Prevention or remedy composition of muscle-loss related disease containing plant mixed extract powder as active ingredient

Abstract

To provide a prevention or remedy composition for muscle loss-related diseases containing mixed extracts of mulberry leaves, cornelian cherry, balloon flower, dried tangerine peel, harygawa, wolfberry, ginger, and yuzu as effective ingredients.SOLUTION: A plant mixed extract of the present invention increases the production of intracellular creatine kinase and exhibits characteristics that enhance the expression of MHC and myogenin that are muscle protein, which increases serum protein concentration and demonstrates an effect of increasing muscle protein concentration. Therefore, the mixed extract of mulberry leaves, cornelian cherry, balloon flower, dried tangerine peel, harygawa, wolfberry, ginger, and yuzu in the present invention can be usefully employed for the prevention or treatment of muscle loss-related diseases.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a composition for preventing or treating diseases associated with muscle loss, comprising a mixed extract of mulberry leaves, Chinese cornus, Chinese bellflower, Uncaria citri peel, Chinese hawthorn, wolfberry, ginger and yuzu as an active ingredient. [Background technology]

[0002] Muscle atrophy can result from factors such as damage to muscle tissue due to mechanical stimuli such as reduced use of the muscle, destruction of muscle due to direct injury or physical factors, impaired ability of muscle cells to recover due to aging, and impaired use of muscle due to damage to the nerves that regulate muscle action (Booth FW., J Appl Physiol Respir Environ Exerc Physiol., 1982). In general, disuse atrophy occurs when muscle strength is lost due to long-term disuse of the muscles in the affected area and surrounding areas caused by injury or accident. It can also occur in the form of myasthenia gravis, progressive muscular dystrophy, myotonic muscular dystrophy, Dusen type, Becker type, limb-girdle type, facioscapulohumeral type, spinal muscular amyotrophy (Berardnig-Hoffmann type, Kugelberg disease, Belander disease), amyotrophic lateral sclerosis (ALS) (Lou Gehrig's disease), and sphinobular muscular atrophy (Kennedy disease), which are caused by muscle disease itself.

[0003] Today, based on the quality of life and lifestyle that has improved, people's concerns are increasingly focused on welfare and leisure compared to the past when people focused on food, clothing and shelter, and they are making efforts to live a healthy life through exercise. In order to achieve this lifestyle, the free control and use of voluntary muscles is a prerequisite, and when voluntary muscles cannot be used due to abnormalities in muscle function, this limits the range of activities of the person, resulting in a decline in the quality of life.

[0004] Generally, the best way to prevent the onset of muscle atrophy in daily life is to prevent muscle loss through continuous muscle use. However, muscle atrophy due to muscle degeneration can also occur when the person is unable to use the muscles regardless of their will, such as due to injury or disability. Therefore, even in such cases, countermeasures that can stimulate the muscles or prevent muscle degeneration and halt muscle atrophy, and research into such measures, are needed. Summary of the Invention [Problem to be solved by the invention]

[0005] The object of the present invention is to provide a composition for preventing or treating diseases associated with muscle loss, comprising a mixed extract of mulberry leaves, Chinese cornus, Chinese bellflower, Unshiu peel, Chinese hawthorn, wolfberry, ginger and yuzu as an active ingredient. [Means for solving the problem]

[0006] According to one embodiment of the present invention, there is provided a composition for preventing or treating diseases associated with muscle loss, comprising a mixed extract of mulberry leaves, Chinese quince, Chinese bellflower, Unshiu peel, Chinese quince, wolfberry, ginger and yuzu as an active ingredient.

[0007] The muscle loss-related disease may include, but is not limited to, one or more selected from the group consisting of muscular atrophy, muscular dystrophy, muscle degeneration, and sacopenia.

[0008] The mixed extract may contain, by total weight, 20-30 wt% of mulberry leaves, 10-20 wt% of Chinese quince, 10-20 wt% of Chinese bellflower, 5-10 wt% of dried citrus fruit peel, 5-10 wt% of Chinese quince, 5-10 wt% of Chinese wolfberry, 5-10 wt% of ginger, and 5-10 wt% of yuzu, but is not limited thereto.

[0009] The mixed extract may be, but is not limited to, a hot water extract.

[0010] The mixed extract may be extracted by an extraction method including, but not limited to, the steps of: 1) mixing mulberry leaves, Chinese quince, Chinese bellflower, dried citrus fruit, Chinese wolfberry, ginger, and yuzu with purified water and grinding the mixture; 2) adding pectinase and cellulase to the grinded mixture to cause an enzymatic reaction; 3) mixing purified water with the enzyme-treated grinded mixture and heating to extract an extract; 4) concentrating the extract; and 5) adding maltodextrin to the concentrate, drying, and powdering the concentrate.

[0011] After step 5), the method may further include a step of mixing powdered extracts of mulberry leaves, Chinese quince, Chinese bellflower, dried citrus fruit, Chinese wolfberry, ginger and yuzu, but is not limited thereto.

[0012] The extract powder may be mixed with 20-30 wt% of mulberry leaves, 10-20 wt% of Chinese quince, 10-20 wt% of Platycodon grandiflorum, 5-10 wt% of Citrus unshiu peel, 5-10 wt% of Chinese hawthorn, 5-10 wt% of Chinese wolfberry, 5-10 wt% of ginger, and 5-10 wt% of yuzu, but is not limited thereto.

[0013] The extract is characterized by inhibiting the production of reactive oxygen species, inhibiting the production of inflammatory cytokines IL-1β and IL-4, increasing the production of PGE2 (Prostaglandin E2) which acts as a defensive factor for the gastric mucosa, inhibiting the secretion of β-hexosaminidase and histamine, increasing the production of intracellular creatine kinase, increasing the expression of muscle differentiation proteins MHC and myogenin, increasing serum protein concentration, and increasing muscle protein concentration.

