A composition for protecting cells exhibiting antioxidant activity, including a myelophycus caespitosus extracts as an active ingredient

Abstract

The present invention relates to a cell-protective composition exhibiting antioxidant activity comprising a rock beard extract. By including the rock beard extract, the composition can exhibit cell-protective activity against oxidative stress, cytotoxicity, and DNA damage inhibition activity, thereby providing a composition with excellent antioxidant activity.

Description

Technology Field

[0001] The present invention relates to a composition for antioxidant activity comprising a rock beard extract and a method for preparing the same. More specifically, the invention relates to a composition that, by including a rock beard extract, can exhibit cell protective activity against oxidative damage caused by oxidative stress and can exhibit strong antioxidant activity through the inhibition of cytotoxicity, DNA damage, and reactive oxygen species generation. Background Technology

[0002] Muscles require large amounts of oxygen to generate energy for contractile activity and are vulnerable to oxidative stress characterized by the abnormal overaccumulation of reactive oxygen species (ROS). Myoblasts, which are embryonic precursors of muscle cells, can differentiate into muscle cells through the myogenesis process of fusing into multinucleated myotubes. While appropriate levels of ROS can regulate cellular signaling pathways necessary for muscle differentiation, excessive ROS accumulation is closely associated with impaired muscle formation; furthermore, myoblast apoptosis caused by excessive ROS production is accompanied by cell cycle arrest and DNA damage, which can contribute to the blockage of muscle differentiation and the induction of muscle atrophy. Therefore, excessive ROS production must be suppressed to protect muscle function.

[0003] Reactive oxygen species (ROS), which are free radicals, are produced when white blood cells are activated during the metabolism of external chemical substances upon absorbing radiant energy such as ultraviolet rays or X-rays, or during inflammatory responses. They are also generated during the normal respiration process of mitochondria within cells. Cells minimize damage caused by these ROS by developing mechanisms to eliminate them. However, if too many ROS are produced compared to the cell's ability to eliminate them, the cell enters a state of oxidative stress. In various inflammatory diseases, ROS amplify inflammatory responses by activating intracellular transcription factors such as NF-κB and AP-1. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a very important transcription factor in protecting cells against oxidative stress and carcinogenesis. It binds to the antioxidant response element (ARE) to activate the transcription of major antioxidant enzymes and phase II detoxifying enzymes, and representative genes associated with this are known to include HMOX1 (Heme oxygenase 1) and FTL (Ferritin light chain). Since Nrf2-dependent cytoprotective responses can protect various organs and tissues, Nrf2 activity has attracted attention for its potential to play a defensive role against various diseases, such as cancer, neurodegenerative diseases, cardiovascular diseases, acute / chronic lung injury, and autoimmune diseases.

[0004] HMOX1 is an important enzyme responsible for maintaining the proper amount of heme within cells and exists as three isoforms. Unlike HMOX2 and HMOX3, which are involved in basic metabolic activities in most cells, HMOX1 is a form induced by various stimuli such as oxidative stress, lipopolysaccharides, melatonin, and adenosine. HMOX1 plays a major role in maintaining tissue homeostasis through the inhibition of oxidative damage, reduction of inflammatory responses, and regulation of cell proliferation.

[0005] Meanwhile, marine resources have recently been receiving significant attention as functional foods and as raw materials for such foods. Among these, there are reports that extracts or components of edible seaweed can inhibit pathological conditions dependent on oxidative stress, such as inflammation, liver damage, dyslipidemia, endothelial dysfunction, and atherosclerosis. The antioxidant activity of the aforementioned marine resources primarily involves ROS scavenging and the activation of intracellular antioxidant signaling pathways.

[0006] The present invention was completed to provide a composition that includes rock beard extract as an active ingredient and can exhibit cell protective activity against cell damage caused by oxidative stress through antioxidant activity. Prior art literature

[0007] (Patent Document 0001) KR 10-2088995 B1(Patent Document 0002) KR 10-2049970 B1 The problem to be solved

[0008] The objective of the present invention is to provide a cell-protective composition that exhibits antioxidant activity by including rock beard extract as an active ingredient.

[0009] In addition, another objective of the present invention is to provide a food composition or a pharmaceutical composition for treating, improving, or preventing cell damage that exhibits a cell-protective effect, comprising the above composition. means of solving the problem

[0010] To achieve the above objective, a cell-protecting composition according to one embodiment of the present invention comprises rock beard extract as an active ingredient and exhibits antioxidant activity.

[0011] The aforementioned cell protection is demonstrated by mitigating cell cycle arrest, DNA damage, and apoptosis caused by oxidative stress.

[0012] The aforementioned oxidative stress refers to the damage caused to normal cells due to an increase in the concentration of reactive oxygen species (ROS).

[0013] The above composition exhibits antioxidant activity due to increased activity of the Nrf2 / HO-1 signaling pathway.

[0014] The above extract is extracted using an extraction solvent selected from the group consisting of water, C1 to C6 lower alcohols, and mixtures thereof.

[0015] A cell-protecting food composition according to another embodiment of the present invention comprises a cell-protecting composition containing rock beard extract as an active ingredient.

[0016] In another embodiment of the present invention, a pharmaceutical composition for treating, improving, or preventing cell damage comprises a cell-protecting composition containing rock beard extract as an active ingredient.

[0018] The present invention will be described in more detail below.

[0020] In the present invention, "prevention" refers to any act of suppressing or delaying the progression of a disease or illness to which the term applies, or one or more symptoms of said disease or illness, by administering a composition of the present invention.

[0021] In the present invention, "improvement" refers to any act in which the disease or illness to which the term applies, or one or more symptoms of said disease or illness, are improved or beneficially altered by the administration of the composition of the present invention.

[0022] In the present invention, "treatment" means reversing, alleviating, inhibiting the progression of, or preventing a disease or illness to which the term applies, or one or more symptoms of said disease or illness, and as used herein, the term "treatment" refers to the act of treating when "treating" is defined as above.

