KR20160124450A - Methods for preparing peptides from Angelica gigas Nakai with multi-functional efficacies of anti-wrinkle and skin lightening - Google Patents

Abstract

The present invention relates to a method for producing a multifunctional peptide having excellent wrinkle alleviating and skin whitening effects from Angelica gigas NAKAI which is an herbal medicinal material. Angelica gigas NAKAI is dried and pulverized, and an Angelica gigas NAKAI protein is extracted by a buffer solution including sodium dodecyl sulfate and glycerin and is hydrolyzed by using a protein decomposing enzyme. Also, a peptide having excellent wrinkle alleviating and skin whitening effects is sorted from peptides fractioned and separated by an extracorporeal ultra-filtration method, so a multifunctional peptide is produced. The method of the present invention can contribute to scientification of herbal medicine, and is expected to be useful in the cosmetic industry in which a multifunctional peptide material is sought for.

Description

TECHNICAL FIELD The present invention relates to a method for preparing an Angelica gigas Nakai with anti-wrinkle and skin lightening,

The present invention relates to a method for producing peptides from angelica orientalis. More specifically, the present invention relates to a method for preparing peptides having excellent wrinkle and skin whitening effect by extracting proteins present in Angelica gigas, hydrolyzing them to make various peptides, and isolating only the double low molecular peptides.

Recently, various peptide cosmetics have been developed and marketed as peptides are introduced as functional cosmetic materials mainly in developed countries. They are mainly characterized by excellent effects on skin aging suppression and wrinkle improvement, and are getting good response in the actual market. At present, various plant-derived peptides have been developed and applied to actual cosmetics. Peptides applied to these cosmetic materials show strong effects within 10 amino acid bases. It is known that the amino acid sequence has a crucial effect on the skin aging effect rather than the peptide size (Lintner, K. (2007), Peptides, Amino Acids and Proteins in Skin Care ?, Cosmetics & Toiletries, 122 (10), 26-34).

Functional peptides currently applied to cosmetics are classified into hydrolyzed peptide complexes and peptides synthesized based on functional sequences, depending on the preparation method. The hydrolyzed peptide mixture is mainly obtained by extracting and hydrolyzing the protein of the plant and isolating and purifying the functional peptide.

Although Korea has a variety of plants, few cases have developed peptides with excellent skin function. In particular, there are excellent medicinal plants that have proven their efficacy over a long history, such as herbal materials, but the development of functional peptides from them is very poor. There have been many researches and developments on physiologically active components of these medicinal plants, but there was not much interest in the development of peptides.

Therefore, there is a continuing need to develop plant-derived peptides, which are well-known as skin-functional materials, from Korean medicinal plants, especially herbal materials. Particularly in the pharmaceutical and cosmetics industries, there is a great need to develop natural peptides derived from oriental herbs, as the commercial use of peptides increases.

[Reference 1] Goldstein, M.; Lintner, K.; Peptides, Amino Acids and Proteins in Skin Care? ; Allured's cosmetics & toiletries: C & T v.122 no.10, pp.26 - 35, 2007, 1530-1338

Accordingly, the present inventors have decided to identify peptides having the effect of improving skin wrinkles and whitening effect from Angelica gigas Nakai, which is typically applied to prescriptions among herbal materials.

In general, methods for producing peptides from plants have not been studied extensively because the protein content of the plant is not high and thus it is not widely used as a protein or peptide source.

Even if a protein is obtained from a plant, it is necessary to hydrolyze it to produce a peptide. There are several methods for hydrolysis, acid alkaline digestion and enzymatic digestion. However, it is preferable to apply a method using an enzyme because the developed peptide is used as a cosmetic material. Protein hydrolytic enzymes are very diverse. They are divided into endo-type and exo-type, depending on the site of action on the protein. Depending on the origin of the enzyme, animal or vegetable hydrolytic enzymes are distinguished. Therefore, hydrolyzing a protein extracted from Angelica gigas Nakai with a peptide having a wrinkle improving and whitening complex function is also a problem to be solved in the present invention.

As a result, the main object of the present invention is to extract a protein from Angelica gigas, which is a representative herbal material, hydrolyze it to produce a peptide, and to separate complex functional peptides showing wrinkle improvement and whitening complex functions in the peptide. In order to attain this object, technical problems such as a protein extraction method, a hydrolysis method and a peptide separation method are solved and the present invention is completed.

As described and confirmed in detail above, it has been made possible to provide peptides having excellent skin wrinkle and whitening effect from Angelica gigas Nakai through the present invention.

