KR20190082524A - Method for preparation of isoquercetin and α-glycosylisoquercetin - Google Patents

Abstract

The present invention relates to a method for the preparation of isoquercetin and α-glycosyl isoquercetin. Specifically, the present invention relates to a method for economically preparing α-glycosyl isoquercetin widely used in food and cosmetic fields as an antioxidant, a color stabilizer, a fragrance change inhibitor, an ultraviolet absorber, and a body fat reducer, by using isoquercetin, high purity of which is prepared by allowing a reaction to proceed while removing free rhamnose from the reaction system, in preparing isoquercetin from rutin by an enzymatic method.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for preparing isoquercetin and alpha-glycosyl isoquercetin,

The present invention relates to a method for producing isoquercetin and? -Glycosyl isoquercetin, and specifically relates to a method for producing isoquercetin and? -Glycosyl isoquercetin which can be widely used in food and cosmetic fields as antioxidants, colorant stabilizers, The present invention relates to a process for producing? -glycosyl isoquercetin and its intermediate isoquercetin with high yield and purity.

Plant-derived flavonoids are yellow-based pigments. Two phenyl groups mediate the C3 chain to form a C6-C3-C6 carbon skeleton, which is linked to glycosides by several saccharides.

Quercetin, like flavonoid compounds, has limitations such as heat instability, instability to oxidation conditions, and low solubility in water, but quercetin glycosides are known to reduce this problem and increase bioavailability. Quercetin-3-glucoside (isoquercetin) is generally known to have a 3-fold increase in bioavailability over quercetin (Hollman PC et al., In man Free Radic Res., 31 (6): 569, 1999; Margreet R. Olthof et al., J. Nutr., 130: 12001203, 2000).

There is also proposed a method of adding a sugar to a flavonoid such as rutin, isoquercetin, hesperidine, naringin and neohesperedine to increase the absorbency (Korean Patent No. 10 -1086441 and Japanese Patent Application Laid-Open No. 2000-78956), and α-glycosyl rutin, in which sugar is transferred to rutin, a main component of flower tree and buckwheat, (Japanese Patent Application Laid-Open No. 2004-59522, Shimoi K. et al., J Agric Food Chem., 51, 2785-2789, 2003).

α-glycosyl isoquercetin significantly reduced the body weight, visceral fat mass, and serum triglyceride, total cholesterol, and free fatty acid concentrations of mice in a study of weight loss in obese-induced mice by high-fat diet (Yeojin Min, et al., J Korean Soc Food Sci Nutr., 45 (4), 474-483 (2016).

On the other hand, isoquercetin is superior to quercetin or rutin in terms of bioabsorbability, but it is difficult to use in liquid products such as food because it is water-insoluble. In order to solve this problem, it has been proposed that a glucose residue from a sugar donor of a substrate is transferred to a glucose residue site of a sugar receptor isoquercetin using cyclodextrin glucano transferase (EC 2.4.1.19) (α-glycosyl isoquercetin). Korean Patent No. 1086441 entitled "Method for preparing α-glycosyl isoquercitrin, its preparation intermediate, and by-product", comprises preparing an isoquercetin having high purity by rutin enzymatic method and then using a glycosyltransferase A method for producing? -Glycosyl isoquercetin is disclosed. At this time, in the step of producing isoquercetin from a routine, the step of treating with an enzyme having a long-lasting cleavage activity is performed in the presence of an edible ingredient such as gelatin or wheat gluten. However, there remains a challenge to economically produce high purity isoquercetin without increasing the yield and further processing when producing isoquercetin from a routine.

The inventors of the present invention have studied about a method for producing? -Glycosyl isoquercetin and its intermediate isoquercetin, and when preparing isoquercetin from an enzyme by an enzymatic method, the produced rhamnose is removed from the reaction system and the reaction is proceeded It was confirmed that high purity isoquercetin can be produced at a high yield and the present invention has been completed.

