KR101525956B1 - preparation method of quercetin or isoquecitrin using β-glucosidase - Google Patents

Abstract

The present invention relates to the use of quercetin glycosides in combination with quercetin by using beta-glucosidase (EC 3.2.1.21) derived from Aspergillus niger (Accession No: KCTC 6906) ) Or isoquercitrin. The method for preparing quercetin or isoquercitrin according to the present invention can be carried out using conventional chemical synthesis techniques, such as quercetin the process can be easily carried out without complicated processes and can be easily purchased on the market as compared with a method of bioconversion into quercetin or isoquercitrin or a method of increasing the content of flavonoid such as quercetin using infrared rays By using the onion as a raw material, the quercetin glycoside, which is contained in a large amount in the onion, is subjected to an enzymatic reaction, so that quercetin or isoquercetin rcitrin and the selective site specificity of the enzyme β-glucosidase (EC 3.2.1.21), it is possible to mass-produce the enzyme with high efficiency in a short time. Therefore, quercetin or isocaine A pharmaceutical composition containing isoquercitrin, a food, a flavoring agent, a beverage, a herbal medicine agent, and a health supplement.

Description

Preparation method of quercetin or isoquecitrin using beta-glucosidase using beta-glucosidase [

The invention Aspergillus and this (Aspergillus quercetin glycoside is prepared as quercetin or isoquercitrin using beta-glucosidase (EC 3.2.1.21), which is derived from the nucleotide sequence (SEQ ID NO: niger , KCTC 6906) The present invention relates to a method of manufacturing a semiconductor device.

Onion is a two-year-old lily belonging to the lily family, the diameter of which is 10cm and is spherical or spherical. In September, a large flower grows from the tip of the flower, many flowers with a bag run in a mountain shape, and the flower is used for food. According to the statistics of the Ministry of Agriculture, Forestry and Fisheries in 2008 (2008 vegetable greenhouse status and vegetable production results), the domestic production of seasoning vegetables is the highest in the world (about 1 million tons) Is an important agricultural product.

Quercetin-3-O-glucoside, which is a quercetin glycoside, quercetin-3,4'-O-glucoside, O-glucoside, quercetin-4'-O-glucoside and the like are known, and a large number of allyl sulfide compounds, such as propylallyldisulfide, Respectively.

Quercetin is rarely contained in edible parts. It is known that qeurcetin has anti-inflammatory activity, brain cell protection activity, anti-cancer activity, and anti-inflammatory activity, because it is contained in a large amount in the brown skin (about 8.3 mg per g of dry matter) Activity, antidiabetic, anti-obesity, antihypertensive, and the like.

Isoquercitrin is quesetin-3-O- beta -D-glucoside, also called isoquercetin. Isoquercetin is an antioxidant, anti-fading and flavor change-inhibiting, UV-blocking, metal chelating actions, and so on. And is known to have pharmacological activities such as diuretic, anti-inflammatory, capillary strengthening, and anti-virus effects. It is also known that the quercetin has a high solubility and has better bioavailability than quercetin or rutin, unlike the insolubility of quercetin. Because of these properties, it has been used as an anti-fading agent (a flavor change-inhibitor) for coloring beverages, which is conventionally used as a material for cosmetics and health foods, as well.

Therefore, various studies on quercetin and isoquercetin having various physiological activities are being carried out, and there is an increasing interest in the continuing interest.

To date, various methods for preparing quercetin and isoquercetin have been developed and are described below.

First, there is known a process for producing isoquercitrin by biotransformation method by treating an enzyme such as hydrolyzate, naringinase or rhamnoseidase from rutin (Patent Document 1 to Patent Document 2).

Next, a method of increasing the content of quercetin by fermenting meju bacillus with onion extracts by increasing the content of quercetin present in onion has been known, and a method of increasing the content of quercetin by adding far infrared rays To increase the content of quercetin (Patent Document 3 to Patent Document 4).

However, the above methods have a problem that expensive equipment or chemicals are required or a long time is required for the production. Therefore, there is an urgent need to develop a new process for producing quercetin or isoquercitrin.

Accordingly, the inventors of the present invention have been studying a new method for producing quercetin or isoquercitrin for reducing the time and cost required for mass production, and have found that the use of Aspergillus niger, Accession No: KCTC 6906) derived from the beta-glucosidase during Days (β-glucosidase, EC 3.2.1.21) is an enzyme Quebec a paroxetine Quebec (quercetin) glycosides present in the paroxetine onion (quercetin) or iso Quebec Citrine (isoquercitrin) (Quercetin) or isoquercitrin (quercetin), thereby completing the present invention.

