Nutritional shake composition containing senior-friendly ingredients

Abstract

The present invention relates to a nutritional shake composition containing an age-friendly ingredient that includes a plant mixed extract powder pretreated with various types of plant powders, which actively participates in protein metabolism and thereby increases muscle production, thereby preventing diseases related to muscle loss.

Description

Technology Field

[0001] The present invention relates to a nutritional shake composition containing an age-friendly ingredient comprising a plant mixed extract powder pretreated with various types of plant powders. Background Technology

[0003] As human life expectancy increases and the population ages, interest in the health of the elderly is growing.

[0004] Accordingly, age-friendly foods such as care food, silver food, and senior food are being developed to prevent diseases caused by food intake or nutritional deficiencies among the elderly.

[0005] In particular, as the incidence of patients with muscle loss among the elderly increases, the importance of preventing it is gradually increasing, and accordingly, shake foods to prevent the aforementioned muscle loss disease are gradually being developed.

[0006] However, most shakes currently on the market intended to prevent muscle loss are manufactured as products containing large amounts of protein or simply mixed with various types of protein. Consequently, they merely provide nutrients for muscle growth and have no substantial effect on diseases related to muscle loss. Therefore, there is currently a demand for shakes that can increase muscle growth by actively participating in protein metabolism. Prior art literature

[0008] Republic of Korea Registered Patent Publication No. 10-2396152 The problem to be solved

[0009] The purpose of the present invention is to manufacture a nutritional shake composition containing an age-friendly ingredient comprising a plant mixed extract powder in which various types of plant powders have been pretreated.

[0010] The objects of the present invention are not limited to those mentioned above, and other unmentioned objects and advantages of the present invention may be understood from the following description and will be more clearly understood by the embodiments of the present invention. Furthermore, it will be readily apparent that the objects and advantages of the present invention can be realized by the means and combinations thereof set forth in the claims. means of solving the problem

[0012] A nutritional shake composition containing an elderly-friendly ingredient according to one embodiment of the present invention contains a main ingredient comprising isolated soy protein, isolated whey protein, and goat milk protein, and an auxiliary ingredient comprising a plant mixed extract powder as an active ingredient.

[0013] The above main ingredient powder contains isolated soy protein, isolated whey protein, goat milk protein, vitamin B1, niacin, pantothenic acid, vitamin B6, biotin, vitamin D, beta-glucan, vitamin K, zinc, calcium, and magnesium.

[0014] The above main ingredient powder comprises 52.5 to 60.5 wt% isolated soy protein, 27.4 to 35.4 wt% isolated whey protein, 3.8 to 11.8 wt% goat milk protein, 0.002 to 0.011 wt% vitamin B1, 0.01 to 0.07 wt% niacin, 0.01 to 0.05 wt% pantothenic acid, 0.005 to 0.013 wt% vitamin B6, 0.01 to 0.03 wt% biotin, 0.02 to 0.1 wt% vitamin D, 0.02 to 0.1 wt% beta-glucan, 0.001 to 0.008 wt% vitamin K, 0.014 to 0.022 wt% zinc, 1.1 to 5.1 wt% calcium, and 0.3 to 1.1 wt% magnesium. Includes.

[0015] The above auxiliary ingredient powder includes roasted brown rice powder, vitamin A, vitamin B2, vitamin C, potassium, oat dietary fiber, plant mixed extract powder, MSM, N-acetylglucosamine, fructooligosaccharide, similar cell powder, and amino acid mixed powder.

[0016] The above auxiliary ingredient powder contains 0.001 to 0.009 wt% of vitamin A, 0.007 to 0.016 wt% of vitamin B2, 0.01 to 0.09 wt% of vitamin C, 1.5 to 2.3 wt% of potassium, 9.7 to 17.7 wt% of oat dietary fiber, 42.7 to 50.7 wt% of roasted brown rice powder, 4.8 to 12.8 wt% of plant mixed extract powder, 4.2 to 12.2 wt% of MSM, 1.7 to 3.7 wt% of N-acetylglucosamine, 12.5 to 20.5 wt% of fructooligosaccharide, 0.1 to 0.9 wt% of lactic acid bacteria dead cell powder, and 0.1 to 0.9 wt% of amino acid mixed powder.

[0017] The above plant mixed extract powder includes a mixed extract of mulberry leaves, Cornus fruit, balloon flower root, dried tangerine peel, Cudrania tricuspidata, goji berries, ginger, and yuzu.

[0018] The above plant mixed extract powder contains 20 to 30 weight% of mulberry leaves, 10 to 20 weight% of Cornus fruit, 10 to 20 weight% of balloon flower root, 5 to 10 weight% of dried tangerine peel, 5 to 10 weight% of Cudrania tricuspidata, 5 to 10 weight% of goji berries, 5 to 10 weight% of ginger, and 5 to 10 weight% of citron, based on the total weight.

