KR20190048794A - Comppsition comprising mori folium extracts for preventing, treating muscular dystrophy - Google Patents

Abstract

The present invention relates to a composition for prevention and improvement of muscle atrophy comprising an extract of Aspergillus oryzae as an active ingredient. According to the composition, by inhibiting the activity of AMP-activated protein kinase (AMPK) , It is possible to prevent and improve symptoms of atrophy.
In particular, the extract of the upper leaves which prevents and improves the symptoms of the atrophy has the advantage that the effect of improving the atrophy is very excellent and there is no side effect due to use.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for preventing and improving muscle atrophy comprising an extract of Aspergillus niger as an effective ingredient. BACKGROUND ART [0002]

The present invention relates to a composition for preventing and ameliorating atrophy of a muscle containing an upper leaf extract as an active ingredient. More specifically, a variety of functional foods for prevention and improvement of muscle atrophy, which contains an active ingredient of a leaf extract that can inhibit muscle atrophy through inhibition of the activity of AMP-activated protein kinase (AMPK) So that it can be used in the field.

Muscle atrophy, a phenomenon caused by intracellular changes such as activation of proteolytic enzymes, ubiquitin conjugation and autophagy, causes aging and malnutrition as well as various diseases such as sepsis and cancer. Is known to be induced.

When muscle atrophy is induced, there are phenomena such as decrease of protein content through muscle protein degradation, decrease of muscle fiber due to reduction of muscle fiber diameter, and reduction of fatigue resistance.

 In particular, the atrophy induced by aging is caused by a decrease in protein synthesis and an increase in protein degradation. As a result, exercise capacity and gait disorder are induced, which not only worsens quality of life significantly but also increases mortality Many researches have been carried out to clarify the related mechanism to overcome this.

AMP-activated protein kinase (AMPK) is a serine-threonine heterotrimeric kinase, an important regulator and energy sensor that regulates energy balance. AMPK activates by Thr172 phosphorylation according to the increase of AMP / ATP ratio, it inhibits the energy consumption process and also regulates glucose and lipid metabolism by inducing glucose uptake and fatty acid oxidation .

Recent studies have shown that AMPK activation activates muscle-specific ubiquitin ligases such as muscle atrophy F-box (MAFbx) / atrogin-1 and muscle RING fingers via the forkhead box O3a (FoxO3a) (MuRF1), which is involved in the activation of the ubiquitin-proteasome pathway, is directly involved in muscle atrophy induced by muscle protein degradation.

In addition, when muscle atrophy is induced, the expression of MyoD and myogenin, which are muscle-specific transcription factors involved in myogenic differentiation, is known to be relatively decreased

Although the pharmacological composition has been developed as a natural material, most of the products have been commercialized using the extract, and thus the exact mechanism of pharmacological action has not been clarified. However, since it is easy to take as an oral preparation and has little toxicity and side effects, .

Under these circumstances, it is urgent to develop a composition of a natural material capable of inhibiting muscle atrophy through inhibition of the activity of AMP-activated protein kinase (AMPK).

(Patent Document 1) KR 10-1770035 B1 (Patent Document 2) KR 10-2013-0005118 A1

It is an object of the present invention to provide a method for preventing or ameliorating atopic dermatitis, comprising the extract of a mulberry leaf which can prevent and improve the symptoms of atrophy through inhibiting the activity of AMP-activated protein kinase (AMPK) And a composition for improvement.

Another object of the present invention is to provide a composition for preventing and improving muscle atrophy comprising the above leaf extract.

The present invention also provides a functional tea or other functional food comprising the composition for preventing or ameliorating atrophy comprising the topical extract as an active ingredient to provide a food having high palatability as well as prevention or improvement of atrophy will be.

In order to accomplish the above object, the composition for preventing and improving muscle atrophy according to one embodiment of the present invention comprises a composition for preventing and improving muscle atrophy comprising an extract of a top leaf extract containing Mori Folium extract as an active ingredient to be.

The topical extract may be extracted with a solvent selected from the group consisting of water, alcohols having 1 to 6 carbon atoms, and mixtures thereof.

The atrophy is caused by at least one of disuse atrophy of muscle, mechanical stimulation member, and denervation of skeletal muscle.

The composition for preventing and improving muscle atrophy may further comprise a silk-tree extract.

The composition for preventing or ameliorating atrophy may further comprise Chinese horned Holly extract.

The pharmaceutical composition according to another embodiment of the present invention may include a composition for preventing and ameliorating atrophy, which comprises a topical extract as an active ingredient.

The food composition according to another embodiment of the present invention may include a composition for preventing and ameliorating atrophy, which comprises a topical extract as an active ingredient.

The tea according to another embodiment of the present invention may include a composition for preventing and improving muscle atrophy comprising an extract of the upper part of the leaf as an active ingredient.

Hereinafter, the present invention will be described in more detail.

The composition for prevention and improvement of muscle atrophy according to one embodiment of the present invention may contain Mori Folium extract as an active ingredient.

Mori Folium is a dried leaf of Moru salba L. It has been used since ancient times for the symptoms of swelling of the body, swelling of the body, cuts, cold sweat, and paralysis. Recent studies have shown that the top lobes include rutin, quercetin, isoquercetin, moracetin, deoxynojirimycin, calistegin, GABA, morusin, And umbelliferone have been reported to have various pharmacological effects such as antibacterial, antioxidant, anti-inflammatory, anti-anxiety, anti-diabetic, hypoglycemic, hyperlipidemic, and anti-obesity have. In particular, rutin, which is one of the major components contained in the leaves, is known to have triglyceride and cholesterol lowering effects. Quercetin is known to reduce DNA damage caused by oxidative stress, And 1-deoxynojirimycin (1-DNJ) has been reported to have a function of inhibiting blood glucose increase through inhibition of? -Glucosidase. As shown in the results of the present study, the top leaf is a functional substance having various pharmacological actions, but there is no study on the effect of inhibiting the atrophy and the molecular biologic mechanism thereof.

