KR101253354B1 - Composition containing flavonol glycosides of green tea for inhibiting protein glycation reaction - Google Patents

Abstract

The present invention contains the green tea extract or flavonol glycosides derived from the green tea extract to inhibit the production of intermediates or advanced products (AGEs; advanced glycation end-products) by protein glycation reaction (protein glycation) reaction It relates to a composition to be. By using this, it can be utilized as a cosmetic, medical or food compositions having a wrinkle improvement effect.

Green tea, saccharification, glycation end product (AGEs), flavonol glycoside, wrinkle improvement

Description

Composition containing flavonol glycosides of green tea for inhibiting protein glycation reaction

The present invention is a composition containing a flavonol glycoside component isolated and purified from green tea extract or green tea extract to inhibit the production of intermediates or end products (AGEs; advanced glycation end-products) by protein glycation reaction (protein glycation) reaction It is about.

Glycation reaction (protein glycation) is the initial reaction of the Mailrad reaction, a non-enzymatic condensation reaction between reducing sugars such as glucose and amino groups of the protein, also called nonenzymatic glycosylation. Many types of reducing sugars, including glucose present in vivo, and α-hydroxy aldose or ketose, are carbonyl compounds, which cause glycation reactions with amino acids and proteins having free amino groups. In addition, the glycosylation reaction is reported to impair the function of biopolymers such as proteins.

In particular, the glycosylation reaction between reducing sugars and proteins in the body, such as glucose and fructose, has been focused on diabetes complications and skin aging. In other words, if this reaction continues, the protein is permanently denatured through cyclization, cleavage, dehydration and oxidation. When diabetes has a high level of blood sugar for a long time, proteins such as collagen and crystalline are denatured to cause diabetes complications.

As a result of studies on substances that inhibit AGE production, a hydrazine compound called aminoguanidine is known as a synthetic therapeutic agent that specifically inhibits AGE production. Reports that the administration of the former aminoguanidine could prevent the development of complications in diabetic experimental animals support the importance of these AGEs in diabetic complications. Although these aminoguanidines are considered non-toxic (LD50 = 1800 mg / kg, rodents), recent studies show that overdose of aminoguanidine produces hydrogen peroxide and inhibits catalase. It can promote the side effects associated with inhibition of catalase (acatalasaemia). Therefore, there has been an effort to find a material having an inhibitory effect on protein glycosylation and ensuring safety.

In Korean Patent No. 2003-60440, "Extracts with Protein Glycation Inhibitory Effect," the protein glycosylation inhibitory effect was compared and searched for 85 plant sources, including domestic and foreign native plants and herbal medicines. In 27 species, it was confirmed that there was an inhibitory effect of at least 10% glycosylation reaction and the composition including the same was specified. Japanese Patent No. 2001-316221A "Anti-Aging Cosmetic" discloses an anti-aging agent and a cosmetic as a maylad reaction inhibitor containing cherry blossoms, Honggyeongcheon, blue egg, bear show, etc., and Japanese Patent No. 2001-58790 " Protein Modification Inhibitors and Methods "discloses formamide oxime derivatives having okisazazole or pharmacologically acceptable salts thereof for the use of protein modification inhibitors. In addition, Japanese Patent No. 2006-63024 "peptides having an affinity for glycosylation end products and adsorbents using the same", to remove glycosylation end products (AGEs) known to be harmful to the human body, peptides, adsorbents and Adsorption apparatus is provided. U.S. Patent US7030133, "Inhibitors of formation of advanced glycation endproducts (AGEs)," discloses that 13 derivatives effectively inhibit glycosylation reactions, thereby inhibiting aging, tooth discoloration, and food protein degeneration.

Green tea is produced by inactivating oxidase present in tea leaves by heat or steam by extracting the shoots or leaves of Camellia sinensis , a plant of Theaceae. As the pharmacological mechanisms of the various components become increasingly known, their value is also recognized by the general public. In particular, recently, the effects of antioxidants, anticancer action, blood cholesterol lowering action, anti-aging action, heavy metal detoxification action, caries prevention and bad breath removal action by polyphenol, which is the main component of green tea, have been attracting much attention. Among these polyphenols, flavonol glycosides and flavone glycosides, etc., are combined to account for about 1-3% of the dry weight of green tea leaves.

