Preparation method of glucosyl modified quercetin derivative

Abstract

The invention discloses a preparation method of a glucosyl modified quercetin derivative. The preparation method of the glucosyl modified isoquercitrin, provided by the invention, comprises the following step: in water, in the presence of enzyme and beta-cyclodextrin, carrying out glycosylation reaction on isoquercitrin and a glycosyl donor, so as to obtain the glucosyl modified isoquercitrin. The preparation method takes water as a solvent, and the product is easy to purify; furthermore, the yield and the purity of the product are relatively high, and the method is suitable for industrial-grade production.

Description

Technical Field

[0001] The present invention relates to a method for preparing flavonoid compounds, and particularly to a method for preparing a glucosyl-modified quercetin derivative. Background Art

[0002] Isoquercitrin is a natural flavonoid compound widely distributed in various plants, including fruits, vegetables, and herbs. This compound has received extensive attention in the scientific community due to its various biological activities such as antioxidant, anti-inflammatory, and anticancer effects (Eur J Pharmacol. 585, 325 - 337)(J Cosmet Dermatol. 20, 2932 - 2939). However, due to the poor solubility of isoquercitrin in common solvent systems such as water, ethanol, and oils, it not only limits its application in the cosmetic field but also restricts its bioavailability and scope of application. Therefore, the development of water-soluble isoquercitrin derivatives can greatly broaden its application range and enhance its efficacy in terms of improving bioavailability.

[0003] In recent years, a multi-glucosyl-modified quercetin derivative (Enzymatically Modified Isoquercitrin, EMIQ) has come into the view of the scientific community. This substance is a derivative obtained by the catalytic action of cyclodextrin glucosyltransferase on isoquercitrin, attaching 1 - 12 glucoside units (Biol Pharm Bull. 2009; 32(12): 2034 - 2040.).

[0004]

[0005] Due to this enzymatic modification, not only is the solubility of EMIQ significantly improved (Food Chem. 2017, 229, 75 - 83), but its bioavailability has also been significantly enhanced (Arch Biochem Biophys. 2010; 501(1): 91 - 97.). This not only expands its application range in cosmetics, pharmaceuticals, and health products but also makes it show more excellent effects in terms of biological activities such as antioxidant properties (Arch Toxicol 84, 143 - 153(2010)). Therefore, the development of an economical and efficient method for preparing EMIQ has become one of the research hotspots in the fields of medicine, health products, and cosmetics.

[0006] So far, two main routes for the preparation of EMIQ have been reported: 1. Using isoquercitrin as the starting material, EMIQ is generated through a one-step enzymatic reaction of glycosyltransferase; 2. Based on rutin, EMIQ is generated through a two-step enzymatic catalytic reaction.

[0007] A literature (Enzyme Microb Technol, 2019, 120: 84-90) describes a preparation method of EMIQ based on isoquercitrin, using the amylosucrase-sucrose / Tris-HCl buffer system, or the α-amylase-starch / citrate phosphate buffer system, or the cyclodextrin glucanotransferase-starch / citrate phosphate buffer system. However, this method uses a large amount of buffer solution, resulting in a high impurity content in the product. At the same time, due to the high water solubility of EMIQ, product purification has become a difficult problem, which greatly limits its industrial application.

[0008] The patent (publication number CN 109438536A) discloses a method for preparing EMIQ using affordable rutin as the starting material through a two-stage reaction. In the first stage, a buffer solution composed of disodium hydrogen phosphate and sodium citrate is used, and rhamnosidase is added to generate isoquercitrin. In the second stage, also in the same disodium hydrogen phosphate-sodium citrate buffer solution, EMIQ is obtained by adding glycosyltransferase. However, this method has multiple problems, making it almost impossible to be industrially applied: 1. The use of a large amount of buffer solution makes product purification very difficult, and this method does not provide an effective way to prepare high-quality products; 2. More strangely, during the synthesis of EMIQ, no glucose donor is added. Theoretically, the transfer of glucose groups is impossible.

[0009] CN101103121A discloses a method for preparing EMIQ using water and corn starch, but when the applicant repeated the preparation method disclosed in this patent, the yield was very low, only 8%. Currently, among the commercially available EMIQ in China, the impurity content is relatively high, and the purity of EMIQ is only 27%. Therefore, a new method for preparing EMIQ needs to be developed. Summary of the Invention

[0010] The technical problem to be solved by the present invention is that in the prior art, EMIQ is difficult to purify, has a low yield, and is not suitable for industrial production. Therefore, the present invention provides a method for preparing a glucosyl-modified quercetin derivative. This preparation method uses water as a solvent, and the product is easy to purify; further, the product has a high yield and purity, and is suitable for industrial production.

[0011] The present invention provides a method for preparing a glucosyl-modified isoquercitrin (i.e., Enzymatically Modified Isoquercitrin, EMIQ), which comprises the following steps: In water, in the presence of an enzyme and β-cyclodextrin, glycosylation reaction is carried out on isoquercitrin and a glycosyl donor to obtain the glucosyl-modified isoquercitrin.

[0012] In a preferred embodiment, the glucosyl-modified isoquercitrin prepared by the above method is a crude product (unpurified), and the crude product further includes unreacted isoquercitrin.

[0013] In a preferred embodiment, the preparation method includes the following steps: in water, in the presence of an enzyme and β-cyclodextrin, the isoquercitrin shown in Formula I and a glycosyl donor are subjected to the glycosylation reaction shown below to obtain the glucosyl-modified isoquercitrin shown in Formula II,

[0014]

[0015] n is selected from one or more of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11.

[0016] In a preferred embodiment, n is at least 1, 2, and 3; for example, n is at least 1, 2, 3, 4, 5, and 6; and further for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11.

[0017] In a preferred embodiment, in the crude product of the glucosyl-modified isoquercitrin (calculated based on the total molar mass of isoquercitrin and glucosyl-modified isoquercitrin being 100%), the molar percentage of isoquercitrin is 0 - 30%; preferably, it is 26% or less; for example, 20%, 22%, 25%, or 26%.

