KR102507494B1 - Composition for preventing, alleviating, or treating muscle weakness diseases comprising celery seed extract as an active ingredient - Google Patents

Abstract

The present invention relates to a composition, for preventing, alleviating, or treating a muscle weakness-related disease, comprising a celery seed extract. The celery seed extract according to the present invention causes a few or no side effects by using a natural product, can increase muscle mass and muscle strength to not only prevent muscle weakness but also inhibit weight gain and body fat increase, and thus can reduce serum lipid profiles and hepatotoxicity markers (GOT and GPT), and thus is expected to be utilized for preventing, relieving, or treating muscle weakness-related diseases, metabolic diseases, liver diseases, or the like.

Description

Composition for preventing, alleviating, or treating muscle weakness diseases comprising celery seed extract as an active ingredient}

The present invention relates to a composition comprising a celery seed extract for preventing, improving or treating muscle weakness-related diseases.

Skeletal muscle is the largest organ in the human body, accounting for 40 to 50% of the total body weight, and plays an important role in various metabolic functions in the body, including energy homeostasis and heat generation. As the human body ages, redistribution of body fat and body proteins occurs as a result of changes in composition. At about age 50, the rate of protein synthesis in muscle cells slows down compared to the rate of decomposition, and muscles begin to degenerate rapidly.

Sarcopenia refers to a state in which about 13 to 24% of one's usual body mass is reduced, and represents a decrease in protein content, fiber diameter, muscle strength production, and fatigue resistance. Sarcopenia is caused by a variety of causes, including sepsis, cancer, renal failure, excess glucocorticoids, denervation, muscle underuse, obesity, and the aging process. Mainly, it can be attributed to the gradual decrease in the quantity and quality of skeletal muscle that occurs as aging progresses.

On the other hand, sarcopenic obesity, in which the amount of body fat increases along with the decrease in muscle mass or strength with aging, has become a problem. Obesity and sarcopenia in the elderly are presumed to act synergistically to exacerbate the risk of functional and metabolic disorders as well as the risk of death, and are suggested to have a strong interaction with each other from an etiological point of view. Therefore, in the case of an overweight person, an obese person, or a person with normal weight but high body fat, metabolic diseases such as diabetes and hypertension can be prevented and treated by increasing muscle mass and reducing body fat. Increased muscle mass increases basal metabolic energy, enabling efficient diet without yo-yo phenomenon. Therefore, exercise, diet, and ergogenic aids are used to increase muscle mass. It contains a chemical compound and is accompanied by fatal side effects.

Accordingly, the inventors of the present invention have tried to develop a therapeutic agent related to muscle weakness that has no side effects and is highly effective. As a result, the present invention was completed by confirming that celery seed extract increases muscle mass and muscle strength in obese or aging mice.

Republic of Korea Patent Publication No. 10-2020-0142107

As a result of efforts to develop active substances derived from natural products capable of preventing, improving or treating muscle weakness-related diseases, the present inventors confirmed the excellent effect of increasing muscle mass and improving muscle metabolism of celery seed extract in obese or aging mice. The invention was completed.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating diseases related to muscle weakness, comprising a celery seed extract as an active ingredient.

In addition, an object of the present invention is to provide a food composition for preventing or improving muscle weakness-related diseases comprising a celery seed extract as an active ingredient.

In addition, an object of the present invention is to provide a feed or feed additive for strengthening muscle strength containing a celery seed extract as an active ingredient.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned can be clearly understood by those skilled in the art from the description below. There will be.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating muscle weakness-related diseases comprising a celery seed extract as an active ingredient.

In addition, the present invention provides a food composition for preventing or improving muscle weakness-related diseases comprising a celery seed extract as an active ingredient.

In addition, the present invention provides a composition for strengthening muscle strength containing a celery seed extract as an active ingredient.

In addition, the present invention provides a feed or feed additive for strengthening muscle strength containing a celery seed extract as an active ingredient.

In one embodiment of the present invention, the celery seed extract is water, alcohol having 1 to 6 carbon atoms, acetone, ether, benzene, chloroform, ethyl acetate ), methylene chloride, hexane, cyclohexane, petroleum ether, subcritical fluid, and may be an extract by one or more solvents selected from the group consisting of supercritical fluid, Not limited to this.

In another embodiment of the present invention, the muscle weakness-related disease may be at least one selected from the group consisting of sarcopenia, muscular dystrophy, muscle dystrophy, and cardiac atrophy, but is not limited thereto.

In another embodiment of the present invention, the sarcopenia may be age-related sarcopenia or obese sarcopenia, but is not limited thereto.

In another embodiment of the present invention, the composition may have one or more of the following characteristics, but is not limited thereto:

(a) increase in muscle mass;

(b) inhibition of muscle mass loss;

(c) increased muscle strength;

(d) inhibition of increase in muscle tissue lipid content; or

(e) inhibition of muscle tissue fibrosis.

In another embodiment of the present invention, the composition may further have one or more of the following characteristics, but is not limited thereto:

(a) inhibition of weight and body fat gain;

(b) inhibition of fibrosis in liver or adipose tissue;

(c) inhibition of plasma lipid concentration increase;

(d) inhibiting the increase in liver tissue lipid content;

(e) inhibition of increase in plasma hepatotoxicity index;

(f) inhibition of lipid peroxide increase and increase of antioxidant activity; or

(g) improvement of intestinal microbial imbalance.

In addition, the present invention is a disease related to muscle weakness, comprising the step of administering a composition containing a celery seed extract to a subject in need thereof; metabolic disease; Or a liver disease prevention or treatment method is provided.

In addition, the present invention is related to muscular weakness of the composition containing the celery seed extract; metabolic disease; or for use in preventing or treating liver disease.

In addition, the present invention relates to muscle weakness-related diseases; metabolic disease; or the use of a celery seed extract for the preparation of a drug for treating liver disease.

In addition, the present invention provides a composition for preventing, improving, or treating metabolic diseases comprising a celery seed extract as an active ingredient.

In addition, the present invention provides a composition for preventing, improving, or treating liver disease comprising a celery seed extract as an active ingredient.

In addition, the present invention provides a muscle strengthening method comprising the step of administering a composition containing a celery seed extract to a subject in need thereof.

In addition, the present invention provides a muscle strengthening use of a composition containing a celery seed extract.

In addition, the present invention provides a use of a celery seed extract for preparing a drug for strengthening muscle strength.

The celery seed extract according to the present invention has no or few side effects by using a natural product and can increase muscle mass and strength, so it not only prevents muscle weakness but also suppresses the increase in body weight and body fat, and improves blood lipid concentration and liver toxicity index (GOT and GPT), it is expected to be useful for preventing, improving, or treating diseases related to muscle weakness, metabolic diseases, or liver diseases.

1 is a diagram schematically illustrating an experimental design using an obesity-induced mouse model for evaluating the effect of celery seed extract on obese muscle loss.
Figure 2a shows the normal diet group (ND), high fat diet group (HFD), and celery seed extract added group (CS) mice after feeding for 20 weeks, gastrocnemius, quadriceps, tibialis anterior , Kidney, and liver were removed and weighed, and this is a diagram showing weight per 100 g of body weight (HFD vs. ND: *p<0.05, **p<0.01, ***p<0.001; HFD vs. CS: #p<0.05, ##p<0.01, ###p<0.001, the same below).
Figure 2b is a normal diet group (ND), high-fat diet group (HFD), and celery seed extract added group (CS) body weight was measured at weekly intervals for 20 weeks, weight change (left) and weight gain inhibition according to breeding period It is a diagram showing the comparison of efficacy (right).
Figure 2c shows the results of food intake, energy intake, and dietary efficiency (FER) measurements of normal diet group (ND), high fat diet group (HFD), and celery seed extract added group (CS) mice. It is a comparative drawing.
Figure 2d is a normal diet group (ND), high-fat diet group (HFD), and celery seed extract added group (CS) mice after 20 weeks of feeding, white adipose tissue (White Adipose Tissue; WAT) was extracted and weight (Epididymal: epididymis, Perirenal: perirenal, Retroperitoneum: retroperitoneum, Mesentric: mesentery, Visceral: visceral, Subcutaneous: subcutaneous, interscapular WAT: interscapular white fat, interscapular BAT: scapula) liver brown fat).
Figure 3a is a diagram showing morphological changes in muscle tissue at 20 weeks after feeding mice of a normal diet group (ND), a high-fat diet group (HFD), and a celery seed extract supplemented group (CS) for 20 weeks (upper : H&E staining/bottom: sirius red staining). Scale bar = 100 μm.
Figure 3b is a view showing the morphological changes of epididymal white adipose tissue at 20 weeks after feeding mice of the normal diet group (ND), the high fat diet group (HFD), and the celery seed extract group (CS) for 20 weeks. (Top: H&E staining/bottom: Masson's trichrome staining). Scale bar = 100 μm.
Figure 3c is a view showing the morphological changes of liver tissue at 20 weeks after feeding mice of the normal diet group (ND), the high-fat diet group (HFD), and the celery seed extract group (CS) for 20 weeks (upper : H&E staining/bottom: Masson's trichrome staining). Scale bar = 100 μm.
Figure 4 shows the results of measuring thigh thickness (top) and tensile force (bottom) at 19 weeks of normal diet group (ND), high fat diet group (HFD), and celery seed extract added group (CS) mice at 4 weeks and 20 weeks. it is a drawing
Figure 5a is a diagram showing the results of measuring the lipid content of muscle tissue of mice of a normal diet group (ND), a high-fat diet group (HFD), and a celery seed extract group (CS) (FA: free fatty acid, TG: neutral fat, CHOL : cholesterol).
Figure 5b is a view showing the results of measuring the lipid content of liver tissue and the activity of lipid metabolism enzyme (PAP) in mice fed a normal diet (ND), a high fat diet (HFD), and a celery seed extract group (CS) (FA: free fatty acids, TG: triglycerides, CHOL: cholesterol).
6 is a diagram showing the comparison of changes in the expression of muscle cell growth-related proteins in mice of a normal diet group (ND), a high-fat diet group (HFD), and a celery seed extract-added group (CS).
7 is a diagram showing comparison of plasma triglyceride (left) and plasma total cholesterol (right) measurement results for each week in mice of a normal diet group (ND), a high-fat diet group (HFD), and a celery seed extract group (CS). .
8 is a diagram showing comparison of plasma glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) measurement results of mice of a normal diet group (ND), a high-fat diet group (HFD), and a celery seed extract supplemented group (CS).
Figure 9a is a diagram showing comparison of paraoxonase (PON) measurement results of plasma and liver tissue of mice of a normal diet group (ND), a high-fat diet group (HFD), and a celery seed extract supplemented group (CS).
FIG. 9B is a diagram showing a comparison of the results of lipid peroxide (TBARS) measurement in red blood cells and liver tissues of mice in a normal diet group (ND), a high-fat diet group (HFD), and a celery seed extract-added group (CS).
FIG. 9C is a diagram showing a comparison of glutathione (GSH) measurement results of red blood cells and liver tissues of mice in a normal diet group (ND), a high-fat diet group (HFD), and a celery seed extract-added group (CS).
10 is a view schematically illustrating an experimental design using an aging mouse model for evaluating the effect of celery seed extract on age-related muscle loss.
11 is a view showing the results of confirming the change in weight according to the breeding period by measuring the body weight of the young mouse group (YC), the aged mouse group (NC), and the celery seed extract group (CS) at weekly intervals for 12 weeks. (YC vs. NC: *p<0.05, **p<0.01, ***p<0.001; NC vs. CS: &p<0.05, &&p<0.01, &&&p<0.001, the same below).
FIG. 12 is a diagram showing comparison of the results of food intake and food efficiency (FER) measurements of a young mouse group (YC), an aged mouse group (NC), and a celery seed extract-added group (CS).
13 shows that after 12 weeks of feeding the experimental diet to the young mouse group (YC), the aged mouse group (NC), and the celery seed extract group (CS), white adipose tissue (WAT) was extracted for each part This is a drawing showing the weight per 100g of body weight by measuring weight (Epididymal: epididymis, Perirenal: perirenal, Retroperitoneum: retroperitoneum, Mesentric: mesentery, Visceral: visceral, Subcutaneous: subcutaneous, interscapular WAT: interscapular white fat, interscapular BAT: interscapular brown fat).
Figure 14a shows after 12 weeks of feeding the experimental diet to the young mouse group (YC), the aged mouse group (NC), and the celery seed extract group (CS), gastrocnemius, quadriceps, tibialis anterior ) is extracted and weighed, and is a drawing showing the weight per 100 g of body weight.
14B is a diagram showing the results of measuring the tensile force at 12 weeks of age in a young mouse group (YC), an aged mouse group (NC), and a celery seed extract-added group (CS).
Figure 14c is a diagram showing the results of thigh thickness measurement at 4 weeks and 12 weeks of a young mouse group (YC), an aged mouse group (NC), and a celery seed extract-added group (CS).
15 is a view showing the results of measuring the lipid content of muscle tissues of young mice (YC), aged mice (NC), and celery seed extract-added group (CS) (FA: free fatty acid, TG: neutral fat, CHOL : cholesterol).
16 is a view showing morphological changes in muscle tissues at 12 weeks after feeding the experimental diet for 12 weeks to a young mouse group (YC), an aged mouse group (NC), and a celery seed extract-added group (CS) ( Top: H&E staining/bottom: sirius red staining). Scale bar = 100 μm.
17 shows the results of confirming the expression of IGF-1 and Myostatin by immunochemical analysis after feeding the experimental diet to the young mouse group (YC), the aged mouse group (NC), and the celery seed extract group (CS) for 12 weeks. is the drawing shown. Scale bar = 100 μm.
Figure 18 shows the expression of muscle atrophy-related genes (FoxO1, FoxO3, Atrogin, and MuRF1) after 12 weeks of feeding the experimental diet to the young mouse group (YC), the aged mouse group (NC), and the celery seed extract-added group (CS). It is a drawing showing the result of confirming the expression.
Figure 19 confirms the expression of muscle fiber proteins (Igf-1R, FoxO1, and sirt3) after feeding the experimental diet to young mice (YC), aged mice (NC), and celery seed extract group (CS) for 12 weeks. This is a diagram showing the result.
20 is a diagram showing the results of examining glucose tolerance after feeding the experimental diet to young mice (YC), aged mice (NC), and celery seed extract-added group (CS) for 12 weeks.
Figure 21a is a view showing the weight per 100 g of body weight by measuring the weight of the liver after feeding the experimental diet to the young mouse group (YC), the aged mouse group (NC), and the celery seed extract-added group (CS) for 12 weeks.
Figure 21b is a view showing the results of measuring liver damage index plasma GOT and GPT after feeding the experimental diet to young mice (YC), aged mice (NC), and celery seed extract-added group (CS) for 12 weeks. .
Figure 21c is a view showing the results of measuring the lipid content of liver tissue after feeding the experimental diet for 12 weeks to a young mouse group (YC), an aged mouse group (NC), and a celery seed extract-added group (CS).
Figure 22a shows the measurement of the glutathione (GSH) content of liver tissue, red blood cells and plasma after feeding the experimental diet to the young mouse group (YC), the aged mouse group (NC), and the celery seed extract group (CS) for 12 weeks. This is a diagram showing the result.
Figure 22b shows superoxide dismutase (SOD) activity in liver tissue and red blood cells after feeding the experimental diet to young mice (YC), aged mice (NC), and celery seed extract-added group (CS) for 12 weeks. It is a drawing showing the result of measuring.
22c shows the content of H ₂ O ₂ in mitochondria, cell matrix and red blood cells after feeding the experimental diet to young mice (YC), aged mice (NC), and celery seed extract group (CS) for 12 weeks. This is a diagram showing the result.
Figure 23 shows the short-chain fatty acids acetic acid, propionate, and butylic acid after feeding the experimental diet to the young mouse group (YC), the aged mouse group (NC), and the celery seed extract-added group (CS) for 12 weeks. (Butyrate) This is a diagram showing the results of measuring the content.
24a and 24b are diagrams showing the results of analyzing intestinal microbes after feeding the experimental diet to a young mouse group (YC), an aged mouse group (NC), and a celery seed extract-added group (CS) for 12 weeks. Figure 24a is a view showing the results of analyzing the intestinal microorganisms involved in colitis and inflammatory response, and Figure 24b is the intestinal microorganisms involved in obesity and short-chain fatty acid production.

