KR20210071218A - Purification method for mass production of Mycosporine-like amino acid porphyra334 - Google Patents

Abstract

The present invention relates to a purification method for mass production of mycosporine-like amino acid Porphyra 334, and by applying direct current to a seaweed extract suspension containing the Porphyra 334 to move the Porphyra 334 having a negative charge in the suspension toward an positive electrode, there is an advantage that the Porphyra 334 can be easily separated and purified.

Description

Purification method for mass production of Mycosporine-like amino acid porphyra334

The present invention relates to a purification method for mass production of Porphyra 334, a mycosporin-like amino acid, wherein direct current is applied to a suspension of seaweed extract containing porphyra 334, and Porphyra 334 exhibiting a negative charge in the suspension It relates to a purification method for mass production of Porphyra 334, a mycosporin-like amino acid that can be easily separated and purified by moving the Porphyra 334 to the positive electrode.

Recently, as the issue of chemical ingredients increases, consumers who check all ingredients when purchasing cosmetics or prefer hypoallergenic natural ingredients are increasing. Accordingly, the cosmetics market is also being replaced with natural materials derived from the sea and plants that exclude chemicals, and cosmetics to which they are applied are being launched. In particular, since most of the sunscreen materials are chemical substances, it is most urgent to replace them with natural materials.

Accordingly, gallic acid, caffeic acid, flavonoid pigments, and aromatic compounds such as phenylalanine, tryptophan, and purine, which are the UV defense mechanisms of terrestrial plants, are being studied as natural sunscreen agents. For example, Korean Patent Publication No. 10-1995-0012443 Cosmetics containing herbal extracts with ultraviolet absorbing function have been disclosed, and mycosporine-like amino acids derived from marine plants or seaweed are known to have a defense mechanism against ultraviolet rays and are being actively studied. . For example, Korean Patent Registration No. 10-0969325 mentions that mycosporin-like amino acid (MMA) has a UV blocking effect, and Korean Patent No. 10-1833895 discloses mycosporin-like amino acid for wound healing containing amino acids. It refers to a composition for external application for skin and a method for preparing the same.

As described above, MMA is a defense material of seaweed generated to prevent damage to cells by UV, etc. Representatively, there are porphyra, shinorine, etc., and the UV absorption ability in the 310-360 nm region is Not only is it high, but it has a small molecular weight and high solubility in water. Among them, porphyra334 has anti-inflammatory and cell mobility promoting effects, and is attracting attention as a marine material that can help skin damage and aging caused by UV rays.

However, the Porphyra 334 purified material is not yet commercially sold, so it is necessary to develop advanced technology for mass production extraction and separation and purification.

Republic of Korea Patent Publication No. 10-1995-0012443 Republic of Korea Patent Registration No. 10-0969325 Republic of Korea Patent No. 10-1833895

The present invention is to solve the above problems, and can produce/extract a large amount of Porphyra 334 of mycosporin-like amino acid, and mass-produce Porphyra 334, a mycosporin-like amino acid that can be easily separated/purified To provide a purification method for

In order to achieve the above object,

One embodiment of the present invention is

preparing a suspension with a seaweed extract containing porphyra334, and introducing the suspension into a chamber including an anode and a cathode;

applying a direct current (DC) to the chamber unit to move the negatively charged Porphyra 334 to the anode in the suspension to separate the Porphyra 334-containing liquid and the Porphyra 334-free liquid in the chamber unit; and

The liquid containing the Porphyra 334 moved to the anode is fluidly moved from the chamber unit to the filtering unit, and removing suspended matter in the liquid containing the Porphyra 334; It provides a purification method of Porphyra 334 comprising a.

In the purification method for mass production of Porphyra 334, a mycosporin-like amino acid, according to an embodiment of the present invention, a direct current is applied to a suspension of seaweed extract containing porphyra334, and a negative charge is obtained in the suspension. By moving Porphyra 334 to the anode, primary separation and filtering can increase purification efficiency, and thus Porphyra 334 can be mass-produced.

