KR20140050314A - Preparation method of quercetin or isoquecitrin using b-glucosidase - Google Patents

Abstract

The present invention relates to a production method of quercetin or isoquercitrin from quercetin glucoside using β-glucosidase (EC 3.2.1.21) derived from Aspergillus niger (deposition number KCTC 6906): which is simple without complex processes compare to existing methods of biologically converting into quercetin or isoquercitrin using a chemical technique or enzymes or increasing the content of flavonoid of the quercetin, etc. using infrared rays; and uses onion, which can be easily obtained from the market, as a raw material for an enzyme reaction of the quercetin glucoside abundantly contained in the onion, thereby economically producing the quercetin or isoquercitrin. [Reference numerals] (AA) Reaction time : 0 hour; (BB) Formula 2; (CC) Reaction time : 2 hours; (DD) Quercetin

Description

Preparation method of quercetin or isoquecitrine using beta-glucosidase {preparation method of quercetin or isoquecitrin using β-glucosidase}

The present invention is Aspergillus Niger ( Aspergillus) Quercetin glycosides are prepared from quercetin or isoquercitrin using beta-glucosidase (EC 3.2.1.21) derived from niger , Accession No .: KCTC 6906. It relates to a manufacturing method.

Onion is a genus of two years belonging to the family Liliaceae, the diameter of the diameter reaches 10cm and is spherical or spherical. In September, a large inflorescence grows at the end of the flower garden, and many flowers with sack run in the form of mountain, and the inflorescence is used for food. In addition, according to the statistics of the Ministry of Food, Agriculture, Forestry and Fisheries in 2008 (2008 Facility Vegetable Greenhouse Status and Vegetable Production Performance), onion has the highest domestic production of seasoned vegetables (about 1 million tons), which is double the onion (about 500,000 tons). Are very important agricultural products.

Onions are mainly composed of quercetin glycosides, quercetin-3-O-glucoside, and quercetin-3,4'-O-glucoside. O-glucoside, quercetin-4'-O-glucoside, etc. are known and contain a large number of allyl sulfide compounds such as propylallyldisulfide, which has a pungent taste. Appeared.

Quercetin is rarely contained in the edible site. It is known to contain a large amount of brown husk (about 8.3 mg per gram of dry matter), and its content reaches tens to hundreds of times of the edible portion, and the quercetin has anti-inflammatory action, protective effect on brain cells, and anticancer. It is a compound reported to exhibit various physiological activities such as action, anti-diabetic, anti-obesity and antihypertensive.

Isoquercitrin is quercetin-3-O-β-D-glucoside, also called isoquercetin. Isoquercetin can be used for antioxidative, anti-fading and flavor change-inhibiting, UV-blocking, metal chelating actions, etc. It is known to have pharmacological activities such as diuretic, anti-inflammatory, capillary strengthening, and anti-virus effects. In addition, unlike the insolubility of quercetin (quercetin) is high solubility, it is known that the bioavailability of the body (quercetin) or rutin (rutin) better. Because of these characteristics, it is conventionally used as a material of cosmetics and health foods, as well as anti-fading agents (flavor change-inhibitor) of the color beverage using the same.

Therefore, various studies are being conducted on quercetin and isoquercetin having various physiological activities, and continuous interest is also increasing.

To date, various manufacturing methods of quercetin and isoquercetin have been developed.

First, a method for producing isoquercitrin through a bioconversion method by treating an enzyme such as hydrolase naringinase or rhamnosidase from rutin is known (Patent Documents 1 to Patent Documents). 2).

Next, as a method of increasing the content of quercetin present in onions, the method of increasing the content of quercetin (quercetin) by the treatment of fermented meju bacteria in onion extract has been known, far infrared to onion It is known to increase the content of quercetin by investigating (Patent Documents 3 to 4).

However, these methods have the problem of requiring expensive equipment or chemicals or long manufacturing time. Therefore, the development of a new method of producing quercetin (quercetin) or isoquercitrin (isoquercitrin) is urgently needed.

Thus, the present inventors have found that the new method of studying the Quebec paroxetine (quercetin) or iso Quebec Citrine (isoquercitrin) for reducing the time and cost required for mass production, Aspergillus and this (Aspergillus niger , Accession No .: KCTC 6906), β-glucosidase (EC 3.2.1.21) enzyme from the quercetin glycosides present in onion quercetin or isoquercitrin By bioconversion, the productivity of quercetin or isoquercitrin was confirmed to be high, and the present invention was completed.