[0023] As used in this specification, the term "active ingredient" means a component that exhibits the intended activity alone or can exhibit activity in combination with a carrier that is inactive itself.

[0024] As used in this specification, the term 'extract' has the meaning commonly used in the art as a crude extract, as described above, but in a broader sense, it also includes fractions obtained by further fractionating the extract. That is, the plant extract includes not only that which is obtained using the extraction solvent described above, but also that which is obtained by additionally applying a purification process thereto. For example, fractions obtained through various additional purification methods, such as a fraction obtained by passing the extract through an ultrafiltration membrane having a certain molecular weight cut-off value, or separation by various chromatographs (designed for separation based on size, charge, hydrophobicity, or affinity), are also included in the plant extract of the present invention.

[0025] The terms used herein are for describing embodiments and are therefore not intended to limit the invention. In this specification, the singular form includes the plural form unless specifically stated otherwise in the text. As used herein, 'comprise' and / or 'comprising' do not exclude the presence or addition of one or more other components, steps, actions, and / or elements to the mentioned components, steps, actions, and / or elements.

[0027] A cell-protecting composition according to one embodiment of the present invention comprises rock beard extract as an active ingredient and exhibits antioxidant activity.

[0028] A cell refers to the basic functional and structural unit of all living organisms. While its size varies depending on functional characteristics, most are measured in micrometers and are difficult to observe with the naked eye.

[0029] Cells consist of cytoplasm surrounded by a cell membrane and contain many biomolecules such as proteins and nucleic acids. Meanwhile, cells take in nutrients and convert them into energy, perform specific functions, are capable of metabolism on their own, and can reproduce as needed. Each cell possesses specific organelles to carry out these various life activities.

[0030] Cells are capable of replication through methods of cell division such as binary fission, mitosis, and meiosis. Furthermore, through metabolism, they form cellular components from absorbed nutrients, generate energy and molecules, and eliminate byproducts. Cells also extract and utilize chemical energy through metabolic pathways, synthesize proteins through the process of protein synthesis, and possess signal transduction systems in response to external or internal stimuli such as pH, temperature, and nutrients.

[0031] Meanwhile, reactive oxygen species (ROS) perform beneficial activities by eliminating bacteria or viruses that have entered the body, but if they are overproduced, they can also attack normal cells, which can cause problems with the normal functioning of cells.

[0032] In particular, among various cells, myoblasts, which are embryonic precursors of muscle cells, can differentiate into muscle cells through the process of muscle formation involving fusion into multinucleated myotubes. While appropriate levels of ROS can regulate cellular signaling pathways necessary for muscle differentiation, excessive ROS accumulation is closely associated with impaired muscle formation. Furthermore, myoblast apoptosis caused by excessive ROS production is accompanied by cell cycle arrest and DNA damage, which can contribute to the blockage of muscle differentiation and the induction of muscle atrophy. Therefore, excessive ROS production must be suppressed to protect muscle function.

[0033] When problems arise in cells due to the overproduction of intracellular reactive oxygen species that perform the various roles mentioned above, the cell cycle may stop, or intracellular DNA damage may occur, or normal cell function may not be maintained.

[0034] In this regard, *Myelophycus caespitosus* is a brown algae belonging to the family Myelophyceae. It grows densely in belt-like colonies on rocks in the mid-intertidal zone, and species inhabiting areas with strong waves sometimes exhibit a twisted body appearance. The plant body consists of long, slender, thread-like branches that grow upright in clumps, reaching a height of 5–15 cm and a thickness of about 1 mm. The dark brown upper section is slightly thicker, becoming lighter brown and tapering toward the base, and it has a short stem. Young individuals are filled with parenchyma cells inside, but the central part of the body becomes hollow as they mature. It is distributed along the southern coast of Korea and in Japan.

[0035] The present invention can provide a composition with excellent cell protective activity by including the above-mentioned rock beard extract, which has excellent antioxidant activity, as an active ingredient.

[0036] More specifically, the rock beard extract of the present invention, which has excellent antioxidant activity, can provide a composition with excellent protective activity against myoblasts.

[0037] The aforementioned cell protection is demonstrated by mitigating cell cycle arrest, DNA damage, and apoptosis caused by oxidative stress.

[0038] The cell cycle is divided into the S phase, during which DNA is doubled; the M phase, during which chromosome partitioning and cell division occur; and the G1 and G2 phases, which are intermediate periods to prepare for these two stages; cells proliferate as the sequence of G1, S, G2, and M phases repeats periodically.

[0039] Oxidative stress causes the cell cycle to stop due to an increase in p21, a Cdk (cyclin-dependent kinase) inhibitor that is a negative regulator of cell cycle progression, and a decrease in cyclin A and cyclin B1, which are positive regulators required for progression from G2 to M phase.

[0040] Meanwhile, DNA exists within the nucleus of a cell and stores the genetic information of the organism. Cells maintain a species by producing daughter cells with the same DNA through the process of division, or by passing on their DNA to offspring through meiosis. However, DNA, which regulates genetic information, is damaged by chemical substances, radiation (UV), and reactive oxygen species (ROS) generated by cellular respiration. This can lead to the formation of cancer in cells or the occurrence of gene mutations during DNA replication, which can affect normal cell growth or cause cell death.

[0041] Furthermore, apoptosis refers to the phenomenon in which cells self-destruct under various stressful conditions. Apoptosis is divided into exogenous apoptosis, initiated by factors outside the cell, and endogenous apoptosis, initiated by factors inside the cell. Among these, when reactive oxygen species are overproduced due to oxidative stress, the depolarization of the mitochondrial membrane leads to the loss of MMPs, causing cytochrome c to be released from the mitochondria into the cytoplasm. This activates the caspase cascade, which is necessary for the mitochondria-mediated endogenous apoptosis pathway, thereby inducing cell death. At this time, cells with less DNA than normal cells during the cell cycle serve as an indicator of apoptosis, and cell death can be confirmed through an increase in sub-G1 during changes in the cell cycle.

