Composition for preventing, alleviating or treating sarcopenia, comprising plantago asiatica extract as active ingredient

Abstract

The present invention relates to a composition for preventing, alleviating or treating sarcopenia, comprising a Plantago asiatica extract as an active ingredient. The Plantago asiatica extract of the present invention has no C2C12 cytotoxicity, has an effect in alleviating muscle loss in a cell experimental model through inhibition of muscle cell differentiation, and has an effect on muscle loss by alleviating skeletal muscle loss in a cachexia-induced muscle loss animal model.

Description

A composition for the prevention, improvement, or treatment of sarcopenia containing Plantago as an active ingredient

[0001] The present invention relates to a composition for the prevention, improvement, or treatment of sarcopenia comprising Plantago major extract as an active ingredient.

[0002] This result was produced with support from the Creative Convergence Research Project 'Discovery of Cachexia-Inducing Factors and Control Technology for Improving Cancer Treatment' (Project No.: 1711201873, CAP21023-000(NSN2113321)) of the National Science and Technology Research Council under the Ministry of Science and ICT, and the Research Operation Expense Support Project 'Development of Technology for Sustainable Utilization of Standard Herbal Medicine Resources' (Project No.: 1711195908, KSN1823320) of the Korea Institute of Oriental Medicine under the Ministry of Science and ICT.

[0003] Sarcopenia is a disease characterized by muscular dysfunction, including a decrease in muscle mass and a decline in muscle endurance, deep breathing function, flexibility, balance, and speed. As it can cause overall physical problems such as falls and difficulties in performing daily activities, interest in the treatment of this disease has been steadily increasing recently. Previously, sarcopenia was considered a concern only in the elderly; however, due to poor dietary habits and an abnormal obsession with appearance leading to indiscriminate dieting without exercise, adolescents and adults are now also exhibiting decreased muscle mass and increased body fat, resulting in a trend toward "skinny fat" body types. Furthermore, factors contributing to muscle loss regardless of age—such as increased sedentary lifestyles and reduced physical activity—are gradually increasing in modern society. Consequently, muscle loss is becoming a very serious issue across all age groups.

[0004] Furthermore, while sarcopenia may inevitably occur as muscle mass and strength decline with the progression of aging, it is also induced by conditions such as cachexia, which is caused by various pathogenic diseases including immunodeficiency disorders and cancer. However, research to date has failed to identify appropriate methods other than exercise to treat or alleviate muscle atrophy (cachexia caused by cancer, and sarcopenia, degenerative muscle atrophy in the elderly). Muscle atrophy resulting from sarcopenia or cachexia affects energy balance and muscle protein breakdown, consequently exacerbating chronic diseases and increasing mortality. Therefore, understanding the mechanisms of muscle atrophy is crucial for clinical control; these mechanisms may include cachexia, a state of significant muscle loss observed in the terminal stages of malignant tumors; muscle wasting caused by acute illnesses such as influenza; or primary muscle diseases.

[0005] Meanwhile, Plantago asiatica L. is a perennial herbaceous plant belonging to the Plantaginaceae family, also known as 'Bui' or 'Chagwarocho'. Commonly found in Korea, China, and Japan, Plantago asiatica grows wild throughout the country, frequently appearing in grasslands, roadsides, and vacant lots. It blooms with white flowers from June to August and produces fruits containing about 6 to 8 black seeds in October. In folk medicine, the young leaves are used for food, while in traditional Korean medicine, the leaves are used as medicinal ingredients known as 'Chajeoncho' and the seeds as 'Chajeonja'. Due to its diverse physiological activities, the leaves have long been used as a remedy for clearing heat, nourishing blood, promoting urination, strengthening the stomach, treating gynecological diseases, and pleuritis, while the seeds, known as Chajeonja, have been used for a long time for their anti-inflammatory and diuretic effects.

