KR102645444B1 - Black Tea Leaf Extract and Manufacturing Method Thereof - Google Patents

Abstract

Disclosed is a black tea extract and a method for producing the same. According to the present disclosure, there is an advantage in that it contains a high content of theabronine and has excellent antioxidant effects, anti-aging effects, anti-inflammatory effects, and excellent skin recovery effects due to stress.

Description

Black Tea Leaf Extract and Manufacturing Method Thereof}

Disclosed herein is a black tea leaf extract and a method for producing the same.

Tea is one of the most consumed beverages worldwide after water. For example, non-fermented tea such as green tea, semi-fermented tea such as oolong tea, and black tea. There is fermented tea, etc. In other words, depending on the presence or absence of fermentation, it is divided into unfermented green tea, fermented tea such as oolong tea, black tea, etc., and depending on the degree of fermentation, it is divided into partially fermented tea, oolong tea, and fully fermented black tea.

The taste and color of these fermented teas vary depending on conditions such as fermentation time and temperature. This is due to the action of oxidative enzymes present in the tea leaves through fermentation, producing theaflavin, thearubigin, and theabronin ( This is because components such as theabrownin) are produced, and the taste and color of fermented tea change due to these components. In other words, the quality of fermented tea, which exhibits a special flavor and color, can be determined by the degree of fermentation (oxidation) of the tea leaves.

However, most of the conventional fermented tea-related technologies are limited to fermentation methods, and no research has been conducted on extraction conditions. Additionally, specific research on specific components of fermented tea has been minimal, and there are many technologies on extraction methods. There is a shortage. Republic of Korea Patent Publication No. 10-2017-0125545 discloses that components of black tea and pu-erh tea inhibit the expression of PTP1B in preadipocytes, but efficient extraction technology for each specific component and excellent effects are required. There is still a need to develop compositions that exhibit this effect.

Republic of Korea Patent Publication No. 10-2017-0125545

The present disclosure was made to solve the above problems, and the purpose of the present disclosure is to provide a black tea leaf extract containing a high content of theabronin and a method for producing the same.

In order to achieve the above-described object of the present disclosure, the present disclosure provides a black tea leaf extract containing a high content of theabronin and a method for producing the same.

According to the present disclosure, there is an advantage in that it contains a high content of theabronine and has excellent antioxidant effects, anti-aging effects, anti-inflammatory effects, and excellent skin recovery effects due to stress.

1 to 5 are graphs showing the results of component analysis of an extract according to an example.
Figure 6 is a graph showing the cytotoxicity evaluation results.
Figure 7 is a graph showing the results of evaluating intracellular reactive oxygen species production.
Figures 8 and 9 are graphs showing the results of evaluating the efficacy of inhibiting MMP-1 expression.
Figure 10 is a graph showing the ABTS radical scavenging ability evaluation results.
Figure 11 is a graph showing the results of evaluating mitochondrial membrane potential recovery ability.
Figures 12 to 14 are graphs showing the results of evaluating the efficacy of inhibiting MMP-1 expression.
Figure 15 is a graph showing the cytotoxicity evaluation results.
Figure 16 is a graph showing the results of evaluating the efficacy of human IL-8 gene expression.

The terms used in this specification are selected from general terms that are currently widely used as much as possible while considering the functions in the present disclosure, but this may vary depending on the intention of a technician working in the field, precedents, or the emergence of new technologies. In addition, in certain cases, there are terms arbitrarily selected by the applicant, and in this case, the meaning will be described in detail in the description of the relevant invention. Therefore, the terms used in this disclosure should be defined based on the meaning of the term and the overall content of this disclosure, rather than simply the name of the term.

Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by a person of ordinary skill in the technical field to which the present invention pertains. Generally understood terms should be interpreted as having the same meaning as the meaning they have in the context of the related technology, and unless clearly defined in the present disclosure, they should not be interpreted in an idealized or excessively formal sense.

Numerical ranges include the numerical values defined in this disclosure. Any maximum numerical limit given throughout this specification includes all lower numerical limits as if the lower numerical limit were clearly written. Every minimum numerical limit given throughout this specification includes every higher numerical limit as if such higher numerical limit were clearly written. All numerical limits given throughout this specification will include all better numerical ranges within the broader numerical range, as if the narrower numerical limits were clearly written.

Hereinafter, the present disclosure will be described in detail with reference to examples and drawings. However, it is obvious that the present invention is not limited to the following examples and drawings.

In one aspect, the present disclosure provides a black tea leaf extract comprising theabrownin in an amount greater than 5% by weight, based on the total weight of the extract.

The “black tea” refers to tea made by fermenting 70% to 85% or more of the buds, leaves, or stems of Camellia sinensis of the Camellia family. In one aspect of the present disclosure, the fermentation is performed using microorganisms in the brewing industry. This means oxidation by oxidizing enzymes contained in leaves, etc., rather than fermentation by . More specifically, repeating the process of sun foraging (letting the buds, leaves, or stems wilt in sunlight), leaving them indoors (leaving them for moisture to evaporate), and asking (the process of wounding the buds, leaves, or stems). It can be done through. At this time, the degree of fermentation increases as the time and number of indoor politics and the time and number of requests are increased.

The “extract” includes all ingredients and substances obtained from natural products, regardless of extraction method, extraction solvent, or extract form. The extraction method may include a process in which components and substances obtained from natural products are extracted and then processed or treated in another method. Specifically, the processing or treatment of the other methods may involve additional fermentation or enzyme treatment of the extract. Therefore, the extract in the present disclosure is a broad concept including fermented products, concentrates, and dried products, and specifically, the extract in the present disclosure may be a fermented product.

The “black tea leaf extract” refers to an extract obtained from black tea obtained by fermenting the black tea leaves, and includes all components and substances obtained from black tea, regardless of extraction method, extraction solvent, or extract form. The extraction method may include a process of treating with heat, acid, base, enzyme, etc., and may also include a process of processing or treating the components and substances obtained from black tea by another method after extraction. You can. Specifically, the processing or treatment of the other methods may include additionally fermenting or enzymatically treating the black tea leaf extract. Therefore, the black tea leaf extract in the present disclosure is a broad concept including fermented products, concentrates, and dried products, and specifically, the extract in the present disclosure may be a fermented product. Additionally, the extraction method may include obtaining the black tea leaf extract by concentrating it under reduced pressure. The reduced pressure concentration may be specifically performed at 35 to 50° C., but is not limited thereto. When vacuum concentration is performed at a temperature in the above range, the synergistic effect of various components in the black tea leaf extract of the present invention is excellent. The reduced pressure concentration is specifically 600mmHg or more, 620mmHg or more, 640mmHg or more, 660mmHg or more, 680mmHg or more, 700mmHg or more, 720mmHg or more, 740mmHg or more; It can be performed under conditions of 760mmHg or less, 740mmHg or less, 720mmHg or less, 700mmHg or less, 680mmHg or less, 660mmHg or less, 650mmHg or less, 640mmHg or less, 620mmHg or less, and 600mmHg or less.

The “black tea leaf extract” according to the extraction of the present disclosure contains theabrownin in a high content of more than 5% by weight based on the total weight of the extract. The “theabronin” is a polyphenol produced by oxidative condensation of catechin components by polyphenol oxidase (polyphenol oxidase or phenolase). More specifically, it is contained in an amount exceeding 5% by weight, 5.5% based on the total weight of the extract. at least 6% by weight, at least 6.5% by weight, at least 7% by weight, at least 7.5% by weight, at least 8% by weight, at least 8.5% by weight, at least 9% by weight, at least 9.5% by weight, at least 10% by weight; It may be included in an amount of 10% by weight or less, 10.5% by weight or less, 11% by weight or less, 11.5% by weight or less, and less than 12% by weight.

However, the “black tea leaf extract” in the present disclosure is a combination of various substances including not only the theabronin component, but also theaflavin, thearubigin, catechin, tannin, etc. Therefore, the effect exerted by the composition of the present disclosure is due to the synergistic effect of the combination of the various substances, and not due to the single theabronine component.

The extraction method for obtaining the “black tea leaf extract” may include heat extraction, cold needle extraction, reflux cooling extraction, or ultrasonic extraction, but is not limited as long as it is an extraction method that is obvious to those skilled in the art. The extraction solvent used to obtain the “black tea leaf extract” may be a C ₁ -C ₆ alcohol extract of black tea leaves, a water extract, etc., and more specifically, the water may be purified water or purified water. , hard water, soft water, natural water, deep ocean water, electrolytic alkaline ion water, electrolytic acid ion water, ion water, and cluster water can be used. However, any extraction solvent that is obvious to those skilled in the art can be used without limitation.

