Application of gallocatechin gallate and isoquercitrin

Abstract

In the present specification, disclosed is a method for preparing a gallocatechin gallate (Gallocatable Gallate), which comprises the steps of: preparing a gallocatechin gallate (Gallocatable Gallate; the present invention relates to uses of a composition for enhancing liver health or for auxiliary protection against chemical liver injury, in particular to uses of a composition for enhancing liver health or for auxiliary protection against chemical liver injury, such as GCG, isoquercitrin, IQC, and to the preparation of a composition for enhancing liver health or for auxiliary protection against chemical liver injury. In one aspect, the composition of the present invention can include a composite component of gallocatechin gallate and isoquercitrin to effectively reduce oxidative stress and chronic inflammatory response occurring in the process of lipid accumulation in liver tissue to improve liver health, and is applied to prevention, improvement or treatment of various liver diseases such as hepatitis, cirrhosis, liver cancer and the like.

Description

Technical Field

[0001] The present specification discloses the use of epigallocatechin gallate and isoquercetin. Background Art

[0002] Liver cells play an important role in regulating the energy homeostasis of our body. They can store the surplus energy that flows in through diet, etc. in the form of high molecular complexes, such as glucose stored as glycogen and fatty acids stored as neutral lipids. In the case of insufficient energy due to fasting, etc., they decompose glycogen and neutral lipids as a stored energy source and send them to the tissues that need nutrients for use. However, when the amount of nutritional supplements is greater than the amount of energy consumed and the energy homeostasis is broken, the surplus energy is stored in fat cells. If it exceeds the limit that fat cells can store, neutral fat will accumulate in other tissues such as liver and muscle in addition to fat cells. In particular, when fat accumulates in hepatocytes and the proportion of fat in a normal liver exceeds 5%, it is called fatty liver. Due to the oxidative stress and chronic inflammatory response generated during the accumulation of lipids in liver tissue, various liver diseases such as hepatitis, cirrhosis, and liver cancer are induced. The liver performs many important functions required for survival, so when liver health deteriorates, the overall metabolism of our body will change and threaten life. In particular, the liver is called a "silent organ". Even if the liver is damaged to some extent, it is not easy to detect abnormal symptoms, so it is difficult to self-detect and also acts as a stumbling block to liver health management. Furthermore, in the case of fatty liver, no special therapeutic agent targeting fatty liver has been developed so far. So far, it needs to be managed through indirect treatment with lipid metabolism abnormality therapeutic agents such as hyperlipidemia (for example, fibrates, statins, etc.) or prevention through diet / lifestyle improvement. The development of targeted therapeutic agents or health functional foods to improve liver health, prevent or treat fatty liver and liver diseases is imminent.

[0003] Prior art literature

[0004] Patent document: (Patent document 1) Korean Patent KR2022-0145604A Summary of the invention

[0005] An object of the present invention is to provide a composition for improving liver health.

[0006] In order to achieve the above object, the present invention provides a composition for improving liver health or for auxiliary protection against chemical liver damage, comprising gallocatechin gallate (GCG) and isoquercetin (IQC) as active ingredients.

[0007] On the one hand, the composition of the present invention can contain a composite component of epigallocatechin gallate and isoquercetin to effectively reduce the oxidative stress and chronic inflammatory response that occur during lipid accumulation in liver tissue, thereby improving liver health, and can be used for the prevention, improvement or treatment of various liver diseases such as hepatitis, cirrhosis, and liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 This graph compares the effect of improving non-alcoholic fatty liver disease according to the composition ratio of a mixture of gallocatechin gallate (GCG) and isoquercetin (IQC) (Adiphenon).

[0009] Figure 2 This graph compares the effects of improving alcoholic fatty liver disease according to the composition ratio of the mixture.

[0010] Figure 3 This graph compares the inhibitory effect on the production of reactive oxygen species in hepatocytes according to the composition ratio of the mixture.

[0011] Figure 4 as well as Figure 5 This graph compares the inhibitory effect of the expression of genes mediating inflammatory response in hepatocytes according to the composition ratio of the mixture.

[0012] Figure 6 This graph compares the inhibitory effect of lipid accumulation in hepatocytes according to the APGQ subdivision ratio.

[0013] exist Figures 1 to 6 The experimental groups with statistically significant differences (P<0.05) are indicated by different marks. DETAILED DESCRIPTION

[0014] Hereinafter, the present invention will be described in detail.

