KR20210101761A - Methods of preparing flavonoid compound using Streptomyces, and the flavonoid compound by them - Google Patents

Abstract

The present invention relates to a method for manufacturing flavonoid compounds using streptomyces and a flavonoid compound manufactured thereby and, more specifically, to method for manufacturing a flavonoid compound having a high absorption rate and efficacy in a human body by reacting Streptomyces coelicolor, which is streptomyces, and chrysin, and to a flavonoid compound prepared therethrough. According to the present invention, the flavonoid compound t can exhibit a higher absorption rate than chrysin, which is one of the flavonoid compounds having a significantly low absorption rate in a human body, and provides excellent physiologically active effects such as antioxidant, anti-cancer, anti-aging, and hormone suppression. The method comprises a step of extracting flavonoid compounds from reactants formed by reacting Streptomyces coelicolor and chrysin.

Description

Method for preparing a flavonoid compound using actinomycetes and a flavonoid compound prepared therethrough {Methods of preparing flavonoid compound using Streptomyces, and the flavonoid compound by them}

The present invention relates to a method for producing a flavonoid compound using an actinomycetes and a flavonoid compound prepared through the method, and more particularly, to the actinomycetes Streptomyces coelicolor and chrysin by reacting with the actinomycetes. It relates to a method for preparing this high flavonoid compound and to a flavonoid compound prepared therethrough.

Flavonoid substance is a generic term for about 4000 yellow-colored compounds widely distributed in the epidermis of fruits, vegetables and herbs, and more than 4000 types of flavonoids have been discovered so far. This flavonoid substance has various effects such as antioxidant, whitening, anticancer, antibacterial, anti-aging, anti-wrinkle, anti-allergy, etc. Among them, the most well-known effect is the antioxidant effect.

Active oxygen is an excessive production of oxygen due to environmental pollution, chemicals, ultraviolet rays, blood circulation disorders, stress, etc., and causes oxidation in the human body. When this happens, the cell membrane, DNA, and all other cellular structures are damaged, and depending on the extent of the damage, the cell loses its function or deteriorates. At the same time, it oxidizes various amino acids in the body, leading to a decrease in protein function. In addition, it can cause mutations or cancer by damaging nucleic acids, causing modifications and liberation of nucleic acid bases, cleavage of bonds, and oxidative degradation of sugars. In addition, physiological functions are lowered, which can cause various diseases and aging.

Flavonoids have antioxidant functions because they inhibit the activation of NF-κB by free radicals, which plays an important role in skin anti-aging. It is expected that flavonoids, which inhibit the function of free radicals, can control not only anti-aging but also endogenous aging. In addition, by inhibiting the activity of tyrosinase, which is known as the most important enzyme in the process of melanin synthesis, it is known to have a whitening effect by inhibiting melanin synthesis, and it is reported that there is almost no toxicity.

Flavonids are divided into chalcone, flavone, flavonol, flavanon, catechin, isoflavone, and isoflavanon according to their structure. Chrysin is a compound of the flavone series, which is contained in large amounts in vegetables, fruits, and wine, and is known to have anticancer effects, and is mainly used as a hormone inhibitor. However, there is a limit to using only chrysin itself because the absorption rate of the human body is significantly reduced.

Accordingly, 'Korea Patent Publication No. 10-2017-0022403' discloses chrysin with increased anti-inflammatory activity by irradiation with radiation and a manufacturing method thereof, but the increased efficacy of chrysin is limited to anti-inflammatory, and through irradiation with radiation There is a problem in that the molecular structure is converted and it is difficult to directly apply it to pharmaceuticals or health functional foods directly related to inflammatory diseases.

In the present invention, a method for preparing a flavonoid compound capable of improving the absorption rate and physiological activity efficacy of chrysin in the human body and its manufacturing based on the research content that the flavonoid compound can increase the physiologically active efficacy and human absorption rate by hydroxylation or O-methylation An object of the present invention is to provide a flavonoid compound prepared through the method.