In order to solve the problems described above, the inventors of the present invention have found that green tea and green tea components, particularly flavonol glycoside components, have excellent safety and inhibitory effect on glycation reaction, while looking for a glycosylation inhibitor that inhibits the AGE. This invention was completed.

It is therefore an object of the present invention to provide a composition which contains green tea extract or flavonol glycoside components derived from green tea extract to inhibit the production of intermediates or end products (AGEs) by the saccharification reaction. In addition, another object of the present invention is to provide a composition having an anti-wrinkle effect by containing a green tea extract or a flavonol glycoside component derived from the green tea extract to inhibit the glycation reaction.

In order to achieve the above object, the present invention provides a composition for inhibiting protein glycosylation reaction containing at least one flavonol glycosides derived from green tea as an active ingredient.

In the composition for inhibiting protein glycosylation of the present invention, the flavonol glycosides are flavonol monosaccharide glycosides, and the flavonol monosaccharide glycosides are flavonol galactose glycosides.

In the composition for inhibiting protein glycosylation of the present invention, the flavonol glycosides are quercetin glycosides.

In addition, in the composition for inhibiting protein glycation reaction of the present invention, the flavonol glycosides are characterized in that it is contained in the form of green tea extract.

In the present invention, the composition for inhibiting the protein glycosylation reaction is characterized by having an anti-wrinkle effect.

In the present invention, the composition is characterized in that the cosmetic composition, a pharmaceutical external preparation composition or an edible composition.

In the present invention, the flavonol glycosides are contained in an amount of 0.0001% to 10% by weight based on the total weight of the composition.

The present invention also provides a topical skin composition comprising at least one flavonol glycosides derived from green tea as an active ingredient.

In the external preparation composition for skin of the present invention, the composition is a cosmetic composition or a pharmaceutical external preparation composition.

Hereinafter, the present invention will be described in detail.

The present invention relates to a composition containing one or more flavonol glycosides derived from green tea as an active ingredient to inhibit the production of intermediates or end products (AGEs; Advanced Glycation End-products) by glycation of proteins. .

At least one flavonol glycoside derived from the green tea is contained in an amount of 0.0001% to 10% by weight based on the total weight of the composition of the present invention. If the content is lower than 0.0001%, it may not show an effective effect. If the content is higher than 10%, there is a problem in formulation stability such as discoloration and odor.

In addition, the use of the composition can be utilized for cosmetics, medicinal and edible.

When applied to medicines, oral or parenteral dosage forms in solid, semi-solid or liquid form by adding a commercially available inorganic or organic carrier using at least one flavonol glycoside derived from green tea according to the present invention as an active ingredient. It may be formulated.

Examples of preparations for oral administration include tablets, pills, granules, soft / hard capsules, powders, fine granules, powders, emulsions, syrups, pellets, and the like. Can be. Preparations for parenteral administration include injections, drops, ointments, lotions, sprays, suspensions, emulsions, suppositories, and the like. In order to formulate the active ingredient of the present invention, it can be easily formulated according to the conventional method, and surfactants, excipients, coloring agents, spices, preservatives, stabilizers, buffers, suspensions, and other commonly used auxiliaries can be suitably used.

The pharmaceutical composition according to the present invention may be administered orally, parenteral, rectal, topical, transdermal, intravenous, intramuscular, intraperitoneal, subcutaneous.

In addition, the dosage of the active ingredient will depend on the age, sex, body weight of the subject to be treated, the particular disease or condition to be treated, the severity of the disease or condition, the route of administration and the judgment of the prescriber. Dosage determination based on these factors is within the level of those skilled in the art, and generally the dosage ranges from 0.001 mg / kg / day to approximately 2000 mg / kg / day. A more preferred dosage is 0.5 mg / kg / day to 2.5 mg / kg / day.