[0019] The above plant mixed extract comprises: 1) a step of mixing purified water with mulberry leaves, Cornus fruit, balloon flower root, dried tangerine peel, Cudrania tricuspidata, goji berries, ginger, and yuzu, respectively, and grinding them; 2) a step of adding an enzyme to the ground material and causing an enzymatic reaction; 3) a step of mixing purified water with the enzyme-treated ground material and heating it to extract an extract; 4) a step of concentrating the extract to prepare a concentrate; and 5) a step of adding dietary fiber to the concentrate and then drying it to powder.

[0020] The enzymes mentioned above are pectinase and cellulase.

[0021] The above dietary fiber is maltodextrin.

[0022] After the above step 5), the method further includes a step of mixing extract powders of mulberry leaves, Cornus fruit, balloon flower, dried tangerine peel, Cudrania tricuspidata, goji berries, ginger, and citron. Effects of the invention

[0024] The present invention includes a plant mixed extract powder pretreated with various types of plant powders, which actively participates in protein metabolism and thereby increases muscle production, which can prevent diseases related to muscle loss.

[0025] In addition to the effects described above, the specific effects of the present invention are described together with the specific details for implementing the invention below. Brief explanation of the drawing

[0027] Figure 1 is a graph showing the survival rate of C2C12 muscle cells according to treatment with H2O2. Figure 2a is a graph showing the viability of C2C12 muscle cells according to MTT assay analysis. Figure 2b is a graph showing the viability of C2C12 muscle cells according to LDH assay analysis. Figure 3 is a graph showing CK activity. Figure 4a is a graph showing Bax expression. Figure 4b is a graph showing Bcl-2 expression. Figure 4c is a graph showing caspase-9 expression. Figure 4d is a graph showing caspase-3 expression. Figure 5a is a graph showing the survival rate of C2C12 cells. Figure 5b is a graph showing the viability of RAW 267.7 cells. Figure 5c is a graph showing the viability of AGS cells. Figure 6a is a graph showing the viability of muscle cells treated with the prototype and H2O2 according to the MTT assay. Figure 6b is a graph showing the viability of muscle cells treated with the prototype and H2O2 according to the LDH assay. Figure 7 is a graph showing the CK activity of muscle cells treated with the prototype and H2O2. Figure 8a is a graph showing the amount of NO in LPS-treated C2C12 cell culture medium. Figure 8b is a graph showing the amount of PGE2 in LPS-treated C2C12 cell culture medium. Figure 9a is a graph showing the amount of IL-6 in LPS-treated C2C12 cell culture medium. Figure 9b is a graph showing the amount of TNF-α in LPS-treated C2C12 cell culture medium. Figure 10 is a graph showing α-Amylase enzyme activity according to the amount of plant mixed extract powder. Figure 11 is a graph showing α-glucosidase enzyme activity according to the amount of plant mixed extract powder. Figure 12 is a graph showing lipase enzyme activity according to the amount of plant mixed extract powder. Figure 13 is a graph showing digestive enzyme activity according to the amount of plant mixed extract powder. Specific details for implementing the invention

[0028] The aforementioned objectives, features, and advantages are described in detail below with reference to the attached drawings, thereby enabling those skilled in the art to easily implement the technical concept of the present invention. In describing the present invention, detailed descriptions of known technologies related to the present invention are omitted if it is determined that such descriptions would unnecessarily obscure the essence of the invention. Hereinafter, preferred embodiments according to the present invention will be described in detail with reference to the attached drawings. In the drawings, the same reference numerals are used to indicate the same or similar components.

[0029] In this specification, terms such as "first," "second," etc. are used to describe various components, but these components are not limited by these terms. These terms are used merely to distinguish one component from another, and unless specifically stated otherwise, the first component may be the second component.

[0030] Additionally, in this specification, the statement that any configuration is disposed on the "upper (or lower)" or "upper (or lower)" of a component may mean not only that any configuration is disposed in contact with the upper (or lower) surface of said component, but also that another configuration may be interposed between said component and any configuration disposed on (or below) said component.

[0031] Furthermore, where it is stated in this specification that one component is "connected," "coupled," or "connected" to another component, it should be understood that while the components may be directly connected or connected to each other, another component may be "interposed" between each component, or each component may be "connected," "coupled," or "connected" through another component.

[0032] Additionally, singular expressions used in this specification include plural expressions unless the context clearly indicates otherwise. In this application, terms such as "composed of" or "comprising" should not be interpreted as necessarily including all of the various components or steps described in the specification, and should be interpreted as meaning that some of the components or steps may not be included, or that additional components or steps may be included.

[0033] Additionally, in this specification, "A and / or B" means A, B, or A and B unless specifically stated otherwise, and "C to D" means C or more and D or less unless specifically stated otherwise.

[0035] A nutritional shake composition containing an elderly-friendly ingredient according to one embodiment of the present invention may include a main ingredient and an auxiliary ingredient powder containing an elderly-friendly ingredient, and may be manufactured by mixing the main ingredient powder and the auxiliary ingredient powder.