[0042] The present invention can exhibit cell-protective activity by inhibiting or mitigating cell cycle arrest, DNA damage, and apoptosis induced by the above-mentioned oxidative stress.

[0043] The aforementioned oxidative stress refers to the damage caused to normal cells due to an increase in the concentration of reactive oxygen species (ROS).

[0044] Reactive oxygen species (ROS) attack the cell membranes of cells, the body's basic units, causing them to lose their original functions, and also attack genes within cells, hindering their regeneration. Consequently, they disrupt signaling pathways or reduce immunity, becoming a cause of disease within the body. Furthermore, by interfering with cell regeneration, they can also cause or accelerate aging.

[0045] The above-mentioned reactive oxygen species are superoxide radicals (O2 - ), hydrogen peroxide (H2O2), hydroxy radical (OH -Includes ) etc.

[0046] It is important to suppress the excessive generation of reactive oxygen species as described above, and this can be achieved through the antioxidant activity of the composition of the present invention.

[0047] The above composition exhibits antioxidant activity due to increased activity of the Nrf2 / HO-1 signaling pathway.

[0048] Nrf2 (nuclear-E2-related factor 2) is a transcription factor capable of enhancing antioxidant capacity by regulating the expression of step 2 detoxification enzymes. Generally, Nrf2 is bound to Keap1 and exists in the cytoplasm; however, under conditions of oxidative stress caused by reactive oxygen species such as ROS, it detaches from Keap1 and joins the nucleus to regulate cell defense mechanisms and function to remove reactive oxygen species or other harmful substances.

[0049] HO-1 is a stress-induced reactive protein and one of the phase II detoxification enzymes regulated by Nrf2, which catalyzes heme oxidation to break it down into biliverdin, free iron, and carbon monoxide, and converts it into bilirubin to produce endogenous antioxidants.

[0050] In other words, when a cell is stimulated by oxidative stress, Nrf2 separates from Keap1 and joins the nucleus, thereby regulating the cell defense mechanism, and HO-1 is converted into bilirubin by catalyzing heme oxidation induced by stress, thereby producing antioxidant substances and exhibiting antioxidant activity.

[0051] That is, the composition of the present invention can exhibit antioxidant activity by increasing the activity of the above Nrf2 / HO-1 signaling pathway.

[0052] The above composition may further include additional natural extracts in addition to the rock bear extract.

[0053] The above natural extract may be selected from the group consisting of Jeju wild rose extract, purple violet extract, and mixtures thereof.

[0054] The aforementioned Jeju wild rose (*Rosa luciae*) is a deciduous broad-leaved shrub belonging to the Rosaceae family (order Rosales) of dicotyledonous plants. It grows in sunny areas at the foot of mountains or in damp streambeds, reaching a height of 1 to 2 meters. The tips of the branches droop downwards, forming a vine-like shape, and the entire plant is covered in thorns. The leaves are alternate and pinnately compound. There are 5 to 9 leaflets, which are oval or inverted egg-shaped and 2 to 3 cm long. Both ends of the leaves are narrow, and the margins of the stipules are nearly smooth. Flowers bloom in May and June in white or light pink, borne in panicles at the tips of new branches. The small flower stalks are hairless or covered with glandular hairs. The sepals are lanceolate and bent backward. The petals are inverted egg-shaped with concave tips and are fragrant. The pistil is hairy. The fruit is round and ripens red in September. The fruit contains several achenes about 3 mm in diameter; they are white and hairy. The young leaves are eaten as a vegetable, and the fruit is used in traditional Korean medicine and folk remedies for edema, arthritis, boils, and other ailments. It is distributed in Korea, Japan, and other regions.

[0055] The aforementioned Viola violacea Makino. is a perennial herb belonging to the family Violaceae in the order Violales of dicotyledonous plants, growing in dry forests. Some varieties are entirely hairless, while others have fine hairs only on the leaf surface. All leaves emerge from the root and are egg-shaped or triangular-egg-shaped. The upper surface of the leaf is dark green but sometimes has white markings, while the underside is reddish-purple with blunt teeth along the margins. Flowers bloom from April to May; they are dark reddish-purple and borne singly at the end of the flower stalk. The flower stalk is 5–8 cm tall, and the lateral lobes of the corolla are hairless. Bracts containing five stamens and one pistil are attached below the center. The fruit is a capsule, 6–7 mm long, and hairless. It is called the Viola violacea because the underside of the leaves is purple. It is distributed in Korea (Jindo, Mt. Halla) and Japan.

[0056] The above composition may provide a composition with improved functionality by including 20 to 40 parts by weight of Jeju wild rose extract and 20 to 40 parts by weight of violet extract per 100 parts by weight of rock bear's beard extract, thereby enhancing antioxidant activity compared to the case where only rock bear's beard extract is included and having a superior cell protective effect.

[0057] Furthermore, the above composition may further include a natural extract selected from the group consisting of *Scutellaria baicalensis* extract, *Aster tataricus* extract, and mixtures thereof.

[0058] The aforementioned *Cerastium holosteoides var. hallaisanense (Nakai) Mizush* is a biennial herb belonging to the Caryophyllaceae family of the Caryophyllales order of dicotyledonous plants, commonly found growing in fields and meadows. Reaching a height of 15–25 cm, it branches out and grows obliquely; it has a dark purplish tint and glandular hairs on the upper parts. The leaves are opposite, egg-shaped or ovate-lanceolate, with smooth margins that narrow at both ends and fine hairs. Flowers bloom from May to July, are white, and borne in cymose inflorescences; after the flowers wither, the tips of the pedicels bend downward. There are five sepals, each about 4.5 mm long. There are also five petals, similar in length to the sepals, which are deeply split into two at the tips. There are 10 stamens, one pistil, and five styles. The fruit is a pale yellowish-brown capsule; it is cylindrical, hangs horizontally, and is about 9 mm long. The seeds are brown and have small, wart-like protrusions. The young shoots are eaten as a vegetable, and it is also used as livestock feed. It is distributed in Korea, Japan, China, and other regions.

