KR102265981B1 - Natural material for enhancing stability vitamin from vitamin tree extract and its manufacturing method - Google Patents

Abstract

The present invention relates to a peptide capable of forming a complex with vitamin C, and can increase the stability of vitamin C by blocking exposure of vitamin C to an external oxidizing environment.

Description

Natural material for enhancing stability vitamin from vitamin tree extract and its manufacturing method

The present invention relates to a peptide capable of forming a complex with vitamin C, and more particularly, to a peptide capable of increasing the stability of vitamin C by blocking the decrease in activity due to exposure of vitamin C to an external oxidizing environment. .

Vitamin C is contained in excess in the vitamin tree, and is a nutrient also known as ascorbic acid. It is known to reduce the risk of chronic diseases and prevent aging by exhibiting powerful antioxidant properties. However, it is also used as a treatment for megadose therapy. The active form of vitamin C is ascorbic acid, and when it is oxidized by external stress such as oxygen or UV, it is converted to the oxidized form, dehydroascorbic acid, and this form is inactive and does not exhibit antioxidant properties.

Therefore, vitamin C is easily inactivated by contact with light and oxygen, and there is a difficulty in thoroughly managing the manufacturing environment when manufacturing products such as medicines, health functional foods, and cosmetics containing vitamin C as an active ingredient. In addition, depending on the storage condition of the product, the activity of vitamin C is easily lost, and it is easily inactivated in the body, so there is a problem that an excessive amount must be consumed to obtain efficacy.

On the other hand, fibroin (fibroin) is a kind of silk protein, a human-friendly natural material, after refining it is used a lot as a cosmetic material by adjusting the size of various molecular weights using acids, neutral salts, enzymes, etc. However, fibroin has not been studied in relation to vitamin C.

One object of the present invention is to protect vitamin C in an active state and to increase stability.

In order to achieve the above object, one aspect provides a peptide comprising the amino acid sequence of the general formula (1):

[General formula 1]

X ₁ -AAX ₂ -X ₃ .

Another aspect provides a complex comprising the peptide and vitamin C.

Another aspect provides an antioxidant composition comprising a complex of the peptide and vitamin C.

Another aspect provides a health functional food using the complex of the peptide and vitamin C.

Another aspect provides a food additive comprising a complex of the peptide and vitamin C.

Another aspect provides a cosmetic composition comprising a complex of the peptide and vitamin C.

One aspect provides a peptide comprising the amino acid sequence of Formula 1 below.

[General formula 1]

X ₁ -AAX ₂ -X ₃

In the above formula, X ₁ , X ₂ , and X ₃ are each independently selected from the group consisting of serine (S), threonine (T), asparagine (N), and glutamine (Q). It may be any one amino acid.

The present inventors observed the active protective effect of vitamin C when vitamin C was mixed with the light chain protein of fibroin, and screened various sites that can successfully protect the active form of vitamin C in the fibroin protein. As a result, it was found that the activity-protecting effect of vitamin C was most excellent in the sequence near the C-term (ID: 251 to 262 amino acid sequence of NP_001037488.1), and the peptide structure of the present invention was found.

The amino acid sequence of Formula 1 is the amino acid sequence of SEQ ID NO: 6. In the amino acid sequence of Formula 1, two alanine sequences, AA, are conserved sequences, and when polar amino acids with similar properties are arranged in the amino acid sequence around them, a complex having a structure similar to vitamin C is formed, and a vitamin with a similar degree It was demonstrated in the description of the present invention that the active-type protective effect of C is expressed. The polar amino acid may be serine, threonine, asparagine, or glutamine, and since these amino acids have similar chemical properties, a similar effect can be expected even when randomly arranged within the sequence defined above.

