KR100665315B1 - New gallic acid-conjugated linoleic acid fatty ester, the preparation method thereof and composition containing the same - Google Patents

Abstract

The present invention relates to novel gallic acid-conjugated linoleic acid fatty esters, methods for their preparation and compositions containing them.

The gallic acid-conjugated linoleic acid fatty ester of the present invention has excellent electron donating ability (radical scavenging), low peroxide value, and low fatty acid saturation, which is excellent in antioxidant activity, and excellent in antibacterial activity against mold and bacteria. , COX-1 and COX-2 inhibition rate is excellent anti-inflammatory effect, tyrosinase inhibition rate is high, so skin whitening effect is excellent.

Therefore, the composition of the present invention can be usefully used for antioxidant, antibacterial, anti-inflammatory and skin whitening.

Description

New gallic acid-conjugated linoleic acid fatty ester, the preparation method about and composition containing the same

1 is a diagram showing the results of thin film chromatography of the gallic acid-conjugated linoleic acid fatty ester of the present invention.

Figure 2 is a view showing the measurement results of the infrared spectrophotometer of the gallic acid-conjugated linoleic acid fatty ester of the present invention.

The present invention relates to novel gallic acid-conjugated linoleic acid fatty esters, methods for their preparation and compositions containing them.

Conjugated linoleic acid (CLA) is a geometrical isomer of linoleic acid, an essential fatty acid. It is also present in ruminant-derived meat products and dairy products (milk, cheese, etc.). CLA is a substance that has not yet been approved for sale in Korea, but since it was first discovered by Professor Parisa of the University of Wisconsin in 1987, more than 1,000 papers have been published in 2002. The major components, trans-10 and cis-12 isomers, are known to reduce body fat. CLA prevents the adsorbed fat from accumulating in adipocytes and uses it as energy in muscle cells, and this algorithm has the effect of reducing body fat. It turns out to be effective.

The physiological functions of CLA are: anti-obesity, lowering blood cholesterol, antioxidant effect to prevent blood LDL-cholesterol oxidation, prevention and treatment of diabetes, anti-cancer effect against breast cancer, skin cancer, stomach cancer, feed efficiency improvement when used as feed, body fat of livestock It is very diverse, such as decreasing, increasing carcass rate, improving meat quality, and inhibiting fatty acidization of meat.

However, CLA itself is highly conjugated, and fatty acidization is likely to develop rapidly.

Therefore, the inventors of the present invention while studying the excellent substance that can lower the fatty acidization of the CLA itself, it was confirmed that the antioxidant, antibacterial, anti-inflammatory and skin whitening effect is excellent in the material prepared by combining gallic acid and CLA and the present invention Completed.

The present invention seeks to provide novel gallic acid-conjugated linoleic acid fatty esters.

In addition, the present invention is to provide a method for preparing the gallic acid-conjugated linoleic acid fatty ester.

In addition, the present invention is to provide an antioxidant, antibacterial, anti-inflammatory and skin whitening composition using the gallic acid-conjugated linoleic acid fatty ester as an active ingredient.

The present invention provides a novel gallic acid-conjugated linoleic acid fatty ester represented by the following formula (1).

In Chemical Formula 1,

R ₁ to R ₄ are each independently hydrogen or conjugated linoleic acid (CLA),

CLA is 9-cis, 11-trans octadecenoic acid; 9-trans, 11-cis octadecenoic acid; 10-cis, 12-trans octadecenoic acid; Or 10-trans, 12-cis octadecenoic acid, provided that R ₁ To R ₄ are all hydrogen).

The present invention also provides a method for preparing the gallic acid-conjugated linoleic acid fatty ester.

The preparation method of the gallic acid-conjugated linoleic acid fatty ester of the present invention

1) dissolving gallic acid and conjugated linoleic acid in an organic solvent in a 1: 3 molar ratio, followed by addition of a base to react;

2) adding ethyl acetate to the reaction solution to separate the layers, and then collecting and concentrating the supernatant.

The organic solvent in step 1) includes one selected from tetrahydrofuran, dimethylformamide and acetonitrile, and in the present invention, tetrahydrofuran is preferable.

The base in step 1) is preferably triethylamine and 1,3-dicyclohexyl carbodiimide (WSCD).

The present invention also provides a composition for antioxidant, antibacterial, anti-inflammatory and skin whitening using the gallic acid-conjugated linoleic acid fatty ester as an active ingredient.

The gallic acid-conjugated linoleic acid fatty esters of the present invention have excellent electron donating ability (radical scavenging), low peroxide value, and low fatty acid saturation.

In addition, the gallic acid-conjugated linoleic acid fatty esters of the present invention are used in four kinds of fungi (Aspergillus flavus, Aspergillus usami, Muce hiemalis, Penicillin verrucolusom), Lactic acid bacteria (Leuconostoc mensenteroides) and bacteria (Listeria ivanova) among 12 kinds of microorganisms. Excellent antibacterial activity.

