KR102751456B1 - Method for producing low-molecular protein hydrolysate from hemp seed defatted meal and composition for preventing or improving muscular atrophy or sarcopenia containing the prepared low-molecular protein hydrolysate - Google Patents

Abstract

The present invention relates to a method for producing a low-molecular-weight protein hydrolysate from defatted hemp seed meal and a composition for preventing or improving muscular atrophy or sarcopenia containing the produced low-molecular-weight protein hydrolysate. Specifically, the present invention comprises: (1) a step of adding distilled water to defatted hemp seed meal, adjusting the pH to 10 to elute proteins, centrifuging to obtain only the supernatant, and then adjusting the pH of the supernatant to 5 to precipitate the eluted proteins to obtain a protein extract of defatted hemp seed meal; (2) a pretreatment step of adding distilled water to the protein extract of the defatted hemp seed meal of step (1), and then sequentially performing ultrasonic treatment and heat treatment; And (3) a method for producing a low-molecular-weight protein hydrolysate of hemp seed defatted meal having activity for improving muscle atrophy or sarcopenia or strengthening muscle, comprising a step of adding a hydrolytic enzyme to a protein extract of hemp seed defatted meal, which has undergone the pretreatment of step (2), and reacting the same, and obtaining a low-molecular-weight protein hydrolysate of hemp seed defatted meal; a food composition for improving muscle atrophy or sarcopenia or strengthening muscle, and a pharmaceutical composition for preventing or treating muscle atrophy or sarcopenia, comprising the low-molecular-weight protein hydrolysate of hemp seed defatted meal produced by the method as an effective ingredient.

Description

Method for producing low-molecular protein hydrolysate from hemp seed defatted meal and composition for preventing or improving muscular atrophy or sarcopenia containing the prepared low-molecular protein hydrolysate}

The present invention relates to the production of a low-molecular-weight protein hydrolysate from defatted hemp seed meal and a composition containing the produced low-molecular-weight protein hydrolysate for preventing or improving muscle atrophy or sarcopenia.

Muscle atrophy is a symptom that appears when muscles become weak, meaning the loss of muscle tissue. The most common cause is lack of exercise, but it is known that muscle atrophy is caused by reduced physical activity due to injury or illness, nutritional deficiencies, and genetic factors. A decrease in protein synthesis in the body due to aging can cause muscle atrophy, and nutritional deficiencies due to dieting are also causes.

In addition, muscle loss and muscle atrophy are closely related, and when looking at the current status of the treatment industry for improving sarcopenia and muscle atrophy due to aging, muscle atrophy due to spinal disease, cancer, aging, etc., or decline in physical function and muscle strength in the elderly due to internal and external problems are major problems in the aging society. Preventing decline in muscle function due to aging, that is, preventing muscle atrophy and maintaining contractile function and muscle elasticity, is an important factor for healthy physical activity and independent daily life in old age.

Therefore, in line with the global trend of an aging society, efforts are needed to maintain the musculoskeletal function of the elderly to increase their quality of life and healthy life expectancy, and research and development of related products to improve muscle strength and muscle atrophy are needed.

As one of the proactive countermeasures to various muscle loss problems, a more fundamental muscle health management method is required through the development of muscle health improvement technology, and the development of functional materials to prevent muscle function decline and recover muscle atrophy can be a countermeasure for preventing and improving diseases related to muscle strength loss due to an aging society.

Meanwhile, in terms of technology developed in relation to improving muscle atrophy, products for improving muscle atrophy using natural extracts such as Schisandra chinensis extract, red sea ginseng enzyme hydrolysate, angelica root extract, Cnidium officinalis extract, peony root extract, and Echinococcus amurense extract are being developed, but their effectiveness is minimal.

Therefore, there is a need to develop a new material that has an excellent muscle atrophy improvement effect and does not cause other side effects while improving muscle strength or muscle atrophy.

Korean Patent Registration No. 10-2403795 Korean Patent Publication No. 10-2022-0134101

Accordingly, in the present invention, a method for producing a low-molecular-weight protein hydrolysate having an excellent muscle strength improvement or muscle atrophy improvement effect was established from defatted hemp seed meal, and the present invention was completed by confirming that the low-molecular-weight protein hydrolysate obtained by the method has an excellent effect of improving muscle atrophy and sarcopenia.

Therefore, the purpose of the present invention is to provide a method for producing a low-molecular-weight protein hydrolysate of hemp seed defatted meal having an activity for improving muscular atrophy or sarcopenia or strengthening muscle, the method comprising the steps of: (1) adding distilled water to defatted hemp seed meal and adjusting the pH to 10 to elute proteins, centrifuging to obtain only the supernatant, and then adjusting the pH of the supernatant to 5 to precipitate the eluted proteins to obtain a protein extract of defatted hemp seed meal; (2) a pretreatment step of adding distilled water to the protein extract of the defatted hemp seed meal of the step (1), and then sequentially performing ultrasonic treatment and heat treatment; and (3) a step of adding a hydrolytic enzyme to the protein extract of the defatted hemp seed meal, of which the pretreatment of the step (2) is completed, and reacting the hydrolysate to obtain a low-molecular-weight protein hydrolysate of the defatted hemp seed meal.

Another object of the present invention is to provide a food composition for improving muscle atrophy or sarcopenia or strengthening muscles, comprising a low molecular weight protein hydrolysate of defatted hemp seed meal produced by the method of the present invention as an effective ingredient.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating muscle atrophy or sarcopenia, comprising a low molecular weight protein hydrolysate of defatted hemp seed meal prepared by the method of the present invention as an effective ingredient.