As a result of efforts to develop active substances derived from natural products capable of preventing, improving or treating muscle weakness-related diseases, the present inventors confirmed the excellent effect of increasing muscle mass and improving muscle metabolism of celery seed extract in obese or aging mice. The invention was completed.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating diseases related to muscle weakness, comprising a celery seed extract as an active ingredient.

In addition, the present invention provides a composition for strengthening muscle strength containing a celery seed extract as an active ingredient.

The "celery seed" of the present invention is a seed of celery, which is a plant of the umbel family Apiaceae, the size of millet, yellowish brown, and has a celery-like fragrance and is used as a spice. Celery seed is characterized by green smell and bitter taste, and is known to be effective in anti-inflammatory, diuretic, sedative, aphrodisiac, anti-rheumatism, and arthritis.

In the present invention, "extract" refers to an extract obtained by the extraction treatment of the celery seeds, a diluted or concentrated liquid of the extract, a dried product obtained by drying the extract, a crude product or a purified product of the extract, or a mixture thereof. It includes extracts of all formulations that can be formed using the extract itself and the extract solution.

The method for extracting celery seeds according to the present invention is not particularly limited, and can be extracted according to a method commonly used in the art. Non-limiting examples of the extraction method include a hot water extraction method, an ultrasonic extraction method, a filtration method, a reflux extraction method, and the like, which may be performed alone or in combination of two or more methods.

In the present invention, the type of extraction solvent used to extract the celery seed is not particularly limited, and according to a conventional method known in the art for extracting an extract from a natural product, that is, a conventional solvent under normal temperature and pressure conditions can be extracted using For example, in the present invention, the celery seed extract may be extracted with at least one solvent selected from the group consisting of water, an organic solvent having 1 to 6 carbon atoms, and a subcritical or supercritical fluid. The organic solvent having 1 to 6 carbon atoms is alcohol having 1 to 6 carbon atoms, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride chloride), hexane, cyclohexane, and petroleum ether, but may be at least one selected from the group consisting of, but is not limited thereto. In the present invention, the celery seed extract may be preferably extracted with ethanol.

In the present invention, the celery seed extract is 70 to 130 g, 70 to 110 g, 70 to 100 g, 70 to 80 g, 80 to 130 g, 100 to 130 g, 110 to 130 g, 90 to 130 g celery seed powder 40 to 95%, 40 to 85%, 40 to 75%, 40 to 65%, 40 to 55%, 40 to 50% in 110 g, 95 to 105 g, 70 g, 80 g, 90 g, or 100 g , 40 to 47%, 40 to 45%, 40 to 43%, 40 to 41%, 50 to 55%, 55 to 65%, 65 to 75%, 68 to 75%, 70 to 75%, 73 to 75% , 65 to 73%, 65 to 70%, 65 to 68%, 75 to 85%, 85 to 95%, 88 to 95%, 90 to 95%, 90 to 92%, 90 to 91%, 40%, 50 %, 60%, 65%, 68%, 69%, 70%, 71%, 72%, 75%, 78%, 80%, or 90% concentration ethanol 700 mL to 1.3 L, 700 mL to 1.1 L, 700 mL to 1 L, 700 mL to 900 mL, 900 mL to 1 L, 1 L to 1.1 L, 1 L to 1.3 L, 1.2 L to 1.3 L, 700 mL, 800 mL, 900 mL, or by adding 1 L 30 to 90 ° C, 30 to 70 ° C, 30 to 50 ° C, 40 to 50 ° C, 50 to 70 ° C, 55 to 75 ° C, 60 to 70 ° C, 60 to 65 ° C, 60 to 63 ° C, 70 to 90 ° C, 1 hour 30 minutes to 3 hours 30 minutes, 1 hour 30 minutes to 3 hours, 1 hour 30 minutes to 2 hours at 80 to 90 °C, 85 to 90 °C, 30 °C, 40 °C, 50 °C, 55 °C, or 60 °C Time, 2 hours to 2 hours 30 minutes, 2 hours 30 minutes to 3 hours, 3 hours to 3 hours 30 minutes, 2 hours, 2 hours 30 minutes, 2 hours 50 minutes, or 3 hours extraction After repeating 1 to 5 times, 1 to 3 times, 1 to 2 times, 2 to 4 times, 4 to 5 times, 1 time, 2 times, or 3 times, the extracted liquid was filtered and concentrated under reduced pressure. It may be lyophilized, but is not limited thereto.

The prepared extract may then be filtered or concentrated or dried to remove the solvent, and both filtration, concentration and drying may be performed. For example, filter paper or a vacuum filter may be used for filtration, a vacuum concentrator may be used for concentration, and a lyophilization method may be used for drying, but is not limited thereto.

In the present invention, "active ingredient" means a component that exhibits the desired activity alone or can exhibit the desired activity together with a carrier having no activity itself.

In the present invention, "disease related to muscle weakness" means any disease in which muscle tissue or muscle cells are reduced or lost. The muscle weakness may be limited to any one muscle, one side of the body, upper limbs or lower limbs, may appear throughout the body, or may appear congenital or acquired. In addition, subjective muscle weakness symptoms including muscle fatigue or muscle pain can be quantified in an objective way through a physical examination.

The muscle weakness-related diseases refer to all diseases that may occur due to muscle weakness, and include, but are not limited to, for example, sarcopenia, muscular atrophy, muscle dystrophy or deep atrophy.

In the present invention, "sarcopenia" refers to a condition in which the body's muscles (muscle mass and strength) are abnormally reduced or weakened for various reasons such as aging and obesity, resulting in poor physical activity. Sarcopenia has far-reaching effects throughout the body, including bones, blood vessels, nerves, liver, heart, and pancreas, beyond the muscles themselves. In particular, since bones maintain density by receiving stress (stimulation) by muscles, sarcopenia and osteoporosis have a very close correlation. In addition, it is known that when muscle is reduced, it interferes with the formation of new blood vessels and nerves, increasing the risk of cognitive decline, fatty liver, and diabetes.

In the present invention, the sarcopenia may be age-related sarcopenia or obese sarcopenia, but is not limited thereto.

The “senescence sarcopenia” refers to a gradual decrease in muscle mass or a gradual weakening of muscle density and function due to aging, which directly causes a decrease in muscle strength, resulting in a decrease in various physical functions and disability. means the state of being able to

The "obesity sarcopenia" refers to a phenomenon in which muscle function is weakened due to the deposition of fat in the muscle due to obesity, a decrease in muscle mass, an increase in mast cell-derived inflammatory factors, and a decrease in the function of mitochondria in the muscle. As a result, when abnormalities in insulin secretion occur due to obesity, energy cannot be properly supplied to cells, which can cause muscle development disorders.

The "amyotrophic disease" is a gradual atrophy of the muscles of the limbs, which causes progressive degeneration of motor nerve fibers and cells in the spinal cord, resulting in amyotrophic lateral sclerosis (ALS) and spinal progressive muscular atrophy , SPMA).

The "muscle dystrophy" refers to a disease in which gradual muscle atrophy and weakness occur, and pathologically refers to a degenerative myopathy characterized by necrosis of muscle fibers.

The “cardiac atrophy” is a condition in which the heart is atrophied by external or internal factors, and appears as a symptom in which myocardial fibers become dry and thin due to starvation, wasting disease, and frailty, causing a decrease in adipose tissue.

As used herein, the term "strengthening muscle strength" refers to enhancement of physical performance, enhancement of maximum endurance, increase in muscle mass, enhancement of muscle recovery, reduction of muscle fatigue, improvement of energy balance, or a combination thereof.

