KR20240103671A - Cell protection composition containing phloroglucinol and exhibiting antioxidant activity - Google Patents

Abstract

The present invention relates to a cell protection composition that contains phloroglucinol as an active ingredient and exhibits antioxidant activity. It contains a phloroglucinol extract to prevent DNA damage caused by oxidative stress, damage to mitochondrial function, and cells. It can exhibit cell protective activity by alleviating death, etc., so it not only has excellent functionality but also has excellent taste, so it can be provided as a food composition or pharmaceutical composition.

Description

Cell protection composition containing phloroglucinol and exhibiting antioxidant activity {CELL PROTECTION COMPOSITION CONTAINING PHLOROGLUCINOL AND EXHIBITING ANTIOXIDANT ACTIVITY}

The present invention relates to a cell protection composition containing phloroglucinol, a phenolic compound. More specifically, by containing phloroglucinol, it has excellent antioxidant activity and can exhibit cell protective activity against oxidative damage caused by oxidative stress. It relates to a composition.

The retina expends excessive energy in forming visual perception and is highly sensitive to oxidative stress. At the same time, the retina acts as a powerful generator of reactive oxygen species (ROS), which are associated with several major retinal diseases, including age-related macular degeneration (AMD), a leading cause of vision loss. Although the pathogenesis and mechanisms of AMD induction are unclear, oxidative stress-related damage to the retinal pigment epithelium (RPE) is recognized as an early event in AMD-like pathology. Appropriate levels of intracellular ROS, including mitochondrial ROS (mtROS), play important physiological roles as regulators of cell signaling pathways. However, excessive accumulation of ROS due to persistent oxidative stress can lead to cell damage and death and contribute to the onset of pathological damage in various organs, including the eyes. Additionally, apoptosis and autophagy of RPE cells caused by excessive ROS production are accompanied by mitochondrial and DNA damage, ultimately contributing to retinal dysfunction. Additionally, because mitochondrial damage in RPE degeneration can induce a cellular defense mechanism known as mitophagy, mitophagy may be a putative therapeutic target in retinal degenerative diseases such as AMD. Therefore, to protect the normal function of the eye, excessive ROS production must be suppressed.

Meanwhile, natural products have long received great attention as a source for drug development. Among them, phenolic compounds derived from natural products with excellent antioxidant activity are attracting attention. Their antioxidant activities mainly involve scavenging of ROS and activation of intracellular antioxidant signaling pathways. Phloroglucinol, a polyphenol trihydroxybenzene with an aromatic phenyl ring and three hydroxyl groups, is a naturally occurring secondary metabolite present in a variety of organisms, including plants, algae, and bacteria. These phenolic compounds are known to have diverse pharmacological potential, such as antibacterial, antispasmodic, antiallergic, antithrombotic, anti-inflammatory, and cancer chemopreventive activities. Recently, the antioxidant potential of phloroglucinol has been verified in several in vitro and in vivo models. For example, Drygalsk reported that phloroglucinol can improve hepatic steatosis and inflammatory responses by enhancing antioxidant defenses and reducing oxidative/nitric damage to cellular macromolecules, and phloroglucinol can improve hepatic steatosis and inflammatory responses in the retinal epithelium, hippocampus, etc. It was confirmed that oxidative damage caused by hydrogen peroxide (H ₂ O ₂ ) treatment and radiation could be blocked by controlling the activity of antioxidant and detoxification enzymes in nerves, renal epithelial cells, and lung fibroblasts. Additionally, phloroglucinol, a ROS scavenger, can attenuate the pathology of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease by regulating synaptic plasticity. Previous studies have shown that phloroglucinol can inhibit DNA damage and apoptosis in HaCaT human keratinocytes exposed to H ₂ O ₂ . Similar results were seen in ultraviolet (UV) B-irradiated keratinocytes and all-trans-retinal-exposed primary rat RPE and mouse photoreceptor cells. Recently, Kuo et al. reported that phloroglucinol can block oxidative cytotoxicity induced by potassium bromate, an AMD inducer, in human RPE ARPE-19 cells by inhibiting ROS production.

However, as there is a lack of research on the protective role of phloroglucinol against cell damage caused by oxidative stress in ARPE-19 (human retinal pigment epithelium) cells, the present invention includes phloroglucinol as an active ingredient. Thus, this invention was completed to provide a composition that can exhibit cell protective activity against oxidative damage caused by oxidative stress.

KR 10-2019-0035474 A KR 10-2050506 B1

The purpose of the present invention is to provide a cell protection composition that exhibits antioxidant activity and contains phloroglucinol as an active ingredient.

In addition, another object of the present invention is to provide a food composition or pharmaceutical composition for cell protection, including a cell protection composition containing phloroglucinol as an active ingredient.

In order to achieve the above object, a cell protection composition according to an embodiment of the present invention exhibits antioxidant activity and contains phloroglucinol as an active ingredient.

The cell protection is achieved by alleviating DNA damage, mitochondrial function damage, and cell death caused by oxidative stress.

The oxidative stress refers to cell damage caused by reactive oxygen species (ROS).

The reactive oxygen species (ROS) includes super oxide radical (O ₂ ^- ), hydrogen peroxide (H ₂ O ₂ ), and hydroxy radical (OH ^- ).

The composition exhibits cytoprotective activity by inhibiting the mitochondria-mediated autophagy process by reducing mtROS production.

A food composition for cell protection according to another embodiment of the present invention includes a composition for cell protection containing phloroglucinol as an active ingredient.

A pharmaceutical composition for treating, improving or preventing cell damage according to another embodiment of the present invention includes a cell protection composition containing phloroglucinol as an active ingredient.

Hereinafter, the present invention will be described in more detail.

In the present invention, 'prevention' refers to any action that suppresses or delays the progression of the disease or disease to which the term applies, or one or more symptoms of the disease or disease, by administering the composition of the present invention.

In the present invention, 'improvement' means any action in which the disease or disease to which the term applies, or one or more symptoms of the disease or disease, is improved or beneficially changed by administration of the composition of the present invention.

In the present invention, unless otherwise stated, 'treatment' refers to reversing, alleviating, inhibiting the progression of, or preventing the disease or disease to which the term applies, or one or more symptoms of the disease or disease. The term 'treatment' used herein refers to the act of treating when 'treating' is defined as above.

As used herein, the term 'active ingredient' refers to an ingredient that can exhibit the desired activity alone or in combination with a carrier that is not active on its own.

The term 'extract' used in this specification has the meaning commonly used in the art as a crude extract, as described above, but in a broad sense also includes fractions obtained by additional fractionation of the extract. In other words, plant extracts include not only those obtained using the above-described extraction solvent, but also those obtained by additionally applying a purification process. For example, fractions obtained by passing the extract through an ultrafiltration membrane with a certain molecular weight cut-off value, separation by various chromatographs (designed for separation according to size, charge, hydrophobicity, or affinity), etc. Fractions obtained through purification methods are also included in the plant extract of the present invention.

