KR20240080219A - Glutamine derivatives having skin whitening and anti-inflammatory activity - Google Patents

Abstract

The present invention relates to a glutamine derivative exhibiting whitening activity or anti-inflammatory activity selected from the group consisting of the following formula (1):
(Formula 1)
In the above formula, R1 represents hydrogen or a saturated aliphatic straight-chain or branched-chain or unsaturated aliphatic having 1 to 30 carbon atoms, or an aromatic or heterocyclic aromatic alkyl group. R2 represents a saturated aliphatic straight-chain or branched-chain or unsaturated aliphatic aliphatic group having 1 to 30 carbon atoms, or an aromatic or heterocyclic aromatic alkyl group.

Description

Glutamine derivatives having skin whitening and anti-inflammatory activity}

This application relates to glutamine derivatives and compositions thereof having skin whitening and anti-inflammatory activities, and more specifically to glutamine derivatives whose efficacy and effectiveness are not inferior to those of RAMALIN derived from natural products.

Ramalin (2-amino-5-(2-(2-hydroxyphenyl)hydrazinyl)-5-oxopentanoic acid; γ-glutamyl-N'-(2-hydroxyphenyl)hydrazide) is from Antarctica. It is a substance extracted from a lichen called Ramalina terebrata, which grows naturally in colonies on King George Island, and has the following chemical formula.

Ramalina terrebreta is a powerful antioxidant produced in an environment where it receives ultraviolet rays directly at -90°C, 2,500m above sea level in Antarctica, where dry and strong winds blow. Ramalin has high potential for use in cosmetic products, as it is widely used in the treatment of oxidation-related diseases, functional foods for anti-aging, and functional cosmetics for wrinkle improvement, but its direct use is low due to its inherent stability problem. (Patent Application Publication No. 10 -2008-0111021) In addition, the RAMALIN derivative derived from RAMALIN structurally belongs to the glutamine derivative and is a substance in which ortho-hydroxy phenylhydrazine is introduced into glutamine. This substance has a higher antioxidant capacity than vitamin C and is active in improving various biological diseases and indications.

Meanwhile, the amino acid L-glutamine is known to exhibit significant anti-inflammatory activity and moderate analgesic activity. In particular, it was effective in suppressing the inflammatory response in various artificially induced inflammations when taken orally, and the advantage of no stomach irritation within the anti-inflammatory dosage range was reported ( Agents Actions . 1981 May;11(3):243-249 )

The amino acid L-glutamic acid and its derivatives are abundant throughout the body and are involved in many metabolic processes. In particular, it is reported to have excellent antioxidant ability. In particular, glutamine is synthesized from glutamic acid and ammonia, and is the main carrier of nitrogen in the body and an important energy source for many cells. An oral formulation of L-glutamine was approved by the FDA in July 2017 for use in sickle cell disease. The oral formulation is sold by Emmaus Medical under the brand name Endari.

Amino acids contribute to self-stabilization through sharing of active hydrogen between carboxylic acid and amine, but in glutamine derivatives (Ramalin) derived from natural products, the active hydrogen in the phenol of the hydroxy phenyl group is easily dehydrogenated by the amine group at the amino acid position of glutamic acid. It is judged to have low stability. Therefore, stability can be induced by converting the amine group at the amino acid position to an amide group to inhibit hydrogen ion sharing. It is known that the method for converting to an amide group can be solved by activating various carboxylic acids and reacting with the amine of the amino acid group (Patent Publication No. 10-2021-0148530).

Ramalin, which is derived from natural products, is well known for its efficacy and effectiveness, but its synthesis method is complicated, so there are limitations in producing it on an industrial scale. However, glutamine derivatives can be synthesized in various ways as RAMALIN derivatives, but if they are to be used for purposes equivalent to RAMALIN, their stabilization is an issue, and it is necessary to confirm their efficacy and effect compared to RAMALIN.

In order to achieve the object of the present invention, in one aspect, the present application provides a glutamine derivative exhibiting whitening activity or anti-inflammatory activity selected from the group consisting of the following formula (1):

(Formula 1)

In the above formula, R1 represents hydrogen or a saturated aliphatic straight-chain or branched-chain or unsaturated aliphatic having 1 to 30 carbon atoms, or an aromatic or heterocyclic aromatic alkyl group. R2 represents a saturated aliphatic straight-chain or branched-chain or unsaturated aliphatic aliphatic group having 1 to 30 carbon atoms, or an aromatic or heterocyclic aromatic alkyl group.