In one embodiment of the present invention, celery seed extract increases muscle mass and strength in a high-fat diet-induced sarcopenic obese mouse model, inhibits fibrosis of muscle tissue, inhibits an increase in lipid content of muscle tissue, and inhibits muscle atrophy It increases the expression of related proteins PGC1α and proteins involved in protein accumulation, MyD88 and Traf6, activates Akt and Pi3k proteins involved in myocyte differentiation and inhibition of apoptosis, as well as suppresses weight and body fat gain, improves liver and adipose tissue It was confirmed that it inhibited fibrosis, suppressed an increase in liver tissue lipid content, suppressed an increase in plasma lipid concentration, suppressed an increase in plasma hepatotoxicity index, suppressed an increase in lipid peroxides, and increased antioxidant capacity (see Example 1). ).

In another embodiment of the present invention, celery seed extract increases muscle mass and strength in an aging mouse model, inhibits fibrosis of muscle tissue, inhibits an increase in lipid content of muscle tissue, and inhibits IGF-1 involved in the synthesis of muscle tissue Increases the expression of Myostatin, which is involved in the breakdown of muscle tissue, reduces the expression of genes related to muscle atrophy, increases the expression of igf-1 R, which is involved in muscle fiber protein synthesis, and is involved in muscle atrophy It not only reduces the expression of FoxO1, but also suppresses the increase in body fat, lowers blood sugar, suppresses the increase in plasma lipid concentration, suppresses the increase in plasma hepatotoxicity index, suppresses the increase in liver tissue lipid content, and increases antioxidant capacity. , increased short-chain fatty acids, and improved intestinal microbial imbalance (see Example Ⅱ).

Accordingly, the celery seed extract can be applied not only to the prevention, improvement, or treatment of muscle weakness-related diseases, but also to the prevention, improvement, or treatment of metabolic diseases or liver diseases.

Therefore, in the present invention, the composition according to the present invention can prevent, improve, or treat muscle weakness-related diseases by satisfying one or more of the following characteristics:

(a) increase in muscle mass;

(b) inhibition of muscle mass loss;

(c) increased muscle strength;

(d) inhibition of increase in muscle tissue lipid content; or

(e) inhibition of muscle tissue fibrosis.

In addition, in the present invention, the composition according to the present invention can prevent, improve, or treat muscle weakness-related diseases, metabolic diseases, or liver diseases by further satisfying one or more of the following characteristics:

(a) inhibition of weight and body fat gain;

(b) inhibition of fibrosis in liver or adipose tissue;

(c) inhibition of plasma lipid concentration increase;

(d) inhibiting the increase in liver tissue lipid content;

(e) inhibition of increase in plasma hepatotoxicity index;

(f) inhibition of lipid peroxide increase and increase of antioxidant activity; or

(g) improving intestinal microbial imbalance.

In the present invention, “improvement of intestinal microbial imbalance” means promoting the proliferation or growth of beneficial bacteria in the intestine and suppressing the proliferation or growth of harmful bacteria in the intestine, while maintaining the balance between beneficial bacteria and harmful bacteria in the intestine. Microorganisms begin to inhabit the organ, and the population reaches equilibrium to form the gut microbiome. Individual microbes constituting the gut microbiome are involved in vitamin supply, infection prevention, and intestinal function, and thus the composition of the gut microbiome. It is known to have a deep relationship with constipation and the occurrence of intestinal-related diseases.

The “intestinal beneficial bacteria” may collectively refer to microorganisms that inhabit the intestines and exert beneficial effects on the human body. For example, intestinal beneficial bacteria may include probiotics. For example, the intestinal beneficial bacteria are not limited thereto, and include Bifidobacterium , Lactobacillus , Lactococcus , Streptococcus , Akkermansia , and Lactococcus. Faecalibacterium , Roseburia , Lachnospiraceae , or Enterococcus . However, in the case of Enterococcus , there are also strains classified as beneficial bacteria and antibacterial activity in the intestine along with Lactobacillus , but some enterococci have high antibiotic resistance due to inflammation caused by vancomycin-resistant enterococci (VRE) However, since it can be classified as a harmful bacteria that causes diseases such as urinary tract infections and infectious endocarditis, additional studies on the probiotic potential and safety of Enterococcus are needed.

The “intestinal harmful bacteria” may collectively refer to microorganisms that live in the intestines and exert harmful effects on the human body, such as enteritis. For example, the intestinal harmful bacteria are not limited thereto, Escherichia coli , Fusobacterium , Clostridium , Staphylococcus , and Desulfovibrio . , Desulfovibrionaceae family, Erysipelatoclostridium genus, Enterorhabdus genus, or Porphyromonas genus.

In the present invention, "short chain fatty acid" is a major metabolite of intestinal microorganisms, and refers to fatty acids having less than 6 carbon atoms. When short-chain fatty acids are increased, the intestinal environment is acidified to activate bowel movements, inhibit fat absorption to reduce the amount of fat in the body, and also reduce blood triglycerides, which can prevent, improve, or treat metabolic diseases such as obesity. According to one embodiment of the present invention, the short-chain fatty acids in the present invention are acetic acid (Acetate), propionic acid (Propionate), and butyrate (Butyrate).

In the present invention, "obesity" refers to a state in which adipose tissue is abnormally increased due to excessive accumulation of energy in the body due to an imbalance between energy intake and consumption. there is. Obesity is caused by a combination of multiple causes rather than a single cause, and causes such as wrong eating habits, decreased activity, emotional factors, and genetic factors, including westernized eating habits, can be cited as causes. In the present invention, the obesity may be sarcopenic obesity, but is not limited thereto.

In the present invention, "sarcomenic obesity" means a state in which the amount of body fat is increased along with a decrease in muscle mass or strength due to aging or obesity, that is, a complex form of obesity and sarcopenia due to conversion of muscle to fat. . For example, it is known that if a person with a similar body mass index has an increased body fat mass and a reduced muscle mass, the risk of functional limitation and metabolic disease is higher than that of a person with a balanced body fat mass and muscle mass.

In the present invention, "metabolic disease" means a condition or disease closely related to obesity or caused by obesity, for example, dyslipidemia; hepatotoxic diseases including drug-induced liver injury, viral liver injury, hepatitis, cirrhosis, liver cancer or hepatic coma; Or it may be fatty liver, but is not limited thereto.

In the present invention, "liver disease" may be any one or more selected from the group consisting of non-alcoholic fatty liver disease and liver cancer.

In the present invention, "Nonalcoholic fatty liver disease (NAFLD)" is one of the types of fatty liver that occurs when fat accumulates in the liver in a patient who has not consumed excessive alcohol, and is a simple disease without an inflammatory reaction. It refers to a wide range of diseases, including fatty liver, progressed by it, inflammatory response of hepatocytes, liver fibrosis and liver cirrhosis. Non-alcoholic fatty liver disease can obtain good results when detected in an early stage, but if not, it can progress to non-alcoholic steatohepatitis (NASH) for various reasons, and can further cause cirrhosis and liver cancer.

The non-alcoholic fatty liver disease is divided into primary and secondary depending on the cause. The primary is caused by hyperlipidemia, diabetes, or obesity, which are characteristic of metabolic syndrome, and the secondary is caused by nutritional causes (rapid weight loss, starvation, intestinal bypass), various It is caused by drugs, toxic substances (poisonous mushrooms, bacterial toxins), metabolic causes, and other factors. The incidence of nonalcoholic fatty liver disease related to diabetes and obesity, which is an important feature of metabolic syndrome, which is a primary factor, is about 50% of diabetic patients and about 76% of obese patients. known to occur.

The non-alcoholic fatty liver disease may be at least one selected from the group consisting of simple fatty liver disease, nutritional fatty liver disease, starvation fatty liver disease, obese fatty liver disease, diabetic fatty liver disease, steatohepatitis, liver fibrosis, liver cirrhosis, and cirrhosis. , but not limited thereto.

The pharmaceutical composition according to the present invention may further include suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. The excipient may be, for example, one or more selected from the group consisting of a diluent, a binder, a disintegrant, a lubricant, an adsorbent, a moisturizer, a film-coating material, and a controlled release additive.

The pharmaceutical compositions according to the present invention are powders, granules, sustained-release granules, enteric granules, solutions, eye drops, elsilic agents, emulsions, suspensions, spirits, troches, perfumes, and limonadese, respectively, according to conventional methods. , tablets, sustained-release tablets, enteric tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, perfusate, It can be formulated and used in the form of an external agent such as a warning agent, lotion, pasta agent, spray, inhalant, patch, sterile injection solution, or aerosol, and the external agent is a cream, gel, patch, spray, ointment, warning agent , lotion, liniment, pasta, or cataplasma may have formulations such as the like.

Carriers, excipients and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, 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.

Corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, di-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, phosphoric acid as additives for tablets, powders, granules, capsules, pills, and troches according to the present invention Calcium monohydrogen, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropylmethyl Excipients such as cellulose (HPMC) 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose phthalate acetate, carboxymethyl cellulose, calcium carboxymethyl cellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethyl cellulose, sodium methyl cellulose, methyl cellulose, microcrystalline cellulose, dextrin Binders such as hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, purified shellac, starch arc, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, and polyvinylpyrrolidone may be used, Hydroxypropyl Methyl Cellulose, Corn Starch, Agar Powder, Methyl Cellulose, Bentonite, Hydroxypropyl Starch, Sodium Carboxymethyl Cellulose, Sodium Alginate, Calcium Carboxymethyl Cellulose, Calcium Citrate, Sodium Lauryl Sulfate, Silicic Anhydride, 1-Hydroxy Propyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, sodium bicarbonate, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, disintegrants such as amylopectin, pectin, sodium polyphosphate, ethyl cellulose, white sugar, magnesium aluminum silicate, di-sorbitol solution, and light anhydrous silicic acid; Calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopod, kaolin, petrolatum, sodium stearate, cacao butter, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogen Added soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, silicic anhydride, higher fatty acid, higher alcohol, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, Lubricants such as starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, light anhydrous silicic acid; may be used.

Additives for the liquid formulation according to the present invention include water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, sucrose monostearate, polyoxyethylene sorbitol fatty acid esters (tween esters), polyoxyethylene monoalkyl ethers, lanolin ethers, Lanolin esters, acetic acid, hydrochloric acid, aqueous ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethyl cellulose, sodium carboxymethyl cellulose, and the like may be used.

In the syrup according to the present invention, a solution of white sugar, other sugars, or a sweetener may be used, and aromatics, coloring agents, preservatives, stabilizers, suspending agents, emulsifiers, thickeners, etc. may be used as necessary.

Purified water may be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. may be used as needed.

Suspension agents according to the present invention include acacia, tragacantha, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906, HPMC 2910, etc. Agents may be used, and surfactants, preservatives, stabilizers, colorants, and fragrances may be used as needed.

Injections according to the present invention include distilled water for injection, 0.9% sodium chloride injection, IV injection, dextrose injection, dextrose + sodium chloride injection, PEG, lactated IV injection, ethanol, propylene glycol, non-volatile oil-sesame oil , solvents such as cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; solubilizing agents such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, twins, nijuntinamide, hexamine, and dimethylacetamide; buffers such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptone, and gums; tonicity agents such as sodium chloride; Stabilizers such as sodium bisulfite (NaHSO ₃ ) carbon dioxide gas, sodium metabisulfite (Na ₂ S ₂ O ₅ ), sodium sulfite (Na ₂ SO ₃ ), nitrogen gas (N ₂ ), ethylenediaminetetraacetic acid; Sulfating agents such as sodium bisulfide 0.1%, sodium formaldehyde sulfoxylate, thiourea, ethylenediamine disodium tetraacetate, acetone sodium bisulfite; analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; Suspending agents such as Siemesis sodium, sodium alginate, Tween 80, aluminum monostearate may be included.