It is an object of the present invention to provide a method for producing high purity isoquercetin from a routine with high yield.

Another object of the present invention is to provide a method for producing? -Glycosyl isoquercetin using the high purity isoquercetin.

According to the present invention, there is provided a process for preparing a compound of formula (I), which comprises: (A) treating a rutin with an enzyme having a long cleavage activity; (B) reacting the produced rhamnose with an enzyme while removing it from the reaction system; And (C) inactivating the enzyme to obtain isoquercetin.

The routine has a purity of 95 to 98%, preferably 95%.

The enzyme having the above-mentioned long-decomposing activity is a naringinase,? -1,6-rhamoxidase, hesperidinase, or pectinase, preferably a naringinase or an agonist.

In the above steps (A) and (B), the enzyme reaction conditions are pH 3.5 to 8, preferably 3.5 to 6, reaction temperature 30 to 80 ° C, and preferably 40 to 80 ° C.

The method for removing rhamnose from the reaction system in the step (B) includes centrifugation; A resin treatment method such as an adsorption method, an ion exchange treatment method, and a gel filtration method; A membrane treatment method such as an ultrafiltration membrane treatment, a reverse osmosis membrane treatment method, and an ion exchange membrane treatment method; Electrodialysis; Solvent recrystallization; And activated carbon treatment may be used. Preferably, centrifugation is used.

(A) a1) dispersing the rutin having a purity of 95 to 98% in water and adjusting pH to 3.5 to 8 using a pH adjusting agent; a2) adding an enzyme having a long-lasting cleavage activity and reacting at 30 to 80 DEG C with stirring;

(B) b1) After 20 to 24 hours of keeping, the mixture is cooled to 20 to 25 DEG C, centrifuged, and the supernatant is discarded. Water is added to the precipitate again and centrifuged to discard the supernatant. b2) dispersing the precipitate in water, adjusting the pH to 3.5 to 8 using a pH adjuster, adding an enzyme having a naringly digesting activity and reacting at 30 to 80 캜 with stirring; b3) After maintaining the temperature for 20 to 24 hours, the mixture is cooled to 20 to 25 DEG C, centrifuged, and the supernatant is discarded. Water is added to the precipitate again and centrifuged to discard the supernatant. b4) dispersing the precipitate in water again and reacting at 30 to 80 DEG C for 20 to 24 hours with no additional enzyme added; And

(C) deactivating the enzyme remaining in the reaction system, washing the precipitate with water, and drying the recovered isoquercetin, thereby recovering the isoquercetin.

According to another aspect of the present invention, there is provided a method for producing? -Glycosyl isoquercetin represented by the following formula, which comprises subjecting isoquercetin prepared by the above production method to glycosylation in the presence of a sugar donor / RTI >

(n은0~12의 정수)

(n is an integer of 0 to 12)

The sugar donor is dextrin, cyclodextrin or starch. The glycoprotein is cyclodextrin glucanotransferase.

Preferably, (A) adding water to the isoquercetin obtained above, adjusting the pH to 5 to 8.5 with a pH adjusting agent, and maintaining the temperature at 30 to 80 ° C for 1 to 2 hours;

(B) a donor is added and dispersed, and a reaction is carried out at pH 5 to 8.5 and 30 to 80 ° C for 20 to 24 hours by adding a sugar transferase; And

(C) deactivating the enzyme, followed by filtration, concentration, and drying to recover the? -Glycosyl isoquercetin, there is provided a process for producing? -Glycosyl isoquercetin.

According to the production method of the present invention, in the production of isoquercetin from an enzyme by an enzymatic method, high-purity isoquercetin can be produced at a high yield by a simple process of enzymatic reaction while removing free rhamnose from the reaction system. There is an advantage that? -Glycosyl isoquercetin can be produced economically.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is an analysis chart showing the glycoside composition ratios of? -Glycosyl isoquercetin prepared according to an embodiment of the present invention.