[0006] Regarding technologies related to Plantago asiatica, Korean Published Patent No. 2022-0077979 discloses a composition for preventing, improving, or treating cancer cachexia containing Uchasingihwan, and Korean Published Patent No. 2024-0112492 discloses a composition for preventing or treating bone loss containing extracts of Osmanthus fragrans, Rehmannia glutinosa, and Plantago asiatica; however, there has not yet been any disclosure of a composition for preventing, improving, or treating sarcopenia containing the Plantago asiatica extract of the present invention as an active ingredient.

[0007] The present invention was derived from the above-mentioned requirements. The present invention provides a composition for the prevention, improvement, or treatment of sarcopenia comprising a Plantago extract as an active ingredient. The present invention was completed by confirming that the Plantago extract of the present invention has no C2C12 cytotoxicity, is effective in improving sarcopenia in a cell experiment model through the inhibition of muscle cell differentiation, and is effective against sarcopenia by improving the reduction of skeletal muscle in a cachexia-derived sarcopenia animal model.

[0008] To achieve the above objective, the present invention provides a health functional food composition for the prevention or improvement of sarcopenia comprising an extract of Plantago asiatica as an active ingredient.

[0009] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of sarcopenia comprising an extract of Plantago asiatica as an active ingredient.

[0010] In addition, the present invention provides a veterinary composition for the prevention or treatment of sarcopenia comprising an extract of Plantago asiatica as an active ingredient.

[0011] In addition, the present invention provides a feed additive for the prevention or improvement of sarcopenia comprising an extract of Plantago asiatica as an active ingredient.

[0012] In addition, the present invention provides a herbal medicine composition for the prevention or treatment of sarcopenia comprising an extract of Plantago asiatica as an active ingredient.

[0013] The present invention relates to a composition for the prevention, improvement, or treatment of sarcopenia comprising a Plantago extract as an active ingredient. The Plantago extract of the present invention has no C2C12 cytotoxicity and is effective in improving sarcopenia in cell experiment models through the inhibition of muscle cell differentiation, and is effective in improving the reduction of skeletal muscle in cachexia-derived sarcopenia animal models.

[0014] Figure 1 shows the results of confirming the cell viability of the Plantago extract of the present invention in myoblasts and myotubes.

[0015] Figure 2 shows an image (A) and a graph (B) quantifying the changes in muscle thickness confirmed by treating a culture medium inducing sarcopenia caused by cancer cachexia and treating with Plantago extract (PA) in an in vitro muscle cell differentiation model. #### indicates that the muscle thickness of the control group (Cachectic media; CM) treated with the culture medium inducing sarcopenia caused by cancer cachexia was statistically significantly reduced compared to the normal group (Normal), with p<0.0001. ** and *** indicate that the reduced muscle thickness of the group treated with the Plantago extract of the present invention was significantly increased compared to the control group (Cachectic media) treated with the culture medium inducing sarcopenia caused by cancer cachexia, with ** being p<0.01 and *** being p<0.001. LiCl is a positive control group treated with lithium chloride.

[0016] Figure 3 shows the results of confirming changes in the weight of the gastrocnemius and tibialis anterior muscles, which are skeletal muscles of the hind leg of mice, after administering the Plantago extract of the present invention to a cachexia model mouse established by transplanting CT26 colorectal cancer cell lines. #### indicates that the muscle weight of the cancer cachexia-induced group was statistically significantly reduced compared to the normal group, with p<0.0001, and * and ** indicate that the reduced muscle weight was significantly increased in the group treated with the Plantago extract of the present invention compared to the cancer cachexia-induced group, with * being p<0.05 and ** being p<0.01.

[0017] The present invention relates to a health functional food composition for the prevention or improvement of sarcopenia comprising an extract of Plantago asiatica as an active ingredient.

[0018] The above-mentioned Plantago extract can be prepared by a method comprising the following steps, but is not limited thereto:

[0019] (1) A step of extracting by adding an extraction solvent to dried plantain;

[0020] (2) A step of filtering the extract of step (1); and

[0021] (3) A step of preparing an extract by drying the filtered extract of step (2).