[0015] The term "prevention" in this specification refers to all actions to inhibit or delay liver disease by applying the pharmaceutical composition. The term "treatment" refers to all actions to improve or favorably change the symptoms of individuals suspected of or suffering from liver disease by applying the pharmaceutical composition.

[0016] The term "improvement" used in the present specification means all actions that make liver diseases better or change favorably by applying the composition.

[0017] In this specification, the term "apply" means providing the composition to an applicable subject by any appropriate method, including administration, application, absorption and ingestion, etc. In this case, the applicable subject means all animals such as humans, monkeys, dogs, goats, pigs or mice to which the composition can be applied.

[0018] The present invention relates to a composition for improving liver health or for auxiliary protection against chemical liver damage, comprising gallocatechin gallate (GCG) and isoquercetin (IQC) as active ingredients.

[0019] In an exemplary implementation, the composition may be a pharmaceutical composition for preventing or treating liver diseases.

[0020] In an exemplary implementation, the composition may be a non-therapeutic oral composition for preventing or improving liver diseases.

[0021] In an exemplary implementation, the composition may be a food composition for auxiliary protection against chemical liver damage.

[0022] In an exemplary implementation example, the epigallocatechin gallate and isoquercetin may be included in a weight ratio of 9:1 or more, 8:2 or more, 7:3 or more, 6:4 or more, 5:5 or more, or 4:6 or more, and may be included in a weight ratio of 1:9 or less or 2:8 or less. For example, the epigallocatechin gallate and isoquercetin may be included in a weight ratio of 9:1 to 1:9, but is not limited thereto.

[0023] In an exemplary implementation example, the epigallocatechin gallate and isoquercetin can be included in a weight ratio of 1:1 or more, 1:1.1 or more, 1:1.2 or more, 1:1.3 or more, 1:1.4 or more, 1:1.5 or more, 1:1.6 or more, 1:1.7 or more, 1:1.8 or less, 1:1.9 or more, 1:2.0 or more, 1:2.1 or more, 1:2.2 or more, 1:2.3 or more, or 1:2.4 or more; and can be included in a weight ratio of 1:9 or less, 1:8 or less, 1:7 or less, 1:6 or less, 1:5 or less, 1:4 or less, 1:3.5 or less, 1:3.4 or less, 1:3.3 or less, 1:3.2 or less, 1:3.1 or less, 1:3 or less, 1:2.9 or less, 1:2.8 or less, or 1:2.7 or less. When the epigallocatechin gallate and isoquercetin are included in the weight ratio, liver health can be effectively improved. For example, the epigallocatechin gallate and isoquercetin can be included in a weight ratio of 1:1 to 1:9 or 1:1.5 to 1:4, but is not limited thereto.

[0024] In an exemplary implementation example, the liver disease may be one or more selected from the group consisting of hepatitis, fatty liver, cirrhosis and liver cancer.

[0025] In an exemplary implementation, the liver disease is alcoholic liver disease.

[0026] In an exemplary implementation, the composition can be characterized as being suitable for use in a subject in need of reducing oxidative stress or inflammation in liver cells.

[0027] In an exemplary implementation, the composition may be characterized as being suitable for a subject in need of reducing the expression of one or more genes selected from the group consisting of IL-1b (interleukin-1b), IL-6 (interleukin-6) and iNOS (inducible nitric oxide synthase).

[0028] In an exemplary implementation, the composition may be characterized as being suitable for use in a subject in need of inhibiting lipid accumulation in hepatocytes.

[0029] In an exemplary implementation example, the daily applicable amount of the active ingredient of the composition can be 5 mg / kg or more, 6 mg / kg or more, 7 mg / kg or more, 8 mg / kg or more, 9 mg / kg or more, 10 mg / kg or more, 20 mg / kg or more, 30 mg / kg or more, 40 mg / kg or more, 50 mg / kg or more, 60 mg / kg or more, 70 mg / kg or more, 80 mg / kg or more, 90 mg / kg or more, 100 mg / kg or more, 1 g / kg or more, 2 g / kg or more, 3 g / kg or more, 4 g / kg or more, 5 g / kg or more or 6 g / kg or more, and can be less than 60 g / kg, less than 59 g / kg, less than 58 g / kg, less than 57 g / kg, less than 56 g / kg, less than 55 g / kg, less than 54 g / kg, less than 53 g / kg, less than 52 g / kg or less than 51 g / kg. It can be that within the applicable amount, the liver health promotion effect is excellent, when it is less than the applicable amount, the liver health promotion effect is slight, and when it is more than the applicable amount, toxicity and other problems occur. The application can also be divided into once a day to several times. For example, it can be applied 2 to 24 times a day, 1 to 2 times in three days, 1 to 6 times a week, 1 to 10 times in two weeks, 1 to 15 times in three weeks, 1 to 3 times in four weeks, or 1 to 12 times a year, but it is not limited to this.