The present invention simplifies the production process of? -Glycosyl isoquercetin and its intermediate isoquercetin, which can be widely used in foods and cosmetics fields as an antioxidant, a pigment stabilizer, an anti-aroma agent, an ultraviolet absorber, To provide an excellent manufacturing method.

Isoquercetin (Quercetin-3-glucoside) represented by the following formula is 3-O-glucoside of quercetin. Can be isolated from a variety of plant species such as mangifera indica, rheum nobile, and sophora japonica.

Isoquercetin is an excellent antioxidant and has a whitening activity, lowering blood pressure and having anticancer activity. In addition, isoquercetin is an intermediate in the production of? -Glycosyl isoquercetin widely used in food and cosmetic fields as an antioxidant, a pigment stabilizer, an anti-aroma agent, an ultraviolet absorber, and a body fat reducing agent. Methods for mass production of isoquercetin include chemical synthesis, enzymatic methods, and the like.

The present invention relates to a method for mass production of isoquercetin by an enzymatic method using as a starting material. The present inventors have found that there is a limit to low yield and purity when preparing isoquercetin by treating a ruminosidase enzyme with conventional routines. In particular, it has been found that the accumulation of free rhamnose in the reaction system inhibits the conversion to isoquercetin.

 According to an aspect of the present invention, there is provided a method for preparing a compound of formula (I), comprising the steps of: (A) treating a rutin with an enzyme having a long degradative activity; (B) reacting the produced rhamnose with an enzyme while removing it from the reaction system; And (C) inactivating the enzyme to obtain isoquercetin.

The routine has a purity of 95 to 98%, preferably 95%. It was confirmed that the conversion rate to isoquercetin was rather high when 95% of low-purity routines were used.

Examples of the enzymes having the above-mentioned long-decomposing activity include naringinase, α-1,6-rhamnosidase (EC 3.2.1.40), hesperidinase or pectinase, preferably α-L L-rhamnosidase and a beta-glucosidase activity are used as the enzyme.

The amount of naringin long-term degrading enzyme used per 1 part by weight of the routine is, for example, the amount of enzyme when the enzyme activity is 150 units / g (1 unit: pH3.5 to produce reducing sugar equivalent to 1 mg of rhamnose for 30 minutes (0.006 to 0.13 part by weight), preferably about 1 to 15 units (0.006 to 0.1 part by weight), more preferably 1 to 10 units ( 0.006 to 0.06 part by weight).

Also, the reaction concentration of the routine is 1 to 20% by weight, preferably 1 to 15% by weight, more preferably 1 to 10% by weight in 100% by weight of the reaction system.

In the above steps (A) and (B), the enzyme reaction conditions are pH 3.5 to 8, more preferably 3.5 to 6, reaction temperature 30 to 80 ° C, and more preferably 40 to 80 ° C. Although the temperature and pH conditions vary depending on the kind of enzyme used, the pH is 3.5 to 8, preferably 3.5 (for example) when using Naringin or "Amano" (150 U / g) ~ 6 range. The reaction temperature condition is also 80 占 폚 or lower, preferably 30 占 폚 to 80 占 폚, and more preferably 40 占 폚 to 80 占 폚, depending on the enzyme used.

The method of removing free rhamnose from the reaction system is not particularly limited, and a common method can be used in combination. Specifically, a centrifugal separation method, various resin treatment methods (adsorption method, ion exchange treatment method, gel filtration method, etc.), membrane treatment method (ultrafiltration membrane treatment, reverse osmosis membrane treatment method, ion exchange membrane treatment method), electrodialysis method, solvent recrystallization method, Preferably, a centrifugation method is used.