[0059] The aforementioned *Justicia procumbens L.* is an annual plant belonging to the family *Justicaceae* in the order *Lambale* of dicotyledonous plants, and it is a common annual that grows in fields. The entire plant is covered with short hairs. The stem is square, highly branched, 10–40 cm tall, and has thick nodes. The leaves are opposite, ovate or oblong-lanceolate, 2–4 cm long and 1–2 cm wide, with smooth margins. The petioles are 2–15 mm long. Flowers bloom from July to September in dense spike inflorescences at the tips of stems and branches, and are pale purple. The calyx is deeply divided into five lobes. The corolla is 7–8 mm long, with the lower lip shallowly divided into three lobes. There are two stamens. The fruit is a capsule and is linear-oblong. It grows commonly in Korea south of the central region. It is widely distributed in temperate regions of Asia.

[0060] The cell-protecting composition of the present invention can provide a composition that exhibits superior antioxidant and cell-protective activity while improving palatability for taste and aroma by further including Jeju wild rose extract and purple violet extract and Japanese knotweed extract in the rock bear's beard extract.

[0061] The above composition may exhibit excellent antioxidant activity and cell protective activity by including 20 to 40 parts by weight of Jeju wild rose extract, 20 to 40 parts by weight of violet extract, 10 to 30 parts by weight of Japanese knotweed extract, and 1 to 5 parts by weight of black cohosh extract, based on 100 parts by weight of rock bear's beard extract.

[0062] According to the above weight range, the functionality of the composition can be maintained or slightly improved by Jeju wild rose, purple-leaved violet, Japanese knotweed, and mouse-tail grass, while the palatability can be improved. In the above range, the mixture of each extract can exhibit an activity that increases the antioxidant effect, and there is no problem with cytotoxicity caused by the use of natural extracts. It can be provided as a food composition or pharmaceutical composition that is suitable for long-term consumption and has improved palatability.

[0063] The above extract is extracted using an extraction solvent selected from the group consisting of water, C1 to C6 lower alcohols, and mixtures thereof.

[0064] The method for preparing the above 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, an alcohol having 1 to 6 carbon atoms, or a mixture of these solvents, and may be an extract obtained by sequentially applying the solvents to a sample.

[0065] The above ultrasonic extraction method is carried out at 30 to 50°C for 0.5 to 2.5 hours, and the extraction solvent is water or 50 to 100% alcohol having 1 to 6 carbon atoms. Specifically, the extraction is carried out at 40 to 50°C for 1 to 2.5 hours, and the extraction solvent is water or 70 to 80% alcohol having 1 to 6 carbon atoms.

[0066] The above leaching method is carried out at 15 to 30°C for 24 to 72 hours, and water or 50 to 100% alcohol having 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 having 1 to 6 carbon atoms.

[0067] The above reflux extraction method is based on 100 mL of water, 10 to 30 g of crushed natural product, a reflux time of 1 to 3 hours, and 50 to 100% of alcohol or water having 1 to 6 carbon atoms. More specifically, based on 100 mL of alcohol or water having 1 to 6 carbon atoms, 10 to 20 g of crushed natural product, a reflux time of 1 to 2 hours, and 70 to 90% of alcohol or water having 1 to 4 carbon atoms.

[0068] The above extraction solvent may be used in an amount of 2 to 50 times the weight of the sample, more specifically 2 to 20 times. For extraction, the sample may be left in the extraction solvent for leaching for 1 to 72 hours, more specifically 24 to 48 hours.

[0069] After extraction, the extract can be fractionated by sequentially applying a new fractionation solvent. The fractionation solvent used for fractionation is one or more selected from the group consisting of water, hexane, butanol, ethylacetic acid, ethyl acetate, methylene chloride, and mixtures thereof, and preferably ethyl acetate or methylene chloride.

[0070] After obtaining the extract or fraction, additional methods such as concentration or freeze-drying may be used.

[0071] A cell-protecting food composition according to another embodiment of the present invention comprises a cell-protecting composition containing rock beard extract as an active ingredient.

[0072] At this time, the amount of the above composition in the food or beverage may be added in an amount of about 0.01 to 15 weight% of the total food weight, and the health drink composition may be added in a ratio of about 0.01 to 10g based on 100 ml, but is not limited thereto.

[0073] When the food composition of the present invention is a beverage composition, there are no special restrictions on other ingredients other than containing the extract as an essential ingredient in the indicated proportion, and various flavoring agents or natural carbohydrates, etc., may be included as additional ingredients, as in ordinary beverages. Examples of the natural carbohydrates described above include monosaccharides, e.g., glucose, fructose, etc.; disaccharides, e.g., maltose, sucrose, etc.; and polysaccharides, such as dextrin, cyclodextrin, etc., and conventional sugars, and sugar alcohols such as xylitol, sorbitol, erythritol, etc. As flavoring agents other than those described above, natural flavoring agents (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) may be used, but are not limited thereto. The proportion of the natural carbohydrate may generally be in the range of about 1 to 20 g per 100 ml of the composition of the present invention, but is not limited thereto.

[0074] In addition to the above, the composition of the present invention may contain various nutritional agents, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc., but is not limited thereto.

[0075] In addition, the composition of the present invention may contain fruit pulp for the production of natural fruit juices, fruit juice beverages, and vegetable beverages, and these ingredients may be used independently or in combination. The proportion of these additives 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, although this is not important, but is not limited thereto.