[0018] In a preferred embodiment, in the crude product of the glucosyl-modified isoquercitrin (calculated based on the total molar mass of isoquercitrin and glucosyl-modified isoquercitrin being 100%), the molar percentage of the component with n = 1 is 10 - 25%; for example, 17%, 18%, or 19%;

[0019] In a preferred embodiment, in the crude product of the glucosyl-modified isoquercitrin (calculated based on the total molar mass of isoquercitrin and glucosyl-modified isoquercitrin being 100%), the molar percentage of the component with n = 2 is 10 - 25%; for example, 14%, 15%, 17%, or 19%.

[0020] In a preferred embodiment, in the crude product of the glucosyl-modified isoquercitrin (calculated based on the total molar mass of isoquercitrin and glucosyl-modified isoquercitrin being 100%), the molar percentage of the component with n = 3 is 10 - 25%; for example, 11% or 12%.

[0021] In a preferred embodiment, in the glycosylation reaction, the volume-mass ratio of water to the isoquercitrin is (50 - 500) mL / g, for example, (100 - 300) mL / g, and further for example, 100 mL / g.

[0022] In a preferred embodiment, the mass ratio of the β-cyclodextrin to the isoquercitrin is (1 - 5):1, such as (2 - 4):1, and more preferably 2:1.

[0023] The enzyme is a glycosyltransferase conventional in such reactions in the art. Preferably, the enzyme is cyclodextrin glycosyltransferase (CGTase) or an α-amylase having cyclodextrin glycosyltransferase activity; the α-amylase having cyclodextrin glycosyltransferase activity may be α-amylase G "Amano" L.

[0024] In a preferred embodiment, the mass ratio of the enzyme to the isoquercitrin is (0.15 - 1):1, such as (0.2 - 0.7):1, and more preferably 0.2:1 or 0.5:1.

[0025] In a preferred embodiment, the glycosyl donor is β-cyclodextrin.

[0026] In a preferred embodiment, the glycosyl donor is starch and not β-cyclodextrin, such as maltodextrin.

[0027] In a preferred embodiment, when the glycosyl donor is starch and not β-cyclodextrin, the mass ratio of the glycosyl donor to the isoquercitrin is (0.1 - 5):1, such as (0.5 - 3):1, and more preferably 0.5:1, 1:1 or 2:1.

[0028] In a preferred embodiment, when the glycosyl donor is starch and not β-cyclodextrin, the mass ratio of the glycosyl donor to the isoquercitrin is (1 - 3):1.

[0029] In a preferred embodiment, the components of the glycosylation reaction further include calcium chloride.

[0030] In a preferred embodiment, the mass ratio of the calcium chloride to the isoquercitrin is (0.01 - 0.1):1, such as (0.01 - 0.05):1, and more preferably 0.02:1.

[0031] In a preferred embodiment, the isoquercitrin and the β-cyclodextrin participate in the glycosylation reaction in the form of a β-cyclodextrin-isoquercitrin inclusion complex, in which the β-cyclodextrin includes the isoquercitrin.

[0032] In a preferred embodiment, the β-cyclodextrin-isoquercitrin inclusion complex is prepared by the following method: in water, the isoquercitrin shown in Formula I and the β-cyclodextrin are subjected to an inclusion reaction to obtain the β-cyclodextrin-isoquercitrin inclusion complex.

[0033] In a preferred embodiment, in the inclusion reaction, the volume-mass ratio of water to isoquercetin is (50 - 500) mL / g, such as (100 - 300) mL / g, and more preferably 100 mL / g.

[0034] In a preferred embodiment, the temperature of the inclusion reaction is 30°C - 90°C, such as 50°C - 85°C; more preferably 60°C, 70°C or 80°C.

[0035] In a preferred embodiment, the time of the inclusion reaction is 2 h - 30 h, such as 4 h - 20 h; more preferably 4 h, 8 h or 16 h.

[0036] In a preferred embodiment, after the inclusion reaction, the following post-treatment steps are further included: drying; preferably, the drying is vacuum drying, such as drying with a rotary evaporator.

[0037] In a preferred embodiment, in the post-treatment step after the inclusion reaction, the temperature of the drying is 30°C - 70°C, such as 50°C - 65°C; more preferably 55°C.

[0038] In a preferred embodiment, the components in the inclusion reaction are composed of the following: water, isoquercetin and β-cyclodextrin.

[0039] In a preferred embodiment, the method for preparing the glucose-modified isoquercetin includes the following steps:

[0040] (1) In water, the isoquercetin and the β-cyclodextrin are subjected to an inclusion reaction, and then dried to obtain a β-cyclodextrin isoquercetin inclusion complex;

[0041] (2) In water, in the presence of an enzyme, the β-cyclodextrin isoquercetin inclusion complex obtained in step (1) is subjected to a glycosylation reaction with a glycosyl donor to obtain the glucose-modified isoquercetin.

[0042] The progress of the glycosylation reaction ends when there is no insoluble matter; preferably, the time of the glycosylation reaction is 12 - 20 h, such as 12 - 18 h, and more preferably 15 h.

[0043] In a preferred embodiment, the temperature of the glycosylation reaction is 35°C - 70°C, such as 45°C - 65°C, and more preferably 50°C - 60°C; even more preferably 55°C.

[0044] When the components of the glycosylation reaction further include calcium chloride, the glycosylation reaction includes the following steps: In water, in the presence of an enzyme and calcium chloride, the β-cyclodextrin isoquercetin inclusion complex obtained in step (1) is subjected to a glycosylation reaction with a glycosyl donor to obtain the glucose-modified isoquercetin.

[0045] In a preferred embodiment, the components in the glycosylation reaction are composed of the following: water, isoquercitrin, β-cyclodextrin, an enzyme, and a glycosyl donor.