According to an embodiment of the present invention, there is provided a method for preparing a pharmaceutical composition, comprising: (A) a1) dispersing a routine with a purity of 95 to 98% in water and adjusting pH to 3.5 to 8 using a pH adjusting agent; a2) adding an enzyme having a long-lasting cleavage activity and reacting at 30 to 80 DEG C with stirring;

(B) b1) After 20 to 24 hours of keeping, the mixture is cooled to 20 to 25 DEG C, centrifuged, and the supernatant is discarded. Water is added to the precipitate again and centrifuged to discard the supernatant. b2) dispersing the precipitate in water, adjusting the pH to 3.5 to 8 using a pH adjuster, adding an enzyme having a naringly digesting activity and reacting at 30 to 80 캜 with stirring; b3) After maintaining the temperature for 20 to 24 hours, the mixture is cooled to 20 to 25 DEG C, centrifuged, and the supernatant is discarded. Water is added to the precipitate again and centrifuged to discard the supernatant. b4) dispersing the precipitate in water again and reacting at 30 to 80 DEG C for 20 to 24 hours with no additional enzyme added; And

(C) deactivating the enzyme remaining in the reaction system, washing the precipitate with water, and drying the recovered isoquercetin, thereby recovering the isoquercetin.

In the above isoquercetin (IQC) production method, IQC was obtained at almost 100% purity after 48 hours when rhamnose free from the reaction system was removed every 24 hours. After 72 hours, 100% purity To obtain isoquercetin. The present invention can take a little longer time than the conventional method, but its yield and purity can be dramatically increased by a simple process in which the reaction is continued while free rhamnose is removed from the reaction system.

Isoquercetin is superior to quercetin and rutin in terms of bioabsorbability, but it is difficult to use in liquid products such as food because it is water-insoluble. According to another aspect of the present invention, there is provided a method for producing α-glycosyl iso-iso-quinazoline represented by the following formula, comprising the step of subjecting isoquercetin prepared by the above- A process for preparing quercetin is provided.

(n은0~12의 정수)

(n is an integer of 0 to 12)

Here, the transferase used for the dividing of IQC is cyclodextrin glucanotransferase (CGTase) (E.C.2.4.1.19). The glycoprotein is a commercially available enzyme. Amylase G "Amano L" (400 U) was used as a commercially available enzyme. The amount of the enzymes was determined by the following formula: Toruzyme 3.0 L (3.0 KNU / g; One Kilo Novo alpha-amylase Unit / g, 1U: Amino Enzyme Inc.), which produces 1 mg of? -cyclodextrin from starch for 1 minute

The amount of enzyme used varies depending on the enzyme, but in the case of Toruzyme 3.0L (3.0 KNU / g, One Kilo Novo alpha-amylase Unit (1 KNU): enzyme amount in decomposing 5.25 g starch per hour, Novozymes) (0.0066 to 0.13 parts by weight), preferably 40 to 350 parts (0.013 to 0.12 parts by weight), and more preferably 70 to 250 parts (0.02 to 0.08 parts by weight) relative to 1 part by weight to be.

Examples of the saccharide donor used for the saccharification include dextrin, cyclodextrin, starch, and the like. The amount to be used is usually about 0.1 to 20 parts by weight, preferably about 0.4 to 15 parts by weight, and more preferably about 0.4 to 10 parts by weight based on 1 part by weight of IQC present in the reaction system.

The reaction conditions vary depending on the kind of the enzyme, but the reaction conditions may vary depending on the kind of the enzyme, for example, at 85 ° C or below, preferably 30-80 ° C, preferably 40-75 ° C, pH 3-10, preferably pH 5-8.5. In the presence of the enzyme. The reaction can be carried out while stirring, or shaking.

The number of bonds of the glucose residue of? -Glycosyl isoquercetin (the number of n in the above formula) is not limited, but is usually 1 to 12, preferably 1 to 7, more preferably 1 to 5. The number of bonds (number of n) of these glucose groups can be arbitrarily adjusted. The molar ratio of 1 to 3 can be increased. For example, after the production of? -Glycosyl isoquercetin, the molar ratio of 1 to 3 can be increased by using various amylases (? -Amylase,? -Amylase, glucoamylase, etc.). The? -Glycosyl isoquercetin may be isolated and purified from the reaction system treated with the amylase. The recovery of? -glycosyl isoquercetin can be carried out by any of a variety of conventional methods, for example, various resin treatment methods (adsorption method, ion exchange method, gel filtration method, etc.), membrane treatment method ), An electrodialysis method, a solvent recrystallization method, an activated carbon treatment method, or the like.