[0022] In step (1) above, the extraction solvent is preferably selected from water, C1-C4 lower alcohols, or a mixture thereof, more preferably water or ethanol, and even more preferably ethanol, but is not limited thereto. In the above manufacturing method, the extraction method may utilize any conventional method known in the art, such as hot water extraction, immersion extraction, reflux cooling extraction, and ultrasonic extraction. It is preferable to extract by adding the extraction solvent in an amount of 1 to 20 times the weight of the Plantago plant. The extraction temperature is preferably 20 to 100°C, but is not limited thereto. In addition, the extraction time is preferably 1 to 5 hours, and more preferably 1 to 3 hours, but is not limited thereto. In step (3) above, drying is preferably performed by reduced pressure drying, vacuum drying, boiling drying, spray drying, or freeze-drying, and more preferably by reduced pressure drying, but is not limited thereto.

[0023] The above sarcopenia may be sarcopenia induced by cachexia, and the above cachexia is preferably cancerous cachexia but is not limited thereto.

[0024] The above-mentioned health functional food composition is preferably prepared in any one formulation selected from powder, granules, pills, tablets, capsules, candies, syrups, and beverages, but is not limited thereto. The health functional food composition of the present invention may be prepared by adding the Plantago extract as is or by mixing it with other foods or food ingredients, and may be appropriately prepared according to conventional methods. Examples of foods to which the Plantago extract may be added include any one form selected from caramel, meat, sausage, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and include all health functional foods in the conventional sense. That is, there are no special restrictions on the types of foods. The above health functional food composition may contain various nutritional supplements, vitamins, minerals (electrolytes), synthetic and natural flavoring agents, coloring agents and promoters (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, it may contain fruit pulp for the production of natural fruit juices and vegetable beverages. The above ingredients may be used independently or in combination.

[0025] The health functional food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients, and the natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. Although the proportion of the natural carbohydrates is not of great importance, it is preferable to include 0.01 to 0.04 g per 100 g of the composition of the present invention, and more preferably 0.02 to 0.03 g, but is not limited thereto. As sweeteners, natural sweeteners such as taumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame may be used.

[0026] In addition, the present invention relates to a pharmaceutical composition for the prevention or treatment of sarcopenia comprising an extract of Plantago asiatica as an active ingredient.

[0027] The above sarcopenia may be sarcopenia induced by cachexia, and the above cachexia is preferably cancerous cachexia but is not limited thereto.

[0028] The pharmaceutical composition containing the Plantago extract according to the present invention is preferably, but is not limited to, any one formulation selected from capsules, powders, granules, tablets, suspensions, emulsions, syrups, and aerosols. In addition to the Plantago extract, it may further include pharmaceutically acceptable carriers, excipients, or diluents. Examples of carriers, excipients, or diluents that may be included in the pharmaceutical composition containing the Plantago extract include lactose, dextrose, sucrose, oligosaccharides, 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, it may be prepared using diluents or excipients such as commonly used fillers, volume expanders, binders, humectants, disintegrants, and surfactants. The preferred dosage of the Plantago extract of the present invention varies depending on the patient's condition and body weight, the severity of the disease, the form of the drug, the route of administration, and the duration, but can be appropriately selected by those skilled in the art. However, for a desirable effect, it is preferable to administer the pharmaceutical composition containing the Plantago extract of the present invention at a dose of 0.0001 to 100 mg / kg per day, preferably 0.001 to 10 mg / kg. Administration may be performed once a day or divided into several doses. The above dosage does not limit the scope of the present invention in any way.

[0029] In addition, the present invention relates to a veterinary composition for the prevention or treatment of sarcopenia comprising an extract of Plantago asiatica as an active ingredient.