In the present invention, tongue refers to a product which is not roasted and roasted without addition of water. In addition, the upper leaves have a higher flavor than the normal upper leaves, so that the flavor can be enhanced and the AMP- Is effective for enhancing the effect of preventing or ameliorating atrophy because the active ingredient inhibiting the activity of protein kinase (AMP-activated protein kinase, AMPK) or its activity is promoted.

In the present invention, " prevention " means any action that inhibits or slows the onset of atrophy by administration of the composition, and " treatment " means that the administration of the composition improves or mitigates muscle atrophy Means

The atrophy may be caused by one or more of disuse atrophy of muscle, mechanical stimulation member, and denervation of skeletal muscle.

The above-mentioned disuse atrophy of muscle means that the muscle thickness is reduced by restricting the activity due to aging, disease, long bed life, casting, and the like. The above leaf extract has excellent improvement effect on insoluble muscle atrophy.

The atrophy is accompanied by various clinical diseases such as peripheral nerve injury, cancer and hematemesis, as well as long-term bed rest, limb gypsum fixation and aging process. In general, muscle atrophy is accompanied by physiological, histochemical and biochemical changes resulting from a decrease in muscle protein, leading to impairment of the inherent function of the skeletal muscle.

The denervation of the skeletal muscle is accompanied by a decrease in muscle mass and muscle strength, a decrease in the thickness of the muscle fiber, a change in the muscle fiber type in which the root fiber is transformed into the inner muscle fiber, ≪ / RTI >

This mechanism of muscle atrophy is due to the increase in apoptosis of muscle cells due to oxidative damage, because mitochondria decrease in mitochondria and increase in ROS production in mitochondria due to a decrease in function in the denervated dominant muscle cells .

In the present invention, the upper leaf extract may be extracted with a solvent selected from the group consisting of water, an alcohol having 1 to 6 carbon atoms, and a mixed solvent thereof. The term 'upper leaf extract' means an extract obtained by extracting upper leaves.

The upper leaf lyophilized product can be eluted with a polar solvent such as an alcohol having a carbon number of C ₁ to C ₆ such as water, ethanol, methanol or the like, or a mixed solvent having a mixing ratio of alcohol and water of 1: 0.1 to 1:10 , Preferably 1: 3 to 5, in a mixed solvent having a mixing ratio of ethanol and water. At this time, the extraction temperature may be an extract extracted at 10 to 100 캜, preferably at room temperature, and for an extraction period of 12 to 4 days, preferably 24 hours.

The extract may be a result obtained by concentration under reduced pressure with a vacuum rotary concentrator. However, it is not limited to the extract of the present invention, which can exhibit the effect of preventing and improving the atrophy of the present invention, but the extract, diluent or concentrate of the extract, A dried product obtained by drying the extract, or a controlled preparation or a purified product thereof.

The upper leaves are superior to the stem, root, and fruit of mulberry trees for prevention and improvement of muscle atrophy. Therefore, it may be preferable to use an ethanol extract for the upper leaves.

More specifically, the ethanol may be 12 to 22 wt%. The extract according to the above range may be most advantageous for obtaining the active ingredient.

On the other hand, the upper leaf extract may be one obtained by preparing an extract using the upper leaves and then fermenting the extract. The fermentation of leaf extract has the advantage of being able to be used stably at a relatively high concentration because of low cytotoxicity.

Examples of the microorganism used for the fermentation include Lactobacillus salivarius, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus rhamnosus, Lactobacillus plantarum Lactobacillus plantus, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus fermentum, Lactobacillus paracasei, Lactobacillus casei, Lactobacillus delbrueckii, Such as Lactobacillus reuteri, Lactobacillus buchneri, Lactobacillus gasseri, Lactobacillus johonsonii, Lactobacillus kefir, and the like, lactic acid bacteria such as lactobacillus kefir, Lactococcus lactis, Lactococcus plantarum, Lactococcus raffinolactis, Enterococcus faecalis, Enterococcus faecium, Streptococcus thermophilus, Streptococcus spp. Bifidobacterium bifidum, Bifidobacterium bifidum, Bifidobacterium bifidum, and Bifidobacterium bifidum, such as Leuconostoc lactis, Leuconostoc mesenteroides, and the like, Bifidobacterium species such as Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium pseudolongum, Bifidobacterium themophilum, Bifidobacteria such as Bifidobacterium adolescentis, and the like, Lactobacillus casei, Lactobacillus rhamnosus, Bifidobacterium bifidum bifidobacterium, Bifidobacterium breve bifidobacterium, and Lactobacillus species such as Lactobacillus casei, Lactobacillus rhamnosus, And Lactobacillus acidophilus. ≪ Desc / Clms Page number 2 >

The composition for preventing or ameliorating atrophy may further comprise a silk-tree extract.

The silk tree is a tree symbolizing a couple's gold thread. It is also called a joining tree, a sum-comer, Yaesu, and Yu Jung-soo. Because of this, I planted a lot of trees growing in mountains and fields in the garden as a garden. Because it was a tree used to make the handle of a swordfish, it is called a silkworm tree.

Also, the stem of the tree is bent or slightly leaned. It is 3 ~ 5m in height, large branches spread sparsely and small branches have ridges. There are 2 or 3 scars of winter snow, but it is hardly visible. Leaves are alternate and double leafy leaves. The small leaf is like a sickle and has a long oval shape with uneven left and right sides and flat edges. The small leaf has a length of 6 to 15 mm and a width of 2.5 to 4.0 mm. There are no hairs on both sides or hairs on the back vein.

The flower is pink to bloom in June or July, and it runs in 15-20 squares on the tip of a small branch. Calyx and corolla are shallowly divided into 5 pieces and turn green. Surgery is about 25, it comes out long and the upper part is scarlet. It is because of the color of the operation that the flower looks red. Fruits are ripened in late September to early October and are flat pods, about 15cm long and contain 5 or 6 seeds. The unusual thing is that the nerves and Mimosa are attached to the external stimuli, but when they are sunny, the leaves are folded facing each other.