[0030] In an exemplary implementation example, the pharmaceutical composition can be provided in all dosage forms suitable for local application. For example, it can be administered orally, transdermally, intravenously, intramuscularly, or subcutaneously. As an example, the pharmaceutical composition can be an injection, a skin external solution, a suspension, an emulsion, a gel, a patch, or a spray, but is not limited thereto. The dosage form can be easily prepared according to the usual methods in the art, and surfactants, molding additives, hydrating agents, emulsifying promoters, suspending agents, salts or buffers for osmotic pressure regulation, colorants, spices, stabilizers, preservatives, preservatives, or other commonly used adjuvants can be appropriately used.

[0031] In an exemplary implementation, the active ingredient of the pharmaceutical composition varies according to the subject's age, sex, weight, pathological condition and its severity, route of administration or the prescriber's judgment. The determination of the appropriate dosage based on these factors is within the level of those skilled in the art.

[0032] In an exemplary implementation example, when the composition is used as an additive for health functional food, it can be added as it is or used together with other food or food ingredients, and can be used appropriately according to the usual method. The mixing amount of the active ingredient can be appropriately determined according to the purpose of use such as prevention, health or treatment. The dosage form of the health functional food can be not only in the form of powder, granules, pills, tablets, capsules, but also any form of common food or beverage is possible.

[0033] In an exemplary implementation example, there is no particular limitation on the types of health functional foods. Examples of foods to which the composition can be added include meat, cakes, noodles, chewing gum, dairy products including ice cream, various soups, beverages, teas, oral liquids, alcoholic beverages, and vitamin complexes, etc., which can include all foods in the usual sense.

[0034] In an exemplary implementation example, when preparing the health functional food or beverage, the composition can be added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, to 100 parts by weight of the raw material. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of health regulation, the amount can be below the range.

[0035] In an exemplary implementation example, the beverage in the health functional food may contain various flavoring agents or natural carbohydrates as additional ingredients like ordinary beverages. The above-mentioned natural carbohydrates may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, sugar alcohols such as xylitol, sorbitol, and erythritol. As sweeteners, natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame may be used. The proportion of the natural carbohydrates may be about 0.01 to 0.04 g per 100 ml of beverage according to the present invention, preferably about 0.02 to 0.03 g, but is not limited thereto.

[0036] In an exemplary implementation example, in addition to the above, the health functional food according to the present invention may also contain various nutrients, vitamins, electrolytes, flavoring agents, colorants, pectin acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerol, alcohol, and carbonating agents used in carbonated beverages. In addition, the health functional food according to the present invention may contain pulp for the preparation of natural fruit juices, fruit juice drinks and vegetable drinks. These ingredients can be used independently or in combination. The proportion of these additives is not limited, but is generally selected within the range of 0.01 to 0.1 parts by weight compared to the health functional food per 100 parts by weight according to the present invention.

[0037] In another aspect, the present invention is a method for improving liver health, comprising administering to a subject a composition comprising an effective amount of epigallocatechin gallate (GCG) and isoquercitrin (IQC).

[0038] In yet another aspect, the present invention is a method for supplementary protection against chemical liver damage, comprising administering to a subject a composition comprising an effective amount of gallocatechin gallate (GCG) and isoquercitrin (IQC).

[0039] In still another aspect, the present invention is a method for preventing, ameliorating or treating liver diseases, comprising administering a composition comprising an effective amount of gallocatechin gallate (GCG) and isoquercitrin (IQC) to a subject.

[0040] In yet another aspect, the present invention is a use of gallocatechin gallate (GCG) and isoquercitrin (IQC) for preparing a composition for improving liver health, auxiliary protection against chemical liver damage, or preventing, improving or treating liver diseases.

[0041] In yet another aspect, the present invention provides gallocatechin gallate (GCG) and isoquercitrin (IQC) for improving liver health, providing auxiliary protection against chemical liver damage, or preventing, improving or treating liver diseases.

[0042] In yet another aspect, the present invention is a non-therapeutic use of gallocatechin gallate (GCG) and isoquercitrin (IQC) for improving liver health, providing auxiliary protection against chemical liver damage, or preventing or improving liver diseases.