[0046] In a preferred embodiment, the components in the glycosylation reaction are composed of the following: water, isoquercitrin, β-cyclodextrin, an enzyme, a glycosyl donor, and calcium chloride.

[0047] In a preferred embodiment, the method for preparing glucosyl-modified isoquercitrin includes the following steps:

[0048] (1) In water, perform an inclusion reaction on isoquercitrin and β-cyclodextrin, and then dry to obtain an inclusion complex of β-cyclodextrin and isoquercitrin;

[0049] (2) In water, in the presence of an enzyme and calcium chloride, perform a glycosylation reaction on the inclusion complex of β-cyclodextrin and isoquercitrin obtained in step (1) with maltodextrin to obtain the glucosyl-modified isoquercitrin;

[0050] In step (1), the volume-to-mass ratio of water to isoquercitrin is (100 - 300) mL / g;

[0051] In step (1), the mass ratio of β-cyclodextrin to isoquercitrin is (2 - 4):1;

[0052] In step (1), the temperature of the inclusion reaction is 70°C - 85°C;

[0053] In step (2), the volume-to-mass ratio of water to isoquercitrin is (100 - 300) mL / g;

[0054] In step (2), the enzyme is cyclodextrin glucosyltransferase or α-amylase with cyclodextrin glucosyltransferase activity;

[0055] In step (2), the mass ratio of the enzyme to isoquercitrin is (0.2 - 0.7):1,

[0056] In step (2), the mass ratio of maltodextrin to isoquercitrin is (1 - 3):1,

[0057] In step (2), the mass ratio of calcium chloride to isoquercitrin is (0.01 - 0.05):1;

[0058] In step (2), the temperature of the glycosylation reaction is 50°C - 60°C.

[0059] After the glycosylation reaction, a post-treatment step may also be included. Preferably, the post-treatment includes one or more of the following steps: decolorization, suction filtration, membrane filtration, concentration, and drying;

[0060] The decolorization mentioned above may be decolorization by adsorption with activated carbon;

[0061] The temperature of the decolorization may be 30°C - 90°C, such as 50°C - 85°C; for another example, 60°C, 70°C or 80°C;

[0062] The time of the decolorization may be 1 - 2 h, such as 1 h;

[0063] The membrane filtration mentioned above may be ultrafiltration membrane filtration; for example, the cut-off molecular weight of the ultrafiltration membrane is 3000.

[0064] On the basis of not violating the common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

[0065] The reagents and raw materials used in the present invention are all commercially available.

[0066] The positive and progressive effects of the present invention are as follows: The preparation method of a quercetin derivative modified with glucosyl provided by the present invention has the advantages that the product is easy to purify, the yield and purity are both relatively high, and it is suitable for industrial production; further, the purity of EMIQ in this product is greater than 21%, and further can reach 50% - 78%, and it can be applied in the food industry or the cosmetics industry. Brief Description of the Drawings

[0067] Figure 1 : HPLC analysis chart of EMIQ prepared in Example 1.

[0068] Figure 2 : HPLC analysis chart of EMIQ prepared in Example 6.

[0069] Figure 3 : HPLC analysis chart of EMIQ prepared in Example 7.

[0070] Figure 4 : HPLC analysis chart of EMIQ prepared in Example 11.

[0071] Figure 5 : Appearance diagram of EMIQ obtained in Example 1 of this application and appearance diagram of the reference product EMIQ. Detailed Description of the Embodiments

[0072] The present invention will be further illustrated by the following examples, but the present invention is not limited to the scope of the described examples. The experimental methods without specific conditions noted in the following examples are carried out according to conventional methods and conditions, or are selected according to the product specifications.

[0073] Isoquercitrin, purchased from Xi'an Shennong, with a content > 95%, batch number SN - 20220618.

[0074] β-Cyclodextrin, purchased from Shandong Xinda Biotechnology Co., Ltd., batch number F2206077.

[0075] Maltodextrin, purchased from Shanghai Senhang Industrial Co., Ltd., batch number 202302082.

[0076] α-Amylase G “Amano” L: α-Amylase G “Amano” L, purchased from Amano Enzyme, batch number CGTV0252203SLK.

[0077] The synthetic route of the present invention is shown as follows:

[0078]

[0079] In the present invention, the molar yield of EMIQ (calculated based on the molar mass of isoquercitrin IQ) refers to using a UV spectrophotometer to detect the absorbance of isoquercitrin IQ (including IQ itself and glycosylated IQ) in the solid powder obtained through operations such as dialysis purification in each example under the condition of 360 nm. Then, according to the standard curve, the number of moles of isoquercitrin IQ (including IQ itself and glycosylated IQ) in the solid powder is obtained. The ratio of this number of moles to the number of moles of the input IQ is the molar yield of EMIQ (calculated based on IQ).

[0080] In the present invention, the purity of EMIQ refers to calculating the molar mass of different glycosylated IQs using the HPLC method, multiplying it by the corresponding molecular weight to obtain the mass of IQ itself and all EMIQs glycosylated to different degrees. The ratio of the sum of this mass to the total mass of the product is the purity of EMIQ.

[0081] In the present invention, the ratio of IQ-n refers to calculating the molar mass of different glycosylated IQs using the HPLC method. The ratio of this molar mass to the sum of the molar masses of IQ itself and all EMIQs glycosylated to different degrees is the ratio of IQ-n.

[0082] In the present invention, the yield of IQ-n refers to multiplying the ratio of IQ-n by the molar yield of EMIQ, which is the molar yield of IQ-n. Multiplying the molar yield by the ratio of the molecular weight of IQ-n to the molecular weight of IQ is the ratio of the mass of IQ-n to the input amount of IQ, which is the yield of IQ-n.

