KR20240077308A - A method for producing the extract of Schizonepeta tenuifolia Briq. with increased hesperetin content. method for producing hesperetin from Schizonepeta tenuifolia Briq., extract and fraction with increased hesperetin content - Google Patents

Abstract

The present invention provides a method for producing an extract with increased hesperetin content. The present invention relates to a method for producing hesperetin from a hesperetin, a hesperetin extract with an increased hesperetin content, an ethyl acetate fraction of the hesperetin extract, and their uses. In the present invention, the hesperetin extract is produced as an Aspergillus kawachi crude enzyme solution. By converting the hesperidin contained in the Hyeonggae extract into hesperetin, a Hyeonggae extract with increased hesperetin content is obtained. The method of the present invention is a new method of producing hesperetin through bioconversion and can be used in various fields such as food, medicine, and cosmetics. In addition, the bioconverted extract containing a large amount of hesperetin obtained according to the present invention, its fractions, and isolated hesperetin can be usefully used in the fields of foods such as functional foods, medicines, and cosmetics.

Description

Method for producing Hyeonggae extract with increased hesperetin content. Method for producing hesperetin from Schizonepeta, extract and fractions with increased hesperetin content {A method for producing the extract of Schizonepeta tenuifolia Briq. with increased hesperetin content. method for producing hesperetin from Schizonepeta tenuifolia Briq., extract and fraction with increased hesperetin content}

The present invention provides a method for producing an extract with increased hesperetin content. The present invention relates to a method for producing hesperetin from molded eggs, a molded bark extract with increased hesperetin content, an ethyl acetate fraction of the molded grain extract, and their uses.

Schizonepeta tenuifolia Briq.) is an annual herb belonging to the Lamiaceae family and its whole herb is used as a medicinal herb. Hyeonggae is distributed in Gyeongbuk, Gangwon, Chungnam, and Gyeonggi-do in Korea and in Jiangsu, Zhejiang, Gangseo, Hubei, and Hebei in China. It has an antipyretic effect, so it is used for sweating and fever in the early stages of a cold, and is often used for sore throat, skin disease, stroke, etc. When roasted black, it has a hemostatic effect, so it is used for uterine bleeding, nosebleeds, stool bleeding, hematemesis, and urine bleeding.

The main components of the mold opening include essential oil components such as d-menthone and d-limonene; Flavonoids include hesperidin and luteolin; Phenolic acids include rosmarinic acid, caffeic acid, and cinnamic acid; and monoterpenes.

Hesperidin is one of the flavonoids and is used to protect capillaries. When hesperidin is hydrolyzed with dilute acid, one molecule each of hesperetin, glucose, and rhamnose is produced.

Hesperetin is one of the flavonoids and exists as hesperidin, a glycoside form bound to a sugar, and has antioxidant and anti-inflammatory activities.

Meanwhile, bio conversion has the same meaning as terms such as biosynthesis and biocatalysis, and refers to a technology for producing a desired product from precursor materials using the enzymatic function of microorganisms. . In other words, it is a technology that induces structural changes in natural biologically active substances through biological methods such as microbial fermentation, enzyme treatment, and/or coenzyme treatment, thereby increasing the content of active ingredients, improving absorption rate, and generating new functional ingredients. Currently, bioconversion is being applied or attempted to be applied in various fields such as food, medicine, and cosmetics, but there is still no known technology for increasing the content of specific active ingredients by applying bioconversion technology in molds.

Republic of Korea Patent Publication No. 10-2017-0051078 Republic of Korea Patent Publication No. 10-2017-0065071

Molecules, 27, 5249, 2022. Nat. Prod. Sci., 14(2), 100-106, 2008. J Biomed Res., 33(4), 250-257, 2019. Chin Med., 25(6), 2011. Nat. Prod. Sci., 25(2), 130-135, 2019. Vietnam J. Chem., 57(4), 438-442, 2019. Korean J. Food Sci. Technol., 29(4), 823-826, 1997. Nat. Prod. Sci., 25(1), 76-80, 2019. Kor. J. Pharmacogn., 47(1), 1-6, 2016. Der Pharma Chemica, 7(2), 1-4, 2015.

The purpose of the present invention is to provide a method for increasing the content of a specific active ingredient contained in a mold shell, a mold shell extract with an increased content of the active ingredient, and an active ingredient isolated therefrom.

In particular, the present invention relates to a method for producing a mold extract in which the content of hesperetin is increased by converting the hesperidin contained in the mold to hesperetin by applying bioconversion technology, and a method for producing hesperetin from the mold. The purpose is to provide extracts and fractions with increased hesperetin content.