1 is a flowchart of a purification method for mass production of Porphyra 334 according to an embodiment of the present invention.
2 is a view schematically showing a purification apparatus of a purification method for mass production of Porphyra 334 according to an embodiment of the present invention.
FIG. 3 is a graph showing the detection results at a measurement wavelength of 210 nm by performing component analysis using HPLC for the separation solution separated by applying a DC current in Example 1 and a solution not subjected to a DC current separation process in Comparative Example 1. FIG. ((a) Example 1, (b) Comparative Example 1).
FIG. 4 is a graph showing the detection result at a measurement wavelength of 330 nm by performing component analysis by HPLC for the separated solution separated by applying a DC current in Example 1 and a solution not subjected to a DC current separation process in Comparative Example 1. FIG. ((a) Example 1, (b) Comparative Example 1).
5 is a graph showing the ESI-Mass spectrum of the corresponding peak fraction obtained by injecting the separated solution separated by applying a DC current in Example 1 and a solution that has not been separated by DC current in Comparative Example 1 into HPLC, respectively. ((a) Example 1, (b) Comparative Example 1).

Since the present invention can have various changes and can have various embodiments, specific embodiments are illustrated in the drawings and described in detail in the detailed description.

However, this is not intended to limit the present invention to specific embodiments, and it should be understood to include all modifications, equivalents and substitutes included in the spirit and scope of the present invention. In describing the present invention, if it is determined that a detailed description of a related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

The terms used in the present application are only used to describe specific embodiments, and are not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly dictates otherwise.

In the present invention, terms such as "comprises" or "have" are intended to designate that the features, numbers, steps, operations, components, parts, or combinations thereof described in the specification exist, but one or more other features It should be understood that this does not preclude the possibility of addition or existence of numbers, steps, operations, components, parts, or combinations thereof.

The present invention relates to a purification method for mass production of Porphyra 334, a mycosporin-like amino acid.

In general, mycosporine-like amino acid is a defense substance of seaweeds that is generated to prevent damage to cells by UV, etc. Representatively, there are porphyra and shinorine, 310 In addition to high UV absorption in the -360 nm region, it has a small molecular weight and high solubility in water. Among them, porphyra334 has anti-inflammatory and cell mobility promoting effects, and is attracting attention as a marine material that can help skin damage and aging caused by UV rays.

However, the Porphyra 334 purified material is not yet commercially sold, so it is necessary to develop advanced technology for mass production extraction and separation and purification.

Accordingly, the present invention provides a purification method for mass production of Porphyra 334, a mycosporin-like amino acid that can be mass-produced/extracted, and can be easily separated/purified, of mycosporin-like amino acid Porphyra 334 .

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

One embodiment of the present invention is

preparing a suspension with a seaweed extract containing porphyra334, and introducing the suspension into a chamber including an anode and a cathode (S100);

A step of separating the Porphyra 334 containing liquid and the Porphyra 334-free liquid in the chamber by applying a direct current (DC) to the chamber part to move the negatively charged Porphyra 334 to the anode in the suspension ( S200); and

The liquid containing Porphyra 334 moved to the anode is fluidly moved from the chamber unit to the filtering unit, and removing suspended matter in the liquid containing Porphyra 334 (S300); It provides a purification method of Porphyra 334 comprising (see FIGS. 1 and 2).

The present invention relates to a purification method for mass production of Porphyra 334, a mycosporin-like amino acid, and can be purified on average from 200 mL/day to 1000 L/day. The purification method according to an embodiment of the present invention can increase the purification rate to increase the purification efficiency, and can continuously purify the Porphyra 334, so that the Porphyra 334 can be mass-produced.

Hereinafter, a step (S100) of preparing a suspension with a seaweed extract containing porphyra334 and introducing the suspension into a chamber including an anode and a cathode will be described.

A suspension is prepared from seaweed extract containing porphyra334.