KR 10-2017-0022403 A KR 10-1915247 B1

In order to solve the above problems, the present invention provides a flavonoid compound having excellent absorption rate and efficacy in the human body by reacting the actinomycetes Streptomyces coelicolor and chrysin and extracting it from a reaction product using actinomycetes, characterized in that An object of the present invention is to provide a method for preparing a flavonoid compound.

Another object of the present invention is to provide a flavonoid compound, which is prepared by a method for preparing a flavonoid compound using actinomycetes.

The present invention in order to solve the above object,

Provided is a method for producing a flavonoid compound using an actinomycete, comprising extracting a flavonoid compound from a reactant formed by reacting an actinomycete, Streptomyces coelicolor, and chrysin.

The Streptomyces coelicolor is characterized in that it is cultured for 3 to 6 days, and the Streptomyces coelicolor and chrysin are reacted for 1 to 6 hours. And, the structure of the flavonoid compound is characterized in that one more hydroxyl group is attached to the structure of the chrysin.

The present invention in order to solve the other object,

It provides a flavonoid compound, characterized in that it is produced by a method for producing a flavonoid compound using an actinomycetes.

The present invention provides a method for producing a flavonoid compound using actinomycetes and a flavonoid compound prepared therefor, thereby exhibiting a higher human absorption rate than chrysin, one of the flavonoid compounds whose absorption rate was significantly lowered by the human body, and has antioxidant, anti-cancer, anti-aging, A physiologically active effect, such as hormone suppression, may exhibit an excellent effect.

1 shows a position to which a functional group added to chrysin can be bound by the manufacturing method according to an embodiment of the present invention.
Figure 2a is an HPLC analysis graph shown by the reaction according to an embodiment of the present invention ( Streptomyces Ceolicolor A3(2)).
Figure 2b is an HPLC analysis graph shown by the reaction according to a comparative example (Streptomyces avermitilis MA4680) of the present invention.
Figure 2c is a graph of HPLC analysis shown by the reaction according to a comparative example (Streptomyces griceus HUT6037) of the present invention.
Figure 2d is a comparative example of the present invention ( Mycobacterium smegmetis ) HPLC analysis graph shown by the reaction according to the.
Figure 2e is an HPLC analysis graph shown by the reaction according to a comparative example (Nocardia farcinica ATCC 3318) of the present invention.
3 is an HPLC analysis graph of chrysin according to a comparative example of the present invention.
Figure 4a is a gas chromatogram graph of the reactant shown by the reaction according to an embodiment of the present invention.
Figure 4b is a mass spectrometry graph of a reactant according to an embodiment and chrysin of a comparative example of the present invention.
Figure 5a is a comparative HPLC analysis graph of the reaction product and apigenin according to an embodiment of the present invention.
Figure 5b is a comparative HPLC analysis graph of the reaction product and baicalein according to an embodiment of the present invention.
Figure 5c is a comparative HPLC analysis graph of a sample prepared by mixing the reaction product and baicalein in a ratio of 1:1 according to an embodiment of the present invention.
6 is a graph showing the reactivity and yield according to the incubation time of actinomycetes according to an embodiment of the present invention.
7 is a graph showing the yield according to the reaction time of actinomycetes and chrysin according to an embodiment of the present invention.

In the present specification, when a part "includes" a certain component, this means that other components may be further included rather than excluding other components unless otherwise stated. And the terminology used in this specification is for describing the embodiments, and is not intended to limit the present invention. In this specification, the singular also includes the plural unless specifically stated in the phrase.

Hereinafter, the present invention will be described in more detail.

According to one aspect, the present invention provides a method for producing a flavonoid compound using an actinomycete, comprising extracting a flavonoid compound from a reactant formed by reacting the actinomycetes Streptomyces coelicolor and chrysin do.

The flavonoid compound prepared according to the method for preparing a flavonoid compound of the present invention may be preferably a compound obtained by hydrating chrysin, and more preferably baicalein. In addition, the structure of the flavonoid compound prepared according to the present invention may have one more hydroxyl group attached to the structure of chrysin, and the position is shown in FIG. 1 .