U.S. Patent No. 7691425 Korea Patent Publication No. 2008-0061997 Korea Patent Publication No. 2010-0011945 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 relates to a process for preparing a quercetin glycoside by reacting a quercetin glycoside with an enzyme β-glucosidase (EC 3.2.1.21) (step 1);

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

(Quercetin or isoquercitrin) represented by the following general formula (1), comprising the step (2) of purifying a product from which the enzyme has been removed in the step (2) by performing a high performance liquid chromatography do.

[Chemical Formula 1]

(Wherein R is hydrogen or a glucoside group)

The method for preparing quercetin or isoquercitrin according to the present invention can be carried out by a conventional chemical synthesis technique, a method of bioconversion into quercetin or isoquercitrin using an enzyme, (Quercetin) and quercetin (quercetin), which can be easily purchased in the market without any complicated process, compared to a method of increasing the content of quercetin such as quercetin ) Glucosidase (EC 3.2.1.21) enzyme can be economically produced not only by the production of quercetin or isoquercitrin but also by utilizing the selective site specificity of beta-glucosidase (EC 3.2.1.21) The pharmaceutical composition comprising quercetin or isoquercitrin, a pharmaceutical composition comprising quercetin or isoquercitrin, Flavors, beverages, herbal medicines, and health supplements.

1 is a graph showing the effect of quercetin-4'-O- beta -D-glucoside of formula (2) on beta-glucosidase (EC 3.2.1.21) enzyme in Experimental Example 2 according to the present invention. < / RTI > is a high performance liquid chromatographic analytical graph showing the conversion to quercetin.
2 is a graph showing the effect of quercetin-3, 4'-O- beta -D-glucoside represented by the general formula (3) on beta-glucosidase (EC 3.2.1.21) enzyme in Experimental Example 2 ≪ / RTI > is a high performance liquid chromatography analytical graph showing the conversion to isoquercitrin.

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

The present invention relates to a process for preparing a quercetin glycoside by reacting a quercetin glycoside with an enzyme β-glucosidase (EC 3.2.1.21) (step 1);

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

(Quercetin) or isoquercitrin represented by the following formula (1), comprising the step of purifying the enzyme-removed product in step 2 by performing high performance liquid chromatography (HPLC) ≪ / RTI >

In Formula 1,

R is hydrogen or a glucoside group.

Hereinafter, the above 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 an enzyme β-glucosidase (EC 3.2.1.21).

More specifically, a quercetin glycoside is reacted with an enzyme β-glucosidase (EC 3.2.1.21) to convert to a quercetin or isoquercitrin represented by the formula (1) . The quercetin glycoside can be provided from various raw materials such as buckwheat and onion, and can be preferably provided from onion.

Aspergillus according to the present invention this scan or (Aspergillus niger, Accession No: KCTC 6906) derived from strain beta-glucosidase during Days (β-glucosidase, EC 3.2.1.21) is an enzyme specific for glucose, which is coupled to Quebec paroxetine (quercetin) 4 'position of the aglycone of glycoside position The quercetin represented by the formula (1) or the isoquercitrin represented by the formula (1) can be produced with high efficiency.

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

(2)

(3)

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

The step 1 according to the present invention preferably performs the reaction under stirring conditions of 100 rpm to 200 rpm. When the stirring condition is less than 100 rpm, the reaction speed is slow, so that it takes a long time to complete the reaction. When the stirring condition is more than 200 rpm, the pulverization phenomenon occurs at high speed rotation, have.

In addition, the step 1 is preferably carried out in a temperature range of 25 ° C to 40 ° C, and more preferably in a temperature range of 27 ° C to 35 ° C. When the temperature is less than 27 ° C, the enzyme does not reach a sufficient temperature for activity, and the reaction rate is lowered. When the temperature exceeds 35 ° C, the enzyme is denatured and enzyme reaction does not occur.

Furthermore, it is preferable that step 1 is carried out for 18 hours or more, and it is more preferable to carry out the reaction for 24 hours. When the reaction is carried out for less than 18 hours, there is a problem that the conversion of quercetin glycoside to quercetin or isoquercitrin represented by the general formula (1) is not completely carried out and the production yield is low. On the other hand, when the reaction was performed for 48 hours, there was no significant difference when the reaction was performed for 24 hours. From this, it can be seen that it is difficult to expect an increase in production efficiency by more than 24 hours (see Experimental Example 1). Therefore, it is preferable that the reaction is carried out for 24 hours, when there is no difference in the conversion of quercetin glycoside to quercetin or isoquercitrin represented by the formula (1) over 24 hours.