United States Patent Application Publication No. 7691425 Republic of Korea Patent Publication No. 2008-0061997 Republic of Korea Patent Publication No. 2010-0011945 Republic of Korea Patent Publication No. 2011-00037469

It is an object of the present invention to provide a process for preparing quercetin or isoquercitrin.

In order to achieve the above object,

The present invention comprises the steps of reacting a quercetin glycoside with beta-glucosidase (EC 3.2.1.21) enzyme (step 1);

Removing the enzyme from the product prepared in step 1 (step 2); And

It provides a method of producing a quercetin (quercetin) or isoquercitrin (isoquercitrin) represented by the formula (1) comprising the step of purifying the product from which the enzyme is removed in step 2 by performing high performance liquid chromatography (step 2) do.

[Chemical Formula 1]

(In Formula 1, R is hydrogen or a glucoside group.)

Quercetin (quercetin) or isoquercitrin manufacturing method according to the present invention is a conventional chemical synthesis technique, a method for bioconversion to quercetin (quercetin) or isoquercitrin (isoquercitrin) by using an enzyme or infrared Compared to the method of increasing the content of flavonoids such as quercetin using a simple process, the process uses a simple and commercially available onion as a raw material, and contains a large amount of quercetin in onions. By enzymatically reacting glycosides, it is possible not only to economically prepare quercetin or isoquercitrin, but also to utilize the selective position specificity of the beta-glucosidase (β 3.2glucosidase, EC 3.2.1.21) enzyme. Since it is easy to mass-produce in a short time with high efficiency, the pharmaceutical composition containing quercetin or isoquercitrin, It can be usefully used in products, flavors, beverages, herbal medicines, and dietary supplements.

1 is a quercetin in Quecetin-4'-O-β-D-glucoside represented by Formula 2 by the beta-glucosidase (EC 3.2.1.21) enzyme in Experimental Example 2 according to the present invention. It is a high performance liquid chromatography analysis graph showing the conversion to (quercetin).
Figure 2 is in the quercetin-3,4'-O-β-D-glucoside represented by the formula (3) by the beta-glucosidase (EC 3.2.1.21) enzyme in Experimental Example 2 according to the present invention A high performance liquid chromatography analysis graph showing the conversion to isoquercitrin.

Hereinafter, a method of preparing quercetin glycoside using quercetin glycoside using beta-glucosidase (EC 3.2.1.21) enzyme or quercetin or isoquercitrin will be described in detail.

The present invention comprises the steps of reacting a quercetin glycoside with beta-glucosidase (EC 3.2.1.21) enzyme (step 1);

Removing the enzyme from the product prepared in step 1 (step 2); And

Preparation of quercetin or isoquercitrin represented by Chemical Formula 1 including the step of purifying the product from which enzyme is removed in step 2 by performing high performance liquid chromatography (HPLC) (step 2) Provide a method.

In Formula 1,

R is hydrogen or a glucoside group.

Hereinafter, the manufacturing method will be described in detail for each step.

First, step 1 according to the present invention is a step of reacting a quercetin glycoside with beta-glucosidase (β 3.2glucosidase, EC 3.2.1.21) enzyme.

More specifically, the quercetin glycoside is reacted with a beta-glucosidase (EC 3.2.1.21) enzyme to be converted to quercetin or isoquercitrin represented by Formula 1 Step. The quercetin glycoside may be provided from various raw materials such as buckwheat and onion, and preferably, may be provided from onion.

Aspergillus niger according to the present invention beta-glucosidase (EC 3.2.1.21) enzyme derived from the strain niger , Accessory No .: KCTC 6906) is site specific for glucose bound to the 4 'position of the aglycone of the quercetin glycoside. Quenchintin (quercetin) or isoquercitrin (isoquercitrin) represented by the formula (1) can be removed with high efficiency.

The quercetin glycoside of step 1 according to the present invention is a quercetin-4'-O-β-D-glucoside represented by the following Chemical Formula 2 or a quercetin-3,4'-O- represented by Chemical Formula 3 β-D-glucoside.

(2)

(3)

Quercetin-4'-O-β-D-glucoside represented by Chemical Formula 2 is a quercetin represented by Chemical Formula 1 through an enzymatic reaction, quercetin-3,4'-O represented by Chemical Formula 3 -β-D-glucoside is converted to isoquercitrin through enzymatic reaction.