[0036] The main ingredient powder may include isolated soy protein, isolated whey protein, goat milk protein, vitamin B1, niacin, pantothenic acid, vitamin B6, biotin, vitamin D, beta-glucan, vitamin K, zinc, calcium, and magnesium.

[0037] The essential components of the above-mentioned main ingredient powder, such as isolated soy protein, isolated whey protein, and goat milk protein, are components of body tissues such as muscles for muscle maintenance, as well as enzymes, hormones, and antibodies, and can transport and store various components through the protein metabolism of vitamin B6, and calcium and magnesium can maintain and activate muscle function through the contraction and relaxation of muscles.

[0038] In this case, calcium, like vitamin K, is closely related to bone health, and since its smooth absorption into the body is facilitated by vitamin D, it can aid in the formation and maintenance of bones, thereby reducing the risk of developing osteoporosis.

[0039] The remaining components, vitamin B1, niacin, pantothenic acid, biotin, beta-glucan, and zinc, can generate energy in the body and promote the metabolism of fats, carbohydrates, and proteins, thereby enhancing immune function.

[0040] Meanwhile, in order to ensure adequate absorption into the body through the optimized content of each ingredient, the main ingredient powder contains 52.5 ~ 60.5 wt% isolated soy protein, 27.4 ~ 35.4 wt% isolated whey protein, 3.8 ~ 11.8 wt% goat milk protein, 0.002 ~ 0.011 wt% vitamin B1, 0.01 ~ 0.07 wt% niacin, 0.01 ~ 0.05 wt% pantothenic acid, 0.005 ~ 0.013 wt% vitamin B6, 0.01 ~ 0.03 wt% biotin, 0.02 ~ 0.1 wt% vitamin D, 0.02 ~ 0.1 wt% beta-glucan, 0.001 ~ 0.008 wt% vitamin K, 0.014 ~ 0.022 wt% zinc, and 1.1 ~ 5.1 wt% calcium. It may contain 0.3 to 1.1 weight% of magnesium. However, the content of the main raw material powder is not limited to what is described above.

[0041] The auxiliary ingredient powder may include roasted brown rice powder, vitamin A, vitamin B2, vitamin C, potassium, oat dietary fiber, plant mixed extract powder, MSM, N-acetylglucosamine, fructooligosaccharide, similar cell powder, and amino acid mixed powder.

[0042] Vitamins A and B2 are involved in the immune system and can improve immunity in the elderly, Vitamin C promotes antioxidant activity in the body to protect cells from harmful free radicals, and potassium can maintain the balance of fluids and electrolytes in the body.

[0043] MSM and N-acetylglucosamine, like the calcium and vitamin K in the main ingredient powder, are closely related to bone health and can promote joint health by aiding in the formation and maintenance of bones.

[0044] Oat dietary fiber, fructooligosaccharides, and similar cell powder can adsorb and eliminate various waste products, cholesterol, and triglycerides from the intestines, thereby promoting intestinal health and improving digestion. Roasted brown rice powder and amino acid mixture powder help with muscle recovery and growth, and can promote energy production in the body.

[0045] As explained earlier regarding the main ingredient powder, meanwhile, in order to ensure adequate absorption into the body through the optimized content of each component, the auxiliary ingredient powder comprises Vitamin A 0.001–0.009 wt%, Vitamin B2 0.007–0.016 wt%, Vitamin C 0.01–0.09 wt%, Potassium 1.5–2.3 wt%, Oat dietary fiber 9.7–17.7 wt%, Roasted brown rice powder 42.7–50.7 wt%, Plant mixed extract powder 4.8–12.8 wt%, MSM 4.2–12.2 wt%, N-acetylglucosamine 1.7–3.7 wt%, Fructooligosaccharide 12.5–20.5 wt%, Lactobacillus dead cell powder 0.1–0.9 wt%, and Amino acid mixed powder 0.1–0.9 wt%. It may contain weight %. However, the content of the main raw material powder is not limited to what is described above.

[0047] Previously, a nutritional shake composition containing an elderly-friendly ingredient according to one embodiment of the present invention was described. Next, a method for manufacturing a plant mixed extract powder included in the auxiliary ingredients will be described in detail.

[0048] Plant mixed extract powder can be prepared by extracting each extract, concentrating it into a powder, and then mixing the powdered extracts in a predetermined ratio.

[0049] Specifically, regarding the manufacturing steps of the plant mixed extract powder, the manufacturing steps may include: 1) mixing purified water with mulberry leaves, Cornus fruit, balloon flower root, dried tangerine peel, Cudrania tricuspidata, goji berries, ginger, and citron, respectively, and grinding them; 2) adding enzymes (pectinase and cellulase) to the ground material to cause an enzymatic reaction; 3) mixing purified water with the enzyme-treated ground material and heating it to extract; 4) concentrating the extract; and 5) adding dietary fiber to the concentrate and then drying it to form a powder.

[0050] 1) In step 1, 50 to 100 weight percent of purified water relative to the total weight of each plant is mixed, and then ground using a grinder.