Next, the step 2 according to the present invention is a step of removing the enzyme from the product prepared through the enzyme reaction in the above step 1 to obtain a product composed of a mixture of quercetin or isoquercitrin represented by the formula (1) . More specifically, in order to facilitate the extraction of quercetin or isoquercitrin represented by the formula (1) present in the reaction product, first, the reaction product is pulverized for 5 to 10 minutes with an ultrasonic pulverizer do. Thereafter, the pulverized product is centrifuged at 4000 rpm to 5000 rpm for about 5 minutes, the supernatant liquid is collected, and the aliquot is filtered to obtain the enzyme-free product.

Next, step 3 according to the present invention is a method for purifying quercetin or isoquercitrin represented by the formula (1) by performing high performance liquid chromatography (HPLC) on a product from which the enzyme has been removed in step 2 .

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

At this time, the concentration gradient of the eluting solvent and the solvent of high performance liquid chromatography (HPLC) can be adjusted depending on the amount of the fraction to be separated or the column size and kind of high performance liquid chromatography (HPLC). Preferably, the elution solvent may be distilled water in which 0.1% formic acid is dissolved and methanol, 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% (5 minutes → 5 minutes), 5% → 80% (5 minutes → 25 minutes), 80% (25 minutes → 30 minutes).

Further, it is preferable to confirm quercetin or isoquercitrin represented by the formula (1) purified through high performance liquid chromatography (HPLC) at a wavelength of 320 nm.

Aspergillus according to the present invention this scan or (Aspergillus A method for producing quercetin or isoquercitrin represented by the formula (1) using an enzyme β-glucosidase (EC 3.2.1.21) derived from a strain, Glucuronidase (EC 3.2.1.21) enzyme by reacting quercetin glycosides, which are abundantly contained in onion, with an easily available onion in the market, Quercetin or isoquercitrin can be produced in a short time in a large amount (see Experimental Example 1).

In addition, the enzyme β-glucosidase (EC 3.2.1.21) has selective site-specificity to remove glucose specifically bound to the 4 'position of the aglycon of the quercetin glycoside Quercetin or isoquercitrin represented by the formula (1) can be produced with high efficiency from the quercetin glycoside (see Experimental Example 2).

Therefore, the process for preparing quercetin or isoquercitrin represented by the formula (1) using an enzyme of β-glucosidase (EC 3.2.1.21) according to the present invention is a method for producing quercetin or isoquercitrin, it is possible to produce quercetin or isoquercitrin represented by the formula (1) with high efficiency in a short period of time by using an enzyme of β-glucosidase (EC 3.2.1.21) quercetin or isoquercitrin, foods, flavors, beverages, herbal medicines, and health 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

The onions used in the present invention were purchased and used in the agricultural products market of Jeongeup city. Aspergillus niger, Accession No: KCTC 6906) derived from the beta-glucosidase during Days (β-glucosidase, EC 3.2.1.21) enzyme (40 U / ml) were purchased from mega jimsa (Megazyme, Wicklow, Ireland). Solvents for high performance liquid chromatography (HPLC) of water and methanol were used from Burdick & Jackson, SK chemicals, Ulsan, ROK, and formic acid was purchased from Sigma.

First, the onion to be reacted with the enzyme solution was prepared, and the onion skin was removed and washed with water. The washed onion was ground using a blender until juice was obtained, and 10 g of the crushed onion was used as a substrate. 0.1 ml of β-glucosidase enzyme solution (40 U / mL) was added to the prepared substrate (10 g wet weight of onion) and reacted at 30 ° C. and 150 rpm for 24 hours. 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 subjected to a centrifugal separation process (4,000 rpm, 5 minutes), followed by filtering the supernatant liquid (0.2 쨉 m) for 5 minutes with an ultrasonic pulverizer for extraction of quercetin compound. The filtered mixture was purified by high performance liquid chromatography (HPLC, Agilent, 1200 series). At this time, the column was a YMC ODS-AM column (5 μm, 4.6 mm × 250 mm), the flow rate was 1.0 ml / min, and the detector was measured at a wavelength of 320 nm using DAD. 5% B (0-5 min), 5% B (0-5 min), and the eluent was dissolved in distilled water (0.1% formic acid) -80% B (5-25 min), 80% B (25-30 min). The amount of the sample injected was adjusted to 10 μl and purified by the above conditions to obtain quercetin-4'-O-β-D-glucoside represented by Chemical Formula 2 or quercetin-quercetin represented by Chemical Formula 3, which is an unreacted quercetin glycoside, 3,4'-O-? -D-glucoside was recovered to obtain quercetin or isoquercitrin represented by the formula (1) as a target compound.