In addition, the gallic acid-conjugated linoleic acid fatty ester of the present invention has excellent COX-1 and COX-2 inhibition rates, and has an excellent anti-inflammatory effect against rheumatoid arthritis, osteoarthritis and other inflammations.

In addition, the gallic acid-conjugated linoleic acid fatty ester of the present invention exhibits a high tyrosinase inhibition rate, and thus has an excellent skin whitening effect.

Therefore, the composition of the present invention can be usefully used for antioxidant, antibacterial, anti-inflammatory and skin whitening.

The composition of the present invention may contain at least one active ingredient exhibiting the same or similar function in addition to the gallic acid-conjugated linoleic acid fatty ester.

The composition of the present invention may be prepared by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration. Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components, as necessary, including antioxidants, buffers, Other conventional additives such as bacteriostatic agents can be added. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Furthermore, it may be preferably formulated according to each disease or component by a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA.

The compositions of the present invention may be administered parenterally (eg, intravenously, subcutaneously, intraperitoneally or topically) or orally, depending on the desired method, and the dosage may be based on the weight, age, sex, health status, The range varies depending on diet, administration time, administration method, excretion rate and severity of disease. The daily dosage is about 20-300 mg / kg, preferably 50-120 mg / kg, of the gallic acid-conjugated linoleic acid fatty ester of Formula 1, more preferably administered once to several times a day.

In addition, the composition of the present invention can be added to health food for the purpose of improving antioxidant, antibacterial, anti-inflammatory and skin whitening. When the gallic acid-conjugated linoleic acid fatty ester of the present invention is used as a food additive, the gallic acid-conjugated linoleic acid fatty ester may be added as it is or may be used together with other food or food ingredients, and may be appropriate according to a conventional method. Can be used. The mixed amount of the active ingredient may be suitably determined depending on the purpose of use (prevention, health or therapeutic treatment). In general, the gallic acid-conjugated linoleic acid fatty ester of the present invention is added in an amount of 1 to 20% by weight, preferably 5 to 10% by weight based on the raw materials in the manufacture of food or beverage. However, in the case of long-term intake for health and hygiene or health control, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety. .

There is no particular limitation on the kind of food. Examples of the food to which the substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, drinks, Alcoholic beverages and vitamin complexes, and the like and include all of the health foods in the conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates, etc. as additional components, as in the general beverage. The above-mentioned natural carbohydrates are glucose, monosaccharides such as fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, sugar alcohols such as xylitol, sorbitol and erythritol. As the sweetening agent, natural sweetening agents such as tautin and stevia extract, synthetic sweetening agents such as saccharin and aspartame, and the like can be used. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols, carbonic acid. Carbonating agents and the like used in beverages. In addition, the composition of the present invention may contain a flesh for preparing natural fruit juice, fruit juice beverage and vegetable beverage. These components can be used independently or in combination. The proportion of such additives is not critical but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

In addition, the composition of the present invention can be prepared by combining the additives commonly used in the field of cosmetic compositions in addition to the gallic acid-conjugated linoleic acid fatty ester, it is not particularly limited in the formulation. That is, the cosmetic composition according to the present invention may have a formulation such as a flexible lotion, astringent lotion, nutrition lotion, eye cream, nutrition cream, massage cream, cleansing cream, cleansing foam, cleansing water, powder, pack or essence.

Hereinafter, preferred examples and experimental examples are presented to help understand the present invention. However, the following Examples and Experimental Examples are provided only to more easily understand the present invention, and the contents of the present invention are not limited by the Examples.

EXAMPLES Preparation of Gallic Acid-Conjugated Linoleic Acid Fatty Ester

Gallic acid used in the present invention was purchased from Sigma (USA) and used, and conjugated linoleic acid (purity 80%, CLA) was supplied from Lipogen Co., Ltd. and used in experiments.

Gallic acid and CLA were dissolved in tetrahydrofuran at a 1: 3 molar ratio, and then triethylamine and WSCD (1,3-dicyclohexyl carbodiimide) were added and reacted with stirring for 24 hours. After the reaction was completed, the mixture was washed by adding distilled water, and washing was repeated twice. After separating the layers by adding ethyl acetate, the supernatants were collected, and the collected supernatants were subjected to thin layer chromatography (Silica Thin Layer Chromatography; 25 DC- Platten, Kiesel gel, 60F254, Merk, Germany, 20 x 20 cm). It was developed to confirm the synthesis. At this time, ethanol: hexane (hexane) was used as a developing solvent in a ratio of 1: 8, and phosphomolbidic acid (PMA, PMA, mixed with 10 g of PMA and 120 ml) was immersed with a dyeing reagent and developed.

Na ₂ SO ₄ was added to the supernatant where the synthesis was confirmed, the water was removed, and the mixture was concentrated under a nitrogen stream. 3 ml of concentrated gallic-conjugated linoleic acid fatty ester solution was inserted into the sample holder and synthesized using an infrared spectrophotometer (FT IR / FT NIR Spectrophotometer, Tensor 37, Bruker OPTIK GmbH) and OPUS spectroscopy software (Version 4). The production of gallic acid-conjugated linoleic acid fatty esters was confirmed.