According to one embodiment, (A) adding water to the isoquercetin obtained above, adjusting the pH to 5 to 8.5 with a pH adjusting agent, and maintaining the temperature at 30 to 80 ° C for 1 to 2 hours;

(B) a donor is added and dispersed, and a reaction is carried out at pH 5 to 8.5 and 30 to 80 ° C for 20 to 24 hours by adding a sugar transferase; And

(C) deactivating the enzyme, followed by filtration, concentration, and drying to recover the? -Glycosyl isoquercetin, there is provided a process for producing? -Glycosyl isoquercetin.

According to the production method of the present invention, isoquercetin, which is an intermediate, can be produced with high yield and high purity by a simple process, so that the final product,? -Glycosyl isoquercetin, can be produced economically.

[Example]

Hereinafter, the present invention will be described in more detail with reference to the following examples. It should be understood, however, that these embodiments are merely examples for the purpose of easily describing the scope and scope of the technical idea of the present invention, and thus the technical scope of the present invention is not limited or changed. It will be apparent to those of ordinary skill in the art that various changes and modifications can be made within the scope of the technical idea of the present invention based on these examples.

Comparative Example 1: Preparation of isoquercetin

30 g of a 95% pure solution of purity was dispersed in 400 mL of water and adjusted to pH 4.5 using a pH adjuster (1N NaOH, 0.01 N HCl). After adding 0.6 g (3 units * 30 g / 150 units = 0.6 g) of a naringin agent (Amano Enzyme Inc. under the trade name of Naringinase Amano, 150 U / g), the mixture was kept at 70 캜 for 24 hours with stirring, Inactivate the remaining enzyme. The resulting precipitate (solid content) was washed with water and dried to recover isoquercetin.

Comparative Example 2: Preparation of isoquercetin

30 g of a 95% pure solution of purity was dispersed in 400 mL of water and adjusted to pH 4.5 using a pH adjuster (1N NaOH, 0.01 N HCl). After adding 0.6 g (3 units * 30 g / 150 units = 0.6 g) of a carrier or agent (Amano Enzyme Inc. under the trade name of Naringinase Amano, 150 U / g), the mixture was maintained at 70 ° C with stirring for 48 hours, Inactivate the remaining enzyme. The resulting precipitate (solid content) was washed with water and dried to recover isoquercetin.

Comparative Example 3: Preparation of isoquercetin

30 g of a 95% pure solution of purity was dispersed in 400 mL of water and adjusted to pH 4.5 using a pH adjuster (1N NaOH, 0.01 N HCl). After adding 0.6 g (3 units * 30 g / 150 units = 0.6 g) of a naringin agent (Amano Enzyme Inc., trade name: Naringinase Amano, 150 U / g) at 70 캜 for 72 hours, Inactivate the remaining enzyme. The resulting precipitate (solid content) was washed with water and dried to recover isoquercetin.

Comparative Example 4: Preparation of isoquercetin

30 g of a 98% pure solution of purity was dispersed in 400 mL of water and adjusted to pH 4.5 using a pH adjuster (1N NaOH, 0.01 N HCl). After adding 0.6 g (3 units * 30 g / 150 units = 0.6 g) of a naringin agent (Amano Enzyme Inc. under the trade name of Naringinase Amano, 150 U / g), the mixture was kept at 70 캜 for 24 hours with stirring, Inactivate the remaining enzyme. The resulting precipitate (solid content) was washed with water and dried to recover isoquercetin.