[0051] 2) In step 2), the enzymes may be pectinase and cellulase. Pectinase may be an enzyme that reduces the size of pectin by hydrolyzing the α-1,4-galacturonic acid bonds, which are the basic structure of pectin, a high-molecular-weight substance, and cellulase may be an enzyme that produces cellulose and glucose by hydrolyzing the β-1,4-glucoside bonds of cellulose, a high-molecular-weight substance. The amount of enzyme added may be 0.01 to 0.9 g of pectinase and 0.01 to 0.9 g of cellulase per 1 kg of ground material, but is not limited thereto.

[0052] In step 2), after adding and mixing Peltinase and cellulase to the ground material, 37 to 45 O The enzyme reaction can be carried out for 15 to 30 hours at C, pH 4.5 to 6.5 while stirring at a rate of 50 ppm or more, and to stop the enzyme reaction, 70 to 80 O The enzyme may also be inactivated by heating at C for 1 to 2 hours.

[0053] In step 3), 50 to 100 weight percent of purified water relative to the total weight of the ground material is further mixed with the enzyme-treated ground material, and 80 to 95 OThe extract can be extracted by heating at a temperature of C for 6 to 7 hours.

[0054] 4) In the next step, the extract is filtered by passing it through a filter, and then concentrated using a vacuum concentrator to form a solid content of 20 to 25 weight%.

[0055] 5) In step 5, 20 to 50% by weight of maltodextrin relative to the total weight of the concentrate is added to each concentrate and stirred, and then dried and powdered using SD (Spray Drying). After powdering, mulberry leaf, Cornus fruit, balloon flower root, dried tangerine peel, Cudrania tricuspidata, goji berry, ginger, and yuzu extract powders can be mixed.

[0056] At this time, in step 5), 20 to 30 weight% of mulberry leaves, 10 to 20 weight% of Cornus fruit, 10 to 20 weight% of balloon flower root, 5 to 10 weight% of dried tangerine peel, 5 to 10 weight% of Cudrania tricuspidata, 5 to 10 weight% of goji berries, 5 to 10 weight% of ginger, and 5 to 10 weight% of citron may be mixed, but are not limited thereto.

[0057] The plant mixed extract powder prepared 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 the concentration of muscle protein in the muscle by increasing the protein concentration in the serum; therefore, the present invention can be usefully used for the prevention or treatment of diseases related to muscle loss.

[0059] <Example> Preparation of plant mixed extract powder

[0060] 1. Preparation of plant mixed extract powder

[0061] Each raw material included in the plant mixed extract powder of the present invention is mulberry leaf, Cornus fruit, balloon flower, dried tangerine peel, Cudrania tricuspidata, goji berry, ginger, and yuzu. In the manufacturing process of the plant mixed extract powder, 100% by weight of purified water is mixed with each raw material and the raw material is ground using a grinder. Then, 0.1g of pectinase and 0.2g of cellulase are added and mixed per 1kg of the ground material. After that, the enzyme reaction is carried out for 15 to 30 hours while stirring at a speed of 50ppm or higher at 37 to 45 OC and pH 4.5 to 6.5 DPTJ, and then the enzyme is inactivated by heating at 70 to 80 OC for 1 to 2 hours.

[0062] Subsequently, in the process of manufacturing the plant mixed extract powder, 100% by weight of purified water relative to the total weight of the enzyme-treated ground material is further mixed, and the extract is extracted by heating at a temperature of 80 to 95 OC for 6 to 7 hours.

[0063] After passing the above extract through a filter and filtering it, the extract was concentrated using a vacuum concentrator to form a solid content of 20 to 25 weight%, then 20 to 50 weight% of maltodextrin was added to the concentrate relative to the total weight of the concentrate and stirred, and then dried using an SD to produce a plant mixed extract powder.

[0064] Each of the above dried powders was mixed as shown in Table 1 below, diluted with distilled water (DW), and used.

[0066] Sangyeop Cornelian cherry balloon flower dermis Cudrania Gujija ginger Yuja bout 25 16.7 16.7 8.3 8.3 8.3 8.3 8.3 100

[0068] 2. Manufacturing of prototypes

[0069] The prototype was prepared by mixing a main ingredient comprising isolated soy protein, isolated whey protein, and goat milk protein with an auxiliary ingredient comprising a plant mixed extract powder, according to the manufacturing method of the present invention.

[0071] <Experimental Example>

[0072] Experimental method

[0073] 1. Cell culture, differentiation, and inflammation induction

[0074] The C2C12, Raw264.7, AGS, and RBL-2H3 cells used in this experiment were cultured in dulbecco's modified eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic, and in Roswell Park Memorial Institute (RPMI) 1640 medium, and cultured in an incubator controlled at 5% CO2 and 37°C.

[0075] Differentiation induction of C2C12 cells was induced for 6 days with medium replacement and treatment with plant mixed extract powder every 2 days, and for this purpose, DMEM was replaced with one containing 2% horse serum (HS).