The results developed on the thin film chromatography are shown in FIG. 1, and the measurement results of the infrared spectrophotometer are shown in FIG. 2.

As shown in Figure 1, it can be seen that the starting materials A (gallic acid) and B (conjugated linoleic acid) developed in a different form from the final product C (gallic acid-conjugated linoleic acid fatty ester). The color development point of the starting material A (gallic acid) did not appear in the final product C (gallic acid-conjugated linoleic acid fatty ester), but in the final product C there was a newly formed color development point.

In addition, as shown in FIG. 2, the peak is confirmed at 1752.65 cm ^-1 , which is a peak appearing in the ester group, so that the gallic acid and the conjugated linoleic acid are combined to form the gallic acid-conjugated linoleic acid fatty ester.

Experimental Example 1 Antioxidant Effect of Gallic Acid-Conjugated Linoleic Acid Fat Ester of the Present Invention

In order to determine whether the gallic acid-conjugated linoleic acid fatty ester of the present invention has antioxidant capacity in human stomach gastric conditions, the following experiment was performed.

1. Preparation of Antioxidant Test Sample

To make gastric acid conditions in the human body, 0.1M gallic acid, 0.3M conjugated linoleic acid and the gallic acid-conjugated linoleic acid fatty ester prepared in the above example were dissolved in ethanol adjusted to pH 2 using 0.1M HCl. After reacting at 37 ° C. for 1 hour, antioxidant activity was measured.

2. Measurement of electron donating ability (1,1-diphenyl-2-picrylhydrazyl, DPPH)

The sample was diluted to a concentration of 1,000 ppm in ethanol and used as a sample for analysis. 1 ml of a 0.2 mM DPPH radical was added to 1 ml of the sample, followed by stirring for 30 minutes at room temperature. The absorbance was measured at 517 nm using a spectrophotometer (UV-1600PC, Shimadzu, Tokyo, Japan).

The results are shown in Table 1.

sample Storage period (days) Standard Deviation 0 3 6 9 0.1M GA 85.21 85.36 84.44 83.12 0.5530 0.3M CLA 1.57 2.48 2.59 2.39 0.2667 GA-CLA 94.61 94.16 93.63 93.50 0.1802 GA-CLA under gastric conditions 40.51 39.41 41.16 36.49 0.8431 Standard Deviation 11.232 11.143 10.986 11.063 ※ GA: Gallic acid, CLA: Conjugated linoleic acid, GA-CLA: Gallic acid-conjugated linoleic acid fatty ester

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As shown in Table 1, the electron donating ability of the gallic acid-conjugated linoleic acid fatty ester (GA-CLA) of the present invention can be seen that the radical scavenging ability is superior to 0.1 mol gallic acid, 0.3 mol conjugated linoleic acid. However, when the gallic acid-conjugated linoleic acid fatty ester of the present invention was reacted under the condition of gastric acid, radical scavenging ability was found to be lowered.

3. Peroxide value (POV) measurement

Peroxide prices were measured by the AOAC method. The sample was diluted to a concentration of 1,000 ppm in soybean oil and used as a sample for analysis.

1 ml of the sample was dissolved by adding 35 ml of a mixture of chloroform and acetic acid (2: 3, v / v), 0.5 ml of saturated potassium iodine (KI) was added thereto, stirred, and then stored in a dark place for 5 minutes. Reacted. 75 ml of distilled water was added to the reacted sample, followed by 1 ml of 1% starch solution, and titrated with 0.005N Na ₂ S ₂ O ₃ (Sodium thiosulfate) solution. Peroxide value was calculated | required by following formula (1) as an end point when coloring by starch is lost.

 The results are shown in Table 2.

Peroxide = (A-B) × N × 1,000 / S

※ A: 0.01N Na ₂ S ₂ O ₃ standard solution usage amount (ml)

B: 0.01 N Na ₂ S ₂ O ₃ standard solution used in the test zone (ml)

N: 0.005N Na ₂ S ₂ O ₃ Standard Solution

   S: sampling amount (ml)

sample Storage period (days) Standard Deviation 0 3 6 9 0.1M GA 5.03 6.03 5.53 7.03 0.310 0.3M CLA 6.65 9.03 4.28 4.28 0.748 GA-CLA 4.40 4.28 3.90 3.28 0.455 GA-CLA under gastric conditions 5.90 9.53 12.53 13.78 1.167 Standard Deviation 0.243 0.612 1.327 1.560 ※ GA: Gallic acid, CLA: Conjugated linoleic acid, GA-CLA: Gallic acid-conjugated linoleic acid fatty ester

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As shown in Table 2, the gallic acid-conjugated linoleic acid fatty ester of the present invention exhibited a low peroxide value of 4.40 throughout the storage period from the beginning of storage. However, when the gallic acid-conjugated linoleic acid fatty ester of the present invention was reacted under the condition of gastric acid, the peroxide value increased rapidly with storage period.