[0030] The veterinary composition of the present invention may further include suitable excipients and diluents according to conventional methods. Examples of excipients and diluents that may be included in the veterinary composition of the present invention 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, cetanol, stearyl alcohol, liquid paraffin, sorbitan monostearate, polysorbate 60, methylparaben, propylparaben, and mineral oil. The veterinary composition according to the present invention may further include fillers, anticoagulants, lubricants, humectants, spices, emulsifiers, preservatives, etc. The veterinary composition according to the present invention may be formulated using methods well known in the art to provide rapid, sustained, or delayed release of the active ingredient after administration to an animal, and the formulation may be in the form of powders, granules, tablets, capsules, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, suppositories, sterile injectable solutions, sterile topical preparations, etc. The effective amount of the veterinary composition according to the present invention may be appropriately selected according to the individual animal. It may be determined according to the severity of the disease or condition, the sensitivity to the active ingredient of the present invention according to the age, weight, health status or sex of the individual, the route of administration, the duration of administration, factors including other compositions combined with or used simultaneously with said composition, and other factors well known in the physiological or veterinary field.

[0031] In addition, the present invention relates to a feed additive for the prevention or improvement of sarcopenia comprising an extract of Plantago asiatica as an active ingredient.

[0032] The feed additive of the present invention corresponds to an auxiliary feed under the Feed Management Act. In the present invention, the term "feed" may refer to any natural or artificial prescribed food, single meal, etc., or the components of said single meal, intended for or suitable for animals to eat, consume, and digest. The types of said feed are not particularly limited, and feeds commonly used in the relevant technical field may be used. Non-limiting examples of said feed include plant-based feeds such as grains, root vegetables, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, meal, or grain by-products; and animal-based feeds such as proteins, inorganic substances, oils and fats, minerals, single-cell proteins, zooplankton, or food waste. These may be used individually or in a mixture of two or more types.

[0033] In addition, the present invention relates to a herbal medicine composition for the prevention or treatment of sarcopenia comprising an extract of Plantago asiatica as an active ingredient.

[0034] The herbal medicine composition according to the present invention refers to a conventional dried medicinal material used in the manufacture of herbal medicine. The herbal medicine composition of the present invention may be an extract obtained by extracting using an extraction solvent selected from water, C1-C4 lower alcohols, or mixtures thereof according to a conventional method, or an extract in powder form obtained by drying thereof, and there are no particular restrictions on the formulation. For example, it may be manufactured in the form of pills, tablets, capsules, or liquids.

[0035] The present invention will be described in more detail below using examples. These examples are solely for the purpose of more specifically explaining the present invention, and it is obvious to those skilled in the art that the scope of the present invention is not limited by them.

[0036]

[0037] Example 1. Preparation of Plantago major extract

[0038] 1 kg of dried Plantago major whole plant was ground using a mixer, and 8 L of 70% (v / v) ethanol was added and ultrasonic extraction was performed at room temperature for 2 hours. The obtained Plantago major extract was filtered through a 53 μm sieve, and the filtered Plantago major extract was concentrated under reduced pressure at 37°C, freeze-dried, and homogenized to prepare a Plantago major extract.

[0039]

[0040] Example 2. Cell viability analysis

[0041] To analyze the cell viability of mouse C2C12 myoblasts treated with Plantago extract, C2C12 cells were placed in a 96-well plate at a rate of 1 × 10⁶ per well. 4 After 24 hours of cell seeding, 3.125, 6.25, 12.5, 25, 50, 100, and 200 µg / ml of Plantago extract were treated for 24 hours. Additionally, DMEM culture medium containing 2% horse serum was used to induce differentiation of myotube cells for 96 hours, followed by the treatment with 3.125, 6.25, 12.5, 25, 50, 100, and 200 µg / ml of Plantago extract for 24 hours. Subsequently, CCK-8 reagent was applied for 2 hours, and the absorbance was measured at a wavelength of 450 nm.

[0042] As a result, as disclosed in Figure 1, it was found that there was no cytotoxicity of the Plantago extract in myoblasts and myotubes.