In order to achieve the above object, in the present invention,

A method for producing a mold extract with increased hesperetin is provided, comprising the step of converting the hesperidin contained in the mold extract into hesperetin by bioconverting the mold extract with Aspergillus kawachi crude enzyme solution.

In the above production method, the bioconversion preferably includes adding Aspergillus kawachi crude enzyme solution to the mold extract and shaking the extract for 15 to 40 hours at 20 to 40°C. Preferably, the shaking reaction is performed at 50 to 200 rpm.

Additionally, in the present invention,

Obtaining a bioconverted Hyeonggeum extract comprising converting the Hesperidin contained in the Hyeonggae extract into hesperetin by bioconverting the Hyeonggea extract with Aspergillus Kawachi crude enzyme solution;

A production method for producing hesperetin from mold extract is provided, comprising the step of isolating hesperetin from the bioconverted mold extract.

In the production method, the bioconversion preferably includes adding Aspergillus kawachi crude enzyme solution to the mold extract and reacting with shaking at 50 to 200 rpm for 15 to 40 hours at 20 to 40 ° C.

In a preferred embodiment, the production method may further include the step of obtaining an ethyl acetate fraction by fractionating the bioconverted extract with ethyl acetate, and in this case, hesperetin is separated from the ethyl acetate fraction.

Additionally, in the present invention,

The present invention provides an extract with increased hesperetin, which is obtained by bioconverting the extract of the extract with Aspergillus kawachi crude enzyme solution to convert the hesperidin contained in the extract into hesperetin.

Additionally, in the present invention,

The ethyl acetate fraction of the Hyeonggae extract with increased hesperetin content obtained by bioconverting the Hyeonggae extract with Aspergillus Kawachi crude enzyme solution to convert the hesperidin contained in the Hyeonggae extract into hesperetin and then fractionating it with ethyl acetate. provides.

Additionally, in the present invention,

Provided is a food composition comprising a Hesperetin extract with an increased hesperetin content or an ethyl acetate fraction of the Hesperetin extract with an increased hesperetin content. Here, food is a concept that includes health functional foods, health supplements, functional foods, and general foods.

According to the present invention, the hesperidin contained in the Hyeonggae extract can be converted to hesperetin by bioconverting the Aspergillus Kawachi crude enzyme solution. In the present invention, the content of hesperetin, which has excellent physiological activity, can be increased in the extract of the extract of the extract, and the extract of the extract with the increased content of the hesperetin can be prepared, and the hesperetin can be produced therefrom in an economical and efficient manner.

According to the method of the present invention, a large amount of hesperetin can be produced from mold opening in a short time at a simpler and lower cost than the existing method.

The bioconverted extract containing a large amount of hesperetin obtained according to the present invention, its fractions, and isolated hesperetin can be usefully used in the fields of foods such as functional foods, medicines, and cosmetics.

Figure 1 is a schematic diagram showing the process of bioconverting a mold extract and separating each component according to a preferred embodiment of the present invention. Compound 1 is caffeic acid, compound 2 is 4-hydroxybenzoic acid, compound 3 is luteolin, compound 4 is cinnamic acid, compound 5 is hesperetin, compound 6 is gallic acid, and compound 7 is hesperidin.
Figure 2 shows the HPLC results confirmed at 210 nm before and after bioconversion of the mold extract. Blue represents the Hyeonggae extract before bioconversion, and red represents the Hyeonggae extract after bioconversion.
Figure 3 shows the HPLC results confirmed at 210 nm of the ethyl acetate fraction of the bioconverted Hyeonggae extract. Compound 1 is caffeic acid, compound 2 is 4-hydroxybenzoic acid, compound 3 is luteolin, compound 4 is cinnamic acid, and compound 5 is hesperetin.
Figure 4 shows the HPLC results confirmed at 210 nm of the butanol fraction of the bioconverted Hyeonggae extract. Compound 6 is gallic acid, and compound 7 is hesperidin.

The present invention provides a method for producing an extract with increased hesperetin content. The present invention relates to a method for producing hesperetin from molded nuts, a molded bark extract with increased hesperetin content, an ethyl acetate fraction of the molded bark extract, and their uses. Hereinafter, the present invention will be described in detail.

The method for producing an extract with increased hesperetin content of the present invention includes the step of bioconverting the extract with Aspergillus kawachi crude enzyme solution to convert the hesperidin contained in the extract into hesperetin.