4. Determination of fatty acid septic (2-Thiobarbituric acid reactive substances, TBARS)

Fatty acid septic was measured by TBARS, and the sample was diluted to an oil emulsion of 1,000 ppm and used as an analysis sample.

The oil emulsion was added to soybean oil (2%) and Triton X-100 0.2% in distilled water, homogenized with a homogenizer, and used in the experiment. To 5 ml of sample, 15 ml of distilled water and 50 µl of butylated hydroxylanisol (BHA) (7.2%) were added, followed by homogenization using a homogenizer (DIAX 900, Heidolph Co., Ltd., Germany). It was. 4 ml of thiobarbituric acid / trichloroacetic acid (TBS / TCA) was added to 2 ml of the homogenized sample, followed by mixing. The mixed sample was reacted in boiling water for 15 minutes and then cooled in ice water for 10 minutes and centrifuged (698.75 xg, 15 minutes). The supernatant centrifuged was measured for absorbance at 532nm.

The results are shown in Table 3.

sample Storage period (days) Standard Deviation 0 3 6 9 0.1M GA 0.098 0.040 0.318 0.279 0.0287 0.3M CLA 0.078 0.170 0.375 0.349 0.0388 GA-CLA 0.061 0.135 0.187 0.191 0.0166 GA-CLA under gastric conditions 0.095 0.131 0.218 0.212 0.0176 Standard Deviation 0.0117 0.0078 0.0239 0.0196 ※ GA: Gallic acid, CLA: Conjugated linoleic acid, GA-CLA: Gallic acid-conjugated linoleic acid fatty ester

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As shown in Table 3, the TBARS value of the gallic acid-conjugated linoleic acid fatty ester of the present invention was the lowest, and the progress of oxidation was also the slowest depending on the storage period. However, when the gallic acid-conjugated linoleic acid fatty ester of the present invention was reacted under the condition of gastric acid, the TBARS value was relatively high at the beginning of storage, but the progress of oxidation was slow depending on the storage period. For 0.1 mole gallic acid and 0.3 mole conjugated linoleic acid, rancidity developed rapidly after 6 days of storage.

As described above, the gallic acid-conjugated linoleic acid fatty ester of the present invention has superior radical scavenging ability, lower peroxide value, and lower fatty acid saturation than 0.1 mol gallic acid and 0.3 mol conjugated linoleic acid, and thus, has an antioxidant effect. great.

In addition, the gallic acid-conjugated linoleic acid fatty ester of the present invention does not completely lose antioxidant properties even in the human stomach environment, it can be seen that the antioxidant effect is excellent.

Experimental Example 2 Antibacterial Effect of Gallic Acid-Conjugated Linoleic Acid Fat Ester of the Present Invention

In order to determine the antimicrobial effect of the gallic acid-conjugated linoleic acid fatty ester of the present invention, the following experiment was performed.

1. Strain Activity

A total of 11 types of microorganisms were used for the experiment, including fungi (4 types), yeast (1 type), lactic acid bacteria (1 type), and pathogenic microorganisms (5 types).

Among the microorganisms, bacteria were taken from the inoculated slope medium and inoculated into 10 ml of the same liquid medium. 0.1 ml of the culture cultured for 24 hours was taken and inoculated in 10 ml of fresh medium, followed by secondary culture for 18 hours, and the culture was used for the experiment.

Yeast was incubated for 72 hours in the same manner as the bacteria and used in the experiment, the fungus was inoculated on the medium plate and used for the experiment 72 hours.

Strains and media used in the experiments are shown in Table 4.

Microbial strain Culture medium Culture temperature (℃) mold Aspergillis flavus (KCCM 11453) PDB & PDA 25 Aspergillis usami (KCTC 6958) PDB & PDA 25 Muco hiemalis (KCCM 12347) PDB & PDA 25 Penicillin verrucolusom (KCTC 6166) PDB & PDA 25 leaven Zygosaccaromyces rouxii (KCTC 7191) PDB & PDA 25 Lactic acid bacteria Leuconostoc mensenteroides (KCTC 3100) MRSB & MRSA 37 Germ Bacillus cereus (KCTC 1012) TSB & TSA 37 Salmonella Typhimurium (KCTC 1925) NB & NA 37 Fserichia coli (KCTC 1682) TSB & TSA 37 Staphilococcus aureus (KCTC 1916) TSB & TSA 37 Listeria ivanova (KCTC 3444) TSB & TSA 30 ※ PDB & PDA: Potato dextrose broth & Potato dextrose agar (Difco) MRSB & MRSA: Lactobacilli MRS broth & Lactobacilli MRS agar (Difco) TSB & TSA: Tryptic soy broth & Tryptic soy agar (Difco) NB & NA: Nutrient broth & Nutrient agar (Difco)

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2. Antimicrobial Activity Test

The antimicrobial activity test of the gallic acid-conjugated linoleic acid fatty ester of the present invention was carried out using a disc diffusion method.