[0076] The inflammatory response of C2C12 cells was induced by treating with 1 mg / ml of LPS along with the medium.

[0078] 2. MTT assay

[0079] In the MTT assay, cells were seeded into 96-well plates containing medium and cultured for 12 hours. Then, the samples were diluted to different concentrations, treated with the cells, and cultured for 24 hours. Next, all the medium was removed, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 5 mg / mL) was diluted to a concentration of 0.5 μg / mL in the medium and 100 μL was added to each well, followed by 4 hours of culture. Finally, all the medium was removed, and 100 μL of dimethyl sulfoxide (DMSO) was added to each well.

[0080] Next, to elute formazan, the treatment solution was reacted at room temperature for 10 minutes, and then the absorbance of the treatment solution was measured at 540 nm using a microplate reader (Versamax, Molecular Devices, San Jose, CA, USA).

[0081] Next, to confirm the protective effect on C2C12 cells, C2C12 cells were treated with a plant mixed extract powder for 3 hours, followed by treatment with 500 μM H2O2 for 24 hours to induce oxidative stress. Subsequently, the MTT assay process was conducted in the same manner as the cytotoxicity evaluation, thereby confirming the cell protective effect.

[0083] 3. Lactate dehydrogenase (LDH) activity

[0084] LDH activity in the culture medium collected after treatment with plant mixed extract powder and H2O2 was completed under the same conditions as the evaluation of the cytoprotective effect on C2C12 cells was assessed using a cytotoxicity detection kit (Roche Diagnostics, Mannheim, Germany) according to the experimental method provided in the kit.

[0086] 4. Creatine kinase (CK) activity

[0087] In the CK activity test method, after the extract and H2O2 treatment were completed under the same conditions as the evaluation of the cytoprotective effect on C2C12 cells, the medium was removed, the cells were collected, and treated with lysis buffer (phosphate buffered saline (PBS) containing 0.1% Tween-20). The supernatant obtained by centrifuging the treatment solution at 4°C and 12,000 rpm for 15 minutes was used to measure CK activity. CK activity was performed using a creatine kinase activity assay kit (abcam, Cambridge, UK) according to the test method provided in the kit.

[0089] 5. Cytokine Measurement

[0090] The concentration of cytokines produced in the culture medium was measured using enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, MN, USA). In the cytokine measurement process, after the sample and TNF-α were treated and cultured, the culture medium was centrifuged to separate the supernatant, stored at -70°C, and then analyzed according to the analysis method provided by the manufacturer. The absorbance of the culture medium was measured using an ELISA reader (Molecular Devices, CA, USA).

[0091] Next, in the cytokine measurement process, a standard curve was obtained using the absorbance of the standard, and the amount of cytokine was quantified using this. Then, cells treated only with the medium were used as a negative control, and cells treated only with TNF-α were used as a positive control to compare the activity with the sample treatment group.

[0093] 6. Western blot assay

[0094] In the Western blot assay, the treated C2C12 muscle cells were washed with PBS, the cells were collected and treated with RIPA buffer for 20 minutes at 4°C, the treated solution was centrifuged at 12,000 rpm for 20 minutes at 4°C, and the protein concentration in the supernatant was quantified using a BCA protein assay kit.

[0095] Next, an equal amount of protein was transferred to a PVDF membrane after electrophoresis using a 10% mini-protean TGX™ precast protein gel. Next, the membrane was blocked with Everyblot blocking buffer at room temperature for 10 minutes, and the primary antibody (1:1000–1:5000) was incubated overnight at 4°C. Then, the PVDF membrane was washed three times for 15 minutes each with 1X tris-buffered saline with 0.1% Tween-20 (TBS-T). Next, the diluted HRP-conjugated secondary antibody (1:1000–1:4000) was incubated at room temperature for 2–3 hours, and the PVDF membrane was washed three times for 15 minutes each with 1X TBS-T. Next, the expression level was analyzed by treating with an enhanced chemiluminescent substrate and observing the protein bands that appeared on the PVDF membrane using Microchemi 4.2 (DNR, Neve Yamin, IS).

[0097] <Experimental Results>

[0098] 1. Protective effect of plant mixed extract powder on muscle cells against oxidative stress

[0100] 1) Muscle cell protective effect

[0101] To confirm the cytotoxicity of H2O2 on C2C12 muscle cells, C2C12 muscle cells were treated with H2O2 at various concentrations for 24 hours, and cytotoxicity was evaluated using an MTT assay. As a result of treating C2C12 with H2O2 at concentrations of 0, 50, 100, 300, 500, and 700 μM for 24 hours, cell viability was 98, 85, 79, 63, and 53% for each concentration, as shown in Figure 1. Therefore, the H2O2 treatment concentration was set to 500 μM to confirm the cytoprotective effect against oxidative stress.

[0102] Next, differentiated C2C12 muscle cells were pretreated with plant mixed extract powder at concentrations of 300, 400, and 500 μg / mL for 4 hours, and then 500 μM H2O2 was added to the medium for 24 hours. The cytoprotective effect of H2O2 was evaluated using MTT assay and LDH assay.