Here, the term "seaweed extract" may mean an extract obtained by drying mycosporin-like amino acids (MAA), pulverizing the dried product, or using various conventionally known extraction methods such as cold water extraction, hot water extraction, and ethanol extraction. have. The extraction method is not particularly limited, and may be, for example, cold extraction, ultrasonic extraction, reflux extraction, or hot water extraction.

Meanwhile, the seaweed extract may be prepared by extraction using water, an organic solvent, or a mixed solvent thereof. The extracted liquid may be used directly or concentrated and/or dried. In case of extraction using an organic solvent, methanol, ethanol, isopropanol, butanol, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, dichloromethane, N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol, or a mixed solvent thereof, an organic solvent, is used, and extraction can be performed at room temperature or heating under conditions in which the active ingredients of seaweed are not destroyed or minimized. Depending on the organic solvent to be extracted, the degree of extraction and loss of the active ingredient of seaweed may differ, so an appropriate organic solvent should be selected and used. For example, in the case of an ethanol extract, preferably 20-50% ethanol extract, in one embodiment, it can be obtained by mixing with 50% ethanol and stirring for 3 days.

In the present invention, the extract may be used after concentration or dilution, and a distillate of the extract may be used.

According to an embodiment of the present invention, after immersing the seaweed powder in a solvent, and then using a method of stirring at a temperature of about 45° C. for about 12 hours, the seaweed extract can be filtered, concentrated and dried to form a powder. For example, after immersing the dried laver in water, it is possible to prepare a laver extract powder.

Then, for purification, about 20 times water is added to the seaweed extract powder and redissolved to prepare a suspension. Then, the suspension is introduced into a chamber including an anode and a cathode.

The chamber part may have any shape as long as it can inject a fluid, and may be, for example, a cylindrical glass material. In addition, the chamber unit may further include a power supply unit in which the anode and the cathode are disposed to be spaced apart from each other, and apply a direct current to the anode and the cathode.

The anode and the cathode may be made of a common material, for example, may be made of copper. In addition, the anode and the cathode may be disposed to surround the inner circumferential surface of the chamber, which is to easily separate the Popira 334 moved to the anode.

Next, a direct current (DC) is applied to the chamber to move the negatively charged Porphyra 334 to the anode in the suspension to separate the Porphyra 334-containing liquid and the Porphyra 334-free liquid in the chamber unit. It includes a step (S200) of doing.

Porphyra 334, a representative material of mycosporin-like amino acid (MAA) to be purified in the present invention, is represented by the following Chemical Formula 1, and has a negative charge (-) in an aqueous solution. At this time, when a direct current is applied to the chamber part, the popira 334 having a negative charge in the suspension may be moved to the anode. In this case, the DC current may apply an average voltage of 100 V.

That is, since the Porphyra 334 has a negative charge in an aqueous solution, it can move toward the anode when a current is applied thereto.

[Formula 1]

At this time, by installing a blocking member in the chamber part, it is possible to separate the liquid containing Popira 334 and the liquid that does not contain Popira 334.

Specifically, the blocking member is installed in the chamber portion and may be detachable between the positive electrode and the negative electrode, and may be a blocking plate for separating the space. In addition, the blocking plate may be a non-conductor.

Next, the liquid containing Porphyra 334 that has moved to the anode is fluidly moved from the chamber unit to the filtering unit, and may include a step (S300) of removing suspended matter in the liquid containing Porphyra 334.

Specifically, the filter unit may be a conventional filter for removing floating matter, and any type of filter that can easily separate the floating matter in the liquid containing Porphyra 334 is irrelevant. For example, the filter may have an average transmittance of 0.2 to 10 μL in a pore size.

And, it includes the step of purifying the liquid containing Porphyra 334 through the filtering step by high performance liquid chromatography (High Performance Liquid Chromatography, HPLC).

Furthermore, the content and components of the purified Porphyra 334 may be analyzed using the high performance liquid chromatography (HPLC).