Bacteria and yeast were applied uniformly after 0.1 ml of activated culture was dropped on agar plate dried overnight at room temperature. A spore suspension was prepared using 80 solutions and 0.1 ml of the suspension was evenly applied to the medium plate.

The microorganisms used in this experiment were the optimum medium according to each strain, the initial concentration of the strain used in the experiment was 10 ⁷ ~ 10 ⁸ CFU / ㎖ level.

Place the filter paper (8.0 mm diameter) in which 20 μl of the gallic acid-conjugated linoleic acid fatty ester prepared in the above example was absorbed on a plate inoculated with each test strain and incubated at an optimum temperature according to the strain for 72 hours, and then around the disc. The antimicrobial activity was measured by the size of the formed transparent zone (mm).

The results are shown in Table 5.

Microbial strain Diameter of the transparent zone (mm) mold Aspergillis flavus (KCCM 11453) 13 (disc diameter 8.0mm) Aspergillis usami (KCTC 6958) 14 Muco hiemalis (KCCM 12347) 10 Penicillin verrucolusom (KCTC 6166) 12 leaven Zygosaccaromyces rouxii (KCTC 7191) No antimicrobial effect Lactic acid bacteria Leuconostoc mensenteroides (KCTC 3100) 12 Germ Bacillus cereus (KCTC 1012) No antimicrobial effect Salmonella Typhimurium (KCTC 1925) No antimicrobial effect Fserichia coli (KCTC 1682) No antimicrobial effect Staphilococcus aureus (KCTC 1916) No antimicrobial effect Listeria ivanova (KCTC 3444) 9

As shown in Table 5, the antimicrobial effects of the gallic acid-conjugated linoleic acid esters of the present invention are fungi (Aspergillus flavus, Aspergillus usami, Muce hiemalis, Penicillin verrucolusom), Lactic acid bacteria (Leuconostoc mensenteroides) and bacteria (Listeria ivanova), etc. Six strains showed antimicrobial activity, and among them, antimicrobial activity was very excellent against fungi and some bacteria.

3. Antimicrobial Effect According to Concentration of Gallic Acid-Conjugated Linoleic Acid Fat Ester of the Present Invention

Antibacterial effect according to the concentration of the gallic acid-conjugated linoleic acid fatty ester of the present invention, the antimicrobial activity using the disc diffusion method of the above 2 only to the strains that showed antimicrobial activity against the gallic acid-conjugated linoleic acid fatty ester Measured.

To the sterilized medium was added a concentration of 0.1 mole gallic acid, 0.3 mole conjugated linoleic acid and the gallic acid-conjugated linoleic acid fatty ester prepared in the above Example and activated microbial culture was added to each medium in 1% ( v / v) was inoculated, and cultured for 72 hours and measured using standard agar culture.

The viable cell counts of yeast, mold and bacteria were counted after 72 hours of incubation at the optimum medium and the optimum temperature of each strain, and the number of microorganisms was expressed as colony forming unit (CFU) per 1 ml of sample. At this time, in order to exclude the antimicrobial activity of ethanol itself used to dissolve the gallic acid-conjugated linoleic acid fatty ester, only ethanol was added as a control concentration, and the minimum count limit for detection was 10 ¹ CFU / mL. .

The results are shown in Table 6.

process Concentration (ppm) mold Lactic acid bacteria Germ Aspergillis flavus Aspergillis usami Muco hiemalis Penicillin verrucolusom Leuconostoc mensenteroides Listeria ivanova Starting point 6.49 6.56 7.60 6.79 6.70 6.48 Control 6.31 7.30 7.20 6.70 9.02 9.31 0.1M GA 10,000 3.96 5.40 4.48 5.81 4.03 3.89 1,000 5.53 5.60 4.70 6.70 7.39 8.06 0.3M CLA 10,000 5.71 5.65 2.78 3.00 3.65 3.49 1,000 7.49 6.60 4.95 6.30 7.93 8.00 GA-CLA 10,000 4.48 Mold not detected Mold not detected 2.95 Mold not detected Mold not detected 1,000 7.08 5.61 5.15 5.90 Mold not detected 7.68

※ GA: Gallic acid, CLA: Conjugated linoleic acid,

   GA-CLA: Gallic acid-conjugated linoleic acid fatty ester

As shown in Table 6, the gallic acid-conjugated linoleic acid fatty ester of the present invention inhibited the growth of 1 to 2 log with respect to the fungus when 1,000 ppm was added, and Aspergillus usami and Muco hiemalis were added when the 10,000 ppm was added. Was completely inhibited. In addition, the growth of lactic acid bacteria (Leuconostoc mensenteroides) was added to 1,000ppm and 10,000ppm completely inhibited the growth. In addition, bacteria (Listeria ivanova) was completely inhibited in the growth when added to 10,000ppm.