In order to achieve the above object, the present invention provides a method for producing a low-molecular-weight protein hydrolysate of hemp seed defatted meal having an activity for improving muscular atrophy or sarcopenia or strengthening muscle, the method comprising: (1) a step of adding distilled water to defatted hemp seed meal and adjusting the pH to 10 to elute proteins, centrifuging to obtain only the supernatant, and then adjusting the pH of the supernatant to 5 to precipitate the eluted proteins to obtain a protein extract of defatted hemp seed meal; (2) a pretreatment step of adding distilled water to the protein extract of the defatted hemp seed meal of the step (1), and then sequentially performing ultrasonic treatment and heat treatment; and (3) a step of adding a hydrolytic enzyme to the protein extract of the defatted hemp seed meal, of which the pretreatment of the step (2) is completed, and reacting the hydrolysate to obtain a low-molecular-weight protein hydrolysate of the defatted hemp seed meal.

In one embodiment of the present invention, the ultrasonic treatment in step (2) may be performed at 250 to 350 W for 10 to 20 minutes, and the heat treatment in step (2) may be performed at 90 to 110°C for 10 to 20 minutes.

In one embodiment of the present invention, the addition and reaction of the hydrolytic enzyme in step (3) may be performed by sequentially treating alcalase and flavourzyme, adding the enzyme in an amount of 3 to 5 wt% relative to the weight of the substrate, and hydrolyzing at a temperature of 45 to 55°C and a pH of 7 to 8 for 1 to 2 hours.

In one embodiment of the present invention, the low-molecular-weight protein hydrolysate of the defatted hemp seed meal may contain a hydrolyzed low-molecular-weight peptide of 3 kDa or less.

In addition, the present invention provides a food composition for improving muscle atrophy or sarcopenia or strengthening muscles, comprising a low molecular weight protein hydrolysate of defatted hemp seed meal produced by the method of the present invention as an effective ingredient.

Furthermore, the present invention provides a pharmaceutical composition for preventing or treating muscle atrophy or sarcopenia, comprising a low molecular weight protein hydrolysate of defatted hemp seed meal produced by the method of the present invention as an effective ingredient.

The present invention establishes a method for producing an optimal low-molecular-weight protein hydrolysate from defatted hemp seed meal having an effect of preventing, improving, and treating muscle atrophy or sarcopenia, and when the low-molecular-weight protein hydrolysate of defatted hemp seed meal produced by the method of the present invention was administered to mice induced with muscle atrophy, it was confirmed that muscle strength and motor skills were improved, weight loss due to muscle strength decline was also improved, and grip strength was enhanced and muscle strengthening effects were excellent. Therefore, the low-molecular-weight protein hydrolysate of defatted hemp seed meal produced by the method of the present invention can be usefully used in the production of functional foods and pharmaceuticals for improving muscle atrophy or sarcopenia and strengthening muscles.

Figure 1 shows the results of HLPC size exclusion chromatography in which the degree of hydrolysis according to the treatment conditions is analyzed for the protein extract of defatted hemp seed meal in one embodiment of the present invention (HPI: hemp seed protein extract, CON: hemp seed protein extract group treated in the same manner without enzyme addition, AF: hemp seed protein extract enzyme treatment group (alcalase and flavozyme), HEAT: hemp seed protein extract heat treatment followed by enzyme treatment, SONIC: hemp seed protein extract sonication followed by enzyme treatment, HEAT+SONIC: hemp seed protein extract heat treatment and sonication followed by enzyme treatment).
Figure 2 is a graph showing the content of low molecular weight (3 kDa or less) peptides and high molecular weight (over 3 kDa) peptides contained in protein hydrolysates obtained under different treatment conditions from protein extracts of defatted hemp seed meal in one embodiment of the present invention, expressed as area % of an HPLC analysis graph.
Figure 3 shows the results of analyzing the content of free amino acids contained in protein hydrolysates obtained under different treatment conditions from protein extracts of defatted hemp seed meal in one embodiment of the present invention using TNBS analysis.
FIG. 4 shows SDS-PAGE gel electrophoresis and Coomassie Brilliant Blue staining images of protein hydrolysates obtained under different treatment conditions from protein extracts of defatted hemp seed meal in one embodiment of the present invention.
FIG. 5 is a schematic diagram of the process for mouse animal testing to confirm the muscle atrophy improvement effect of the low-molecular-weight protein hydrolysate of defatted hemp seed meal according to the present invention in one embodiment of the present invention.
Figure 6 shows the results of measuring the change in body weight and muscle strength by period for each mouse in the normal group (NC), muscle atrophy-induced group (DEX), hemp seed defatted protein extract administration group (HPI), hemp seed defatted protein hydrolysate low-dose administration group (250 mg/kg administration group: HPH-L), and hemp seed defatted protein hydrolysate high-dose administration group (500 mg/kg administration group: HPH-H).
Figure 7 shows the results of measuring the amount of autonomous exercise for each mouse in the normal group (NC), the muscle atrophy-induced group (DEX), and the high-dose administration group of the hemp seed defatted protein hydrolysate of the present invention (500 mg/kg administration group: HPH-H).
Figure 8 shows the muscle photographs and muscle weight measurement results for each mouse in the normal group (NC), muscle atrophy-induced group (DEX), hemp seed defatted protein extract administration group (HPI), hemp seed defatted protein hydrolysate low-dose administration group (250 mg/kg administration group: HPH-L), and hemp seed defatted protein hydrolysate high-dose administration group (500 mg/kg administration group: HPH-H).
Figures 9 and 10 show the results of microscopic observation of muscle fibers and measurement of the cross-sectional area of muscle fibers in the calf muscles (Figure 9) and thigh muscles (Figure 10) isolated from each mouse of the normal group (NC), muscle atrophy-induced group (DEX), hemp seed defatted meal protein extract administration group (HPI), hemp seed defatted meal protein hydrolysate low-dose administration group (250 mg/kg administration group: HPH-L), and hemp seed defatted meal protein hydrolysate high-dose administration group (500 mg/kg administration group: HPH-H).
Figure 11 shows the results of isolating calf muscles from each mouse of the normal group (NC), muscle dystrophy-induced group (DEX), hemp seed defatted meal protein extract administration group (HPI), hemp seed defatted meal protein hydrolysate low-dose administration group (250 mg/kg administration group: HPH-L), and hemp seed defatted meal protein hydrolysate high-dose administration group (500 mg/kg administration group: HPH-H), obtaining protein lysates, and analyzing the expression levels of proteins involved in muscle breakdown (MuRF1, MAFbx) and muscle synthesis (Myf5, MyoD, MyHC) via Western blotting.
Figure 12 shows the results of isolating thigh muscles from each mouse of the normal group (NC), muscle dystrophy-induced group (DEX), hemp seed defatted meal protein extract administration group (HPI), hemp seed defatted meal protein hydrolysate low-dose administration group (250 mg/kg administration group: HPH-L), and hemp seed defatted meal protein hydrolysate high-dose administration group (500 mg/kg administration group: HPH-H), obtaining protein lysates, and analyzing the expression levels of proteins involved in muscle breakdown (MuRF1, MAFbx) and muscle synthesis (Myf5, MyoD, MyHC) via Western blotting.
Figure 13 shows the results of isolating calf muscles from each mouse of the normal group (NC), muscle atrophy-induced group (DEX), hemp seed defatted meal protein extract administration group (HPI), hemp seed defatted meal protein hydrolysate low-dose administration group (250 mg/kg administration group: HPH-L), and hemp seed defatted meal protein hydrolysate high-dose administration group (500 mg/kg administration group: HPH-H), obtaining protein lysates, and analyzing the expression levels of apoptosis-related proteins via Western blotting.