In the method for producing a flavonoid compound of the present invention, Streptomyces coelicolor can be cultured for 3 to 6 days, and Streptomyces coelicolor and chrysin are used for 1 to 6 hours. can react.

According to another aspect, the present invention provides a flavonoid compound, which is prepared by a method for preparing a flavonoid compound using an actinomycetes.

The description of the flavonoid compound of the present invention is the same as or similar to that described above with respect to the preparation method of the flavonoid compound using the actinomycetes, and thus will be omitted.

The present invention will be described in more detail by the following examples. However, the following examples are intended to illustrate the present invention, and the scope of the present invention is not limited by the examples. It is provided to complete the disclosure of the present invention, and to completely inform those of ordinary skill in the art to which the present invention pertains to the scope of the invention.

<Example>

Example 1 - Preparation of substrate (chrysine)

In the present invention, chrysin (5,7-dihydroxyflavone), a kind of flavone family of flavonoids, was used as an example of a substrate.

Chrysin has a molecular formula of C ₁₅ H ₁₀ O ₄ and a molecular weight of 254.24. Due to its poor solubility in ethanol and methanol, which are organic solvents, it was dissolved in DMSO (Demethylsulfoxide) and then dissolved in methanol. The chemical formula of God is shown.

Example 1 - Culture of microorganisms ( Streptomyces Ceolicolor A3(2))

First, R2YE medium was prepared for culturing Streptomyces Ceolicolor A3(2), a type of actinomycetes. Put 103 g of sucrose, 10 g of glucose, 10.12 g of _{MgCl 2} ㆍ6H _{2 O, 0.25 g of K 2} SO ₄ , 5 g of yeast extract, and 0.1 g of difco casamino acid into a 1 liter bottle, add 800 ml of distilled water, and use a sterilizer. and sterilized at 121 ° C., 1.5 bar for 30 minutes, then TES buffer (5.73%) adjust to pH 7.4 100 ml, KH ₂ PO ₄ (0.5%) 10 ml, CaCl ₂ 2H ₂ O (3.68%) 80 ml, 15 ml of L-proline (20%), 2 ml of trace element solution, and 5 ml of NaOH (1N) were further added to prepare a medium.

Then, the sterilized medium was inoculated with the added bacteria, and cultured by stirring in a shaker incubator at 220 rpm. At this time, the culture temperature was 28℃, and glass beads were used to prevent aggregation of cells during culture.

Example 2 - Preparation of flavonoid compounds

A flavonoid compound was prepared by reacting the chrysin solution, which is the substrate prepared according to Example 1, and the actinomycete culture solution cultured according to Example 2 above.

Chrysin was diluted in methanol to a concentration of 10 mM, and actinomycetes and chrysin were reacted using PBS buffer (pH 7.4). Ethyl acetate was used to extract the compound produced after the reaction for each reaction time, and the reacted reactant and ethyl acetate were mixed in a volume ratio of 1:1 to extract the product for 3 hours. After extraction, ethyl acetate was evaporated using a hood.

Comparative Example 1 - Preparation of substrate (chrysine)

In order to compare and analyze the chrysin prepared according to Example 1 with Example 3 in which the microorganism was reacted, chrysin was prepared in the same manner as in Example 1.

Comparative Example 2 - Microbial-substrate reaction ( Streptomyces avermitilis MA4680)

In order to culture another actinomycetes, Streptomyces avermitilis MA4680, a medium was prepared in the same manner as in Example 2, and then the bacteria were inoculated into the sterilized medium, and cultured by stirring in a shaker incubator at 220 rpm. At this time, the incubation temperature was 28 °C.

After culturing the bacteria, the reaction product was extracted by reacting with chrysin in the same manner as in Example 2.