Comparative Example 5: Preparation of isoquercetin

30 g of a 98% pure solution of purity was dispersed in 400 mL of water and adjusted to pH 4.5 using a pH adjuster (1N NaOH, 0.01 N HCl). After adding 0.6 g (3 units * 30 g / 150 units = 0.6 g) of a carrier or agent (Amano Enzyme Inc. under the trade name of Naringinase Amano, 150 U / g), the mixture was maintained at 70 ° C with stirring for 48 hours, Inactivate the remaining enzyme. The resulting precipitate (solid content) was washed with water and dried to recover isoquercetin.

Comparative Example 6: Preparation of isoquercetin

30 g of a 98% pure solution of purity was dispersed in 400 mL of water and adjusted to pH 4.5 using a pH adjuster (1N NaOH, 0.01 N HCl). After adding 0.6 g (3 units * 30 g / 150 units = 0.6 g) of a naringin agent (Amano Enzyme Inc., trade name: Naringinase Amano, 150 U / g) at 70 캜 for 72 hours, Inactivate the remaining enzyme. The resulting precipitate (solid content) was washed with water and dried to recover isoquercetin.

Test Example 1: Purity analysis of isoquercetin

The content of recovered isoquercetin was analyzed by the following method.

Isoquercetin (IQC) assay method

1) Preparation of standard solution: Approximately 20 mg of IQC standard product is accurately weighed and filled with methanol in a 50 mL volumetric flask.

2) Preparation of test solution: 20 mg of specimen is precisely weighed and filled with methanol in a 50 mL volumetric flask.

<HPLC analysis conditions>

1) Column: C18 column (4.6 m x 250 mm, 5 m)

2) mobile phase: Acetonitrile: water = 18: 82

3) Detector and wavelength: UV at 254 nm

4) Flow rate: 0.8 mL / min

5) Injection amount: 10 μl

6) Column temperature: room temperature

The results are shown in Tables 1 and 2 below.

Routine purity Comparative Example 1
(24 hours) Comparative Example 2
(48 hours) Comparative Example 3
(72 hours) IQC content (%) 95% 42% 52% 53%

Routine purity Comparative Example 4
(24 hours) Comparative Example 5
(48 hours) Comparative Example 6
(72 hours) IQC content (%) 98% 30% 39% 42%

As can be seen in the above table, it can be seen that the conversion of IQC is higher when the starting material is 95% of a relatively low purity. However, it can be seen that the increase in conversion rate over time is not large.

Example 1: Preparation of isoquercetin

30 g of a 95% pure solution of purity was dispersed in 400 mL of water and adjusted to pH 4.5 using a pH adjuster (1N NaOH, 0.01 N HCl). 0.6 g (3 units * 30 g / 150 units = 0.6 g) of a carrier or agent (Amano Enzyme Inc. under the trade name of Naringinase Amano, 150 U / g) was added and kept at 70 캜 with stirring for 24 hours. After cooling to 25 ° C, the supernatant is discarded, water is added to the precipitate again, centrifuged, and the supernatant is discarded.

The precipitate was again dispersed in 400 mL of water and adjusted to pH 4.5 using a pH adjusting agent (1N NaOH, 0.01N HCl), and 0.3 g of Naringin or 0.3 g (1.5 units * 30/150 = 0.3 g) And the enzyme remaining in the reaction system is deactivated at 95 ° C for 24 hours.

The obtained precipitate (solid content) was washed with water, and then dried to recover isoquercetin.

Example 2: Preparation of isoquercetin

30 g of a 95% pure solution of purity was dispersed in 400 mL of water and adjusted to pH 4.5 using a pH adjuster (1N NaOH, 0.01 N HCl). 0.6 g (3 units * 30 g / 150 units = 0.6 g) of a carrier or agent (Amano Enzyme Inc. under the trade name of Naringinase Amano, 150 U / g) was added and kept at 70 캜 with stirring for 24 hours. After cooling to 25 ° C, centrifuge and discard the supernatant, add water again to the precipitate, centrifuge and discard the supernatant.