[0076] A functional food composition may be provided including the above-mentioned cell-protecting composition, and a health functional food may be provided by including the same. The "health functional food" referred to in the present invention includes health supplement foods that are manufactured or processed by methods such as extraction, concentration, purification, or mixing of specific components contained in food ingredients, or by using specific components as raw materials for the purpose of health supplementation. Additionally, it includes all foods designed and processed to fully exert biological regulatory functions on the body, such as biological defense, regulation of biological rhythms, and prevention and recovery of disease, which are possessed by food components, and which also possess functions related to disease prevention and recovery.

[0077] In another embodiment of the present invention, a pharmaceutical composition for treating, improving, or preventing cell damage comprises a cell-protecting composition containing rock beard extract as an active ingredient.

[0078] Specifically, the above pharmaceutical composition can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as external preparations, suppositories, and sterile injectable solutions, each according to conventional methods.

[0079] In the present invention, carriers, excipients, and diluents that may be included in the pharmaceutical composition may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulating, the composition is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

[0080] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the extracts and fractions thereof. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Liquid dosage forms for oral administration include suspensions, liquid formulations, emulsions, syrups, etc., and may contain various excipients, such as humectants, sweeteners, flavorings, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin.

[0081] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As bases for suppositories, Witepsol, Macrogol, Tween 61, cacao oil, laurin oil, glycerogelatin, etc. may be used.

[0082] The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" in this invention refers to an amount sufficient to treat or prevent a disease with a reasonable benefit / risk ratio applicable to medical treatment or prevention. The effective dose level may be determined based on factors including the severity of the disease, drug activity, the patient's age, weight, health, gender, the patient's sensitivity to the drug, the time of administration of the composition of the present invention used, the route of administration and elimination rate, the duration of treatment, drugs combined or used concurrently with the composition of the present invention, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered alone or in combination with known immunotherapies. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects, taking all of the above factors into consideration. Effects of the invention

[0084] The present invention can provide a cell-protective composition that exhibits antioxidant activity by including rock beard extract as an active ingredient.

[0085] The present invention can provide a food or pharmaceutical composition that is highly functional and palatable by including a rock beard extract as an active ingredient, exhibiting antioxidant activity, and including a cell-protective composition. Brief explanation of the drawing

[0086] Figure 1 shows the activity of the present invention in improving the reduction of cell viability by H2O2 treatment of the rock beard extract. Figure 2 shows the inhibitory effect of the rock beard extract of the present invention on H2O2-induced cell cycle arrest in C2C12 cells. Figure 3 shows the effect of the rock beard extract of the present invention on inhibiting ROS production and DNA damage in H2O2-treated C2C12 cells. Figure 4 shows the effect of the rock beard extract of the present invention on reducing H2O2-induced mitochondrial dysfunction in C2C12 cells. Figure 5 shows the cell-protective activity of the rock beard extract of the present invention inducing apoptosis and the Nrf2 activation effect in H2O2-treated C2C12 cells. Figure 6 shows the experimental results regarding the preventive effect of the HO-1 inhibitor ZnPP against H2O2-induced cytotoxicity caused by rock beard extract in C2C12 cells. Figure 7 is a diagram showing the role of the present invention's rock beard extract in oxidative damage to C2C12 cells. Specific details for implementing the invention

[0087] Hereinafter, embodiments of the present invention are described in detail so that those skilled in the art can easily implement the invention. However, the present invention may be embodied in various different forms and is not limited to the embodiments described herein.

[0089] [Experimental Example 1: Antioxidant Effect and Cell Protective Activity of Rock Beard Extract]

[0090] 1. Materials and Methods

[0091] 1-1. Cell Culture and Treatment

[0092] C2C12 mouse myoblasts were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured using the same method as previously described. Stock solution of MEMC (M. caespitosus methanol extract) provided by Jeju Technopark (Jeju, South Korea) and H2O2 (Thermo Fisher Scientific, Waltham, MA, USA) were prepared by dissolving them in dimethyl sulfoxide (Sigma-Aldrich Co., St. Louis, MO, USA). These were diluted to appropriate concentrations in the culture medium and used to treat the cells. C2C12 cells were maintained for 24 hours in a medium containing MEMC and H2O2 at indicated concentrations, or pretreated with MEMC, N-acetyl-L-cysteine ​​(NAC, Thermo Fisher Scientific), and zinc protoporphyrin IX (ZnPP, Sigma-Aldrich Co.). They were treated with H2O2 for 24 hours prior to treatment. To investigate the blocking effect of MEMC on ROS generation induced by H2O2, C2C12 cells were pretreated with MEMC and NAC for 1 hour, followed by treatment with H2O2 for 1 hour.

[0094] 1-2. Cell Viability Analysis

[0095] To investigate the cell viability of C2C12 cells cultured under various conditions, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) assay was performed. Morphological changes in the cells were observed using an inverted-phase contrast microscope (Olympus, Tokyo, Japan).

[0097] 1-3. Flow Cytometry

[0098] To investigate the cell cycle distribution of cells cultured under different conditions, collected cells were fixed with 70% ethanol. Cells were stained with propidium iodide (PI, Thermo Fisher Scientific). The frequency of cells corresponding to each cell cycle was calculated using a flow cytometer (Becton Dickinson, San Jose, CA, USA). For the quantitative evaluation of ROS production by 2',7'-dichlorofluorescein diacetate (DCF-DA) fluorescence staining, cells were stained with 10 μM DCF-DA (Becton Dickinson), and the intensity of DCF fluorescence reflecting ROS production was analyzed using a flow cytometer. To investigate the levels of mitochondrial membrane potential (MMP), cells treated with H2O2 in the presence or absence of MEMC were stained with 5,5',6,6'-tetrachloro-1,1'3,3'-tetraethyl-imidacarbosyan iodide (JC-1, Thermo Fisher Scientific). The percentage of JC-1 monomers using a flow cytometer was expressed to indicate cells that had lost MMP. For the quantitative evaluation of apoptotic cells, cells were collected and fixed with 75% ethanol. After staining with annexin V-fluorescein isothiocyanate and PI (Abcam, Inc., Cambridge, UK), annexin V-positive cells were considered to be apoptotic cells as previously described.