[0014] The mixed extract may be orally administered in an amount of 500 to 1000 mg / kg once a day, but is not limited thereto.

[0015] In yet another embodiment of the present invention, there is provided a health functional food for improving diseases associated with muscle loss, comprising a mixed extract of mulberry leaves, Chinese cornus, Chinese bellflower, dried citrus fruit peel, Chinese quince, wolfberry, ginger and yuzu as an active ingredient. Effect of the Invention

[0016] The mixed extract of mulberry leaves, Cornus fruit, Platycodon grandiflorum, Unshiu peel, Chinese laurel wort, wolfberry, ginger and yuzu of the present invention exhibits the characteristics of inhibiting the production of active oxygen, inhibiting the production of inflammatory cytokines IL-1β and IL-4, increasing the production of PGE2 (Prostaglandin E2) which acts as a defense factor for the gastric mucosa, inhibiting the secretion of β-hexosaminidase and histamine, increasing the production of intracellular creatine kinase, and increasing the expression of muscle differentiation proteins MHC and Myogenin, and exhibits the effect of increasing serum protein concentration and increasing muscle protein concentration in muscles, and therefore the mixed extract of mulberry leaves, Cornus fruit, Platycodon grandiflorum, Unshiu peel, Chinese laurel wort, wolfberry, ginger and yuzu of the present invention can be usefully used for the prevention or treatment of muscle loss-related diseases. [Brief description of the drawings]

[0017] [Figure 1] 1 is a graph showing a standard curve of α-amylase on substrate at 540 nm. [Diagram 2]1 is a graph showing the residual enzyme activity in an alpha-amylase activity assay of the mixed plant extract powder of the present invention. [Diagram 3] 1 is a graph showing a standard curve of lipase on substrate at 405 nm. [Figure 4] 1 is a graph showing the residual enzyme activity in a lipolytic activity assay of a mixed plant extract powder. [Diagram 5] 1 is a graph showing a standard curve of trypsin versus substrate at 405 nm. [Figure 6] 1 is a graph showing the residual enzyme activity in a proteolytic activity assay of a plant mixture extract powder. [Figure 7] 1 is a graph showing the effect of mixed plant extract powder on cell viability of animal cells. [Figure 8] 1 is a graph showing the effect of mixed plant extract powder on NO production in LPS-induced Raw264.7 cells. [Figure 9] FIG. 1 is a graph showing the effect of mixed plant extract powder on inflammatory cytokine production in LPS-induced Raw264.7 cells. [Figure 10] 1 is a graph showing the effect of mixed plant extract powder on IL-4 production in anti-DNPIgE-sensitized RBL-2H3 cells. [Figure 11] 1 is a graph showing the effect of mixed plant extract powder on PGE2 production in AGS cells. [Figure 12] 1 is a graph showing the effect of mixed plant extract powder on degranulation in anti-DNPIgE-sensitized RBL-2H3 cells. [Figure 13] 1 is a graph showing the effect of mixed plant extract powder on creatine kinase activity in C2C12 cells. [Figure 14] 1 is a graph showing the effect of the mixed plant extract powder on the expression of myogenin and MHC in C2C12 cells. [Figure 15]1 is a graph showing the effect of the mixed plant extract powder on mouse body weight. The left column shows the measurement results of the mouse body weight on each day for the control group, the center column shows the group that ingested 500 mg / kg of the mixed plant extract (experimental group 1), and the right column shows the group that ingested 1000 mg / kg of the mixed plant extract (experimental group 2). [Figure 16] 1 is a graph showing the effect of mixed plant extract powder on weight gain in mice. [Figure 17] 1 is a graph showing the effect of mixed plant extract powder on the weight gain rate of mice. [Figure 18] 1 is a graph showing the effect of the mixed plant extract on food intake, daily weight gain, and feed efficiency in mice. [Figure 19] 1 is a graph showing the effect of a mixed plant extract on food intake, daily weight gain, and feed efficiency in mice. [Figure 20] 1 is a graph showing the effect of a mixed plant extract on food intake, daily weight gain, and feed efficiency in mice. [Figure 21] 1 is a graph showing the effect of mixed plant extract powder on liver tissue weight, kidney weight, and spleen weight in mice. [Figure 22] This is also a graph showing the effect of the mixed plant extract powder on the liver tissue weight, kidney weight, and spleen weight of mice. [Diagram 23] This is also a graph showing the effect of the mixed plant extract powder on the liver tissue weight, kidney weight, and spleen weight of mice. [Figure 24] 1 is a graph showing the thigh muscle protein content of mice that were administered a mixed plant extract powder for 30 days. [Diagram 25] 1 is a graph showing the serum protein content of mice that were administered a mixed plant extract powder for 30 days. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] In this specification, when an element is said to be "located on" another element, this does not only include the case where one element is in contact with the other element, but also the case where another element is present between the two elements. When a part in this specification is said to "include" a certain element, this does not mean to exclude other elements, but means that it may further include other elements, unless specifically stated to the contrary.

[0019] The present invention will be described in detail below.

[0020] According to one embodiment of the present invention, there is provided a composition for preventing or treating diseases associated with muscle loss, comprising a mixed extract of mulberry leaves, Chinese quince, Chinese bellflower, Unshiu peel, Chinese quince, wolfberry, ginger and yuzu as an active ingredient.

