METHOD FOR PREVENTING OXIDATION OF POLYPHENOL BY MEANS OF APTAMER, MATERIAL THEREOF, AND USE THEREOF

Abstract

The present invention relates to a method for preventing oxidation of polyphenols, materials thereof, and uses thereof, and more particularly, it relates to a method of preventing oxidation of catechins by treating catechins with aptamers, aptamers having such activity with preventing oxidation of catechins, and it relates to its application to various fields such as cosmetics, foods, and pharmaceuticals using the aptamer. The aptamer of the present invention has an antioxidant effect of catechins, so the aptamer of the present invention can be applied in various fields such as cosmetics, foods, and pharmaceuticals that require inhibition of oxidation of catechins.

Claims

1. A method of preventing oxidation of catechins by treating aptamers with catechins.

2. The method of claim 1, wherein the catechin is one of the material selected from the group consisting of catechin (C), epicatechin (EC), gallocatechin (GC), epigallocatechin (EGC), catechin gallate (CG), epicatechin gallate (ECG), gallocatechin gal. Rate (GCG) and epigallocatechin gallate (EGCG).

3. The method of claim 1, wherein the aptamer consists of one nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NOs: 1 to 9.

4. An aptamer that prevents oxidation of catechins.

5. The aptamer of claim 4, wherein the catechin is one of the material selected from the group consisting of catechin (C), epicatechin (EC), gallocatechin (GC), epigallocatechin (EGC), catechin gallate (CG), epicatechin gallate (ECG), gallocatechin gal. Rate (GCG) and epigallocatechin gallate (EGCG).

6. The aptamer of claim 4, wherein the aptamer consists of one nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NOs: 1 to 9.

7. A cosmetic composition comprising the aptamer of claim 4 as an active ingredient.

8. The cosmetic composition according to claim 7, wherein the aptamer comprises one nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NOs: 1 to 9.

9. A food composition comprising the aptamer of claim 4 as an active ingredient.

10. The food composition according to claim 9, wherein the aptamer consists of one nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NOs: 1 to 9.

11. The food composition according to claim 9, wherein the food is a food selected from the group consisting of beverages, confectionery, candy, dairy products, gum, paste, bread, and ice cream.

12. A pharmaceutical composition comprising the aptamer of claim 4 as an active ingredient.

13. The pharmaceutical composition according to claim 12, wherein the aptamer consists of one nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NOs: 1 to 9.

Description

DESCRIPTION OF DRAWINGS

[0044] FIG. 1 is a graph showing the effect of delaying EGCG oxidation in aptamer K1 (SEQ ID NO: 1) to K9 (SEQ ID NO: 9) of the present invention. Nine types of aptamer K from aptamer K1 to aptamer K9 that bind to EGCG were prepared in the same manner as described above. After 20 μM aptamer K1 to K9 and 500 μM EGCG were reacted for 30 minutes, respectively, WST-1 assay was performed. The degree of delaying EGCG oxidation of each aptamer K was compared,

[0045] FIG. 2 is a graph of the dissociation constant (K.sub.D) for EGCG of the aptamer of the present invention.

[0046] FIGS. 3 and 4 are graphs showing the effect of the aptamers K4 and K5 of the present invention on EGCG oxidation delay, as can be seen from the figure, in the case of EGCG, it is rapidly oxidized and reduced CYST-1, but in the case of EGCG aptamer K4 or 1(5, it was confirmed that the oxidation of EGCG was delayed depending on the concentration and the reduction of WST-1 was delayed.

[0047] FIG. 5 is a graph comparing the effects of aptamers K4 and K5 of the present invention on EGCG oxidation delay.

MODE FOR INVENTION

[0048] Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are described with the intention of illustrating the present invention, and the scope of the present invention is not to be construed as being limited by the following examples.

Example 1: DNA Aptamer Selection and Sequence Analysis

[0049] Egcg Selex:

[0050] Eight rounds of SELEX against EGCG were performed using a DNA library consisting of −10.sup.15 unique oligonucleotides.

