A method for improving aglycone conversion efficiency by using a eutectic solvent

Abstract

The application discloses a method for improving aglycone conversion efficiency by using a eutectic solvent and belongs to the technical field of green extraction. The method for improving aglycone conversion efficiency by using a eutectic solvent is characterized by comprising the following steps: uniformly mixing kaempferol-3-O-rutinoside, a eutectic solvent solution and a rutin degrading enzyme solution, performing an enzymolysis reaction and obtaining an enzymolysis solution; then, purifying the enzymolysis solution to obtain aglycone kaempferol; wherein the eutectic solvent in the eutectic solvent solution is one of betaine-ethylene glycol, choline chloride-urea, choline chloride-malic acid, choline chloride-urea-acetamide. The method is green and efficient, the prepared aglycone kaempferol has a high yield of more than 45% and a high purity of more than 95%.

Description

Technical Field

[0001] This invention relates to a method for improving aglycone conversion efficiency using a eutectic solvent, belonging to the field of green extraction technology. Background Technology

[0002] Flavonoids typically exist as flavonoid glycosides linked to glycosyl groups, with a small portion existing in a free state (aglycone). Generally, flavonoid glycosides have lower physiological activity than flavonoid aglycones, and flavonoid aglycones have higher bioavailability than their corresponding glycosides. Human studies have shown that flavonoid aglycones have higher bioavailability than flavonoid glycosides. Numerous studies have also reported that the in vitro antioxidant and glycosidase inhibitory activities of flavonoid glycosides are significantly lower than those of their corresponding aglycones. Therefore, the efficient production and preparation of flavonoid aglycones is of greater significance.

[0003] Currently, the main methods for preparing flavonoid aglycones are acid hydrolysis, high-pressure hydrolysis, and enzymatic catalysis. Acid hydrolysis is highly polluting and produces many impurities; high-pressure hydrolysis requires harsh reaction conditions and easily generates small-molecule impurities, making subsequent aglycone purification and separation difficult. Enzymatic catalysis offers mild reaction conditions and strong regioselectivity, and is widely used in aglycone preparation. However, enzymatic catalysis is greatly limited by substrate concentration; when the substrate concentration exceeds 2 mg / mL, a large amount of precipitate is generated, leading to low reaction efficiency in subsequent synthesis reactions and hindering industrial application.

[0004] Eutectic solvents, as a novel type of green solvent, exhibit high solubility for lipid-soluble components. Based on these properties, eutectic solvents are widely used in the extraction processes of active compounds (such as polyphenols and carotenoids) from raw materials in food and traditional Chinese medicine. However, their application in the catalytic preparation of flavonoid aglycones has not yet been reported. Summary of the Invention

[0005] [Technical Issues]

[0006] The method of preparing aglycone kaempferol using ordinary water medium and enzyme-assisted catalysis suffers from poor solubility and low enzyme activity, resulting in low yield and purity.

[0007] Eutectic solvents were not used to synthesize the aglycone kaempferol.

[0008] [Technical Solution]

[0009] To address the aforementioned issues, this invention employs a specific raw material (kaempferol-3-O-rutin glycoside), a specific eutectic solvent solution, and a specific rutin-degrading enzyme solution for enzymatic hydrolysis to obtain the aglycone kaempferol. The method of this invention is green and efficient, and the prepared aglycone kaempferol has high yield and purity.

[0010] The first objective of this invention is to provide a method for improving aglycone conversion efficiency using a eutectic solvent, comprising the following steps:

[0011] Kaempferol-3-O-rutin glycoside, eutectic solvent solution, and rutin-degrading enzyme solution were mixed evenly and subjected to enzymatic hydrolysis to obtain the hydrolysate; then purified to obtain the aglycone kaempferol.

[0012] The eutectic solvent in the eutectic solvent solution is one of betaine-ethylene glycol, choline chloride-urea, choline chloride-malic acid, or choline chloride-urea-acetamide.

[0013] In one embodiment of the present invention, the molar ratio of betaine to ethylene glycol in betaine-ethylene glycol is 1:1-2.

[0014] In one embodiment of the present invention, the molar ratio of choline chloride to urea in the choline chloride-urea mixture is 1:1.5-2.5.