In another aspect, the present application provides a glutamine derivative characterized in that it is selected from the group consisting of the following compounds 2 to 10:

In another aspect, the present application provides a glutamine derivative characterized by inhibiting melanin production.

In another aspect, the present application provides a glutamine derivative that inhibits the activity of tyrosinase or the production of IL-6, an inflammatory cytokine.

Meanwhile, the present application provides a glutamine derivative characterized by having a whitening effect or anti-inflammatory effect at a concentration of 0.001% to 5.0%.

Additionally, in another aspect, the present application provides a glutamine derivative characterized in that the N-terminus has increased stability by an amide group. Also provided is a glutamine derivative characterized by increased stability due to an alkyl group at the O-terminus.

In another aspect, the present application provides a composition for treating skin diseases or a cosmetic composition for skin whitening, characterized in that it contains a glutamine derivative as an active ingredient.

According to the present invention, glutamine derivatives have an excellent effect on skin whitening by suppressing melanin production, and have excellent stability, so they are useful in the composition of cosmetic compositions for skin whitening, and in the composition of skin disease treatment compositions through their anti-inflammatory effect. useful. Accordingly, it can be confirmed that the glutamine derivative of the present invention is stabilized and its efficacy and effectiveness are not reduced compared to RAMALIN.

Figure 1 is a graph showing the results of measuring cytotoxicity of glutamine derivatives in HaCaT cells.
Figure 2 is a numerical graph of LDH expressed in HaCaT cells after treatment with glutamine derivatives.
Figure 3 is a graph showing the DPPH scavenging ability of glutamine derivatives.
Figure 4 is a graph showing the amount of GSH after treatment with glutimine derivatives in HaCaT cells.
Figure 5 is a graph showing the amount of SOD analyzed in HaCaT cells after treatment with glutimine derivatives.
Figure 6 is a graph for analyzing the amount of ROS produced after treating glutimine derivatives in HaCaT cells.
Figure 7 is a comparative diagram of the amount of inflammatory cytokine (IL-6) produced after treatment with glutamine derivatives in HaCaT cells.
Figure 8 is a graph showing the effect of glutamine derivatives on the tyrosinase inhibition rate in the B16F10 meanoma cell line.
Figure 9 is a graph showing the melanin content of glutamine derivatives in HaCaT cells.
Figure 10 is a graphical representation of stability confirmation for 180 days at 25°C.

The present inventors focused on the skin whitening effect of a glutamic acid-conjugated derivative whose stability was secured through an amide group at the N-terminus and conducted extensive experiments. As a result, it was discovered that some glutamine derivatives in compounds 2 to 10 inhibit melanin production, are effective in skin whitening, and exhibit anti-inflammatory activity, leading to the present invention.

In addition, the present inventors prepared a group of candidate glutamine derivatives with guaranteed stability by introducing protective groups into the O-terminus and N-terminus of the glutamine derivatives, and prepared the candidate glutamine derivatives. Then, among these, candidates with excellent melanin production inhibitory activity are screened to provide the glutamine derivative of the present invention, which is not inferior in efficacy and effectiveness compared to natural product-derived RAMALIN.

The present invention provides a glutamine derivative exhibiting whitening activity or anti-inflammatory activity selected from the group consisting of the following formula (1):

(Formula 1)

In the above formula, R1 represents hydrogen or a saturated aliphatic straight-chain or branched-chain or unsaturated aliphatic having 1 to 30 carbon atoms, or an aromatic or heterocyclic aromatic alkyl group. R2 represents a saturated aliphatic straight-chain or branched-chain or unsaturated aliphatic aliphatic group having 1 to 30 carbon atoms, or an aromatic or heterocyclic aromatic alkyl group.