[0083] HPLC conditions in the present invention: The mobile phase consists of phase A (0.4% phosphoric acid) and phase B (acetonitrile: methanol, with a ratio of 10:1). The detection wavelength is 360 nm, the column temperature is 35 °C, and the injection volume is 10 μL. The elution program is as follows (the proportion of mobile phase B in the mobile phase): 0.01 s - 5.5%, 2 min - 13.2%, 4 min - 8.7%, 30 min - 18.7%, 32 min - 19.3%, 34 min - 23.1%, 40 min - 29.3%, 41 min - 5.5%.

[0084] Example 1:

[0085] Weigh 1 g of isoquercitrin, add 100 mL of water, add 2 g of β-cyclodextrin, heat up to 80 °C with stirring, and react for 8 h. Under a vacuum of -0.095 MPa and at 55 °C, after evaporating to dryness under reduced pressure using a rotary evaporator, add 100 ml of water, 1 g of maltodextrin, 0.2 g of α-Amylase G “Amano” L, and 0.02 g of calcium chloride, and stir and react at 55 °C for 15 h. Add 0.02 g of activated carbon, keep warm and decolorize for 1 hour, filter by suction. The filtrate is filtered through a ultrafiltration membrane with a molecular weight cut-off of 3000 under a pressure of 0.4 mPa to remove macromolecules, and then concentrated and dried on the permeate side to obtain EMIQ. The absorbance of the EMIQ finished product is measured at 360 nm using a UV spectrophotometer, and the yield is calculated to be 93% (based on isoquercitrin). HPLC is used to analyze the purity and the content of each component of the prepared EMIQ, as shown in Table 1 or Figure 1 as shown

[0086] Example 2:

[0087] Weigh 1 g of isoquercitrin, add 100 mL of water, add 1 g of β-cyclodextrin, heat up to 80 °C with stirring, and react for 8 h. Under a vacuum of -0.095 MPa and at 55 °C, after evaporating to dryness under reduced pressure using a rotary evaporator, add 100 ml of water, 1 g of maltodextrin, 0.2 g of α-Amylase G “Amano” L, and 0.02 g of calcium chloride, and stir and react at 55 °C for 15 h. Add 0.02 g of activated carbon, keep warm and decolorize for 1 hour, filter by suction. The filtrate is filtered through a ultrafiltration membrane with a molecular weight cut-off of 3000 under a pressure of 0.4 mPa to remove macromolecules, and then concentrated and dried on the permeate side to obtain EMIQ. The absorbance of the EMIQ finished product is measured at 360 nm using a UV spectrophotometer, and the yield is calculated to be 46% (based on isoquercitrin). HPLC is used to analyze the purity and the content of each component of the prepared EMIQ, as shown in Table 1.

[0088] Example 3:

[0089] Weigh 1 g of isoquercitrin, add 100 mL of water, add 2 g of β-cyclodextrin, heat up to 70 °C with stirring, react for 8 h, under a vacuum of -0.095 MPa and at 55 °C, after evaporating to dryness under reduced pressure using a rotary evaporator, add 100 ml of water, 1 g of maltodextrin, 0.2 g of α-Amylase G “Amano” L, 0.02 g of calcium chloride, and stir and react at 55 °C for 15 h. Add 0.02 g of activated carbon, keep warm for decolorization for 1 h, filter by suction, use an ultrafiltration membrane with a molecular weight cut-off of 3000 to filter the filtrate under a pressure of 0.4 mPa, remove macromolecules by membrane filtration, concentrate the permeate side, and dry to obtain EMIQ. The absorbance of the EMIQ finished product is measured at 360 nm using a UV spectrophotometer, and the yield is calculated to be 78% (based on isoquercitrin). HPLC is used to analyze the purity and the content of each component of the prepared EMIQ, as shown in Table 1 specifically.

[0090] Example 4:

[0091] Weigh 1 g of isoquercitrin, add 100 mL of water, add 2 g of β-cyclodextrin, heat up to 60 °C with stirring, react for 8 h, under a vacuum of -0.095 MPa and at 55 °C, after evaporating to dryness under reduced pressure using a rotary evaporator, add 100 ml of water, 1 g of maltodextrin, 0.2 g of α-Amylase G “Amano” L, 0.02 g of calcium chloride, and stir and react at 55 °C for 15 h. Add 0.02 g of activated carbon, keep warm for decolorization for 1 h, filter by suction, use an ultrafiltration membrane with a molecular weight cut-off of 3000 to filter the filtrate under a pressure of 0.4 mPa, remove macromolecules by membrane filtration, concentrate the permeate side, and dry to obtain EMIQ. The absorbance of the EMIQ finished product is measured at 360 nm using a UV spectrophotometer, and the yield is calculated to be 53% (based on isoquercitrin). HPLC is used to analyze the purity and the content of each component of the prepared EMIQ, as shown in Table 1 specifically.

[0092] Example 5:

[0093] Weigh 1 g of isoquercitrin, add 100 mL of water, add 2 g of β-cyclodextrin, heat up to 80 °C with stirring, react for 4 h, under a vacuum of -0.095 MPa and at 55 °C, after evaporating to dryness under reduced pressure using a rotary evaporator, add 100 ml of water, 1 g of maltodextrin, 0.2 g of α-Amylase G “Amano” L, 0.02 g of calcium chloride, and stir and react at 55 °C for 15 h. Add 0.02 g of activated carbon, keep warm for decolorization for 1 h, filter by suction, use an ultrafiltration membrane with a molecular weight cut-off of 3000 to filter the filtrate under a pressure of 0.4 mPa, remove macromolecules by membrane filtration, concentrate the permeate side, and dry to obtain EMIQ. The absorbance of the EMIQ finished product is measured at 360 nm using a UV spectrophotometer, and the yield is calculated to be 62% (based on isoquercitrin). HPLC is used to analyze the purity and the content of each component of the prepared EMIQ, as shown in Table 1 specifically.