The terminology used herein is for describing embodiments and is therefore not intended to limit the invention. As used herein, singular forms also include plural forms, unless specifically stated otherwise in the context. As used in the specification, 'comprise' and/or 'comprising' refers to the presence of one or more other components, steps, operations and/or elements. or does not rule out addition.

The cell protection composition according to an embodiment of the present invention exhibits antioxidant activity and contains phloroglucinol as an active ingredient.

Phloroglucinol, one of the phenolic compounds derived from natural products, corresponds to the polyphenol trihydroxybenzene, which has an aromatic phenyl ring and three hydroxyl groups. It is a naturally occurring secondary metabolite commonly present in a variety of organisms, including plants, algae, and bacteria, and is known to have diverse pharmacological potential, such as antibacterial, antispasmodic, antiallergic, antithrombotic, anti-inflammatory, and cancer chemopreventive activities. there is.

The cell protection composition of the present invention can exhibit cell protection activity through antioxidant activity by containing the above-described phloroglucinol as an active ingredient.

More specifically, the cell protective composition of the present invention may exhibit cytoprotective activity against retinal cells.

The retina expends a lot of energy to form visual perception, is highly sensitive to oxidative stress, and acts as a powerful generator of reactive oxygen species (ROS), which are associated with several major retinal diseases, including age-related macular degeneration (AMD), a leading cause of vision loss. Do it. Oxidative stress-related damage to the retinal pigment epithelium (RPE) is found to occur early in AMD-like pathology. Meanwhile, an appropriate level of intracellular ROS, including mitochondrial ROS (mtROS), plays an important physiological role as a regulator of cell signaling pathways, but excessive accumulation of ROS due to persistent oxidative stress can lead to cell damage and death. It can contribute to the initiation of pathological damage to various organs, including

Additionally, apoptosis and autophagy of retinal pigment epithelial (RPE) cells due to excessive ROS production can be accompanied by mitochondrial and DNA damage, ultimately leading to retinal dysfunction, and mitochondrial damage in RPE degeneration is a cellular defense mechanism known as mitophagy. Because it can induce mitophagy, it could be a putative therapeutic target in retinal degenerative diseases such as AMD. Therefore, excessive ROS production must be suppressed to protect the normal function of the eye.

That is, the composition of the present invention can exhibit cytoprotective activity by containing phloroglucinol as an active ingredient, and can especially protect retinal cells from active oxygen.

The cell protection is achieved by alleviating DNA damage, mitochondrial function damage, and cell death caused by oxidative stress.

DNA exists in the nucleus of a cell and stores the genetic information of a living organism. Through the process of division, cells create daughter cells with the same DNA as themselves, or reproductive cells pass on their own DNA to their children through meiosis. species will be maintained. However, DNA, which controls genetic information, is damaged by chemicals, radiation (UV), and reactive oxygen species (ROS) generated by cellular respiration, which can lead to the formation of cancer in cells or gene mutations during the DNA replication process. This may affect the normal growth of cells or cause cell death.

In addition, apoptosis refers to a phenomenon in which cells die on their own under various stress situations. Apoptosis is divided into extrinsic cell death, which starts from factors existing outside the cell, and intrinsic cell death, which starts from factors inside the cell. . Among them, when reactive oxygen species are overgenerated due to oxidative stress, MMPs are lost by depolarizing the mitochondrial membrane, and cytochrome c is released from the mitochondria to the cytoplasm, which activates the caspase cascade required for the mitochondria-mediated intrinsic apoptosis pathway, causing cell death. do. At this time, during the cell cycle, there is less DNA than in normal cells, which is an indicator of apoptosis, and cell death can be confirmed by an increase in sub-G1 in cell cycle changes.

Meanwhile, mitochondria can undergo membrane depolarization by reactive oxygen species (ROS), which contributes to the activation of the mitochondria-mediated intrinsic apoptotic pathway, resulting in the collapse of MMPs, indicating mitochondrial dysfunction, and consequently leading to cytoplasmic release of cytochrome c. I do it. The above-mentioned cytochrome c is a component that can inhibit cell death by causing decomposition of caspase-dependent proteins such as PARP by activating the caspase cascade required for the endogenous cell death pathway. As a result, depolarization of the membrane occurs due to active oxygen, and cytoplasmic cytochrome c of cytochrome c occurs. Inhibiting release can prevent mitochondrial dysfunction.

Accordingly, the cell protective composition containing phloroglucinol of the present invention exhibits antioxidant activity and may exhibit cell protective activity through inhibition of DNA damage, protection from mitochondrial function damage, and inhibition of cell death.

Meanwhile, oxidative stress refers to cell damage caused by reactive oxygen species (ROS).

The reactive oxygen species (ROS) includes super oxide radical (O ₂ ^- ), hydrogen peroxide (H ₂ O ₂ ), and hydroxy radical (OH ^- ).

Reactive oxygen species (ROS) attack the cell membrane of cells, the basic unit of the body, causing loss of original cell function, and attack genes within cells, interfering with cell regeneration. As a result, it destroys the signaling system or reduces immunity, causing disease in the body. Additionally, it may interfere with cell regeneration and cause or accelerate aging. Therefore, it is important to suppress excessive production of active oxygen as described above.

Therefore, the cell protection composition of the present invention exhibits antioxidant activity, thereby preventing damage to cells caused by free radicals, thereby exhibiting cytoprotective activity, and suppressing the causes of various diseases caused by cell damage.

The composition exhibits cytoprotective activity by inhibiting the mitochondria-mediated autophagy process by reducing mtROS production.

Mitochondria are known to be organelles that produce ROS, but they also have the function of neutralizing them, and mitochondria can control vascular cell function through fine regulation of reactive oxygen species. Among the mitochondrial mechanisms, electrons generated in the energy generation process pass through the electron transport chain and arrive at complex 5, the fifth mitochondrial enzyme. Among the electrons reduced in this process, 1-3% are incompletely combined with oxygen molecules. This produces superoxide and hydrogen peroxide (H ₂ O ₂ ), which are called mitochondrial ROS (mtROS).

An appropriate amount of ROS corresponding to physiological levels plays an important role in cell survival, but when ROS exceeding this level is generated, a mechanism to neutralize it is activated in the mitochondria, and mitochondrial function can be maintained.

However, when exposed to reactive oxygen species (ROS) due to oxidative stress, cells suffer oxidative damage, which causes a decrease in mitochondrial protein expression and a decrease in ATP production capacity, resulting in mitochondrial dysfunction, thereby meeting physiological standards. This causes excessive mtROS increase.