In the above formula, R1 and R2 represent a straight-chain or branched alkyl group having 1 to 30 carbon atoms, preferably 1 to 15 carbon atoms, more preferably 1 to 6 carbon atoms, and most preferably 1 to 4 carbon atoms. As a specific example, R1 is methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isophene. Tyl group, cetyl, palmityl, myristyl, stearyl, lauryl, oleyl group, etc. are mentioned. Additionally, Bn represents a benzyl group. Additionally, specific examples of the aromatic or heterocyclic aromatic substituent for R2 include phenyl, pyridyl, thiazole, imidazole, etc. with or without substituents.

The glutamine derivative will exhibit a whitening effect or anti-inflammatory effect at a concentration of 0.001% to 5.0%, preferably at a concentration of 0.01% to 4.0%. Below 0.001% concentration these desired effects will not be achieved and above 5.0% concentration it will be uneconomical.

The glutamine derivative of the present invention can be manufactured into a skin whitening cosmetic composition. The glutamine derivative used as an active ingredient in the composition of the present invention is selected from the group consisting of compounds 2 to 10 described above. When the composition of the present invention is applied topically to the skin, a skin whitening effect can be achieved due to a high skin penetration rate. The composition of the present invention suppresses melanin production, so it brightens skin color, maintains consistent skin tone, and is effective in removing skin pigment and age spots. The composition containing the glutamine derivative according to the present invention as an active ingredient can be applied to cosmetic compositions such as gel type, skin type, cream type, and ointment type, but is not limited to these.

Additionally, the present invention can be provided as a preparation containing a glutamine derivative selected from the group consisting of the above-mentioned compounds 2 to 10 as an active ingredient and one or more pharmaceutically acceptable carriers or excipients. Accordingly, the formulation may include pharmaceutically acceptable carriers, diluents, excipients, or combinations thereof, as needed. These agents facilitate administration of the active ingredient into an organism.

Hereinafter, the present invention will be described in more detail through examples. However, those skilled in the art will fully understand that the following examples are provided as examples and that the present application is not limited thereto.

Example

Example 1 (Synthesis of Compound 1)

Compound 1 is identical to RAMALIN derived from natural products and is known in Prior Document 1, Patent Application Publication No. 10-2008-0111021 mentioned in the background art, so it is omitted here.

Example 2 (Synthesis of Compound 2)

Into a 3-neck round bottom flask, 10 g of 5-(benzyloxy)-5-oxo4--pentadecane-amidopentanoic acid compound derived from palmitic acid and 200 ml of MC were added, cooled to 0°C, and triethylamine. (TEA) 2.55 g was added for 30 minutes and all was dissolved. The reaction solution was cooled to 15°C, activated by adding 2.74 g of ethyl chloroformate (ECF) over 20 minutes, and stirred for 2 hours while maintaining the temperature below 0°C to prepare an activated glutamic acid intermediate where R1 is ethyl.

Then, in a new reactor, 5.80 g of ortho-benzylhydrazine hydrochloride (compound 3) of the formula 3 was dissolved in 100 ml of MC, and 2.34 g of triethylamine (TEA) was added for 20 minutes while maintaining the temperature below 0°C, and the solution was activated. Add the glutamic acid intermediate compound solution for 1 hour, raise the temperature to rt, and stir for 10 hours. When the reaction is complete, 100ml of H2O is added to the reaction organic layer and washed, then washed once with 100ml of 1N HCl and once with 100ml of Sat.NaHCO3. The organic layer is dried over Na2SO4, concentrated under reduced pressure to remove the solvent, and dissolved in MeOH. Treated with activated carbon. After filtration through Celite, the methanol solvent was removed again through reduced pressure distillation, and crystallized using MC/Hex (methylene chloride/hexane) to produce 6.75 g (yield: 40.7%) of Intermediate Compound 2 in the protective group state, a glutamine derivative precursor. Obtained with a purity of 96.1%.