In the case of using the above extracts of japanese squirrel, the effect of inhibiting the activity of AMP-activated protein kinase (AMPK) is increased according to the synergistic effect according to the mixing use. Therefore, it is possible to exert an excellent effect in prevention and improvement of muscle atrophy with a smaller amount.

The composition for preventing or ameliorating atrophy may further comprise Chinese horned Holly extract.

It is also called aphids. It grows on the shore of a low mountain on the beach. It is 2 ~ 3m in height and has no branches. Leaves are slanting, thick, shiny, oval hexagonal, and each point is sharp. Flowers bloom in 4 or May, and there are fragrance, and 5 or 6 of them hang on a leaf axil. The pistil has no style, and the stigma is slightly elevated to divide into 4 pieces and become black. Fruits are round and have a diameter of 8 to 10 mm and ripen red in 9 or October. There are four seeds, ovate and mature.

Leaves are used for strong winds, tangles, etc. Fruits are used for consonants, The breeding takes the seeds ripe in autumn and sows in spring. It is distributed in South Korea (South Jeolla Province) and South China.

In the case of using the above extracts of rhododendron japonica and Rhododendron japonica in combination, the active ingredient having the effect of preventing or ameliorating atrophy is synergistically caused by the mutual reaction of the extracts, It is possible to exhibit a synergistic effect over a simple combination of extracts.

Preferably, the composition for preventing or ameliorating atrophy comprises 1 to 15 parts by weight of a rhizome extract and 10 to 40 parts by weight of a rhododendron extract in 100 parts by weight of the leaf extract. In the above range, a synergistic effect with a critical significance is expressed by the synergistic effect due to the interaction of the respective extracts, and when the concentration is outside the above range, the synergistic effect is significantly lowered or hardly occurs.

More preferably, the composition for preventing or ameliorating atrophy comprises 1 to 15 parts by weight of a rhizome extract, 10 to 40 parts by weight of a rhododendron extract, and 1 to 15 parts by weight of a rhizome extract, based on 100 parts by weight of the leaf extract .

In the above range, the effect of inhibiting the activity of AMP-activated protein kinase (AMPK) is activated due to a higher synergistic effect due to the mixing action of the respective extracts, so that prevention and improvement of muscle atrophy can be enhanced have.

The Kirilow indigo (Kirilow indigo) grows frequently on the shore, about 1m high and has many shoots from its roots. Several stalks are rising and there are line-shaped projections on the branches. Leaves are alternate and odd numbered haploid leaf. Small leaves are 7 ~ 11 thick, round, elliptical or inverted egg-shaped, with hairs on both sides. A red-purple flower about 2cm in length blooms in May or June, and it forms a flower-like flower on the axilla. On the other hand, calyx is about 3mm long and there is hairs on the outer surface of the board (旗.). The fruit is in the shape of a string with a cone and ripens in October. It is distributed all over the country excluding Manchuria, China and North Hamgyong Province. It is also used for beekeeping and farming. It is called "coreana". It is called "coreana." It is a species similar to a flower, and its ear is twice as long as a leaf.

Preferably, the composition for preventing or ameliorating atrophy further comprises a Yellow melilot extract, a Pachysandra terminalis extract, and a Hosta longipes extract. When the above extract is further included, the sweetness of the upper leaves is neutralized and the flavor is improved by mixing with other extracts, so that a food composition having higher palatability can be provided.

Yellow melilot is also called Mitsutosugi or Melitoseus. It is the northern part of China. It grows in low-lying meadows. It is 60 to 90 cm high and branches are split and smelled when dried. There are hairs in my childhood, but they disappear later. Leaves are alternate phyllotaxis of three small leaves. Leaves are long oval or inverted, and have small sawtooth edges. The flower blooms in July or August and it is yellow, and it runs tightly with gunshot at the tip of the branch or the axilla. Calyx has hairy hairs. The fruit is egg-shaped, has no hair, and is black. The entire paste is used for heat, heat, nephritis, etc. It is called white algae (M. alba) that flowers are smaller and white. Everything that grows as a grass resource grows wild and grows.

The Pachysandra terminalis grows in the shade of the tree, with the main stem hanging sideways, the tip is straight, green, and initially there is hairy hair but gradually disappears. It grows up to about 30cm in height. Leaves are alternate but they are gathered in the upper part and look like inverted egg, with sawtooth on the upper part. Leaf hairs on leaf vein and narrowed lower part to become petiole. Flowers also bloom in April or May and are white and run on aquatic inflorescences. The female flower runs slightly under the flower head and the male flower runs on the upper part. Calyx is divided into 4 pieces and there are no petals. The stamen is divided into 3 to 5 and the style is divided into 2 pieces. The fruit is ovate, egg-shaped and has no hairs on the surface. It is distributed in Korea, Japan, Sakhalin Island, China.

Hosta longipes grow well in mountain streams or wet places and are 30 to 40 cm high. All leaves grow from the roots and grow at an angle. Leaves are elliptical, pointed end, and have 8 or 9 veins. The flower is light purple and blooms in July or August. It is biased to one side and runs on a gun. The flower stem is 30 to 40cm long. The bract is thin, purplish white and almost similar to the peduncle. The corolla is divided into 6 pieces, and the pieces are bent back a little, and 6 stamens and 1 pistil come out of the flower for a long time. Fruit is long oval with capsule. Seeds are black with wings on the edge. It is edible for soft season and planted for ornamental purposes. Wild species are distributed in Korea, Japan, and China. Vivicula is a kind of horticultural variety and has been popular as a garden plant in foreign countries.