The present invention is characterized in that it establishes a method for producing a low-molecular-weight protein hydrolysate of defatted hemp seed meal having muscle strengthening activity or improving muscle atrophy or sarcopenia.

Specifically, the method for producing a low-molecular-weight protein hydrolysate of defatted hemp seed meal having the activity of improving muscle atrophy or sarcopenia or strengthening muscle according to the present invention comprises: (1) a step of adding distilled water to defatted hemp seed meal and adjusting the pH to 10 to elute proteins, centrifuging to obtain only the supernatant, and then adjusting the pH of the supernatant to 5 to precipitate the eluted proteins to obtain a protein extract of defatted hemp seed meal; (2) a pretreatment step of adding distilled water to the protein extract of the defatted hemp seed meal of step (1), and then sequentially performing ultrasonic treatment and heat treatment; and (3) a step of adding a hydrolytic enzyme to the protein extract of the defatted hemp seed meal, which has undergone the pretreatment of step (2), and reacting the same to obtain a low-molecular-weight protein hydrolysate of the defatted hemp seed meal.

A more detailed description of the above method is as follows.

First, a protein extract from defatted hemp seed meal is obtained.

The above hemp seed is a seed of the hemp plant and is considered a superfood because it contains many nutrients such as protein and amino acids. It is low in calories and rich in protein, and contains more unsaturated fatty acids such as omega 3 and omega 6 than mackerel, and also contains various essential amino acids.

Accordingly, as studies on the useful physiological activities of hemp seeds are reported, interest in the production of functional materials using them is increasing. However, there has been no report yet on materials that can prevent, improve, and treat muscle atrophy or sarcopenia using defatted hemp seeds.

In addition, the above-mentioned defatted residue refers to the residue of seeds after oil is squeezed, and in the present invention, the defatted residue of hemp seeds was used.

The step of obtaining a protein extract from defatted hemp seed meal includes the steps of adding distilled water to defatted hemp seed meal, adjusting the pH to 10 to elute the protein, centrifuging to obtain only the supernatant, and then adjusting the pH of the supernatant to 5 to precipitate the eluted protein.

Here, distilled water can be added in an amount equivalent to 20 times the weight of the hemp seed defatted meal, and the pH is adjusted to 10 so as to be a basic condition to elute proteins from the hemp seed defatted meal. The eluted proteins are obtained as a supernatant through centrifugation, and the pH of the supernatant is adjusted to 5 so as to be an acidic condition to precipitate the eluted proteins, thereby obtaining a protein extract of the hemp seed defatted meal.

Additionally, the protein extract of the defatted hemp seed meal, which has been eluted and precipitated by adjusting the pH to 5, can be centrifuged again to obtain a precipitated protein extract, and then the pH can be adjusted to 7 and freeze-dried.

When the step of obtaining a protein extract from the defatted hemp seed meal as described above is completed, a pretreatment step is performed in which distilled water is added to the protein extract from the defatted hemp seed meal, and then ultrasonic treatment and heat treatment are sequentially performed.

When adding distilled water to the protein extract of the defatted hemp seed meal to perform the above pretreatment step, the distilled water is added in an amount corresponding to 20 times the weight of the protein extract of the defatted hemp seed meal.

In addition, the pretreatment can be performed sequentially by ultrasonic treatment and heat treatment, and it is preferable that the ultrasonic treatment is performed at 250 to 350 W for 10 to 20 minutes, and the heat treatment in step (2) is performed at 90 to 110°C for 10 to 20 minutes.

In one embodiment of the present invention, the ultrasonic treatment was performed at 300 W for 15 minutes, and the heat treatment was performed at 100°C for 15 minutes.

If the above ultrasonic treatment and heat treatment are performed outside the range of conditions described above, problems such as decreased hydrolytic activity or protein denaturation resulting in decreased physiological activity may occur.

Next, after the pretreatment is completed, a hydrolytic enzyme is added to the protein extract of the pretreated hemp seed defatted meal and reacted to obtain a low-molecular-weight protein hydrolysate of the hemp seed defatted meal.

Adding and reacting the above hydrolytic enzyme is carried out by sequentially treating alcalase and flavourzyme, and the enzyme is added in an amount of 3 to 5 wt% relative to the weight of the substrate, and hydrolysis can be carried out at a temperature of 45 to 55°C and a pH of 7 to 8 for 1 to 2 hours.

In one embodiment of the present invention, an alcalase enzyme was added in an amount of 4 wt% based on the substrate to a protein extract of pretreated hemp seed defatted meal, and hydrolysis was performed at a temperature of 50°C and pH 8 for 1 hour. After the reaction was completed, a flavourzyme enzyme was added in an amount of 4 wt% based on the substrate, and hydrolysis was performed at a temperature of 50°C and pH 7 for 2 hours.