[0100] 1-4. Protein Isolation and Immunoblotting

[0101] Total protein for immunoblotting was extracted as previously described. Specifically, cytoplasmic and mitochondrial fractions were separated using a mitochondrial fraction kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to Immobilon®-P PVDF membranes (Merck Millipore, Bedford, MA, USA). The membranes were blocked with 5% non-fat milk powder, reacted with the primary antibody, and then incubated with a secondary antibody conjugated with HRP (horseradish peroxidase). The membrane-bound antibodies were visualized using enhanced chemiluminescence solution (Thermo Fisher Scientific). The primary and HRP-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), Cell Signaling Technology (Beverly, MA, USA), or Abcam, Inc. (Cambridge, UK). Actin was used as a loading control for total protein. Cytochrome oxidase subunit 4 (COX IV) was used as an internal control for mitochondrial proteins.

[0103] 1-5. Comet Analysis and Nuclear Staining

[0104] DNA damage analysis was performed using a Comet assay kit purchased from Trevigen, Inc. (Gaithersburg, MD, USA) according to the manufacturer's protocol. Morphological identification of apoptotic nuclei was determined by 4,6-diamidino-2-phenolindole (DAPI) staining (Sigma-Aldrich CO.). Fluorescence images were examined and captured using a fluorescence microscope (Carl Zeiss, Oberkochen, Germany) after Comet analysis and DAPI staining.

[0106] 1-6. HO-1 Activity Analysis

[0107] To measure HO-1 activity, the amount of bilirubin produced from heme was evaluated using the HO-1 enzyme-linked immunosorbent assay kit (Abcam, Inc.). HO-1 activity was expressed as a fold change relative to the control group according to the manufacturer's instructions.

[0109] 1-7. Statistical Analysis

[0110] All statistical analyses were performed using GraphPad Prism (version 5.0) (Graphpad Inc., San Diego, CA, USA). Data are expressed as mean ± standard deviation (SD). 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.

[0112] 2. Experimental Results

[0113] 2-1. Restoration of H2O2-induced cell viability in MEMCs

[0114] To evaluate the inhibitory effect of MEMC on H2O2-mediated oxidative stress, we first investigated the effect of MEMC on the viability of C2C12 cells using an MTT assay. As shown in Figure 1A, MEMC did not induce significant cytotoxicity at concentrations up to 50 mg / ml. Therefore, 50 μg / ml of MEMC was used as the optimal concentration. The viability of C2C12 cells treated with H2O2 decreased in a concentration-dependent manner, and the concentration inducing cytotoxicity was set at 1 mM H2O2, which reduced cell viability by approximately half (Figure 1B). Next, we evaluated the inhibitory effect of MEMC on H2O2-mediated cytotoxicity and found that MEMC significantly restored the reduction in cell viability and morphological changes induced by H2O2 (Figures 1C and 1D). This indicates that MEMC pretreatment can improve H2O2-induced cytotoxicity. In addition, pretreatment with NAC, a free radical scavenger, completely suppressed H2O2-induced cytotoxicity (Fig. 1C).

[0116] 2-2. Inhibition of H2O2-induced cell cycle arrest in MEMC

[0117] We investigated whether the loss of cell viability in H2O2-treated C2C12 cells was associated with disruption of cell cycle progression and induction of apoptosis. As shown in Figures 2A and 2B below, the frequency of cells in the G2 / M phase and the apoptotic sub-G1 phase increased with H2O2 treatment but was inhibited in the presence of MEMC or NAC. Additionally, the expression of p21, a cyclin-dependent kinase (Cdk) inhibitor, was not regulated in H2O2-treated cells. Meanwhile, the expression levels of cyclin A and cyclin B1 were inhibited, whereas there was no change in the expression levels of Cdk2 and Cdk1 in H2O2-treated cells (Figure 2C). However, the upregulation of p21 expression by H2O2 was significantly reduced by MEMC treatment, whereas the downregulation of cyclin A and cyclin B1 by H2O2 conversely increased. These results indicate that the decrease in viability of C2C12 cells exposed to H2O2 is attributed to apoptosis accompanied by G2 / M phase arrest of the cell cycle, which is effectively inhibited by MEMC.

[0119] 2-3. Removal of H2O2-induced ROS accumulation and DNA damage in MEMC

[0120] To determine whether the blocking ability of MEMC against H2O2-mediated cytotoxicity is directly related to antioxidant activity, the protective effect of MEMC against H2O2-induced ROS generation was investigated using DCF-DA staining. Flow cytometry results showed that ROS generation in C2C12 cells treated with hydrogen peroxide for 1 hour increased approximately tenfold compared to untreated control cells, whereas it was significantly reduced upon pretreatment with NAC or MEMC (Figures 3A and 3B). Additionally, an increase in comet tail moment (DNA shift) and phosphorylation of γH2AX (p-γH2AX), an indicator of DNA damage, were observed in H2O2-treated cells (Figures 3C and 3D). However, the increased DNA shift and phosphorylation of γH2AX caused by H2O2 treatment were significantly attenuated in the presence of MEMC, indicating that oxidative damage to DNA can be protected by MEMC.

[0122] 2-4. Protection of MEMC from H2O2-induced mitochondrial dysfunction

[0123] To evaluate whether MEMC could protect against H2O2-induced mitochondrial damage, MMPs were estimated after JC-1 staining. Flow cytometry results (Figures 4A and 4B) showed that the frequency of JC-1 monomers significantly increased, while the frequency of JC-1 aggregates decreased in H2O2-treated cells, demonstrating the loss of MMPs and resulting mitochondrial dysfunction. Additionally, cytochrome c expression levels increased in the cytoplasm but decreased in the mitochondria after H2O2 treatment (Figure 4C). H2O2 treatment also induced a decrease in Bcl-2 expression and an increase in Bax expression through the cleavage of poly(ADP-ribose) polymerase (PARP) (Figure 4D). However, these changes did not occur in cells pretreated with MEMC, suggesting that MEMC can inhibit H2O2-induced mitochondrial damage.