Hyeonggae extract is not limited to the extraction method and can be prepared using extraction methods known in the art. Preferably, an extraction method acceptable for the food, pharmaceutical, and cosmetic fields is used depending on the end use. For example, using various organic solvents such as water, alcohols with 1 to 5 carbon atoms (e.g. methanol, ethanol, propanol, butanol, etc.), acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane, and cyclohexane. It can be extracted using solvent extraction, supercritical extraction using reduced pressure and high temperature using carbon dioxide, or ultrasonic extraction.

Preferably, a solvent extraction method may be used in which a solvent is added to the mold opening and then extracted. At this time, the solvent is not particularly limited, and can preferably be selected from water and a polar organic solvent having 1 to 5 carbon atoms. More preferably, it can be selected from water and an alcohol compound having a carbon skeleton of 2 to 4 carbon atoms. Here, ‘water’ is a concept that includes distilled water, purified water, and bottled water. Preferably, water from which impurities have been removed, such as distilled water or purified water, is used. The solvent may preferably be used in a volume 3 to 10 times the mold opening weight, but is not limited thereto. In a preferred embodiment, the extract is extracted twice at room temperature for about 36 to 60 hours using 70% ethanol as a solvent, then filtered, and the ethanol extract thus obtained is concentrated under reduced pressure and used as an extract. However, the method of obtaining the mold extract is not limited to this, and if necessary, the mold extract obtained as above can be freeze-dried and used. The reduced-pressure concentrated extract or freeze-dried extract obtained as above can be used by suspending it in distilled water, for example.

Aspergillus kawachi ( Aspergillus ) extract obtained as above. kawachii ) Bioconversion using crude enzyme solution.

In the present invention, 'Aspergillus kawachi crude enzyme solution' is defined as a supernatant obtained by filtering and centrifuging the culture medium in which Aspergillus kawachi was cultured. The Aspergillus kawachi crude enzyme solution preferably includes the steps of culturing Aspergillus kawachi in a medium containing wheat bran and distilled water at 25-35°C for 2-8 days; Adding sodium phosphate buffer to the culture medium and leaving it at 0-10°C for 10-30 hours; And it can be obtained by a method comprising the step of obtaining Aspergillus kawachi crude enzyme solution, which is the supernatant, by filtration and centrifugation.

Aspergillus kawachi crude enzyme solution is added to the above mold extract and reacted to perform bioconversion. The mold extract is preferably used by suspending the vacuum-concentrated mold extract or the freeze-dried mold extract obtained above in water.

Preferably, the Aspergillus Kawachi crude enzyme solution obtained in the same manner as above may be added to the concentrated reduced-pressure extract obtained as above in a volume of 5 to 20 times the weight of the extract. Alternatively, the Aspergillus Kawachi crude enzyme solution can be added to a suspension in which the reduced-pressure concentrated Hyeonggae extract is suspended in water and reacted. In this case, the ratio can be calculated using the reduced-pressure concentrated Hyeonggae extract and the same ratio as above. However, the mixing ratio of the extract and Aspergillus kawachi crude enzyme solution is not limited to this, and the mixing ratio depends on the manufacturing method of the mold extract or Aspergillus kawachi crude enzyme solution, temperature, humidity, surrounding environment, season, etc. during the enzyme reaction. It can be adjusted according to the conditions and, if necessary, outside the above range.

The reaction is preferably performed at 20 to 40°C with shaking for 15 to 40 hours, and more preferably at 50 to 200 rpm. Particularly preferably, bioconversion is performed by shaking at 100±20 rpm for 24±4 hours at 30±5°C. However, the temperature and time are not limited to these, and a person skilled in the art can appropriately adjust the temperature and time to ensure that the enzyme reaction takes place well.

When the extract is bioconverted in this way, as shown in the test example below and in Figure 2, the hesperidin contained in the extract is converted to hesperetin, thereby obtaining an extract with an increased hesperetin content.

The method for producing hesperetin from the mold of the present invention includes:

Obtaining a bioconverted Hyeonggeum extract comprising converting the Hesperidin contained in the Hyeonggea extract into hesperetin by bioconverting the Hyeonggea extract with Aspergillus Kawachi crude enzyme solution; It includes the step of isolating hesperetin from the bioconverted extract.

The steps for obtaining the bioconverted Hyeonggae extract are the same as described in the ‘Method for producing a Hyeonggae extract with increased hesperetin content’ above.

The step of separating hesperetin from the bioconverted extract was performed using silica gel column chromatography in the experimental example below. However, for mass production, those skilled in the art can separate and purify hesperetin from the bioconverted mold extract by applying known separation methods using the chemical and physical properties of the compound.