[0015] In one embodiment of the present invention, the molar ratio of choline chloride to malic acid in choline chloride-malic acid is 1:0.5-1.5.

[0016] In one embodiment of the present invention, the molar ratio of choline chloride, urea and acetamide in choline chloride-urea-acetamide is 1:0.5-1.5:0.5-1.5.

[0017] In one embodiment of the present invention, the ratio of kaempferol-3-O-rutin glycoside, eutectic solvent solution, and rutin degrading enzyme solution is 1g:3-45mL:0.5-1.5mL.

[0018] In one embodiment of the present invention, the solvent of the eutectic solvent solution is water, with a volume fraction of 15-25%.

[0019] In one embodiment of the present invention, the rutin-degrading enzyme solution is prepared by dissolving rutin-degrading enzyme powder in water, with a concentration of 0.5-1.5 mg / mL.

[0020] In one embodiment of the present invention, the enzyme activity of rutin-degrading enzyme is 15-30 U / mg; the preparation method of rutin-degrading enzyme is as follows: defatted buckwheat flour is mixed with acetate buffer, extracted overnight, frozen and centrifuged, and the supernatant is the crude enzyme solution of rutin-degrading enzyme; then separated, extracted, concentrated and dried to obtain rutin-degrading enzyme.

[0021] In one embodiment of the present invention, the enzymatic hydrolysis reaction is carried out at a pH of 4-8 and a temperature of 20-50°C for 1-60 minutes.

[0022] In one embodiment of the present invention, purification is performed by D101 macroporous resin chromatography; wherein, column chromatography is performed by D101 macroporous adsorption resin column chromatography, eluting with water, discarding the first 1.5-2 column volumes of water eluent, then eluting with 2 column volumes of ethanol aqueous solution (volume fraction of 80%), concentrating to obtain pure kaempferol.

[0023] The second objective of this invention is to prepare the aglycone kaempferol using the method described herein.

[0024] The third objective of this invention is the application of the aglycone kaempferol described herein in the food or pharmaceutical fields.

[0025] [Beneficial Effects]

[0026] (1) The eutectic solvent used in this invention is a green solvent with better dissolving performance and can avoid the destruction of enzyme activity by conventional organic reagents.

[0027] (2) Compared with conventional synthesis methods of aglycone kaempferol, the present invention uses an enzyme catalytic system composed of betaine-ethylene glycol, which can increase the relative efficiency of aglycone kaempferol synthesis to 170.76%.

[0028] (3) The yield of aglycone kaempferol in this invention can reach up to 1.62 times that of conventional systems.

[0029] (4) The aglycone kaempferol prepared by this invention has a high yield, reaching more than 45%; and a high purity, reaching more than 95%. Attached Figure Description

[0030] Figure 1 This is a diagram illustrating the mechanism by which kaempferol-3-O-rutin glycoside is hydrolyzed into the aglycone kaempferol under the catalysis of rutin-degrading enzymes.

[0031] Figure 2 This is a mass spectrometry infographic of the aglycone kaempferol obtained in Example 7.

[0032] Figure 3 This is the mass spectrometry ion chromatogram of Example 7 after enzymatic reaction in the betaine-ethylene glycol system.

[0033] Figure 4 This is the mass spectrometry ion chromatogram of the enzyme reaction in the pure water system in Comparative Example 3. Detailed Implementation

[0034] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0035] Test method:

[0036] 1. Quantitative method for the aglycone kaempferol:

[0037] Instruments: Ultra-high performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometer (UPLC-Q-TOF-MS, Waters Corporation, USA); Column: CORTECS C18+ (2.7μm, 2.1×150mm);

[0038] Liquid chromatography conditions: Phase A: acetonitrile; Phase B: 0.1% formic acid in water; Flow rate: 0.3 mL / min; Column temperature: 45℃;

[0039] Gradient elution program: Phase B is maintained at 97% from 0 min to 0.5 min, Phase B decreases from 97% to 45% from 0.5 min to 10 min, Phase B decreases from 45% to 100% from 10 min to 10.5 min, Phase B is maintained at 100% from 10 min to 12 min, and then Phase B is restored to 97% for operation.