[0021] The mulberry leaves referred to in this invention are the leaves of mulberry (Morus alba Linne) or mountain mulberry (Moraceae). They are also known as winter mulberry leaves. The leaves are egg-shaped or broad egg-shaped, some of which are split into 3 to 5 pieces, and are 8 to 15 cm long and 7 to 13 cm wide. The upper surface is yellowish green or light yellowish brown. The tip of the leaf is pointed, the lower part is heart-shaped, and the edge of the leaf is flat. The veins protrude from the lower surface, and the small veins are net-shaped, with hairs on top. The texture is easy to break and light. It has almost no odor and a light taste, but is slightly bitter and astringent.

[0022] The Citrus Unshiu Peel is the dried skin of the mature fruit of the Rutaceae family, Citrus unshiu Markovich. It is an aromatic stomachic medicine used for gastritis and indigestion, as well as for cough suppressant and expectorant.

[0023] The mixed extract may contain, by total weight, 20-30 wt% of mulberry leaves, 10-20 wt% of Chinese quince, 10-20 wt% of Chinese bellflower, 5-10 wt% of dried citrus fruit peel, 5-10 wt% of Chinese quince, 5-10 wt% of Chinese wolfberry, 5-10 wt% of ginger, and 5-10 wt% of yuzu, but is not limited thereto.

[0024] The plant mixed extract of the present invention exhibits the characteristic of increasing the production of intracellular creatine kinase and increasing the expression of muscle differentiation proteins MHC (myosin heavy chain) and myogenin, thereby increasing serum protein concentration and increasing muscle protein concentration in muscles, and therefore can be usefully used in the prevention or treatment of muscle loss-related diseases.

[0025] The muscle loss-related disease may be one or more selected from the group consisting of muscular atrophy, muscular dystrophy, muscle degeneration, and sacopenia, but is not limited thereto.

[0026] The muscle atrophy is preferably a muscle atrophy caused by loss of muscle tissue due to disuse of the muscle, a muscle atrophy caused by a disease of the muscle itself, or a muscle atrophy caused by damage to the nerves that control the muscle. More preferably, the muscle atrophy caused by loss of muscle tissue due to disuse of the muscle is disuse atrophy, the muscle atrophy caused by a disease of the muscle itself is myasthenia gravis or muscular dystrophy, and the muscle atrophy caused by damage to the nerves that control the muscle is spinal muscular amyotrophy, amyotrophic lateral sclerosis, or sphinobular muscular atrophy, but is not limited thereto.

[0027] The mixed extract may be prepared by extracting the extracts of each plant, concentrating and powdering them, and then mixing the powdered extracts in a predetermined ratio.

[0028] Specifically, the extract may be extracted by an extraction method including, but not limited to, the steps of: 1) mixing mulberry leaves, Chinese quince, Chinese bellflower, dried citrus fruit, Chinese hawthorn, wolfberry, ginger, and yuzu with purified water and grinding the mixture; 2) adding pectinase and cellulase to the grinding mixture to cause an enzymatic reaction; 3) mixing the enzyme-treated grinding mixture with purified water and heating to extract an extract; 4) concentrating the extract; and 5) adding maltodextrin to the concentrate, drying, and powdering the concentrate.

[0029] In the crushing step, each plant may be mixed with purified water in an amount of 50 to 100% by weight based on the total weight thereof and crushed using a crusher.

[0030] Next, pectinase and cellulase are added to the pulverized material to cause an enzymatic reaction.

[0031] The pectinase is an enzyme that hydrolyzes α-1,4-galacturonic acid bonds, which are the basic structure of pectin, a polymeric substance, to reduce the size of pectin, and the cellulase is an enzyme that hydrolyzes β-1,4-glucoside bonds of cellulose, a polymeric substance, to produce cellulose and glucose. 0.01 to 0.9 g of pectinase and 0.01 to 0.9 g of cellulase can be added per 1 kg of the pulverized material, but the amount is not limited thereto.

[0032] Pectinase and cellulase are added to the pulverized material and mixed, and then the mixture is subjected to an enzymatic reaction at 37 to 45° C., pH 4.5 to 6.5, and agitated at a rate of 50 ppm or more for 15 to 30 hours.

[0033] Thereafter, in order to terminate the enzyme reaction, the mixture is heated at 70 to 80°C for 1 to 2 hours to inactivate the enzyme.

[0034] In the extraction step after the enzyme reaction step, the enzyme-treated pulverized material is further mixed with purified water in an amount of 50-100% by weight based on the total weight of the pulverized material, and heated at a temperature of 80-95° C. for 6-7 hours to obtain an extract. The mixed plant extract of the present invention is a hot water extract.

[0035] After the extraction step, a concentration step can be performed. The extract is filtered through a filter and then concentrated using a vacuum concentrator to form a solid content of 20 to 25 wt%.

[0036] Then, maltodextrin is added to each of the concentrates in an amount of 20 to 50 wt % based on the total weight of the concentrates, and the mixture is stirred and dried using a spray drying (SD) to be powdered.

[0037] After the powdering step, the method may further include a step of mixing with extract powders of mulberry leaves, Chinese quince, Chinese bellflower, dried orange peel, Chinese wolfberry, ginger and yuzu, but is not limited thereto.

[0038] The extract powder may be mixed with 20-30 wt% of mulberry leaves, 10-20 wt% of Chinese quince, 10-20 wt% of Platycodon grandiflorum, 5-10 wt% of Citrus unshiu peel, 5-10 wt% of Chinese hawthorn, 5-10 wt% of Chinese wolfberry, 5-10 wt% of ginger, and 5-10 wt% of yuzu, but is not limited thereto.

[0039] The mixed plant extract powder mixed by the above extraction method exhibits the characteristics of increasing the production of intracellular creatine kinase and increasing the expression of muscle differentiation proteins MHC and Myogenin, and has the effect of increasing serum protein concentration and increasing muscle protein concentration in muscles. Therefore, the mixed extract of mulberry leaves, Chinese quince, Chinese bellflower, dried unripe citrus fruit, Chinese wolfberry, ginger and yuzu of the present invention can be usefully used for the prevention or treatment of muscle loss-related diseases.