In one embodiment, the peptide may further include 0 to 5 amino acids each independently at the N-terminus and the C-terminus in the peptide of Formula 1 . Amino acids that may be further included may be polar, non-polar, acidic or basic amino acids, preferably arginine (R), tyrosine (Tyrosine, Y), isoleucine (Isoleucine, I), alanine (Alanine, A), It may be selected from the group consisting of valine (V), and histidine (H), but is not limited thereto.

In one embodiment, the peptide further comprises an amino acid sequence of RYIA (Arg-Tyr-Ile-Ala) at the N-terminus in the peptide of Formula 1, and VHV (Val-His-Val) at the C-terminus. It may be composed of an amino acid sequence further comprising a sequence. More specifically, the peptide may be composed of the amino acid sequence of SEQ ID NO: 7.

In one embodiment, the peptide may be composed of an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5.

The peptide according to one embodiment may have a molecular weight of 1200 to 1500 g/mol, 1250 to 1450 g/mol, 1250 to 1400 g/mol, or 1300 to 1360 g/mol. An appropriate molecular weight may be important for the peptide to be properly absorbed into the body, to form a stable dissolved state without precipitation in a solvent, and not to induce an unnecessary immune response in the body.

One aspect provides a complex comprising the peptide and vitamin C.

The complex has a structure in which hydrophobic amino acids form a loop, and vitamin C is located in a helix structure surrounded by acidic amino acids, as shown in FIG. 1 . Since vitamin C is located in the helix structure, it is possible to maintain the active structure without exposure to oxidizing conditions such as light or oxygen. _{In the peptide sequence, the X 1} -AAX ₂ -X ₃ portion of Formula 1, which is a sequence formed by two alanine and surrounding polar amino acids, plays an important role as a vitamin C-protecting site.

In one embodiment, the peptide and vitamin C may form a complex in a molar ratio of 1:1. In the complex, the peptide and vitamin C may be electrostatically bound. Here, the peptide may refer to one of the above-defined peptides, and may refer to a combination of one or more selected from among the above-defined peptides.

Another aspect provides an antioxidant composition comprising a complex composed of a peptide and vitamin C according to an embodiment.

The term "antioxidant" is to block exposure of cells to oxidative stress, and the composition for antioxidants may be used to treat or prevent diseases caused by oxidative stress, such as aging, cancer, metabolic diseases, immune system diseases, and degenerative diseases. .

The antioxidant composition may be a pharmaceutical composition. When the composition is used as a pharmaceutical composition, the composition may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is, for example, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone. , cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, but not limited thereto.

In addition, the composition may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like.

The composition can be used orally, parenterally, or as a skin patch, and in the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, mucosal administration and eye drop administration, etc., A suitable dosage may be appropriately adjusted under the guidance of a specialist in consideration of factors such as formulation, administration mode, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity of the patient.

The composition may be provided in various dosage forms depending on the purpose of administration, for example, in the form of solutions, suspensions, syrups or emulsions in oil or aqueous medium, or extracts, powders, powders, granules, tablets or capsules, It is not limited thereto.

Another aspect provides a health functional food comprising a complex composed of a peptide and vitamin C according to an embodiment.

Examples of the health functional food include, but are not limited to, nutritional supplements, nutritional supplements, pharmaceutical food, health food, nutraceutical, designer food, food additives, and the like.

Another aspect provides a food additive comprising a complex composed of a peptide and vitamin C according to an embodiment.

Since vitamin C has antioxidant properties, it can be used as an antioxidant or preservative in food, and because it has a sour taste, it can be used as an acidifier. Thus, the complex can be used as a food additive such as an antioxidant, a preservative, or an acidifying agent. Such foods include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages and vitamin complexes, etc. but is not limited thereto.

Another aspect provides a cosmetic composition comprising a complex composed of a peptide and vitamin C according to an embodiment.

The cosmetic composition may be in the form of skin, serum, lotion, emulsion, cream, essence, lotion, foundation, pack, soap, surfactant-containing cleansing, spray, or powder, but is not limited thereto.