According to one embodiment of the present disclosure, the black tea leaf extract is a hard water extract of black tea leaves. “Hardness” is the sum of the amounts of calcium (Ca) and magnesium (Mg) contained in 1L of water and converted to the amount of calcium carbonate (CaCO ₃ ). Depending on the level, it is divided into soft water and hard water. It is classified into (hard water). However, the standards for classifying soft water and hard water are different for each organization. More specifically, according to the standards presented by the World Health Organization (WHO), hardness of 60 mg/L or less is considered 'soft water' and hard water. 60~120mg/L is classified as 'moderately hard water', hardness between 120~180mg/L is classified as 'hard water', and hardness over 180mg/L is classified as 'very hard water'. According to the standards presented by the Korea Water Resources Corporation, hardness of 75 mg/L or less is ‘soft water’, hardness of 75 to 150 mg/L is ‘moderately hard water’, hardness of 150 to 300 mg/L is ‘hard water’, and hardness of 300 mg/L or more is ‘strong water’. It is classified as ‘hard water’.

The “hard water”, which is the extraction solvent used in the present disclosure, has a hardness of 180 mg/L or more, 190 mg/L or more, 200 mg/L or more, 210 mg/L or more, 220 mg/L or more, 230 mg/L or more, 240 mg/L or more, More than 250 mg/L, more than 260 mg/L, more than 270 mg/L, more than 280 mg/L, more than 290 mg/L, more than 291 mg/L, more than 300 mg/L; 230mg/L or less, 240mg/L or less, 250mg/L or less, 260mg/L or less, 270mg/L or less, 280mg/L or less, 290mg/L or less, 291mg/L or less, 300mg/L or less, 310mg/L or less, It may be 320 mg/L or less, 330 mg/L or less, 340 mg/L or less, and 350 mg/L or less. More specifically, the hardness of the hard water is 218.23 mg/L to 325.74 mg/L.

The hard water contains one or more minerals selected from the group consisting of calcium, magnesium, potassium, and sodium, and the minerals are contained in an amount of 79.7 mg/L to 130.3 mg/L based on the total volume of the hard water. More specifically, 75 mg/L or more, 79.7 mg/L or more, 80 mg/L or more, 85 mg/L or more, 90 mg/L or more, 95 mg/L or more, 100 mg/L or more, 105 mg/L or more. or more, 110 mg/L or more, 115 mg/L or more, 120 mg/L or more, 125 mg/L or more, 130 mg/L or more; 80 mg/L or less, 85 mg/L or less, 90 mg/L or less, 95 mg/L or less, 100 mg/L or less, 105 mg/L or less, 110 mg/L or less, 115 mg/L or less, 120 mg /L or less, 125 mg/L or less, 130 mg/L or less, 130.3 mg/L or less, 135 mg/L or less, and 140 mg/L or less.

More specifically for each mineral component, the calcium is contained in an amount of 65 mg/L to 100 mg/L based on the total volume of hard water. More specifically, 65 mg/L or more, 67.5 mg/L or more, 70 mg/L or more, 72.5 mg/L or more, 75 mg/L or more, 77.5 mg/L or more, 80 mg/L or more, 82.5 mg/L or more. more; 77.5 mg/L or less, 80 mg/L or less, 82.5 mg/L or less, 85 mg/L or less, 87.5 mg/L or less, 90 mg/L or less, 92.5 mg/L or less, 95 mg/L or less, 97.5 mg /L or less, 100 mg/L or less.

Based on the total volume of hard water, magnesium is contained in an amount of 10 mg/L to 50 mg/L. More specifically, 10 mg/L or more, 12.5 mg/L or more, 15 mg/L or more, 17.5 mg/L or more, 20 mg/L or more, 22.5 mg/L or more, 25 mg/L or more, 26 mg/L or more. or more, 27.5 mg/L or more; 25 mg/L or less, 26 mg/L or less, 27.5 mg/L or less, 30 mg/L or less, 32.5 mg/L or less, 35 mg/L or less, 37.5 mg/L or less, 40 mg/L or less, 42.5 mg /L or less, 45 mg/L or less, 47.5 mg/L or less, and 50 mg/L or less.

Based on the total volume of hard water, the potassium is contained in an amount of more than 0 mg/L to 2 mg/L. More specifically, greater than 0 mg/L, greater than or equal to 0.1 mg/L, greater than or equal to 0.2 mg/L, greater than or equal to 0.3 mg/L, greater than or equal to 0.4 mg/L, greater than or equal to 0.5 mg/L, greater than or equal to 0.6 mg/L, and greater than or equal to 0.7 mg/L. or more, 0.8 mg/L or more, 0.9 mg/L or more, 1.0 mg/L or more; 1.0 mg/L or less, 1.1 mg/L or less, 1.2 mg/L or less, 1.3 mg/L or less, 1.4 mg/L or less, 1.5 mg/L or less, 1.6 mg/L or less, 1.7 mg/L or less, 1.8 mg /L or less, 1.9 mg/L or less, 2.0 mg/L or less.

Based on the total volume of hard water, the sodium is contained in an amount of 6 mg/L to 17 mg/L. More specifically, 6 mg/L or more, 6.25 mg/L or more, 6.5 mg/L or more, 6.75 mg/L or more; 6.25 mg/L or less, 6.5 mg/L or less, 6.75 mg/L or less, 7 mg/L or less, 7.5 mg/L or less, 8 mg/L or less, 8.5 mg/L or less, 9 mg/L or less, 9.5 mg /L or less, 10 mg/L or less, 10.5 mg/L or less, 11 mg/L or less, 11.5 mg/L or less, 12 mg/L or less, 12.5 mg/L or less, 13 mg/L or less, 13.5 mg/L Below, 14 mg/L or less, 14.5 mg/L or less, 15 mg/L or less, 15.5 mg/L or less, 16 mg/L or less, 16.5 mg/L or less, 17 mg/L or less, less than 18 mg/L .

Meanwhile, another standard for classifying water according to the components it contains is total dissolved solids (TDS), which refers to solid substances including minerals such as calcium, sodium, magnesium, and iron in water. This means the amount dissolved. Solid substances mainly exist in an ionic state. Total dissolved solids are classified as low if they are 0 to 250 mg/L, medium if they are 250 to 800 mg/L, and high if they are more than 800 mg/L.

The “hard water”, which is the extraction solvent used in the present disclosure, has a total dissolved solids of 300 mg/L or more, 325 mg/L or more, 350 mg/L or more, 357 mg/L or more, 375 mg/L or more, 400 mg/L or more, 425 mg/L or more. L or more, 450 mg/L or more, 475 mg/L or more, 500 mg/L or more; It may be 375 mg/L or less, 400 mg/L or less, 425 mg/L or less, 450 mg/L or less, 475 mg/L or less, 500 mg/L or less, 512 mg/L or less, 525 mg/L or less, and 550 mg/L or less. More specifically, the total dissolved solids are 357 mg/L to 512 mg/L.

According to one embodiment of the present disclosure, the black tea leaf extract is a hot water extract of black tea leaves. The hot water extraction temperature may be in the temperature range of 30°C to 90°C. More specifically, 30°C or higher, 35°C or higher, 40°C or higher, 45°C or higher, 50°C or higher, 55°C or higher, 60°C or higher, 65°C or higher, 70°C or higher, 75°C or higher, 80°C or higher; It may be 80°C or lower, 85°C or lower, 90°C or lower, 95°C or lower, and 100°C or lower.

According to one embodiment of the present disclosure, the black tea leaf extract is obtained by extraction for more than 1 hour. The upper limit of the extraction time is not particularly limited, but can be appropriately set by a person skilled in the art considering the yield increase rate, etc.

In one aspect, the present disclosure provides a composition comprising the black tea leaf extract or theabronin isolated from the black tea leaf extract as an active ingredient. According to one embodiment of the present disclosure, a composition is provided having at least one use selected from the group consisting of antioxidant, anti-aging, anti-inflammatory, and recovery of skin damaged due to stress. Additionally, the present disclosure provides a pharmaceutical composition, cosmetic composition, or health food composition containing the black tea extract as an active ingredient.

According to one embodiment of the present disclosure, the composition is a composition for preventing, improving, or treating various diseases and inflammation related to oxidation and/or aging. Additionally, according to one embodiment of the present disclosure, the composition is a composition for recovering skin damaged due to stress.

The “antioxidation” refers to inhibiting oxidation of cells by highly reactive free radicals or reactive oxygen species (ROS) due to oxidative stress caused by intracellular metabolism or the influence of ultraviolet rays, It includes removing free radicals or reactive oxygen species and reducing damage to cells caused by them.