After the above hydrolysis reaction is completed, an additional step of inactivating the enzymes by heat treatment at a temperature of 90°C for 15 minutes can be performed.

A low-molecular-weight protein hydrolysate of hemp seed defatted meal can be obtained from the defatted hemp seed meal by the method described above.

The inventors of the present invention analyzed the characteristics of the hydrolysate contained in the final protein hydrolysate obtained by the method of the present invention, and confirmed through HPLC size exclusion chromatography analysis that a large amount of low-molecular-weight peptides, specifically low-molecular-weight peptides of 3 kDa or less, were contained in the hydrolysate due to enzyme treatment. In particular, the group that underwent pretreatment with ultrasonication and heat treatment and then enzyme treatment showed the highest content of low-molecular-weight peptides of 3 kDa or less compared to the other experimental groups, and confirmed that the free amino acid content was also very high.

Through this, it was found that pretreatment by ultrasonic treatment and heat treatment could further increase the degree of hydrolysis.

Furthermore, the inventors of the present invention confirmed whether the low molecular weight protein hydrolysate of defatted hemp seed meal produced by the method of the present invention has the activity of improving, preventing or treating muscle atrophy or sarcopenia.

To this end, when the low-molecular-weight protein hydrolysate of the defatted hemp seed meal of the present invention was administered to mice in which muscle atrophy or sarcopenia was induced by administering dexamethasone, it was shown that the body weight decreased due to muscle atrophy or sarcopenia was recovered, grip strength was improved, and reduced exercise volume was also improved, and it was shown that there was an effect of increasing muscle mass and strengthening muscles.

In addition, in another embodiment of the present invention, it was confirmed that muscle fiber reduction symptoms due to muscle atrophy or muscle loss were improved by administration of the low molecular weight protein hydrolysate of the defatted hemp seed meal of the present invention, and muscle fiber reduction was effectively inhibited. In addition, this effect was shown to be more effective in the group treated with the low molecular weight protein hydrolysate of the defatted hemp seed meal obtained by hydrolysis treatment than in the group treated with the protein extract extracted from the defatted hemp seed meal (before hydrolysis treatment; HPI).

In addition, in another embodiment of the present invention, it was analyzed whether the low molecular weight protein hydrolysate of hemp seed defatted meal of the present invention affects the expression of proteins involved in muscle decomposition and muscle synthesis.

As a result, in the group treated with the low-molecular-weight protein hydrolysate of hemp seed defatted meal of the present invention, the protein expression levels of MuRF1 and MAFbx, which are ubiquitin ligase E3 transferases that induce muscle atrophy, were found to decrease, whereas the expression of Myf5, MyoD, and MyHC, which are involved in muscle differentiation and synthesis, was found to increase.

Therefore, through these results, the inventors of the present invention were able to determine that the low-molecular-weight protein hydrolysate of defatted hemp seed of the present invention can prevent, improve, and treat muscle diseases such as muscle atrophy or sarcopenia by inhibiting the expression of proteins that induce muscle atrophy and simultaneously promoting the expression of proteins that induce muscle differentiation and muscle synthesis.

Furthermore, the inventors of the present invention analyzed whether the low molecular weight protein hydrolysate of defatted hemp seed of the present invention also affects the expression of proteins related to apoptosis in muscle tissue.

As a result, in the muscle cells of the group in which muscle atrophy was induced, the expression levels of Bax, known to induce apoptosis, and cleaved caspase-3, which occurs when apoptosis occurs, were found to be significantly increased compared to the normal group, whereas in the group treated with the low-molecular-weight protein hydrolysate of hemp seed defatted meal of the present invention, the expression levels of Bax and cleaved caspase-3 were found to be suppressed.

At the same time, in the group in which muscle atrophy was induced, the expression of Bcl-2, which is known to have the function of inhibiting apoptosis, was found to be decreased compared to the normal group, whereas in the group treated with the low-molecular-weight protein hydrolysate of hemp seed defatted meal of the present invention, the expression of Bcl-2 was found to be increased.

These results imply that the low-molecular-weight protein hydrolysate of hemp seed defatted meal of the present invention can effectively suppress muscle cell death caused by muscle atrophy or sarcopenia by regulating the expression of proteins related to muscle cell death.

Through the above results, the inventors of the present invention were able to find out that a composition containing a low molecular weight protein hydrolysate of the defatted hemp seed meal of the present invention can be used as a material for improving, preventing or treating muscle atrophy or sarcopenia.

Therefore, the present invention can provide a food composition for improving muscle atrophy or sarcopenia or strengthening muscles, which comprises a low-molecular-weight protein hydrolysate of defatted hemp seed meal produced by the method of the present invention as an effective ingredient.

The food composition for improving muscle atrophy or sarcopenia or strengthening muscles according to the present invention may contain, in addition to containing the low-molecular-weight protein hydrolysate of defatted hemp seed meal as an effective ingredient, various flavoring agents or natural carbohydrates as additional ingredients, like a conventional food composition.

Examples of the above-mentioned natural carbohydrates are monosaccharides, for example, glucose, fructose, etc.; disaccharides, for example, maltose, sucrose, etc.; and polysaccharides, for example, dextrin, cyclodextrin, etc., common sugars, and sugar alcohols, for example, xylitol, sorbitol, erythritol, etc. As the above-mentioned flavoring agent, natural flavoring agent (thaumatin), stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agent (saccharin, aspartame, etc.) can be advantageously used.

The food composition of the present invention can be formulated and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gum, candy, ice cream, alcoholic beverages, vitamin complexes, and health supplements.

The above food composition may contain, in addition to the effective ingredient of the present invention, various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickeners (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the food composition of the present invention may contain fruit pulp for the production of natural fruit juice and fruit juice drinks and vegetable drinks.

In addition, the present invention provides a health functional food for improving muscle atrophy or sarcopenia or strengthening muscles, which contains a low molecular weight protein hydrolysate of defatted hemp seed meal prepared by the method of the present invention as an effective ingredient.