[0040] The compositions of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

[0041] Each of the compositions can be formulated and used in the form of external preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, and sterile injection solutions by conventional methods, and preferably has the dosage form of creams, gels, patches, sprays, ointments, plasters, lotions, liniments, pastes, or cataplasms.

[0042] Examples of carriers, excipients and diluents that can be included in the composition containing the plant mixed extract powder as an active ingredient include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, amorphous cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoic acid, propylhydroxybenzoic acid, talc, magnesium stearate and mineral oil. When formulated, it is prepared using a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, surfactant, etc. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations are prepared by mixing the extract with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc.

[0043] In addition to simple excipients, lubricants such as magnesium stylate talc are also used. Oral liquid preparations include suspensions, tolerable liquids, milks, syrups, etc., and may contain various excipients such as wetting agents, sweeteners, flavorings, preservatives, etc. in addition to water and liquid paraffin, which are commonly used simple diluents.

[0044] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include witepsol, macrogol, Tween 61, cacao butter, laurin butter, and glycerogelatin.

[0045] The composition of the present invention can be formulated in various ways according to the administration route with the pharma- ceutically acceptable carrier by a method known in the art. As used herein, the term "pharma- ceutically acceptable" refers to a non-toxic composition that is physiologically acceptable and does not inhibit the action of an active ingredient when administered to a human, and does not generally cause gastrointestinal disorders, allergic reactions such as dizziness, or similar reactions.

[0046] In yet another embodiment of the present invention, there is provided a health functional food for improving diseases associated with muscle loss, comprising a mixed extract of mulberry leaves, Chinese cornus, Chinese bellflower, dried citrus fruit peel, Chinese quince, wolfberry, ginger and yuzu as an active ingredient.

[0047] The health functional foods of the present invention include, for example, folk remedies such as tea, jelly, juice, extract, and drink intended to improve organ function.

[0048] The health functional food of the present invention processed in such various forms has no side effects on the human body, is easy to take, and can be stored for a long period of time.

[0049] There is no particular limitation on the type of food. Examples of foods to which the plant mixed extract powder can be added include meat, sausage, bread, chocolate, candy, snacks, sweets, pizza, ramen, other noodles, gum, ice cream, ice cream, dairy products including milk, milk substitutes, cream, butter, buttermilk, yogurt, cheese, various soups, beverages, tea, energy drinks, alcoholic beverages, and vitamin complexes, including all health foods in the usual sense.

[0050] The health drink composition of the present invention may contain various flavorings or natural carbohydrates as additional ingredients, like ordinary drinks. The natural carbohydrates mentioned above are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As sweeteners, natural sweeteners such as thaumatin and stevia extract, and synthetic sweeteners such as saccharin and aspartame can be used.

[0051] The present invention will be described in detail below with reference to examples and experimental examples. However, the following examples and experimental examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples and experimental examples.

[0052] Example: Production of mixed plant extract powder The raw materials used in this plant mixture powder are mulberry leaves, Chinese quince, Chinese bellflower, dried citrus fruit peel, Chinese hawthorn, wolfberry, ginger and yuzu.

[0053] Each of the raw materials is mixed with purified water at a weight ratio of 100% and pulverized using a pulverizer. 0.1 g of pectinase and 0.2 g of cellulase are added per 1 kg of the pulverized material, mixed, and the enzyme reaction is carried out for 15 to 30 hours at 37 to 45°C, pH 4.5 to 6.5, and stirred at a speed of 50 ppm or more. Then, the enzyme is inactivated by heating at 70 to 80°C for 1 to 2 hours.

[0054] The enzyme-treated pulverized material is further mixed with purified water in an amount of 100% by weight based on the total weight of the pulverized material, and heated at a temperature of 80 to 95° C. for 6 to 7 hours to obtain an extract.

[0055] The extract is filtered through a filter and then concentrated using a vacuum concentrator to a solid content of 20 to 25 wt %.

[0056] Maltodextrin is added to the concentrate in an amount of 20 to 50 wt % based on the total weight of the concentrate, stirred, and then dried using SD.

[0057] The dried powders were mixed as shown in Table 1 below and diluted with distilled water (DW) before use.

[0058] [Table 1]

[0059] <Experimental Example> 1. Digestive enzyme activity analysis (1) α-Amylase glycolytic enzyme activity In order to analyze the digestive function-related sugar digestive enzyme activity of the prepared mixed plant extract powder, amylase (glycolytic enzyme)-like activity, which is an indicator of sugar digestion and absorption, was analyzed.

[0060] Various concentrations of samples were mixed with salivary α-amylase enzyme solution (5 unit / mL, in 50 mM potassium phosphate buffer) and reacted at room temperature for 5 minutes, then 1% starch, which was a substrate solution, was added and stirred, and reacted at room temperature for 5 minutes. Then, 3,5-dinitrosalicylic acid (DNS) solution was added and boiled at 100℃ for 5 minutes to develop color, then cooled, DW was added and stirred, and the activity was evaluated by measuring the absorbance at 550 nm using a microplate reader (Molecular Devices, Sunnyvale, CA, USA).

[0061] For comparative analysis, the negative control group was treated with vehicle and the positive control group was treated with α-amylase.

[0062] The experimental group was carried out using the plant mixed extract powder at concentrations of 10 μg, 20 μg, 50 μg, 100 μg, and 200 μg, and the OD540 absorbance of α-amylase at 0.5, 1, 2, 5, and 10 mU was used to create a standard curve (Figure 1).