In a preferred embodiment of the present invention, the bioconverted extract is fractionated with ethyl acetate and then hesperetin is separated from the ethyl acetate fraction.

In addition, in addition to hesperetin, caffeic acid, 4-hydroxybenzoic acid, luteolin, cinnamic acid, gallic acid, etc. can also be effectively separated from the bioconverted extract.

In addition, the present invention relates to an extract with an increased hesperetin content obtained by converting the hesperidin contained in the extract into hesperetin by bioconverting the extract with Aspergillus kawachi crude enzyme solution. The description of each configuration is the same as above.

In addition, the present invention bioconverts the mold extract with Aspergillus kawachi crude enzyme solution to convert the hesperidin contained in the mold extract into hesperetin and then fractionates it with ethyl acetate to produce a mold with increased hesperetin content. It relates to the ethyl acetate fraction of the extract. The description of each configuration is the same as above.

'Hesperetin extract with increased hesperetin content', 'ethyl acetate fraction of hesperetin extract with increased hesperetin content' obtained according to the present invention, and 'hesperetin' obtained by separation by the method of the present invention. As one of the flavonoids, it can be usefully used in fields such as food, medicine, and cosmetics.

The present invention also relates to a food composition comprising the above-described extract with an increased hesperetin content or an ethyl acetate fraction of the extract with an increased hesperetin content.

In the present invention, 'food' is defined to include health functional foods, health supplements, functional foods, and general foods. Therefore, the food composition may be in the form of any one of health functional foods, health supplements, functional foods, and general foods.

In a food composition, the extract or fraction may be included alone as an active ingredient, added to other active ingredients, or used together with other foods or food ingredients, and may be used appropriately according to conventional methods of using food materials. The extract or fraction can be expected to have the efficacy of hesperetin, one of the flavonoids, as well as the efficacy of the mold.

There is no particular limitation on the type of food containing the extract or fraction. Examples of foods include meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, beverages, alcoholic beverages, and vitamins. complexes, etc.

In addition, when the extract or fraction is used in the form of a beverage, various flavoring agents or natural carbohydrates may be included as additional ingredients, like ordinary beverages. Natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as textrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As a sweetener, natural sweeteners such as thaumatin and stevia extract or synthetic sweeteners such as saccharin and aspartame can be used.

In addition, the composition containing the extract or fraction may optionally contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, It may include stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the composition of the present invention may contain pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These ingredients can be used independently or in combination.

The present invention will be described in detail below through examples. These examples illustrate the present invention and the scope of the present invention is not limited thereto.

<Example 1>

Preparation of Hyeonggae Extract

Hyeonggae was purchased and used at the herbal medicine market. 3 kg of mold lid and 10 liters of 70% ethanol were mixed and extracted twice for 2 days at room temperature. The extract was filtered, and the filtrate was concentrated under reduced pressure to obtain a mold extract.

<Example 2>

Preparation of Aspergillus Kawachi crude enzyme solution

In a 500 ml Erlenmeyer flask, 10 g of wheat bran and 10 ml of distilled water were mixed to allow the wheat bran to absorb moisture well, sterilized at 121°C for 15 minutes, then inoculated with Aspergillus kawachi and cultured at 30°C for 3 days.

The culture was suspended in 100mM sodium phosphate buffer (pH 7.0) and left at 4°C for 18 hours. After leaving, it was filtered through gauze, and the filtrate was refrigerated and centrifuged at 10,000 rpm for 15 minutes to obtain the supernatant, Aspergillus kawachi crude enzyme solution.

<Example 3>

of hesperetin Separation and purification

300 g of the mold extract of Example 1 was suspended in 3 liters of distilled water to obtain a suspension of the mold extract. 3 liters of the Aspergillus kawachi crude enzyme solution of Example 2 was added to the Hyeonggae extract suspension and shaken at 100 rpm at 30°C for 24 hours to bioconvert the Hyeonggae extract.

The reaction product was fractionated with each solvent of n-hexane, ethyl acetate, and n-butanol and concentrated using a rotary vacuum concentrator to obtain each fraction (37.8 g).

The ethyl acetate fraction (37.8 g) was subjected to silica gel column chromatography with increasing polarity using a mixed solvent of normal hexane and ethyl acetate as a developing solvent in a concentration gradient (v/v) from 9:1 to 0:100. It was separated into 14 fractions (HGE-1 to 14).