The “anti-aging” refers to the effect of suppressing the aging phenomenon of cells, and more specifically, the anti-aging may be skin anti-aging. The aging may be aging that occurs naturally in cells over time, or may be photoaging caused by sunlight. In particular, it may be photoaging caused by ultraviolet rays. In addition, the aging may be induced by oxidative stress within cells, which can occur due to various causes, which may lead to cell growth inhibition or apoptosis and inhibit the synthesis of various fiber proteins that make up skin cells. and the expression of decomposition enzymes may be increased. In particular, depending on ultraviolet irradiation, the expression of genes such as SIRT1, AQP3, Col1a1, fibronectin, and elastin is suppressed in skin cells, and the expression of MMP-1 and MMP-2 genes is promoted, which can cause photoaging of skin cells and the development of wrinkles. .

The term “anti-inflammatory” refers to the effect of suppressing inflammation occurring in the body. The term “inflammation” refers to a defensive response of biological tissue against injury occurring locally in the body, and more specifically, it may be inflammation of the skin. Inflammatory diseases prevented, improved or treated by the present disclosure include allergic inflammatory diseases, acute or chronic inflammatory diseases, chronic bronchitis and related obstructive airway diseases, dermatitis, upper respiratory tractitis and rhinitis, arthritis or inflammatory bowel disease, pain, arteriosclerosis, It includes diseases including heart disease, multiple sclerosis, Parkinson's disease, Alzheimer's disease, or colon cancer, and preferably includes dermatitis.

The “damaged skin” refers to skin damaged by external physical damage, penetration of chemicals, bacteria, mold, viruses, etc., exposure to ultraviolet rays, loss of skin moisture, wrinkles due to aging, skin pigmentation, and stress hormones. This means damage to skin tissue or cells from a clinical and cosmetic point of view. According to one embodiment of the present disclosure, the damage is due to a stress phenomenon.

The above “stress” refers to physical and psychological burden, and is known to be the cause of various mental and physical diseases. When humans experience physical and psychological stress due to stimulation from the internal and external environment, glucocorticoid synthetic proteins within the human body use cholesterol to produce stress hormones such as cortisol. These stress hormones are the main causes of aging, dryness, pigmentation, and skin barrier dysfunction. The black tea leaf extract of the present disclosure contains a high content of theabronin, thereby improving skin barrier function and restoring damaged skin.

The “prevention” refers to any action that suppresses or delays a disease or disease by administering the composition. The term “improvement” means any action that results in at least a reduction in the severity of the parameters associated with the condition being treated, such as symptoms. The “treatment” refers to any action that improves or beneficially changes the symptoms of a disease by applying the pharmaceutical composition. The above “recovery” means regaining the continuity that the skin has lost.

According to one embodiment of the present disclosure, the daily application amount of the black tea leaf extract is 0.2 mg/kg to less than 100 mg/kg. More specifically, the daily application amount is 0.2 mg/kg or more, 0.3 mg/kg or more, 0.4 mg/kg or more, 0.5 mg/kg or more, 0.6 mg/kg or more, 0.7 mg/kg or more, 0.8 mg/kg or more, 0.9 mg/kg or more. mg/kg or more, 1.0 mg/kg or more, 1.25 mg/kg or more, 2 mg/kg or more, 2.5 mg/kg or more, 5 mg/kg or more, 10 mg/kg or more, 20 mg/kg or more, 25 mg/ kg or more, 30 mg/kg or more, 35 mg/kg or more, 40 mg/kg or more, 45 mg/kg or more, 50 mg/kg or more, 55 mg/kg or more, 60 mg/kg or more, 65 mg/kg or more , 70 mg/kg or more, 75 mg/kg or more, 80 mg/kg or more, 85 mg/kg or more, 90 mg/kg or more, 95 mg/kg or more; 20 mg/kg or less, 25 mg/kg or less, 30 mg/kg or less, 35 mg/kg or less, 40 mg/kg or less, 45 mg/kg or less, 50 mg/kg or less, 55 mg/kg or less, 60 mg /kg or less, 65 mg/kg or less, 70 mg/kg or less, 75 mg/kg or less, 80 mg/kg or less, 85 mg/kg or less, 90 mg/kg or less, 95 mg/kg or less, 100 mg/kg It may be less than, but is not limited to this. The above application may be applied once or several times a day. For example, 2 to 24 times per day, 1 to 2 times per 3 days, 1 to 6 times per week, 1 to 10 times per 2 weeks, 1 to 15 times per 3 weeks, 1 to 3 times per 4 weeks. It may be applied once or 1 to 12 times a year.

According to one embodiment of the present disclosure, the daily application amount of theabronin isolated from the black tea leaf extract is 0.1 mg/kg to less than 100 mg/kg. More specifically, the daily application amount is 0.1 mg/kg or more, 0.3 mg/kg or more, 0.4 mg/kg or more, 0.5 mg/kg or more, 0.6 mg/kg or more, 0.7 mg/kg or more, 0.8 mg/kg or more, 0.9 mg/kg or more. mg/kg or more, 1.0 mg/kg or more, 1.25 mg/kg or more, 2 mg/kg or more, 2.5 mg/kg or more, 5 mg/kg or more, 10 mg/kg or more, 20 mg/kg or more, 25 mg/ kg or more, 30 mg/kg or more, 35 mg/kg or more, 40 mg/kg or more, 45 mg/kg or more, 50 mg/kg or more, 55 mg/kg or more, 60 mg/kg or more, 65 mg/kg or more , 70 mg/kg or more, 75 mg/kg or more, 80 mg/kg or more, 85 mg/kg or more, 90 mg/kg or more, 95 mg/kg or more; 20 mg/kg or less, 25 mg/kg or less, 30 mg/kg or less, 35 mg/kg or less, 40 mg/kg or less, 45 mg/kg or less, 50 mg/kg or less, 55 mg/kg or less, 60 mg /kg or less, 65 mg/kg or less, 70 mg/kg or less, 75 mg/kg or less, 80 mg/kg or less, 85 mg/kg or less, 90 mg/kg or less, 95 mg/kg or less, 100 mg/kg It may be less than, but is not limited to this. The above application may be applied once or several times a day. For example, 2 to 24 times per day, 1 to 2 times per 3 days, 1 to 6 times per week, 1 to 10 times per 2 weeks, 1 to 15 times per 3 weeks, 1 to 3 times per 4 weeks. It may be applied once or 1 to 12 times a year.

The “application” means providing the composition to the subject to be applied by any suitable method, and includes administration, application, absorption, and ingestion. At this time, the target of application refers to all animals to which the composition can be applied, such as humans, monkeys, dogs, goats, pigs, or rats.

According to one embodiment of the present disclosure, the content of the black tea leaf extract may vary depending on the formulation, use, number of applications, and application route. In particular, when the composition of the present disclosure is manufactured as a parenteral formulation such as an injection, or an oral formulation such as food, pills, and syrup, the content of the active ingredient of the present disclosure determines the intestinal absorption rate and digestibility of the composition. It may vary depending on etc. However, when the composition of the present disclosure is prepared as an external skin preparation and applied topically, considering the daily application amount, it may be applied in an amount of 120 mg to less than 6000 mg for a subject weighing 60 kg. Assuming that the daily application amount is 100 g, the content of the active ingredient of the present disclosure may be included in an amount of 0.12% by weight to less than 6.0% by weight based on the total weight of the composition.

More specifically, 0.12% by weight or more, 0.15% by weight or more, 0.3% by weight or more, 0.6% by weight or more, 1.2% by weight or more, 1.5% by weight or more, 1.8% by weight or more, 2.1% by weight or more, 2.4% by weight or more, 2.7% by weight. % or more, 3.0% by weight or more, 3.3% by weight or more, 3.6% by weight or more, 3.9% by weight or more, 4.2% by weight or more, 4.5% by weight or more, 4.8% by weight or more, 5.1% by weight or more, 5.4% by weight or more, 5.7% by weight % more; 1.2% by weight or less, 1.5% by weight or less, 1.8% by weight or less, 2.1% by weight or less, 2.4% by weight or less, 2.7% by weight or less, 3.0% by weight or less, 3.3% by weight or less, 3.6% by weight or less, 3.9% by weight or less, It may be 4.2% by weight or less, 4.5% by weight or less, 4.8% by weight or less, 5.1% by weight or less, 5.4% by weight or less, 5.7% by weight or less, and less than 6.0% by weight, but is not limited thereto.