[0043]

[0044] Example 3. Confirmation of the effect of Plantago extract on improving muscle thickness induced by cachexia

[0045] Mouse myoblasts (C2C12 cells) were purchased from ATCC in the United States. The purchased C2C12 cells were cultured at 37°C under 5% CO2 conditions in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin.

[0046] 3×10 in a 12-well plate 5 After inoculating mouse C2C12 myoblasts and culturing them for 24 hours, DMEM culture medium containing 2% horse serum was applied to the myoblasts to induce differentiation into myotubes for 96 hours.

[0047] Subsequently, CT26 colorectal cancer cell culture medium (Cachectic media) and Plantago extract were added to the differentiated myotube cells and cultured for 48 hours, and the effect of improving / treating muscle loss in the myotube cells was confirmed through cell immunofluorescence staining.

[0048] As a result, as disclosed in Figure 2, the muscle thickness of the control group (Cachectic media) treated with the culture medium inducing sarcopenia caused by cancer cachexia was statistically significantly reduced compared to the normal group (Normal), but the muscle thickness of the group treated with the Plantago extract of the present invention was significantly increased compared to the control group (Cachectic media) treated with the culture medium inducing sarcopenia caused by cancer cachexia.

[0049]

[0050] Example 4. Confirmation of sarcopenia inhibitory effect in a cancer cell transplanted cachexia animal model

[0051] A cachexia model was constructed using 10-week-old BALB / C female mice with CT26 colorectal cancer cell line transplantation. All mice were randomly assigned 5 per cage under conditions of a 12-hour light-dark cycle, 30–70% humidity, and 20–26°C. Muscle weight was measured after oral administration of Plantago extract at a dose of 50 or 100 mg / kg for 24 days.

[0052] As a result, as disclosed in Figure 3, the weight of the gastrocnemius and tibialis anterior muscles of the cancer cachexia-induced group was statistically significantly reduced compared to the normal group, but the muscle weight of the group administered the Plantago extract of the present invention was significantly increased compared to the cancer cachexia-induced group.

[0053]

[0054] [Statistical Analysis]

[0055] Statistical analysis was performed using the GraphPad Prism 9 program. The experimental results were expressed as mean ± standard error (SEM). For significance analysis, # was set to p<0.05, ## to p<0.01, ### to p<0.001, * to p<0.05, ** to p<0.01, and *** to p<0.001, and multivariate analysis of variance was performed using multiple comparisons.

Claims

A health functional food composition for the prevention or improvement of sarcopenia containing Plantago asiatica extract as an active ingredient. A health functional food composition for the prevention or improvement of sarcopenia, characterized in that, in claim 1, the extraction solvent of the above-mentioned Plantago extract is water, a C1-C4 lower alcohol, or a mixture thereof. A health functional food composition for the prevention or improvement of sarcopenia, characterized in that, in claim 1, the sarcopenia is induced by cachexia. A health functional food composition for the prevention or improvement of sarcopenia, characterized in that, in paragraph 3, the cachexia is cancerous cachexia. A health functional food composition for the prevention or improvement of sarcopenia according to claim 1, characterized in that the composition is manufactured in any one formulation selected from powder, granules, pills, tablets, capsules, candies, syrups, and beverages. A pharmaceutical composition for the prevention or treatment of sarcopenia comprising an extract of Plantago asiatica as an active ingredient. A pharmaceutical composition for the prevention or treatment of sarcopenia, characterized in that, in claim 6, the sarcopenia is induced by cachexia. A pharmaceutical composition for the prevention or treatment of sarcopenia according to claim 6, characterized in that the composition further comprises, in addition to the active ingredient, a pharmaceutically acceptable carrier, excipient, or diluent. A veterinary composition for the prevention or treatment of sarcopenia comprising an extract of Plantago asiatica as an active ingredient. A feed additive for the prevention or improvement of sarcopenia containing Plantago asiatica extract as an active ingredient. A herbal medicine composition for the prevention or treatment of sarcopenia containing an extract of Plantago asiatica as an active ingredient.

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