[0094] Example 6:

[0095] Weigh 1 g of isoquercitrin, add 100 mL of water, add 2 g of β-cyclodextrin, heat up to 80 °C with stirring, react for 16 h, under the conditions of a vacuum of -0.095 MPa and 55 °C, after evaporating to dryness under reduced pressure with a rotary evaporator, add 100 ml of water, 1 g of maltodextrin, 0.2 g of α-Amylase G “Amano” L, and 0.02 g of calcium chloride, and stir and react at 55 °C for 15 h. Add 0.02 g of activated carbon, keep warm and decolorize for 1 hour, filter by suction, the filtrate is filtered through a ultrafiltration membrane with a molecular weight cut-off of 3000 under a pressure of 0.4 mPa, remove macromolecules, concentrate the permeate side, and dry to obtain EMIQ. The absorbance of the EMIQ finished product is measured at 360 nm with a UV spectrophotometer, and the yield is calculated to be 95% (based on isoquercitrin). HPLC is used to analyze the purity and the content of each component of the prepared EMIQ, as shown in Table 1 and Figure 2 as follows.

[0096] Example 7:

[0097] Weigh 1 g of isoquercitrin, add 100 mL of water, add 2 g of β-cyclodextrin, heat up to 80 °C with stirring, react for 8 h, under the conditions of a vacuum of -0.095 MPa and 55 °C, after evaporating to dryness under reduced pressure with a rotary evaporator, add 100 ml of water, 1 g of maltodextrin, 0.5 g of α-Amylase G “Amano” L, and 0.02 g of calcium chloride, and stir and react at 55 °C for 15 h. Add 0.02 g of activated carbon, keep warm and decolorize for 1 hour, filter by suction, the filtrate is filtered through a ultrafiltration membrane with a molecular weight cut-off of 3000 under a pressure of 0.4 mPa, remove macromolecules, concentrate the permeate side, and dry to obtain EMIQ. The absorbance of the EMIQ finished product is measured at 360 nm with a UV spectrophotometer, and the yield is calculated to be 97% (based on isoquercitrin). HPLC is used to analyze the purity and the content of each component of the prepared EMIQ, as shown in Table 1 and Figure 3 as follows.

[0098] Example 8:

[0099] Weigh 1 g of isoquercitrin, add 100 mL of water, add 2 g of β-cyclodextrin, heat up to 80 °C with stirring, react for 8 h, under a vacuum of -0.095 MPa and at 55 °C, after evaporating to dryness under reduced pressure with a rotary evaporator, add 100 ml of water, 1 g of maltodextrin, 0.2 g of α-Amylase G “Amano” L, 0.02 g of calcium chloride, and stir and react at 45 °C for 15 h. Add 0.02 g of activated carbon, keep warm and decolorize for 1 h, filter by suction, use an ultrafiltration membrane with a molecular weight cut-off of 3000 to filter the filtrate to remove macromolecules under a pressure of 0.4 mPa, concentrate the permeate side, and dry to obtain EMIQ. The absorbance of the EMIQ finished product is measured at 360 nm with a UV spectrophotometer, and the yield is calculated to be 76% (based on isoquercitrin). HPLC is used to analyze the purity and the content of each component of the prepared EMIQ, as shown in Table 1 specifically.

[0100] Example 9:

[0101] Weigh 1 g of isoquercitrin, add 100 mL of water, add 2 g of β-cyclodextrin, heat up to 80 °C with stirring, react for 8 h, under a vacuum of -0.095 MPa and at 55 °C, after evaporating to dryness under reduced pressure with a rotary evaporator, add 100 ml of water, 1 g of maltodextrin, 0.2 g of α-Amylase G “Amano” L, 0.02 g of calcium chloride, and stir and react at 65 °C for 15 h. Add 0.02 g of activated carbon, keep warm and decolorize for 1 h, filter by suction, use an ultrafiltration membrane with a molecular weight cut-off of 3000 to filter the filtrate to remove macromolecules under a pressure of 0.4 mPa, concentrate the permeate side, and dry to obtain EMIQ. The absorbance of the EMIQ finished product is measured at 360 nm with a UV spectrophotometer, and the yield is calculated to be 61% (based on isoquercitrin). HPLC is used to analyze the purity and the content of each component of the prepared EMIQ, as shown in Table 1 specifically.

[0102] Example 10:

[0103] Weigh 1 g of isoquercitrin, add 100 mL of water, add 2 g of β-cyclodextrin, heat up to 80 °C with stirring, react for 8 h, under a vacuum of -0.095 MPa and at 55 °C, after evaporating to dryness under reduced pressure with a rotary evaporator, add 100 ml of water, 0.5 g of maltodextrin, 0.2 g of α-Amylase G “Amano” L, 0.02 g of calcium chloride, and stir and react at 55 °C for 15 h. Add 0.02 g of activated carbon, keep warm and decolorize for 1 h, filter by suction, use an ultrafiltration membrane with a molecular weight cut-off of 3000 to filter the filtrate to remove macromolecules under a pressure of 0.4 mPa, concentrate the permeate side, and dry to obtain EMIQ. The absorbance of the EMIQ finished product is measured at 360 nm with a UV spectrophotometer, and the yield is calculated to be 80% (based on isoquercitrin). HPLC is used to analyze the purity and the content of each component of the prepared EMIQ, as shown in Table 1 specifically.

[0104] Example 11:

[0105] Weigh 1 g of isoquercitrin, add 100 mL of water, add 2 g of β-cyclodextrin, heat up to 80 °C with stirring, react for 8 h, under a vacuum of -0.095 MPa and at 55 °C, after evaporating to dryness under reduced pressure with a rotary evaporator, add 100 ml of water, 2 g of maltodextrin, 0.2 g of α-Amylase G “Amano” L, and 0.02 g of calcium chloride, and stir and react at 55 °C for 15 h. Add 0.02 g of activated carbon, keep warm for decolorization for 1 hour, filter by suction, the filtrate is filtered through a ultrafiltration membrane with a molecular weight cut-off of 3000 under a pressure of 0.4 mPa, after removing macromolecules by membrane filtration, concentrate the permeate side, and dry to obtain EMIQ. The absorbance of the EMIQ finished product is measured at 360 nm with a UV spectrophotometer, and the yield is calculated to be 94% (calculated based on isoquercitrin). HPLC is used to analyze the purity and the content of each component of the prepared EMIQ, as shown in Table 1 and Figure 4 as follows.