Step 1 according to the present invention is preferably carried out under a stirring condition of 100 rpm to 200 rpm. If the stirring condition is less than 100 rpm, the reaction rate is slow, the problem is that it takes a lot of time to complete the reaction, if it exceeds 200 rpm, the grinding phenomenon occurs due to the high-speed rotation, the enzyme reaction does not occur sufficiently have.

In addition, the step 1 is preferably performed in a temperature range of 25 ℃ to 40 ℃, more preferably carried out in a temperature range of 27 ℃ to 35 ℃. If the temperature is less than 27 ℃, the enzyme does not reach a sufficient temperature to have the activity, there is a problem that the reaction rate is lowered, if it exceeds 35 ℃, there is a problem that the enzyme reaction does not occur due to denaturation of the enzyme.

Furthermore, in step 1, the reaction is preferably carried out for 18 hours or more, more preferably for 24 hours. When the reaction is performed for less than 18 hours, the conversion of the quercetin glycoside to the quercetin (quercetin) or isoquercitrin represented by the formula (1) is not completely achieved, resulting in a poor production yield. On the other hand, when the reaction was carried out for 48 hours compared with the results when the reaction was carried out for 24 hours it was found that there is no significant difference. From this, it can be seen that it is difficult to expect an increase in manufacturing efficiency by a time exceeding 24 hours (see Experimental Example 1). Therefore, if there is no difference in the conversion rate from the quercetin glycoside to the quercetin or isoquercitrin represented by the formula (1) when 24 hours or more, the reaction is preferably performed for 24 hours.

Next, step 2 according to the present invention removes the enzyme from the product prepared by the enzymatic reaction in step 1 to form a product consisting of a mixture of quercetin (quercetin) or isoquercitrin represented by the formula (1) It is a step to get. More specifically, in order to facilitate the extraction of quercetin or isoquercitrin represented by Formula 1 present in the reaction product, first, the reaction product is pulverized for 5 to 10 minutes by an ultrasonic mill. do. Thereafter, the milled product is centrifuged at 4000 rpm to 5000 rpm for about 5 minutes, the supernatant is separated, and the aliquot is filtered to obtain a product free of enzyme.

Next, step 3 according to the present invention performs high performance liquid chromatography (HPLC) on which the enzyme is removed in step 2 to purify quercetin (quercetin) or isoquercitrin represented by Formula 1 Step.

The high performance liquid chromatography (HPLC) for purification may use a C18 column (5 μm, 4.6 mm × 250 mm), but is not limited thereto.

At this time, the concentration gradient of the eluting solvent and the solvent of the high performance liquid chromatography (HPLC) can be used according to the amount of the fraction to separate or the column size and type of the high performance liquid chromatography (HPLC). Preferably, the eluting solvent may be distilled water and methanol in which 0.1% formic acid is dissolved, and the concentration gradient of the solvent may be eluted while maintaining or changing the concentration of methanol for a predetermined time as follows:

5% (0 minutes → 5 minutes), 5% → 80% (5 minutes → 25 minutes), 80% (25 minutes → 30 minutes).

In addition, the identification of quercetin or isoquercitrin represented by Chemical Formula 1 purified through high performance liquid chromatography (HPLC) is preferably measured at a wavelength of 320 nm.

Aspergillus niger according to the present invention niger , Accession No .: Method for preparing quercetin or isoquercitrin represented by Chemical Formula 1 using beta-glucosidase (EC 3.2.1.21) enzyme derived from strain KCTC 6906 Silver is reacted with a beta-glucosidase (β 3.2-glucosidase, EC 3.2.1.21) enzyme, which is a large amount of onion, using a commercially available onion. Quercetin or isoquercitrin can be produced in large quantities in a short time (see Experimental Example 1).

In addition, the beta-glucosidase (EC 3.2.1.21) enzyme has a selective position specificity that specifically removes glucose bound to the 4 'position of the aglycone of quercetin glycosides. From quercetin glycosides, quercetin or isoquercitrin represented by Formula 1 can be prepared with high efficiency (see Experimental Example 2).

Therefore, the method for preparing quercetin or isoquercitrin represented by Chemical Formula 1 using beta-glucosidase (EC 3.2.1.21) enzyme according to the present invention is beta-glucosidase By using the enzyme (β-glucosidase, EC 3.2.1.21), it is possible to manufacture quercetin or isoquercitrin represented by the formula (1) with high efficiency in a short time, so that mass production is easy, quercetin) or isoquercitrin (isoquercitrin) can be usefully used in pharmaceuticals, foods, flavors, beverages, herbal preparations, dietary supplements.