The health functional food of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of improving muscle atrophy or sarcopenia or strengthening muscles.

In the present invention, “health functional food” refers to a food manufactured and processed using raw materials or ingredients having functionality useful to the human body according to Act No. 6727 on Health Functional Foods, and means consumption for the purpose of obtaining useful effects for health purposes such as regulating nutrients for the structure and function of the human body or physiological effects.

The health functional food of the present invention may contain conventional food additives, and its suitability as a food additive is determined by the specifications and standards for the relevant item according to the general provisions and general test methods of the Food Additive Code approved by the Ministry of Food and Drug Safety, unless otherwise specified.

Items listed in the above "Food Additives Codex" include, for example, chemically synthesized substances such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, high-molecular-weight pigment, and guar gum; and mixed preparations such as sodium glutamate preparations, alkaline agents added to noodles, preservative preparations, and tar color preparations.

For example, a health functional food in tablet form can be made by mixing the active ingredient of the present invention with excipients, binders, disintegrants, and other additives, granulating the mixture using a conventional method, adding a lubricant, etc., and then compressing and molding the mixture, or by directly compressing and molding the mixture. In addition, the health functional food in tablet form can contain a maturing agent, etc., if necessary.

Among health functional foods in capsule form, hard capsules can be manufactured by filling a mixture of the effective ingredient of the present invention with additives such as excipients into a conventional hard capsule, and soft capsules can be manufactured by filling a mixture of the effective ingredient of the present invention with additives such as excipients into a capsule base such as gelatin. The soft capsules may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative, etc., as necessary.

A health functional food in the form of a ring can be prepared by mixing the effective ingredient of the present invention with an excipient, a binder, a disintegrant, etc., and molding the mixture using a conventionally known method. If necessary, the mixture can be coated with white sugar or another coating agent, or the surface can be coated with a substance such as starch or talc.

A health functional food in granular form can be manufactured into a granular form by mixing the effective ingredient of the present invention with an excipient, a binder, a disintegrant, etc. using a conventionally known method, and may contain a flavoring agent, a flavoring agent, etc., as needed.

The above health functional foods may include beverages, meat, chocolate, food, confectionery, pizza, ramen, other noodles, gum, candy, ice cream, alcoholic beverages, vitamin complexes, and health supplements.

In addition, the present invention can provide a pharmaceutical composition for preventing or treating muscle atrophy or sarcopenia, which comprises a low molecular weight protein hydrolysate of defatted hemp seed meal produced by the method of the present invention as an effective ingredient.

The pharmaceutical composition of the present invention can be manufactured using pharmaceutically suitable and physiologically acceptable excipients in addition to the above-mentioned effective ingredients, and the excipients can include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, glidants, or flavoring agents.

The above pharmaceutical composition may be preferably formulated as a pharmaceutical composition by additionally including one or more pharmaceutically acceptable carriers in addition to the above-described effective ingredients for administration.

The pharmaceutical composition may be in the form of granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops or injectable solutions. For example, for formulation in the form of tablets or capsules, the active ingredient may be combined with an oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. In addition, suitable binders, lubricants, disintegrants and coloring agents may also be included in the mixture, if desired or necessary. Suitable binders include, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, natural and synthetic gums such as corn sweetener, acacia, tracheacanth or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like. Acceptable pharmaceutical carriers for compositions formulated as liquid solutions include sterile and biocompatible ones, such as saline, sterile water, Ringer's solution, buffered saline, albumin injection, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components can be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents can be added as needed. In addition, diluents, dispersants, surfactants, binders, and lubricants can be additionally added to formulate the compositions as injectable formulations such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets. Furthermore, the compositions can be preferably formulated according to each disease or component using a method appropriate to the field, such as that disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA.

The dosage of the pharmaceutical composition of the present invention will vary depending on the age, sex, and weight of the subject to be treated, the specific disease or pathological condition to be treated, the severity of the disease or pathological condition, the dosage intensity, and the judgment of the prescriber. Determination of the dosage based on these factors is within the level of those skilled in the art, and generally the dosage ranges from 0.01 mg/kg/day to about 2,000 mg/kg/day.

A more preferred dosage is 1 mg/kg/day to 500 mg/kg/day. Administration may be once a day or divided into several doses. The above dosage does not limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention can be administered to mammals such as mice, livestock, and humans by various routes. All modes of administration can be envisaged, for example, oral, rectal, or intravenous, intramuscular, subcutaneous, intrauterine, epidural, or intracerebrovascular injection.

Hereinafter, the present invention will be described in more detail by way of examples. These examples are only intended to explain the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

<Example 1>

Preparation of low molecular weight protein hydrolysate from defatted hemp seed meal

<1-1> Extraction of protein from defatted hemp seed meal

The defatted hemp seed residue purchased on the market was mixed with distilled water at a weight ratio of 1:20 (defatted seed residue: distilled water), 2N NaOH was added, and the pH was adjusted to 10 to elute the protein. Afterwards, the residue was centrifuged to remove precipitated impurities, and only the supernatant was collected, 2N HCl was added, and the pH was adjusted to 5, which is the isoelectric point, and then freeze-dried.

<1-2> Ultrasonic, heat and hydrolysis treatment of protein extract from defatted hemp seed meal

Distilled water was added to the protein extract of the defatted hemp seed obtained in the above <1-1> and mixed (protein extract: distilled water = 1:20 weight ratio), and then pretreatment was performed by dividing into each group in Table 1 below. At this time, the pretreatment was performed in the order of ultrasonic treatment and then heat treatment, and after all pretreatments were completed, hydrolysis through enzyme treatment was performed.