Accordingly, cells damaged by oxidative stress accumulate ROS and may exhibit mitochondrial dysfunction due to mitochondrial damage, such as autophagy, mitophagy, and DNA damage.

Accordingly, in the present invention, by including a cell protection composition as an active ingredient, mitochondrial function can be preserved through inhibition of mtROS production, resulting in cell protection activity.

Preferably, the cell protection composition of the present invention may further improve antioxidant and cell protection activities by further including natural extracts in addition to phloroglucinol.

The natural extract may be any one selected from the group consisting of Jeju wild rose extract, purple leaf violet extract, and mixtures thereof, and may include both the Jeju wild rose extract and purple leaf violet extract.

The Jeju starling (Rosa luciae) is a deciduous broad-leaved shrub of the dicotyledonous Rosaceae family. It grows in sunny areas at the foot of mountains or in moist river areas, and is 1 to 2 m tall. The ends of the branches droop downward, forming a vine shape, and there are thorns all over the plant. The leaves are alternate and pinnately compound. There are 5 to 9 small leaves, shaped like an oval or an upside-down egg, and are 2 to 3 cm long. Both ends of the leaves are narrow and the edges of the stipules are almost smooth. Flowers bloom in white or light pink from May to June and hang in panicles at the ends of new branches. The peduncle is hairless or has glandular hairs. The calyx pieces are lanceolate and bent backwards. The petals are shaped like an upside-down egg, have concave ends, and are fragrant. Hair grows on the style. The fruit is round in shape and ripens red in September. The fruit contains several achenes about 3 mm in diameter, which are white and hairy. The young leaves are eaten as a vegetable and the fruits are used as medicine for edema, arthritis, and boils in oriental and folk medicine. Distributed in Korea, Japan, etc.

The purple leaf violet (Viola violacea Makino.) is a perennial plant of the dicotyledonous plant Violaceae family, growing in dry forests. Some have no hair all over or have fine hair only on the surface of the leaf. The leaves all come from the roots and are egg-shaped or triangular egg-shaped. The surface of the leaf is dark green, but sometimes has white patterns, and the back is reddish-purple, with dull sawtooth edges. Flowers bloom from April to May, are dark red-purple, and hang one at a time at the end of the peduncle. The peduncle is 5 to 8 cm high, the side segments of the corolla are hairless, and the bracts with 5 stamens and 1 pistil are located below the center. The fruit is a capsule, 6 to 7 mm long, and has no hairs. Because the back of the leaf is purple, it is called purple leaf violet. Distributed in Korea (Jindo, Hallasan) and Japan.

The cell protection composition has excellent antioxidant activity by further comprising phloroglucinol and Jeju wildflower extract and purple leaf violet extract, and can exhibit a cell protection effect by alleviating cell DNA damage, mitochondrial function damage, and cell death caused by oxidative stress. there is.

In particular, it was confirmed that the effect was the best when mixing 10 to 30 parts by weight of Jeju starling extract and 10 to 30 parts by weight of purple leaf violet extract with respect to 100 parts by weight of phloroglucinol.

Even more preferably, the composition of the present invention may further include any one selected from the group consisting of a chinensis root extract, a cypress extract, or a mixture thereof.

The Cerastium holosteoides var. hallaisanense (Nakai) Mizush) is a biennial herb of the dicotyledonous plant Centrophyllaceae and is commonly grown in fields or fields. It is 15 to 25 cm tall, has branched branches, grows at an angle, is black and purple in color, and has glandular hairs on the upper part. The leaves are opposite each other, egg-shaped or egg-shaped, lanceolate, have smooth edges, are narrow at both ends, and have fine hairs. The flowers bloom from May to July and are white, hung on cyme inflorescences, and after the flowers fade, the ends of the florets bend downward. There are 5 calyx pieces and the length is about 4.5mm. There are also 5 petals, similar in length to the calyx, and split deeply into 2 at the end. There are 10 stamens, 1 pistil, and 5 styles. The fruit is a light yellowish brown capsule, cylindrical, runs horizontally, and is about 9mm long. The seeds are brown and have small wart-like protrusions. The young shoots are eaten as a vegetable and also used as feed for livestock. Distributed in Korea, Japan, China, etc.

The above-mentioned Justicia procumbens L. is an annual herb of the Justicia procumbens family of the dicotyledonous plant order, and is an annual herb that commonly grows in the fields. Short hair grows all over. The stem is square, has many branches, is 10-40cm high, and has thick nodes. The leaves are opposite, ovate or long oval-lanceolate, 2-4 cm long, 1-2 cm wide, and have smooth edges. The petiole is 2-15mm. Flowers grow densely in spikes at the ends of stems and branches from July to September and are light purple in color. The calyx is deeply divided into 5 branches. The corolla is 7-8mm long, and the lower lip is shallowly divided into three segments. There are two surgeries. The fruit is a capsule and has a long oval shape. It commonly grows in the central and southern regions of Korea. Widely distributed in temperate regions of Asia.

The cell protection composition of the present invention further contains phloroglucinol as well as Jeju starling extract, purple leaf violet extract, seaweed extract, and rat's tail extract, thereby enhancing the cell protection effect through the excellent antioxidant activity of phloroglucinol. It can be improved and the palatability of compositions prepared including it can also be improved.

In particular, based on 100 parts by weight of phloroglucinol, 10 to 30 parts by weight of Jeju starling extract, 10 to 30 parts by weight of purple leaf violet extract, 5 to 15 parts by weight of sorghum extract, and 5 to 15 parts by weight of rat's tail extract. When included, the activity may be superior compared to when it is outside the above weight range.

It can be seen that the effect is improved according to the mixing of the active ingredients of the natural extract according to the specific weight part mixing, and the preference of the composition of the present invention is improved by the inherent flavor of the natural extract.

The extract is extracted using an extraction solvent selected from the group consisting of water, C ₁ to C ₆ lower alcohols, and mixtures thereof.

The method of preparing the extract may be a conventional extraction method in the art, such as ultrasonic extraction, leaching, and reflux extraction. Specifically, it may be an extract obtained by extracting a natural product from which foreign substances have been removed by washing and drying with water, alcohol having 1 to 6 carbon atoms, or a mixed solvent thereof, and may be an extract obtained by sequentially applying the solvents to the sample.

The ultrasonic extraction method proceeds at 30 to 50°C for 0.5 to 2.5 hours, and the extraction solvent is water or 50 to 100% alcohol with 1 to 6 carbon atoms. Specifically, extraction is performed at 40 to 50°C for 1 to 2.5 hours, and the extraction solvent is water or 70 to 80% alcohol with 1 to 6 carbon atoms.