YMC ODS-A column using the 9th fraction (HGE-9) (3.5 g) among the 14 fractions as a developing solvent and a mixed solvent of water and methanol with a concentration gradient (v/v) from 7:3 to 0:100. Chromatography was performed and separated into 11 small fractions (HGE-9-1~11).

Hesperetin (Compound 5) (679.5 mg) in the form of brown powder was obtained from the 8th subfraction among 11 subfractions.

<Example 4>

of Sinnamsan Mountain Separation and purification

The ethyl acetate fraction (37.8 g) obtained in Example 3 was grown on a silica gel column while increasing polarity using a mixed solvent of normal hexane and ethyl acetate as a developing solvent at a concentration gradient (v/v) from 9:1 to 0:100. Chromatography was performed and separated into 14 fractions (HGE-1 to 14).

YMC ODS-A column using the 6th fraction (HGE-6) (5.0g) among the 14 fractions as a developing solvent and a mixed solvent of water and methanol with a concentration gradient (v/v) from 5:5 to 4:6. Chromatography was performed to separate it into four small fractions (HGE-6-1~4).

Among the four subfractions, the third subfraction (HGE-6-3) (0.2g) was subjected to Sephadex LH-20 column chromatography using 100% methanol as a developing solvent, resulting in three Separated into small fractions.

Cinnamic acid (compound 4) (39.4 mg) in the form of white powder was isolated from the second of the three subfractions.

<Example 5>

4- of hydroxybenzoic acid Separation and purification

The ethyl acetate fraction (37.8 g) obtained in Example 3 was grown on a silica gel column while increasing polarity using a mixed solvent of normal hexane and ethyl acetate as a developing solvent at a concentration gradient (v/v) from 9:1 to 0:100. Chromatography was performed and separated into 14 fractions (HGE-1 to 14).

YMC ODS-A column using the 9th fraction (HGE-9) (3.5 g) among the 14 fractions as a developing solvent and a mixed solvent of water and methanol with a concentration gradient (v/v) from 7:3 to 0:100. Chromatography was performed and separated into 11 small fractions (HGE-9-1~11).

Among the 11 subfractions, the 1st subfraction (HGE-9-1) (0.3g) was used as a developing solvent and a mixed solvent of normal hexane and ethyl acetate was used at a concentration gradient (v/v) from 8:2 to 0:100. Column chromatography on silica gel was performed with increasing polarity to separate it into five small fractions.

4-hydroxybenzoic acid (compound 2) (39.3 mg) in the form of brown powder was isolated from the third of the five subfractions.

<Example 6>

Separation and purification of caffeic acid and luteolin

The ethyl acetate fraction (37.8 g) obtained in Example 3 was grown on a silica gel column while increasing polarity using a mixed solvent of normal hexane and ethyl acetate as a developing solvent at a concentration gradient (v/v) from 9:1 to 0:100. Chromatography was performed and separated into 14 fractions (HGE-1 to HGE-14).

YMC ODS-A column using the 12th fraction (HGE-12) (5.1 g) among the 14 fractions as a developing solvent with a concentration gradient (v/v) of water and methanol from 7:3 to 0:100. Chromatography was performed and separated into 10 small fractions (HGE-12-1~10).

Among the 10 subfractions, caffeic acid (compound 1) (183.8 mg) in the form of brown powder was isolated from the second subfraction, and luteolin (compound 3) (205.1 mg) in the form of brown powder was isolated from the sixth fraction.

<Example 7>

Isolation and purification of gallic acid

The butanol fraction (48.9 g out of 67.4 g) obtained in Example 3 was used as a developing solvent in a mixed solvent of normal hexane and ethyl acetate at a concentration gradient (v/v) from 9:1 to 0:100 to increase polarity. Silica gel column chromatography was performed and separated into 9 fractions (HGB-1 to 9).

YMC ODS-A column using the 5th fraction (HGB-5) (4.0g) among the 9 fractions as a developing solvent and a mixed solvent of water and methanol with a concentration gradient (v/v) from 7:3 to 4:6. Chromatography was performed and separated into 12 small fractions (HGB-5-1~12).

Among the 12 subfractions, the second subfraction (HGB-5-2) (719.0 mg) was used as a developing solvent, and a mixed solvent of ethyl acetate and methanol was polarized with a concentration gradient (v/v) from 100:0 to 0:100. Silica gel column chromatography was performed while increasing to separate into three small fractions.

Gallic acid (Compound 6) (49.4 mg) in the form of yellow powder was isolated from the first of the three subfractions.