1H-NMR (DMSO-d6, 400MHz, δ/ppm) is 9.73(d, 1H), 8.22(d, 1H), 7.3~7.5 (m, 10H), 6.56-6.90(m, 5H), 5.14(s) , 1H), 5.11(s, 1H), 4.21(m, 1H), 2.27(m, 2H), 2.10(m, 2H), 2.05~1.75(m, 2H), 1.48 (m, 2H), 1.22( m, 26H), 0.85 (t, 3H)

Example 3 (Compound-3 synthesis)

(Diazonium salt synthesis)

Add 2.15 g of 2-(benzyloxy)aniline and 2.6 g of H ₂ O to a 3-neck round bottom flask and stir at rt. HCl diluted with 3 g of 35% HCl and 2.3 g of H ₂ O was added to rbf at rt for 30 minutes and then cooled to 0°C. NaNO ₂ was dissolved using 1.73 g of H ₂ O and then added for 1 hour while maintaining the temperature below 5°C to prepare diazonium salt.

(Diazonium reduction)

Add 3.4 g of SnCl2, 6.5 g of H ₂ O, 3.2 g of NaCl, and 7.6 g of H ₂ O to a 3-neck round bottom flask and dissolve at -10 degrees. Add ammonia water to make pH 6.0, then raise the temperature to 20℃ and adjust pH to 6.0 again. Maintain the temperature at 60℃, and add the diazonium salt aqueous solution prepared above for 40 minutes. Raise the temperature to 75℃, stir for 1 hour, cool to 5℃, and add 7.6 g of 35% HCl for 1 hour while maintaining 5℃. At this time, the pH was maintained around 0 to 1. The resulting solid was concentrated and removed. The remainder contained 1.66 g of red crystalline hydrazine, 72.1% yield, 99.3% purity.

1H-NMR (DMSO-d6, 400 MHz, δ/ppm) is 9.31 (d, 1H), 8.17 (m, 4H), 7.3-7.5 (m, 5H), 6.52-6.71 (m, 2H),

Example 4 (Compound-4 synthesis)

During the synthesis of Compound-1, the O-allyl-N-Cbz-benzyl ester intermediate (compound 4) was synthesized in the same manner.

Next, 2 g of O-allyl-N-Cbz-benzyl ester intermediate, 10% Pd/C, and 50v of MeOH were added and stirred at room temperature. H2 at atmospheric pressure is injected into the reactor and stirred for 10 hours. The catalyst is removed through Celite filtration. The organic layer was concentrated, and when a solid was formed, the concentration was stopped, and a white solid glutamine derivative (Formula 1) was obtained through a flash silica gel column using methanol/MC (1/4) as a mobile phase. (Yield: 73%, purity 98.5%)

1H-NMR (DMSO-d6, 400 MHz, δ/ppm) is 9.73 (d, 1H), 9.38 (s, 1H), 8.02 (d, 1H), 6.56-6.70 (m, 4H), 4.15 (m, 1H) ), 2.55(m, 2H), 2.21(m, 2H), 2.05~1.75(m, 2H), 1.48 (m, 2H), 1.23(m, 24H), 0.85 (t, 3H)

Examples 5 to 10 (Synthesis of Compounds 5 to 10)

Compounds 5 to 10 were synthesized with reference to Examples 1 to 4, along with Prior Art Document 1, and their properties are listed in the table below.

First, to secure a basis for judging stability through a study on the compatibility of the glutamine derivatives synthesized above, 10 types of glutamine derivatives were stored in a solid state at room temperature (25°C) and evaluated, and the most stable was evaluated. Stability evaluation was performed on the good compound No. 4 as a representative of the change in solution pH.

(a) Stability plan

Secure data by evaluating storage stability at 25°C (analysis by 3day, 5day, 10day, 15day, 30day, 3month, 6month)

(B) Selection of stability samples

After 6 months of storage in the company's own 25°C stability chamber and -20°C freezer, one candidate with excellent antioxidant effect and stability was selected, and the purity of the frozen storage sample and the 25°C stability sample was requested from a public certification agency for HPLC purity analysis.

(c) Stability evaluation

The evaluation standard was selected as a stable and valid result when the purity was 95% or higher.

(D) Stability evaluation results

Stability at 25°C of more than 95% was secured in all samples except for compounds (GHP)-1 and 3, and in the case of compounds (GHP)-5 and 6, the purity tended to decrease over time. In the case of compounds (GHP)-2, 4, 7, 8, 9, and 10, the purity remained above 98% even after 6 months. Figure 10 is a graphical representation of stability confirmation for 180 days at 25°C.

Among them, Compound-4, which has the best stability and excellent antioxidant activity, was selected and an official test was conducted on samples stored frozen at -20℃ and samples stored at 25℃.