Hereinafter, the present invention will be described more specifically with reference to Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the present invention is not limited to the following Examples and Experimental Examples.

< Example  1> Quercetin ( quercetin ) or Of isoquercitrin  Manufacturing-1

Onions used in the present invention were purchased from the agricultural product market in Jeongeup-si. Aspergillus The beta-glucosidase (β 3.2-glucosidase, EC 3.2.1.21) enzyme (40 U / ml) derived from niger , Accession No .: KCTC 6906 was purchased from Megazyme (Wicklow, Ireland). Water and methanol were used as solvents for high performance liquid chromatography (HPLC) from Burdick & Jackson (SK chemicals, Ulsan, ROK), and formic acid was purchased from Sigma.

First, onions were prepared to react with the enzyme solution, and then the onion skin was removed and washed with water. The washed onion was pulverized until a juice using a blender, 10 g of the crushed onion was used as a substrate. 0.1 ml of beta-glucosidase enzyme solution (40 U / mL) was added to the prepared substrate (10 g wet weight of onion), and then reacted at 30 ° C. for 24 hours at 150 rpm. The reaction was then stopped by sampling 1 g (wet weight) from the product and adding 100% methanol (10 mL). The mixture (product / methanol) was impacted with an ultrasonic mill for 5 minutes to extract the quercetin compound, subjected to centrifugation (4,000 rpm, 5 minutes), and then the supernatant was aliquoted and filtered (0.2 μm). The filtered mixture was purified by high performance liquid chromatography (HPLC, Agilent, 1200 series). At this time, the column was YMC ODS-AM column (5um, 4.6mm × 250mm), the flow rate is 1.0 ml / min, the detector was measured at 320 nm wavelength using DAD. The mobile phase solvent used as the elution solvent was distilled water (0.1% formic acid), the mobile phase solvent B was methanol, and the solvent gradient was eluted at a constant mixing ratio as follows: 5% B (0-5 minutes), 5 -80% B (5-25 minutes), 80% B (25-30 minutes). The injection amount of the sample was 10 μl, which was purified under the above conditions, and the quercetin-4′-O-β-D-glucoside represented by Formula 2, which is an unreacted quercetin glycoside, or the Quecetin- 3,4'-O-β-D-glucoside was recovered, and quercetin or isoquercitrin represented by Chemical Formula 1 as a target compound was obtained.

< Example  2> Quercetin ( quercetin ) or Of isoquercitrin  Manufacturing-2

Instead of performing the reaction for 24 hours, except for 48 hours, the same method as in Example 1 was carried out in the same manner as Quecetin (Quercetin glycoside) represented by the formula (2) -O-β-D-glucoside or quercetin-3,4'-O-β-D-glucoside represented by formula (3) is recovered, and quercetin or isoquecitrin represented by formula (1) as the target compound (isoquercitrin) was obtained.

< Example  3> Quercetin ( quercetin )

Quecetin-4'-O-β-D-glucoside represented by Formula 2, a quercetin monosaccharide, was purchased from Extrasynthese, Genay, France, and dissolved in 100 mM phosphate buffer (pH 7.0). The final concentration is 100 μg / ml. This solution was used as a substrate solution for enzyme reaction, and the reaction solution was added to the prepared substrate solution (1 ml) by adding an enzyme solution (1 ml, 40 U / ml). At this time, it was reacted for 2 hours at 30 ℃. Then, 100% methanol (1.1 ml) was added to extract quercetin from the product (1.1 ml) and ground for 5 minutes with an ultrasonic mill, followed by centrifugation at 4000 rpm for 5 minutes, Filtration (0.2 μm). The filtered mixture (product / methanol) was subjected to high performance liquid chromatography (HPLC) under the same conditions as in Example 1 to obtain quercetin represented by Chemical Formula 1 as a target compound.

< Example  4> Isoquitoline ( isoquercitrin )

Instead of using quercetin-4'-O-β-D-glucoside represented by formula (2) which is a quercetin monosaccharide, quercetin-3,4'-O-β-D- represented by formula (3), which is a quercetin disaccharide Except for purchasing and using glucoside from Extrasynthese, Genay, France, the same procedure as in Example 3 was carried out to obtain isoquercitrin represented by Chemical Formula 1 as a target compound.

