 7 days, heme degraded or transformed. However, in the deep eutectic solvent, heme was stored for 28 days and the peak area only decreased by 8%.

[0036] It can be seen from Figure 3 and Figure 4 that when 5 μg / mL and 10 μg / mL heme were stored for 4 weeks, although the overall trend of the decrease in the heme peak area was not significant, the peak area of heme decreased more slowly in the deep eutectic solvent. After 28 days of storage, the peak area decrease rates were 11% and 5.3% respectively, while in the sodium hydroxide solution, the peak area decrease rates of heme were 28.1% and 23.1% respectively.

[0037] It can be seen from this that compared with the commonly used alkaline solution, the deep eutectic solvent is more suitable for the storage of heme and can extend the storage time of heme.

Claims

1. A method for efficiently dissolving hemoglobin, characterized in that: The steps include: (1) Preparation of a low eutectic solvent: Mix a hydrogen bond donor and a hydrogen bond acceptor in a molar ratio of 1:0.5 to 1:8 and heat in a water bath to prepare a low eutectic solvent; (2) Dissolution: Add hemoglobin to the prepared low eutectic solvent, mix hemoglobin and the low eutectic solvent thoroughly at a solvent ratio of 1-3.5 mg / mL, heat in a water bath at 30-90°C, heat and dissolve for 5-30 min, and stir continuously to allow the hemoglobin to fully dissolve in the low eutectic solvent.

2. The method for efficiently dissolving heme according to claim 1, characterized in that: The hydrogen bond donor is any one of urea, oxalic acid, levulinic acid, glycine, L-proline, ethylene glycol, 1,2-propylene glycol, glycerol, 1,4-butanediol, triethylene glycol or polyethylene glycol 200.

3. The method for efficiently dissolving heme according to claim 2, characterized in that: The hydrogen bond acceptor is any one of tetraethylammonium bromide, tetrabutylammonium bromide, tetrabutylammonium chloride, betaine or choline chloride.

4. The method for efficiently dissolving heme according to claim 1, characterized in that: The stirring rate in step (2) is 50-500 rpm.

5. The method for efficiently dissolving heme according to claim 1, characterized in that: In the step (1), the water bath heating temperature is 25-80° C., and the heating time is 10-240 min.

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