Comparative Example 3 - Microbial-substrate reaction ( Streptomyces griceus HUT6037)

In order to culture another actinomycetes, Streptomyces griceus HUT6037 , a medium was prepared in the same manner as in Example 2, and then the bacteria were inoculated into the sterilized medium, and cultured by stirring in a shaker incubator at 220 rpm. At this time, the incubation temperature was 28 °C.

After culturing the bacteria, the reaction product was extracted by reacting with chrysin in the same manner as in Example 2.

Comparative Example 4 - Microbial-substrate reaction ( Mycobacterium smegmetis )

In order to culture another actinomycetes, Mycobacterium smegmetis , a medium was prepared in the same manner as in Example 2, and then the bacteria were inoculated into the sterilized medium, and cultured by stirring in a shaker incubator at 220 rpm. At this time, the culture temperature was 28℃, and glass beads were used to prevent aggregation of cells during culture.

After culturing the bacteria, the reaction product was extracted by reacting with chrysin in the same manner as in Example 2.

Comparative Example 5 - Microbial-substrate reaction ( Nocardia farcinica ATCC 3318)

In order to culture another actinomycete type, Nocardia farcinica ATCC 3318, a medium was prepared in the same manner as in Example 2, and then the bacteria were inoculated into the sterilized medium, and cultured by stirring in a shaker incubator at 220 rpm. At this time, the incubation temperature was 37°C.

After culturing the bacteria, the reaction product was extracted by reacting with chrysin in the same manner as in Example 2.

<Experimental example>

Experimental Example 1 - HPLC analysis test

High Performance Liquid Chromatography (HPLC) was used to compare and analyze the types and amounts of substances before and after the reaction between the microorganism and the substrate, chrysin.

After the reaction between actinomycetes and chrysin, 200 μl of methanol was added to the reaction product in which ethyl acetate was vaporized to prepare a sample, and analysis was performed using HPLC of Acme 9000 Younglin Instrument model and Eclipse plus C18 column (Agilent).

As the analysis conditions, Acetonitrile and 2% acetic acid were flowed at a ratio of 47:53 at a rate of 1 ml per minute, and 20 μl of the detector was injected at a wavelength of 254 nm.

Experimental Example 2 - GC-Mass (Gas Chromatography - Mass Spectrometry) analysis test

After the formation of a new material after the reaction was confirmed according to Experimental Example 1, GC-Mass was used to measure the molecular weight of the produced material.

After the reaction between actinomycetes and chrysin, 50 μl of BSTFA (N,O-bis(trimethylsilyl)trifluoroacetamide) was added to the reaction product in which ethyl acetate was vaporized and reacted in a water bath at 70 ° C for 30 minutes. is substituted for the group. A lot of TMS derivatives are used in GC-Mass analysis to make GC-Mass analysis easier.

For GC-Mass, Shimadzu's QP5000 model was used, and RESTEK's Rtx-5 was used for the column. As the mobile phase, helium gas was flowed at a rate of 1 ml per minute, and the temperature of the injector and detector was fixed at 250 °C. As for the temperature change of the column, wait at 60 ℃ for 3 minutes, raise the temperature to 250 ℃ at 30 ℃ per minute, and wait for 10 minutes. Then, the temperature was raised to 275 °C at a rate of 1 °C per minute and then used in the form of waiting for 3 minutes. The analysis conditions of the mass were used in the form of electron impact ionzation, and 1 μl was injected and analyzed.

<Results and evaluation>

Result 1 - Reaction result of actinomycetes and chrysin

The reactants of each actinomycete and chrysin according to Example 2 and Comparative Examples 2 to 5 were comparatively analyzed according to Experimental Example 1, and the results are shown in FIGS. 2A to 2E, and comparison with unreacted chrysin. For this, an HPLC analysis graph of chrysin according to Comparative Example 1 is shown in FIG. 3 .

First, looking at the reaction results according to an embodiment of the present invention, in the graph of FIG. 3 and FIG. 2a showing the results of the reaction product generated after the reaction of 1.5 mM pure substrate chrysin was analyzed, the substrate retention time of about 11 minutes It can be seen that the peak is significantly reduced after 4 hours of reaction, and it can be confirmed that a new material is generated between 5-6 minutes of retention time.