The cosmetic composition may further include an appropriate carrier to enhance the skin absorption rate and improve the feeling of use. For example, when the cosmetic composition is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide, etc. may further include, but is not limited thereto.

When the cosmetic composition is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, additional chlorofluorohydrocarbon, propane/ propellants such as butane or dimethyl ether, but are not limited thereto.

When the cosmetic composition is a solution or emulsion, a solvent, solubilizer or emulsifier may be used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylglycol oil, glycerol fatty esters, fatty acid esters of polyethylene glycol or sorbitan, but not limited thereto.

When the cosmetic composition is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline Cellulose, aluminum metahydroxide, bentonite, agar or tracanth may be used, but is not limited thereto.

When the cosmetic composition is for cleansing, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, and alkylami Dobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative or ethoxylated glycerol fatty acid ester may be used, but is not limited thereto.

In the pharmaceutical composition, health functional food and cosmetic composition comprising the complex, the active ingredient is vitamin C, and the peptide may serve as a carrier that does not have a physiologically effective effect. Although the peptide liberates vitamin C and exists alone in the body, it is not cytotoxic as a part of fibroin, and can be easily decomposed and excreted by the action of an enzyme in the body.

Pharmaceutical compositions, health functional foods and cosmetic compositions including the complex may not contain additional antioxidants or preservatives for protecting vitamin C. In addition, the preservation power of the active form of vitamin C is increased, so that the effective period of the composition can be increased by 2 times or more, more specifically by 4 times compared to the case where vitamin C alone is included without the complex. In addition, by preventing the loss of the active form of vitamin C during manufacturing, it is possible to achieve a sufficient effective content as ascorbic acid even if the composition is prepared using a small amount of vitamin C, and has excellent effects and saves materials. It can be economical

Another aspect provides a method for preparing a peptide-vitamin C complex, comprising mixing the peptide and vitamin C according to an embodiment.

The step of mixing the peptide and vitamin C may be to prepare an aqueous solution by mixing with an aqueous solvent such as water. The aqueous solution may mean that the peptide and vitamin C are dissolved in a solution such as water, saline, or a buffer solution. The peptide and vitamin C may be mixed in a molar ratio of preferably 1:1, and may be reacted by depositing at 10 to 15° C. for 15 to 45 minutes or stirring.

The preparation method may further include a step of drying in order to increase the concentration of the peptide-vitamin C complex. The drying step includes, but is not limited to, hot air drying, freeze drying, and the like. When the drying is performed by freeze-drying, it may be more preferable because the risk of denaturing the peptide is small.

The peptide according to an aspect forms a complex with vitamin C to enhance the stability of vitamin C to light or oxygen, so that the activity of vitamin C can be maintained for a long time. Therefore, by using the peptide, it is possible to enhance the activity of vitamin C in the body and improve storage stability.

1 is an image showing a three-dimensional structure in which a peptide according to an aspect forms a complex with vitamin C.
2 is an image showing the three-dimensional structure of the five peptides of Table 1.
3 is a graph showing the protective effect of vitamin C active type against UV of the peptide of the present invention.
Figure 4 is a graph showing the antioxidant activity protective effect of vitamin C against UV of the peptide of the present invention.

Hereinafter, examples and the like will be described in detail to help the understanding of the present invention. However, the embodiments according to the present invention may be modified in various other forms, and the scope of the present invention should not be construed as being limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

[Preparation Example 1] Preparation of vitamin C stabilizing peptide and structural analysis of peptide-vitamin C complex

Peptides according to one embodiment were synthesized through de novo synthesis with the amino acid sequence shown in Table 1 below, and had a purity of 98% or more, and after freeze-drying, diluted with water as needed and used.

number order Molecular Weight (g/mol) DL-1 (SEQ ID NO: 1) RYIAQAASQVHV 1342 DL-2 (SEQ ID NO: 2) RYIASAAQSVHV 1301 DL-3 (SEQ ID NO: 3) RYIANAANQVHV 1355 DL-4 (SEQ ID NO: 4) RYIATAATQVHV 1329 DL-5 (SEQ ID NO: 5) RYIAQAANTVHV 1342