In addition, the enzyme treatment was performed sequentially with two types of enzymes in the order of alcalase and flavourzyme, and the alcalase treatment was added in an amount of 4 wt% relative to the substrate, and the treatment was performed at 50°C, pH 8 for 1 hour. After the reaction was completed, the flavourzyme enzyme was added in an amount of 4 wt% relative to the substrate, and the treatment was performed at 50°C, pH 7 for 1 hour. After the two types of enzyme treatment reactions were completed, the enzymes were inactivated by heat treatment at 90°C for 15 minutes. Afterwards, the samples with completed reactions were freeze-dried and used in subsequent experiments.

<Example 2>

Characterization of protein hydrolysate prepared from defatted hemp seed meal

<2-1> HPLC size exclusion analysis

The characteristics of the protein hydrolysate derived from defatted hemp seed meal manufactured through the above Example <1-2> were analyzed. First, the size of each protein hydrolysate was analyzed through HPLC column chromatography analysis. Shodex SUGAR KS-804 (8.0 mm × 300 mm, 7 μm) was used as the column for sample analysis using HPLC (High Performance Liquid Chromatography, Waters, USA), and the sample injection amount was 20 μL and detected with a refractive index detector (RI). 100% distilled water was used as the mobile phase, and the analysis was performed under the conditions of Table 2 below.

As a result, as shown in Fig. 1, the groups treated with enzymes such as alcalase and flavourzyme showed an increase in peaks corresponding to low-molecular-weight peptides compared to the group not treated with enzymes, and in particular, it was confirmed that the peaks of low-molecular-weight peptides corresponding to 3 kDa or less increased significantly.

In addition, the inventors of the present invention converted the area% values into a graph to analyze the trend of low-molecular-weight peptides of 3 kDa or less and high-molecular-weight peptides of more than 3 kDa in the hydrolysates of each experimental group based on the HPLC analysis results.

As a result, as shown in Fig. 2, in the experimental groups that underwent enzymatic treatment with alcalase and flavourzyme, the proportion of low-molecular-weight peptides increased, while the proportion of high-molecular-weight peptides decreased. In particular, in the group that underwent pretreatment with ultrasound and heat treatment before enzymatic treatment, the content of low-molecular-weight peptides of 3 kDa or less was found to be the highest, while the content of high-molecular-weight peptides exceeding 3 kDa was found to be the lowest.

These results imply that when performing pretreatments including ultrasonication and heat treatment prior to enzyme treatment when producing protein hydrolysate from defatted hemp seed meal, the hydrolysis efficiency of the enzyme increases, allowing the highest amount of low-molecular-weight peptides of 3 kDa or less to be obtained.

<2-2> TNBS (2,4,6-trinitrobenzene sulfonic acid) analysis

Next, for each sample used in the above Example <2-1>, TNBS analysis was performed to measure the content of free amino acids contained in the sample. The analysis can analyze the content of free amino acids contained in the sample by quantitatively measuring free amino groups. Specifically, for TNBS analysis, the sample and standard reagent solutions were dissolved in 0.1 M sodium bicarbonate pH 8.5 at 20 to 200 ug/ml. 250 ul of a 0.01% (w/v) TNBSA solution was mixed with 500 ul of each sample and standard reagent solution. Afterwards, the reaction was performed in an incubator at 37°C for 2 hours, and then 250 ul of 10% SDS and 125 ul of 1 N HCl were added to each reaction solution to stop the reaction. The free amino acids were analyzed by measuring the absorbance at a wavelength of 335 nm, and the absorbance values of the samples were expressed as values by substituting them into the leucine standard curve.

As a result of the analysis, as shown in Fig. 3, the group that underwent hydrolysis by enzyme treatment showed an increase in the content of free amino acids and an increase in the degree of hydrolysis compared to the group that was not treated with enzyme, and it was confirmed that the degree of hydrolysis increased further in the group that underwent pretreatment with ultrasound and heat treatment.

<2-3> SDS-PAGE analysis

Furthermore, the inventors of the present invention analyzed the degree of hydrolysis of protein hydrolysate prepared from defatted hemp seed meal for each sample used in Example <2-1> through SDS-PAGE electrophoresis. SDS-PAGE analysis used a 15% gel, and after each sample was diluted to a concentration of 10 mg/ml, 20 ul was loaded onto the gel, and electrophoresis was performed at 85 V for about 2 hours, followed by staining using Coomassie Brilliant Blue reagent and decolorization using 30% methanol containing 10% acetic acid.

As a result, as shown in Fig. 4, a dark protein band was confirmed in the hemp seed protein extract (HPI) group that was not subjected to enzyme treatment, whereas in the groups that were subjected to hydrolysis through enzyme treatment, protein bands were barely observed. These results imply that all proteins contained in the protein extract of defatted hemp seed meal were effectively hydrolyzed into low-molecular-weight peptides by enzyme treatment.

<Example 3>

Analysis of the muscle atrophy protective effect of low-molecular-weight protein hydrolysate obtained from defatted hemp seed meal

<3-1> Preparing the mouse experimental group

Based on the results of the above examples, the protein extract of defatted hemp seed meal, which has the highest degree of hydrolysis and therefore contains a large amount of low-molecular-weight peptides, was pretreated with ultrasonic and heat treatment, and a hydrolysate obtained through enzyme treatment (Production Example 4, protein hydrolysate of defatted hemp seed meal) was used to analyze whether it has a muscle atrophy protection effect.

Experimental animals were purchased from Pyeongtaek Co., Ltd. (Korea), 7-week-old male C57BL/6N mice, and were raised under controlled temperature (23±2℃), humidity (50±5%), and a 12-h light/dark cycle. After a 1-week acclimation period, all experimental animals were randomly divided into the following groups: 1) normal control group, 2) dexamethasone administration group, 3) dexamethasone + protein extract of defatted hemp seed meal (500 mg/kg) administration group, 4) dexamethasone + protein hydrolysate of defatted hemp seed meal (250 mg/kg) administration group, and 5) protein hydrolysate of defatted hemp seed meal (500 mg/kg) administration group. During the experimental period (28 days), the protein extract of the defatted hemp seed meal or the protein hydrolysate of the defatted hemp seed meal was orally administered daily, and each control group was fed the same amount of saline. To induce muscle atrophy in the mice, 2 weeks after the start of oral administration, dexamethasone dissolved in saline was intraperitoneally administered daily for 2 weeks at a concentration of 10 mg/kg body weight, and the control group was treated only with saline in the same manner. During the experimental period, all experimental animals were allowed to freely consume their normal diet and drinking water, and after anesthesia by injection of avertin (2,2,2-tribromoethanol; Sigma-Aldrich Co.), blood was collected by cardiac bleed method, and a total of four leg muscles (quadriceps femoris, tibialis anterior, gastrocnemius, and peroneus longus) of each mouse were excised and stored at -40℃. A schematic diagram of the animal experiment described above is shown in Fig. 5.