<Example 8>

Isolation and purification of hesperidin

The butanol fraction (48.9 g out of 67.4 g) obtained in Example 3 was used as a developing solvent and a mixed solvent of normal hexane and ethyl acetate was used on a silica gel column while increasing the polarity with a concentration gradient (v/v) from 9:1 to 0:100. Chromatography was performed and it was separated into 9 fractions (HGB-1 to 9).

YMC ODS-A column using the 5th fraction (HGB-5) (4.0g) among the 9 fractions as a developing solvent and a mixed solvent of water and methanol with a concentration gradient (v/v) from 7:3 to 4:6. Chromatography was performed and separated into 12 small fractions (HGB-5-1~12).

YMC used the 12th subfraction (HGB-5-12) (121.5 mg) among the 12 subfractions as a developing solvent using a mixed solvent of water and methanol with a concentration gradient (v/v) from 5:5 to 4:6. ODS-A column chromatography was performed and separated into four small fractions.

Hesperidin (Compound 7) (21.0 mg) in the form of yellow powder was isolated from the second of the four subfractions.

A schematic diagram of the process of bioconverting the extracts of Examples 1 to 8, preparing fractions, and separating each compound is shown in Figure 1. In Figure 1, Compound 1 is caffeic acid, Compound 2 is 4-hydroxybenzoic acid, Compound 3 is luteolin, Compound 4 is cinnamic acid, Compound 5 is hesperetin, Compound 6 is gallic acid, and Compound 7 is hesperidin.

<Experimental Example 1>

Nuclear magnetic resonance (NMR) analysis

Nuclear magnetic resonance (NMR) analysis was performed on the compounds isolated from the bioconverted Hyeonggae extract.

The compounds of Examples 3 to 8 were used as samples.

¹ H NMR and ¹³ C NMR analysis were performed using a JEOL ECX-500 spectrometer (JEOL Ltd., Japan).

As a result of structural analysis through ¹ H and ¹³ C NMR, each compound was identified as follows. Structural analysis was performed by comparison with previously published literature:

(1) Caffeic acid

Caffeic acid (Compound 1) : Brown powder; ¹ H-NMR (500MHz, CD ₃ OD) δ 7.46 (1H, d, J =15.8Hz, H-7), 7.03 (1H, d, J =2.0Hz, H-2), 6.91 (1H, dd, J =8.0, 2.0Hz, H-6), 6.78 (1H, d, J =8.0Hz, H-5), 6.24 (1H, d, J =15.8Hz, H-8). ; ¹³ C NMR (125MHz, CD ₃ OD) δ 172.8 (C-9), 149.1 (C-4), 146.8 (C-7), 145.6 (C-3), 128.3 (C-1), 122.7 (C- 6), 117.8 (C-5), 116.6 (C-8), 115.1 (C-2).

(2) 4-hydroxybenzoic acid

4-Hydroxybenzoic acid (Compound 2): Brown powder; ¹ H-NMR (500MHz, CD ₃ OD) δ 7.87 (2H, d, J = 8.9Hz, H-2, 6), 6.82 (2H, d, J = 8.9Hz, H-3, 5).; ¹³ C NMR (125 MHz, CD ₃ OD) δ 170.4 (C-7), 163.5 (C-4), 133.1 (C-2, 6), 123.0 (C-1), 116.2 (C-3, 5).

(3) Luteolin

Luteolin (Compound 3) : Brown powder; ¹ H-NMR (500MHz, DMSO- d ₆ ) δ 7.41 (1H, dd, J= 2.2, 8.2Hz, H-6'), 7.39 (1H, d, J= 2.2Hz, H-2'), 6.89 (1H, d, J= 8.2Hz, H-5'), 6.66 (1H, s, H-6), 6.45 (1H, d, J= 2.0Hz, H-8), 6.19 (1H, d, J = 2.0Hz, H-6).; ¹³ C NMR (125 MHz, DMSO- d ₆ ) δ 181.6 (C-4), 164.1 (C-7), 163.9 (C-2), 161.2 (C-5), 157.4 (C-9), 149.6 (C) -4'), 145.6 (C-3'), 121.5 (C-1'), 119.0 (C-6'), 116.0 (C-5'), 113.3 (C-2'), 103.7 (C-10) ), 103.0 (C-3), 98.8 (C-6), 93.9 (C-8).