[0106] Example 12:

[0107] Weigh 1 g of isoquercitrin, add 100 mL of water, add 2 g of β-cyclodextrin, heat up to 80 °C with stirring, react for 8 h, under a vacuum of -0.095 MPa and at 55 °C, after evaporating to dryness under reduced pressure with a rotary evaporator, add 100 ml of water, 1 g of maltodextrin, 0.2 g of α-Amylase G “Amano” L, and stir and react at 55 °C for 15 h. Add 0.02 g of activated carbon, keep warm for decolorization for 1 hour, filter by suction, the filtrate is filtered through a ultrafiltration membrane with a molecular weight cut-off of 3000 under a pressure of 0.4 mPa, after removing macromolecules by membrane filtration, concentrate the permeate side, and dry to obtain EMIQ. The absorbance of the EMIQ finished product is measured at 360 nm with a UV spectrophotometer, and the yield is calculated to be 72% (calculated based on isoquercitrin). HPLC is used to analyze the purity and the content of each component of the prepared EMIQ, as shown in Table 1.

[0108] Example 13:

[0109] Weigh 1 g of isoquercitrin, add 100 mL of water, add 2 g of β-cyclodextrin, heat up to 30 °C with stirring, react for 8 h, under a vacuum of -0.095 MPa and at 55 °C, after evaporating to dryness under reduced pressure using a rotary evaporator, add 100 ml of water, 1 g of maltodextrin, 0.2 g of α-Amylase G“Amano”L, 0.02 g of calcium chloride, and stir and react at 55 °C for 15 h. Add 0.02 g of activated carbon, keep warm and decolorize for 1 h, filter by suction, and use an ultrafiltration membrane with a molecular weight cut-off of 3000 to filter the filtrate under a pressure of 0.4 mPa to remove macromolecules, then concentrate the permeate side and dry to obtain EMIQ. The absorbance of the EMIQ finished product is measured at 360 nm using a UV spectrophotometer, and the yield is calculated to be 31% (based on isoquercitrin). HPLC is used to analyze the purity and the content of each component of the prepared EMIQ, as shown in Table 1 specifically.

[0110] Example 14:

[0111] Weigh 1 g of isoquercitrin, add 100 mL of water, add 2 g of β-cyclodextrin, heat up to 80 °C with stirring, react for 8 h, under a vacuum of -0.095 MPa and at 55 °C, after evaporating to dryness under reduced pressure using a rotary evaporator, add 100 ml of water, 0.2 g of α-Amylase G“Amano”L, 0.02 g of calcium chloride, and stir and react at 55 °C for 15 h. Add 0.02 g of activated carbon, keep warm and decolorize for 1 h, filter by suction, and use an ultrafiltration membrane with a molecular weight cut-off of 3000 to filter the filtrate under a pressure of 0.4 mPa to remove macromolecules, then concentrate the permeate side and dry to obtain EMIQ. The absorbance of the EMIQ finished product is measured at 360 nm using a UV spectrophotometer, and the yield is calculated to be 41% (based on isoquercitrin). HPLC is used to analyze the purity and the content of each component of the prepared EMIQ, as shown in Table 1 specifically.

[0112] Example 15:

[0113] Weigh 1 g of isoquercitrin, add 100 mL of water, add 2 g of β-cyclodextrin, heat up to 80 °C with stirring, react for 8 h. Add 0.2 g of α-Amylase G“Amano”L, 0.02 g of calcium chloride, 1 g of soluble starch, and stir and react at 55 °C for 15 h. Add 0.02 g of activated carbon, keep warm and decolorize for 1 h, filter by suction, and use an ultrafiltration membrane with a molecular weight cut-off of 3000 to filter the filtrate under a pressure of 0.4 mPa to remove macromolecules, then concentrate the permeate side and dry to obtain EMIQ. The absorbance of the EMIQ finished product is measured at 360 nm using a UV spectrophotometer, and the yield is calculated to be 28% (based on isoquercitrin). HPLC is used to analyze the purity and the content of each component of the prepared EMIQ, as shown in Table 1 specifically.

[0114] Comparative Example 1:

[0115] Weigh 1 g of isoquercitrin, add 100 mL of water, heat it to 80 °C with stirring, react for 8 h, under a vacuum of -0.095 MPa and at 55 °C, evaporate to dryness under reduced pressure using a rotary evaporator, then add 100 ml of water, 1 g of maltodextrin, 0.2 g of α-Amylase G“Amano”L, and 0.02 g of calcium chloride, and stir and react at 55 °C for 15 h. Add 0.02 g of activated carbon, keep warm and decolorize for 1 hour, filter by suction, use an ultrafiltration membrane with a molecular weight cut-off of 3000 to filter out macromolecules from the filtrate under a pressure of 0.4 mPa, concentrate the permeate side, and dry to obtain EMIQ. Measure the absorbance of the EMIQ finished product at 360 nm using a UV spectrophotometer, and calculate the yield to be 14% (based on isoquercitrin). Use HPLC to analyze the purity and the content of each component of the prepared EMIQ, as shown in Table 1 specifically.

[0116] As can be seen from Comparative Example 1, in the absence of β-cyclodextrin, the yield of EMIQ decreased significantly.