According to one embodiment of the present disclosure, the content of theabronin isolated from the black tea leaf extract is 0.006% by weight to less than 6.0% by weight, or 0.06% by weight to less than 6.0% by weight based on the total weight of the composition. You can. More specifically, 0.006% by weight, 0.06% by weight or more, 0.075% by weight or more, 0.12% by weight or more, 0.15% by weight or more, 0.3% by weight or more, 0.6% by weight or more, 1.2% by weight or more, 1.5% by weight or more, 1.8% by weight. or more, 2.1% by weight or more, 2.4% by weight or more, 2.7% by weight or more, 3.0% by weight or more, 3.3% by weight or more, 3.6% by weight or more, 3.9% by weight or more, 4.2% by weight or more, 4.5% by weight or more, 4.8% by weight or more, 5.1% by weight or more, 5.4% by weight or more, 5.7% by weight or more; 1.2% by weight or less, 1.5% by weight or less, 1.8% by weight or less, 2.1% by weight or less, 2.4% by weight or less, 2.7% by weight or less, 3.0% by weight or less, 3.3% by weight or less, 3.6% by weight or less, 3.9% by weight or less, It may be 4.2% by weight or less, 4.5% by weight or less, 4.8% by weight or less, 5.1% by weight or less, 5.4% by weight or less, 5.7% by weight or less, and less than 6.0% by weight, but is not limited thereto.

According to one embodiment of the present disclosure, the composition is a pharmaceutical composition. In this case, it may contain a pharmaceutically acceptable carrier. The carrier is used to include an excipient, diluent, or auxiliary agent. The carriers include, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl chloride. It may be selected from the group consisting of rolidone, water, physiological saline, buffer solutions such as PBS, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate, and mineral oil. The composition may include fillers, anti-coagulants, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, or combinations thereof.

The pharmaceutical composition is administered in a pharmaceutically effective amount. The above “pharmaceutically effective amount” means an amount sufficient to prevent or treat a disease with an applicable reasonable benefit/risk ratio, and the effective dose level depends on the type of patient's disease, the severity of the disease, the activity of the drug, and the drug's It can be determined based on factors including sensitivity, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other well-known pharmaceutical factors.

The pharmaceutical composition may be formulated as an oral dosage form (e.g., powder, tablet, capsule, syrup, pill, or granule), or a parenteral dosage form (e.g., injection).

The pharmaceutical composition may be prepared as a systemic formulation or a topical formulation, and may be especially applied to the skin as an external skin preparation. The skin external preparation may be in the form of a liquid, ointment, cream, lotion, spray, patch, gel, or aerosol. When used as an external skin agent, additionally fatty substances, organic solvents, solubilizers, thickening and gelling agents, emollients, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or non-ionic. Such as emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, blocking agents, humectants, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or any other ingredients commonly used in external skin preparations. May contain adjuvants commonly used in the field of dermatology. Additionally, the ingredients may be introduced in amounts commonly used in the field of dermatology. The composition for topical skin application may further include an agent that increases transdermal absorption, for example, dimethyl sulfoxide, dimethyl acetamide, dimethyl formamide, surfactant, azone, alcohol, acetone, propylene glycol, or polyethylene glycol.

The pharmaceutical composition may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art. Additionally, the pharmaceutical composition may be used alone or in combination with surgery, radiation therapy, hormone therapy, chemotherapy, and methods using biological response regulators.

According to one embodiment of the present disclosure, the composition is a health food composition. The health food composition may further include a physiologically acceptable carrier. The type of carrier is not particularly limited, and any carrier commonly used in the art can be used.

The health food composition contains preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), disinfectants (bleaching powder, high bleaching powder, sodium hypochlorite, etc.), and antioxidants (butylhydroxyanisole (BHA), butylhydroxyanisole). toluene (BHT), etc.), colorants (tar color, etc.), coloring agents (sodium nitrite, sodium nitrite, etc.), bleaching agents (sodium sulfite), seasonings (MSG monosodium glutamate, etc.), sweeteners (dulcine, cyclemate, saccharin, Food additives such as sodium, etc.), flavorings (vanillin, lactones, etc.), leavening agents (alum, D-potassium hydrogen tartrate, etc.), strengtheners, emulsifiers, thickeners (grease), coating agents, gum base agents, foam suppressants, solvents, improvers, etc. (food additives) may be included. The additives can be selected depending on the type of food and used in an appropriate amount.

The food composition may include additional ingredients that are commonly used in food compositions to improve smell, taste, vision, etc. For example, it may include vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, pantothenic acid, etc. Additionally, minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), and chromium (Cr); and amino acids such as lysine, tryptophan, cysteine, and valine.

According to one embodiment of the present disclosure, the composition is a cosmetic composition. The cosmetic composition can be formulated by conventional methods. In the formulation of external skin preparations, information disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA can be referred to, and in the formulation of cosmetic compositions, in The Cosmetic, Toiletry and Fragrance Association. You can refer to the information disclosed in the International Cosmetic Ingredient Dictionary published by the International Cosmetic Ingredient Dictionary.

Specifically, the cosmetic composition can be prepared in a general emulsified formulation and solubilized formulation. For example, lotion such as softening lotion or nourishing lotion; Emulsions such as facial lotion and body lotion; Creams such as nutritional cream, moisture cream, eye cream, etc.; essence; cosmetic ointment; spray; gel; pack; sunscreen; makeup base; Foundation, such as liquid type, solid type, or spray type; powder; Make-up removers such as cleansing creams, cleansing lotions, and cleansing oils; Alternatively, it can be formulated as a cleanser such as cleansing foam, soap, or body wash. Additionally, the skin external preparation may be formulated as an ointment, patch, gel, cream, or spray.

In each formulation, the cosmetic composition may be appropriately mixed with other ingredients in addition to the black tea leaf extract within the range that does not impair the purpose according to the present disclosure, depending on the type of formulation or purpose of use.

The cosmetic composition may contain fatty substances, organic solvents, solubilizers, thickeners, gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, and fragrances commonly used in the industry depending on the quality or function of the final product. , surfactants, water, ionic or non-ionic emulsifiers, fillers, sequestering agents, chelating agents, preservatives, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic activators, commonly used in cosmetics. It may additionally contain auxiliaries commonly used in the fields of cosmetology or dermatology, such as any other ingredients.

In another aspect, the present disclosure provides a method for producing the black tea leaf extract, comprising the step of hot water extracting the black tea leaves in hard water, and according to one embodiment of the present disclosure, the extraction The step may be performed for a period of time greater than 1 hour. The upper limit of the extraction time is not particularly limited, but can be appropriately set by a person skilled in the art considering the yield increase rate, etc.

In another aspect, the present disclosure provides a black tea leaf extract or the black tea in the preparation of a composition having one or more uses selected from the group consisting of antioxidant, anti-aging, anti-inflammatory, and recovery of skin damaged due to stress phenomenon. Provided is the use of theabronin isolated from leaf extract.

In another aspect, the present disclosure provides antioxidant, anti-aging, anti-inflammatory and Provided is one or more methods selected from the group consisting of restoring skin damaged due to stress phenomena.

In another aspect, the present disclosure provides a black tea leaf extract or theabronin isolated from the black tea leaf extract for use in recovering skin damaged due to oxidation, anti-aging, anti-inflammatory and stress phenomena.

{Example}

Hereinafter, the present disclosure will be described in detail through examples. However, the following examples are merely examples to aid the overall understanding of the present disclosure, and the content of the present disclosure is not limited to the following examples.

<Preparation Example 1> Preparation of extracts of Example 1, Comparative Example 1 and Comparative Example 2

1. Preparation of extract of Example 1

3g of black tea leaves (Osulloc Co., Ltd.) that have undergone more than 80% fermentation contain water (sodium: 6.5mg/L, potassium: 1.0mg/L, calcium: 80mg/L, magnesium: 26mg/L; total dissolved solids are 511.7mg/L) Add 125 mL of hard water (Evian mineral water, Evian ^® , France), which is medium level and hardness is 304 mg/L, and extract by heating to 95°C for 10 minutes. After removing the black tea leaves and filtering, Black tea leaf extract powder was prepared by evaporation to dryness using a rotary vacuum concentrator.

2. Preparation of extracts of Comparative Example 1 and Comparative Example 2

Example 1 and Example 1, except that as Comparative Example 1, a Seonhyang product (fermented tea with about 10 to 20% fermentation) was used, and as Comparative Example 2, a Ruthyang product (fermented tea with about 30 to 40% fermentation) was used. Black tea leaf extract powder was prepared in the same manner.

<Test Example 1> Comparison of theaflavin, thererubigin, and theabronin contents 1

1. Preparation of measurement samples

6 mL each of the extracts of Example 1, Comparative Example 1, and Comparative Example 2 were mixed with 6 mL of ethyl acetate and then vortexed for 5 minutes to form an upper ethyl acetate layer (A layer) and a lower aqueous layer (D). layers). 3.2 mL of the A layer was mixed with 3.2 mL of 2.5% sodium bicarbonate (NaHCO ₃ ) and vortexed for 30 seconds to separate the upper ethyl acetate layer (C layer).