Preferably, the composition for preventing or ameliorating atrophy comprises 2 to 3 parts by weight of an electric sphygmomanometer extract, 3 to 12 parts by weight of a preservative extract, and 3 to 12 parts by weight of a non-vegetable extract, based on 100 parts by weight of the leaf extract .

By the above range, it is possible to provide a functional food composition which is excellent in palatability and has an excellent effect of preventing and improving muscle atrophy by improving the inhibition of the activity of AMP-activated protein kinase (AMPK) Can be provided. In particular, it has a high advantage of being highly palatable and widely spread.

More preferably, the composition for preventing or ameliorating atrophy comprises 1 to 15 parts by weight of a rhizome extract, 10 to 40 parts by weight of a rhododendron extract, and 1 to 15 parts by weight of a rhizome extract, based on 100 parts by weight of the leaf extract, 1 to 3 parts by weight of an electric sphagnum extract, 3 to 12 parts by weight of a succulent superabundance, and 3 to 12 parts by weight of a non-avocado extract.

In the above range, the effect of inhibiting the activity of AMP-activated protein kinase (AMPK) is greatly enhanced by the synergistic effect due to the mixing action of each extract, thereby maximizing the effect of preventing and improving muscle atrophy The flavor according to each combination is excellent and the palatability is very high, so that it can be provided as a food composition having high functionality and palatability.

The pharmaceutical compositions of the present invention may comprise a pharmaceutically acceptable carrier. Compositions comprising a pharmaceutically acceptable carrier can be of various oral or parenteral formulations. In the case of formulation, it can be prepared using diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants and the like which are usually used. Solid formulations for oral administration may include tablet pills, powders, granules, capsules and the like, which may contain one or more excipients such as starch, calcium carbonate, sucrose or lactose lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent and the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

In addition, the pharmaceutical composition of the present invention may be formulated into tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations and suppositories And can have any one of the formulations selected.

The topical extract of the present invention has been used for edible and medicinal purposes from the past, and there is no particular limitation on the dosage of the topical extract of the present invention. The amount of the extract of the topical extract of the present invention, , The severity of the disease, and the like. In general, the leaf extract is preferably administered in an amount of about 10 to 1000 mg per kg of body weight, and more preferably about 50 to 500 mg per kg of body weight. The pharmaceutical composition comprising the extract of the present invention as an active ingredient is prepared in consideration of an effective dose range, and the unit dosage formulations thus formulated are classified according to the judgment of an expert who monitors or observes the administration of the drug, , Or may be administered several times at regular intervals.

The present invention also provides a quasi-drug composition for preventing or ameliorating atrophy involving a topical extract as an active ingredient. The production and composition of the above leaf extract are as described above. More specifically, the topical extract of the present invention can be added to a quasi-drug composition for the purpose of preventing or treating atrophy.

In the present invention, the term 'quasi-drug' means a fiber, a rubber product or the like used for the purpose of treating, alleviating, treating or preventing a disease of a human or an animal, a weak action against the human body, Used for the purpose of diagnosing, curing, alleviating, treating or preventing diseases of human or animal, which are not machinery, similar products, products for sterilization, insecticide and similar uses for the prevention of infectious diseases. Machinery, or apparatus, and that is not an apparatus, machine or apparatus of an article used for the purpose of causing pharmacological effects on the structure or function of a person or animal.

When the topical extract of the present invention is used as a quasi-drug additive, the topical extract may be directly added or used in combination with other quasi-drugs or quasi-drugs, and may be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be appropriately determined depending on the purpose of use.

The quasi-drug composition of the present invention is not limited thereto, but may be a disinfectant cleaner, shower foam, gagrin, wet tissue, detergent soap, hand wash, humidifier filler, mask, ointment or filter filler.

The present invention also provides a health functional food composition for preventing or ameliorating atrophy involving an extract of a topical composition as an active ingredient. The production and composition of the above leaf extract are as described above. More specifically, the topical extract of the present invention can be added to a health functional food composition for the purpose of preventing or treating atrophy.

When the leaf extract of the present invention is used as an additive for health functional food, the leaf extract can be used as it is or can be used in combination with other health functional food or health functional food ingredient and can be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be appropriately determined depending on the purpose of use.

There is no particular limitation on the kind of the health functional food of the present invention. Examples of the health functional food to which the above extraction mixture can be added include meat products, sausages, breads, chocolates, candies, snacks, confectionery, pizza, ramen noodles, other noodles, gums, dairy products including ice cream, , A drink, an alcoholic beverage, and a vitamin complex, and may include foods used as food for animals, which may include all health functional foods in the conventional sense. In addition to the above, the health functional food composition of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, , Alcohols, carbonating agents used in carbonated drinks, and the like. It may also contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks.

The present invention also provides a method for treating atrophy, comprising the step of administering the composition to a subject suspected of atrophy other than human.

In the present invention, the suspicious individual of the atrophy refers to all animals in which the atrophy has occurred or can develop, and by administering the pharmaceutical composition containing the leaf extract of the present invention to suspect individuals with atrophic diseases, have.

In the present invention, 'administering' means introducing the pharmaceutical composition of the present invention to a suspect individual with an appropriate atrophy by any appropriate method, and the administration route may be administered through various oral or parenteral routes as long as it can reach the target tissue .

The method of treatment of the present invention may include administering a pharmaceutical effective amount of a pharmaceutical composition comprising the leaf extract as an active ingredient. The appropriate total daily dose may be determined by the treatment within the scope of appropriate medical judgment, and may be administered once or several times. For purposes of the present invention, however, the specific therapeutically effective amount for a particular patient will depend upon the nature and extent of the reaction to be achieved, the particular composition, including whether or not other agents are used, the age, weight, Sex and diet of the patient, the time of administration, the route of administration and the rate of administration of the composition, the duration of the treatment, the drugs used or concurrently used with the specific composition, and similar factors well known in the medical arts.