Hereinafter, the present invention will be described in more detail with reference to the following specific examples, which are merely illustrative of the present invention and the scope of the present invention is not limited thereto.

[ Example  1-8] Preparation of Green Tea Extract and from Green Tea Extract Flavonol  Isolation and Purification of Glycoside Components

Green tea extract is prepared according to the following procedure. First, 1kg of green tea samples (Jeju-do green tea, first tea, first tea, steamed tea, yellow tea) are shaken at 25 ° C. for 12 hours with a 16:64:20 H ₂ O / MeOH / CH ₂ Cl ₂ mixed solvent (100 rpm). Thereafter, only the liquid is stored and the extraction is repeated once more. The two extracts thus obtained are combined and concentrated. Add 1: 2 H ₂ O / MeOH and extracted twice with hexane (hexane), and concentrated completely to make a green tea extract.

The separation and purification of flavonol glycoside components from green tea is as follows. The sample used for the component separation was the same as the sample used in the preparation of the extract, the extraction was extracted four times using 3L 3:27:70 H ₂ O / MeOH / CH ₂ Cl ₂ mixed solvent on the basis of 1kg of green tea. The extract was concentrated, extracted four times with 2 L of hexane, and the remaining water layer was extracted four times with 2 L butanol. Each of the three layers thus obtained (hexane layer, butanol layer, and water layer) was 130 g, 300 g, and 60 g. A portion of the butanol layer was first divided into 11 portions using C-18 reverse phase chromatography. Each of these was again separated into pure components using silica gel chromatography or HPLC. The hexane layer was divided into 35 parts using silica gel chromatography and separated again into pure components using silica gel or HPLC. Identification of each component isolated was carried out by NMR (1H, 13C, COZY, HSQC, HMBC, NOESY) experiment, LC-MS (ESI).

Table 1 below shows the main component list and content of the green tea extract, and the name and content of the main flavonol glycoside isolated from the extract of the butanol layer.

division ingredient content
green tea
Of extract
Major Components
(g / kg of tea)
Example 1 Epigallocatechin Gallate (EGCG)  50 ± 10 ECG  4 ± 0.3 EGC 28 ± 6 EC 11.4 ± 3.3 Caffeine 24 ± 3.8 Flavonol glycosides
(Flavonol glycosids) 25 ± 5.3


Flavonol glycosides
detach
(mg / kg of tea)


Example 2 Camperol galactose glycosides
(Kaempferol galactoside) 11 Example 3 Camperol Glucose Glycosides
(Kaempferol glucoside) 50.5 Example 4 Camperol Glucosyl-Rhamnose Glycoside
(Kaempferol glucosyl-rhamnoside) 31.4 Example 5 Quercetin galactose glycosides
(Quercetin galactoside) 30 Example 6 Quercetin glucose glycosides
(Quercetin glucoside) 20.1 Example 7 Myricetin Galactose Glycoside
(Myricetin galactoside) 9.3 Example 8 Myricetin Glucose Glycoside
(Myricetin glucoside) 19

[ Test Example  1] Protein Glycation ( protein glycation ) Determination of reaction inhibition effect

Protein glycosylation reaction of green tea extract and flavonol glycoside components obtained in Examples 1 and 2 by performing a saccharification reaction using albumin protein (Bovine Serum Albumin, "BSA") obtained from bovine serum as a reaction substrate and measuring the degree of fluorescence change Inhibitory effect was measured.

First, add 0.2 ml of BSA solution (10 mg / ml), 0.4 ml of glucose solution (225 mg / ml), 0.3 ml of PBS buffer (pH 7.4), and 0.1 ml of sample solution, and place it in a 1.5 ml eppendorf tube. Reaction was carried out for 24 hours. At this time, each extracted sample was added to the sample solution for each concentration, and the fluorescence generation inhibitory effect of each sample was determined by comparing the degree of fluorescence. Fluorescence was measured by 360 nm excitation and 460 nm emission using a fluorometer. The reaction concentration and AGE production inhibitory effect of each sample are shown in Table 2. Inhibition rate was calculated by the following formula using each measurement of fluorescence intensity and is the average value of each of three replicates.