Meanwhile, 1.6 mL each of the extracts of Example 1, Comparative Example 1, and Comparative Example 2 were mixed with 1.6 mL of butanol (n-butanol) and then vortexed for 3 minutes to separate the lower aqueous layer (layer B). did.

5 mL of solution (solution E _A ) was prepared by adding 95% ethanol to 0.8 mL of layer A.

0.4 mL of saturated oxalic acid solution, 1.2 mL of distilled water, and 3 mL of 95% ethanol were added to 0.4 mL of layer B to prepare a 5 mL solution (solution E _B ).

95% ethanol was added to 0.8 mL of layer C to prepare 5 mL of solution (solution E _C ).

0.4 mL of saturated oxalic acid solution, 1.2 mL of distilled water, and 3 mL of 95% ethanol were added to 0.4 mL of layer D to prepare a 5 mL solution (solution E _D ).

2. Measurement method

The contents of theaflavin, thererubigin, and theabronin were measured at a wavelength of 380 nm using a UV-vis spectrophotometer using the formula below.

[Equation 1]

The results are shown in Table 1 and Figure 1 below (unit: weight % based on the dry weight of the extract).

Classification (weight%) Theaflavin Dear Biggin Deabronin Comparative Example 1 0.005 3.507 1.693 Comparative Example 2 0.124 5.413 3.927 Example 1 0.283 6.553 8.608

3. Measurement results

As can be seen from Table 1 and Figure 1, the contents of theaflavin, thererubigin, and theabronin are all higher in Example 1 compared to Comparative Examples 1 and 2, especially the content of theabronin. In the case of Example 1, there was a significant increase. From this, it can be seen that the extract of black tea leaves, which are tea leaves that have undergone more than 80% of fermentation, contains high amounts of theaflavin, thererubigin, and theabronin, and in particular, the content of theabronin is very high. there is.

<Preparation Example 2> Preparation of extracts of Example 2, Comparative Example 3 and Comparative Example 4

1. Preparation of extract of Example 2

3g of black tea leaves with more than 80% fermentation are mixed with water (sodium: 6.5mg/L, potassium: 1.0mg/L, calcium: 80mg/L, magnesium: 26mg/L; total dissolved solids are 511.7mg/L (average) 125 mL of hard water (Evian mineral water, Evian ^® , France) with a hardness of 304 mg/L (medium) was added and extracted by heating to 95°C for 20 minutes. After removing the black tea leaves, filtering, and using a rotary vacuum concentrator. By evaporating and drying, black tea leaf extract powder was prepared.

2. Preparation of extracts of Comparative Example 3 and Comparative Example 4

As Comparative Example 3, distilled water (hardness and total dissolved solids: 0) was used, and as Comparative Example 4, soft water (sodium: about 5.6 mg/L, potassium: about 2.45 mg/L, calcium: about 2.9 mg/L, magnesium : approximately 1.9 mg/L; hardness 18.7 mg/L; total dissolved solids 400 mg/L; black tea leaf extract powder was prepared in the same manner as in Example 2, except that Samdasoo ^® , Kwangdong Pharmaceutical, Korea) was used.

<Test Example 2> Comparison of theaflavin, thererubigin, and theabronin contents 2

1. Preparation of measurement samples

For the extracts of Example 2, Comparative Example 3, and Comparative Example 4, measurement samples were prepared in the same manner as in Test Example 1.

2. Measurement method

The contents of theaflavin, thererubigin, and theabronin were measured in the same manner as in Test Example 1. The results are shown in Table 2 and Figure 2 below (unit: weight % based on the dry weight of the extract).

Classification (weight%) Theaflavin Dear Biggin Deabronin Comparative Example 3 0.027 5.273 4.614 Comparative Example 4 0.027 4.363 4.231 Example 2 0.040 3.495 5.648

3. Measurement results

As can be seen from Table 2 and Figure 2, the content of theaflavin is higher in Example 2 than in Comparative Examples 3 and 4, and in particular, the content of theabronin is significantly higher in Example 2. showed an increase. From this, it can be seen that when black tea leaves are extracted using high mineral content, high hardness, and high hardness water, an extract containing a high content of theabronin can be obtained.

<Preparation Example 3> Preparation of extracts of Example 3, Comparative Example 5 and Comparative Example 6

1. Preparation of extract of Example 3

Black tea leaf extract powder was prepared in the same manner as in Preparation Example 2, except that a new variety of black tea leaves (Jangwon No. 2, Osulloc Farm) with high amino acid and catechin content was used.

2. Preparation of extracts of Comparative Example 5 and Comparative Example 6

As Comparative Example 5, distilled water (hardness and total dissolved solids: 0) was used, and as Comparative Example 6, soft water (sodium: about 5.6 mg/L, potassium: about 2.45 mg/L, calcium: about 2.9 mg/L, magnesium) was used. : approximately 1.9 mg/L; hardness 18.7 mg/L; total dissolved solids 400 mg/L; black tea leaf extract powder was prepared in the same manner as in Preparation Example 2 using Samdasoo ^® , Kwangdong Pharmaceutical, Korea.

<Test Example 3> Comparison of theaflavin, thererubigin, and theabronin contents 3

1. Preparation of measurement samples

For the extracts of Example 3, Comparative Example 5, and Comparative Example 6, measurement samples were prepared in the same manner as in Test Example 1.

2. Measurement method

The contents of theaflavin, thererubigin, and theabronin were measured in the same manner as in Test Example 1. The results are shown in Table 3 and Figure 3 below (unit: weight % based on the dry weight of the extract).

Classification (weight%) Theaflavin Dear Biggin Deabronin Comparative Example 5 0.117 3.086 4.746 Comparative Example 6 0.112 3.190 4.953 Example 3 0.019 1.149 9.336

3. Measurement results

As can be seen from Table 3 and Figure 3, the content of theabronine in Example 3 showed a significant increase compared to Comparative Examples 5 and 6. From this, it can be seen that when black tea leaves are extracted using high mineral content, high hardness, and high hardness water, an extract containing a high content of theabronin can be obtained. In particular, compared to Test Example 2, it can be seen that when using a new variety of black tea leaves (Jangwon No. 2, Osulloc Farm), an extract containing a higher content of theabronin can be obtained.

<Preparation Example 4> Preparation of extracts of Example 4 and Comparative Examples 7 to 9

1. Preparation of extract of Example 4

3g of black tea leaves with more than 80% fermentation are mixed with water (sodium: 6.5mg/L, potassium: 1.0mg/L, calcium: 80mg/L, magnesium: 26mg/L; total dissolved solids are 511.7mg/L (average) Add 125 mL of hard water (Evian mineral water, Evian ^® , France) with a hardness of 304 mg/L and extract by heating to 95°C for 60 minutes. After removing the black tea leaves, filtering, and using a rotary vacuum concentrator. By evaporating and drying, black tea leaf extract powder was prepared.

2. Preparation of extracts of Comparative Examples 7 to 9

As Comparative Example 7, Jeju lava seawater (sodium: about 18 mg/L, potassium: about 22 mg/L, calcium: about 62 mg/L, magnesium: about 9 mg/L; hardness 200 mg/L; total dissolved solids 93 mg/L; Jeju Black tea leaf extract powder was prepared in the same manner as in Example 4, except that it was extracted for 20 minutes with Lava Water ^® , Orion, Korea.

As Comparative Example 8, soft water (sodium: about 5.6 mg/L, potassium: about 2.45 mg/L, calcium: about 2.9 mg/L, magnesium: about 1.9 mg/L; hardness 18.7 mg/L; total dissolved solids 400 mg/L) L; Samdasoo ^® , Kwangdong Pharmaceutical, Korea) was used, and as Comparative Example 9, Jeju lava seawater (sodium: about 18 mg/L, potassium: about 22 mg/L, calcium: about 62 mg/L, magnesium: about 9 mg/L; Black tea leaf extract powder was prepared in the same manner as in Example 4, except that hardness 200 mg/L; total dissolved solids 93 mg/L; Jeju Lava Water ^® , Orion, Korea) was used.

<Test Example 4> Comparison of theaflavin, thererubigin, and theabronin contents 4

1. Preparation of measurement samples

For the extracts of Example 2, Example 4, Comparative Example 4, and Comparative Examples 7 to 9, measurement samples were prepared in the same manner as in Test Example 1.

2. Measurement method

The contents of thererubigin and theabronin were measured in the same manner as in Test Example 1. The results are shown in Table 4 and Figure 4 below (unit: weight % based on the dry weight of the extract).