[0063] The experimental group's mixed plant extract powder had absorbances of 0.17004±0.00367, 0.17318±0.00367, 0.17997±0.00540, 0.21063±0.00632, and 0.24227±0.00727 at 540 nm at concentrations of 10 μg, 20 μg, 50 μg, 100 μg, and 200 μg, respectively.

[0064] The absorbance of the negative control group, in which the vehicle was used instead of the sample, was 0.16557 ± 0.00, and α-amylase was detected at concentrations of 0.18389, 0.20415, 0.23928, 0.27075, and 0.29813 at 0.5-10 mU. The α-amylase-like activity of the plant mixed extract powder was confirmed using a standard curve utilizing absorbance confirmed at concentrations of 0.5-10 mU. The experimental group treated with 10 μg of plant mixed extract powder had an α-amylase-like activity of 0.111892 ± 0.099316 mU, the experimental group treated with 20 μg had an α-amylase-like activity of 0.196757 ± 140416 mU, the experimental group treated with 20 μg had an α-amylase-like activity of 0.38027 ± 0.145922 mU, the experimental group treated with 50 μg had an α-amylase-like activity of 1.208919 ± 0.170781 mU, and the experimental group treated with 200 μg had an α-amylase-like activity of 2.064054 ± 0.196435 mU (Figure 2).

[0065] In conclusion, 0.1-2.1mU of α-amylase-like activity was confirmed from 10-200μg of the plant mixed extract powder, and the α-amylase digestive enzyme activity increased in proportion to the concentration of the plant mixed extract powder. From this, it is expected that the plant mixed extract powder can be involved in the promotion of sugar digestion and sugar absorption-related functionality through its sugar decomposition enzyme-like activity.

[0066] (2) Lipase fat-decomposing enzyme activity In order to analyze the fat and lipid digestive enzyme activity related to digestive function of the prepared mixed plant extract powder, lipase-like activity was examined using a lipolytic activity assay.

[0067] Spectrophotometric assay was performed using p-nitrophenyl butyrate (PNPB) substrate (10 mM). Various concentrations of lipase treated with samples were added to the substrate solution at 10 mU and incubated at 60℃ for 15 min. p-nitrophenol (PNP) was measured at 405 nm for 10 min and analyzed by Vmax or absorbance. Measurement was performed using a microplate reader (Molecular Devices, Sunnyvale, CA, USA).

[0068] For comparative analysis, the negative control was vehicle, the positive control was lipase-treated, and the experimental groups were treated with plant extract powder at concentrations of 10 μg, 20 μg, 50 μg, 100 μg, and 200 μg. The OD405 absorbance of 0.5, 1, 2, 5, and 10 mU of lipase was used to generate a standard curve (Figure 3).

[0069] The experimental plant extract powder had absorbances of 0.01735±0.00087, 0.04639±0.00232, 0.05816±0.00291, 0.08443±0.00422, and 0.09021±0.00451 at 405 nm in the order of concentrations of 10μg, 20μg, 50μg, 100μg, and 200μg. The absorbance of the negative control group, which was treated with the vehicle instead of the sample, was 0.0±0.00, and lipase was confirmed at concentrations of 0.5 to 10mU, with values ​​of 0.06983, 0.08542, 0.11829, 0.17570, and 0.24756.

[0070] The lipase-like activity of the plant mixed extract powder was confirmed using a standard curve utilizing absorbance confirmed at concentrations of 0.5 to 10 mU. The results showed that the experimental group treated with 10 μg of plant mixed extract powder had a lipase-like activity of 0.0 ± 0.0 mU, the experimental group treated with 20 μg had a lipase-like activity of 0.334994 ± 0.033268 mU, the experimental group treated with 50 μg had a lipase-like activity of 0.885734 ± 0.041709 mU, the experimental group treated with 100 μg had a lipase-like activity of 2.114956 ± 0.060549 mU, and the experimental group treated with 200 μg had a lipase-like activity of 2.385413 ± 0.064694 mU (Figure 4).

[0071] In conclusion, 0.3-2.4mU of lipase-like activity was confirmed from 10-200μg of plant mixed extract powder, and lipase digestive enzyme activity increased in proportion to the concentration of plant mixed extract powder. From this, it is expected that the plant mixed extract powder may be involved in the promotion of fat digestion and fat absorption-related functions through its lipolytic enzyme-like activity.

[0072] (3) Protease activity In order to analyze the digestive function-related protein digestive enzyme activity of the prepared mixed plant extract powder, trypsin (protease)-like activity was examined using a trypsin-specific substrate.

[0073] A spectrophotometric assay was performed using a specific trypsin substrate (1-10 mM). 1 mU of trypsin treated with various concentrations of samples was added to the substrate solution and incubated at 60°C for 15 minutes, after which the absorbance was analyzed by measuring p-nitrophenol (PNP) at 405 nm for 10 minutes. Measurements were performed using a microplate reader (Molecular Devices, Sunnyvale, CA, USA).

[0074] For comparative analysis, the negative control group was treated with vehicle, and the positive control group was treated with trypsin. The experimental groups were treated with the plant mixed extract powder at concentrations of 10 μg, 20 μg, 50 μg, 100 μg, and 200 μg. The OD405 absorbance of 0.5, 1, 2, 5, and 10 mU of trypsin was used to create a standard curve (Figure 5).