<3-2> Weight and grip strength analysis

According to the present invention, it was analyzed whether the low-molecular-weight protein hydrolysate of defatted hemp seed meal can suppress weight loss and muscle strength loss in a muscular atrophy animal model. To this end, body weight measurement and grip strength analysis were performed on each mouse experimental group prepared in the above <3-1>. The above analysis was measured on days 0, 7, 14, 21, 23, 25, and 28 after a 1-week adaptation period, and the grip strength was measured in kgf, using a stainless steel mesh plate attached to the measuring device. After confirming that the experimental animal grabbed the mesh plate of the experimental tool with all legs, the grip strength was measured by pulling the tail at a constant speed until the grip was released. After obtaining three measurement values for each animal, the average value was calculated.

As a result, as shown in Fig. 6, the phenomenon of weight loss in mice due to muscular atrophy was most effectively suppressed in the group (HPH-L) administered the low-molecular-weight protein hydrolysate of the defatted hemp seed meal manufactured by the present invention (6a), and the grip strength analysis also showed that the group administered the low-molecular-weight protein hydrolysate of the defatted hemp seed meal of the present invention was superior, and the group administered the low-molecular-weight protein hydrolysate at a concentration of 500 mg/kg showed a better grip strength effect than the normal control group.

<3-3> Analysis of the effect of suppressing the decrease in exercise volume

An experiment was conducted to confirm whether the low-molecular-weight protein hydrolysate of defatted hemp seed meal according to the present invention can improve symptoms of decreased exercise capacity observed in a muscular atrophy animal model. The experiment was conducted by measuring voluntary wheel running, and was conducted on the 21st day of adaptation to the mouse animal model, and the number of times the mouse freely ran on the wheel for 24 hours was measured.

As a result, as shown in Fig. 7, in the case of mice in which muscle atrophy was induced by administration of dexamethasone, the amount of exercise was found to be decreased compared to the normal group, whereas in the group administered the low-molecular-weight protein hydrolysate of the defatted hemp seed meal of the present invention (500 mg/kg administration group: HPH-H), it was confirmed that the symptoms of decreased exercise amount shown in the mice with muscle atrophy were improved.

<3-4> Analysis of muscle loss inhibition effect

Furthermore, the inventors of the present invention measured the muscle weight of each mouse experimental group and observed the muscles to confirm whether the low-molecular-weight protein hydrolysate of defatted hemp seed meal can suppress muscle loss. Muscle photography and weight measurement were performed simultaneously with dissection after the experimental period (28 days). Photography was performed by fixing the camera and maintaining a constant distance between muscles, and the weight measurement value was corrected to body weight.

As a result, as shown in Fig. 8, the results of examining four muscles (quadriceps femoris, tibialis anterior, gastrocnemius, and soleus) isolated from each mouse showed that, compared to the normal group, the group in which sarcopenia was induced by dexamethasone administration showed muscle loss in all four muscles. Meanwhile, in the case of the group administered the low-molecular-weight protein hydrolysate of the defatted hemp seed meal of the present invention (HPH-L; 250 mg/kg and HPH-H; 500 mg/kg), muscle loss was improved and muscle mass was improved again to a level similar to the normal level.

<Example 4>

Analysis of the effect of low molecular weight protein hydrolysate obtained from defatted hemp seed meal on muscle fiber reduction

In the above examples, the calf and thigh muscles were separated from the mouse experimental group used in the experiment. The calf and thigh muscles were refrigerated in 10% formalin immediately after dissection for tissue fixation, and for tissue staining, each muscle was thinly sliced into 4–5 μm thicknesses and stained in the order of hematoxylin for 2 minutes and eosin for 1 minute. Afterwards, photographs were taken under an optical microscope (Leica Microsystems, Wetzlar, Germany) for histological analysis, and the cross-sectional area (CSA) of the muscle fibers was quantified using Image j software (64-bit Java 1.8.0_172).

As a result, it was shown that the decrease in muscle fibers was significantly increased in the calf and thigh muscles of mice in which muscle atrophy was induced compared to the normal group, whereas the group administered the low-molecular-weight protein hydrolysate obtained from the defatted hemp seed meal of the present invention showed effective inhibition of the decrease in muscle fibers, and in particular, it was confirmed that the group treated with the hydrolyzed low-molecular-weight protein hydrolysate showed more effective inhibition of muscle fiber decrease in both the calf and thigh than the group treated with the protein extract extracted from the defatted hemp seed meal (before hydrolysis treatment; HPI), and it was shown that the decrease in muscle fibers was inhibited in a concentration-dependent manner by the low-molecular-weight protein hydrolysate treatment (Figs. 9 and 10).

Through the above results, the inventors of the present invention were able to find out that the low molecular weight protein hydrolysate of defatted hemp seed meal produced by the method of the present invention can effectively improve, prevent or treat muscle atrophy or muscle loss.

<Example 5>

Analysis of the effect of low-molecular-weight protein hydrolysate obtained from defatted hemp seed meal on the expression of proteins related to muscle breakdown and muscle synthesis

The effect of the low-molecular-weight protein hydrolysate obtained from the defatted hemp seed meal according to the present invention on the expression of proteins related to muscle decomposition and muscle synthesis was performed through Western blot analysis.