[0040] The content of the aglycone kaempferol was calculated using a standard curve.

[0041] Raw materials used in the examples:

[0042] Kaempferol-3-O-rutin standard was purchased from Yuan Ye Biotechnology Co., Ltd.; CAS code: 17650-84-9;

[0043] Rutin-degrading enzyme: It was prepared according to the literature (Zhang Yuwei, Li Jie, Yuan Yong, et al. Purification, enzymatic properties and partial primary structure analysis of rutin-degrading enzyme from buckwheat seeds. Chinese Journal of Biotechnology, 2017, 33(5):796-807), and the enzyme activity was 20.84 U / mg;

[0044] Preparation of eutectic solvents:

[0045] Hydrogen bond donors and hydrogen bond acceptors were mixed in an appropriate molar ratio (Table 1) and placed in a reaction flask. The mixture was then magnetically stirred at 80°C until a homogeneous liquid was formed.

[0046] Table 1

[0047]

[0048]

[0049] Example 1

[0050] A method for improving aglycone conversion efficiency using a eutectic solvent includes the following steps:

[0051] Mix 100g of kaempferol-3-O-rutin glycoside, 400mL of 20% choline chloride-urea (molar ratio 1:2) aqueous solution, and 100mL of 1mg / mL rutin-degrading enzyme aqueous solution evenly. Carry out the enzymatic hydrolysis reaction at pH 5 and 30℃ for 1min (observe the reaction rate in a short time). Immediately place the reactants in a boiling water bath to terminate the enzymatic reaction.

[0052] The content of aglycone kaempferol is the concentration of aglycone kaempferol in the system after the reaction is complete.

[0053] Example 2

[0054] In Example 1, the choline chloride-urea (molar ratio 1:2) was changed to choline chloride-malic acid (molar ratio 1:1), while all other aspects remained the same as in Example 1.

[0055] Example 3

[0056] In Example 1, the choline chloride-urea (molar ratio 1:2) was changed to choline chloride-urea-acetamide (molar ratio 1:1:1), while all other aspects remained the same as in Example 1.

[0057] Example 4

[0058] In Example 1, the choline chloride-urea (molar ratio 1:2) was changed to betaine-ethylene glycol (molar ratio 1:2), while all other aspects remained the same as in Example 1.

[0059] Comparative Example 1

[0060] In Example 1, the choline chloride-urea (molar ratio 1:2) was adjusted to the eutectic solvent numbered 5-25 in Table 1, while other aspects remained the same as in the Example.

[0061] Comparative Example 2

[0062] The choline chloride-urea (molar ratio 1:2) aqueous solution in Example 1 was changed to pure water, while other aspects remained the same as in Example 1.

[0063] The obtained product was subjected to performance testing, and the test results are as follows:

[0064] Table 2

[0065]

[0066]

[0067] Note: The synthesis efficiency of each eutectic solvent treatment group is expressed as the relative synthesis efficiency of the blank group.

[0068] Relative synthesis efficiency (%) = content of kaempferol in the eutectic solvent treatment / content of kaempferol in the blank treatment × 100%.

[0069] from Figure 1 It can be seen that when the eutectic solvents are betaine-ethylene glycol, choline chloride-urea, choline chloride-malic acid, and choline chloride-urea-acetamide, the relative synthesis efficiency is over 100%. Other eutectic solvents have lower relative synthesis efficiency. Therefore, betaine-ethylene glycol, choline chloride-urea, choline chloride-malic acid, and choline chloride-urea-acetamide will be selected for the reaction system in the future.

[0070] Example 5

[0071] A method for improving aglycone conversion efficiency using a eutectic solvent includes the following steps:

[0072] Mix 100g of kaempferol-3-O-rutin glycoside, 400mL of 20% betaine-ethylene glycol (molar ratio 1:2) aqueous solution, and 100mL of 1mg / mL rutin-degrading enzyme aqueous solution evenly, and carry out enzymatic hydrolysis reaction at pH 8 and 40℃ for 30min to obtain enzymatic hydrolysate;

[0073] The enzymatic hydrolysate was subjected to macroporous adsorption resin D101 column chromatography, eluted with water, and the first two column volumes of water eluent were discarded. Then, it was eluted with two column volumes of ethanol-water solution (80% by volume), and concentrated to obtain pure kaempferol.