(4) Sinnamsan Mountain

Cinnamic acid (Compound 4): White powder; ¹ H-NMR (500MHz, CD ₃ OD) δ 7.67 (1H, d, J= 16.1Hz, H-1'), 7.59 (2H, m, H-2, 6), 7.41(3H, m, H- 3, 4, 5), 6.48(1H, d, J= 16.1Hz, H-2').; ¹³ C NMR (125MHz, CD ₃ OD) δ 171.6 (C-3'), 145.5 (C-1'), 136.2 (C-1), 131.3 (C-4), 130.1 (C-3, 5), 129.5 (C-2, 6), 120.8 (C-2').

(5) Hesperetin

Hesperetin (Compound 5): White powder; ¹ H-NMR (500MHz, CD ₃ OD) δ 6.95 (1H, d, J= 2.1Hz, H-2'), 6.94 (1H, d, J= 8.3Hz, H-5'), 6.91 (1H, dd, J= 8.3, 2.1Hz, H-6'), 5.91 (1H, d, J= 2.2Hz, H-6), 5.89 (1H, d, J= 2.2Hz, H-8), 5.32 (1H , dd, J= 3.0, 12.6Hz, H-2), 3.86 (3H, s, O-CH ₃ ), 3.07 (1H, dd, J= 12.6, 17.2Hz, H-3a), 2.72 (1H, dd , J =3.0, 17.2Hz, H-3b).; ¹³ C NMR (125 MHz, CD ₃ OD) δ 197.8 (C-4), 168.6 (C-5), 165.6 (C-7), 164.9 (C-9), 149.5 (C-3'), 147.9 (C -4'), 133.2 (C-1'), 119.2 (C-2'), 114.7 (C-5'), 112.7 (C-6'), 103.5 (C-10), 97.2 (C-6) , 96.4 (C-8), 80.4 (C-2), 56.6 (O-CH3), 44.2(C-3).

(6) Gallic acid

Gallic acid (Compound 6): Yellow powder; ¹ H-NMR (500 MHz, CD ₃ OD) δ 7.07 (2H, s, H-3, H-5).; ¹³ C NMR (125 MHz, CD ₃ OD) δ 170.5 (COOH), 146.5 (C-3, C-5), 139.7 (C-4), 122.1 (C-1), 110.4 (C-2, C-6) ).

(7) Hesperidin

Hesperidin (Compound 7) : white powder; ¹ H-NMR (500MHz, DMSO- d ₆ ) δ 6.92-6.88 (3H, m, H-2', H-5', H-6'), 6.13-6.11 (2H, m, H-6, H -8), 5.49 (1H, dd, J=12.3, 2.9Hz, H-2), 4.94 (1H, t, J=7.5Hz, H-1''), 4.51 (1H, br s, H-1 ), 3.79 (3H, s, 4'-OCH3), 3.60-3.15 (9H, m, H-2''-H-6'', H-2'''-H-5'''), 2.77 (1H, dd, J=3.2, 17.2Hz, H-3b), 1.08 (3H, d, J=4.6Hz, 6'''-CH3).; ¹³ C NMR (125MHz, DMSO- d ₆ ) δ 197. (C-4), 165.4 (C-7), 163.3 (C-5), 162.8 (C-9), 148.3 (C-4'), 146.6 (C-5'), 131.2 (C-1'), 118.2 (C-2'), 114.3 (C-6'), 112.3 (C-3'), 103.6 (C-10), 100.8 (C- 1'''), 99.7 (C-1''), 96.7 (C-6), 95.9 (C-8), 78.7 (C-2), 76.5 (C-3''), 75.8 (C-5) ''), 73.2 (C-2''), 72.4 (C-4'''), 70.9 (C-3'''), 70.5 (C-2'''), 69.9 (C-4'') ), 68.6 (C-5'''), 66.2 (C-6''), 55.9 (4'-OCH3), 42.2 (C-3), 18.1 (C-6''').

As a result of structural identification through NMR of the Hyeonggae extract before and after bioconversion, it was confirmed that the content of hesperidin decreased and the content of hesperetin increased.

<Experimental Example 2>

HPLC analysis

HPLC analysis was performed on the compounds isolated from the bioconverted extract.

The compounds of Examples 3 to 8 were used as samples.

High performance liquid chromatography (HPLC) analysis was performed using an Agilent 1260 series (Agilent Inc., Palo Alto, CA, USA) instrument equipped with a diode array detect (DAD) and a SHIMADZU HPLC system (Shimadzu Scientific Instruments, Columbia, USA). MD) was carried out by attaching a C18 column (YMC Pack Pro C18, 5㎛, 250mm × 4.6mm). Open column chromatography was performed using silica gel 60 (70-230 mesh ASTM, Merck, Darmstadt, Germany), C ₁₈ reversed-phase silica gel (12 nm, S-150 μm or S-75 μm, YMC), and Sephadex ( Sephadex) LH-20 (Pharmacia) was used.