Inhibition rate = 100-{(sample-blank value) / (negative control-blank value)} X100

division Ingredients Concentration (ug / ml) Inhibition rate (%) Comparative Example 1 Untreated group (voice control) - 0

Comparative Example 2


Aminoguanidine HCl
(Aminoguanidine HCl)
(Positive control)
0.3 18.1 ± 3.8 One 35.3 ± 3.0 3 29.7 ± 4.5 10 48.5 ± 0.7 30 56.9 ± 7.9

Example 1



Green tea extract

0.3 25.3 ± 3.2 One 33.4 ± 4.5 3 51.2 ± 0.4 10 65.1 ± 3.5 30 78.3 ± 10.1

Example 2



Camperol galactose glycosides

0.3 36.7 ± 2.4 One 36.7 ± 2.4 3 44.9 ± 2.3 10 68.2 ± 2.9 30 92.6 ± 17.5

Example 3



Camperol Glucose Glycosides

0.3 43.9 ± 6.1 One 41.1 ± 7.2 3 42.3 ± 12.5 10 65.7 ± 11.6 30 92.8 ± 2.0

Example 4



Camperol Glucosyl-Rhamnose Glycoside

0.3 44.8 ± 3.3 One 50.1 ± 6.2 3 36.2 ± 9.8 10 50.7 ± 12.0 30 88.4 ± 4.4

Example 5



Quercetin galactose glycosides

0.3 41.1 ± 0.8 One 39.8 ± 5.2 3 52.4 ± 3.6 10 79.5 ± 0.1 30 95.0 ± 0.1

Example 6


Quercetin glucose glycosides


0.3 42.3 ± 0.9 One 55.3 ± 2.0 3 46.9 ± 3.4 10 76.0 ± 0.1 30 112.0 ± 0.6

Example 7



Myricetin Galactose Glycoside

0.3 30.4 ± 4.7 One 43.4 ± 2.1 3 49.2 ± 3.5 10 67.9 ± 0.1 30 87.3 ± 4.6

Example 8



Myricetin Glucose Glycoside

0.3 37.5 ± 4.2 One 45.9 ± 0.7 3 44.7 ± 1.3 10 59.9 ± 3.5 30 83.2 ± 2.7

As can be seen in Table 2, a single flavonol glycoside compound component (Examples 2 to 8) or green tea extract (Example 1) isolated and purified from green tea is an intermediate or final product by glycosylation of protein. It was confirmed that significantly inhibit the production of (AGEs). That is, seven kinds of flavonol glycosides, 3-O glycosides of kaempferol, quercetin, and myricetin, which are flavonoids, are all positive control aminoguanidine-HCl (aminoguanidine-HCl). , Showed a superior protein glycosylation inhibitory effect than Comparative Example 2).

This inhibitory effect is superior to monosaccharide glycosides (Example 2-3) than disaccharide glycosides (Example 4), galactose glycosides (Examples 2, 5, 7) are superior to glucose glycosides (Examples 3, 6, 8) Among the entire group, quercetin glycosides (Examples 5 and 6) were the best. In addition, the same inhibitory effect was obtained in the green tea extract (Example 1), which may be due to the role of flavonol glycosides contained in the green tea extract or synergistic action with catechins (EGCG, ECG, EGC, EC).

Example 9-11 Preparation of Flavonol Glycoside Mixture-Containing External Preparation Composition

Flavonol glycoside mixtures to be used in the following tests were prepared by the following procedure. First, extraction was performed four times using 3 L of 3:27:70 H ₂ O / MeOH / CH ₂ Cl ₂ mixed solvent based on 1 kg of green tea. The extract was concentrated, extracted four times with 2 L of hexane, and the remaining water layer was extracted four times with 2 L butanol. Each of the three layers thus obtained (hexane layer, butanol layer, and water layer) was 130 g, 300 g, and 60 g. Part of the butanol layer was obtained by fractionating a flavonol glycoside mixture by C-18 reverse phase chromatography (about 10% yield).