Afterwards, a test was conducted to evaluate the effect of the compounds 2 to 10 synthesized above, which had secured stability, on improving antioxidant properties and whitening of skin cells.

- Exam purpose

The purpose is to determine candidate materials suitable for commercialization by evaluating the impact of stable glutamine derivative materials (9 types) on improving antioxidant and whitening of skin cells (HaCaT).

- Trial design

go. Experimental cells: HaCaT cells (human skin keratinocytes)

me. experimental material

1) Reference material: Glutamine derivative #1 (Ramalin)

2) 9 types of glutamine derivatives

all. evaluation

1) Cytotoxicity: MTT, LDH

2) Antioxidant efficacy: DPPH scavenging ability, GSH, SOD, ROS

3) Anti-inflammatory effect: inflammatory cytokine (IL-6)

4) Whitening efficacy: Tyrosinase, Melanin

The test items and experimental methods for glutamine derivatives are briefly summarized as follows:

1) Check cell viability (cytotoxicity) using HaCaT keratinocytes

Here, the cell culture medium was used by adding 10% fetal bovine serum, 1% penicillium, and streptomycin to MEM medium, and cultured in an incubator (Sanyo, Japan) at 37°C and 5% CO2. The medium was replaced with fresh medium once every 48 hours.

(1-1) MTT assay (3-(4,5-dimethythiazol-2-yl)-2-5-diphenytetrazolium bromide) is measured by applying the reduction method.

- It uses the principle that dehydrogenase within the mitochondria reduces yellow water-soluble tetrazolium salt [3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide] (MTT) into water-insoluble dark purple MTT formazan crystals. Measure absorbance at an appropriate wavelength (mainly 500 to 600 nm).

(1-2) Measure using LDH (latate dehydrogenase) leakage assay.

- This test method measures the activity of lactate dehydrogenase and is characterized by being fast, simple, and highly reliable.

- Lactate dehydrogenase generally cannot pass through the cell membrane and is therefore not released out of the cell. However, if the cell membrane is damaged or the cell dies, it is released into the medium. Therefore, the amount of lactate dehydrogenase in the medium is proportional to the number of dead or injured cells.

2) Verification of antioxidant activity of glutamine derivatives

(2-1) Evaluation using DPPH (1,1-diphenyl-2picrylhydrazyl) assay

- The DPPH method measures the ability of DPPH, a stable free radical, to react with a hydrogen donor (H-donor).

(2-2) Evaluation using ROS (reactive oxygen species) assay

- The degree of occurrence is measured using DCFH-DA (Dichlorofluorescin diacetate) staining method.

- DCFH-DA, a hydrophobic substance, penetrates the cell membrane and is then decomposed into non-fluorescent DCFH by being acetylated by intracellular esterase. At this time, when DCFH is oxidized by ROS, it is converted to DCF, which fluoresces green. This fluorescence intensity is measured to evaluate the intracellular ROS concentration.

(2-3) Evaluation using SOD (Superoxide Dismutase) assay

- Measures the scavenging activity rate of superoxide anion radical (O ₂ ).

(2-4) Evaluation using GSH ( Glutathione) assay

- Glutathione is a major substance within cells and consists of glutamic acid, cysteine, and glycine.

- Function to protect cells and tissues by acting as an antioxidant against oxidative stress

- Glutathione peroxidase plays a role in lowering lipid peroxidation in cells using GSH reducing agent.

3) Verification of anti-inflammatory activity of glutamine derivatives

(3-1) The amount of cytokines secreted in the culture medium of HaCat cells was measured using an ELISA kit.

- By checking the production amount of pro-inflammatory cytokines such as IL-6, the anti-inflammatory effect in many cells such as macrophages, mast cells, T cells, epithelial cells, and airway smooth muscle cells is confirmed.

4) Verification of whitening effect of glutamine derivatives

(4-1) In vitro tyrosinase inhibition assay

- Tyrosinase is an enzyme involved in the most important initial rate-determining step in the melanin biosynthesis pathway in the human body. By inhibiting the activity of this enzyme, it has the effect of inhibiting melanin production. This test is a method to evaluate the effect of the test sample on inhibiting tyrosinase activity by reacting the test sample, purified tyrosinase, and the substrate tyrosine in vitro.