The suppository according to the present invention includes cacao butter, lanolin, witapsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, subanal, cottonseed oil, peanut oil, palm oil, cacao butter + Cholesterol, Lecithin, Lannet Wax, Glycerol Monostearate, Tween or Span, Imhausen, Monolen (Propylene Glycol Monostearate), Glycerin, Adeps Solidus, Buytyrum Tego-G -G), Cebes Pharma 16, Hexalide Base 95, Cotomar, Hydroxycote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hyde Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Masupol, Masupol-15, Neosupostal-N, Paramound-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), suppository type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecobi (W, R, S, M, Fs), testosterone triglyceride base (TG-95, MA, 57) and The same mechanism can be used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient, for example, starch, calcium carbonate, sucrose, etc. ) or by mixing lactose and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

Liquid preparations for oral administration include suspensions, solutions for oral administration, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. there is.

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents.

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is the type of patient's disease, severity, activity of the drug, It may be determined according to factors including sensitivity to the drug, administration time, route of administration and excretion rate, duration of treatment, drugs used concurrently, and other factors well known in the medical field.

The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by a person skilled in the art to which the present invention belongs.

The pharmaceutical composition of the present invention can be administered to a subject by various routes. All modes of administration can be envisaged, eg oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal space (intrathecal) injection, sublingual administration, buccal administration, intrarectal insertion, vaginal It can be administered by intraoral insertion, ocular administration, otic administration, nasal administration, inhalation, spraying through the mouth or nose, dermal administration, transdermal administration, and the like.

The pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient together with various related factors such as the disease to be treated, the route of administration, the age, sex, weight and severity of the disease of the patient.

In addition, the present invention provides a method for preventing or treating muscle weakness-related diseases, comprising administering a composition containing a celery seed extract to a subject in need thereof.

In addition, the present invention provides a use of a composition containing a celery seed extract for preventing or treating muscle weakness-related diseases.

In addition, the present invention provides a use of the celery seed extract for preparing a drug for treating muscle weakness-related diseases.

In the present invention, "individual" means a subject in need of treatment of a disease, and more specifically, a human or non-human primate, mouse, rat, dog, cat, horse, cow, etc. of mammals.

Administration in the present invention means providing a given composition of the present invention to a subject by any suitable method.

In the present invention, "prevention" refers to any action that suppresses or delays the onset of a desired disease, and "treatment" means that a desired disease and its associated metabolic abnormalities are improved or treated by administration of the pharmaceutical composition according to the present invention. It means any action that is advantageously changed.

In another aspect of the present invention, the present invention provides a food composition for preventing or improving muscle weakness-related diseases comprising a celery seed extract as an active ingredient.

In the present invention, "improvement" refers to all actions that reduce the parameters related to the desired disease, for example, the degree of symptoms, by administration of the composition according to the present invention. For the prevention or improvement of metabolic disease or liver disease, it can be used before or after the onset of the disease, simultaneously with or separately from a drug for treatment.

When the celery seed extract of the present invention is used as a food additive, the celery seed extract may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment). In general, when preparing food or beverage, the celery seed extract of the present invention may be added in an amount of 15% by weight or less, or 10% by weight or less based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount greater than the above range.

There is no particular limitation on the type of food. Examples of foods to which the above substances can be added include meat, sausages, bread, chocolates, candies, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice creams, various soups, beverages, tea, drinks, There are alcoholic beverages and vitamin complexes, and includes all health functional foods in a conventional sense.

The health beverage composition according to the present invention may contain various flavoring agents or natural carbohydrates as additional components, like conventional beverages. The aforementioned natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrins and cyclodextrins, and sugar alcohols such as xylitol, sorbitol and erythritol. As the sweetener, natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame may be used. The proportion of the natural carbohydrate is generally about 0.01-0.20 g, or about 0.04-0.10 g per 100 mL of the composition of the present invention.

In addition to the above, the composition of the present invention contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonic acid It may contain a carbonation agent used in beverages and the like. In addition, the composition of the present invention may contain fruit flesh for preparing natural fruit juice, fruit juice beverages and vegetable beverages. These components may be used independently or in combination. The ratio of these additives is not critical, but is generally selected in the range of 0.01-0.20 parts by weight per 100 parts by weight of the composition of the present invention.

In the present invention, the food composition may be a health functional food composition, but is not limited thereto.

In the present invention, "health functional food" is the same term as food for special health use (FoSHU), and means food with high medical and medical effects processed to efficiently display bioregulatory functions in addition to nutritional supply. However, the food may be prepared in various forms such as tablets, capsules, powders, granules, liquids, pills, etc. in order to obtain useful effects for preventing or improving muscle weakness-related diseases, metabolic diseases, or liver diseases.

In the present invention, the "health functional food composition" is characterized in that it is formulated as one selected from the group consisting of tablets, pills, powders, granules, powders, capsules and liquid formulations, including one or more of carriers, diluents, excipients and additives. to be

The health functional food of the present invention can be prepared by a method commonly used in the art, and can be prepared by adding raw materials and components commonly added in the art during the preparation. In addition, unlike general drugs, there is an advantage in that there is no side effect that may occur when taking a drug for a long time by using food as a raw material, and it can be excellent in portability.

In addition, the present invention can provide a feed or feed additive for strengthening muscle strength containing a celery seed extract as an active ingredient in another aspect of the present invention.

In the present invention, "feed" means a substance that supplies organic or inorganic nutrients necessary for maintaining the life of an animal. The feed includes nutrients such as energy, protein, lipid, vitamins, and minerals required by animals such as livestock, and includes grains, roots and fruits, food processing by-products, algae, fibers, oils, starches, It may be vegetable feed such as grain by-products or animal feed such as proteins, inorganic materials, oils, minerals, oils, and single cell proteins, but is not limited thereto.

In the present invention, "feed additive" means a substance added to feed to improve productivity or health of animals, and is not particularly limited thereto, but is not particularly limited thereto, but is not particularly limited thereto, such as amino acids, vitamins, enzymes, and flavors for growth promotion and disease prevention. Agents, silicate agents, buffers, extractants, oligosaccharides, and the like may be further included.

The content of the celery seed extract included in the feed or feed additive for muscle strength is not particularly limited thereto, but may be 0.001 to 1% (w / w), preferably 0.005 to 0.9% (w / w) And, most preferably, it may be 0.01 to 0.5% (w / w).

Hereinafter, a preferred embodiment is presented to aid understanding of the present invention. However, the following examples are provided to more easily understand the present invention, and the content of the present invention is not limited by the following examples.

[Example]

Example I. Effect of Celery Seed on Obesity-induced Muscle Reduction

< Experiment materials and methods>

1. Preparation of celery seed extract

Celery seed powder used in the experiment was purchased from ES Food Raw Materials Co., Ltd. and used. 1 L of 70% ethanol was added to 100 g of celery seed powder, the raw material, and extraction was repeated three times for 3 hours at 60 ° C. The extracted liquid was filtered, concentrated under reduced pressure, and freeze-dried. A celery seed extract powder was obtained, stored frozen, and then used for dietary preparation. The extraction yield was 10.02%.

2. Experimental animal models

2-1. laboratory animal

In order to confirm the effect of intake of celery seed extract, an experiment was designed and conducted as shown in the schematic diagram shown in FIG. 1 using a high-fat diet-induced sarcopenic obese mouse model. Specifically, 4-week-old male C57BL/6J mice (JA BIO, Korea) were purchased from JoongA Bio and used. After adaptation by providing a diet in the form of pellets for 1 week, the mice were fed a normal diet (ND), a high-fat diet (HFD; 20% fat, 1% cholesterol) and celery by a randomized complete block design. They were divided into seed extract added groups (CS, HFD + 0.1% (w/w) celery seed ethanol extract), and fed experimental diets and raised for 20 weeks. In the animal breeding room, constant conditions were maintained with a constant temperature (24 ± 2 ℃), constant humidity (50 ± 5%) and a photoperiod of 12 hour intervals (AM 6:00 ~ PM 18:00), and individual cages were placed one by one. Experimental diet and drinking water were provided ad libitum.

2-2. composition of the experimental diet

The experimental diet composition of the ND group, the HFD group and the CS group is shown in Table 1 below. In the ND group, AIN-76 semisynthetic diet was prepared and fed as a normal diet group, and in the HFD group, lard and cholesterol were added to the AIN-76 diet to provide 20% fat and 1% cholesterol. A high-fat diet containing was prepared and fed. The CS group was fed a high-fat diet with the addition of celery seed extract at a dose of 0.1% of the diet.

3. Sample collection

3-1. blood sample collection

After fasting for 12 hours every 4 weeks during the breeding period, blood lipid concentration was measured by tail blood sampling without anesthesia. After fasting for 12 hours at the end of breeding, they were anesthetized with isoflorane (5 mg/kg body weight, Baxter, USA), and blood was collected from the inferior vena cava. The collected blood was treated with heparin, centrifuged at 3,000 rpm and 4° C. for 15 minutes, and plasma was collected and stored at -70° C. until sample analysis.

3-2. Tissue sample collection

Liver, kidney, epididymal white fat, perirenal white fat, interscapular white fat, brown fat, and muscle tissues obtained by sacrificing mice after blood was collected from the abdominal inferior vena cava in 3-1. above were mixed with saline (0.9% saline). solution) After rinsing several times in the solution, the surface water was removed and weighed, divided according to the purpose of the experiment, and rapidly cooled in liquid nitrogen and stored at -70 ° C until analysis.

4. Measurement of thigh thickness and whole-limb grip strength

During the breeding period, the thigh thickness of the mice was measured at 4-week intervals using calipers.

After adapting to the measuring device for 3 days to measure the tensile force of the mice at the 19th week of feeding the experimental diet, the tensile force was measured continuously for 5 days. Place the mouse on top of the grid of the measuring device and hold the grid with all four feet. Hold the mouse's body and the grid while keeping the mouse's body parallel to the grid and gently and slowly pull it backwards. value was recorded.

5. Plasma lipid concentration analysis

Plasma triglyceride (TG), total cholesterol, and HDL-cholesterol concentrations were measured using an enzyme kit from Asan Pharm Co., Seoul, Korea. Free fatty acid (FA) content was measured using a test solution for measuring free fatty acid (Non-Esterified Fatty Acid, NEFA kit, Shinyang Chemical) using the principle of color development using an enzymatic method. Plasma apolipoprotein A-1 (Apo A-1) and apolipoprotein B (apolipoprotein B; Apo B) concentrations were measured by a kit for measuring Apo A-1 and Apo B (Nitto Prefecture Co., Ltd., Tokyo, Japan) was used.

6. Measurement of tissue lipid content

To measure lipid content, liver and muscle tissue lipids were extracted according to the method of Folch et al. (1957), and then the extract was volatilized with nitrogen gas at 37 °C and diluted with isopropanol. For quantification, enzymatic reagent was mixed with 3 mM cholic acid as an emulsifier and 0.5% Triton X-100 to remove turbidity during color development, and lipid components were extracted. quantified.

7. Enzyme activity measurement

7-1. Erythrocyte Enzyme Source Isolation

According to the method of McCord and Fridovich (1969), the heparinized blood was centrifuged at 3,000 rpm and 4° C. for 15 minutes to completely remove plasma and buffy coat, and then washed three times with 0.9% physiological saline. The washed red blood cells were lysed with the same amount of distilled water and used for the measurement of antioxidant enzyme activity.

7-2. Liver tissue enzyme source isolation

For the isolation of enzyme sources in liver tissue, the isolation method conducted by Hulcher et al. (1973) was partially modified and applied. Add a buffer solution containing 0.1 M triethanolamine, 0.02 M ethylenediamine tetracetate (EDTA, pH 7.4), and 0.002 M dithiothreitol (DTT) to 0.5 g of liver tissue and homogenize with a glass teflon homogenizer (Glascol, 099C K44, USA) in an ice-cooled state. After that, centrifugation was performed at 3,000 rpm and 4 °C for 15 minutes, and only the supernatant was centrifuged again at 13,000 rpm and 4 °C for 15 minutes. Among them, the precipitate of the lower layer separated from the supernatant was used as a mitochondrial fraction, and the supernatant was obtained by using an ultracentrifuge (Beckman, Optima TLX-120, USA) for 1 hour at 32,500 rpm and 4 ° C. The cytosol fraction of was obtained. For the microsome fraction, the same buffer solution was added to the pellet separated from the cytoplasmic fraction in the supernatant, followed by ultracentrifugation at 33,000 rpm at 4 ° C for 40 minutes, and then the pellet was dissolved in 1 ml buffer and stored at -70 ° C. and then used for analysis and protein quantification.