[0125] 2-5. H2O2-induced apoptosis-reducing activity of MEMC

[0126] DAPI staining and flow cytometry were performed to confirm that MEMC prevents H2O2-induced apoptosis. As shown in Figures 5A and 5B, characteristic morphological changes of apoptosis, including chromatin condensation and nuclear fragmentation, were clearly observed in C2C12 cells exposed to H2O2. However, MEMC pretreatment significantly attenuated these nuclear morphological changes. In parallel, flow cytometry results following annexin V / PI staining showed that significantly more apoptosis was induced in H2O2-treated cells than in control cells (Figures 5C and 5D). However, the induction of apoptosis by H2O2 was significantly attenuated in cells present with MEMC, appearing similar to the reduction in the sub-G1 cell population.

[0128] 2-6. Activation of the Nrf2 / HO-1 signaling pathway in H2O2-treated cells by MEMC

[0129] We investigated whether the activation of the Nrf2 / HO-1 signaling pathway correlated with the antioxidant capacity of MEMC. As shown in Figure 5E, the levels of Nrf2 and its phosphorylated form (p-Nrf2, Ser40) were slightly increased by treatment with MEMC or H2O2 alone. However, their expression levels were significantly increased in cells treated with H2O2 after MEMC pretreatment, whereas the level of Kelch-like ECH-associated protein-1 (Keap1), a negative regulator of Nrf2, was much lower. Furthermore, the expression level and activity of HO-1 protein were significantly promoted in cells treated with H2O2 and MEMC together compared to cells treated with H2O2 and MEMC alone (Figures 5E and 5F), indicating that Nrf2-mediated HO-1 was further increased by MEMC.

[0131] 2-7. Activation of HO-1 Related to Mitigation of H2O2-Induced Cytotoxicity in MEMC

[0132] We evaluated whether the activation of HO-1 by MEMC in H2O2-treated C2C12 cells was directly related to the cytotoxic blocking effect of MEMC using ZnPP, a selective inhibitor of HO-1. As shown in Figures 6A and 6B, the blocking effect of MEMC on apoptosis induced in cells exposed to H2O2 was significantly offset in the presence of ZnPP. At the same time, pretreatment with ZnPP significantly eliminated the antiproliferative effect of MEMC in H2O2-stimulated cells (Figure 6C). Consequently, these results demonstrated that Nrf2-mediated activation of HO-1 serves as an upstream signal for the inhibitory action of MEMC on H2O2-induced cytotoxicity in C2C12 cells.

[0134] Through the experimental results above, it was confirmed that MEMC can alleviate cell cycle arrest, DNA damage, and apoptosis in C2C12 myoblasts by acting as a ROS scavenger and mitigating H2O2-induced mitochondrial damage. In addition, MEMC, an activator of Nrf2, can contribute to blocking oxidative damage by promoting the activity of HO-1 (Fig. 7), which indicates that MEMC has high potential for maintaining myoblast function against oxidative damage.

[0136] [Preparation Example 1: Cell protection composition containing a complex extract]

[0137] 1. Preparation of composition

[0138] Preparation of Jeju wild rose extract

[0139] After washing the Jeju rosehip with water, freeze-dried it. Subsequently, freeze-dried A was ground using a blender, steeped in 80% ethanol at room temperature for 48 hours, and then filtered to produce Jeju rosehip extract (RE).

[0140] Preparation of other natural extracts

[0141] In the same manner as the above-mentioned Jeju wild rose extract (RE), only the natural materials were replaced with violets, ragweed, and ragweed to produce violet extract (VE), ragweed extract (CE), and ragweed extract (JE).

[0142] Preparation of complex extract

[0143] Complex extracts (MX 1 to MX 13) were prepared by mixing the Jeju wild rose extract (RE), purple violet extract (VE), Japanese knotweed extract (CE), and black cohosh extract (JE) with the rock rose extract (MEMC) used in Experimental Example 1 in the weight ranges of Table 1 below.

[0144] MX1 MX2 MX3 MX4 MX5 MX6 MX7 MX8 MX9 MX10 MX11 MX12 MX13 MEMC 100 100 100 100 100 100 100 100 100 100 100 100 100 RE - 30 - 10 20 30 40 50 30 30 30 30 30 VE - - 30 10 20 30 40 50 30 30 30 30 30 CE - - - - - - - - 1 10 20 30 40 JE - - - - - - - - 0.1 1 3 5 10

[0145] (Unit: parts by weight)

[0146] [Experimental Example 2: Antioxidant Effect and Cell Protective Activity of Complex Extract]

[0147] To demonstrate the cell-protective effect of the composition of the present invention, the same experiment as Experimental Example 1 was conducted, and only the extract was replaced with the composition of the present invention.

[0148] In addition, the above results were presented by fixing the index of the rock beard extract (MEMC) at 3 and showing the comparative results, with the index evaluated as a number between 1 and 10. A higher number indicates superior antioxidant and cytoprotective activity, and the results are comprehensively shown in Table 2 below.