[0043] The following examples are given to more specifically describe the configuration and effects of the present invention. However, the following examples are provided for the purpose of illustration only to help understanding of the present invention, and the scope and range of the present invention are not limited thereto.

[0044] [Example]

[0045] [Example 1]

[0046] Comparison of the inhibitory effect of gallocatechin gallate (GCG) and isoquercitrin (IQC) on lipid accumulation in hepatocytes by treatment with a mixture of gallocatechin gallate (GCG) and isoquercitrin (IQC) (Adiphenon)

[0047] Fatty liver, as the name implies, is a symptom of excessive accumulation of neutral lipids in liver tissue (hepatocytes). Whether a mixture of gallocatechin gallate (GCG) and isoquercitrin (IQC) (Adiphenon) can inhibit lipid accumulation in hepatocytes was studied. Specifically, HepG2 human hepatoma cell lines purchased from ATCC were cultured in Roswell Park Memorial Institute-1640 medium (Sigma Aldrich) containing fetal bovine serum (Gibco (Gibco)) and 1% penicillin / streptomycin (P / S; Sigma Aldrich). In order to confirm the fatty liver improvement effect of the composition ratio of GCG and IQC, HepG2 cells were pretreated with the complexes of various ratios (GCG:IQC = 10:0 to 0:10) at a total concentration of 100 μg / ml for 2 hours, and then oleic acid (500 μM; Sigma Aldrich; Figure 1 ) or ethanol (2 mM; Sigma Aldrich; Figure 2 ) for 72 hours to induce fatty liver. Thereafter, the cells were washed twice with Phosphate Buffered Saline (PBS; Sigma Aldrich), fixed in 10% formaldehyde (Sigma Aldrich) solution for 5 minutes, and then the accumulation of neutral lipids in the hepatocytes was confirmed with Nile Red (Sigma Aldrich; 300nM) solution, which is specifically stained for neutral fat. The amount of accumulated neutral fat was quantified using Tecan Infinite M200 Microplate Reader (Tecan M200 multifunctional microplate reader, ex 495nm, em 585nm).

[0048] The results, such as Figure 1 as well as Figure 2 As shown, when the mixture of various composition ratios was treated, the lipid accumulation in the hepatocytes was significantly reduced regardless of the fatty liver inducing factor, and this reducing effect was most excellent when GCG:IQC=3:7 to 4:6.

[0049] [Example 2]

[0050] Effect of the mixture on alleviating oxidative stress and chronic inflammatory response in hepatocytes

[0051] A large amount of reactive oxygen is produced in the process of decomposing alcohol, and excessive fat intake increases the concentration of free fatty acids. The intrinsic immune response that protects cells from external harmful substances mainly starts with the binding of TLR4 and lipopolysaccharides. TLR4 secretes various inflammatory factors through signal transduction systems such as NF-κB and AP-1. However, TLR4 not only recognizes lipopolysaccharides as endotoxins, but also recognizes free fatty acids. Therefore, even in the absence of external harmful substances, the presence of excessive free fatty acids can induce undesirable immune responses (for example, chronic inflammation). The long-term immune response has become the main cause of secondary liver diseases such as cirrhosis, liver fibrosis and liver cancer. Therefore, the alleviation of oxidative stress and inflammatory response can help prevent and improve fatty liver and related liver diseases.

[0052] In this regard, additional experiments were performed to study whether the mixture can improve the oxidative stress and inflammatory response induced during the production of fatty liver. Specifically, HepG2 cells were pre-treated with a mixture of various composition ratios at a concentration of 100 μg / ml for 2 hours, and then treated with oleic acid (500 μM) or ethanol (2mM) for 24 hours. After preparing the same experimental group twice, a part of the cells were washed twice with PBS as in Example 1 and fixed with 10% formaldehyde. After that, the cells were washed twice more with PBS to remove the residual formaldehyde, and H2-DCFDA (20 μM; Invitrogen by Thermo Fisher Scientific) that can sense reactive oxygen was treated for 30 minutes. The amount of reactive oxygen generated was observed using Tecan Infinite M200 Pro Microplate Reader (Tecan M200 Pro multifunctional microplate reader, ex 492nm, em517nm).