Looking at the reaction results according to one comparative example of the present invention, Comparative Examples 2, 3, and 5 (Figs. 2b, 2c, and 2e) confirmed that a new peak was formed compared with Fig. 3, which is a graph of chrysin, but It was confirmed that there was little reactivity with chrysin because a very weak amount of reactant was formed compared to the reduced amount of chrysin. In the case of Comparative Example 4 (FIG. 2d), no new peak was formed, so it was also confirmed that there was no reactivity with chrysin.

Result 2 - GC-Mass analysis result

The new peak material from Example 2, which showed the best reactivity according to Result 1, was confirmed according to Experimental Example 2 (reaction time = 2 hours), and the results are shown in FIGS. 4A to 4B .

Referring to FIG. 4a showing the gas chromatogram of the reactant, it can be seen that the peak of the newly generated material appears. There are several methods of analyzing chrysin using GC-Mass, but as a result of analyzing various methods to establish the analysis conditions, it was possible to establish a method in which chrysin and newly produced products are best analyzed. .

Through mass spectrometry, the molecular weight of chrysine and the newly formed material could be known, and that the newly formed material was a structure with one more hydroxyl group attached to the chrysine structure could be known from the molecular weight. Referring to FIG. 4b showing this, when mass spectrometry is used by substituting a TMS derivative for chrysine, a molecular weight of 383 is shown, but it can be confirmed that the newly created material has a molecular weight of 471 by increasing 88 from this, which is This is a result interpreted as the formation of one more hydroxyl group in God.

Result 3 - Confirmation of the reaction product of actinomycetes and chrysin

Through Result 2, it was possible to confirm the reaction product in which one more hydroxyl group is present in chrysine, and in order to confirm what kind of material this product is, apigenin and baicalein, which have one more hydroxyl group in the chrysine structure, were used in Experimental Example 1 above. Through comparative analysis, it was compared with the retention time of the product.

All reaction times were 2 hours. The HPLC analysis of apigenin and the product is compared and shown in Figure 5a, the HPLC analysis of baicalein and the product is compared and shown in Figure 5b, and the sample to be made by mixing baicalein and the product in a ratio of 1:1 is analyzed by HPLC. is shown in Figure 5c. As a result, it was confirmed that the reaction product produced according to an embodiment of the present invention is baicalein.

Result 4 - Yield according to incubation time and reaction time of actinomycetes

The reactivity with chrysin according to the incubation time of the actinomycetes according to Example 1 and the yield according to the reaction time of the reaction product (baicalein) according to Example 2 were comparatively analyzed according to Experimental Example 1.

First, after culturing actinomycetes for 3 to 6 days, reacting with chrysin for 6 hours, and calculating the yield of the product, a graph is shown in FIG. All of these turned out to be the best.

In addition, a comparative graph of the yield of the product according to the reaction time of 1 to 6 hours is shown in FIG. 7, and as a result of examining this, a yield of up to 12% was shown in 4 hours of the reaction, and thereafter, the appeared to be decomposed.

Claims (5)

A method for producing a flavonoid compound using an actinomycete, comprising extracting a flavonoid compound from a reaction product formed by reacting an actinomycete, Streptomyces coelicolor, and chrysin.
The method of claim 1,
The Streptomyces coelicolor (Streptomyces coelicolor) is a method for producing a flavonoid compound using actinomycetes, characterized in that the culture for 3 to 6 days.
The method of claim 1,
The Streptomyces coelicolor (Streptomyces coelicolor) and chrysin (chrysin) is a method for producing a flavonoid compound using actinomycetes, characterized in that the reaction for 1 to 6 hours.
2. The method of claim 1
The structure of the flavonoid compound is a method for producing a flavonoid compound using actinomycetes, characterized in that one more hydroxyl group is attached to the structure of the chrysin.
5. A flavonoid compound, characterized in that it is prepared by the method according to any one of claims 1 to 4.

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