The leaching method is carried out at 15 to 30°C for 24 to 72 hours, and water or 50 to 100% alcohol with 1 to 6 carbon atoms is used as the extraction solvent. More specifically, it is carried out at 20 to 25°C for 30 to 54 hours, and the extraction solvent is water or 70 to 80% alcohol with 1 to 6 carbon atoms.

The reflux extraction method is based on 100 mL of water and alcohol with 1 to 6 carbon atoms, 10 to 30 g of pulverized natural product, reflux time of 1 to 3 hours, and 50 to 100% of alcohol or water with 1 to 6 carbon atoms. More specifically, based on 100 mL of alcohol with 1 to 6 carbon atoms or 100 mL of water, 10 to 20 g of pulverized natural product, 1 to 2 hours of reflux time, and 70 to 90% of alcohol or water with 1 to 4 carbon atoms.

The extraction solvent can be used in an amount of 2 to 50 times, more specifically, 2 to 20 times the weight of the sample. For extraction, the sample may be left for 1 to 72 hours, more specifically, 24 to 48 hours for leaching in the extraction solvent.

After extraction, the extract can be fractionated by sequentially applying a new fractionation solvent. The fractionating solvent used during fractionation is at least one selected from the group consisting of water, hexane, butanol, ethyl acetic acid, ethyl acetate, methylene chloride, and mixtures thereof, and is preferably ethyl acetate or methylene chloride.

After obtaining the extract or fraction, additional methods such as concentration or freeze-drying can be used.

A food composition for cell protection according to another embodiment of the present invention includes a composition for cell protection containing phloroglucinol as an active ingredient.

At this time, the amount of the composition in the food or beverage can be about 0.01 to 15% by weight of the total weight of the food, and the health drink composition can be added at a rate of about 0.01 to 10g based on 100 ml, but is not limited thereto. .

When the food composition of the present invention is a beverage composition, other than containing the extract as an essential ingredient in the ratio indicated, there are no particular restrictions on other ingredients, and various flavoring agents or natural carbohydrates may be contained as additional ingredients like ordinary beverages. You can. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sucrose, etc.; and polysaccharides, such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used, but are not limited to these. The ratio of the natural carbohydrate may generally range from about 1 to 20 g per 100 ml of the composition of the present invention, but may not be limited thereto.

In addition to the above, the composition of the present invention contains various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its It may contain salt, organic acid, protective colloidal thickener, Ph adjuster, stabilizer, preservative, glycerin, alcohol, carbonating agent used in carbonated beverages, etc., but may not be limited thereto.

In addition, the composition of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks, and these ingredients can be used independently or in combination. The proportion of these additives is not critical, but may be selected in the range of about 0.1 to about 20 parts by weight per 100 parts by weight of the composition of the present invention, but is not limited thereto.

It is possible to provide a functional food composition including the cell protection composition, and by including it, a health functional food can be provided. In the present invention, a "health functional food" refers to a food made from a specific ingredient as a raw material or food for the purpose of health supplementation. It includes health supplements as foods manufactured and processed by methods such as extraction, concentration, purification, and mixing of specific ingredients contained in raw materials, and other food ingredients include biological defense, regulation of biological rhythm, and disease prevention and recovery. Foods designed and processed to fully exert bioregulatory functions on the living body, including those that also have functions related to disease prevention and disease recovery, etc.

A pharmaceutical composition for treating, improving or preventing cell damage according to another embodiment of the present invention includes a cell protection composition containing phloroglucinol as an active ingredient.

Specifically, the pharmaceutical composition may be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, external preparations, suppositories, and sterile injection solutions, respectively, according to conventional methods. You can.

In the present invention, carriers, excipients, and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, and calcium phosphate. , calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include the extract and its fractions with at least one excipient such as starch, calcium carbonate, It is prepared by mixing sucrose, lactose, and gelatin. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, and syrups, and may contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives in addition to the commonly used simple diluents such as water and liquid paraffin. there is.

Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurin, glycerogenatin, etc. can be used.

The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount, and the term "pharmaceutically effective amount" in the present invention refers to the amount used to treat or prevent a disease with a reasonable benefit/risk ratio applicable to medical treatment or prevention. It refers to a sufficient amount, and the effective dose level is determined by the severity of the disease, the activity of the drug, the patient's age, weight, health, gender, the patient's sensitivity to the drug, the administration time, route of administration, and excretion rate of the composition of the present invention used. Factors including the duration of time, drugs used in combination or concurrently with the compositions of the invention used, and other factors well known in the medical field. The pharmaceutical composition of the present invention can be administered alone or in combination with a known immunotherapeutic agent. It is important to consider all of the above factors and administer the amount that will achieve the maximum effect with the minimum amount without side effects.

The present invention can provide a cell protection composition that exhibits antioxidant activity and contains phloroglucinol as an active ingredient.

In addition, another object of the present invention is to provide a food composition or pharmaceutical composition for cell protection that has excellent functionality and high preference by including a cell protection composition containing phloroglucinol as an active ingredient.

Figure 1 shows the activity of phloroglucinol in suppressing H ₂ O ₂ -induced viability reduction and cytotoxicity of ARPE-19 cells.
Figure 2 shows the anti-apoptotic activity of phloroglucinol in ARPE-19 cells treated with H ₂ O ₂ .
Figure 3 shows the inhibitory activity of phloroglucinol on ROS production induced by H ₂ O ₂ in ARPE-19 cells.
Figure 4 shows the activity of phloroglucinol in alleviating DNA damage caused by H ₂ O ₂ in ARPE-19 cells.
Figure 5 shows the H ₂ O ₂ -mediated mtROS production removal activity of phloroglucinol in ARPE-19 cells.
Figure 6 shows the protective activity of phloroglucinol against H ₂ O ₂ -induced mitochondrial damage and cytoplasmic release of cytochrome c in ARPE-19 cells.
Figure 7 shows the activity of phloroglucinol in attenuating H ₂ O ₂ induced autophagy in ARPE-19 cells.
Figure 8 shows the protective effect of phloroglucinol against oxidative damage in human RPE ARPE-19 cells.

Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily implement it. However, the present invention may be implemented in many different forms and is not limited to the embodiments described herein.