The precipitate was again dispersed in 400 mL of water and adjusted to pH 4.5 using a pH adjusting agent (1N NaOH, 0.01N HCl), and 0.3 g of Naringin or 0.3 g (1.5 units * 30/150 = 0.3 g) And the mixture is further stirred for 24 hours. The mixture is cooled to 25 DEG C by centrifugation, and the supernatant is discarded. Water is added to the precipitate again, centrifuged, and the supernatant is discarded.

The precipitate is dispersed again in 400 mL of water, and the mixture is maintained at 70 DEG C for 24 hours without further administration of naringin or an additional agent, and the enzyme remaining in the reaction system at 95 DEG C is inactivated.

The resulting precipitate (solid content) was washed with water and then dried to recover 22.5 g of isoquercetin. The recovery rate is 99%.

Test Example 2: Purity analysis of isoquercetin

The content of recovered isoquercetin in the same manner as in Test Example 1 was analyzed.

The results are shown in Table 3 below.

Routine purity Example 1
(48 hours) Example 2
(72 hours) IQC content (%) 95% 96% 100%

As shown in Table 3, when the reaction was performed while removing rhamnose free from the reaction system every 24 hours of the enzymatic reaction, the IQC content was 96% after 48 hours (Example 1), and after 72 hours Showed an IQC content of 100%. It was confirmed that the results are significantly different from those of Comparative Example 2 (52%) and Comparative Example 3 (53%) in Table 1, which were subjected to enzyme reaction without removal of free rhamnose.

Example 3: Preparation of? -Glycosyl isoquercetin

Water (300 mL) was added to 20 g of isoquercetin obtained in Example 2, adjusted to pH 8.5 with a pH adjuster (1N NaOH, 0.01 N HCl), and maintained at 75 DEG C for about one hour. Then, 10 g of starch was added and dispersed, and 1 g of a transfer unit enzyme Toruzyme 3.0 L (3.0 KNU / g, Novozymes) of 200 units (15 g * 200 unit / 3000 U = 1 g) per 1 part of the substrate (sugar donor) After the addition, the reaction was carried out at pH 8.0 and 75 ° C for 24 hours. The obtained reaction solution was kept at 95 DEG C for 30 minutes to inactivate the enzyme. Filtered, concentrated and spray dried. 29 g of? -glycosyl isoquercetin glycoside was obtained.

Test Example 3: Content analysis of? -Glycosyl isoquercetin

The content was analyzed by the following method, that is, the content of isoquercetin (α-glycosyl isoquercetin) in Japanese enzymes treated with food additives.

The α-glycosyl isoquercetin obtained so that the absorbance is in the range of 0.3 to 0.7 is precisely weighed and dissolved in 50 ml of water to make 100 ml. 1 ml of this solution is accurately taken and 0.085% phosphoric acid solution is added to make 100 ml as the test solution .

Separately, the routine standard is dried for 2 hours at 135 ° C. Approximately 0.2 g is precisely weighed and dissolved in 80 ml of methanol, heated and dissolved. Methanol is added to make 100 ml. Then 1 ml of this solution is diluted to 100 ml with 0.085% phosphoric acid Standard solution.

Absorbance A1 and A2 were measured at a wavelength of 351 nm using 0.085% phosphoric acid as a reference solution, and the content of enzyme-treated isoquercetin (in terms of rutin conversion) was determined according to the following equation, Quercetin content.

A: Contents of enzyme treatment routines (amount converted to rutin) (%)

A1: absorbance of test solution

A2: Absorbance of standard solution

S: Amount of sample (mg)

R: Weight of routine reference (mg)

Analysis by the above method revealed that the content of? -Glycosyl isoquercetin was about 70%.

Test Example 4: Analysis of glycoside composition ratio of? -Glycosyl isoquercetin

0.1 g of α-glycosyl isoquercetin was dissolved in 20% ethanol, and the solution was taken up to 100 mL. The solution was filtered through a 0.45 μm membrane filter and used as a test solution.