As an example, using high-pressure reverse-phase liquid chromatography (HPLC) (Shimadzu-LC20A, Seattle, WA, USA) with diode array detection (DAD)-SPD M20A detector (Quantum Northwest, Seattle, WA, USA) can be analyzed.

In this case, the column used as the stationary phase of the high-performance liquid chromatography (HPLC) may be any conventionally available one. Preferably, Octadecylsilanized silica gel (3 μm), Acclaim surfactant (4.6 mm id X 150 mm L., 5 μm, Dionex) or Capcellpak C18 (4.6 mm id X 150 mm L., 5 μm, Shiseido) can be used. , more preferably Octadecylsilanized silica gel can be used.

In the purification method for mass production of Porphyra 334, a mycosporin-like amino acid, according to an embodiment of the present invention, a direct current is applied to a suspension of seaweed extract containing porphyra334, and a negative charge is obtained in the suspension. By moving Porphyra 334 to the anode, primary separation and filtering can increase purification efficiency, and thus Porphyra 334 can be mass-produced.

Porphyra 334 produced by the above-described purification method can be concentrated about 4 times compared to the prior art, and the same effect as when performing 1 cycle of Porphyra 334 by HPLC can be obtained through the example of performing 4 cycles of the prior art. can be checked

Hereinafter, the present invention will be described in more detail by way of Examples and Experimental Examples.

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

<Production Example>

Preparation Example 1. Preparation of seaweed extract powder

After weighing 1 kg of dried laver, 40 times water is added and stirred. Then, it is extracted while stirring at 45° C. for 12 hours.

After filtration, the extract is concentrated and dried to form a powder.

<Example>

Separation of porphyra 334 through direct current (DC)

For purification, add 20 times water to the dried laver extract powder and re-dissolve. Then, the extraction suspension is administered to a chamber manufactured so that a direct current flows.

Then, a direct current (DC) of 100V is applied to separate the Popira 334. Specifically, since Porphyra 334 has a negative charge (-) in the aqueous solution, when a current flows in the aqueous solution, it moves to the + electrode.

Direct current is applied for about 30 minutes, and a blocking plate is installed between the + and - electrodes in the chamber to separate both sides.

<Comparative example>

Comparative Example 1.

In the same manner as in Example 1, 20 times water was added to the seaweed extract powder and re-dissolved.

<Experimental example>

Experimental Example 1. HPLC component analysis of Porphyra 334-1

The separated solution separated by applying a DC current in Example 1 and the solution that was not subjected to the process of separating by DC current in Comparative Example 1 were subjected to HPLC analysis under the following analysis conditions, the areas of all peaks were obtained, and then the main peak Purity was confirmed by the area ratio of

Specific analysis conditions are as follows.

division Analysis conditions HPLC instrument type Waters 1525 Pump Column 5㎛ octadecylsilanized silica gel solvent conditions - Solvent A: Water (containing 0.1 % TFA (Trifluoro acetic acid)) - Solvent B: Acetonitrile (containing 0.1 % TFA (Trifluoro acetic acid)) measurement wavelength 210 nm (UV lamp) flow rate 1 mL/min

As a result of the component analysis of the liquid separated by applying a DC current in Example 1 and the liquid separated by DC current in Comparative Example 1, the HPLC analysis peak results as shown in FIG. 3 and Table 2 were obtained.

Peak name RT Area % Area Height Example 1 Porphyra334_210nm 11.692 9118826 54.15 446297 Comparative Example 1 Porphyra334_210nm 12.047 2106210 12.61 108062

Referring to FIG. 3 and Table 2, the peak area of Porphyra 334 purified in Example 1 was 9118826, and it was measured to be 54.15% of the total peak area. On the other hand, Porphyra 334 purified through Comparative Example 1 had a peak area of 2106210, which was measured to be 12.61% of the total peak area.

This indicates that the peak area of Porphyra 334 in the solution containing Porphyra 334 separated by applying DC increases by about 4.3 times.