[0149] MX1 MX2 MX3 MX4 MX5 MX6 MX7 MX8 MX9 MX10 MX11 MX12 MX13 Recovery from reduced cell viability caused by H2O2 3 3.5 3.3 3.8 5.5 6.5 7.5 7.0 8.0 8.5 9.0 8.8 8.3 Inhibition of cell cycle arrest and protection against DNA damage 3 3.5 3.4 3.7 5.0 6.0 7.4 7.0 8.1 8.6 8.9 8.5 8.0 Protecting against mitochondrial damage 3 3.3 3.5 3.7 5.3 6.0 7.4 7.2 8.3 8.6 8.8 8.5 7.8 Apoptosis reduction activity 3 3.2 3.3 3.6 5.5 6.5 7.0 7.0 8.0 8.5 9.0 8.8 7.7 Activate Nrf2 / HO-1 signal transmission path 3 3.3 3.4 3.7 5.4 6.6 7.2 6.9 7.9 8.3 9.0 8.8 7.6 Activation of HO-1 related to cytotoxicity mitigation 3 3.2 3.5 3.8 5.3 6.5 7.1 6.7 8.0 8.3 9.1 8.9 7.8

[0150] (Unit: Index)

[0151] Referring to Table 2 above, it can be seen that MX2 and 3, which contain Jeju wild rose extract (RE) and purple violet extract (VE) respectively in addition to the single extract rock bear extract (MEMC), exhibit slightly improved cell protective activity and antioxidant activity compared to the case containing only rock bear extract (MEMC).

[0152] In addition, it can be confirmed that the effect is further enhanced in the case of MX5 to 7, which contain the three types of the above-mentioned natural products in a specific content range.

[0153] Furthermore, it was confirmed that MX9 to 13, which additionally contain extracts of *Scutellaria baicalensis* (CE) and *Erythronium japonicum* (JE) in addition to *Scutellaria baicalensis*, *Rosa multiflora*, and *Viola japonica*, exhibited enhanced activity, and in this case, it was found that MX10 to 12, which contain a specific amount of the extract, also exhibited enhanced activity.

[0154] That is, in the case of a composition comprising 20 to 40 parts by weight of Jeju wild rose extract and 20 to 40 parts by weight of purple-leaved violet with respect to 100 parts by weight of rock bear's beard extract, and furthermore, in the case of a composition comprising 20 to 40 parts by weight of Jeju wild rose extract, 20 to 40 parts by weight of purple-leaved violet, 10 to 30 parts by weight of Japanese knotweed extract and 1 to 5 parts by weight of Japanese knotweed extract with respect to 100 parts by weight of rock bear's beard extract, it can be confirmed that the antioxidant and cell-protective activities are excellently enhanced by the mixing of the active ingredients of the natural products according to the mixing of natural extracts. Therefore, it has been confirmed through experiments that the present invention can have excellent antioxidant and cell-protective activities by including rock bear's beard extract, and can exhibit even more enhanced activity by additionally including the above natural extracts.

[0156] [Experimental Example 3: Effect of Improving the Palatability of the Composition of the Present Invention]

[0157] Tea beverages containing each of the above composite compositions (MX1 to MX13) were prepared, and 40 adult men and women in their 20s to 50s were asked to taste them and evaluate their aroma and flavor, with the preference being rated on an index of 1 to 10. For relative evaluation, the MX1 (ME) of the present invention, extracted by immersion in water, was fixed at an index of 3 and evaluated using relative index values. The results were aggregated, averaged, and rounded to the second decimal place to be comprehensively presented in Table 3 below.

[0158] 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 Overall 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

[0159] (Unit: Index)

[0160] Referring to Table 3 above, it can be seen that the preference is increased in the case of MX2 and MX3, which additionally include Jeju wild rose extract (RE) and purple violet extract (VE), respectively, compared to MX1, which includes only the rock bear extract (MEMC) of the present invention.

[0161] In the case of MX4 to MX8, which are included in the form of complex extracts rather than as single extracts, the effect is further enhanced. This means that by using a mixture of natural extracts, the unique scent of a single natural product is mitigated, and a better palatability can be achieved through the harmony of taste and scent.

[0162] In addition, in the case of MX9 to MX13 which further contain *Scutellaria baicalensis* extract (CE) and *Scutellaria baicalensis* extract (JE), the palatability was further improved, which can be seen as being due to the alleviating effect on the unique scent of *Scutellaria baicalensis* extract resulting from the mixed use of natural extracts.

[0163] In particular, it can be seen that when mixed with 20 to 40 parts by weight of Jeju wild rose extract (RE), 20 to 40 parts by weight of violet extract (VE), 10 to 30 parts by weight of Japanese knotweed extract (CE), and 1 to 5 parts by weight of black cohosh extract (JE) relative to 100 parts by weight of rock bear extract (MEMC), which is a preferred weight part, the overall preference for taste and aroma is the best, thereby providing a highly preferred composition.

[0164] Therefore, it was confirmed that a composition containing the above-mentioned mixed composition can be provided as a food or pharmaceutical composition with high functionality and palatability, as it includes natural extracts, has no issues with side effects, exhibits cell protective effects and antioxidant activity, and has excellent evaluations regarding taste and aroma.

[0166] Although 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 by those skilled in the art using the basic concept of the present invention as defined in the following claims also fall within the scope of the present invention.

Claims

Claim 1 A cell-protective composition exhibiting antioxidant activity, comprising, with respect to 100 parts by weight of rock rose extract, 20 to 40 parts by weight of Jeju wild rose extract, 20 to 40 parts by weight of violet extract, 10 to 30 parts by weight of Japanese knotweed extract, and 1 to 5 parts by weight of black cohosh extract as active ingredients. Claim 2 A cell-protecting composition according to claim 1, wherein cell protection is achieved by alleviating cell cycle arrest, DNA damage, and apoptosis caused by oxidative stress. Claim 3 In claim 2, the above oxidative stress refers to cell damage caused by reactive oxygen species (ROS), a cell-protecting composition. Claim 4 In claim 1, the composition is a cell-protective composition exhibiting antioxidant activity due to increased activity of the Nrf2 / HO-1 signaling pathway. Claim 5 A cell-protecting composition according to claim 1, wherein the extract is extracted using an extraction solvent selected from the group consisting of water, C1 to C6 lower alcohols, and mixtures thereof. Claim 6 A cell-protecting food composition comprising a composition according to any one of claims 1 to 4. Claim 7 delete

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