7-3. Measurement of lipid metabolism enzyme activity (Phosphatidate phosphohydrolase)

Phosphatidate phosphohydrolase (PAP) activity was measured according to the method of Walton et al. (1985). 0.05 M Tris-HCl (pH 7.0), 1.25 mM Na ₂ -EDTA, 50 μl of a reaction solution with and without addition of 1 mM MgCl ₂ was dissolved in a 0.9% NaCl solution, and 50 μl of a substrate containing 1 mM phosphatidate and phosphatidylcholine was added. After addition, 0.1 mL of the microsome fraction was added to initiate the reaction. After reacting at 37 ℃ for 15 minutes, 0.1 mL of 1.8 MH ₂ SO ₄ was added to stop the reaction, and then 0.13% sodium dodecyl sulfate (SDS) solution 0.1 mL and 1.25% ascorbic acid (ascorbic acid) and 0.32% 0.25 mL each of % ammonium molybdate was added, reacted and developed at 45 °C for 20 minutes, and then absorbance was measured at 820 nm.

7-4. Measurement of antioxidant enzyme activity

7-4-a. Measurement of Paraoxonase activity

Paraoxonase (PON) activity was measured by modifying and supplementing the method of Mackness et al. (1991). As a reaction solution, 30 μl of plasma and liver tissue microsomes as enzyme sources were added to 940 μl of 0.1 M Tris-HCl buffer (pH 8.0) containing 2 mM CaCl ₂ , and 100 mM paraoxon (O,O-diethyl-Op- 30 μl of nitrophenylphosphate, Sigma Chemical Co.) was added to measure the increase in absorbance of p-nitrophenol (extinction coefficient: 17,000 M ⁻¹ cm ⁻¹ ) generated at 25° C. and 405 nm for 90 seconds.

7-4-b. Glutathione (GSH) content measurement

Glutathione content included both oxidized and reduced GSH, and was measured by modifying and supplementing the method of Fiala et al. (1976). 0.5 g of liver tissue was added to a buffer solution containing 0.1 M triethanolamine, 0.02 M EDTA (pH 7.4), and 0.002 M DTT, and homogenized with a glass teflon homogenizer (Glascol, 099C K44, USA) in an ice-cooled state. 0.3 mL of distilled water and 0.5 mL of 4% sulfosalicylic acid were added to 0.2 mL of the homogenate, followed by centrifugation at 25,000 rpm and 4 °C for 10 minutes to obtain a supernatant. 2.7 mL of 0.1 M disulfide reagent (5.5'-dithiobis + 0.1 M sodiumphosphate buffer, pH 8.0) was added to 0.3 mL of the supernatant, reacted for 20 minutes at room temperature, and absorbance was measured at 412 nm.

8. Measurement of red blood cell and liver tissue lipid peroxides (TBARS) content

8-1. Measurement of red blood cell lipid peroxide content

Red blood cell lipid peroxide content was measured using the method of Tarladgis et al. (1964). 3 mL of 5% triochloroacetic acid (TCA) and 1 mL of 0.06 M thiobarbituric acid (TBA) were added to 50 μl of red blood cells and reacted at 80 °C for 90 minutes. After cooling to room temperature and centrifuging at 2,000 rpm and 25 °C for 15 minutes, the supernatant was taken and absorbance was measured at 535 nm. Lipid Peroxidation (MDA) standard solution hydrolyzed tetramethoxypropane (TMP), and the amount of TBA reactant at 267 nm was calculated as MDA extinction coefficient. That is, 1 mmol of TMP was dissolved in 100 mL of a 0.01 N HCl solution, reacted at 50 °C for 60 minutes, cooled to room temperature, and TMP was hydrolyzed with MDA. MDA standard solution (1×10 ^-4 M) was prepared by diluting 1 mL of the hydrolyzed TMP solution in 100 mL of 0.01 M Na ₃ PO ₄ (pH 7.0) buffer. After obtaining the absorption spectrum of the MDA standard solution at 267 nm and correcting it by calculating the exact concentration from the extinction coefficient, a TBA-MDA chromopore standard curve was obtained. From this curve, the amount of the TBA reactant was calculated as the MDA extinction coefficient. .

8-2. Measurement of liver tissue lipid peroxide content

Liver tissue lipid peroxide content was measured by adding a buffer solution containing 0.1 M triethanolamine, 0.02 M EDTA (pH 7.4), and 0.002 M DTT to 0.5 g of liver tissue using the method of Ohkawa et al. was homogenized with a glass teflon homogenizer (Glascol, 099C K44, USA). Mix 0.2 mL of the homogenate, 0.2 mL of 8.1% sodium dodecyl sulfate (SDS) solution, and 0.6 mL of distilled water, leave it at room temperature for 5 minutes, add 1.5 mL of 20% acetate (pH 3.5) buffer and 1.5 mL of 0.8% TBA to 95 mL. It was reacted for 1 hour at °C. After the reaction, the sample was cooled to room temperature, 1 mL of distilled water and 5 mL of n-butanol:pyridine (15:1) solution were added, centrifuged at 3,000 rpm, 20 ° C for 15 minutes, and the absorbance of the supernatant was measured at 532 nm. . The MDA standard solution hydrolyzed TMP, and the amount of TBA reactant at 267 nm was calculated as the MDA extinction coefficient.

9. Morphological analysis of tissue

For morphological observation of tissue, parts of liver, epididymal white fat and muscle tissues excised from mice were fixed in 10% formaldehyde solution for 24 hours, exchanged with the same solution twice, dehydrated with 2-fold ethanol, and paraffinized. After embedding in paraffin, 5 μm-thick tissue sections treated with poly-L-lysine were prepared, stained with hematoxylin eosin (H&E), and observed under an optical microscope at 200x magnification. To stain collagen and muscle fibers, liver and adipose tissue were stained with Masson's trichrome (connective tissue was stained blue, nuclei were stained dark red, and cytoplasm was stained pink), and muscle tissue was stained with sirius red (muscle fibers were stained yellow and collagen was stained red). stained) was performed and observed with an optical microscope at 200x magnification.

10. Western blot measurement

0.1 g of muscle tissue was homogenized by adding 3 mm beads and 1 ml of lysis buffer (T-PER buffer, Thermo Scientific, Rockford, IL, USA), and then centrifuged at 14,000 rpm for 15 minutes to collect only the supernatant. It was transferred to a new tube, and the protein was extracted and used in the experiment. Protein quantification was performed using Quick Start™ Bradford Reagent (Bio-rad, Hercules, CA, USA). The same amount of protein was subjected to electrophoresis (SDS-PAGE) on SDS-polyacrylamide gel. After transferring the protein separated on the gel through electrophoresis to a polyvinylidene fluoride (PVDF) membrane (Merck Millipore, New Jersey, USA), 5% skim milk/Tris-buffered saline with tween 20 (TBST; 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20) for 1 hour at room temperature, and reacted with the primary antibody overnight at 4°C. Primary antibodies are anti-PGC1α (1:1000, abcam, ab191838), anti-Akt (1:1000, cell signaling, #9272), anti-phospho Akt (1:1000, cell signaling, #4058), anti- Pi3k (1:1000, cell signaling, #4292), anti-phospho Pi3k (1:1000, cell signaling, #4228), anti-mTOR (1:1000, cell signaling, #2983), anti-MyD88 (1: 1000, cell signaling, #4283), anti-Sirt1 (1:1000, cell signaling, #3931), and anti-Traf6 (1:1000, abcam, ab33915) were diluted in 5% BSA and used as a loading control. Alpha tubulin (1:1000, cell signaling, #2125) was used. For the secondary antibody, horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (1:5000; Cell Signaling, #7074S) was diluted in 5% skim milk and reacted at room temperature for 1 hour. After each reaction, it was washed three times for 10 minutes with TBST buffer and then proceeded. The washed membrane was visualized using an Enhanced Chemiluminescent (ECL) kit (super-signal west pico plus, 34580, Thermo Scientific, Rockford, IL, USA) that develops bands, and G-box (50S; BI System Co.) It was quantified and analyzed using .

11. Statistical analysis

Experimental results were calculated using SPSS package program versuib 25.0 (Statistical Paskage for the Social Sciences, SPSS Inc., Chicago), one of the computer statistical programs. Student's t-test was conducted to test the significance between the ND group and the HFD group and between the HFD and CS groups. All results were expressed as mean±S.E (standard error).

<Experiment result>

1. Analysis of tissue weight change and body weight change by celery seed extract

1-1. Changes in muscle and organ weight

In order to analyze changes in muscle and organ weight, gastrocnemius muscle, femoris muscle, tibialis anterior muscle, kidney and liver were extracted and weighed, and then expressed as weight per 100 g of body weight and compared.

As a result, as shown in FIG. 2a, in the case of muscle, the weight of muscle tissue by all parts of the HFD group was significantly reduced compared to the ND group, while the weight of the gastrocnemius and femoral muscles of the CS group was significantly greater than that of the HFD group increased. In the case of kidney, the weight of the HFD group decreased significantly compared to the ND group, while the weight of the kidney increased significantly in the CS group compared to the HFD group. In the case of the liver, it was confirmed that the weight of the HFD group was significantly increased compared to the ND group, whereas the weight of the liver was significantly decreased in the CS group than the HFD group. Through the above results, it was confirmed that the celery seed extract had an effect of increasing muscle mass, suppressing an increase in liver weight, and suppressing a decrease in kidney weight.

1-2. weight change

To investigate the effect of intake of celery seed extract on weight gain, body weight and food intake were measured once a week during the breeding period.

As a result, as shown in FIG. 2b, in the case of the HFD group, the body weight increased sharply compared to the ND group, whereas in the case of the CS group, the experimental diet was fed from the 12th week. there was.

In addition, when the body weight gain during the 20 weeks was converted into the weight gain per day (g/day), it was confirmed that the weight gain in the CS group was significantly suppressed compared to the HFD group.

On the other hand, as shown in FIG. 2c, there was no significant difference between the CS group and the HFD group in the daily average food intake and energy intake, but the dietary efficiency representing the weight gain for energy intake ( Food efficiency ratio (FER) was found to be significantly increased in the HFD group with high energy density compared to the ND group with low energy density, whereas significantly decreased in the CS group compared to the HFD group. From the above results, it can be seen that the celery seed extract has an effect of significantly reducing body weight in an environment where the same energy is consumed.

1-3. Adipose tissue weight change

In order to analyze the change in the weight of the adipose tissue, the adipose tissue of the mouse was removed and the weight was measured, and then expressed as a weight per 100 g of body weight and compared.

As a result, as shown in FIG. 2d, in the case of the HFD group, since the weight of adipose tissue by all parts significantly increased compared to the ND group, it was confirmed that obesity was induced by the high-fat diet. On the other hand, in the CS group supplemented with celery seed extract, the weights of perirenal and mesenteric white adipose tissue corresponding to visceral fat decreased significantly, and the weight of visceral fat and total white adipose tissue decreased. From the above results, it can be seen that the celery seed extract has an effect of reducing body fat by significantly reducing the weight of white adipose tissue that stores energy.

2. Analysis of the effect of celery seed extract on morphological changes in liver tissue, adipose tissue and muscle tissue

2-1. muscle tissue

As a result of observing the gastrocnemius muscle of the mouse by H&E staining, as shown in FIG. 3a, the shape and structure of the muscle cell bundle were irregular in the HFD group compared to the ND group, but in the case of the CS group, the shape of the muscle cells was normal It was confirmed that it was improved (upper part of FIG. 3a).