(4-2) Melanin production inhibition test

- Use the B16F10 meanoma cell line.

- Evaluate the effect of whitening ingredients on inhibiting melanin production in cells

- Culture the cells and quantify the amount of melanin inside the cells or the total amount of melanin inside and outside the cells and compare them with the blank sample solution.

Hereafter, the results according to the test items and experimental methods are presented in detail. Here, glutamine derivatives #1 to #10 shown in the table are merely used as indicated during the research process for convenience and correspond to compounds 1 to 10 of the present specification.

First, the cytotoxicity measurement results of glutamine derivatives in HaCaT cells are shown in Table 1 and Figure 1 below.

(Data are expressed as mean ± ^# p < 0.05, ^## p < 0.01 and ^### p < 0.001 compared to control (CON)).

Figure 1 shows the results of MTT analysis of RAMALIN (#1) and glutamine derivatives (#2 to #10) in HaCaT cells. Seven types of glutamine derivatives (#2, #3, #4, #5, #7, #8, #10) showed similar or better cell survival rates than RAMALIN (#1). Through these results, seven types of glutamine derivatives without cytotoxicity were obtained.

Next, the levels of LDH expressed in HaCaT cells after treatment with glutamine derivatives are shown in Table 2 and Figure 2 below.

Figure 2 shows the results of LDH analysis in HaCaT cells of RAMALIN (#1) and four types of glutamine derivatives (#4, #7, #8, #10). Four types of glutamine derivatives (#4, #7, #8, #10) showed similar or better LDH levels compared to RAMALIN (#1), and the results of two types of glutamine derivatives (#4, #8) were particularly good. did. Through this, it was confirmed through LDH analysis that the four new glutanim derivatives (#4, #7, #8, #10) selected for their antioxidant efficacy had no cytotoxicity compared to RAMALIN.

Next, the DPPH scavenging ability of glutamine derivatives is shown in Table 3 and Figure 3.

Figure 3 shows the results of analysis of the DPPH scavenging ability of RAMALIN (#1) and glutamine derivatives (#2 to #10). The DPPH scavenging ability of two types of glutamine derivatives (#7, #10) showed better antioxidant efficacy than vitamin C, and the efficacy of three types of glutamine derivatives (#4, #5, #8) was similar to that of RAMALIN (#1). Results were at a similar level. Through these results, five types of glutamine derivatives with high antioxidant activity were identified, and based on the results of analysis of stability, solubility, cytotoxicity, and antioxidant activity efficacy, four types of glutamine derivatives (#4, #7, #8, #10) were identified. ) were selected.

Next, the amount of GSH after treatment of glutimine derivatives in HaCaT cells is shown in Table 4 and Figure 4 below.

(Data are expressed as mean ± ^# p < 0.05, ^## p < 0.01 and ^### p < 0.001 compared to control (CON)).

Figure 4 shows the GSH analysis results of RAMALIN (#1) and glutamine derivative candidates (#4, #7, #8, #10). As the concentration of glutamine derivative increased up to 10㎍/ml, the expression level of the antioxidant GSH tended to increase, and as the concentration increased beyond that, the expression level of GSH tended to decrease. All four types of glutamine derivatives (#4, #7, #8, #10) showed a significant increase in GSH expression compared to CON, and two types of glutamine derivatives (#7, #10) showed a significant increase in GSH expression level compared to RAMALIN (# It showed a similar level of antioxidant efficacy as 1).

Meanwhile, the amount of SOD analyzed after treatment of glutimine derivatives in HaCaT cells is shown in Table 5 and Figure 5 below.

(Data are expressed as mean ± ^# p < 0.05, ^## p < 0.01 and ^### p < 0.001 compared to control (CON)).

Figure 5 shows the SOD analysis results of RAMALIN (#1) and glutamine derivative candidates (#4, #7, #8, #10). As the concentration of glutamine derivative increased up to 10㎍/ml, the expression level of the antioxidant enzyme SOD tended to increase, and when the concentration increased beyond that, the expression level of SOD tended to decrease. The SOD expression levels of two glutamine derivatives (#7, #8) showed a significant increase compared to CON, and the increase was similar to or higher than that of RAMALIN (#1).