[0103] As confirmed by the MTT assay, compared to the untreated group, the cell viability of the group treated with H2O2 alone decreased to 61%, but the cell viability of the group treated with plant mixed extract powder increased to 68%, 79%, and 87% (Fig. 2a).

[0104] In addition, as confirmed by the LDH assay, the LDH activity increased to 180% in the group treated with H2O2 alone compared to the untreated group, but decreased to 166%, 154%, and 131% in the group treated with plant mixed extract powder (Fig. 2b).

[0106] 2) Effects on CK activity

[0107] In the experiment on the effect on CK activity, differentiated C2C12 muscle cells were pretreated with plant mixed extract powder at concentrations of 300, 400, and 500 μg / mL for 4 hours, and after treating the medium with 500 μM H2O2 for 24 hours, the activity of CK was measured to confirm the effect of the plant mixed extract powder on CK activity increased by oxidative stress.

[0108] Compared to the untreated group, CK production increased to 286% in the group treated with H2O2 alone, but CK activity decreased to 259%, 220%, and 181% in the group treated with plant mixed extract powder (Fig. 3).

[0110] 3) Effects on apoptosis-related protein expression

[0111] In the experiment on the effect on the expression of apoptosis-related proteins, to determine whether the cytoprotective effect of the plant complex extract against oxidative stress in differentiated C2C12 muscle cells was due to the inhibition of the apoptosis pathway, differentiated C2C12 muscle cells were pretreated with the plant mixed extract powder at concentrations of 300, 400, and 500 μg / mL for 4 hours, then the medium was treated with 500 μM H2O2 for 24 hours, and the level of related protein expression was confirmed using western blot.

[0112] First, in this experiment, changes in Bax and Bcl-2 expression were examined to determine whether the apoptosis inhibitory effect of H2O2 is associated with the Bcl-2 family. It was confirmed that compared to the untreated group, the group treated with H2O2 alone showed an increase in Bax expression and a decrease in Bcl-2 expression. In the group treated with the plant mixed extract powder, Bax expression decreased by 18%, 17%, and 41% compared to the group treated with H2O2 alone (Fig. 4a), while Bcl-2 expression increased by 200%, 1,325%, and 2,125% (Fig. 4b).

[0113] In addition, in this experiment, changes in the expression of caspase-9 and -3 were examined to determine whether the changes were due to the blockade of caspase, an enzyme that acts as a key regulator of apoptosis induced by H2O2. While the expression of caspase-9 and -3 increased in the group treated with H2O2 alone compared to the untreated group, in the group treated with plant mixed extract powder, the expression of caspase-9 decreased by 0.3%, 24%, and 41% compared to the group treated with H2O2 alone (Fig. 4c), and the expression of caspase-3 decreased by 15%, 36%, and 60% (Fig. 4d).

[0115] 2. In-vitro (cell experiment) efficacy evaluation of the prototype regarding muscle loss and digestive activity

[0117] 1) Effect on cell viability

[0118] In the experiment on the effect on cell viability, the plant mixed extract was applied at different concentrations to confirm the cytotoxicity of the prototype on animal cells, and cytotoxicity was evaluated using the MTT assay. For this purpose, the prototype was diluted in the medium at concentrations of 0, 50, 100, 300, 500, 700, 1000, 1500, and 2000 μg / mL and treated for 24 hours.

[0119] The viability of C2C12 cells was 103, 108, 105, 106, 103, 104, 100, and 100% at different concentrations, and did not decrease up to a concentration of 2000 μg / mL (Fig. 5a).

[0120] The viability of Raw264.7 cells was 105, 103, 102, 103, 101, 100, 100, and 92% at different concentrations, and decreased at a concentration of 2000 μg / mL (Fig. 5b).

[0121] The viability of AGS cells was 100, 103, 106, 105, 107, 103, 101, and 101% at different concentrations, and did not decrease up to a concentration of 2000 μg / mL (Fig. 5c).

[0123] 2) Muscle cell protective effect of the prototype

[0124] In the experiment on the protective effect of the prototype on muscle cells, to confirm the cell protective effect against oxidative stress caused by H2O2, differentiated C2C12 muscle cells were pretreated with the prototype at concentrations of 500, 700, and 900 μg / mL for 3 hours, then treated with 500 μM H2O2 in the medium for 24 hours, and confirmed through MTT assay and LDH assay.

[0125] As confirmed by the MTT assay, compared to the untreated group, the cell viability of the group treated with H2O2 alone decreased to 59%, but the cell viability of the groups treated with the prototype increased to 67%, 73%, and 83%, and the control group treated with Selex (900 μg / mL) showed a cell viability of 63% (Fig. 6a).

[0126] In addition, as confirmed by the LDH assay, the LDH activity increased to 172% in the group treated with H2O2 alone compared to the untreated group, but decreased to 159%, 139%, and 127% in the group treated with the prototype, and the LDH activity was higher than that of the prototype at 165% in the control group treated with Selex (900 μg / mL) (Fig. 6b).