Furthermore, as a result of sirius red staining for histological visualization of collagen fibers in muscle tissue, more collagen stained in muscle tissue was observed in the HFD group than in the ND group, but in the case of the CS group, fibrosis of muscle tissue due to high-fat diet was shown to be suppressed (bottom of FIG. 3a).

2-2. adipose tissue

As shown in FIG. 3B, as a result of observing epididymal white adipose tissue (Epididymal WAT) by H&E staining, it was observed that the size of fat cells in the HFD group was larger than that in the ND group, and the size of fat cells in the CS group was larger than that of the HFD group. It was confirmed that is small (upper part of Fig. 3b).

Furthermore, as a result of Masson's trichrome staining to examine the effect of celery seed extract on fibrosis of adipose tissue, fibrotic connective tissue was observed in adipocytes of the HFD group more prominently than that of the ND group. In the case of, it was found that the fibrosis of adipose tissue due to the high-fat diet was suppressed (bottom of FIG. 3b).

2-3. liver tissue

As a result of H&E staining of the liver tissue, as shown in FIG. 3c, in the case of the HFD group, the largest amount of lipid droplet was accumulated around the portal vein of the liver tissue, but supplemented with celery seed extract In the case of one CS group, the number of fat cells was reduced compared to the HFD group.

In addition, Masson's trichrome staining was performed to examine the effect of celery seed extract on liver tissue fibrosis. As a result, in the case of the HFD group, a large amount of connective tissue was accumulated around the portal vein, but in the case of the CS group, liver due to the high-fat diet It was confirmed that tissue fibrosis was inhibited.

3. Analysis of the effect of celery seed extract on thigh thickness and muscle strength

As a result of measuring the thigh thickness of the mouse, as shown in FIG. 4, the thickness of the hind leg thigh measured at the 4th week of feeding the experimental diet was significantly higher in the HFD group than in the ND group, but there was no significant difference between the CS group and the HFD group Didn't show up. However, at the 20th week of feeding the experimental diet, it was confirmed that the thigh thickness of the right and left hind limbs of the CS group increased significantly compared to the HFD group.

In addition, as a result of the tensile force measurement conducted at the 19th week of the experimental diet, there was no significant difference between the ND group and the HFD group, but in the case of the CS group, it was confirmed that the tensile force was significantly increased compared to the HFD group. Therefore, from the above results, it was confirmed that the celery seed extract had the effect of improving the thigh thickness reduced by the high-fat diet and increasing muscle strength.

4. Analysis of the effect of celery seed extract on tissue lipid content

In order to analyze the effect of the celery seed extract on the lipid content of tissue, the results of comparing free fatty acid, triglyceride and cholesterol contents per unit weight of tissue are shown in FIG. 5 .

4-1. Lipid content of muscle tissue

As shown in Figure 5a, it was confirmed that the triglyceride, free fatty acid and cholesterol contents of the muscle tissue were significantly increased in the HFD group, whereas the free fatty acid and cholesterol contents in the CS group were significantly decreased compared to the HFD group. From the above results, since the celery seed extract suppresses the increase in the lipid content of muscle tissue, the above 1-1. and 3., it was found that the increased muscle weight and thickness were not due to lipid accumulation.

4-2. Lipid content of liver tissue and activity of lipid metabolism enzymes

As a result of measuring the activity of phosphatidate phosphohydrolase (PAP), an enzyme related to triglyceride synthesis in liver tissue, as shown in Figure 5b, the HFD group was observed to be significantly higher than the ND group, and the increased PAP activity due to the high-fat diet was confirmed to be significantly reduced by supplementing with celery seed extract.

In addition, in the case of the HFD group consuming a high-fat diet, the lipid content of liver tissue increased significantly compared to the ND group consuming a normal diet, but in the case of the CS group supplemented with celery seed extract, free fatty acids in liver tissue And it was confirmed that the total cholesterol content was significantly reduced compared to the HFD group. From the above results, it was confirmed that the celery seed extract reduced the lipid content of liver tissue.

5. Analysis of the effect of celery seed extract on the expression of proteins related to muscle cell growth

To confirm the effect of celery seed extract on the cell growth mechanism of muscle tissue, the protein expression level of the Pi3k/Akt/mTOR pathway, which promotes muscle cell differentiation and is involved in muscle fiber protein accumulation, was measured.

As a result, as shown in FIG. 6, PGC1α (peroxisome proliferator-activated receptor gamma coactivator 1-alpha) protein involved in suppressing inflammation and muscle atrophy in muscle tissue and MyD88, a parent factor of the Pi3k/Akt/mTOR pathway ( Myeloid differentiation primary response 88) and Traf6 (Tumor necrosis factor receptor associated factor 6) proteins showed a significant increase in expression in the CS group compared to the HFD group.

On the other hand, in the case of Akt (Protein kinase B) and Pi3k (phosphoinositide 3-kinase) proteins involved in myocyte differentiation and apoptosis inhibition, it was confirmed that the expression of pAkt and pPi3k proteins, which are activated forms of Akt and Pi3k, increased. From the above results, it was confirmed that the celery seed extract has an effect of improving muscle metabolism.

6. Analysis of the effect of celery seed extract on plasma lipid concentration

6-1. Plasma triglyceride and total cholesterol concentrations

As a result of measuring the plasma triglyceride and total cholesterol concentrations once a week during the experimental period, the total cholesterol concentrations of the HFD group increased significantly compared to the ND group after 4 weeks of feeding the experimental diet, as shown in FIG. 7, CS In the case of the group, it was confirmed that plasma total cholesterol concentration decreased significantly compared to the HFD group at the 16th week of feeding the experimental diet.

On the other hand, triglyceride concentration did not show a significant difference between groups.

6-2. Plasma lipid concentration

Free fatty acids, triglycerides, total cholesterol concentrations, and nonHDL cholesterol concentrations in plasma were significantly decreased in the CS group compared to the HFD group, while the ratio of HDL cholesterol to total cholesterol (HDL-C/Total C ratio; HTR ) was significantly increased in the CS group compared to the HFD group. Furthermore, it was confirmed that the levels of atherogenic intex (AI) and plasma apolipoprotein B (Apo B) were significantly lower in the CS group than in the HFD group. Therefore, it was found from the above results that the celery seed extract reduced the plasma lipid concentration.

Measurements Experimental groups ND HFD CS Free fatty acids (mol/L) 1.14±0.04 1.08±0.02 0.99±0.02 ^# Triglycerides (mmol/L) 1.02±0.12 1.00±0.06 0.80±0.04 ^# Total cholesterol (mmol/L) 3.86±0.22 7.61±0.56 ^*** 5.27±0.14 ^## HDL-cholesterol (mmol/L) 0.94±0.06 1.68±0.05 ^*** 1.45±0.09 nonHDL-cholesterol (mmol/L) 2.92±0.17 5.93±0.52 ^*** 3.82±0.08 ^# HTR (%) 24.47±0.84 22.88±1.36 27.48±1.23 ^# AI 3.11±0.14 3.45±0.23 2.69±0.18 ^# Apo A-1 (mg/ml) 0.29±0.02 0.35±0.03 0.37±0.03 Apo B (mg/ml) 0.22±0.01 0.40±0.05 ^* 0.24±0.02 ^# Apo B / Apo A-1 0.78±0.07 1.13±0.11 ^* 0.66±0.05 ^##

7. Analysis of the effect of celery seed extract on plasma hepatotoxicity index

In order to confirm the effect of celery seed extract on hepatotoxicity, the activities of glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT), which are closely related to liver damage, were analyzed using an enzyme kit from Asan Pharm Co., Seoul, Korea. was measured using

In addition, as shown in FIG. 8, in the case of the HFD group, plasma GOT and GPT levels were significantly increased compared to the ND group, and in the case of the CS group, plasma GOT and GPT levels were significantly decreased compared to the HFP group Confirmation did From the above results, it was confirmed that the celery seed extract had an effect of inhibiting hepatotoxicity.

8. Analysis of the effect of celery seed extract on antioxidant-related biomarkers

8-1. Paraoxonase (PON) activity

As a result of comparison of PON activity that inhibits oxidation of plasma lipoprotein particles, as shown in FIG. 9a, in the case of plasma PON activity, the HFD group showed a tendency to decrease compared to the ND group, whereas the CS group showed a tendency to decrease compared to the HFD group It was found that there was a significant increase compared to In addition, in the case of liver tissue PON activity, the HFD group showed the lowest activity, and it was confirmed that the CS group increased significantly compared to the HFD group.

8-2. Lipid peroxides (Thiobarbituricacid reactive substance; TBARS)

As a result of comparison of TBARS of red blood cells and liver tissue, as shown in FIG. 9B, in the case of red blood cell TBARS, the HFD group significantly increased compared to the ND group, but the CS group significantly decreased compared to the HFD group. . In addition, in the case of liver tissue TBARS, it was confirmed that the HFD group increased significantly compared to the ND group, but the CS group showed a decreasing tendency.

8-3. Glutathione (GSH)

As a result of comparing the content of GSH, an antioxidant-related biomarker, in red blood cells and liver tissue, as shown in FIG. It was confirmed that it increased compared to the HFD group. From the above results, it was confirmed that the celery seed extract had the effect of suppressing the increase in lipid peroxides and increasing the antioxidant activity.

Example II. Effect of Celery Seed on Geriatric Muscle Loss

<Experiment materials and methods>

1. Preparation of celery seed extract

Celery seed powder was purchased from ES Food Ingredients Co., Ltd. and used. After adding 1 L of 70% ethanol to 100 g of celery seed powder as a raw material, extraction was repeated three times for 3 hours at 60 ° C, and the extracted solution was filtered, concentrated under reduced pressure, and freeze-dried. The celery seed alcohol extract powder was obtained and stored frozen and used for dietary preparation, and the yield of the extract was 10.02%.

2. Experimental animal models

2-1. laboratory animal

As experimental animals, 8-week-old male C57BL/6J mice and 50-week-old male C57BL/6J mice (JA BIO, Korea) were purchased from JoongA Bio and used. As shown in FIG. 10, after adaptation by providing a diet in the form of pellets for 2 weeks, the experimental animals were divided into young mouse groups (YC, Young control, n = 8) and aged mouse groups (NC, Negative control, n = 7) and aging mice were divided into groups provided with a diet supplemented with celery seed extract (CS, 0.1% (w/w) celery seed ethanol extract, n = 7), and fed with an experimental diet for 12 weeks. . In the animal breeding room, constant conditions were maintained with a constant temperature (24 ± 2 ℃), constant humidity (50 ± 5%), and a photoperiod of 12 hours apart (AM 6:00 ~ PM 18:00). and drinking water were provided ad libitum. During the 12-week experimental period, food intake and body weight were measured once a week, and the value obtained by dividing daily weight gain by daily energy intake was used as food efficiency ratio (FER).

2-2. composition of the experimental diet

The composition of the experimental diet is as shown in Table 3. AIN-93G diet was prepared and fed to all groups, and CS group was fed by adding celery seed extract at a dose of 0.1% of the diet to the normal diet.

Ingredient (g) YC
(AIN-93G diet) NC
(AIN-93G diet) CS
(AIN-93G diet+celery seed 0.1%) Casein (from milk) 200.0 200.0 200.0 Corn Starch 397.5 397.5 397.5 Sucrose 100.0 100.0 100.0 Maltodextrin 132.0 132.0 132.0 Cellulose 50.0 50.0 50.0 Soybean Oil 70.0 70.0 70.0 Mineral Mix, AIN-93G-MX 35.0 35.0 35.0 Vitamin Mix, AIN-93-VX 10.0 10.0 10.0 TBHQ, antioxidants 0.014 0.014 0.014 L-Cystine 3.0 3.0 3.0 cholin Bitartrate 2.5 2.5 2.5 Celery Seed Extract 1.00 Total (g) 1000.01 1000.04 1001.04

3. Sample collection

At the end of breeding, the experimental animals were fasted for 12 hours, anesthetized using isoflurane (5 mg/kg body weight, Baxter, USA), and blood was collected from the inferior vena cava. The collected blood was treated with heparin, centrifuged at 3,000 rpm and 4 °C for 15 minutes, and plasma was collected and stored at -70 °C until sample analysis.