Next, to analyze the amount of ROS produced after treating glutimine derivatives in HaCaT cells, the results are shown in Table 6 and Figure 6 below.

(Data are expressed as mean ± ^# p < 0.05, ^## p < 0.01 and ^### p < 0.001 compared to control (CON)).

Figure 6 shows the results of analysis of ROS production of RAMALIN (#1) and glutamine derivative candidates (#4, #7, #8, #10). All four types of glutamine derivatives (#4, #7, #8, #10) showed a significant inhibitory effect on ROS production compared to CON, and the levels were similar to those of RAMALIN (#1).

Meanwhile, to compare the amount of inflammatory cytokine (IL-6) produced after treating glutamine derivatives in HaCaT cells, they are shown in Table 7 and Figure 7.

(Data are expressed as mean ± ^# p < 0.05, ^## p < 0.01 and ^### p < 0.001 compared to control (CON)).

Figure 7 shows the results of analysis of inflammatory cytokine (IL-6) expression levels of RAMALIN (#1) and glutamine derivative candidates (#4, #7, #8, #10). Three types of glutamine derivatives (#7, #8, #10) showed a significant inhibitory effect on inflammatory cytokine (IL-6) production compared to CON, and the levels were similar to those of RAMALIN (#1).

Additionally, Figure 8 shows the effect of glutamine derivatives on the tyrosinase inhibition rate in the B16F10 meanoma cell line in Table 8 and Figure 8 below.

(Data are expressed as mean ± ^# p < 0.05, ^## p < 0.01 and ^### p < 0.001 compared to control (CON)).

Figure 8 shows the results of Tyrosinase inhibition rate analysis of RAMALIN (#1) and glutamine derivative candidates (#4, #7, #8, #10). Tyrosinase inhibition rate tended to increase as the concentration of glutamine derivatives increased up to 100 ㎍/ml, and the three types of glutamine derivatives (#7, #8, #10) showed higher inhibition of tyrosinase production than RAMALIN (#1). showed results. Through these results, it was confirmed that the three types of glutamine derivatives (#7, #8, #10) had high whitening efficacy.

Finally, the melanin content of glutamine derivatives in HaCaT cells is shown in Table 9 and Figure 9 below.

(Data are expressed as mean ± ^# p < 0.05, ^## p < 0.01 and ^### p < 0.001 compared to control (CON)).

Figure 9 shows the results of melanin expression level analysis of RAMALIN (#1) and glutamine derivative candidates (#4, #7, #8, #10). As the concentration of the glutamine derivative increased to 100 ㎍/ml, the melanin expression level tended to increase, and glutamine derivative #7 showed a melanin expression level similar to that of RAMALIN (#1). Two types of glutamine derivatives (#8, #10) also showed a lower effect than RAMALIN (#1), but a significant inhibitory effect on melanin expression compared to CON. Through these results, it was confirmed that three types of glutamine derivatives (#7, #8, #10) have high whitening efficacy.

In this experiment, the cytotoxicity, antioxidant activity, anti-inflammatory, and whitening efficacy of nine glutamine derivatives with improved stability were evaluated.

Cytotoxicity was confirmed through MTT and LDH analysis, and as the concentration of glutamine derivative increased up to 10 ㎍/ml, cell viability increased and then decreased from a certain concentration, reaching a concentration lower than 10 ㎍/ml at concentrations of 50 and 100 ㎍/ml. This value was shown for most materials. Through MTT and LDH analysis, seven types of materials (#2, #3, #4, #5, #7, #8, #10) that were similar or better than Ramalin (#1) were identified. In particular, glutamine derivative #4 maintained a cell viability of over 90% even at a concentration of 100 μg/ml.

Antioxidant activity was confirmed through analysis of DPPH scavenging ability, GSH, SOD, and ROS, and for IC50 values, vitamin C and RAMALIN (# It showed similar levels to 1). In the case of GSH expression levels, the GSH expression levels of two types of glutamine derivatives (#7, #10) showed good levels similar to those of RAMALIN (#1), and the remaining two types of glutamine derivatives (#4, #8) Although the value was slightly lower than Ramalin (#1), it was confirmed to have good antioxidant efficacy.