[Experimental Example 1: Antioxidant and cell protective activity of phloroglucinol]

1. Materials and Methods

1-1. Cell culture and treatment

ARPE-19 cells (CRL-2302), human retinal pigment epithelial cells, were purchased from American Type Culture Collection (Manassas, VA, USA) and incubated with 10% fetal bovine serum and 1% penicillin-streptomycin (WELGENE Inc., Gyeongsan, Korea). Cultured in supplemented Dulbecco's Modified Eagle Medium/F-12. To investigate the beneficial effects of phloroglucinol on oxidative damage, cells were incubated in medium containing desired concentrations of phloroglucinol and H ₂ O ₂ (Thermo Fisher Scientific, Inc., Waltham, MA, USA) for 24 hours. by incubation for an hour or phloroglucinol, N-acetyl-L-cysteine (NAC), (2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2- Oxoethyl) triphenylphosphonium chloride (Mito-TEMPO) and/or 3-methyladenine (3-MA, Sigma-Aldrich Co., St. Louis, MO, USA) treated with H ₂ O ₂ for 24 hours. It was treated for 1 hour beforehand. To investigate the blocking effect of phloroglucinol on ROS production induced by H ₂ O ₂ , cells were pretreated with phloroglucinol, NAC, and Mito-TEMPO for 1 h, followed by 1 h with H ₂ O ₂ processed for a while. To obtain fluorescence images of ROS production, H2AX expression, and autophagic vacuoles, cells cultured on coverslips were stimulated with H ₂ O ₂ in the presence or absence of phloroglucinol, NAC, and/or Mito-TEMPO. After treatment, cells were washed with phosphate-buffered saline and fluorescently stained.

1-2. Cell viability analysis

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) analysis was performed to determine the viability of ARPE-19 cells cultured under various conditions. Specifically, after the necessary experimental treatments, cells were incubated with MTT solution (Thermo Fisher Scientific, Inc.) for 3 hours. The insoluble formazan product formed was dissolved in dimethyl sulfoxide (DMSO, Thermo Fisher Scientific, Inc.) and analyzed at 570 nm using an enzyme-linked immunosorbent assay (ELISA) microplate reader (Molecular Device Co., Sunnyvale, CA, USA). The absorbance was read. Cell viability was expressed as a percentage of untreated control cells.

1-3. Cytotoxicity assay

To assess cytotoxicity, lactate dehydrogenase (LDH) release was detected using the LDH cytotoxicity assay kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Specifically, culture medium obtained from conditions treated with H ₂ O ₂ in the presence or absence of phloroglucinol was transferred to a 96-well plate, and the amount of released LDH was measured at 490 nm with an ELISA microplate reader.

1-4. Quantitative Assessment of Apoptosis

Annexin V-Fluorescein Isothiocyanate (FITC) Apoptosis Detection Kit was purchased from Abcam Inc. (Cambridge, UK) and used for quantitative assessment of apoptosis-inducing cells upon treatment with phloroglucinol and/or H ₂ O ₂ . After treatment, the collected cells were suspended in annexin binding buffer containing Annexin V-FITC and propidium iodide (PI) according to the manufacturer's instructions. Then, the fluorescence of 10,000 events was acquired using a flow cytometer (Becton Dickinson, San Jose, CA, USA). Annexin V-positive cells were considered as apoptosis-inducing cells as previously described.

1-5. DNA fragmentation analysis

To observe fragmented DNA, an apoptosis marker, the cell pellet was suspended in the lysis solution described previously. The supernatant was incubated with RNase A and proteinase K (Sigma-Aldrich Co.). DNA was precipitated with isopropyl alcohol (Sigma-Aldrich Co.). The extracted DNA was fractionated using a 1.0% agarose gel, stained with ethidium bromide (EtBr, Thermo Fisher Scientific, Inc.), and the DNA fragmentation pattern, a characteristic of apoptosis, was visualized under ultraviolet light.

1-6. Protein isolation and Western Blotting

Total proteins used for Western blot analysis were extracted as previously described. Cytosolic and mitochondrial proteins were isolated using the Mitochondrial Fractionation Kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Specifically, proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to Immobilon ®-P PVDF membrane (Merck Millipore, Bedford, MA, USA). The membrane was then incubated with specific primary antibodies overnight and then reacted with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) for 1 hour at room temperature. Immune complexes were visualized with enhanced chemiluminescence reagents (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Densitometric analysis of the data was performed using ImageJ® software (v1.48, NIH, Bethesda, MD, USA). Primary antibodies were from Santa Cruz Biotechnology, Inc. and Cell Signaling Technology (Beverly, MA, USA).

1-7. Caspase-3 activity analysis

Caspase 3 activity was quantified using the Caspase-3 Colorimetric Assay Kit (Abcam, Inc.). That is, an aliquot of the cytoplasmic extract was mixed with the fluorescent substrate of caspase-3 and acetyl-Asp-Glu-Val-Asp-chromophore-p-nitroanilide (Ac-DVAD-pNa) in the buffer provided in the kit. Enzyme-catalyzed release of pNa was monitored at 405 nm using an ELISA microplate reader. The activity of caspase-3 was presented compared to the control group.

1-8. Evaluation of ROS generation

The levels of intracellular ROS and mtROS production were detected using the fluorescent probes 2′,7′-dichlorofluorescein diacetate (DCF-DA) and MitoSOX (Sigma-Aldrich Co.), respectively. After exposure to H ₂ O ₂ with or without phloroglucinol, NAC, and/or Mito-TEMPO, cells were reacted with DCF-DA and MitoSOX to assess intracellular and mitochondrial superoxide levels, respectively, using flow cytometry. In parallel, fluorescence images of DCF-DA- and MitoSOX-stained cells cultured on coverslips were observed with a fluorescence microscope (Carl Zeiss, Oberkochen, Germany) at the Core Facility Center for Tissue Regeneration at Dong-eui University (Busan, Korea).

1-9. Comet assay

After appropriate treatment, the inhibitory effect of phloroglucinol on H ₂ O ₂ -induced DNA damage was determined using comet assay (single cell gel electrophoresis). Specifically, collected cells were suspended in 1% low melting point agarose and then spread on comet slides according to the manufacturer's protocol of the commercially available Comet Assay Kit (Trevigen, Inc., Gaithersburg, MD, USA). After DNA denaturation, electrophoresis was performed, and the slides were stained with an asymmetric cyanine dye. The resulting images were acquired with a fluorescence microscope.

1-10. γH2AX immunofluorescence analysis

Immunofluorescence assay was applied to analyze the expression of phosphorylated histone H2AX (p-γH2AX) in cells treated or not with phloroglucinol or NAC before adding H ₂ O ₂ . After treatment, cells were fixed with formaldehyde, permeabilized with Triton X-100 solution (Thermo Fisher Scientific, Inc.), and blocked with bovine serum albumin solution (Sigma-Aldrich Co.). Afterwards, cells were probed with anti-p-γH2AX antibody (Cell Signaling Technology, Inc.) and then reacted with Alexa Fluor 555-conjugated secondary antibody (Thermo Fisher Scientific, Inc.). For nuclear counterstaining, cells were immersed in 4',6-diamidino-2-phenylindol (DAPI) solution (Sigma-Aldrich Co.). Afterwards, p-γH2AX and DAPI fluorescence images were captured using a fluorescence microscope.