Each of the prepared peptides was mixed with vitamin C in a molar ratio of 1:1 as an aqueous solution, and reacted at 10-15° C. for 30 minutes to form a complex. Then, the three-dimensional structure of the complex was predicted using PepFold3.5 software, and the images are shown in FIGS. 1 and 2. As a result, the peptide of the present invention has a structure that wraps vitamin C inside, so that vitamin C is removed from the outside. A structure not exposed to the environment was formed, and it was confirmed that all peptides had a similar structure.

[Test Example 1] Confirmation of vitamin C stabilization effect of peptides

In order to confirm the vitamin C stabilizing effect of the peptide prepared in Preparation Example 1, the DL-1 peptide of Table 1 was mixed with 1M vitamin C to form a complex as in Preparation Example 1, but the concentration of the peptide was 0.1 M to Various additions were made at 10 M. Then, the uncomplexed vitamin C and the prepared complex were exposed to UV-a wavelength in a clean bench for experiments, respectively, and ascorbic acid (AA), an active form of vitamin C, and a form inactivated by UV Phosphorus dehydroascorbic acid (DHA) was quantified by HPLC, and calculated as relative activity (%) with respect to the initial activity. UV was irradiated for 8 hours, and samples were taken every 2 hours. The results are shown in FIG. 3 and Table 2.

0 h 2 h 4 h 6 h 8 h relative activity
(%) AA (1M) Vehicle 100 ± 7.42 80 ± 4.32 20 ± 7.32 8 ± 1.8 0 ± 0 DL-1
+
AA (1M) 0.1 M 100 ± 4.34 88 ± 6.2 34 ± 6.4 18 ± 1.23 7 ± 1.32 1M 100 ± 10.82 95 ± 3.2 92 ± 5.82 90 ± 5.76 88 ± 3.42 10M 100 ± 4.16 96 ± 2.34 90 ± 4.87 90 ± 8.32 90 ± 8.32

When vitamin C was mixed with the peptide in a molar ratio of 1:0.1, it exhibited an activity almost similar to that of uncomplexed vitamin C, but when mixed in a 1:1 ratio, vitamin C was hardly oxidized for 8 hours of UV irradiation. On the other hand, when vitamin C and peptide were mixed in a molar ratio of 1:1, they were already saturated, and even when the peptide was mixed in a molar ratio of 1:10, there was no difference in relative activity. Therefore, it can be seen that vitamin C and peptide form a complex in a molar ratio of 1:1.

[Test Example 2] Confirmation of vitamin C activity protective effect of peptides 1

In order to confirm whether the peptide of the present invention can protect the antioxidant activity of vitamin C from loss while the active form is changed to the inactive DHA form after UV-a irradiation, DPPH analysis was performed. As a control group, vitamin C without UV-a irradiation was used. Specifically, 1M of AA type vitamin C was mixed with 0.1 to 10 M of DL-1 peptide, irradiated with UV-a for 4 hours, treated with DDPH reagent, and incubated at 37°C to measure the degree of color change of the reagent. was measured and converted into relative activity. The results are shown in Figure 4 and Table 3.

AA(1M) + DL-1 control
(untreated group) DL-1 concentration 0 0.1 One 10 AA (1M) Relative activity (%) 17 23 98 84 100

As in Test Example 1, when vitamin C was mixed with the peptide in a molar ratio of 1:0.1, the protective effect on the activity of vitamin C was not observed, but when vitamin C and the peptide were mixed in a molar ratio of 1:1 or more, it was close to 100% It was confirmed that vitamin C exhibits a protective effect on antioxidant activity.