[0075] The experimental plant extract powder had absorbances of 0.01663±0.00083, 0.02169±0.00108, 0.04428±0.00221, 0.07372±0.00369, and 0.11075±0.00554 at 405 nm in the order of concentrations of 10μg, 20μg, 50μg, 100μg, and 200μg. The negative control group, which was treated with the vehicle instead of the sample, had an absorbance of 0.0±0.00, and trypsin was confirmed to have absorbances of 0.04097, 0.05524, 0.07319, 0.10575, and 0.12694 at concentrations of 0.5-10mU. The trypsin-like activity of the plant mixed extract powder was confirmed using a standard curve using the absorbance confirmed at concentrations of 0.5-10 mU of trypsin. The experimental group treated with 10 μg of plant mixed extract powder had a trypsin-like activity of 0.111674 ± 0.007516 mU, the experimental group treated with 20 μg had a trypsin-like activity of 0.260879 ± 0.009802 mU, the experimental group treated with 20 μg had a trypsin-like activity of 0.926994 ± 0.020011 mU, the experimental group treated with 50 μg had a trypsin-like activity of 1.795097 ± 0.033316 mU, and the experimental group treated with 200 μg had a trypsin-like activity of 2.887007 ± 0.050051 mU (Figure 6).

[0076] In conclusion, trypsin-like activity of 0.1-2.9mU was confirmed from 10-200μg of plant mixed extract powder, and trypsin digestive enzyme activity increased in proportion to the concentration of plant mixed extract powder. This suggests that the protease-like activity of plant mixed extract powder may be involved in the promotion of protein digestion and protein absorption-related functions.

[0077] 2. Activity analysis using animal cells (1) Cell viability analysis To examine the cytotoxicity of each extract, an MTT assay was performed using animal cell lines. Each cell line was cultured in a 96-well plate and treated with the extract at concentrations of 0 to 1000 μg / ml for 24 hours, and the change in cell viability was observed (Figure 7).

[0078] Referring to Figure 7, the concentrations were 100, 102, 99, 100, 100, 99, 99% in RAW264.7 cells, 101, 98, 102, 92, 102, 103, 94% in RBL-2H3 cells, 100, 102, 105, 103, 101, 103, 100% in AGS cells, and 99, 104, 101, 103, 100, 109, 113% in C2C12 cells. Therefore, the following experiments were performed in a non-toxic concentration range.

[0079] (2) Measurement of NO production amount NO is an inflammatory mediator that plays a role in eliminating bacteria and tumors, but it is also involved in almost all inflammatory responses and can induce disease.

[0080] The amount of NO produced in cultured RAW264.7 cells after treatment with LPS and a mixed plant extract powder was analyzed using Griess reagent. The effect on NO production was measured, and the inhibitory effect was greater with increasing extract concentration. Treatment with 1ug / ml LPS alone showed 89μM, while pretreatment with the extract at 300, 400, and 500ug / ml and then treatment with LPS showed 81, 62, and 43μM, respectively, confirming the inhibitory effect of the extract on NO production (Figure 8). As it inhibits NO production induced by LPS, it is believed to have an anti-inflammatory effect through the inhibition of such inflammatory mediators.

[0081] (3) Cytokine measurement Cytokines are factors that regulate the activity, proliferation, and differentiation of immune cells and mediate inflammatory responses. Macrophages secrete cytokines to mediate various inflammatory responses and suppress the production of NO and PGE. 2 To confirm the inhibitory effect on the production of IL-1β and TNF-α secreted by RAW264.7 cells, ELISA was used (Figure 9).

[0082] In the case of IL-1β, the level was 321pg / ml when treated with LPS 1ug / ml alone, whereas the levels were 294, 201, and 187pg / ml when treated with LPS after pretreatment with plant extract powder at 300, 400, and 500ug / ml, confirming the inhibitory effect of plant extract powder on IL-1β production. In the case of TNF-α, the level was 639pg / ml when treated with LPS 1ug / ml alone, whereas the levels were 510, 384, and 296pg / ml when treated with LPS after pretreatment with plant extract powder at 300, 400, and 500ug / ml, confirming the inhibitory effect of the extract on TNF-α production. ELISA was used to confirm the inhibitory effect on the production of IL-4 secreted by RBL-2H3 cells (Figure 10). In the case of IL-4, when treated with anti-DNP-IgE alone, the level was 151pg / ml, whereas when treated with 400, 500, and 600ug / ml of mixed plant extract powder, the levels were 125, 103, and 64pg / ml, respectively, confirming the inhibitory effect of mixed plant extract powder on IL-4 production. It is believed that the anti-inflammatory effect is exerted by suppressing the secretion of inflammatory cytokines.

[0083] (4) Measurement of prostaglandin E2 As for protective factors against stomach damage, we confirmed the effect on the production of PGE2, which is produced in the arachidonic acid cascade and acts as a defensive factor for the gastric mucosa. AGS cells were treated with the mixed plant extract powder, and the amount of PGE2 secreted was quantified. When treated with concentrations of 0, 300, 400, and 500 μg / ml, PGE2 production increased at 83, 109, and 154 pg / ml (Figure 11). This result confirmed that the increase was concentration-dependent. It was suggested that the extract exerts a protective effect on the stomach by enhancing gastric mucosa defensive factors.

[0084] (5) β-hexosaminidase and histamine secretion inhibitory activity The effect of each extract on β-hexosaminidase secretion was measured using RBL-2H3 cells. DNP-IgE was administered to induce secretion. In the group treated with DNP-IgE alone, secretion increased by 218%, whereas secretion was inhibited by 185, 173, and 149% when the extract was administered at concentrations of 400, 500, and 600 μg / ml (Figure 12A). The effect of the extract on histamine secretion was measured using RBL-2H3 cells. DNP-IgE was administered to induce secretion. In the group treated with DNP-IgE alone, secretion increased by 75 pg / ml, whereas secretion was inhibited by 69, 61, and 39 pg / ml when the extract was administered at concentrations of 400, 500, and 600 μg / ml (Figure 12B). It was suggested that the cell protective effect can be exhibited through the inhibition of β-hexosaminidase and histamine.