In the case of SOD expression levels, there was a tendency to increase as the concentration of glutamine derivatives increased up to 10㎍/ml, and SOD levels increased compared to CON in all four types of glutamine derivatives (#4, #7, #8, #10). It was a trend. The SOD expression levels of two glutamine derivatives (#4, #7) showed a significant increase in SOD compared to CON, and the increase was similar to or better than that of RAMALIN (#1).

In the case of ROS production levels, all four types of glutamine derivatives (#4, #7, #8, #10) showed a significant increase inhibition effect compared to CON, and the levels were similar to those of RAMALIN (#1). It showed significant antioxidant efficacy.

Anti-inflammatory activity was confirmed through analysis of inflammatory cytokine (IL-6) expression level, and the inflammatory cytokine (IL-6) production amount of four types of glutamine derivatives (#4, #7, #8, #10) was compared to CON. It showed a significant increase-inhibiting effect, and its levels showed a good level of significant antioxidant efficacy similar to that of Ramalin (#1).

The whitening effect was confirmed through analysis of tyrosinase inhibition rate and melanin expression level. Tyrosinase inhibition rate showed a tendency to increase as the concentration of glutamine derivative increased up to 100 ㎍/ml, and 3 types of glutamine derivatives (#7 , #8, #10) showed better results than Ramalin (#1). The amount of melanin expression tended to increase as the concentration of glutamine derivative increased up to 100 ㎍/ml, and glutamine derivative #7 showed similar or better results than RAMALIN (#1) at high concentration.

As a result of a comprehensive review, four final candidates for glutamine derivatives (#4, #7, #8, #10) can be recommended as materials suitable for commercialization.

In addition, according to the final test results, as a result of confirming cytotoxicity in HaCaT cells, seven of the nine glutamine candidate groups (#6, #9) except two showed similar or better cell viability values compared to RAMALIN.

As a result of confirming the antioxidant efficacy (DPPH scavenging ability), four types of glutamine derivatives (#4, #7, #8, #10) showing IC50 values similar to those of Ramalin were derived, and as a result of GSH, SOD, and ROS analysis, Ramalin It shows similar or better antioxidant efficacy levels compared to other products.

As a result of confirming inflammatory cytokine (IL-6), the IL-6 production level of three types of glutamine derivatives (#7, #8, #10) showed a significant increase inhibitory effect compared to CON, and the level was similar to that of RAMALIN. It shows better anti-inflammatory efficacy.

As a result of confirming the whitening efficacy (Tyrosinade, Melanin), the three glutamine candidates (#7, #8, #10) show better whitening efficacy than RAMALIN.

Claims (8)

A glutamine derivative exhibiting whitening or anti-inflammatory activity selected from the group consisting of the following formula (1):
(Formula 1)
In the above formula, R1 represents hydrogen or a saturated aliphatic straight-chain or branched-chain or unsaturated aliphatic having 1 to 30 carbon atoms, or an aromatic or heterocyclic aromatic alkyl group. R2 represents a saturated aliphatic straight-chain or branched-chain or unsaturated aliphatic aliphatic group having 1 to 30 carbon atoms, or an aromatic or heterocyclic aromatic alkyl group. The glutamine derivative according to claim 1 or 2, wherein the glutamine derivative is selected from the group consisting of the following compounds 2 to 10.


The glutamine derivative according to claim 1 or 2, wherein the glutamine derivative inhibits melanin production. The glutamine derivative according to claim 1 or 2, wherein the glutamine derivative inhibits the activity of tyrosinase or the production of IL-6, an inflammatory cytokine. The glutamine derivative according to claim 1 or 2, wherein the glutamine derivative has a whitening effect or an anti-inflammatory effect at a concentration of 0.001% to 5.0%. The glutamine derivative according to claim 1 or 2, wherein the glutamine derivative has increased stability due to an amide group at its N-terminus. The glutamine derivative according to claim 1 or 2, wherein in the group consisting of glutamine derivatives, the O-terminus has increased stability due to an alkyl group. The composition for treating skin diseases or the cosmetic composition for skin whitening according to claim 1 or 2, comprising the glutamine derivative as an active ingredient.