Therefore, it can be seen that the gallic acid-conjugated linoleic acid fatty ester of the present invention has superior antimicrobial effect than 0.1 mol gallic acid and 0.3 mol conjugated linoleic acid.

Experimental Example 3 : Anti-inflammatory Effect of Gallic Acid-Conjugated Linoleic Acid Fat Esters of the Present Invention

In order to determine the anti-inflammatory effect of the gallic acid-conjugated linoleic acid fatty ester of the present invention, the following experiment was performed.

Cyclooxygenase (COX) enzymes are in the first and second process of biosynthesis of prostaglandins based on arachidonic acid. The enzyme exists in two forms. First, COX-1 is expressed systematically to maintain normal physiological function, and prostaglandins produced by this enzyme play a protective role. COX-2 is expressed under the influence of external stimuli such as mitogens, oncogenes, tumor promoters, growth factors, and the like. COX-2 is an isomer involved in inflammation, and induction of COX-2 causes the production of prostaglandins at the site of inflammation. Thus, selective inhibition of COX-2 has the same therapeutic effect as nonsteroidal anti-inflammatory drugs. Several selective COX-2 inhibitors such as celecoxib, valdecoxib, parecoxib, rofecoxib, etoricoxib, lumiracoxib, and the like Is used in clinical practice and is mainly used as an inhibitor of inflammation of rheumatoid arthritis or osteoarthritis. COX-2 inhibitors are also used to prevent and treat cancer or to reduce the risk of Alzheimer's or Parkinson's disease. However, commercial drugs using these COX-2 inhibitors have many side effects, and it has been reported that COX-2 knockout mice also express inflammation (Buttar, NS, Wang, KK 2000. The "aspirin" of the new millennium: cyclooxygenase-2 inhibitors.Mayo. Clin. Proc. 75: 1027-1038.), And also both COX-1 and COX-2 enzymes are involved in inflammation and some COX enzymes It is reported that further contribution to the action depends on the type of inflammation, the site of expression and the stimulation of inflammation (Hu, KO 2003. Cyclooxygenase-2 (COX-2) -prostanoid pathway and liver diseases.Prostaglandiins, Leukotrienes and Essential Fatty acids 69: 329-337.).

COX-1 and COX-2 inhibitory effects were determined by measuring prostaglandin E ₂ (PGE ₂ ) using a COX inhibitor screening kit (Catalog No. 560131, Cayman Chemicals, Ann Arbor, Michigan, USA).

100 mM Tris-HCl buffer [containing pH 8.0, 1 μM heme and COX-1 (ovine) or COX-2 (human recombination)] was preheated in a 37 ° C. water bath for 10 minutes and then reacted by injecting 10 μl of arachidonic acid. Was initiated. At this time, the final concentration of the reaction solution was 100 μM. After 2 minutes, 1 M HCl was injected to terminate the reaction, and PGE ₂ was quantified by ELISA. The sample was dissolved in DMSO and distilled water, and then the concentration was adjusted using 100 mM phosphate buffer (pH 7.4). The reaction solution was transferred to a 96-well plate coated with mouse anti-rabbit IgG and tracer prostagalndin acetylcholine esteras and a primary antibody (mouse anti PGE ₂ ) were added. The plate was left at room temperature for 12 hours before the reaction solution was removed and washed with 10 mM phosphate buffer containing 0.05% Tween 20. Ellman's reagent (200 μl) was added to each well and allowed to stand for 60 minutes at room temperature in the dark so that the OD value of the control wells was 0.3-0.8 at 412 nm. Standard curves were drawn using PGE ₂ and quantitatively analyzed using wells to which samples were added. The results showed the inhibitory effect as a percentage of the treatment compared to the control (sample containing only solvent). In addition, the IC ₅₀ value was also obtained by the following equation.

The results are shown in Table 7.

% Inhibition of COX-1 (or COX-2) = [(I _c -S _c ) / I _c ] × 100

※ I _c : Concentration of PGs (prostagladins) before sample processing

S _c : concentration of PGs (prostagladins) after sample treatment

Concentration of PGs (I _c ) before sample treatment for COX-1 = 3.44

Concentration of PGs (I _c ) before sample treatment for COX-2 = 2.18

Cyclooxygenase Inhibitory Effect of the Gallic Acid-Conjugated Linoleic Acid Fatty Ester of the Present Invention (Anti-inflammatory Effect) density % Inhibition  COX-1 COX-2 Gallic acid-conjugated linoleic acid fatty ester 0.01 (μM) 55 10 0.1 (μM) 64 21 1.0 (μM) 67 28 10 (μM) 74 32

As shown in Table 7, the gallic acid-conjugated linoleic acid fatty ester of the present invention had a 74% COX-1 enzyme inhibition rate and a 32% COX-2 enzyme inhibition rate at a low concentration of 10 μM. This anti-inflammatory effect is higher than celecoxib currently used as an anti-inflammatory agent.