In the present situation that there is no peptide material derived from native Korean native plants, the present invention will be an opportunity to provide a composite functional material excellent in the cosmetics industry. In addition, by introducing the proteomics study, it is expected that the supply of angelica peptides, which have been proven scientifically to improve wrinkles and whitening, will contribute to the scientific development of oriental medicine and it will be useful also in related industries looking for materials with excellent wrinkle and whitening effect do.

In the present invention, when a protein is extracted from Angelica gigas, which is a representative oriental material, and hydrolyzed to produce a Angelica giganta peptide, it is possible to increase the efficiency of protein extraction from Angelica gigas Nakai, isolate only the peptides that are effective for improving skin wrinkles and whitening, An inventive method for producing peptide materials for cosmetics having excellent functional effects was conducted.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Protein Extraction from Angelicae

Protein is extracted from Angelica gigas Nakai. In order to increase the efficiency of extraction, Angelicae gigantis was crushed to make microparticles with an average size of 100 micrometers. Tris buffer solution composed of 0.5% of SDS (sodium dodecyl sulfate) and 10% of glycerin was used as an extract.

The angelic microparticles and buffer were extracted with stirring. The extracted Angelica gigantis was recovered by freezing with acetone. As shown in the table, the extraction efficiency increased with extraction time, and after 6 hours, the extraction efficiency decreased.

Extraction efficiency of Angelica gigas Nakai Extraction time (Hour) The protein (g) extracted from 5 g of the Angelica microparticles Extraction yield (%) 2 0.33 6.6 4 0.41 8.2 6 0.49 9.8 8 0.51 10.2 10 0.52 10.4

Enzymatic Hydrolysis of Angelica gigas Nakai

The Angelica keiskei karyotype protein extracted in Example 1 was hydrolyzed. Trypsin and papain were used as hydrolytic enzymes. Trypsin is a representative animal-derived hydrolyzing enzyme, and papain is a plant protease derived from papaya. During the hydrolysis, the ratio of angiotensin protein to enzyme was 25: 1, the enzyme reaction temperature was 37 ° C, and the reaction time was 20 hours.

Even under the same conditions, hydrolysis efficiency varied depending on the type of hydrolytic enzyme. The following table shows the hydrolytic enzymatic degradation efficiency of Angelica gigas Nakai.

Hydrolysis efficiency of Angelica gigas due to enzyme type Hydrolytic enzyme type Trypsin Papain Trypsin + papain (1: 1) Results of hydrolysis at 37 ° C for 20 hours
Protein degradation efficiency of Angelica gigas Nakai (%) 76 68 81

Fractionation of Angelicae Peptide

The angelic peptides were prepared through Example 2, but the peptides can be called crude peptides without purification. They have various amino acid sequences and sizes. Peptides were fractionated from these Crude peptides.

The fractionation was carried out by ultra-filtration. Applicable membranes were fractionated under 500 Da, 500-1000 Da, 1.0-10.0 KDa, and 10.0 kDa on MWCO (Molecular Weight Cut Off) basis. The peptides fractionated according to the molecular weight were evaluated for the effect of improving wrinkles and whitening of skin as in Examples 4 and 5.

Effect of Angelicae peptides on skin wrinkles

Dangguet peptides were administered to cultured human dermal fibroblast cells and the identification of intracellular proteins was evaluated to evaluate the wrinkle - improving effect. The wrinkle improvement effect of Angelica gigas Nakai was measured by analyzing the changes of various proteins involved in the wrinkle reduction mechanism among the intracellular proteins. More specifically, the angiopeptide was administered to the cells and cultured for 3 days. Then, changes in intracellular proteins were analyzed by 2D-PAGE (two-dimensional polyacryl amide gel electrophoresis). The mechanism of wrinkle improvement was evaluated by collagen synthesis, collagen degradation, regulation by cytokine, interaction with ECM (extra cellular matrix), and increase of antioxidant enzyme.

Cell culture: Human skin fibroblast (Hs 68) was purchased from ATCC. The cells were cultured in DMEM (WelGene lnc.) Supplemented with 10% fetal bovine serum (Gibco) and 1% antibiotic-antimycotic (Gibco) at 37 ° C and 5% CO ₂ Respectively.

Analysis of Proteomic Segregation Expressed by Administration of Angelicae Peptide Fractions: Each of the separated angelica peptides isolated in Example 3 was dissolved in a small amount of carrier, diluted with DMEM, and administered to Fibroblast at various concentrations. After incubation for 48 hours, proteomes expressed in fibroblast Respectively.