Classification (weight%) Dear Biggin Deabronin Comparative Example 4 4.363 4.231 Comparative Example 7 4.878 4.965 Example 2 3.495 5.648 Comparative Example 8 5.484 7.722 Comparative Example 9 5.223 5.833 Example 4 2.778 10.349

3. Measurement results

As can be seen from Table 4 and Figure 4, the content of theabronine in Example 4 showed a significant increase compared to Example 2, Comparative Example 4, and Comparative Examples 7 to 9. From this, it can be seen that when black tea leaves are extracted using high mineral content, high hardness, and high hardness water, an extract containing a high content of theabronin can be obtained. Additionally, it can be seen that when the extraction time is increased from 20 minutes to 60 minutes, an extract containing a higher content of theabronin can be obtained.

<Preparation Example 5> Preparation of extract of Example 5

3g of black tea leaves with more than 80% fermentation are mixed with water (sodium: 6.5mg/L, potassium: 1.0mg/L, calcium: 80mg/L, magnesium: 26mg/L; total dissolved solids are 511.7mg/L (average) Add 125 mL of hard water (Evian mineral water, Evian ^® , France) with a hardness of 304 mg/L and extract by heating to 80°C for 15 hours. After removing the black tea leaves, filtering, and using a rotary vacuum concentrator. By evaporating and drying, black tea leaf extract powder was prepared.

<Test Example 5> Comparison of theaflavin, thererubigin, and theabronin contents 5

1. Preparation of measurement samples

For the extracts of Examples 4 and 5, measurement samples were prepared in the same manner as in Test Example 1.

2. Measurement method

The contents of thererubigin and theabronin were measured in the same manner as in Test Example 1. The results are shown in Table 5 and Figure 5 below (unit: weight % based on the dry weight of the extract).

Classification (weight%) Dear Biggin Deabronin Example 4 2.778 10.349 Example 5 0.401 7.909

<Test Example 6> Cytotoxicity evaluation 1

1. Prepare ingredients

Theaflavin, thererubigin, and theabronin were separated from the black tea leaf extract of Example 1, and then dissolved in distilled water to prepare stock solutions (H-TF, H-TR, and H-TB, respectively). . A stock solution was prepared by dissolving theabrolin, which is of natural origin and not from black tea leaf extract, in distilled water (N-TB).

2. Evaluation method

Human Skin Fibroblast Cell (Hs68 Cell) was inoculated into a 96 well plate at 1.0 x 10 ⁴ cells/mL and then cultured for 24 hours under cell culture conditions (5% CO ₂ , 37°C). Next, the materials (H-TF, H-TR, H-TB, and N-TB) were added to the cell culture medium at different concentrations (0.1, 1, 10, 100 ppm) and cultured for 24 hours. Washed with DPBS (Dulbecco's Phosphate-Buffered Saline), replaced with culture medium containing 10% EZ-Cytox solution, cultured under cell culture conditions for l hour, and then measured absorbance at 450 nm with an ELISA reader (Tecan Infinite M200 Pro). did. The results are shown in Table 6 and Figure 6 below.

Cell viability (%) 0.1 ppm 1 ppm 10ppm 100ppm H-TF 101.93±6.42 97.39±4.21 82.73±3.37 57.71±3.71 H-TR 105.87±1.73 101.08±2.15 92.46±3.28 51.87±7.96 H-TB 96.94±4.68 102.03±5.17 92.68±4.55 59.15±2.97

3. Evaluation results

As can be seen from Table 6 and Figure 6, Hs68 cells showed a cell survival rate of 82.73% when treated with 10 ppm of H-TF and 57.71% when treated with 100 ppm; When treated with 100 ppm H-TR, the cell survival rate was 51.87%; When treated with 100 ppm of H-TB, the cell survival rate was 59.15% and it was judged to be cytotoxic. Meanwhile, it can be seen that the cell survival rate is highest when treated with theabronin (H-TB) isolated from the black tea leaf extract of the present disclosure in the 10 ppm content condition and the 100 ppm content condition.

<Test Example 7> Evaluation of intracellular reactive oxygen species production

1. Prepare ingredients

The same material as that used in Test Example 6 was used.

2. Evaluation method

HaCaT cells, a human keratinocyte cell line, were inoculated into a 96 well plate at 0.5 x 10 ⁴ cells/mL and then cultured for 24 hours under cell culture conditions (5% CO ₂ , 37°C). Next, the cells were washed with HBSS (Hank's balanced salt solution), then changed to medium containing the above materials (H-TF, H-TR, and H-TB) and positive control ascorbic acid diluted, and then incubated for 24 hours. cultured for some time. Afterwards, the cells were washed with HBSS, treated with 50 μM of DCF (2',7'-Dichlorofluorescin diacetate), blocked from light, and incubated for 30 minutes under cell culture conditions. After washing twice with HBSS, the cells were filled with 30 μL of HBSS to prevent them from drying out, and irradiated with UVB 50 mJ/cm ² . After incubation for 1 hour, fluorescence (excitation at 485 nm, emission at 535 nm) was measured with an ELISA reader (Tecan Infinite M200 Pro). The results are shown in Table 7, Table 8 and Figure 7 below (###p<O.0Ol vs. (-)UVB cells; *p<O.05, **p<O.Ol, *** p<O.OOl vs. UVB treated cells, n=3).

DCF fluorescence (%) (-)UVB UVB treatment 0mM 0mM 0.5mM 1mM ascorbic acid 100.00±2.93 271.26±21.39
** 246.88±15.23
* 162.53±12.45
**

DCF fluorescence (%) 0.1ppm 1 ppm 10ppm 100 ppm H-TF 349.47±13.28 271.26±8.64 268.60±14.37 213.81±15.41
** H-TR 280.60±18.32 224.17±18.64
* 218.10±16.59
** 192.29±8.70
** H-TB 271.39±19.97 260.36±9.74 220.92±13.57
** 206.02±0.30
**

3. Evaluation results

To measure intracellular reactive oxygen species production, the fluorescent probe DCF-DA was used. As can be seen from Table 7, Table 8 and Figure 7, when HaCaT cells were irradiated with UVB 50mJ/cm ² , the generation of reactive oxygen species (ROS) increased by 196.24%, and in the UVB irradiated group, In comparison, it was confirmed that the generation of reactive oxygen species was significantly reduced by 49.36% when treated with 0.5 mM ascorbic acid and by 133.70% when treated with 1 mM ascorbic acid. For H-TF, 82.43% at 100 ppm treatment; For H-TR, 72.06% at 1 ppm treatment, 78.14% at 10 ppm treatment, 103.94% at 100 ppm treatment; In the case of H-TB, a significant reduction of active oxygen radicals was observed by 75.32% when treated at 10 ppm and by 90.22% when treated at 100 ppm. From this, it can be seen that the composition containing the black tea leaf extract of the present disclosure or theabronin isolated from the black tea leaf extract as an active ingredient can exhibit antioxidant efficacy.

<Test Example 8> Evaluation of efficacy of inhibiting MMP-1 expression 1

1. Prepare ingredients

The same material as that used in Test Example 6 was used.

2. Evaluation method

Human skin fibroblast cells (Hs68 Cell) were inoculated into a 48-well plate at 2.0 x 10 ⁴ cells/mL and then cultured for 24 hours under cell culture conditions (5% CO ₂ , 37°C). Afterwards, the medium was removed, washed with DPBS (Dulbecco's Phosphate-Buffered Saline), and the materials (H-TF, H-TR, and H-TB) were diluted by concentration in DMEM medium without FBS and pretreated for 6 hours. . The medium was removed, filled with 100 μL of DPBS, and then irradiated with UVB 30 mJ/cm ² . Afterwards, the test substance was diluted in DMEM medium without FBS, treated, and cultured for 48 hours. After collecting the medium from the cultured cells, the secretion amount of MMP-1 was measured using the Human MMP-1 ELlSA kit at 450 nm with an ELlSA reader (Tecan Infinite M200 Pro). Cells attached to the bottom of the plate were washed with DPBS, lysed with 1N NaOH, and the amount of protein was measured through BCA assay. The amount of MMP-1 secreted per certain protein was corrected and expressed as a percentage (%). The results are shown in Tables 9 to 12 below and Figures 8 and 9 (###p<O.0Ol vs. (-)UVB cells; *p<O.05, **p<O.Ol, ***p<O.OOl vs. UVB treated cells, n=3).