< Comparative Example  1> Quercetin ( quercetin ) or Of isoquercitrin  Produce

Instead of performing the reaction for 24 hours, except that the reaction was performed for 6 hours, Quecetin-4 'represented by Formula 2, which is an unreacted quercetin glycoside, which was carried out in the same manner as in Example 1 above. -O-β-D-glucoside or quercetin-3,4'-O-β-D-glucoside represented by formula (3) is recovered, and quercetin or isoquecitrin represented by formula (1) as the target compound (isoquercitrin) was obtained.

< Comparative Example  2> Quercetin ( quercetin ) or Of isoquercitrin  Produce

Instead of performing the reaction for 24 hours, except that the reaction was carried out for 12 hours, Quecetin-4 ′ represented by Formula 2, which is an unreacted quercetin glycoside by performing the same method as Example 1, was used. -O-β-D-glucoside or quercetin-3,4'-O-β-D-glucoside represented by formula (3) is recovered, and quercetin or isoquecitrin represented by formula (1) as the target compound (isoquercitrin) was obtained.

< Experimental Example  1> over time Quercetin ( quercetin ) or Isoquecitrine ( isoquercitrin  Manufacturing efficiency evaluation

Preparation to prepare quercetin glycosides with quercetin or isoquercitrin represented by Formula 1 using beta-glucosidase (EC 3.2.1.21) according to the present invention. In order to evaluate the production efficiency over time of the method was carried out the following experiment.

Onion juice used in the present invention was filtered (0.2 μm) to prepare an onion juice solution. The prepared onion juice was subjected to high performance liquid chromatography to quantify the content of quercetin glycosides present in the onion juice, which was used as an untreated group. In addition, the amount of quercetin or isoquercitrin represented by Formula 1 prepared in Examples 1 to 2 and Comparative Examples 1 to 2 and isoquercitrin obtained during the reaction and unreacted quercetin The recovery amount of the quercetin-4'-O-β-D-glucoside represented by formula (2) or the quercetin-3,4'-O-β-D-glucoside represented by formula (3) was compared. The results are shown in Table 1 below.

Reaction time Concentration (㎍ / g onion wet weight) Compound 1 Compound 2 Compound 3 Compound 4 Example 1 24 hours 83 161 19 30 Example 2 48 hours 82 160 19 28 Comparative Example 1 6 hours 24 22 99 155 Comparative Example 2 12 hours 71 26 77 128 Untreated group 0 hours 0 0 135 176

As shown in Table 1, the method for preparing quercetin or isoquercitrin represented by Formula 1 according to the present invention is Quecetin-4'-O- represented by Formula 2 present in onions. Preparation of quercetin or isoquecitrin represented by Formula 1 through bioconversion of β-D-glucoside or Quecetin-3,4'-O-β-D-glucoside represented by Formula 3 I can see that. In addition, when the reaction time has elapsed for 24 hours, the amount of preparation is 83 μg / g of quercetin represented by Formula 1, and 160 μg / g of isoquercitrin, and even if time passes further, quercetin (quercetin) or isoquercitrin (isoquercitrin) can be seen that there is no big change any more. From this, the method for producing quercetin or isoquercitrin according to the present invention is a beta-glucosidase (β 3.2-glucosidase, EC 3.2.1.21) enzyme is a quercetin glycoside present in onion It can be seen that the bioconversion to produce a quercetin (quercetin) or isoquercitrin (isoquercitrin) represented by the formula (1), when the reaction is carried out for more than 24 hours, the quercetin (quercetin) or iso represented by the formula (1) It can be seen that the manufacturing yield of isoquercitrin is the highest.

Therefore, the preparation method of preparing quercetin or isoquercitrin represented by Chemical Formula 1 using beta-glucosidase according to the present invention (EC 3.2.1.21) is readily available on the market. Using a commercially available onion as a raw material, it is represented by Chemical Formula 1 by reacting quercetin glycosides contained in onions with beta-glucosidase (EC 3.2.1.21) enzyme for 24 hours. Since it is easy to mass-produce quercetin or isoquercitrin that are quercetin, a pharmaceutical composition comprising a quercetin or isoquercitrin represented by Formula 1, a food, a flavor, a beverage It can be useful in herbal medicines and dietary supplements.

< Experimental Example  2> beta- Glucosidase  Enzyme Bioconversion

Beta-glucosidase (β-glucosidase, EC 3.2.1.21) enzyme according to the present invention bioqueries the quercetin (quercetin) glycosides prepared by quercetin (quercetin) or isoquercitrin represented by the formula (1) In order to verify that the following experiment was performed.