[0117] Comparative Example 2:

[0118] Weigh 1 g of isoquercitrin, add 100 mL of 0.5% sodium dihydrogen phosphate-citrate buffer solution, heat it to 60 °C with stirring, add 0.5 g of α-Amylase G“Amano”L, and react for 12 h. Add 0.01 g of activated carbon, keep warm and decolorize for 1 hour, filter by suction, use an ultrafiltration membrane with a molecular weight cut-off of 3000 to filter out macromolecules from the filtrate under a pressure of 0.4 mPa, concentrate the permeate side, and dry to obtain EMIQ. Measure the absorbance of the EMIQ finished product at 360 nm using a UV spectrophotometer, and calculate the yield to be 0% (based on isoquercitrin). Use HPLC to analyze the purity and the content of each component of the prepared EMIQ, as shown in Table 1 specifically.

[0119] As can be seen from Comparative Example 2, in the absence of any glucose donor, EMIQ cannot be obtained.

[0120] Comparative Example 3:

[0121] Weigh 1 g of isoquercitrin, add 100 mL of 0.5% sodium dihydrogen phosphate-citrate buffer solution, heat it to 55 °C with stirring, add 0.5 g of α-Amylase G“Amano”L, add 3 g of maltodextrin, and react for 12 h. Add 0.01 g of activated carbon, keep warm and decolorize for 1 hour, filter by suction, use an ultrafiltration membrane with a molecular weight cut-off of 3000 to filter out macromolecules from the filtrate under a pressure of 0.4 mPa, concentrate the permeate side, and dry to obtain EMIQ. Measure the absorbance of the EMIQ finished product at 360 nm using a UV spectrophotometer, and calculate the yield to be 19% (based on isoquercitrin).

[0122] Comparative Example 4:

[0123] Weigh 1 g of isoquercitrin, add it to 100 ml of 100 mM sodium acetate buffer (pH 5.0), heat it to 55 °C with stirring, add 0.5 g of α-Amylase G “Amano” L, add 3 g of maltodextrin, and react for 12 h. Add 0.01 g of activated carbon, keep warm for decolorization for 1 h, filter by suction, and use an ultrafiltration membrane with a molecular weight cut-off of 3000 on the filtrate. Under a pressure of 0.4 mPa, after removing macromolecules by membrane filtration, concentrate on the permeate side and dry to obtain EMIQ. The absorbance of the EMIQ finished product is measured at 360 nm with a UV spectrophotometer, and the yield is calculated to be 18% (based on isoquercitrin).

[0124] Comparative Example 5 (CN101103121A - Suntory): Take 10 g of isoquercitrin and 40 g of corn starch, add them to 500 ml of water to make the components evenly dispersed. Add 15 g of α-amylase G “Tianye” L to the feed liquid, adjust the pH = 7.25, and react at 60 °C for 24 h. Filter by suction, collect the filtrate, add 0.02 g of activated carbon, keep warm for decolorization for 1 h, filter by suction, and use an ultrafiltration membrane with a molecular weight cut-off of 3000 on the filtrate. Under a pressure of 0.4 mPa, after removing macromolecules by membrane filtration, concentrate on the permeate side and dry to obtain EMIQ. The absorbance of the EMIQ finished product is measured at 360 nm with a UV spectrophotometer, and the yield is calculated to be 8% (based on isoquercitrin).

[0125] In Examples 1 - 15 and Comparative Examples 1 - 2, the data of the mass, purity of EMIQ, and the content of the main components in the prepared EMIQ are shown in Table 1.

[0126] Table 1

[0127]

[0128]

[0129] IQ - 1 is isoquercitrin, and IQ - 2 - 12 respectively correspond to glucose-modified isoquercitrin containing 1 - 11 glucose residues.

[0130] Effect Example 1: Comparison of product appearance

[0131] The EMIQ products prepared in Examples 1 - 15 of this application have a yellow powder appearance; the commercially available EMIQ is yellowish-brown, as Figure 5 shown.

Claims

1. A preparation method of glucosyl-modified isoquercitrin, characterized in that, It includes the following steps: In water, in the presence of an enzyme and β-cyclodextrin, glycosylation reaction is carried out on isoquercitrin and a glycosyl donor to obtain the glucose-modified isoquercitrin.

2. The preparation method of the glucose-modified isoquercitrin according to claim 1, wherein, It includes the following steps: In water, in the presence of an enzyme and β-cyclodextrin, glycosylation reaction is carried out on the isoquercitrin shown in Formula I and a glycosyl donor as shown in the following formula to obtain the glucose-modified isoquercitrin shown in Formula II. n is selected from one or more of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11; preferably, the n is at least 1, 2, and 3; for example, the n is at least 1, 2, 3, 4, 5, and 6; for another example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11.

3. The preparation method of the glucosyl-modified isoquercetin according to claim 1 or 2, characterized in that, The isoquercitrin participates in the glycosylation reaction in the form of a β-cyclodextrin-isoquercitrin inclusion complex.

4. The preparation method of the glucose-modified isoquercitrin according to claim 3, wherein, The β-cyclodextrin-isoquercitrin inclusion complex is prepared by the following method: In water, inclusion reaction is carried out on the isoquercitrin shown in Formula I and β-cyclodextrin to obtain the β-cyclodextrin-isoquercitrin inclusion complex.

5. The preparation method of the glucose-modified isoquercitrin according to claim 4, wherein, It satisfies one or more of the following conditions: (1) In the inclusion reaction, the volume-mass ratio of the water to the isoquercitrin is (50 - 500) mL / g, for example (100 - 300) mL / g; (2) The temperature of the inclusion reaction is 30°C - 90°C, for example 50°C, 60°C, 70°C, 80°C, or 85°C; (3) After the inclusion reaction, the following post-treatment steps are further included: drying; preferably, the drying is vacuum drying, for example, drying with a rotary evaporator; the drying temperature can be 30°C - 70°C, for example 50°C - 65°C; for another example 55°C; and (4) The components in the inclusion reaction are composed of the following: water, isoquercitrin, and β-cyclodextrin.