According to Muria et al., Celecoxib requires a concentration of 19 μM for COX-1 inhibition rate of 50% (IC ₅₀ ) and 35 nM for COX-2 (Murias et al. (2004)). Resveratrol analogues as selective cycloxygenase-2 inhibitors: synthesis and structure-activity relationship. Bioorganic & Medical Chemistry 12: 5571-5578.]. The celecoxib of COX-1 and COX-2 in (IC ₅₀₎ and gallic acid of the present invention as compared to (IC ₅₀₎ of the COX-1 and COX-2 in the conjugate gated linoleic acid, fatty esters, celecoxib a (IC ₅₀₎ of the COX-1 in the conjugate gated linoleic acid, fatty esters is high inhibition rate of more than 900-fold with 10 nM (0.01 μM) - a (IC ₅₀₎ was 19 μM, whereas gallic acid of the present invention the COX-1 Indicated. (IC ₅₀₎ of the COX-2 in also celecoxib is 35 nM, whereas gallic acid of the present invention - (IC ₅₀₎ of the COX-2 of conjugate gated linoleic acid fatty ester is at least 900-fold greater inhibition by 6.2 μM Indicated.

Therefore, the novel gallic acid-linoleic acid fatty esters of the present invention have excellent COX-1 and COX-2 inhibition rates, and thus, are effective in rheumatoid arthritis, osteoarthritis and other inflammations.

Experimental Example 4 : Skin whitening effect of the gallic acid-conjugated linoleic acid fatty ester of the present invention

In order to determine the skin whitening effect of the gallic acid-conjugated linoleic acid fatty ester of the present invention, the tyrosinase inhibition rate was measured.

Tyrosinase inhibition experiments consisted of 100 mL / mL mushroom tyrosinase 0.2 mL, 10 mM DOPA (L-3,4-dihydroxyphenyl-alanine) 0.4 mL as substrate, phosphate buffer (0.07 M, sodium phosphate buffer, pH). 6.8) 0.2 ml of the sample solution was added to 0.2 ml of the mixed solution and reacted at 37 ° C. for 30 minutes to measure absorbance at 475 nm. The change in dopachrome is shown in Table 8 by converting the difference in absorbance between the sample solution-free addition and the sample solution addition into the inhibition rate.

Tyrosinase Inhibition Rate (%)-0.1% Mixture Conjugated Linoleic Acid 55.7 Gallic acid-conjugated linoleic acid fatty ester 63.6

As shown in Table 8, it was confirmed that the tyrosinase inhibition rate of the gallic acid-conjugated linoleic acid fatty ester of the present invention was 63.5%. Therefore, the gallic acid-conjugated linoleic acid fatty ester of the present invention can be usefully used as a cosmetic material for skin whitening.

Examples of preparations for the compositions of the present invention are illustrated below.

Formulation example  One  : Preparation of Pharmaceutical Formulations

Powder

2 g of gallic acid-conjugated linoleic acid fatty acid ester

1g lactose

The above ingredients were mixed and filled in airtight cloth to prepare a powder.

2. Tablet

Gallic acid-conjugated linoleic acid fatty ester of formula 1 500 mg

Corn Starch 100mg

Lactose 100mg

2 mg magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

3. Capsule

Gallic acid-conjugated linoleic acid fatty ester of formula 1 500 mg

Corn Starch 100mg

Lactose 100mg

2 mg magnesium stearate

After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.

4. Injection solution

Gallic acid-conjugated linoleic acid fatty ester of formula 1 500 mg

Maintain citrate buffer pH 3.5

Dextrose appearance

The sterilized 20 ml injection vial was filled with gallic acid-conjugated linoleic acid fatty ester, citrate sodium salt, citric acid and dextrose, and sealed with an aluminum cap. It was prepared by dissolving the above mixture using distilled water for injection and then diluting with an appropriate volume of distilled water for injection.

Formulation example  2  : Preparation of Beverages

1.carbonated drinks

5-10% of sugar, 0.05-0.3% citric acid, 0.005-0.02% caramel, 0.1-1% of vitamin C are mixed, and 79-94% purified water is mixed to make syrup, and the syrup is 85-98 After sterilizing at 20 ° C. for 20 seconds to 180 seconds, the mixture was mixed with cooling water at a ratio of 1: 4, and 0.5 to 0.82% of carbonic acid gas was injected to prepare a carbonated beverage containing gallic acid-conjugated linoleic acid ester of Chemical Formula 1.

2. Health drink

Homogeneously blends minor ingredients such as liquid fructose (0.5%), oligosaccharides (2%), sugars (2%), salts (0.5%), water (75%) and gallic acid-conjugated linoleic acid esters After the instant sterilization it was packaged in a small packaging container such as glass bottles, plastic bottles to prepare a healthy beverage.

3. Vegetable Juice

0.5 g of the gallic acid-conjugated linoleic acid fatty ester of Formula 1 was added to 1,000 ml of tomato or carrot juice to prepare a vegetable juice for health promotion.