<HPLC analysis conditions>

1) Column: C18 column (4.6 m x 250 mm, 5 m)

2) mobile phase: Acetonitrile: water = 18: 82

3) Detector and wavelength: UV at 254 nm

4) Flow rate: 0.8 mL / min

5) Injection amount: 10 μl

6) Column temperature: room temperature

As a result of the above analysis, the ratio of glycoside composition of? -Glycosyl isoquercetin was confirmed by the following HPLC chromatogram peak area ratio (Area%) (Table 4, Fig. 1).

IQC glycoside HPLC Peak Area% IQC + G0 28.2784 IQC + G1 21.5898 IQC + G2 18.9976 IQC + G3 11.1584 IQC + G4 7.7730 IQC + G5 5.1781 IQC + G6 3.3910 IQC + G7 1.8061 IQC + G8 0.9923 IQC + G9 0.5212 IQC + G10 0.3140

Claims (9)

(A) treating the rutin with an enzyme having a long degradative activity and reacting;
(B) reacting the produced rhamnose with an enzyme while removing it from the reaction system; And
(C) inactivating the enzyme to obtain isoquercetin. The method of claim 1, wherein the routine has a purity of 95 to 98%. The method for producing isoquercetin according to claim 1, wherein the enzyme reaction conditions in the steps (A) and (B) are pH 3.5 to 8 and a reaction temperature is 30 ° C to 80 ° C. The method according to claim 1, wherein the step (b) removes rhamnose from the reaction system by centrifugation; A resin treatment method such as an adsorption method, an ion exchange treatment method, and a gel filtration method; A membrane treatment method such as an ultrafiltration membrane treatment, a reverse osmosis membrane treatment method, and an ion exchange membrane treatment method; Electrodialysis; Solvent recrystallization; And an activated charcoal treatment method. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt; The method according to claim 1,
(A) a1) dispersing a routine with a purity of 95 to 98% in water and adjusting the pH to 3.5 to 8 using a pH adjuster; a2) adding an enzyme having a long-lasting cleavage activity and reacting at 30 to 80 DEG C with stirring;
(B) b1) After 20 to 24 hours of keeping, the mixture is cooled to 20 to 25 DEG C, centrifuged, and the supernatant is discarded. Water is added to the precipitate again and centrifuged to discard the supernatant. b2) dispersing the precipitate in water, adjusting the pH to 3.5 to 8 using a pH adjuster, adding an enzyme having a naringly digesting activity and reacting at 30 to 80 캜 with stirring; b3) After maintaining the temperature for 20 to 24 hours, the mixture is cooled to 20 to 25 DEG C, centrifuged, and the supernatant is discarded. Water is added to the precipitate again and centrifuged to discard the supernatant. b4) dispersing the precipitate in water again and reacting at 30 to 80 DEG C for 20 to 24 hours with no additional enzyme added; And
(C) deactivating the enzyme remaining in the reaction system, washing the precipitate with water, and drying the recovered isoquercetin to recover isoquercetin. A process for producing? -Glycosyl isoquercetin represented by the following formula, which comprises the step of subjecting isoquercetin prepared by the production method of any one of claims 1 to 5 to a glycosyltransferase treatment in the presence of a sugar donor.

(n is an integer of 1 to 12) 7. The method according to claim 6, wherein the saccharide donor is dextrin, cyclodextrin or starch. [Claim 7] The method according to claim 6, wherein the glycoprotein is cyclodextrin glucanotransferase. The method according to claim 6,
(A) adding water to the isoquercetin prepared by the method of any one of claims 1 to 5, adjusting the pH to 5 to 8.5 with a pH adjusting agent, and maintaining the temperature at 30 to 80 ° C for 1 to 2 hours;
(B) a donor is added and dispersed, and a reaction is carried out at pH 5 to 8.5 and 30 to 80 ° C for 20 to 24 hours by adding a sugar transferase; And
(C) inactivating the enzyme, followed by filtration, concentration, and drying to recover the? -Glycosyl isoquercetin.

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