6. The preparation method of the glucose-modified isoquercitrin according to claim 5, characterized in that, It includes the following steps: (1) In water, inclusion reaction is carried out on the isoquercitrin and the β-cyclodextrin, and then drying is carried out to obtain the β-cyclodextrin-isoquercitrin inclusion complex; (2) In water, in the presence of an enzyme, glycosylation reaction is carried out on the β-cyclodextrin-isoquercitrin inclusion complex obtained in step (1) and a glycosyl donor to obtain the glucose-modified isoquercitrin.

7. The preparation method of the glucosyl-modified isoquercitrin according to claim 1 or 2, characterized in that, The glycosylation reaction satisfies one or more of the following conditions: (1) The volume-mass ratio of the water to the isoquercitrin is (50 - 500) mL / g, for example (100 - 300) mL / g; (2) The mass ratio of the β-cyclodextrin to the isoquercitrin is (1 - 5):1, for example (2 - 4):1; (3) The enzyme is cyclodextrin glucosyltransferase or α-amylase with cyclodextrin glucosyltransferase activity; the α-amylase with cyclodextrin glucosyltransferase activity can be α-amylase G "Tianye" L; (4) The mass ratio of the enzyme to the isoquercitrin is (0.15 - 1):1, for example 0.2:1, 0.5:1, or 0.7:1; (5) The glycosyl donor is β-cyclodextrin; or the glycosyl donor is starch and not β-cyclodextrin, such as maltodextrin; when the glycosyl donor is starch and not β-cyclodextrin, the mass ratio of the glycosyl donor to isoquercitrin can be (0.1 - 5):1, such as 0.5:1, 1:1, 2:1 or 3:1; (6) The components of the glycosylation reaction further include calcium chloride; the mass ratio of the calcium chloride to isoquercitrin can be (0.01 - 0.1):1, such as (0.02 - 0.05):1; (8) The temperature of the glycosylation reaction is 35°C - 70°C, such as 45°C, 50°C, 55°C, 60°C, 65°C; and (9) After the glycosylation reaction, post-treatment is also included, and the post-treatment includes one or more of the following steps: decolorization, suction filtration, membrane filtration, concentration and drying; the decolorization can be decolorization by adsorption with activated carbon; the temperature of the decolorization can be 30°C - 90°C, such as 50°C, 60°C, 70°C, 80°C or 85°C; the membrane filtration can be ultrafiltration membrane filtration; for example, the cut-off molecular weight of the ultrafiltration membrane is 3000.

8. The preparation method of the glucose-modified isoquercitrin according to claim 6, characterized in that, When the components of the glycosylation reaction further include calcium chloride, the glycosylation reaction includes the following steps: in water, in the presence of an enzyme and calcium chloride, the β-cyclodextrin-isoquercitrin inclusion complex obtained in step (1) is subjected to a glycosylation reaction with a glycosyl donor to obtain the glucose-modified isoquercitrin.

9. The preparation method of the glucose group-modified isoquercitrin according to claim 1, wherein, It satisfies one or both of the following conditions: (1) The components in the glycosylation reaction are composed of the following: water, isoquercitrin, β-cyclodextrin, enzyme, glycosyl donor and calcium chloride; and (2) The glucose-modified isoquercitrin obtained by the preparation method of the glucose-modified isoquercitrin as described in claim 1 is a crude product, and the crude product further includes unreacted isoquercitrin; Preferably, the preparation method of the glucose-modified isoquercitrin as described in claim 1 satisfies one or more of the following conditions: (1) In the crude product of the glucose-modified isoquercitrin (calculated based on the total molar mass of isoquercitrin and glucose-modified isoquercitrin being 100%), the molar percentage of isoquercitrin is 0 - 30%; preferably, it is 26% or less; such as 20%, 22%, 25% or 26%; (2) In the crude product of the glucose-modified isoquercitrin (calculated based on the total molar mass of isoquercitrin and glucose-modified isoquercitrin being 100%), the molar percentage of the component with n = 1 is 10 - 25%; such as 17%, 18% or 19%; (3) In the crude product of the glucose-modified isoquercitrin (calculated based on the total molar mass of isoquercitrin and glucose-modified isoquercitrin being 100%), the molar percentage of the component with n = 2 is 10 - 25%; such as 14%, 15%, 17% or 19%; and In the crude product of the glucose-modified isoquercitrin (calculated with the total molar mass of isoquercitrin and glucose-modified isoquercitrin being 100%), the molar percentage of the component with n = 3 is 10 - 25%; for example, 11% or 12%.

10. The preparation method of the glucose-modified isoquercetin according to claim 1, characterized in that, It includes the following steps: (1) In water, perform an inclusion reaction on isoquercitrin and β-cyclodextrin, and then dry to obtain the β-cyclodextrin-isoquercitrin inclusion complex; (2) In water, in the presence of an enzyme and calcium chloride, perform a glycosylation reaction on the β-cyclodextrin-isoquercitrin inclusion complex obtained in step (1) and maltodextrin to obtain the glucose-modified isoquercitrin; In step (1), the volume-mass ratio of the water to the isoquercitrin is (100 - 300) mL / g; In step (1), the mass ratio of the β-cyclodextrin to the isoquercitrin is (2 - 4):1; In step (1), the temperature of the inclusion reaction is 70°C - 85°C; In step (2), the volume-mass ratio of the water to the isoquercitrin is (100 - 300) mL / g; In step (2), the enzyme is cyclodextrin glucosyltransferase or α-amylase with cyclodextrin glucosyltransferase activity; In step (2), the mass ratio of the enzyme to the isoquercitrin is (0.2 - 0.7):1; In step (2), the mass ratio of the maltodextrin to the isoquercitrin is (1 - 3):1; In step (2), the mass ratio of the calcium chloride to the isoquercitrin is (0.01 - 0.05):1; In step (2), the temperature of the glycosylation reaction is 50°C - 60°C.

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