[0053] The remaining experimental groups were used for mRNA expression analysis to observe inflammatory responses. RNA was extracted using the TaKaRa MiniBESTUniversal RNA Extraction Kit (Takara Bio), and then quantified using the NanoQuant add-on of Tecan Infinite M200 (Tecan). The same amount (1 mg) of RNA was then synthesized into cDNA using the RevertAid 1st-strand cDNA Synthesis Kit (Thermo Fisher Scientific). In order to observe the expression of genes involved in inflammatory response, the coding sequence of the target gene was used as a template, and the primers were designed and prepared using the NCBIP Primer BLAST (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ) service (Bioneer). The synthesized cDNA and primers were used to observe the expression of the corresponding gene using CFX96 thermocycler (Bio-Rad).

[0054] The results, such as Figure 3 , Figure 4 as well as Figure 5 As shown, the active oxygen and inflammatory response effects of the mixtures according to various composition ratios were determined, and the effects were most excellent at a ratio of 3:7 to 4:6.

[0055] [Example 3]

[0056] Comparison of effects according to mixture ratio

[0057] There are many causes of fatty liver, such as alcoholic fatty liver caused by excessive drinking and non-alcoholic fatty liver caused by excessive nutritional intake due to diet. Regardless of the inducing factors, fatty liver is accompanied by oxidative stress and chronic inflammation during its development, so oleic acid, which induces excessive lipid accumulation in cells, is used from the following examples. In order to study the optimal ratio of each Adiphenon composition ratio to show the best synergistic effect, the experiment of Example 1 was repeated after various treatments with a ratio of GCG:IQC=4:6 (about 1:1.5) to 2:8 (about 1:4).

[0058] The results, such as Figure 6 As shown, the GCG:IQC ratio between 1:2.4 and 1:2.7 showed the best lipid accumulation inhibition effect.

[0059] The present disclosure is additionally illustrated by the following implementation examples which do not limit the claims.

[0060] Implementation Example 1. Use of gallocatechin gallate (GCG) and isoquercetin (IQC) for preparing a composition for promoting liver health or for auxiliary protection against chemical liver damage.

[0061] Implementation Example 2. As the use of Implementation Example 1, the composition is a pharmaceutical composition for preventing or treating liver diseases.

[0062] Implementation Example 3. As the use of Implementation Example 1 or 2, the liver disease is one or more selected from the group consisting of hepatitis, fatty liver, cirrhosis and liver cancer.

[0063] Implementation Example 4. As the use of Implementation Examples 1 to 3, the liver disease is alcoholic liver disease.

[0064] Implementation Example 5. As the use of Implementation Example 1, the composition is a food composition for auxiliary protection against chemical liver damage.

[0065] Implementation Example 6. As the use of Implementation Examples 1 to 5, the composition comprises gallocatechin gallate (GCG) and isoquercitrin (IQC) in a weight ratio of 9:1 to 1:9.

[0066] Implementation Example 7. As the use of Implementation Examples 1 to 6, the composition is characterized in that it is suitable for a subject in need of reducing oxidative stress or inflammation in liver cells.

[0067] Implementation Example 8. As the use of Implementation Examples 1 to 7, the composition is characterized in that it is suitable for a subject in need of reducing the expression of one or more genes selected from the group consisting of IL-1b, IL-6 and iNOS.

[0068] Implementation Example 9. As the use of Implementation Examples 1 to 8, the composition is characterized in that it is suitable for a subject in need of inhibiting lipid accumulation in hepatocytes.

Claims

1. A use of gallocatechin gallate (GCG) and isoquercetin (IQC) for preparing a composition for promoting liver health or for auxiliary protection against chemical liver damage.

2. The use according to claim 1, wherein The composition is a pharmaceutical composition for preventing or treating liver diseases.

3. The use according to claim 1, wherein The composition is a food composition for auxiliary protection against chemical liver damage.

4. The use according to claim 2, wherein The liver disease is one or more selected from the group consisting of hepatitis, fatty liver, cirrhosis and liver cancer.

5. The use according to claim 2, wherein The liver disease is alcoholic liver disease.

6. The use according to claim 1, wherein The composition includes gallocatechin gallate (GCG) and isoquercitrin (IQC) in a weight ratio of 9:1 to 1:

9.

7. The use according to claim 1, characterized in that The composition is suitable for a subject in need of reducing oxidative stress or inflammation in liver cells.

8. The use according to claim 7, characterized in that The composition is suitable for a subject in need of reducing the expression of one or more genes selected from the group consisting of IL-1b, IL-6 and iNOS.

9. The use according to claim 1, characterized in that The composition is suitable for a subject in need of inhibiting lipid accumulation in hepatocytes.

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