MMP-1 (%) (-)UVB UVB treatment 0mM 0mM 1mM 10mM retinoic acid 100.00±9.49 463.00±13.67
*** 351.95±22.95
** 223.29±14.85
***

MMP-1 (%) 0.001ppm 0.01ppm 0.1 ppm 1 ppm H-TF 242.81±13.68 252.68±3.49 235.43±17.52 226.84±9.24 H-TR 258.82±3.88 240.05±10.89 222.80±10.07 203.38±12.02 H-TB 248.18±14.63 228.18±9.20 224.14±11.54 190.89±8.79
*

MMP-1 (%) (-)UVB UVB treatment 0mM 0mM 1mM 10mM retinoic acid 100.00±3.39 262.21±13.63
*** 217.78±19.44
** 156.38±12.70
***

MMP-1 (%) 1 ppm 5 ppm 10 ppm 25 ppm 50 ppm H-TB 217.89±9.62
* 213.19±1.62
** 222.82±2.05
** 208.68±3.75
** 179.73±1.30
***

3. Evaluation results

As can be seen from Table 9, Table 10, and Figure 8, MMP-l secretion increased by 126.03% when HS68 cells were irradiated with 30 mJ/cm ² of UVB, 32.69% when treated with 1 μM retinoic acid, and 101.58% when treated with 10 μM UVB. It was confirmed that there was a significant decrease compared to the investigation group. In the case of H-TB, it decreased by 35.14% when treated with 1 ppm, showing a significant difference compared to the UVB irradiated group; In the case of H-TF and H-TR, no reduction effect was confirmed.

Meanwhile, as can be seen from Table 11, Table 12 and Figure 9, when H-TB was evaluated at five concentrations (1, 5, 10, 25, 50 ppm), the reduction effect was observed in all treated concentration groups. However, at concentrations above 5 ppm, the amount of protein decreased to less than 80% compared to the UVB irradiated group, so it is judged to be cytotoxic.

From this, in the composition containing the black tea leaf extract of the present disclosure or theabronin isolated from the black tea leaf extract as an active ingredient, by reducing MMP-1, which promotes collagen decomposition, anti-aging effect, especially skin anti-aging, skin It can be seen that it can exert anti-wrinkle effects.

<Test Example 9> Evaluation of ABTS radical scavenging ability

1. Prepare ingredients

3g of black tea leaves (Osulloc Co., Ltd.) that have undergone more than 80% fermentation contain water (sodium: 6.5mg/L, potassium: 1.0mg/L, calcium: 80mg/L, magnesium: 26mg/L; total dissolved solids are 511.7mg/L) Add 125 mL of hard water (Evian ^® , France), which is a medium level of L and have a hardness of 304 mg/L, and extract by heating to 80°C for 15 hours. After removing the black tea leaves and filtering, Black tea leaf extract powder was prepared by evaporation to dryness using a rotary vacuum concentrator.

Meanwhile, after separating theabronin from the black tea leaf extract, it was dissolved in distilled water to prepare a 100,000 ppm stock solution (TB).

2. Evaluation method

Potassium persulfate was dissolved to 2.45mM in a 7.4mM ABTS solution and then reacted in the dark for 24 hours. After adjusting this with distilled water so that the absorbance at 734 nm is 0.7, 180 μL was taken, 20 μL of the black tea leaf extract of the present disclosure or theabronin isolated from the black tea leaf extract was added at each concentration, and reacted at room temperature in the dark for 10 minutes. Absorbance was measured at 734 nm. The results are shown in Figure 10.

3. Evaluation results

Measurement of antioxidant activity using ABTS radicals is a method in which ABTS free radicals generated by reaction with potassium persulfate are removed by antioxidants in the extract, thereby discoloring the radical's unique color, blue-green. As can be seen from Figure 10, in the case of the black tea leaf extract of the present disclosure and theabronin isolated therefrom, the ABTS radical scavenging ability was concentration-dependent and significantly increased. More specifically, the IC50 value of ascorbic acid (L-ascorbic acid), which was a positive control, was 4.90 ppm, the IC50 value of black tea leaf extract was 10.60 ppm, and the IC50 value of theabronin was 13.21 ppm. It was confirmed that ABTS radicals were significantly eliminated when both black tea leaf extract and theabronin were treated in excess of 1.25 ppm.

From this, it can be seen that the composition containing the black tea leaf extract of the present disclosure or theabronin isolated from the black tea leaf extract as an active ingredient can exhibit antioxidant efficacy.

In particular, since the black tea leaf extract exhibits superior antioxidant efficacy compared to theabronin isolated from the black tea leaf extract, the effect exerted by the composition of the present disclosure is not due to the single theabronin component, but to the black tea leaf extract. It can be seen that it is due to a synergistic effect caused by a combination of various substances included.

<Test Example 10> Evaluation of mitochondrial membrane potential recovery ability

1. Prepare ingredients

The same material as that used in Test Example 9 was used.

2. Evaluation method

HaCaT cells, a ^human keratinocyte cell line, were inoculated into a black ⁹⁶ well plate at 2.0 The test materials were treated in DMEM medium containing no. 0.1% DMSO was used as the vehicle, and as a positive control, one treated with 2 ppm of epigallocatechin gallate (EGCG) was used. 24 hours after treatment with the test material, mitochondrial membrane potential was measured through JC-10 staining, and mitochondrial mass was measured through Mitotracker staining. The results are shown in Figure 11.

3. Evaluation results

As can be seen from Figure 11, it was confirmed that the damaged mitochondrial membrane potential was restored by the black tea leaf extract of the present disclosure and theabronin isolated therefrom. When the black tea leaf extract of the present disclosure and theabronin isolated therefrom were treated, the mitochondrial content in cells was not reduced and showed no significant change. Therefore, it can be seen that the black tea leaf extract of the present disclosure and theabronin isolated therefrom have anti-aging effects against UVB and cortisol through restoration of mitochondrial membrane potential.

In particular, in the case of black tea leaf extract, despite being contained in a smaller amount than isolated theabronin, it exhibits anti-aging efficacy similar to or better than isolated theabronin, so the effect exerted by the composition of the present disclosure is single. It can be seen that it is not caused by a single theabronin component, but by a synergistic effect caused by a combination of various substances contained in the black tea leaf extract.

<Test Example 11> Evaluation of efficacy of inhibiting MMP-1 expression 2

1. Prepare ingredients

The same material as that used in Test Example 9 was used.

2. Evaluation method

NHDF cells, which are normal human dermal fibroblasts, were seeded on a dish at 5.0 x 10 ⁵ cells/mL and then cultured for 24 hours under cell culture conditions (5% CO ₂ , 37°C). Afterwards, UVB 16mJ/cm ² was irradiated, and the test material was treated with DMEM medium. 0.1% DMSO was used as the vehicle, and as a positive control, one treated with 2 ppm of epigallocatechin gallate (EGCG) was used. A group that was not irradiated with ultraviolet rays was used as a negative control group. After collecting the medium from the cultured cells, the secretion amount of MMP-1 was measured using the Human MMP-1 ELlSA kit and the absorbance at 450 nm with an ELlSA reader (Tecan Infinite M200 Pro). The results are shown in Figure 12 (***p<O.OOl vs. (-)UVB cells or UVB treated cells, n=3). Meanwhile, the results of confirming the anti-aging effect of theabronin isolated from the black tea leaf extract of the present disclosure at a lower concentration are shown in Figure 13 (***p<O.OOl vs. (-)UVB cells; +++ p<O.OOl vs. UVB treated cells, n=3).

3. Evaluation results

As can be seen from Figure 12, when NHDF cells were irradiated with UVB 16mJ/cm ² , MMP-l secretion increased, and when treated with the black tea leaf extract of the present disclosure and theabronin isolated therefrom, MMP was increased compared to the UVB irradiated group. -l It was confirmed that secretion was significantly reduced. In addition, as can be seen from Figure 13, it was confirmed that MMP-l secretion was significantly reduced even in the case of theabronin at a lower concentration (0.1 to 2 ppm). From this, in the composition containing the black tea leaf extract of the present disclosure or theabronin isolated from the black tea leaf extract as an active ingredient, by reducing MMP-1, which promotes collagen decomposition, anti-aging effect, especially skin anti-aging, skin It can be seen that it can exert anti-wrinkle effects.

<Test Example 12> Evaluation of efficacy of inhibiting MMP-1 expression 3

1. Prepare ingredients

The same material as that used in Test Example 9 was used.

2. Evaluation method

HaCaT cells, a human keratinocyte cell line, were seeded on a dish at 1.0 x 10 ⁶ cells/mL and then cultured for 24 hours under cell culture conditions (5% CO ₂ , 37°C). Afterwards, UVB 18mJ/cm ² was irradiated, and the test material was treated with DMEM medium. 0.1% DMSO was used as the vehicle, and as a positive control, one treated with 2 ppm of epigallocatechin gallate (EGCG) was used. A group that was not irradiated with ultraviolet rays was used as a negative control group. After collecting the medium from the cultured cells, the secretion amount of MMP-1 was measured using the Human MMP-1 ELlSA kit and the absorbance at 450 nm with an ELlSA reader (Tecan Infinite M200 Pro). The results are shown in Figure 14 (***p<O.OOl vs. (-)UVB cells; ++p<O.Ol, +++p<O.OOl vs. UVB treated cells, n=3 ).