According to the composition of the present invention, it is possible to prevent and improve the symptoms of atrophy by inhibiting the activity of AMP-activated protein kinase (AMPK) which regulates energy balance.

In particular, the extract of the upper leaves which prevents and improves the symptoms of the atrophy has the advantage that the effect of improving the atrophy is very excellent and there is no side effect due to use.

Therefore, when using the leaf extract of the present invention for preventing and improving the symptoms of the atrophy according to the present invention, as an antibacterial effect of the leaf of the present invention, or for improving the muscle atrophy effect, And various pharmaceutical preparations such as pharmaceutical preparations can be manufactured, and thus, it is possible to widely utilize the top leaves.

FIG. 1 shows the result of counting the number of living cells using a hemocytometer after treating AICAR with C2C12 myoblasts for 24 hours and trypan blue staining.
FIG. 2 is a result of MTT assay to examine the degree of inhibition of AICAR-induced growth of C2C12 myoblasts treated with AICAR for 24 hours.
FIG. 3 shows the results of DAPI staining to identify changes in the morphology of nuclei treated with AICAR in C2C12 myoblasts for 24 hours and induce apoptosis.
FIG. 4 shows the results of flow cytometry after treating AICAR with C2C12 myoblasts for 24 hours and staining with annexin V-FITC / PI.
FIG. 5 is a graph showing the results of AICAR-induced apoptosis by staining cells with ITC-conjugated Annexin V and PI for DNA flow cytometry. Annexin V ⁺ / PI ^- cells and each annexin V ⁺ / PI ^- cells.
Figure 6 relates to the level of expression of atrophy-treated proteins by AICAR treatment of C2C12 myofibroblasts.
Figure 7 relates to the level of expression of atrophy-treated proteins by AICAR treatment of C2C12 myoblasts.
FIG. 8 shows results of morphological characteristics of C2C12 canaliculus cells according to AICAR treatment.
Fig. 9 shows the results of C2C12 root canal cell myofilamus according to AICAR treatment.
Figure 10 shows the effect of muscle-specific ubiquitin ligases and muscle-specific transcription factors on the expression of muscle-specific ubiquitin ligases in association with AICAR-induced atrophy, As shown in FIG.
Figure 11 shows the effect of muscle-specific ubiquitin ligases and muscle-specific transcription factors on the expression of muscle-specific ubiquitin ligases in association with AICAR-induced atrophy, As shown in FIG.
FIG. 12 is a graph showing the effect of AICAR-induced AICAR-induced apoptosis on C2C12 root canaliculus cells. The results were shown in FIG. 12, The results of this study are as follows.
FIG. 13 is a graph showing the effect of AICAR-induced AICAR-induced apoptosis on C2C12 root canal cells. After setting the conditions without cytotoxicity according to AICAR treatment, the C2C12 muscle fiber diameter As shown in FIG.
FIG. 14 is a graph showing the effect of AICAR-induced AICAR-induced apoptosis on C2C12 root canaliculus cells. After setting the conditions without cytotoxicity according to AICAR treatment, And the microscopic photographs of the muscle fiber diameter.
Fig. 15 shows the result of measuring the diameter of a muscle fiber in a randomly selected field using an image analysis program.
FIG. 16 relates to the results of confirming the change of genes associated with the inhibition of atrophy by the extract of the upper leaves.

Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily carry out the present invention. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

[Preparation Example: Preparation of leaf extracts]

To obtain ethanol extract of Mori Folium (EEMF), 1 L of ethanol was added per 100 g of the upper leaves, and the mixture was stirred at 60 ° C. and 150 rpm for 3 days, and centrifuged at rpm for 20 minutes to separate only the supernatant. The separated supernatant was filtered with a Whatman filter (No. 2), and the filtrate was concentrated under reduced pressure to obtain a solid component. The supernatant was finely ground with a mortar and sealed and stored in a -70 ° C cryocooler. In the experiment, 100 mg / ml was prepared by using dimethyl sulfoxide (DMSO, Amresco, Solon, OH, USA) and diluted in the medium to the appropriate concentration.

[Experimental Example: Test for improving artificial muscle atrophy using AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide]

1. Experimental Method

Experimental material

In this experiment, Dulbecco's modified eagle's medium (DMEM), fetal bovine serum (FBS), horse serum (HS), penicillin / Streptomycin (PS) was purchased from WELGENE (Daegu, Korea), and AICAR and 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (3- (Sigma-Aldrich Chemical Co., St. Louis, MO, USA) was purchased from Sigma-Aldrich Chemical Co., AMPK, p-AMPK, and acetyl-coA carboxylase, which are primary antibodies used for protein analysis, were purchased from Bioneer (Daejeon, Korea) ACC), p-ACC, FoxO3a, pFoxO3a, MAFbx / atrogin-1, MuRF1, MyoD, myogenin and actin as well as horseradish peroxidase Horseradish peroxidase (HRP) -conjugated anti-mouse and anti-rabbit antibodies were purchased from Santa Cruz Biotechnology Inc., Santa Cruz, Calif., USA Respectively.

Induction of C2C12 myoblast differentiation and atrophy

C2C12 myofibroblasts were purchased from the American Type Culture Collection (Manassas, Va., USA) and cultured in DMEM supplemented with 10% FBS and 100 units / ml PS at 37 ° C and 5% CO 2 Lt; / RTI > In order to differentiate C2C12 myoblasts into canaliculus cells, 100% confluent cells were replaced every 2 days with differentiation medium containing 90% DMEM, 2% HS and 100 unit / ml PS, AICAR at the appropriate concentration was treated for 24 hours to induce atrophy. In order to confirm the morphological change during the atrophy process, we observed the myotube diameter (myotube diameter) in order to confirm the degree of the atrophy induced by using an inverted microscope (Carl Zeiss, Gottingen, Germany) And then analyzed using an image analysis program.