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

The reaction was carried out in the same manner as in Example 1, except that the reaction was carried out for 24 hours instead of 48 hours to obtain quercetin-4 ', an unreacted quercetin glycoside represented by the formula (2) -O-? - D-glucoside represented by the formula (1) or quercetin-3, 4'-O-? -D-glucoside represented by the formula (3) is recovered and quercetin or isoquitoline (isoquercitrin).

< Example  3> Quercetin ( quercetin )

Quercetin-4'-O-? -D-glucoside represented by the formula (2) as a quercetin monosaccharide was purchased from Extrasynthese (Genay, France) and dissolved in a 100 mM phosphate buffer solution , So that the final concentration is 100 占 퐂 / ml. This solution was used as a substrate solution for the enzyme reaction, and the enzyme solution (1 ml, 40 U / ml) was added to the prepared substrate solution (1 ml) for reaction. At this time, the reaction was carried out at 30 ° C for 2 hours. Then, 100 ml of methanol (1.1 ml) was added to extract quercetin from the product (1.1 ml), pulverized with an ultrasonic grinder for 5 minutes, centrifuged at 4000 rpm for 5 minutes, Filtration (0.2 탆). 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 the formula (1).

< Example  4> Isoquitoline ( isoquercitrin )

Instead of using the quercetin-4'-O- beta -D-glucoside represented by the formula (2) as a quercetin monosaccharide, the quercetin-3,4'-O- Glucoside was purchased from Extrasynthese (Genay, France) and used in the same manner as in Example 3 to obtain isoquercitrin represented by the formula (1).

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

The quercetin-4-quercetin glycoside represented by the formula (2), which was an unreacted quercetin glycoside, was obtained by carrying out the same procedure as in Example 1, except that the reaction was carried out for 6 hours instead of 24 hours. -O-? - D-glucoside represented by the formula (1) or quercetin-3, 4'-O-? -D-glucoside represented by the formula (3) is recovered and quercetin or isoquitoline (isoquercitrin).

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

The reaction was carried out in the same manner as in Example 1, except that the reaction was carried out for 12 hours instead of performing the reaction for 24 hours to obtain quercetin-4 'represented by the formula (2) as an unreacted quercetin glycoside, -O-? - D-glucoside represented by the formula (1) or quercetin-3, 4'-O-? -D-glucoside represented by the formula (3) is recovered and quercetin or isoquitoline (isoquercitrin).

< Experimental Example  1> Over time Quercetin ( quercetin ) or Isoquitricin ( isoquercitrin  Manufacturing efficiency evaluation

The preparation of quercetin glycoside as quercetin or isoquercitrin represented by the general formula (1) using beta-glucosidase (EC 3.2.1.21) according to the present invention. The following experiments were conducted to evaluate the manufacturing efficiency over time of the method.

The onion juice used in the present invention was filtered (0.2 탆) to prepare an onion juice. The produced onion juice was subjected to high performance liquid chromatography to quantify the amount of quercetin glycoside present in the onion juice and used as a non - treated group. In addition, the amount of quercetin or isoquercitrin represented by the formula (1) prepared in Examples 1 to 2 and Comparative Examples 1 to 2 and the amount of unreacted quercetin O-beta-D-glucoside represented by the formula (2) or quercetin-3, 4'-O- beta -D-glucoside represented by the formula (3) , And 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 quercetin or isoquercitrin represented by the formula (1) according to the present invention is a quercetin-4'-O- (quercetin) or quercitrin represented by the formula (1) through bioconversion of β-D-glucoside or quercetin-3, 4'-O-β-D- . When the reaction time is 24 hours, the production amount of quercetin and quercitrin represented by the formula (1) is 83 占 퐂 / g and isoquercitrin is 160 占 퐂 / g, respectively, it can be seen that the production amount of quercetin or isoquercitrin is not changed much more. The method for producing quercetin or isoquercitrin according to the present invention is characterized in that the enzyme β-glucosidase (EC 3.2.1.21) is a quercetin glycoside existing in onion (Quercetin) or isoquercitrin represented by the general formula (1) can be prepared by biotransformation of the quercetin or isoquercitrin. When the reaction is carried out for more than 24 hours, quercetin or isoquercitrin represented by the general formula It can be seen that the production yield of isoquercitrin is the highest.