Proteomic extraction and quantification: The fibroblast cells were washed with PBS, centrifuged and resuspended in lysis buffer (7 M Urea, 2 M Thiourea, 4% CHAPS, 1% DTT, 2% Carrier ampholyte, 40 μl / ml PIC, 1 μl / , 0.002% BPB) and centrifuged at 30 ° C for 5 hours. The protein supernatant was obtained by centrifugation. The Bradford method was used for protein determination.

2D-PAGE: Isoelectric focusing and equlibration were performed and two-dimensional electrophoresis was performed. Second-dimension SDS-PAGE was performed on a Hoefer SE600 system (Amersham Bioscience, Sweden), making 180 x 240 x 1.5 mm, 11.5% acrylamide homogenous gel. Stacking was carried out at 5 mA / gel for 40 min and for electrophoresis at 35 mA / gel for 6 h. Protein staining was performed using silver staining. The stained gel was scanned and the protein spot image was analyzed using Image Master2D (Amersham Bioscience, Sweden).

Proteome pattern profiling: For the comparative analysis of proteome expression pattern by administration of angiopeptide fractions, the results of 2D-PAGE without administration of the fractions and the results of administration were compared and analyzed. The effects of the fractions on the wrinkle-reducing mechanism are evaluated and summarized in the following table. As shown in the table, fraction of peptides having molecular weight of 500 Da or less among the fractions of Angelica gigas Nakai had the best wrinkle-reducing effect.

Effects of Angelicae Peptide fractions on the improvement of skin wrinkles Related to skin wrinkle improvement
Effect on protein Peptide fraction
500 Da or less Peptide fraction
500-1000 Da Peptide fraction
1.0-10.0 kDa Peptide fraction
10.0 kDa or more Collagen synthesis type1 collagen 90% up 25% up 25% up - type2 collagen 45% up 20% up - - proxy 4 hydroxylase 320% up 50% up 20% up 25% up p-disulfide isomerase 25% up - - - Collagen degradation IL 1-a 260% down 65% down - NF-kappa B 40% down - - - Cytokine
control TGF-beta 290% up 50% up 35% up 30% up TNF-a 150% up 35% up - - Interaction with ECM alpha-actinin 80% up 25% up - - integrin-beta 60% up - - - Antioxidant enzyme vimentin - - - -

Skin Whitening Effect of Angelicae Peptide

Angelica peptides were administered to the cultured B16F1 melanoma cells and the skin whitening effect was evaluated by identifying the intracellular proteins. The changes in various proteins involved in the whitening mechanism among the proteins in the melanoma cells were analyzed to evaluate the whitening effect of Angelica gigas Nakai.

The specific method is the same as in Example 4, except that the cells used for evaluation were changed from fibroblast to B16F1 melanoma cells.

Proteome pattern profiling: For the comparative analysis of proteome expression pattern by administration of angiopeptide fractions, the results of 2D-PAGE without administration of the fractions and the results of administration were compared and analyzed. The effects of the fractions on the skin whitening mechanism are evaluated and summarized in the following table. As shown in the table, peptide fractions having a molecular weight of 500 Da or less in the Angelica peptides fractions had a skin whitening effect, and the peptides having larger molecular weights had little effect.

Skin whitening effect of Angelicae peptides fractions Skin Whitening Related
Effect on protein Peptide fraction
500 Da or less Peptide fraction
500-1000 Da Peptide fraction
1.0-10.0 kDa Peptide fraction
10.0 kDa or more Tyrosinase
Expression α-MSH 45% down - - - MITF 50% down - - - Tyrosinase 25% down 10% down - - Tyrosinase
activation SCF - - - - PKC-β 35% down - - - Melanin
produce TRP-1 20% down - - - TRP-2 35% down 20% down - - Melanosome
Active and moving DCT 70% down - - - DKK1 - - - -

Claims (5)

Peptides having excellent skin wrinkle and whitening effect were selected from the peptides separated by the ultrafiltration method by extracting proteins with a buffering solution, digesting the plants with a buffer solution, and screening the complex functional peptides Lt; / RTI > The method for producing a complex functional peptide according to claim 1, wherein the plant is a medicinal herb ginger, and has excellent skin wrinkle and whitening effect. The method according to claim 1, wherein the buffer solution is a tris buffer solution containing sodium dodecyl sulfate and glycerin, thereby improving the skin wrinkle and whitening effect. The method for producing a complex functional peptide according to claim 1, wherein the protease is animal-derived trypsin and plant-derived papain. The method according to claim 1, wherein the complex functional peptide is a low molecular peptide that passes through a MWCO (Molecular Weight Cut-off) 500 Da membrane, and has excellent skin wrinkle and whitening effect.