Measurement of cell viability using a hemocytometer

To determine the survival rate of C2C12 myofibroblasts by treatment with AICAR and EEMF, AICAR and EEMF were treated at appropriate concentrations in C2C12 cells in 100% confluent state. After a certain period of time, the medium was removed and the cells were treated with 0.05% trypsin-ethylene diamine tetraacetic acid (EDTA, Sigma-Aldrich Chemical Co.) to suspend the cells. The cells were centrifuged at 2,000 rpm for 5 minutes The supernatant was removed. The cells were washed with 1 ml of phosphate buffered saline (PBS) and 0.5% trypan blue solution (Gibco BRL, Grand Island, NY, USA) for 2 min. And applied to a hemocytometer. Live cells were counted using an inverted microscope.

Growth inhibition assay using MTT assay

In order to confirm the degree of inhibition of AICAR and EEMF-induced growth in C2C12 myofibroblasts, the cells were treated with the same method as above, and the medium was subcutaneously dispensed with 2 ml of 0.5 mg / ml MTT CO ₂ incubator. After 2 hours, the MTT reagent was removed and DMSO was added in an appropriate amount. All of the generated formazin was dissolved in the wells, and 200 μl of the solution was transferred to a 96-well plate. The plate was transferred to an ELISA reader (Molecular Devices , Sunnyvale, CA, USA) at 540 nm.

Quantitative measurement of apoptosis by staining of annexin V-fluorescein isothiocyanate (FITC)

For the quantitative analysis of apoptosis induction by AICAR treatment in C2C12 myofibroblasts, the prepared cells were collected, and then subjected to anabsin V binding buffer containing 10 mM HEPES / NaOH, pH 7.4, 140 mM NaCl and 2.5 mM CaCl ₂ (Annexin V-FITC) and propidium iodide (PI) were suspended in an annexin V binding buffer (Becton Dickinson, San Jose, Calif., USA) Lt; / RTI > for 20 minutes. After completion of the reaction, the cells were subjected to flow cytometry (FACS Calibur, Becton Dickinson), and the cells (annexin V + / PI-) induced apoptosis were analyzed by fluorescence reaction.

Confirmation of induction of apoptosis by DAPI staining

In order to observe morphologic changes of nuclei that were induced when apoptosis was induced, the cultured cells were treated with a fixing solution (3.7% formaldehyde in PBS) at various concentrations of AICAR for 24 hours And fixed at room temperature for 10 minutes. After the fixation, the cells were attached to a slide glass and incubated at room temperature for 10 minutes with 0.2% Triton X-100 (Amresco). Then, 2.5 μg / ml of 4 ', 6-diamidino-2-phenylindole 4 ', 6-diamidino-2-phenylindole, DAPI, Sigma-Aldrich Co.) for 15 minutes. After staining, the morphology of cancer cells was observed at 400x magnification using a fluorescence microscope (Carl Zeiss).

Analysis of protein expression by Western blot analysis

In order to compare the expression of genes related to the regulation of the atrophy, the cells were lysed in a lysis buffer (25 mM Tris-Cl (pH 7.5), 250 mM NaCl, 5 mM EDTA, 1% NP- 1 mM phenylmethylsulfonylfluoride (PMSF), 5 mM dithiothreitol (DTT)], and the reaction was carried out at 4 ° C. for 1 hour to separate the proteins. Then, Bio-Rad protein quantitation reagent Bio-Rad Laboratory, Hercules, Calif., USA). The proteins mixed with the Laemmli sample buffer (Bio-Rad Laboratory) were separated by electrophoresis using sodium dodecyl sulphate-polyacrylamide gel, Cellulose membrane (nitrocellulose membrane, Schleicher and Schuell, Keene, NH, USA) was transferred. Protein-transferred membranes were treated with primary and secondary antibodies and reacted with enhanced chemiluminescence (ECL) solution (Amersham Life Science Corp., Arlington Heights, IL, USA) Expression changes of specific proteins were analyzed.

Analysis of mRNA by reverse transcriptase polymerase chain reaction (RT-PCR)

Total RNA was isolated and quantified using TRIzol reagent (Invitrogen Co., Carlsbad, CA, USA) in the same conditions. Each primer, DEPC water and ONE-STEP RT-PCR PreMix Kit (Intron, Seongnam, Korea) was added and amplified using a Mastercycler gradient (Eppendorf, Hamburg, Germany). 1% agarose gel was prepared with 1 × TAE buffer to confirm the quantitative difference between the PCR products, and the PCR product corresponding to each primer per well was loaded with a gel loading solution, After electrophoresis at 100 V, After electrophoresis, the gel separated from the DNA was stained with EtBr and confirmed under UV.

Statistical analysis

Duncan's multiple range tests were performed at p <0.05 after ANOVA to test the significance of all experimental results. The results were mean ± standard deviation, SD).

2. Test results

Effect of AICAR on survival and growth of C2C12 myofibroblast

To investigate the effect of AICAR used to induce atrophy on the survival and growth of C2C12 cells, various concentrations of AICAR were treated for 24 hours and then examined using trypan blue staining and MTT assay. The number of viable cells was counted using a hemocytometer after trypan blue staining. As shown in FIG. 1, the survival rate was not significantly decreased until 1 mM, Showed a decrease in survival rate with increasing treatment concentration. The results of MTT assays were also shown in FIG. 2 to determine the extent of AICAR-induced growth inhibition under the same conditions as described above. As shown in the results, the growth inhibition induced by AICAR was not significantly changed up to 1 mM treatment group but it was strongly induced from 1.5 mM treatment group.