Table 3 below shows the blending components and contents of the external preparation composition containing the flavonol glycoside mixture prepared in the above process (Example 9-11) and the external preparation composition containing the flavonol glycoside mixture (Comparative Example 3). .

Compounding ingredient Example 9
(weight%) Example 10
(weight%) Example 11
(weight%) Comparative Example 3
(weight%) Purified water To 100 To 100 To 100 To 100 Flavonol Glycoside Mixture 0.001 0.05 2.5 - Vegetable hydrogenated oil 1.50 1.50 1.50 1.50 Stearic acid 0.60 0.60 0.60 0.60 Glycerol stearate 1.00 1.00 1.00 1.00 Cetaleyl Alcohol 2.00 2.00 2.00 2.00 Polyglyceryl-10 Pentastearate & Behenyl Alcohol & Sodium Stearoyl Lactylate 1.00 1.00 1.00 1.00 Arachidil Behenyl Alcohol & Arachidil Glucoside 1.00 1.00 1.00 1.00 Cetylaryl Alcohol & Cetearyl Glucoside 2.00 2.00 2.00 2.00 PEG-100 Stearate & Glycerol Olate & Propylene Glycol 1.50 1.50 1.50 1.50 Caprylic / Carric Triglycerides 11.00 11.00 11.00 11.00 Cyclomethicone 6.00 6.00 6.00 6.00 Preservatives and Incense Suitable amount Suitable amount Suitable amount Suitable amount Triethanolamine 0.10 0.10 0.10 0.10

[ Test Example  2] Flavonol  Wrinkle improvement effect of glycosides

Animal experiment model was used to confirm the effect of improving the production of wrinkles by inhibition of protein glycosylation of the prepared flavonol glycoside mixture-containing external preparation composition. Sixty hairless mice were raised in a constant temperature and humidity room and divided into four groups of 15 rats in each group. The compositions of Comparative Example 3 and Examples 9 to 11 were applied to the back region daily for 8 weeks, respectively, and a replica of the dorsal portion was floated using silicone, followed by a visometer (SV600, C & K electronics GmbH, Germany) was used to measure the depth of wrinkles and to evaluate the degree of wrinkle improvement. The results are shown in Table 4.

division Corrugation depth (mm) Example 9 0.37 ± 0.04 Example 10 0.31 ± 0.03 Example 11 0.25 ± 0.01 Comparative Example 3 0.41 ± 0.02

As can be seen from Table 4, the external preparation composition containing the flavonol glycoside mixture (Examples 9 to 11) has a much better antiwrinkle effect in the animal compared to the external preparation composition (Comparative Example 3) which does not contain the flavonol glycoside mixture. It was confirmed that the excellent, the improvement effect was confirmed to increase depending on the concentration of the flavonol glycoside mixture contained in the external preparation composition.

As described above, the composition of the present invention contains flavonol glycosides or green tea extracts, which are separated and purified from green tea extracts, to inhibit the production of intermediates or end products (AGEs) by protein glycosylation. In addition, it can be used as a cosmetic, medical or food compositions having a wrinkle improvement effect using this.

Claims (10)

delete At least one flavonol glycosides derived from green tea (flavonol glycosides) comprising a flavonol monosaccharide glycoside as an active ingredient, cosmetic composition for improving wrinkles. The cosmetic composition for improving wrinkles according to claim 2, wherein the flavonol monosaccharide glycoside is a flavonol galactose glycoside. delete The cosmetic composition for improving wrinkles according to claim 2, wherein the flavonol glycoside is contained in the form of green tea extract. delete The cosmetic composition for improving wrinkles according to any one of claims 2, 3, and 5, wherein the flavonol glycoside is contained in an amount of 0.0001% to 10% by weight based on the total weight of the composition. delete delete delete