Beta-glucoside of quercetin-4'-O-β-D-glucoside represented by formula (2) which is a quercetin glycoside or quercetin-3,4'-O-β-D-glucoside represented by formula (3) The reaction products before and after the reaction of Example 3 to Example 4 reacted with a sidase (β-glucosidase, EC 3.2.1.21) enzyme were aliquoted, and high-performance liquid chromatography of the separated reactants was measured. The measured graph was compared with the measured graph of high performance liquid chromatography of the standard to verify the quercetin glycoside, quercetin and isoquercitrin represented by the formula (1). At this time, the quercetin represented by the formula (1) of the standard of each compound was purchased from Sigma (Sigma, St. Louis, MO, USA) and used, and is represented by the formula (2) which is a quercetin glycoside Quercetin-4'-O-β-D-glucoside or Quecetin-3,4'-O-β-D-glucoside represented by Formula 3 and isoquercitrin represented by Formula 1 are (Extrasynthese, Genay, France) was used. The verified results are shown in FIGS. 1 to 2.

As shown in FIGS. 1 and 2, after 2 hours of reaction time, the quercetin-4′-O-β-D-glucoside represented by Formula 2 is a quercetin, and the quercetin-3 represented by Formula 3 It can be seen that the 4'-O-β-D-glucoside is all converted to isoquercitrin. From this, Aspergillus niger according to the present invention beta-glucosidase (EC 3.2.1.21) enzyme derived from niger , Accession No .: KCTC 6906) site-specific glucose linked to the 4 'position of the aglycone of the quercetin glycoside It can be seen that it has a selective position specificity to remove.

Therefore, the preparation method of preparing quercetin or isoquercitrin represented by Chemical Formula 1 using beta-glucosidase according to the present invention (EC 3.2.1.21) is readily available on the market. By using the commercially available onion as a raw material, the enzymatic reaction of quercetin glycosides contained in a large amount of onion in the onion for 24 hours, economically of the quercetin or isoquecitriline In addition to the preparation, the quercetin represented by the formula (1) can be easily produced in a short time with high efficiency by using the selective position specificity of the beta-glucosidase (β 3.2-glucosidase) enzyme. Or it may be usefully used in pharmaceutical compositions, foods, flavors, beverages, herbal preparations, dietary supplements containing isoquercitrin (isoquercitrin).

Claims (7)

Reacting the quercetin glycoside with beta-glucosidase (EC 3.2.1.21) enzyme (step 1);
Removing the enzyme from the product prepared in step 1 (step 2); And
Preparation of quercetin or isoquercitrin represented by Chemical Formula 1 including the step of purifying the product from which enzyme is removed in step 2 by performing high performance liquid chromatography (HPLC) (step 2) Way:
[Chemical Formula 1]

(1)
R is hydrogen or a glucoside group).
The method of claim 1,
Beta-glucosidase (EC 3.2.1.21) enzyme of step 1 is Aspergillus niger , Accession No .: KCTC 6906) A method for producing a quercetin (quercetin) or isoquercitrin represented by the formula (1), characterized in that derived from.
The method of claim 1,
The quercetin glycoside of step 1 is a quercetin-4'-O-β-D-glucoside represented by the following formula (2) derived from onion or a quercetin-3,4'-O- represented by formula (3) Method for preparing quercetin (quercetin) or isoquercitrin represented by the formula (1) characterized in that β-D-glucoside:
(2)

(3)

.
The method of claim 1,
The step 1 is a method for producing a quercetin (quercetin) or isoquercitrin (isoquercitrin) represented by the formula (1), characterized in that carried out under a stirring condition of 100 rpm to 200 rpm.
The method of claim 1,
Step 1 is a method for producing a quercetin (quercetin) or isoquercitrin (isoquercitrin) represented by the formula (1), characterized in that carried out in a temperature range of 25 ℃ to 35 ℃.
The method of claim 1,
Step 2 is a method for producing a quercetin (quercetin) or isoquercitrin (isoquercitrin) represented by the formula (1) characterized in that the reaction product by ultrasonic milling and centrifugation.
The method of claim 1,
The high performance liquid chromatography (HPLC) of step 3 changes the concentration of methanol sequentially from 0% → 5 minutes for 5%, 5 minutes → 25 minutes for 5% → 80%, 25 minutes → 30 minutes for 80%. Method of producing a quercetin (quercetin) or isoquercitrin (isoquercitrin) represented by the formula (1) characterized in that it is carried out.

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