Liver, kidney, epididymal white fat, perirenal white fat, interscapular white/brown fat, and muscle tissue were removed from the experimental animals, rinsed several times in a saline solution (0.9% saline solution), and then removed and weighed. Divided according to the purpose of the experiment, it was rapidly cooled in liquid nitrogen and stored at -70 ℃ until analysis.

4. Measurement of Thigh Thickness and Tensile Force

During the experiment period, the thigh thickness of the mouse was measured at 4-week intervals using a caliper.

In order to measure the tensile force of the experimental animals at the 12th week of feeding the experimental diet, the tensile force was measured continuously for 5 days after adaptation in the measuring device for 3 days.

Specifically, place the experimental animal on top of the grid of the measuring device and have it grasp the grid with all four feet, hold the mouse's body and the tail while keeping it parallel to the grid, and gently and slowly pull it backwards. The maximum muscle strength value of the force was recorded.

5. Biochemical analysis

5-1. Plasma lipid concentration analysis

Plasma triglyceride, total cholesterol, and HDL-cholesterol concentrations were measured using an enzyme kit from Asan Pharm Co., Seoul, Korea. Free fatty acid content was measured using a test solution for measuring free fatty acid (Non-Esterified fatty acid, NEFA kit, Shinyang Chemical) using the principle of a coloring method using an enzymatic method.

5-2. Tissue lipid content measurement

Lipid content of liver and muscle tissue was extracted according to the method of Folch et al. (1957), and then the extract was volatilized with nitrogen gas at 37 °C and diluted with isopropanol. For quantification, the enzyme reagent was mixed with 3 mM cholic acid as an emulsifier and 0.5% Triton X-100 to remove turbidity during color development.

5-3. Enzyme Source Separation

5-3-a. Erythrocyte Enzyme Source Isolation

Erythrocytes were centrifuged at 3,000 rpm and 4 °C for 15 minutes according to the method of McCord and Fridovich (1969) to completely remove plasma and buffy coat, and then washed three times with 0.9% physiological saline. The washed red blood cells were lysed with the same amount of distilled water and used for the measurement of antioxidant enzyme activity.

5-3-b. Liver tissue enzyme source isolation

For the isolation of enzyme sources in liver tissue, the isolation method conducted by Hulcher et al. (1973) was partially modified and applied. 0.5 g of liver tissue was homogenized with a glass teflon homogenizer (Glascol, 099C K44, USA) in an ice-cooled state by adding a buffer solution containing 0.1 M triethanolamine, 0.02 M ethylenediamine tetracetate (EDTA, pH 7.4), and 0.002 M dithiothreitol (DTT). After that, centrifugation was performed at 3,000 rpm and 4 °C for 15 minutes, and only the supernatant was centrifuged again at 13,000 rpm and 4 °C for 15 minutes. Among them, the precipitate of the lower layer separated from the supernatant was used as the mitochondrial fraction, and the supernatant was used for 1 hour at 32,500 rpm, 4 ℃ using an ultracentrifuge (Beckman, Optima TLX-120, USA). cytosol) fraction was obtained. For the microsomal fraction, the same buffer solution was added to the pellet separated from the cytoplasmic fraction of the supernatant, followed by ultracentrifugation at 33,000 rpm and 4 ° C for 40 minutes, and then the pellet was dissolved in 1 ml buffer solution and stored at -70 ° C for analysis. and protein quantification.

5-4. Measurement of antioxidant enzyme activity

5-4-a. Determination of glutathione content

Glutathione (GSH) content includes both oxidized GSH and reduced GSH, and was measured by modifying and supplementing the method of Fiala et al. (1976). 0.5 g of liver tissue was added to a buffer solution containing 0.1 M triethanolamine, 0.02 M EDTA (pH 7.4), and 0.002 M DTT, and homogenized with a glass teflon homogenizer (Glascol, 099C K44, USA) in an ice-cooled state. 0.3 mL of distilled water and 0.5 mL of 4% sulfosalicylic acid were added to 0.2 mL of the homogenate, followed by centrifugation at 25,000 rpm and 4 °C for 10 minutes to obtain a supernatant. 2.7 mL of 0.1 M disulfide reagent (5.5'-dithiobis + 0.1 M sodiumphosphate buffer, pH 8.0) was added to 0.3 mL of the supernatant, reacted for 20 minutes at room temperature, and absorbance was measured at 412 nm.

5-4-b. Measurement of superoxide dismutase (SOD) activity

SOD is an enzyme that catalyzes the decomposition of superoxide anion radical (O ² - ) into H ₂ O ₂ and O ₂ . The activity measurement was performed by modifying and supplementing the method of Marklund et al. (1974) to obtain pyrogallol in an alkaline state. ) was measured using the degree of color development by autoxidation. Add 0.1 mL of liver tissue and red blood cell enzyme source and 0.1 mL of 7.2 mM pyrogallol solution to 1.5 mL of 50 mM Tris-hydroxymethyl-aminomethane buffer (pH 8.5) containing 10 mM EDTA, react at 25 ° C for 10 minutes, and then add 50% of 1 N HCl solution. The reaction was terminated by the addition of μL, and the change in absorbance was measured at 420 nm (Beckman 650 spectrophotometer, USA) to calculate the SOD unit for cytosolic protein and red blood cell hemoglobin required to prevent autoxidation of pyrogallol (SOD unit /mg cytosolic protein, SOD unit/g hemoglobin).

6. Intraperitoneal glucose tolerance test

After the mice were fasted for 12 hours, a glucose solution was intraperitoneally administered at 0.5 g per kg body weight, and blood was collected through the tail vein after 0, 30, 60, and 120 minutes, respectively, and measured using a glucometer.

7. Liver tissue toxicity analysis by measuring plasma GOT and GPT activity

GOT and GPT activities, which are closely related to hepatocellular damage, were measured using an enzyme kit from Asan Pharm Co., Seoul, Korea.

8. Morphological analysis of tissue cells

For morphological observation of the tissue, part of the muscle tissue excised at the time of animal sacrifice was fixed in 10% formaldehyde solution for 24 hours, exchanged twice with the same solution, dehydrated with 2-fold ethanol, embedded in paraffin, and poly- Tissue sections with a thickness of 5 μm treated with L-lysine were prepared, stained with H&E, and observed under an optical microscope at 200x magnification. To stain muscle fibers, sirius red staining was performed and observed under an optical microscope at 200x magnification. For immunochemical analysis, Igf-1R and Myostatin were stained by Immunohistochemistry (IHC) staining and observed under a light microscope at 400x magnification.

9. Real-time PCR

RNA was isolated from muscle tissue and cDNA was synthesized using the Prime Script Real time reagent kit (Takara, Japan). cDNA was diluted in RNAse free water, stored at -20 ° C, and used when performing Real-time RT-PCR. For real-time RT-PCR gene expression analysis, SYBR Green PCR kit (Takara, Japan) was used. Primers capable of analyzing the expression of each gene were synthesized by Genotech Co., Ltd. (Daejeon, Korea). At this time, the threshold cycle (Ct) was analyzed by monitoring the fluorescence signal for each cycle, and the mRNA expression between each experimental group was quantitatively analyzed with the CFX96 Real time system (Bio-rad, USA). The primers used are shown in Table 4.

10. Western blot measurement

0.1 g of frozen muscle tissue was homogenized by adding 3 mm beads and 1 ml of lysis buffer (T-PER buffer, Thermo Scientific, Rockford, IL, USA), centrifuged at 14,000 rpm for 15 minutes, and only the supernatant was collected for new It was transferred to a tube, and the protein was extracted and used in the experiment. Protein quantification was performed using Quick Start™ Bradford Reagent (Bio-rad, Hercules, CA, USA). The same amount of protein was subjected to electrophoresis (SDS-PAGE) on SDS-polyacrylamide gel. After transferring the protein separated on the gel through electrophoresis to a polyvinylidene fluoride (PVDF) membrane (Merck Millipore, New Jersey, USA), 5% skim milk/Tris-buffered saline with tween 20 (TBST; 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20) for 1 hour at room temperature and reacted with primary antibody overnight at 4 °C. The secondary antibody was diluted in 5% skim milk of horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (1:5000; Cell Signaling, #7074S) and reacted at room temperature for 1 hour. After each reaction, it was washed three times for 10 minutes with TBST buffer and then proceeded. The washed membrane was visualized using an Enhanced Chemiluminescent (ECL) kit (super-signal west pico plus, 34580, Thermo Scientific, Rockford, IL, USA) that develops bands, and G-box (50S; BI System Co.) It was quantified and analyzed using .

11. Analysis of short-chain fatty acids

A certain amount of the obtained biofeces was weighed, diluted with tertiary distilled water according to the ratio, sufficiently stirred, and then centrifuged at 13,000 rpm, 3 min, room temperature (RT).

After transferring 150 μL of the supernatant to a vial for short-chain fatty acid analysis, 150 μL of GC buffer was dispensed. It was sealed with a short-chain fatty acid analysis cap and analyzed for 10 minutes using an Agilent Headspace 7697A + Agilent GC 7890 B + Flame lpnization Detector equipped with an HP-lnnowax column. The short-chain fatty acids to be analyzed were prepared and mixed at the optimum concentration for analysis, followed by serial dilution with tertiary distilled water, and analysis was repeated 5 times for each concentration, starting with a low concentration, to derive the results.

12. Intestinal microorganism analysis method

For biofecal bacterial DNA extraction, Mag-Bind ^® Universal Pathogen Kit (Omega Bio-tek) was used. According to the manufacturer's protocol, fecal samples were suspended in SLX-Mlus Buffer, then ground, separated and washed. The extracted fecal microbial DNA was amplified with 16S Amplicon PCR Forward Primer (5'-TCGTCGGCAGCGTCAGATGTATAAGAGACAGCCTACGGGNGGCWGCAG-3') and 16S Amplicon PCR Reverse Primer (5'-GTCTCGTGGGCTCGGAGATGTGTATAAGACCAGAGTATCACTACH). PCR was performed at 95 °C for 3 minutes, 8 cycles of 95 °C for 30 seconds, 55 °C for 30 seconds, 72 °C for 30 seconds, and 72 °C for 5 minutes, and maintained at 4 °C for the PCR reaction. After the cleanup step, the concentration of the library was checked using Qubit 4.0 (ThermoFisher Scientific) with 1×dsDNA HS Assay Solution (ThermoFisher Scientific) and then sequenced using the Illumina Miseq system. Reads were aligned using a unique barcode for each PCR product. Sequencing results were analyzed using the Qiime2 bioinformatics pipeline, and taxonomic assignments were performed with the Silva reference database.

13. Statistical Analysis

Experimental results were calculated using SPSS package program version 25.0 (Statistical Package for the Social Sciences, SPSS Inc., Chicago), one of the computer statistical programs. Student's t-test was conducted to test the significance between each YC group and the NC group and between the NC and CS groups. All results were expressed as mean±S.E (standard error).

<Experiment result>

1. Weight change by celery seed extract

The average weight change during 12 weeks of feeding the experimental diet is shown in FIG. 11 . There was a significant difference in body weight between the YC and NC groups from before the start of the experiment to the end of the experiment, but there was no significant difference in body weight and weight gain between the NC and CS groups.

2. Effect of Celery Seed Extract on Dietary Intake and Dietary Efficiency

As shown in Figure 12, the average food intake per day (g / day) increased in the NC group compared to the YC group, but there was no significant difference between the CS and NC groups. Food efficiency ratio (FER) increased significantly in the YC group, where weight increased due to growth, compared to the NC group, which had little weight change, and there was no significant difference between the NC and CS groups.