[0085] (6) Creatine kinase activity Creatine kinase is an important enzyme that is responsible for supplying ATP through the decomposition of creatine in various cells, especially muscle cells. The present inventors treated C2C12 cells with the plant mixed extract powder of the present invention to confirm the degree of differentiation by checking creatine kinase, an enzyme related to muscle growth. When treated with 0, 400, 500, and 600 μg / ml concentrations, the intracellular creatine kinase was 22, 34, 49, and 60 IU / I / mg protein, and the production of intracellular creatine kinase increased (FIG. 13). This suggests that the mixed extract induces muscle growth.

[0086] (7) Expression of genes related to muscle cell differentiation To confirm the regulatory effect of the extract on muscle differentiation in C2C12 cells, the protein expression of muscle differentiation markers myogenin and MHC was examined using Western blot (Figure 14). Myogenin expression increased in a concentration-dependent manner after treatment with the extract compared to cells not treated with the extract. MHC expression also increased in a concentration-dependent manner with treatment with the extract. This suggests that the extract can promote muscle differentiation by increasing the expression of muscle differentiation proteins MHC and myogenin.

[0087] 3. Analysis of weight regulation (gain) effects using experimental animals (1) Analysis of weight regulation (gain) effects In order to analyze the effects of the mixed plant extract powder on weight control (weight gain) and muscle loss improvement, the mixed plant extract powder was orally administered at 500mg / kg and 1000mg / kg daily for 30 days.

[0088] In this experiment, 30 8-week-old male white ICR mice were purchased and used, and the experiment was carried out in three groups through a control group and experimental group design. The control group was given only normal food and saline instead of the sample for comparative analysis. Experimental group 1 was orally administered the plant mixed extract powder at 500mg / kg / day, and experimental group 2 was orally administered the plant mixed extract powder at 1000mg / kg / day.

[0089] Body weight was measured at the same time until the end of the diet, and the average body weight, body weight gain rate, and average daily gain were analyzed. Food intake was measured at the same time until the end of the diet, and the average daily intake was analyzed. Based on the body weight and intake measured until the end of the diet, the feed efficiency (G / F) = average daily gain (ADG) / average daily intake (ADFI) was analyzed.

[0090] The weight of the experimental animals was measured in the animal breeding room for 30 days after the 4-week adaptation period. The control group 1, which was fed only normal food and saline, had an average weight of 31.5±1.0g at the beginning, 34.7±0.6g after 5 days, 34.1±0.6g after 10 days, 34.3±0.0g after 15 days, 36.2±0.0g after 20 days, 36.0±0.6g after 25 days, and 36.1±0.6g after 30 days, resulting in an average weight gain of 4.6g.

[0091] Experimental group 1, which received the mixed plant extract powder at a concentration of 500 mg / kg, had an initial average body weight of 31.3 ± 1.0 g, 5 days later it was 35.0 ± 1.0 g, 10 days later it was 34.3 ± 0.6 g, 15 days later it was 34.7 ± 0.6 g, 20 days later it was 36.7 ± 1.5 g, 25 days later it was 36.0 ± 1.0 g, and 30 days later it was 36.3 ± 0.6 g, resulting in a final average weight gain of 5.0 g.

[0092] In experimental group 2, which received the mixed plant extract powder at a concentration of 1000 mg / kg, the average body weight was 32.0 ± 1.0 g at first, 36.7 ± 0.6 g after 5 days, 36.0 ± 1.0 g after 10 days, 35.3 ± 0.6 g after 15 days, 38.0 ± 1.0 g after 20 days, 38.0 ± 1.0 g after 25 days, and 38.3 ± 2.3 g after 30 days, resulting in an average weight gain of 6.3 g (Figures 15 and 16).

[0093] The weight gain was analyzed based on the average weight before and after feeding and the sample. In control group 1, which was fed only normal food and saline, the weight gain after feeding was 4.6g compared to the average weight before feeding. In experimental group 1, which was fed plant mixed extract powder at a concentration of 500mg / kg, the weight gain after feeding the sample was 5.0g, and in experimental group 2, which was fed plant mixed extract powder at a concentration of 1000mg / kg, the weight gain after feeding the sample was 6.3g. The weight gain rate calculated from this was confirmed to be 14.6%, 16.0%, and 19.7% for control group 1, experimental group 1, and experimental group 2, respectively (Figure 17). From these results, it was confirmed that the weight gain effect of the experimental group was higher than that of the control group, and it was analyzed that the weight gain rate of experimental group 1 was about 1.4% higher than that of the control group, and that of experimental group 2 was about 5.1% higher.

[0094] The daily average intake of control group 1, which was given only normal food and saline, was measured to be 8.47±0.34g. The daily average intake of experimental group 1, which received the plant extract powder at a concentration of 500mg / kg, was measured to be 8.31±0.33g, and the daily average intake of experimental group 2, which received the plant extract powder at a concentration of 1000mg / kg, was measured to be 8.09±0.32g (Figure 18).

[0095] The average daily gain of control group 1, which was fed only normal food and saline, was measured to be 0.16±0.01g. The average daily gain of experimental group 1, which was fed 500mg / kg of mixed plant extract powder, was measured to be 0.17±0.01g, and the average daily gain of experimental group 2, which was fed 1000mg / kg of mixed plant extract powder, was measured to be 0.21±0.01g (Figure 19).

[0096] Feed efficiency was calculated based on the average daily weight gain and average daily intake for the control group and each experimental group. The feed efficiency of control group 1, which was fed only normal feed and saline, was measured as 0.552 ± 0.051. The feed efficiency of experimental group 1, which was fed 500 mg / kg of mixed plant extract powder, was measured as 0.601 ± 0.030, and the feed efficiency of experimental group 2, which was fed 1000 mg / kg of mixed plant extract powder, was measured as 0.783 ± 0.039 (Figure 20).