[0128] 3) Effects on CK activity

[0129] In the experiment on the effect on CK activity, differentiated C2C12 muscle cells were pretreated with the prototype at concentrations of 500, 700, and 900 μg / mL for 3 hours, and then 500 μM H2O2 was added to the medium for 24 hours and the activity of CK was measured to confirm the effect of the plant mixed extract powder on CK activity increased by oxidative stress.

[0130] Compared to the untreated group, CK production increased to 271% in the group treated with H2O2 alone, but CK activity decreased to 248%, 200%, and 171% in the group treated with the prototype, and CK activity was higher than that of the prototype at 251% in the control group treated with Selex (900 μg / mL) (Fig. 7).

[0132] 4) Inhibitory activity on NO and PGE2 production

[0133] In the experiment on the inhibitory activity of NO and PGE2 production, the inhibitory effect of the prototype on the production of NO and PGE2 was measured to confirm the effect of suppressing the inflammatory response. In this experiment, muscle cells were pretreated with the prototype at concentrations of 500, 700, and 900 μg / mL, and then treated with LPS to analyze the amounts of NO and PGE2 present in the cell culture medium.

[0134] In the case of NO, NO production increased rapidly to 12 μM in the untreated group and 67 μM in the group treated with LPS alone, but NO production decreased to 53, 40, and 29 μM when treated with the prototype, and NO production was higher than that of the prototype in the control group treated with Selex (900 μg / mL) at 59 μM (Fig. 8a).

[0135] In the case of PGE2, NO production increased rapidly to 530 pg / mL in the untreated group and 530 pg / mL in the group treated with LPS alone, but PGE2 production decreased to 488, 133, and 318 pg / mL when treated with the prototype, and PGE2 production was higher than that of the prototype in the control group treated with Selex (900 μg / mL) at 510 pg / mL (Fig. 8b).

[0137] 5) Inhibitory activity against inflammatory cytokine secretion

[0138] In the inflammatory cytokine secretion inhibitory activity test, to investigate the effect on inflammatory cytokine production in C2C12 cells induced with an inflammatory response using LPS, C2C12 cells were pretreated with the prototype at concentrations of 500, 700, and 900 μg / mL, and inflammatory cytokines were measured using ELISA after treatment with LPS.

[0139] In the case of IL-6, the production of IL-6 increased rapidly to 59 pg / mL in the untreated group and 1402 pg / mL in the group treated with LPS alone, but when treated with the prototype, the production of IL-6 decreased to 1140, 1092, and 708 pg / mL, and in the control group treated with Selex (900 μg / mL), the production of IL-6 was higher at 1382 pg / mL compared to the prototype (Fig. 9a).

[0140] In the case of TNF-α, the production of TNF-α increased rapidly to 48 pg / mL in the untreated group and 1239 pg / mL in the group treated with LPS alone, but when treated with the prototype, the production of TNF-α decreased to 992, 721, and 529 pg / mL, and in the control group treated with Selex (900 μg / mL), the production of TNF-α was higher at 1108 pg / mL compared to the prototype (Fig. 9b).

[0142] 3. Analysis of digestive activity of prototype containing plant mixed extract powder (α-Amylase, α-Glucosidase, Lipolytic activity)

[0144] In order to analyze the digestive enzyme activity of the prototype containing the plant mixed extract powder, the experiment was designed with a negative control (NC) group that was not treated with the prototype for each enzyme-specific substrate, and an experimental group that was treated with the prototype at each concentration (50, 100, 200, 400 μg) for each enzyme-specific substrate.

[0145] As a result of treatment with each enzyme-specific substrate under each group's conditions, the prototype using the plant mixed extract powder showed α-Amylase enzyme activity of 0.51 ± 0.01 mU at 50 μg, 1.42 ± 0.04 mU at 100 μg, 1.82 ± 0.02 mU at 200 μg, and 2.33 ± 0.05 mU at 400 μg, and

[0146] It showed α-Glucosidase enzyme activity of 0.85 ± 0.02 mU at 50 μg, 2.36 ± 0.05 mU at 100 μg, 2.22 ± 0.09 mU at 200 μg, and 2.44 ± 0.10 mU at 400 μg, and

[0147] Lipase enzyme activity of 0.031 ± 0.001 mU at 50 μg, 0.042 ± 0.002 mU at 100 μg, 0.054 ± 0.002 mU at 200 μg, and 0.072 ± 0.002 mU at 400 μg was observed (Fig. 10).

[0148] The α-Amylase enzyme activity of the prototype using the plant mixed extract powder increased to 17.62 ± 0.51% at 50 μg, 26.42 ± 1.26% at 100 μg, 30.75 ± 1.22% at 200 μg, and 42.04 ± 2.80% at 400 μg (Fig. 11), and

[0149] The α-glucosidase enzyme activity of the prototype using the plant mixed extract powder increased to 5.22 ± 0.35% at 50 μg, 16.20 ± 0.54% at 100 μg, 19.34 ± 1.00% at 200 μg, and 26.22 ± 1.30% at 400 μg (Fig. 12).