TABLE-US-00001 TABLE 1 No. Name Sequence (5′.fwdarw.3′) of mer SEQ ID CGAGTGAACGACGAGGCGCGTCACACT 27 NO: 1 SEQ ID GCACGGCACAACGGGCGCGCCTCCATGCTGTTC 33 NO: 2 SEQ ID GACCAACGGAAGCGCGGCACCACAACGGT 29 NO: 3 SEQ ID CGACGACAGGAGGTGCGGCCCCGGCAGACC 30 NO: 4 SEQ ID ACGCATGCCGGGCGCGCTCCCTGTCGTCC 29 NO: 5 SEQ ID CGAGTCAGTGCGAGGCGCTCCCCTGTCGGT 30 NO: 6 SEQ ID GGTCTGCCGGGGCCGCACCTCCTGTCGTCG 30 NO: 7 SEQ ID CGAACAGCATGGAGGCGCGCCCGTTGTGCC 30 NO: 8 SEQ ID GGCACGCAGTGTGACGCGCCTCGTCGTTCA 30 NO: 9

[0051] Table 1 is a table showing the aptamer sequence specifically binding with EGCG.

Example 2: Effect of the Aptamer of the Present Invention on EGCG Oxidation Delay

[0052] The effect of the aptamers K1 to K9 of the present invention on EGCG oxidation delay was performed in the same manner as in Example 2, and the results are shown in FIG. 1.

[0053] As can be seen from FIG. 1, when K1 to K9 aptamer was treated, a significant effect of delaying EGCG oxidation of about 30-40% was observed.

[0054] The aptamer (aptamers of SEQ ID NOs: 1 to 9) of the present invention reacting specifically with the EGCG obtained through the SELEX was dissolved in a folding buffer (1 mM MgCl2 in PBS) and then dissolved at 95° C. After boiling for a minute, the temperature was gradually lowered to room temperature to form a tertiary structure.

[0055] After reacting 500 uM EGCG and one of the prepared 20 μM aptamers K1 to K9 and for 30 minutes, WST-1 assay was performed. The degree of EGCG oxidation delay of each aptamer K was compared.

[0056] As can be seen from FIG. 1, the group treated with one of the aptamers of K1 to K9 exhibited about 20% to 40% of the EGCG oxidation delay effect compared to the control without treatment.

Example 3: Effect of Aptamer K4 on EGCG Oxidation Delay

[0057] Aptamer K4, which specifically reacts with EGCG obtained through SELEX, was dissolved in folding buffer (1 mM MgCl2 in PBS), boiled at 95° C. for 5 minutes, and then gradually lowered to room temperature to form a tertiary structure.

[0058] 500 mM EGCG and 5, 10, and 20 uM of the prepared aptamer K4 were reacted at room temperature for 30 minutes, respectively, and then WST-1 assay was performed. Prepared sample and WST-1(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was reacted to measure the formazan produced and with the plate reader, 440 nm absorbance was measured for 16 hours.

[0059] In the case of EGCG, it was rapidly oxidized and reduced WST-1, but in the case of EGCG+aptamer K4, the oxidation of EGCG was delayed (about 33% at 20 uM) depending on the concentration of aptamer K4, indicating that the reduction of WST-1 was delayed (FIG. 3).

Example 4: Effect of Aptamer K5 on EGCG Oxidation Delay

[0060] The aptamer K5 (aptamer of SEQ ID NO: 5) that specifically reacts with EGCG obtained through the SELEX was prepared in the same manner as described above and was tested for the effect of delaying EGCG oxidation.

[0061] In the case of EGCG, it was rapidly oxidized and reduced WST-1, but in the case of EGCG+aptamer K5, the oxidation of EGCG was delayed (about 33% at 20 uM) depending on the concentration of aptamer K5, indicating that the reduction of WST-1 was delayed (FIG. 4).

Example 5: Comparison of the Effect of Aptamers K4 and K5 on EGCG Oxidation Delay

[0062] Aptamers K4 and K5 binding to the EGCG were prepared in the same manner as described above. 20 μM aptamer K4, or aptamer K5, and 500 μM EGCG were reacted for 30 minutes, respectively, followed by WST-1 assay. It was confirmed that the EGCG oxidation delay effect of 20 μM aptamer K4 and aptamer K5 was similar (FIG. 5).