[0074] Example 6

[0075] The volume fraction of the betaine-ethylene glycol (molar ratio 1:2) aqueous solution in Example 5 was adjusted to 10%, while other aspects remained the same as in Example 5.

[0076] Example 7

[0077] The pH of the enzymatic hydrolysis in Example 5 was adjusted to 4, while other aspects remained the same as in Example 5.

[0078] Example 8

[0079] The enzymatic hydrolysis temperature in Example 5 was adjusted to 25°C, while other aspects remained the same as in Example 5.

[0080] Example 9

[0081] The amount of betaine-ethylene glycol (molar ratio 1:2) aqueous solution in Example 5 was adjusted to 200 mL, while other aspects remained the same as in Example 5.

[0082] Comparative Example 3

[0083] In Example 5, the betaine-ethylene glycol (molar ratio 1:2) aqueous solution was changed to pure water, while other aspects remained the same as in Example 5.

[0084] Comparative Example 4

[0085] In Example 5, the betaine-ethylene glycol (molar ratio 1:2) aqueous solution was changed to an ethanol aqueous solution with a volume fraction of 20%, while other aspects remained the same as in Example 5.

[0086] The obtained product was subjected to performance testing, and the test results are as follows:

[0087] Table 3

[0088] example Kaempferol content (mg / mL) in enzyme reaction extract Mass of kaempferol (g) Purity (%) of pure kaempferol Example 5 151.11 50.15 95.68 Example 6 138.53 45.28 96.63 Example 7 166.78 55.95 95.26 Example 8 154.52 50.08 97.00 Example 9 149.12 50.50 96.11 Comparative Example 3 109.28 34.32 96.38 Comparative Example 4 118.12 40.23 93.26

[0089] Figure 1 This is a diagram illustrating the mechanism by which kaempferol-3-O-rutin glycoside is hydrolyzed into the aglycone kaempferol under the catalysis of rutin-degrading enzymes.

[0090] Figure 2 This is a mass spectrum infographic of the aglycone kaempferol obtained in Example 7. From... Figure 2 It can be seen that the product prepared in Example 7 is indeed aglycone kaempferol. The mass spectrometry information of aglycone kaempferol obtained in other examples and comparative examples is consistent with that in Example 7, proving that the product prepared by the system of the present invention is aglycone kaempferol.

[0091] Figure 3 This is a mass spectrometry ion chromatogram after the enzymatic reaction in the betaine-ethylene glycol system. Figure 4 The mass spectrometry ion chromatogram after the enzyme reaction in the pure water system revealed the following: Figure 4 The kaempferol content obtained from the pure water system was significantly lower than that from the betaine-ethylene glycol system, indicating that the eutectic system can effectively improve the conversion efficiency of aglycone kaempferol.

[0092] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for improving the conversion efficiency of aglycone by using a deep eutectic solvent, characterized in that, Comprising the following steps: Mix kaempferol-3-O-rutinoside, a solution of a deep eutectic solvent, a solution of rutin-degrading enzyme in a ratio of 1g:3-4mL:0.5-1.5mL, and then carry out enzymatic reaction under the conditions of pH 4-8 and 25-40℃ for 30-60min to obtain an enzymatic hydrolysate; then purify to obtain aglycone kaempferol; The solvent of the solution of the deep eutectic solvent is water, and the volume fraction is 15%-25%. The solution of rutin-degrading enzyme is prepared by dissolving rutin-degrading enzyme powder in water, and the concentration is 0.5-1.5mg / mL. The deep eutectic solvent in the solution of the deep eutectic solvent is betaine-ethylene glycol; the molar ratio of betaine to ethylene glycol in betaine-ethylene glycol is 1:1-2.

2. The method of claim 1, wherein, The purification is D101 macroporous resin chromatography.

3. The method of claim 2, wherein, The chromatography method is: loading the enzymatic hydrolysate onto a D101 macroporous resin chromatography column, eluting with water, discarding the first 1.5-2 times column volume of water eluate, then eluting with 2 times column volume of 80% ethanol aqueous solution, concentrating to obtain pure aglycone kaempferol.

Images