AICAR induced apoptosis in C2C12 myoblasts

The results of FIGS. 1 and 2 were examined to determine whether the survival rate and proliferation inhibition of C2C12 cells by AICAR treatment were related to the induction of apoptosis. As shown in FIG. 3, DAPI staining was performed to confirm the morphological change of the nucleus when apoptosis was induced. No change was observed in the cells cultured under the normal condition, but the concentration of chromatin condensation Induced the formation of apoptotic bodies. For quantitative comparison of AICAR-induced apoptosis in C2C12 cells, cells cultured under the same conditions as above were stained with annexin V-FITC / PI and then subjected to flow cytometry analysis. As can be seen from the results of FIG. 4, the degree of apoptosis induction did not significantly increase until 1 mM AICAR treated group, but rapidly increased from 1.5 mM AICAR treated group to about 37.32% in 2 mM AICAR treated cells Apoptosis was induced. These results suggest that the decrease of survival rate and inhibition of growth of C2C12 cells by high concentration of AICAR are caused by apoptosis. Therefore, up to 1 mM of AICAR was considered to have no cytotoxicity to C2C12 myofibroblasts, and subsequent experiments were conducted at a concentration of 1 mM.

Effect of AICAR on expression of muscle-associated genes in C2C12 myoblasts

AMPK is an energy balance regulator and energy sensor that regulates glucose and lipid metabolism through blood glucose absorption and fatty acid oxidation induction as well as inhibition of energy consumption process. In addition, AMPK is also known to be involved in muscle atrophy by directly participating in muscle protein degradation through activation of the ubiquitin-proteasome pathway. When AMPK is activated, muscle-specific ubiquitin ligases (MAFbx / atrogin-1, F-box type E3 ligase), which are specifically expressed in skeletal muscle via FoxO3a, Specific activation of the ubiquitin-proteasome pathway through the increase of MuRF1, an E3 ligase of the Ring Finger type, to induce muscle-specific reverse transcription factors involved in myogenic differentiation transcription factors (MyoD and myogenin), leading to muscle atrophy. Therefore, the expression of AMPK and the atrophy-related gene in C2C12 cells was confirmed by AICAR treatment. As shown in FIGS. 6 and 7, AICAR was found to increase the phosphorylation of ACC acting in the lower stages of AMPK and AMPK in a concentration-dependent manner, and it also increased the expression of FoxO3a. In addition, increased expression of muscle-specific ubiquitin ligases, MAFbx / atrogin-1 and MuRF1, as well as muscle-specific transcription factors MyoD and myogenin, Of the cells. These results suggest that AICAR may be involved in muscle atrophy by modulating the expression of atrophy-related genes via AMPK and FoxO3a.

Effect of AICAR on muscle atrophy in C2C12 canalicular cells

The muscle atrophy is induced by aging, malnutrition, and various diseases. It induces muscle protein degradation through ubiquitin-proteasome pathway activation, thereby reducing the total muscle mass through reduction of muscle fiber diameter and diameter. . The aim of this study was to investigate whether AICAR could be used as an inducer of muscle contraction. For this purpose, morphological features and the expression of genes related to atrophy were examined in the case of AICAR treated with no cytotoxicity on C2C12 canaliculus cells. As shown in FIGS. 8 and 9, the morphological characteristics of the C2C12 canaliculus cells and the diameter of the myofiber according to the AICAR treatment were examined. As a result, the number and diameter of the muscle fibers were significantly decreased depending on the AICAR treatment concentration. Next, how do they affect the expression of muscle-specific ubiquitin ligases and muscle-specific transcription factors in relation to AICAR induced atrophy? MRNA and protein 10 and 11, respectively. As can be seen from the results, when AICAR was treated after inducing differentiation into myotubes from myoblasts, the expression of MyoD and myogenin increased with increasing expression of MAFbx / atrogin-1 and MuRF1 . These results suggest that AICAR, an AMPK activator, induces atrophy of C2C12 canaliculus cells, and thus may be used as an inducer of atrophy.

Effect of EEMF on AICAR-induced atrophy in C2C12 canalicular cells

(EEMF) in C2C12 canaliculus cells induced by AICAR, an AMPK activator. As shown in FIGS. 12 and 13, the morphological characteristics of the C2C12 canaliculus cells and the diameters of the myofibers were examined after setting the conditions without cytotoxicity according to the EEMF and AICAR treatments. As a result, the number and diameter of the myofiber decreased Induced EEMF treatment, but it was confirmed that EEMF could inhibit atrophy. In addition, as shown in FIG. 16, EEMF inhibited the phosphorylation of AMPK and ACC by AICAR treatment, and the expression of FoxO3a was also decreased. EEMF also reduced the expression of MAFbx / atrogin-1 and MuRF1 by AICAR treatment and restored the expression of reduced MyoD and myogenin. These results suggest that EEMF inhibits AIKAR-induced atrophy. This phenomenon can be explained by the fact that ubiquitin-proteasome induced by inhibition of FoxO3a, a transcription factor through AMPK inhibition, Interception of pathways played an important role.

[Preparation Example 2: Preparation of mixed extract]

1. Preparation of rape tree extract (AF)

The silkworms were washed, dried and then ground. The pulverized material was mixed with purified water and maintained at 98-100 ° C for 2 hours. After cooling, it was filtered with Watman filter paper to obtain a filtrate.

2. Manufacture of other plant extracts

(BF), CF, DF, EF and FF were prepared by the same method as that of A. japonica extract.

3. Preparation of mixed extract

(AF), a bergamot (BF), a ground bug extract (CF), an electric bug extract (DF), a succulent extract (EF) and a viviparous extract (FF) The mixture as shown in Table 1 was mixed to prepare a mixed extract.

MX1 MX2 MX3 MX4 MX5 MX6 MX7 MX8 MX9 MX10 MF 100 100 100 100 100 100 100 100 100 100 AF 0.5 One 10 15 20 10 10 10 10 10 BF 5 10 25 40 50 25 25 25 25 25 CF 0.5 One 10 15 20 10 10 10 10 10 DF - - - - - 0.5 One 2 3 5 EF - - - - - One 3 10 12 16 FF - - - - - One 3 10 12 16

(Unit: parts by weight)

[Experimental Example 2: prevention of muscle atrophy of mixed extracts and confirmation of improvement effect]

1. Arthritis inhibitory effect experiment

In order to confirm the synergistic effect by the interaction with the use of the mixed extract, the inhibition of phosphorylation of AMPK and ACC and the decrease of expression of FoxO3a in relation to the MF extract and the muscle atrophy Respectively.