Figure 2 compares the HPLC results at 210 nm of the mold extract before and after bioconversion. Blue represents the Hyeonggae extract before bioconversion, and red represents the Hyeonggae extract after bioconversion. It can be confirmed that the type or content of ingredients has changed due to bioconversion.

Figure 3 compares the HPLC results at 210 nm of the ethyl acetate fraction of the bioconverted Hyeonggae extract. Compound 1 is caffeic acid, compound 2 is 4-hydroxybenzoic acid, compound 3 is luteolin, compound 4 is cinnamic acid, and compound 5 is hesperetin.

Figure 4 compares the HPLC results at 210 nm of the butanol fraction of the bioconverted Hyeonggae extract. Compound 6 is gallic acid, and compound 7 is hesperidin.

As a result of comparing the HPLC analysis results before and after bioconversion of the Hyeonggae extract, it was confirmed that the content of hesperidin decreased and the content of hesperetin increased.

In the present invention, a large amount of hesperetin can be produced from mold opening in a short time at a simpler and lower cost than existing methods. The present invention is a new method of producing hesperetin through bioconversion and can be used in various fields such as food, medicine, and cosmetics.

In addition, the bioconverted extract containing a large amount of hesperetin obtained according to the present invention, its fractions, and isolated hesperetin can be usefully used in the fields of foods such as functional foods, medicines, and cosmetics.

The method for producing the extract with increased hesperetin content of the present invention described above. The method of producing hesperetin from mold, the extract of mold with increased hesperetin content, the ethyl acetate fraction of mold extract, and their uses are exemplary, and those of ordinary skill in the art to which the present invention pertains will understand. From this, it will be apparent that various modifications and other equivalent embodiments are possible. Therefore, it will be well understood that the present invention is not limited to the forms mentioned in the description of the invention above. The true technical protection scope of the present invention shall be determined by the technical spirit of the claims, and is understood to include all modifications, equivalents and substitutes within the spirit and scope of the present invention defined by the claims of the present invention. It has to be. In addition, terms or words used in this specification and claims should not be construed as limited to their usual or dictionary meanings, and the inventor appropriately defines the concept of terms in order to explain his or her invention in the best way. It should be interpreted with meaning and concept consistent with the technical idea of the present invention based on the principle that it can be done.

Claims (9)

A method for producing an extract with increased hesperetin, comprising the step of converting the hesperidin contained in the extract to hesperetin by bioconverting the extract with Aspergillus kawachi crude enzyme solution. According to paragraph 1,
The bioconversion is,
A method for producing an extract with increased hesperetin, comprising adding Aspergillus kawachi crude enzyme solution to the extract and shaking it for 15 to 40 hours at 20 to 40 ° C. According to paragraph 2,
A method for producing a mold extract with increased hesperetin content, characterized in that the shaking reaction is performed at 50 to 200 rpm. Obtaining a bioconverted Hyeonggeum extract comprising converting the Hesperidin contained in the Hyeonggae extract into hesperetin by bioconverting the Hyeonggea extract with Aspergillus Kawachi crude enzyme solution;
A method of producing hesperetin from the bioconverted mold extract, comprising the step of isolating hesperetin from the bioconverted mold extract. According to clause 4,
The bioconversion is,
A method of producing hesperetin from mold extract, comprising adding Aspergillus Kawachi crude enzyme solution to the mold extract and reacting with shaking at 50 to 200 rpm for 15 to 40 hours at 20 to 40°C. According to clause 4 or 5,
Further comprising the step of fractionating the bioconverted extract with ethyl acetate to obtain an ethyl acetate fraction,
A method for producing hesperetin from mold opening, characterized in that hesperetin is separated from the ethyl acetate fraction. A Hyeonggae extract with increased hesperetin content obtained by bioconverting the Hyeonggae extract with Aspergillus Kawachi crude enzyme solution to convert the hesperidin contained in the Hyeonggae extract into hesperetin. The ethyl acetate fraction of the Hyeonggae extract with increased hesperetin content obtained by bioconverting the Hyeonggae extract with Aspergillus Kawachi crude enzyme solution to convert the hesperidin contained in the Hyeonggae extract into hesperetin and then fractionating it with ethyl acetate. . A food composition comprising the extract of the hesperetin content of claim 7 or the ethyl acetate fraction of the extract of the extract of the claim 8 with the increased hesperetin content.