To this end, RIPA buffer was added to the calf and thigh muscles (samples stored at -40℃ or lower) collected from each mouse experimental group, homogenized, and centrifuged to obtain the supernatant, and the concentration of protein contained in the supernatant was quantitatively analyzed using the BCA protein assay method. Afterwards, electrophoresis was performed on 8% or 12% SDS-PAGE, and the proteins were transferred to 0.45 μm polyvinylidene difluoride membranes. After the membranes were blocked with 3% BSA and 0.1% Tween-20 solution for 1 hour at room temperature, primary antibodies were added, and reacted overnight at 4°C, washed with TBS-T, and horseradish peroxidase secondary antibodies were added and reacted for 1 hour. The expressed protein levels were then confirmed using a Fusion Solo 6S EDGE imaging system (Vilber, Marne-la-Vallee, France) and quantified using Fusion-capt software (Vilber).

The primary antibodies used at this time were Myf5 (ab125078; Abcam, Cambridge UK), MyoD (MA1-41017; Invitrogen, USA), MHC (sc-376157; Santa Cruz Biotechnology, USA), MuRF1 (ab183094; Abcam, Cambridge, UK), and MAFbx (ab168372; Abcam, Cambridge, UK).

The analysis results showed that in the case of calf muscles, the protein expression levels of MuRF1 and MAFbx, which are ubiquitin ligase E3 transferases that induce muscle atrophy, were decreased in a treatment concentration-dependent manner in the group treated with hemp seed protein hydrolysate (HPH), whereas the expression of Myf5, MyoD, and MyHC, which are involved in muscle differentiation and synthesis, was increased (Fig. 11).

In addition, in the case of thigh muscles, similar to the results for calf muscles, the protein expression levels of muscle atrophy regulatory factors were found to be decreased in the group treated with hemp seed protein hydrolysate (HPH), whereas the expression levels of proteins involved in muscle differentiation and synthesis were found to be increased (Fig. 12).

In addition, the results as above were confirmed to be significantly increased in the group treated with hemp seed protein hydrolysate (HPH) compared to the group treated with hemp seed defatted protein extract (HPI), which is a sample before hemp seed protein hydrolysis.

<Example 6>

Analysis of the effect of low-molecular-weight protein hydrolysate obtained from defatted hemp seed meal on the expression of apoptosis-related proteins

Furthermore, the inventors of the present invention analyzed the effect of low-molecular-weight protein hydrolysate obtained from the defatted hemp seed meal of the present invention on the expression of proteins related to apoptosis.

To this end, Western blot was performed in the same manner as in <Example 5> for calf muscle samples obtained using the same method, except that Bax (2772, Cell signaling, USA), Bcl-2 (ab692; Abcam, Cambridge UK), and Cleaved Caspase-3 (Asp175, Cell signaling, USA) were used as primary antibodies, respectively, to analyze the expression levels of Bax, Bcl-2, and Cleaved Caspase-3 proteins.

As a result, as shown in Fig. 13, in the case of the muscle atrophy-induced group (DEX), the expression of caspase-3, an apoptosis-inducing factor, was found to significantly increase compared to the normal group (NC), whereas in the groups treated with the hemp seed protein hydrolysate of the present invention (HPH-L, HPH-H), the expression of caspase-3 was found to decrease, and the expression of Bax, an apoptosis-inducing factor, was found to be effectively inhibited, whereas the expression of Bcl-2, known as a factor that inhibits apoptosis, was found to increase. On the other hand, in the group treated with the hemp seed defatted protein extract, which is a sample before hemp seed protein hydrolysis (HPI), no significant effect, similar to the group treated with the hemp seed protein hydrolysate, was found.

These results imply that the low-molecular-weight protein hydrolysate obtained from the defatted hemp seed meal according to the present invention can effectively inhibit cell death occurring in areas where muscle atrophy or sarcopenia has occurred, thereby maintaining muscle mass and improving muscle strength.

The present invention has been described with reference to preferred embodiments thereof. Those skilled in the art will appreciate that the present invention may be implemented in modified forms without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated by the claims, not the foregoing description, and all differences within the scope equivalent thereto should be interpreted as being included in the present invention.

Claims (6)

(1) A step of adding distilled water to defatted hemp seed meal, adjusting the pH to 10 to elute protein, centrifuging to obtain only the supernatant, and then adjusting the pH of the supernatant to 5 to precipitate the eluted protein to obtain a protein extract of defatted hemp seed meal;
(2) a pretreatment step of sequentially performing ultrasonic treatment and heat treatment after adding distilled water to the protein extract of the defatted hemp seed meal of step (1); and
(3) A step of adding a hydrolytic enzyme to the protein extract of the defatted hemp seed meal, which has undergone the pretreatment of step (2) above, and reacting the same to obtain a low-molecular-weight protein hydrolysate of the defatted hemp seed meal,
In the above step (2), ultrasonic treatment is performed at 250 to 350 W for 10 to 20 minutes, and heat treatment in the above step (2) is performed at 90 to 110°C for 10 to 20 minutes.
The step (3) of adding and reacting the hydrolytic enzyme is characterized by sequentially treating and reacting alcalase and flavourzyme, adding the enzyme in an amount of 3 to 5 wt% relative to the substrate weight, and hydrolyzing at a temperature of 45 to 55°C and pH 7 to 8 for 1 to 2 hours.
A method for producing a low-molecular-weight protein hydrolysate of defatted hemp seed meal having muscle strengthening activity or improving muscle atrophy or sarcopenia. delete delete In the first paragraph,
A method characterized in that the low-molecular-weight protein hydrolysate of the above defatted hemp seed meal contains a hydrolyzed low-molecular-weight peptide of 3 kDa or less. A food composition for improving muscle atrophy or sarcopenia or strengthening muscles, comprising a low-molecular-weight protein hydrolysate of defatted hemp seed meal manufactured by the method of claim 1 as an effective ingredient. A pharmaceutical composition for preventing or treating muscle atrophy or sarcopenia, comprising a low molecular weight protein hydrolysate of defatted hemp seed meal manufactured by the method of claim 1 as an effective ingredient.