1-11. 8-Hydroxy-2'-Deoxyguanosine (8-OHdG) measurement

To measure 8-OHdG, the OxiSelect Oxidative DNA Damage ELISA Kit (Cell Biolabs, San Diego, USA), a deoxyriboside form of 8-oxoGuanine, was used. That is, DNA was extracted from cells cultured under the same conditions as above. Afterwards, the DNA of each isolated sample was digested with DNase I (Sigma-Aldrich Co.). Then, the absorbance of the ELISA reaction was measured at 450 nm according to the protocol provided in the kit.

1-12. Mitochondrial membrane potential (MMP) measurements

MMP levels were monitored by staining with the fluorescent carbocyanine probe 5,5',6,6'-tetrachloro-1,1'3,3'-tetraethyl-imidacarbocyano iodide (JC-1). did. For this analysis, cells treated with H ₂ O ₂ in the presence or absence of phloroglucinol or Mito-TEMPO were stained with JC-1 solution (Thermo Fisher Scientific, Inc.). The percentage of JC-1 monomers was analyzed by flow cytometry to indicate cells that had lost MMP.

1-13. Autophagy detection

Formation of autophagosomes was assessed using the CYTO-ID® Autophagy Detection Kit purchased from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). First, cells were collected for quantitative analysis of autophagy induction. Afterwards, the Cyto-ID staining procedure was performed according to the manufacturer's instructions. Specifically, cells cultured under various conditions were washed with the analysis buffer included in the kit and fixed with paraformaldehyde. Afterwards, the fluorescently labeled cells were analyzed by flow cytometry. Next, cells were subjected to CYTO-ID staining followed by DAPI staining to monitor autophagosome and nuclear localization. To monitor the location of autophagosomes and nuclei, cells were further subjected to CYTO-ID staining followed by DAPI staining. Autophagic signals (green) and nuclear signals (blue) were collected by fluorescence microscopy.

1-14. statistical analysis

All statistical analyzes were performed using GraphPad Prism (Graphpad Inc., San Diego, CA, USA). Statistical differences were determined by one-way analysis of variance using the Tukey test. Statistical significance was considered when the p value was less than 0.05. All data are expressed as mean ± standard deviation (SD) ( ^* p 0.05, ^** p 0.01 and ^*** p 0.001 vs. unstimulated control; ^## p 0.01 and ^### p 0.001 vs. H ₂ O ₂ treatment alone, ^&&& p 0.05 vs. phloroglucinol ⁺ Mito-TEMPO group).

2. Experimental results

2-1. Recovers decreased cell viability and increased cytotoxicity caused by H ₂ O ₂ of phloroglucinol

MTT analysis was performed to select the concentration of H ₂ O ₂ that can cause oxidative damage in ARPE-19 cells. As expected, H ₂ O ₂ treatment significantly inhibited cell viability in a dose-dependent manner (Figure 1A). H ₂ O ₂ at a concentration of 0.5mM reduced cell viability to about 60% of the control (untreated cells). Therefore, the cytotoxic concentration of hydrogen peroxide was set at 0.5mM. In addition, in an experiment to determine the appropriate concentration range of phloroglucinol to evaluate the cytotoxicity inhibitory effect of H ₂ O _2, phloroglucinol was found to have no significant effect on cell survival up to a concentration of 20 μg/ml. appeared (Figure 1B). Therefore, the highest optimal concentration of phloroglucinol was determined to be 20 μg/ml. Afterwards, the inhibitory effect of phloroglucinol on cytotoxicity induced by H ₂ O ₂ was evaluated and it was found that phloroglucinol significantly restored the decrease in cell viability induced by H ₂ O ₂ (Figure 1C). At the same time, pretreatment with NAC, a ROS scavenger used as a positive control _, completely inhibited the downregulation of cell viability in response to H ₂ O ₂ , _thereby reducing the oxidative stress induced by H ₂ O ₂ It has been proven that it can mediate a decrease in cell viability. Further analysis of the protective effect of phloroglucinol using the LDH leakage assay showed that phloroglucinol and NAC significantly reduced H ₂ O ₂ -induced LDH release into the cell culture medium (Figure 1D).

2-2. Inhibition of H ₂ O ₂ induced apoptosis by Phloroglucinol

We investigated whether the loss of cell survival and induction of cytotoxicity were related to the induction of cell death in ARPE-19 cells exposed to H ₂ O ₂ . As can be seen in Figures 2A and B, flow cytometry results after annexin V/PI staining showed that significantly more apoptosis was induced in H ₂ O ₂ treated cells than in untreated control cells. However, induction of apoptosis by H ₂ O ₂ was significantly weakened in cells treated with phloroglucinol. Afterwards, DNA fragmentation assay was performed to confirm whether phloroglucinol prevents cell death caused by hydrogen peroxide (H ₂ O ₂ ). As shown in Figure 2C, H ₂ O ₂ treated cells exhibited DNA laddering and oligonucleosome-sized DNA fragments. This pattern was not observed in untreated control cells. However, this pattern was significantly attenuated in cells preincubated with phloroglucinol. H ₂ O ₂ treatment also inhibited the Bcl-2/Bax ratio, which is known to be related to the activation of caspase-3 and the cleavage of PARP (poly(ADP-ribose) polymerase), a representative substrate protein of activated caspase-3. . However, these changes were greatly ameliorated in cells pretreated with phloroglucinol (Figure 2D, Figure 2E). These results suggest that phloroglucinol can effectively reduce H2O2-induced ARPE-19 cell death by regulating apoptosis regulators.

2-3. Elimination of H ₂ O ₂ induced ROS generation by Phloroglucinol

To confirm the antioxidant properties of phloroglucinol, intracellular ROS production was measured using the DCFH-DA probe. As shown in Figures 3A and B, flow cytometry results showed that intracellular superoxide production in cells treated with H ₂ O ₂ was significantly increased compared to untreated control cells, whereas it was significantly inhibited when pretreated with phloroglucinol. . In addition, almost no ROS was generated in cells treated with phloroglucinol alone. H ₂ O ₂ treatment was unable to increase ROS levels in cells treated with phloroglucinol even in the presence of NAC, indicating that phloroglucinol may act as a ROS scavenger. These results were confirmed through fluorescence microscopy of cells stained with DCF-DA. Pretreatment with phloroglucinol was shown to significantly eliminate H ₂ O ₂ -induced DCF fluorescence intensity (Figure 3C).