3. Effect of Celery Seed Extract on Adipose Tissue Weight

As a result of measuring adipose tissue weight per unit body weight, as shown in FIG. 13, epididymal white adipose tissue, perirenal white adipose tissue, mesenteric white adipose tissue, visceral white adipose tissue, interscapular white adipose tissue, and total white adipose tissue of the NC group. The weight of adipose tissue was significantly increased compared to the YC group, and with the supplementation of celery seed extract, epididymal white adipose tissue, perirenal white adipose tissue, mesenteric white adipose tissue, visceral adipose tissue, interscapular white adipose tissue, and total white adipose tissue were observed. It was confirmed that the weight of the tissue was significantly reduced.

4. Effects of Celery Seed Extract on Muscle Tissue Weight, Tensile Force and Hind Thigh Thickness

Changes in weight and strength of muscle tissue per 100 g of body weight are shown in FIGS. 14a and 14b. After the end of the experiment, the gastrocnemius muscle, femoral muscle, and tibialis anterior muscle of the experimental animals were removed and measured. As shown in FIG. 14a, the weight of the gastrocnemius muscle, tibialis anterior muscle, and total muscle tissue in the NC group was significantly reduced compared to the YC group, In the CS group, gastrocnemius muscle and total muscle mass decreased by aging were significantly increased compared to the NC group.

As a result of the tensile force measurement performed at the 12th week of the experiment, as shown in FIG. 14b, the tensile force of the NC group was significantly reduced compared to that of the YC group, while the reduced tensile force of the CS group was normalized to the level of the YC group. .

As a result of measuring the thigh thickness of the hind limbs during the experimental diet feeding period, as shown in FIG. 14c, no significance was observed in all groups at the 4th week of feeding the experimental diet, but at the 12th week of feeding the experimental diet, the thickness of the right and left hind limbs of the CS group All of them were found to be significantly increased compared to the NC group.

5. Effect of Celery Seed Extract on Muscle Tissue Lipid Content

As a result of comparing free fatty acid (FA), triglyceride (TG) and cholesterol (CHOL) contents per unit weight of muscle tissue, as shown in FIG. 15, the neutral fat, free fatty acid and cholesterol contents of muscle tissue were significant in the NC group. It was significantly increased in the CS group compared to the NC group with the supplementation of celery seed extract.

6. Morphological analysis of muscle tissue

As a result of H&E staining for morphological analysis of the gastrocnemius muscle, as shown in Figure 16, the shape and structure of the muscle cell bundle appeared irregular in the NC group compared to the YC group, but normal levels due to the supplementation of celery seed extract improvement could be observed. Sirius red staining was performed for histological visualization of collagen fibers in muscle tissue. Collagen stained in muscle tissue was significantly observed in the NC group compared to the YC group, and it was confirmed that the fibrosis of the muscle tissue was suppressed by the supplementation of the celery seed extract in the CS group.

7. Immunochemical analysis of muscle tissue

As a result of confirming the effect of celery seed extract on the expression levels of IGF-1 and Myostatin, which are involved in the synthesis and degradation of muscle tissue, as shown in FIG. It was confirmed that the expression of IGF-1, which was reduced by the supplementation of celery seed extract, was increased, whereas the expression was low between cells.

In addition, it was confirmed that the expression of Myostatin decreased to a level similar to that of the YC group by supplementing with celery seed extract.

8. Effect of celery seed extract on muscle function-related gene expression

As a result of comparing the expression of muscle atrophy-related genes (FoxO1, FoxO3, Atrogin, and MuRF1) in the gastrocnemius muscle, as shown in FIG. 18, the expression of all muscle atrophy-related genes significantly increased in the NC group compared to the YC group. It was confirmed that it was significantly reduced by the supplementation of celery seed extract.

9. Effect of celery seed extract on muscle fiber protein expression

In order to confirm the growth mechanism of muscle tissue cells by celery seed extract supplementation, the protein expression level of the Igf-1 R pathway, which promotes muscle cell differentiation and is involved in muscle fiber protein accumulation, was measured. As a result, as shown in FIG. 19, the expression of the Igf-1 R (Igf-1 Receptor) protein involved in muscle fiber protein synthesis was most strongly expressed in the CS group, whereas it was confirmed that the expression level decreased in the NC group. In the case of FoxO1, which is involved in inflammatory metabolism and muscular atrophy in muscle tissue, it was most clearly expressed in the NC group compared to the YC group, and it was confirmed that the expression decreased in the CS group.

In addition, it was confirmed that the expression of Sirt3 protein, which regulates antioxidant metabolism in mitochondria, increased in the CS group compared to the NC group.

10. Intraperitoneal glucose tolerance test

As a result of the glucose tolerance test conducted at the 12th week of experimental diet feeding, as shown in FIG. 20, when 30 minutes elapsed after intraperitoneal glucose injection, the blood sugar in all groups increased, and after 120 minutes, compared to the NC group, the YC group and the YC group It was confirmed that the blood sugar of the CS group was significantly decreased and showed a normal value.

11. Effect of Celery Seed Extract on Plasma Lipid Concentration

Table 5 shows plasma total cholesterol and other lipid concentrations measured in plasma obtained by sacrificing mice after feeding the experimental diet for 12 weeks. As shown in Table 5, total plasma cholesterol concentration and nonHDL cholesterol concentration in the CS group were significantly decreased compared to the NC group. On the other hand, the HDL cholesterol concentration and the ratio of HDL cholesterol to total cholesterol (HDL-C/Total C ratio; HTR) were significantly increased in the CS group compared to the NC group.

Measurements Experimental groups YC NC CS Free fatty acid (mmol/L) 0.73±0.03 0.76±0.04 0.69±0.05 Triglycerides (mmol/L) 0.93±0.06 1.01±0.15 1.00±0.11 Total cholesterol (mmol/L) 4.80±0.08 ^*** 5.71±0.11 4.85±0.30 ^& HDL-cholesterol (mmol/L) 1.15±0.03 1.18±0.07 1.23±0.07 nonHDL-cholesterol (mmol/L) 3.68±.0.08 ^** 4.96±0.26 3.93±0.17 ^& HTR (%) 23.36±0.65 ^* 19.92±1.16 25.61±1.22 ^&

12. Effects of Celery Seed Extract on Liver Tissue Weight, Liver Toxicity Index and Lipid Content

As for the liver tissue weight per 100 g of body weight, as shown in FIG. 21a, hypertrophy was observed in the NC group compared to the YC group.

GOT and GPT, indicators of liver toxicity, are enzymes present in hepatocytes, and high levels of GOT and GPT in plasma generally indicate liver damage. As shown in FIG. 21B, plasma GOT and GPT levels were significantly increased in the NC group compared to the YC group, and significantly decreased by the supplementation of celery seed extract.

In addition, as shown in Figure 21c, it was confirmed that the liver tissue lipid content of the NC group was significantly increased compared to the YC group, whereas the liver tissue lipid content of the CS group was significantly decreased compared to the NC group.

13. Antioxidant related biomarkers

As a result of comparing the GSH content, which is an antioxidant-related biomarker, as shown in FIG. 22a, it was confirmed that the GSH content of liver tissue, red blood cells, and plasma decreased in the NC group, but increased to the level of the YC group by supplementing with celery seed extract.

In addition, SOD metabolic activity in liver tissue and erythrocytes was significantly decreased in the NC group compared to the YC group, as shown in FIG.

As shown in FIG. 22c , the contents of liver tissue (mitochondria and cytosol) and red _blood cells were significantly increased in the NC group compared to the _YC group, and significantly decreased by the supplementation of celery seed extract.

14. Gut microbiome analysis

Figure 23 shows the results of comparative analysis on short-chain fatty acid content and microbial flora using live mouse feces collected at 11 to 12 weeks of the breeding period. As a result of the short-chain fatty acid analysis, as shown in FIG. 23, the YC group showed a higher value than the NC group, and in particular, it was confirmed that a significant difference was shown in acetate (acetate). In addition, it was shown that supplementation of celery seed extract normalized the content of short-chain fatty acids decreased due to aging to the level of the YC group.

Intestinal microflora analysis showed the results analyzed at the genus stage. As a result of analyzing the microorganisms associated with the occurrence of inflammatory bowel disease and colitis, as shown in FIG. 24a, it was confirmed that the increased number due to aging with the supplement of celery seed extract was normalized to the level of the YC group. In addition, as shown in Figure 24b, microorganisms related to intestinal microbial imbalance ( Desulfovibrionaceae family, Erysipelatoclostridium genus) were significantly increased in the NC group compared to the YC group It was significantly decreased in the CS group compared to the NC group. Obesity/metabolic disease-related microorganisms ( Lactobacillus genus) were significantly increased in the CS group compared to the NC group. As a result of microbial analysis related to short-chain fatty acid synthesis, Lachnospiraceae , which is involved in acetate production, tended to decrease in NC group compared to YC group, and Roseburia genus celery Supplementation with seed extract showed an increasing trend.

The above description of the present invention is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting.

Claims (13)

A pharmaceutical composition for the prevention or treatment of at least one muscle weakness-related disease selected from the group consisting of sarcopenia, muscular dystrophy, muscle dystrophy and cardiac atrophy, comprising a celery seed extract as an active ingredient.
According to claim 1,
The celery seed extract is water, alcohol having 1 to 6 carbon atoms, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride , Hexane (hexane), cyclohexane (cyclohexane), petroleum ether (petroleum ether), subcritical fluid, characterized in that the extract by one or more solvents selected from the group consisting of, and supercritical fluid, a pharmaceutical composition.
delete According to claim 1,
Characterized in that the sarcopenia is age-related sarcopenia or obese sarcopenia, the pharmaceutical composition.
According to claim 1,
The pharmaceutical composition, characterized in that the composition has the effect of one or more of the following characteristics:
(a) increase in muscle mass;
(b) inhibition of muscle mass loss;
(c) increased muscle strength;
(d) inhibition of increase in muscle tissue lipid content; or
(e) inhibition of muscle tissue fibrosis.
According to claim 5,
The pharmaceutical composition, characterized in that the composition further has the effect of one or more of the following features:
(a) inhibition of weight and body fat gain;
(b) inhibition of fibrosis in liver or adipose tissue;
(c) inhibition of plasma lipid concentration increase;
(d) inhibiting the increase in liver tissue lipid content;
(e) inhibition of increase in plasma hepatotoxicity index;
(f) inhibition of lipid peroxide increase and increase of antioxidant activity; or
(g) improvement of intestinal microbial imbalance.
A food composition for preventing or improving one or more muscle weakness-related diseases selected from the group consisting of sarcopenia, muscular dystrophy, muscle dystrophy and cardiac atrophy, comprising a celery seed extract as an active ingredient.
According to claim 7,
The celery seed extract is water, alcohol having 1 to 6 carbon atoms, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride , Hexane (hexane), cyclohexane (cyclohexane), petroleum ether (petroleum ether), subcritical fluid, characterized in that the extract by one or more solvents selected from the group consisting of, and supercritical fluid, food composition.
delete According to claim 7,
The sarcopenia is characterized in that aging sarcopenia or obese sarcopenia, food composition.
According to claim 7,
The food composition, characterized in that the composition has the effect of one or more of the following characteristics:
(a) increase in muscle mass;
(b) inhibition of muscle mass loss;
(c) increased muscle strength;
(d) inhibition of increase in muscle tissue lipid content; or
(e) inhibition of muscle tissue fibrosis.
According to claim 11,
The food composition, characterized in that the composition further has the effect of one or more of the following features:
(a) inhibition of weight and body fat gain;
(b) inhibition of fibrosis in liver or adipose tissue;
(c) inhibition of plasma lipid concentration increase;
(d) inhibiting the increase in liver tissue lipid content;
(e) inhibition of increase in plasma hepatotoxicity index;
(f) inhibition of lipid peroxide increase and increase of antioxidant activity; or
(g) improvement of intestinal microbial imbalance.
A feed or feed additive for strengthening muscle strength containing celery seed extract as an active ingredient.

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