3. Evaluation results

As can be seen from Figure 14, even in HaCaT cells, in the case of lower concentrations (0.1 to 2 ppm) of theabronin, MMP-l secretion was confirmed to be significantly reduced. From this, in the composition containing the black tea leaf extract of the present disclosure or theabronin isolated from the black tea leaf extract as an active ingredient, by reducing MMP-1, which promotes collagen decomposition, anti-aging effect, especially skin anti-aging, skin It can be seen that it can exert anti-wrinkle effects.

<Test Example 13> Cytotoxicity evaluation 2

1. Prepare ingredients

The same material as that used in Test Example 9 was used.

2. Evaluation method

HaCaT cells ^, a human keratinocyte cell line, were inoculated into a black ⁹⁶ well plate at 2.0 0.1% DMSO was used as the vehicle, and as a positive control, one treated with 2 ppm of epigallocatechin gallate (EGCG) was used. A group that was not irradiated with ultraviolet rays was used as a negative control group. Each well was treated with CCK8 (Cell Counting Kit 8) reagent. Afterwards, absorbance was measured at 450 nm with an ELISA reader (Tecan Infinite M200 Pro). The results are shown in Figure 15 (***p<O.OOl vs. UVB treated cells, n=3).

3. Evaluation results

As can be seen from Figure 15, EGCG, a positive control, showed cytotoxicity at a concentration of 2 ppm (71.5% compared to UVB unirradiated Vegicle), but the black tea leaf extract of the present disclosure or the tea leaf extract isolated from the black tea leaf extract In the case of theabronine, it did not show cytotoxicity.

<Test Example 14> Evaluation of human IL-8 gene expression efficacy

1. Prepare ingredients

The same material as that used in Test Example 9 was used.

2. Evaluation method

HaCaT cells ^, a human keratinocyte cell line, were inoculated into a black ⁹⁶ well plate at 2.0 0.1% DMSO was used as the vehicle, and as a positive control, one treated with 2 ppm of epigallocatechin gallate (EGCG) was used. A group that was not irradiated with ultraviolet rays was used as a negative control group. Afterwards, RT-PCR was performed for expression of the human IL-8 gene using primers specific for human IL-8. The results are shown in Figure 16 (***p<O.OOl vs. (-)UVB cells or UVB treated cells, n=3).

2. Evaluation results

As can be seen from Figure 16, when the black tea leaf extract of the present disclosure and theabronin isolated therefrom were treated, the expression level of IL-8 mRNA was confirmed to be significantly reduced compared to the UVB irradiated group. From this, it can be seen that the composition containing the black tea leaf extract of the present disclosure or theabronin isolated from the black tea leaf extract as an active ingredient exhibits excellent anti-inflammatory efficacy by significantly reducing the expression of IL-8.

Hereinafter, formulation examples of the composition according to the present invention will be described. However, pharmaceutical compositions, health food compositions, and cosmetic compositions can be applied in various formulations, and this is not intended to limit the present invention, but is only intended to provide a detailed explanation.

[Formulation Example 1] Preparation of ointment

An ointment was prepared by a conventional method according to the composition shown in Table 13 below.

ingredient Content (% by weight) Black tea leaf extract of Example 4 3.0 Cetostearyl alcohol 1.0 Self-emulsifying monostearic acid 2.0 stearic acid 1.0 beeswax 4.0 squalane 7.0 Monostearin Glycerin 3.0 Sorbitan monostearate 1.0 Polysorbate 80 3.0 glycerin 5.0 propylene glycol 4.0 Spices Appropriate amount Vaseline remaining amount

[Formulation Example 2] Preparation of a drink A drink was prepared by a conventional method according to the composition shown in Table 14.

ingredient Content (g) Black tea leaf extract of Example 4 0.2 glucose 10 citric acid 2 Purified water 188

[Formulation Example 3] Preparation of flexible lotion Soft lotion was prepared in a conventional manner according to the composition shown in Table 15.

ingredient Content (% by weight) Black tea leaf extract of Example 4 3.0 glycerin 5.0 1,3-butylene glycol 3.0 ethanol 5.0 Polyoxyethylene nonylphenyl ether 0.5 Spices Appropriate amount antiseptic Appropriate amount Purified water remaining amount

[Formulation Example 4] Preparation of astringent lotion An astringent lotion was prepared in a conventional manner according to the composition shown in Table 16.

ingredient Content (% by weight) Black tea leaf extract of Example 4 3.0 glycerin 3.0 citric acid 0.1 ethanol 10.0 Polyoxyethylene oleyl ether 1.0 Sorbitol 2.0 Spices Appropriate amount antiseptic Appropriate amount Purified water remaining amount

[Formulation Example 5] Preparation of cream A cream was prepared in a conventional manner according to the composition shown in Table 17.

ingredient Content (% by weight) Black tea leaf extract of Example 4 3.0 glycerin 5.0 stearic acid 8.0 squalane 5.0 Self-emulsifying glycerin monostearate 2.5 Polyoxyethylene sorbitan monostearate 1.5 propylene glycol 4.0 Stearyl glycyrrhetinate 0.2 vaseline 2.0 antioxidant Appropriate amount Spices Appropriate amount antiseptic Appropriate amount Purified water remaining amount

Although the present invention has been described in connection with the above-mentioned preferred embodiments, various modifications and variations can be made without departing from the spirit and scope of the invention. Accordingly, the appended claims will include such modifications and variations as long as they fall within the spirit of the present invention.

Claims (14)

As a black tea leaf extract,
The black tea leaves are tea leaves in which fermentation has progressed by more than 80%, the extract is a hard water extract of black tea leaves, the extract is obtained by extraction performed for less than 1 hour, and the hardness of the hard water is 218.23 mg/L. 325.74 mg/L, the extract contains theabronin in an amount exceeding 5% by weight based on the total weight of the extract, and the extract has the same weight ratio of theabronin to thereubigin except that it is a soft water extract. higher than the extracted control group, or
The black tea leaves are tea leaves in which fermentation has progressed to 80% or more, and the extract is a hard water extract of black tea leaves. The extract is obtained by extraction performed for more than 1 hour to less than 15 hours, and the hardness of the hard water is 218.23. mg/L to 325.74 mg/L, and the extract contains theabronin in an amount of 8% by weight or more based on the total weight of the extract, except that the extract is a soft water extract in which the weight ratio of theabronin to thereubigin is and higher than that of the equally extracted control group,
Black tea leaf extract. According to paragraph 1,
The black tea leaf extract is a hot water extract of black tea leaves. According to paragraph 1,
The hard water contains calcium, magnesium, potassium, and sodium,
Based on the total volume of hard water,
The calcium is contained in an amount of 65 mg/L to 100 mg/L,
The magnesium is contained in an amount of 10 mg/L to 50 mg/L,
The potassium is contained in an amount of more than 0 mg/L to 2 mg/L,
The black tea leaf extract contains the sodium in an amount of 6 mg/L to 17 mg/L. A cosmetic composition comprising the black tea leaf extract of any one of claims 1 to 3 as an active ingredient. According to paragraph 4,
A cosmetic composition, wherein the black tea leaf extract is included in an amount of 0.12% by weight or more and less than 6% by weight based on the total weight of the composition. According to paragraph 4,
A cosmetic composition wherein the daily application amount of theabronin is 0.1 mg/kg or more to less than 100 mg/kg. According to paragraph 4,
The composition is a cosmetic composition having one or more uses selected from the group consisting of antioxidant, anti-aging, anti-inflammatory, and recovery of skin damaged due to stress. A health food composition comprising the black tea leaf extract of any one of claims 1 to 3 as an active ingredient. According to clause 8,
A health food composition, wherein the black tea leaf extract is included in an amount of 0.12% by weight or more and less than 6% by weight based on the total weight of the composition. According to clause 8,
A health food composition wherein the daily application amount of theabronin is 0.1 mg/kg or more to less than 100 mg/kg. According to clause 8,
The composition is a health food composition having one or more uses selected from the group consisting of antioxidant, anti-aging, anti-inflammatory, and recovery of skin damaged due to stress. A method for producing the black tea leaf extract of any one of claims 1 to 3,
A method for producing black tea leaf extract, comprising the step of hot water extracting black tea leaves from hard water. According to clause 12,
Said extraction step of the extract comprising theabronin in an amount greater than 5% by weight based on the total weight of the extract is carried out for a time of less than 1 hour,
A method of producing the extract, wherein the extraction step of the extract containing theabronin in an amount of 8% by weight or more based on the total weight of the extract is performed for a period of time from 1 hour to 15 hours. delete