2-4. Removal of DNA damage caused by H ₂ O ₂ of Phloroglucinol

We evaluated whether phloroglucinol could prevent DNA damage induced by H ₂ O ₂ in ARPE-19 cells. The blocking effect of phloroglucinol on DNA damage induced by H ₂ O ₂ treatment was first investigated using the comet assay. As expected, an increase in comet tail moment was clearly observed in H ₂ O ₂ treated cells, indicating that DNA damage was induced by H ₂ O ₂ treatment (Figure 4A). To confirm this result, the expression of γH2AX was analyzed. Immunofluorescence results showed that the fluorescence intensity of γH2AX in the nuclei of H ₂ O ₂ treated cells clearly increased compared to the nuclei of untreated cells. Additional tests to quantify oxidative damage to nucleic acids also showed a significant increase in 8-OHdG levels upon exposure to H ₂ O ₂ (Figure 4C). However, the increase in DNA migration, γH2AX expression, and 8-OHdG/8-oxoGuanine ratio caused by H ₂ O ₂ treatment was significantly attenuated in the presence of NAC and phloroglucinol, suggesting that phloroglucinol is resistant to oxidation by H ₂ O ₂ This suggests that it can attenuate enemy DNA damage.

2-5. Phloroglucinol's H ₂ O ₂ -induced mtROS production reduction activity

MitoSOX-red, a mitochondrial superoxide-specific dye, was used to determine whether mitochondria are a major source of ROS induced by H ₂ O ₂ and whether phloroglucinol can be inhibited. As shown in Figure 5A, strong red fluorescence intensity was evident in ARPE-19 cells treated with H ₂ O ₂ but not in untreated control cells or cells treated with phloroglucinol alone. However, the fluorescence intensity induced by H ₂ O ₂ was suppressed in the presence of phloroglucinol. It was further removed in cells pretreated with phloroglucinol and Mito-TEMPO, a mitochondria-targeting antioxidant. These results were consistent with the results of flow cytometry, which directly measured the frequency of MitoSOX-red positive cells (Figure 5B, Figure 5C). In addition, phloroglucinol inhibited H ₂ O ₂ -induced accumulation of PINK1 and PARKIN, known as major autophagy proteins in mitochondria. These results suggest that phloroglucinol may contribute to the inhibition of mitophagy through its role as a scavenger of mtROS induced by H ₂ O ₂ in ARPE-19 cells.

2-6. Phloroglucinol's protective activity against mitochondrial dysfunction caused by H- ₂ O ₂

To determine whether phloroglucinol can prevent H _{-2O2 -} induced mitochondrial damage, MMPs were estimated after JC-1 staining. Flow cytometry results (FIG. 6A, FIG. 6B) showed that the frequency of JC-1 monomers was significantly increased, while the frequency of JC-1 aggregates was decreased in H- _{2O2 -} treated cells, indicating that H- _2O2 induces the breakdown of MMPs _. It indicates that it was done. However, these changes were significantly attenuated by phloroglucinol pretreatment. In addition, when phloroglucinol and Mito-TEMPO were used together in pretreatment, the loss of MMPs induced by H- ₂ O ₂ was almost completely reduced to the control level. Additionally, after H- ₂ O ₂ treatment, the expression level of cytochrome c increased in the cytoplasm but decreased in the mitochondria. Phloroglucinol pretreatment was able to restore these changes (Figure 6C). These results show that blocking H- ₂ O ₂ -induced mtROS production by phloroglucinol can preserve mitochondrial function.

2-7. Inhibitory effect of H ₂ O ₂ induced autophagy of Phloroglucinol

The effect of phloroglucinol on H ₂ O ₂ induced autophagy was evaluated in ARPE-19 cells. As shown in Figures 7A and B, flow cytometry using Cyto-ID tracking dye, which can monitor autophagic vacuoles, showed that H ₂ O ₂ significantly induced autophagy. However, pretreatment with phloroglucinol or 3-MA, a selective autophagic inhibitor, drastically reduces H ₂ O ₂ -induced autophagy in cells. Consistent with these results, the formation of Cyto-ID puncta was enhanced in response to H ₂ O ₂ , whereas it was almost completely reduced after phloroglucinol pretreatment (Figure 7C). Afterwards, H ₂ O ₂ -induced autophagy was confirmed by detecting autophagy biomarkers such as microtubule-associated protein-1 light chain-3 (LC3), Beclin-1, and p62 by immunoblotting. As shown in Figure 7D, H ₂ O ₂ enhanced the conversion of C3-I to LC3-II and induced Beclin-1 expression, but downregulated the expression of p62. However, these changes induced by H ₂ O ₂ were all eliminated by phloroglucinol, which supports the results of flow cytometry analysis that H ₂ O ₂ -mediated autophagy can be protected by phloroglucinol. there is.

Experimental results show that phloroglucinol can protect RPE cells from oxidative damage induced by H ₂ O ₂ , thereby reducing DNA damage, mitochondrial damage, and cell death, and It was confirmed that survival can be improved. In addition, it was confirmed that the cytoprotective effect of phloroglucinol can be achieved by controlling mitochondria-mediated autophagy through blocking mtROS production (Figure 8).

[Preparation Example 2: Cell protection composition containing complex extract]

1. Preparation of composition

Preparation of Jeju wild rose extract

Jeju starlings were washed in water and then freeze-dried. Afterwards, the freeze-dried A was pulverized with a blender, leached at room temperature for 48 hours using 80% ethanol, and the sample was filtered to prepare Jeju wild rose extract (RE).

Preparation of other natural extracts

In the same manner as the Jeju wild rose extract (RE), only natural products were replaced with purple leaf violet, purple leaf violet, and rat's tail, to produce purple leaf violet extract (VE), black and white violet extract (CE), and rat's tail extract (JE). did.

Preparation of complex extract

In addition to the rock mulberry extract (MEMC) used in Experimental Example 1, the Jeju wild rose extract (RE), purple leaf violet extract (VE), cypress root extract (CE), and rat's tail extract (JE) were added in parts by weight as shown in Table 1 below. Complex extracts (MX 1 to MX13) were prepared by mixing within the range.

MX1 MX2 MX3 MX4 MX5 MX6 MX7 MX8 MX9 MX10 MX11 MX12 MX13 phloroglucinol 100 100 100 100 100 100 100 100 100 100 100 100 100 R.E. - 20 - One 10 20 30 40 20 20 20 20 20 VE - - 20 One 10 20 30 40 20 20 20 20 20 C.E. - - - - - - - - One 5 10 15 20 J.E. - - - - - - - - One 5 10 15 20

(Unit: parts by weight)

[Experimental Example 2: Antioxidant effect and cell protective activity of complex extract]

In order to show the cell protection effect of the composition of the present invention, the same experiment as Experimental Example 1 was performed, and only the extract was replaced with the composition of the present invention.

In addition, the above results showed the comparative results by fixing phloroglucinol at an index of 3, and the index was evaluated as a number between 1 and 10. The higher the number, the better the antioxidant activity and cell protection activity, and the results are comprehensively shown in Table 2 below.