4. Fruit Juice

Health promotion fruit juice was prepared by adding 0.1 g of gallic acid-conjugated linoleic acid fatty ester of Formula 1 to 1,000 ml of apple or grape juice.

Formulation example  3  : Cosmetics  Preparation of the formulation

1. Flexible cosmetics (content: wt%)

Gallic acid-conjugated linoleic acid fatty ester of formula 1 0.01

Glycerin 3.0

Butylene Glycol 2.0

Propylene Glycol 2.0

Carboxy Vinyl Polymer 0.1

Ethanol 10.0

Triethanolamine 0.1

Preservative, Micro Pigment, Micro-Fragrance, Micro Purified Water Balance System 100.0

2. Nutritional Cosmetics (Content: Weight%)

Gallic acid-conjugated linoleic acid fatty ester of formula 1 0.01

Beeswax 4.0

Polysorbate 60 1.5

Solbitan Sesquioleate 0.5

Floating paraffin 5.0

Squalane 5.0

Caprylic / Capric Triglycerides 5.0

Glycerin 3.0

Butylene Glycol 3.0

Propylene Glycol 3.0

Carboxy Vinyl Polymer 0.1

Triethanolamine 0.2

Preservative, Micro Pigment, Micro-Fragrance, Micro Purified Water Balance System 100.0

3. Nutrition Cream (Content: Weight%)

Gallic acid-conjugated linoleic acid fatty ester of formula 1 0.005

Beeswax 10.0

Polysorbate 60 1.5

Solbitan Sesquioleate 0.5

Floating paraffin 10.0

Squalane 5.0

Caprylic / Capric Triglycerides 5.0

Glycerin 5.0

Butylene Glycol 3.0

Propylene Glycol 3.0

Triethanolamine 0.2

Preservative, Micro Pigment, Micro-Fragrance, Micro Purified Water Balance System 100.0

4. Massage cream (content: wt%)

Gallic acid-conjugated linoleic acid fatty ester of formula 1 0.005

Beeswax 10.0

Polysorbate 60 1.5

Solbitan Sesquioleate 0.8

Floating paraffin 40.0

Squalane 5.0

Caprylic / Capric Triglycerides 4.0

Glycerin 5.0

Butylene Glycol 3.0

Propylene Glycol 3.0

Triethanolamine 0.2

Preservative, Micro Pigment, Micro-Fragrance, Micro Purified Water Balance System 100.0

5. Pack (Content: Weight%)

Gallic acid-conjugated linoleic acid fatty ester of formula 1 0.005

Polyvinyl Alcohol 13.0

Sodium Carboxymethylcellulose 0.2

Allantoin 0.1

Ethanol 5.0

Nonylphenyl Ether 0.3

Preservative, Micro Pigment, Micro-Fragrance, Micro Purified Water Balance System 100.0

The gallic acid-conjugated linoleic acid fatty ester of the present invention has excellent electron donating ability (radical scavenging), low peroxide value, and low fatty acid saturation, which is excellent in antioxidant activity, and excellent in antibacterial activity against mold and bacteria. , COX-1 and COX-2 inhibition rate is excellent anti-inflammatory effect, tyrosinase inhibition rate is high, so skin whitening effect is excellent.

Therefore, the composition of the present invention can be usefully used for antioxidant, antibacterial, anti-inflammatory and skin whitening.

Claims (9)

Gallic acid-conjugated linoleic acid fatty esters represented by Formula 1 below: <Formula 1>

In Chemical Formula 1, R ₁ to R ₄ are each independently hydrogen or conjugated linoleic acid (CLA), CLA is 9-cis, 11-trans octadecenoic acid; 9-trans, 11-cis octadecenoic acid; 10-cis, 12-trans octadecenoic acid; Or 10-trans, 12-cis octadecenoic acid, except when R ₁ to R ₄ are all hydrogen. 1) dissolving gallic acid and conjugated linoleic acid in an organic solvent in a 1: 3 molar ratio, followed by addition of a base to react; 2) adding ethyl acetate to the reaction solution to separate the layers, and then collecting and concentrating the supernatant. A method for producing a gallic acid-conjugated linoleic acid ester of claim 1 comprising a. The method of claim 2, wherein the organic solvent in step 1) is selected from tetrahydrofuran, dimethylformamide, and acetonitrile according to claim 1, wherein the gallic acid-conjugated linoleic acid fatty ester is prepared. The method according to claim 2, wherein the base in step 1) is triethylamine and WSCD (1,3-dicyclohexyl carbodiimide), wherein the gallic acid-conjugated linoleic acid fatty ester of claim 1 is used. delete The antimicrobial composition containing the gallic acid-conjugated linoleic acid fatty ester of Claim 1. The anti-inflammatory composition containing the gallic acid-conjugated linoleic acid fatty ester of Claim 1. 8. The composition of claim 7, wherein the composition is useful for the prevention and treatment of rheumatoid arthritis or osteoarthritis. A composition for skin whitening comprising the gallic acid-conjugated linoleic acid fatty ester of claim 1.

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