In the same manner as described above, the inhibition of phosphorylation of AMPK and ACC and the expression level of FoxO3a were evaluated in comparison with the results of the above leaf extracts in the experimental group to which each of the mixed extracts (MX 1 to MX 5) was administered for 3 weeks. In order to directly reveal the effects of each of the mixed extracts, the leaf extract and each of the mixed extracts were administered at a dose of 10 mg / kg once a day by reducing the dose to 10% of the previous dose.

 In addition, the inhibition of phosphorylation of AMPK and ACC and the degree of expression of FoxO3a in the above leaf extract (MF) were fixed at an index of 5, and the result of using the above mixed extracts was expressed as an index of 1 to 10 as compared with this. The higher the number, the better the inhibitory activity and the better the activity to improve the muscle atrophy.

The results are shown in Table 2 below.

MF MX1 MX2 MX3 MX4 MX5 AMPK inhibition of phosphorylation 5 5 8 9 9 6 Inhibition of ACC phosphorylation 5 4 8 9 8 6 Fox03a inhibitory activity 5 4 9 9 9 6

(Unit: index)

Referring to Table 2 above, it can be seen that the synergistic effect according to the use of the mixed extract is exhibited. In particular, the inhibition of phosphorylation of AMPK and ACC and the inhibition of FoxO3a expression are greatly increased by MX 2 to MX 4.

Therefore, it can be seen that the synergistic effect of the combination of the extracts in the range of MX 2 to MX 4 is higher than that of the simple combination due to the synergistic action by mixing the respective extracts.

Thus, it can be seen that the phosphorylation inhibition activity of AMPK and ACC and the inhibitory activity of FoxO3a expression can be exhibited in a smaller amount than in the case of using the mixed extract. Therefore, it is possible to prevent and ameliorate the effect of the atrophy by relatively low concentration, and thus it is possible to produce a product containing the composition for preventing or improving muscle atrophy, which can be taken for a long time with few side effects.

2. Sensory Evaluation

The flavor and taste sensory properties of each composition were evaluated so that the composition containing the above leaf extract (MF) and each of the mixed extracts could be provided as a functional food composition.

Therefore, the extracts of the leaf extracts (MF) and the mixed extracts MX 3 and MX 6 to MX 10 were prepared as tea beverages, and the sensory evaluation of 20 adult male and female was carried out. The sensory evaluation was evaluated based on the fragrance and taste based on the palatability, and the results are shown in Table 3.

On the other hand, the higher the number is, the higher the palatability.

MF MX3 MX6 MX7 MX8 MX9 MX10 incense 5 One 2 7 9 9 5 flavor 4 One 3 9 8 9 4 Purity 4.5 One 2.5 7.5 8.5 9.0 4.5

(Unit: index)

Referring to the above Table 3, it can be seen that the upper leaf extract has higher flavor and taste than the normal leaf tea because the flavor and taste of the upper leaf extract increases and the mingling taste unique to the upper leaf is improved and the overall fragrance is enhanced have.

However, when the mixed extracts according to MX 3 were used, it was found that when the extracts of Zagyubi extract, Taurine Vermicelli extract and Zygomycota extract were contained, the inhibitory effect against degenerative arthritis was increased, .

However, as shown in Table 3, in the case where the sphagnum extract, Suho supernatant, and the Leviticus extract are mixed in a certain range, even though the mixture of the Zagoubia extract, It can be seen that the taste is improved.

Especially, referring to MX 7 to MX 9, when a certain range of Zygomyceta japonica extract, Morus alba extract, and Zygomyceta japonica extract is mixed, the fragrance of the mulberry leaves is maintained, and the mulberry tree extract, And flavor and flavor of tea beverages are greatly improved according to the mutual combination of the respective extracts.

Therefore, when the mixed extract of the above-mentioned range is used, it is possible to greatly enhance the inhibitory effect on the degenerative arthritis including the extracts of Araliacea japonica extract, Rhododendron vine leaf extract, They can be widely spread as functional foods or functional foods with higher palatability.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, Belong to the scope of

Claims (8)

Containing an extract of Mori Folium as an active ingredient,
The upper leaf extract was obtained by extracting the upper leaves with a solvent
A composition for prevention and improvement of muscle atrophy comprising an extract of a leaf as an active ingredient. The method according to claim 1,
The topical extract is extracted with a solvent selected from the group consisting of water, alcohols having 1 to 6 carbon atoms, and mixtures thereof
A composition for prevention and improvement of muscle atrophy comprising an extract of a leaf as an active ingredient. The method according to claim 1,
The atrophy is caused by at least one of disuse atrophy of muscle, mechanical stimulation member, and denervation of skeletal muscle
A composition for prevention and improvement of muscle atrophy comprising an extract of a leaf as an active ingredient. The method according to claim 1,
The composition for prevention and improvement of muscle atrophy further includes a silk-tree extract
A composition for prevention and improvement of muscle atrophy comprising an extract of a leaf as an active ingredient. 5. The method of claim 4,
The composition for prevention and improvement of muscle atrophy further comprises Chinese horned Holly extract
A composition for prevention and improvement of muscle atrophy comprising an extract of a leaf as an active ingredient. A composition for prevention and improvement of muscle atrophy comprising an extract of a leaflet according to any one of claims 1 to 5 as an active ingredient
A pharmaceutical composition. A composition for prevention and improvement of muscle atrophy comprising an extract of a leaflet according to any one of claims 1 to 5 as an active ingredient
Food composition. A composition for prevention and improvement of muscle atrophy comprising an extract of a leaflet according to any one of claims 1 to 5 as an active ingredient
Tea (tea).

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