[Test Example 3] Confirmation of vitamin C activity protective effect of peptides 2

It was confirmed whether the protective effect on vitamin C activity of DL-1 confirmed in Test Example 2 was reproduced in peptides having homology.

Specifically, five polypeptides having the amino acid sequence of Table 1 were mixed with vitamin C in a molar ratio of 1:1, and a complex was formed as in Test Example 1, irradiated with UV-a as in Test Example 2, followed by DPPH analysis to perform antioxidant Activity was confirmed. The results are shown in Table 4.

AA sole DL-1 DL-2 DL-3 DL-4 DL-5 Relative activity
(%) 20 ± 4.73 90 ± 7.42 80 ± 4.32 84 ± 7.32 88 ± 1.8 78 ± 5.08

As a result, as confirmed in Test Example 2, when vitamin C forms a complex with the polypeptide of the present invention, it was confirmed that the antioxidant activity was maintained even when irradiated with UV-a.

SEQUENCE LISTING <110> PLUSNATURE <120> Natural material for enhancing stability vitamin from vitamin tree extract and its manufacturing method <130> PN0222 <160> 7 <170> PatentIn version 3.2 <210> 1 <211> 12 <212> PRT <213> <220> <223> engineered from fibroin <400> 1 Arg Tyr Ile Ala Gln Ala Ala Ser Gln Val His Val 1 5 10 <210> 2 <211> 12 <212> PRT <213> <220> <223> engineered from fibroin <400> 2 Arg Tyr Ile Ala Ser Ala Ala Gln Ser Val His Val 1 5 10 <210> 3 <211> 12 <212> PRT <213> <220> <223> engineered from fibroin <400> 3 Arg Tyr Ile Ala Asn Ala Ala Asn Gln Val His Val 1 5 10 <210> 4 <211> 12 <212> PRT <213> <220> <223> engineered from fibroin <400> 4 Arg Tyr Ile Ala Thr Ala Ala Thr Gln Val His Val 1 5 10 <210> 5 <211> 12 <212> PRT <213> <220> <223> engineered from fibroin <400> 5 Arg Tyr Ile Ala Gln Ala Ala Asn Thr Val His Val 1 5 10 <210> 6 <211> 5 <212> PRT <213> <220> <223> Xs are independently selected from the group consisting of Ser, Thr, Asp and Glu. <220> <221> misc_feature <222> (1)..(1) <223> Xaa can be selected from the group consisting of Ser, Thr, Asp and Glu. <220> <221> misc_feature <222> (4)..(5) <223> Xaa can be selected from the group consisting of Ser, Thr, Asp and Glu. <400> 6 Xaa Ala Ala Xaa Xaa 1 5 <210> 7 <211> 12 <212> PRT <213> <220> <223> Xs are independently selected from the group consisting of Ser, Thr, Asp and Glu. <220> <221> misc_feature <222> (5)..(5) <223> Xaa can be selected from the group consisting of Ser, Thr, Asp and Glu. <220> <221> misc_feature <222> (8)..(9) <223> Xaa can be selected from the group consisting of Ser, Thr, Asp and Glu. <400> 7 Arg Tyr Ile Ala Xaa Ala Ala Xaa Xaa Val His Val 1 5 10

Claims (12)

A peptide comprising the amino acid sequence of Formula 1 below:
[General formula 1]
X ₁ -AAX ₂ -X ₃
In the above formula, X ₁ , X ₂ , and X ₃ are each independently selected from the group consisting of serine (S), threonine (T), asparagine (N), and glutamine (Q). is an amino acid,
The peptide is a peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. delete delete delete delete A complex comprising the peptide of claim 1 and vitamin C. The complex according to claim 6, wherein the peptide and vitamin C are included in a molar ratio of 1:1. The complex according to claim 6, wherein the vitamin C electrostatically binds to the peptide. delete A health functional food comprising the complex of claim 6. A food additive comprising the complex of claim 6. A cosmetic composition comprising the complex of claim 6.

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