[0150] The lipase enzyme activity of the prototype using the plant mixed extract powder increased by 6.45 ± 0.18% at 50 μg, 10.23 ± 0.65% at 100 μg, 17.61 ± 0.99% at 200 μg, and 38.29 ± 2.61% at 400 μg (Fig. 13).

[0151] Accordingly, the digestive enzyme activity of the prototype using the plant mixed extract powder increased in proportion to the concentration, and an increase of 25 to 47% was confirmed at the highest concentration.

[0153] Although the present invention has been described above with reference to the illustrated drawings, the present invention is not limited by the embodiments and drawings disclosed in this specification, and it is obvious that various modifications can be made by a person skilled in the art within the scope of the technical concept of the present invention. Furthermore, even if the effects of the configuration of the present invention were not explicitly described while explaining the embodiments of the present invention above, it is natural to acknowledge that the effects predictable by said configuration should also be recognized.

Claims

Claim 1 A nutritional shake composition containing a component that prevents muscle loss, comprising as active ingredients a main ingredient powder containing 60.5 wt% isolated soy protein, 35.4 wt% isolated whey protein, 11.8 wt% goat milk protein, 0.011 wt% vitamin B1, 0.07 wt% niacin, 0.05 wt% pantothenic acid, 0.013 wt% vitamin B6, 0.03 wt% biotin, 0.1 wt% vitamin D, 0.1 wt% beta-glucan, 0.008 wt% vitamin K, 0.022 wt% zinc, 5.1 wt% calcium, and 1.1 wt% magnesium. Claim 2 delete Claim 3 delete Claim 4 In claim 1, the above auxiliary ingredient powder comprises a nutritional shake composition containing ingredients that prevent muscle loss, including roasted brown rice powder, vitamin A, vitamin B2, vitamin C, potassium, oat dietary fiber, plant mixed extract powder, MSM, N-acetylglucosamine, fructooligosaccharide, similar cell powder, and amino acid mixed powder. Claim 5 In claim 4, the above auxiliary ingredient powder comprises a nutritional shake composition containing ingredients that prevent muscle loss, wherein the auxiliary ingredient powder comprises 0.001 to 0.009 wt% vitamin A, 0.007 to 0.016 wt% vitamin B2, 0.01 to 0.09 wt% vitamin C, 1.5 to 2.3 wt% potassium, 9.7 to 17.7 wt% oat dietary fiber, 42.7 to 50.7 wt% roasted brown rice powder, 4.8 to 12.8 wt% plant mixed extract powder, 4.2 to 12.2 wt% MSM, 1.7 to 3.7 wt% N-acetylglucosamine, 12.5 to 20.5 wt% fructooligosaccharide, 0.1 to 0.9 wt% lactic acid bacteria dead cell powder, and 0.1 to 0.9 wt% amino acid mixed powder. Claim 6 In claim 5, the above plant mixed extract powder comprises a nutritional shake composition containing a component that prevents muscle loss, comprising a mixed extract of mulberry leaves, Cornus fruit, balloon flower, dried tangerine peel, Cudrania tricuspidata, goji berries, ginger, and yuzu. Claim 7 In claim 6, the above plant mixed extract powder comprises a nutritional shake composition containing ingredients that prevent muscle loss, comprising 20 to 30 weight% of mulberry leaves, 10 to 20 weight% of Cornus fruit, 10 to 20 weight% of balloon flower root, 5 to 10 weight% of dried tangerine peel, 5 to 10 weight% of Cudrania tricuspidata, 5 to 10 weight% of goji berries, 5 to 10 weight% of ginger, and 5 to 10 weight% of citron, based on the total weight. Claim 8 In claim 6, the above-mentioned plant mixed extract powder is a nutritional shake composition containing a component that prevents muscle loss, prepared through a manufacturing method comprising: 1) mixing purified water with mulberry leaves, Cornus fruit, balloon flower root, dried tangerine peel, Cudrania tricuspidata, goji berry, ginger, and citron, respectively, and grinding them; 2) adding an enzyme to the ground material and causing an enzymatic reaction; 3) mixing purified water with the enzyme-treated ground material and heating it to extract an extract; 4) concentrating the extract to prepare a concentrate; and 5) adding dietary fiber to the concentrate and then drying it to form a powder. Claim 9 In claim 8, the above enzyme is pectinase and cellulase, and the nutritional shake composition comprises a component that prevents loss of the area. Claim 10 In claim 8, the dietary fiber is a nutritional shake composition containing a component that prevents the loss of maltodextrin. Claim 11 In claim 8, the above plant mixed extract powder is a nutritional shake composition containing a component that prevents muscle loss, prepared through a manufacturing method comprising the step of mixing extract powders of mulberry leaves, Cornus fruit, balloon flower, dried tangerine peel, Cudrania tricuspidata, goji berry, ginger, and citron after step 5).

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