Therefore, the production method of quercetin or isoquercitrin represented by the formula (1) using β-glucosidase (EC 3.2.1.21) according to the present invention is easy to produce in the market Glucosidase (EC 3.2.1.21) enzyme by using a commercially available onion as a raw material and reacting a quercetin glycoside, which is contained in a large quantity in an onion for 24 hours, with an enzyme of the formula (1) A pharmaceutical composition comprising quercetin or isoquercitrin represented by the formula (1), a food, a flavoring agent, a drink or the like, which is easy to mass-produce quercetin or isoquercitrin, , Herbal medicines, and health supplements.

< Experimental Example  2> Beta- Glucosides  Bioconversion reaction of enzyme

The β-glucosidase (EC 3.2.1.21) enzyme according to the present invention bioconverts a quercetin glycoside to produce quercetin or isoquercitrin represented by the formula (1) The following experiment was conducted.

Quercetin-4'-O-? -D-glucoside represented by the formula (2) which is quercetin glycoside or quercetin-3,4'-O-? -D-glucoside represented by the formula (3) The reaction products before and after the reaction in Examples 3 to 4, which were reacted with the enzyme β-glucosidase (EC 3.2.1.21), were aliquoted and the separated reaction products were subjected to high performance liquid chromatography. The measured graph was compared with a measurement graph of high performance liquid chromatography of a reference material to verify quercetin glycoside, quercetin and isoquercitrin represented by formula (1). The quercetin represented by the formula (1) was purchased from Sigma (St. Louis, Mo., USA) and used as a quercetin glycoside represented by the formula (2) Quercetin-4'-O-? -D-glucoside or quesetin-3,4'-O-? -D-glucoside represented by the formula (3) and isoquercitrin represented by the formula (1) (Extrasynthese, Genay, France). The results of the verification are shown in Figs. 1 and 2. Fig.

As shown in FIGS. 1 and 2, quesetin-4'-O-β-D-glucoside represented by the formula (2) is quercetin after 2 hours of reaction, quercetin- , 4 ' -O- [beta] -D-glucoside are all converted to isoquercitrin. From this, Aspergillus according to the present invention this scan or (Aspergillus The enzyme β-glucosidase (EC 3.2.1.21), derived from the niger (Accession No: KCTC 6906), is an enzyme that binds glucose at the 4'-position of the aglycon of the quercetin glycoside, As shown in Fig.

Therefore, the production method of quercetin or isoquercitrin represented by the formula (1) using β-glucosidase (EC 3.2.1.21) according to the present invention is easy to produce in the market The quercetin glycoside, which is contained in a large amount in the onion for 24 hours, can be enzymatically reacted using commercially available onion as a raw material, thereby economically producing quercetin or isoquercitrin represented by the formula (1) (Quercetin) represented by the general formula (1) can be obtained because it is possible to produce a large amount of sample with high efficiency in a short time by utilizing the selective site specificity of the enzyme of β-glucosidase (EC 3.2.1.21) Or isoquercitrin, foods, flavors, beverages, herbal medicines, and health supplements.

Claims (7)

The step of reacting quercetin-4'-O -? - D-glucoside represented by the following formula 2 with? -Glucosidase (EC 3.2.1.21) enzyme as a quercetin glycoside (step 1 );
Removing the enzyme from the product prepared in step 1 (step 2); And
(Step 3) of purifying the enzyme-removed product in step 2 by performing high performance liquid chromatography (HPLC).
(2)

The step of reacting quercetin-3, 4'-O-? -D-glucoside represented by Formula 3 with? -Glucosidase (EC 3.2.1.21) enzyme as a quercetin glycoside One);
Removing the enzyme from the product prepared in step 1 (step 2); And
A method for producing isoquercitrin comprising purifying an enzyme-removed product in step 2 by performing high performance liquid chromatography (HPLC) (step 3)
(3)

3. The method according to claim 1 or 2,
Wherein the β-glucosidase (EC 3.2.1.21) enzyme of step 1 is derived from Aspergillus niger (Accession No. KCTC 6906).
3. The method according to claim 1 or 2,
Wherein step 1 is carried out under stirring conditions of 100 rpm to 200 rpm.
3. The method according to claim 1 or 2,
Wherein step 1 is carried out in a temperature range of from 25 캜 to 35 캜.
3. The method according to claim 1 or 2,
Wherein the step (2) comprises subjecting the reaction product to ultrasonic pulverization and centrifugation.
3. The method according to claim 1 or 2,
The high performance liquid chromatography (HPLC) of the above step 3 sequentially changed the concentration of methanol from 5% for 5 minutes to 5% for 80 minutes, 25 minutes for 30 minutes to 80% for 5 minutes, &Lt; / RTI &gt;

Images