Experimental Example 2. HPLC component analysis of Porphyra 334-2

Except that the measurement wavelength was 330 nm, the separated solution separated by applying a DC current in Example 1 in the same manner as in Experimental Example 1 and the solution that was not separated by DC current in Comparative Example 1 were analyzed by HPLC. Analysis was performed.

And, the results are shown in Figure 4 and Table 3.

Peak name RT Area % Area Height Example 1 Porphyra334_330nm 11.707 72211044 98.02 2607874 Comparative Example 1 Porphyra334_330nm 12.030 19505155 79.81 995204

Referring to FIG. 4 and Table 3, the peak area of Porphyra 334 purified in Example 1 was 72211044, and it was measured to be 98.02% of the total peak area. On the other hand, Porphyra 334 purified through Comparative Example 1 had a peak area of 19505155, which was measured to be 79.81% of the total peak area.

This indicates that the peak area of Porphyra 334 in the solution containing Porphyra 334 separated by applying DC increases by about 3.7 times.

Experimental Example 3. ESI-Mass spectrum molecular weight measurement of Porphyra 334

When the separated solution separated by applying a DC current in Example 1 was analyzed by HPLC, the separated solution of Example 1 and the extract of Comparative Example 1 were respectively injected into HPLC to confirm whether the corresponding peak was Porphyra 334. After fractionation of the peak portion, mass spectrometry (ESI-Mass spectrum) was measured in positive mode, and the results are shown in FIG. 5 .

5 is a graph showing the ESI-Mass spectrum of the corresponding peak fraction obtained by injecting the separated solution separated by applying a DC current in Example 1 and a solution that has not been separated by DC current in Comparative Example 1 into HPLC, respectively. ((a) Example 1, (b) Comparative Example 1).

Referring to FIG. 5 , in both Example 1 and Comparative Example 1, a molecular weight peak of [M+H] + 347 was confirmed for Porphyra 334, and it was confirmed that the corresponding peak was Porphyra 334.

Claims (8)

preparing a suspension with a seaweed extract containing porphyra334, and introducing the suspension into a chamber including an anode and a cathode;
applying a direct current (DC) to the chamber unit to move the negatively charged Porphyra 334 to the anode in the suspension to separate the Porphyra 334-containing liquid and the Porphyra 334-free liquid in the chamber unit; and
The liquid containing Porphyra 334 moved to the anode is fluidly moved from the chamber unit to the filtering unit, and removing suspended matter in the liquid containing Porphyra 334; Purification method of Porphyra 334 comprising a.
According to claim 1,
Purifying the liquid containing Porphyra 334 through the filtering step by High Performance Liquid Chromatography (HPLC); Purification method of Porphyra 334 further comprising a.
3. The method of claim 2,
A method for purifying Porphyra 334, characterized in that the content and components of the purified Porphyra 334 are analyzed using High Performance Liquid Chromatography (HPLC).
According to claim 1,
The method for purifying Porphyra 334, characterized in that the volume of the seaweed extract suspension containing the purified Porphyra 334 is an average of 200 mL/day to 1000 L/day.
According to claim 1,
Seaweeds containing porphyra334 are: Cyanophyta, Cryptophyta, Chrysophyta, Euglenophyta, Bacillariophyta, Phaeophyta, Red algae Porphyra 334 purification method, characterized in that at least one selected from the group consisting of green algae (Chlorophyta) and axle algae (Charophyta).
According to claim 1,
In the chamber unit, the anode and the cathode are disposed to be spaced apart from each other,
Porphyra 334 purification method, characterized in that it further comprises a power supply for applying a direct current to the anode and the cathode.
According to claim 1,
Porphyra 334 purification method, characterized in that the anode and the cathode are disposed to surround the inner peripheral surface of the chamber.
According to claim 1,
In the step of separating the liquid containing Porphyra 334 and the liquid containing no Porphyra 334 in the chamber part, Porphyra 334 tablet, characterized in that a blocking member is installed to be detachably between the anode and the cathode in the chamber part Way.

Images