[0097] As a result, the feed efficiency of the experimental group was higher than that of the control group, and the feed efficiency increased in proportion to the concentration in the experimental group. This is because although the total feed amount in the experimental group was lower than that in the control group, the average weight gain rate was higher, and a relatively higher feed efficiency value was calculated than in the control group.

[0098] After the feeding, the weights of the excised tissues were measured and analyzed for comparison. The weights of the liver tissue were 2.68±0.04g in the control group, 2.49±0.08g in experimental group 1, which received 500mg / kg of the plant mixture extract powder, and 2.51±0.08g in experimental group 2, which received 1000mg / kg of the plant mixture extract powder (Figure 21). The weights of the kidneys were 0.38±0.02g in the control group, 0.39±0.02g in experimental group 1, and 0.34±0.03g in experimental group 2 (Figure 22). The weights of the spleen were 0.14±0.02g in the control group, 0.11±0.02g in experimental group 1, and 0.12±0.01g in experimental group 2 (Figure 23).

[0099] The weights of the liver and spleen increased in proportion to the concentration of the mixed plant extract powder, but were slightly lower than the control group. The weight of the kidneys was slightly lower or at the same level in the experimental group compared to the control group.

[0100] (2) Analysis of muscle protein content Muscle protein concentrations in the thigh muscles of experimental animals were measured and analyzed at the end of the experiment. Thigh muscle tissues were excised from mice, crushed, and dissolved in lysis buffer. Protein was quantified using a BCA kit and measured using a microplate reader (Molecular Devices, Sunnyvale, CA, USA).

[0101] At the end of the experiment, the average thigh muscle protein concentration of the control group (general diet) was measured to be 1.082 ± 0.087 μg / mg, the average thigh muscle protein concentration of experimental group 1, which consumed 500 mg / kg of plant mixed extract powder, was 1.135 ± 0.091 μg / mg, and the average thigh muscle protein concentration of experimental group 2, which consumed 1000 mg / kg of plant mixed extract powder, was 1.141 ± 0.091 μg / mg (Figure 24).

[0102] In addition, after the diet was completed, the serum protein concentration in the blood was measured and analyzed for comparison. The serum protein concentration in the control group was 5.241±0.419μg / mg, that in experimental group 1 was 5.262±0.368μg / mg, and that in experimental group 2 was 5.265±0.474μg / mg (Figure 25).

[0103] These results suggest that intake of the mixed plant extract powder slightly increased or maintained serum protein concentration, while increasing muscle protein concentration in thigh muscles from 1.082μg / mg to 1.135-1.141μg / mg, confirming its potential as a material that can improve muscle loss by increasing muscle protein concentration in a concentration-proportional manner.

Claims

1. A composition for preventing or treating diseases associated with muscle loss, comprising a mixed extract of mulberry leaves, Chinese cornus, Chinese bellflower, dried citrus fruit, Chinese hawthorn, wolfberry, ginger and yuzu as an active ingredient.

2. The composition according to claim 1, wherein the muscle loss-related disease is one or more selected from the group consisting of muscular atrophy, muscular dystrophy, muscle degeneration and sacopenia.

3. The composition according to claim 1, wherein the mixed extract comprises, by total weight, 20-30 wt% of mulberry leaf, 10-20 wt% of Chinese quince, 10-20 wt% of Platycodon grandiflorum, 5-10 wt% of Citrus unshiu peel, 5-10 wt% of Chinese quince, 5-10 wt% of Chinese wolfberry, 5-10 wt% of ginger, and 5-10 wt% of yuzu.

4. The composition of claim 1, wherein the mixed extract is a hot water extract.

5. The mixed extract comprises: 1) mixing mulberry leaves, Chinese quince, Chinese bellflower, dried citrus fruit, Chinese hawthorn, Chinese wolfberry, ginger and yuzu with purified water and grinding the mixture; 2) adding pectinase and cellulase to the pulverized material to cause an enzymatic reaction; 3) mixing the enzyme-treated ground material with purified water and heating to extract an extract; 4) concentrating the extract; 5) adding maltodextrin to the concentrate, drying and powdering the concentrate.

6. The composition according to claim 5, further comprising the step of mixing, after step 5), extract powders of mulberry leaves, Chinese quince, Chinese bellflower, dried citrus fruit, Chinese wolfberry, ginger and yuzu.

7. The composition according to claim 6, characterized in that it is a mixture of 20-30 wt% mulberry leaves, 10-20 wt% Chinese quince, 10-20 wt% Platycodon grandiflorum, 5-10 wt% Citrus unshiu peel, 5-10 wt% Hariguwa, 5-10 wt% Wolfberry, 5-10 wt% ginger and 5-10 wt% Yuzu.

8. The composition described in claim 1, characterized in that the extract inhibits the production of reactive oxygen species, inhibits the production of inflammatory cytokines IL-1β and IL-4, increases the production of PGE2 (prostaglandin E2) which acts as a defensive factor for the gastric mucosa, inhibits the secretion of beta-hexosaminidase and histamine, increases the production of intracellular creatine kinase, increases the expression of muscle differentiation proteins MHC and myogenin, increases serum protein concentration, and increases muscle protein concentration in muscles.

9. The composition according to claim 1, wherein the mixed extract is orally administered at 500-1000 mg / kg once a day.

10. A health functional food for improving muscle loss-related diseases, containing a mixture of extracts of mulberry leaves, Chinese quince, Chinese bellflower, dried tangerine peel, Chinese hawthorn, wolfberry, ginger and yuzu as active ingredients.

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