MX1 MX2 MX3 MX4 MX5 MX6 MX7 MX8 MX9 MX10 MX11 MX12 MX13 Recovery of decreased cell viability and increased toxicity caused by H2O2 3 3.5 3.3 6.0 6.5 7.5 7.4 6.3 8.0 8.5 9.0 8.8 8.3 Apoptosis reduction activity by H2O2 3 3.5 3.4 6.2 7.0 7.4 7.3 6.6 8.1 8.6 8.9 8.5 8.0 Elimination of ROS production by H2O2 3 3.3 3.5 5.9 6.3 7.4 7.2 6.0 8.3 8.6 8.8 8.5 7.8 Removal of DNA damage by H2O2 3 3.2 3.3 6.0 6.5 7.0 7.0 6.2 8.0 8.5 9.0 8.8 7.7 Reduced mtROS production by H2O2 3 3.3 3.4 5.8 6.4 7.2 6.9 6.0 7.9 8.3 9.0 8.8 7.6 Protective effect against mitochondrial dysfunction caused by H2O2 3 3.5 3.6 5.6 6.6 7.2 7.0 6.2 8.2 8.5 9.0 8.8 7.7 Autophagy inhibition effect due to H2O2 3 3.2 3.5 5.9 6.3 7.1 6.7 6.0 8.0 8.3 9.1 8.9 7.8

(Unit: index)

Referring to Table 2 above, compared to the case containing only phloroglucinol, in the case of MX2 and 3, which further contain Jeju wild rose extract (RE) and purple leaf violet extract (VE), respectively, the case containing only phloroglucinol Compared to this, it shows slightly improved cell viability, toxicity increasing activity, apoptosis reducing activity, ROS generation removal activity, DNA damage inhibition activity, mtROS generation inhibition activity, and mitochondrial disorder protection effect, and shows antioxidant and cytoprotective activity through autophagy inhibition effect. can confirm.

In addition, it was confirmed that in the case of MX5 to 7, which contain natural extracts such as Jeju wild rose extract and purple leaf violet extract in a specific content range in addition to the phloroglucinol, the effect is further improved compared to cases outside the above weight part range. You can.

Furthermore, it was confirmed that in the case of MX9 to 13, which further contains the phloroglucinol, as well as the Jeju starling extract and the purple violet extract, as well as the phloroglucinol extract (CE) and the rat's tail extract (JE), slightly improved activity was observed. It was found that even in this case, the activity was more improved than in the case of MX10 to 12 contained in a specific extender.

That is, with respect to 100 parts by weight of phloroglucinol, a composition comprising 10 to 30 parts by weight of Jeju starling extract and 10 to 30 parts by weight of purple leaf violet, and further, with respect to 100 parts by weight of phloroglucinol, 10 to 30 parts by weight of Jeju starling extract. If it contains 30 parts by weight, 10 to 30 parts by weight of purple leaf violet, 5 to 15 parts by weight of dandelion root extract, and 5 to 15 parts by weight of rat's tail columbine extract, antioxidant and cell It can be confirmed that the protective activity is significantly improved, and therefore, the present invention confirms that the antioxidant and cytoprotective activity of phloroglucinol is excellent, and further improved activity can be exhibited by further including the above-mentioned natural extract. This was confirmed through experiment.

[Experimental Example 3: Effect of improving preference of the composition of the present invention]

Tea drinks containing each of the above complex compositions (MX1 to MX13) were prepared, and 40 adult men and women in their 20s to 50s were tasted and then evaluated for flavor and taste to evaluate preference with an index of 1 to 10. did. For relative evaluation, the MX1(ME) of the present invention extracted by immersion in water was fixed to an index of 3 to evaluate it as a relative index value. The results were synthesized and averaged, and then rounded to two decimal places to obtain a comprehensive result in Table 3 below. It is expressed as

MX1 MX2 MX3 MX4 MX5 MX6 MX7 MX8 MX9 MX10 MX11 MX12 MX13 taste 3.0 4.0 4.5 5.0 6.0 6.0 6.5 5.5 7.0 8.5 9.0 9.0 8.0 incense 3.0 4.0 4.0 5.2 6.3 6.5 6.6 5.1 7.5 8.5 9.0 9.0 7.5 Synthesis
preference 3.0 4.0 4.3 5.1 6.2 6.3 6.6 5.3 7.3 8.5 9.0 9.0 7.8

(Unit: index)

Referring to Table 3, it can be seen that preference increases in the case of MX2 and MX3, which additionally contain Jeju wild rose extract (RE) and purple leaf violet extract (VE), respectively, compared to MX1, which contains only phloroglucinol of the present invention. there is.

In the case of MX4 to MX8, which are included in the form of a complex extract rather than as a single extract, the effect is further improved. This is because the unique scent of a single natural product is alleviated by using a mixture of natural extracts, making it more effective through harmony of taste and aroma. This means that it can show excellent preference.

In addition, in the case of MX9 to MX13, which additionally contain snail herb extract (CE) and cypress extract (JE), preference was further improved, which was achieved by alleviating the unique scent of the rock beard extract due to the mixed use of natural extracts. It can be seen that this is due to an effect.

In particular, for 100 parts by weight of phloroglucinol, which is a preferred weight part, 10 to 30 parts by weight of Jeju wild rose extract (RE), 10 to 30 parts by weight of violet leaf violet extract (VE), and 5 to 15 parts by weight of eucalyptus extract (CE). It can be seen that when mixing 5 to 15 parts by weight of JE and JE, the overall preference for taste and aroma is the best, making it possible to provide a composition with high preference.

Therefore, it was confirmed that the composition containing the above mixed composition exhibits cell protection effect and antioxidant activity while being excellent in taste and aroma evaluation, making it possible to provide it as a food composition or pharmaceutical composition with high functionality and preference.

Although the preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements made by those skilled in the art using the basic concept of the present invention defined in the following claims are also possible. falls within the scope of rights.

Claims (7)

Contains phloroglucinol as an active ingredient
showing antioxidant activity
Composition for cell protection. According to clause 1,
Cell protection is achieved by alleviating DNA damage, mitochondrial function damage, and cell death caused by oxidative stress.
Composition for cell protection. According to clause 1,
The oxidative stress is cell damage caused by reactive oxygen species (ROS).
Composition for cell protection. According to clause 1,
The reactive oxygen species (ROS) includes super oxide radical (O ₂ ^- ), hydrogen peroxide (H ₂ O ₂ ), and hydroxy radical (OH ^- ).
Composition for cell protection. According to clause 1,
The composition exhibits cytoprotective activity by inhibiting the mitochondria-mediated autophagy process through reducing mtROS production.
Composition for cell protection. Comprising the composition according to any one of claims 1 to 5.
Food composition for cell protection. Comprising the composition according to any one of claims 1 to 5.
Pharmaceutical composition for treating, improving or preventing cell damage.