KR20150117514A - Pharmaceutical composition comprising Oenothera odorata extract for preventing or treating muscle atrophy in microgravity or nervous damage - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating muscle atrophy due to neural damage or microgravity, containing an extract of Oenothera odorata as an active ingredient. The extract of Oenothera odorata inhibits the apoptosis of muscle cells or tissues by increasing expression of heat shock proteins, which have cell protective ability and antioxidative effects in disused muscles or in a no-load condition supposing a space environment, thereby exhibiting effects of anti-muscle atrophy and thus being useful for treating, preventing, or ameliorating muscle atrophy due to neural damage or microgravity.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for preventing or treating muscular atrophy caused by micro-gravity or nerve damage containing Oenothera odorata extract as an active ingredient,

The present invention is heat shock protein Microgravity or nerve injury containing evening primrose (Oenothera odorata) extract of promoting the expression of a (heat shock protein, HSP), and inhibited the muscle atrophy via antioxidant effects, such as muscle cell death inhibition, as an active ingredient To a pharmaceutical composition for preventing or treating muscle atrophy.

Muscle atrophy is caused by a decrease in muscle use induced by several factors such as no load, space flight, aging, muscle disorders, nerve damage, etc. (Booth FW, J Appl Physiol Respir Environ Exerc Physiol ., 1982). In particular, the various stressors have been attracting worldwide attention as causes of various diseases as well as atrophy (MCKinnell IW., And Rudnicki MA., Cell , 2004).

Heat Shock Protein (HSP) in muscle cells has been shown to protect cells at the level of muscle cells against oxidative stress in vivo, and this mechanism is known to be involved in superoxide dismutase 1 (superoxide dismutase 1, SOD1) (Arya R., et al., J Biosci ., 2007).

To date, there have been no systematic studies on the biological changes of muscle atrophy caused by various factors. If the body is in a microgravity or no-load state, muscle tissue that has been constantly contracting in response to gravity on the ground causes severe atrophy. For example, after 17 days of astronaut flight, the astroseal muscles decreased in cross-section by 15% and muscle strength by 34% (Widrick JJ et al., J Appl Physiol ., 2001). On the other hand, the administration of vitamin E in the 2-week resting state resulted in a 32% reduction in the amount of thiobarbituric acid in the rat, and superoxide dismutase (SOD) , Catalase and antioxidant enzymes were not increased, but the expression of heat shock protein was significantly increased (Servais S et al., Free RadicBiol Med ., 2007). This phenomenon is due to the antioxidative action of vitamin E and the inhibition of proteolytic enzyme activity thereby suppressing muscle atrophy.

In addition, when the body temperature was elevated to 41 ° C immediately before switching from rehabilitation to reloading, and the thermal shock was applied for 60 minutes, the recovery of muscle atrophy was studied. The recovery of muscle mass and protein amount of the soleus muscle was significantly (Selsby JT et al., Am J Physiol Regulintegr Comp Physiol ., 2005). In addition, the expression of heat shock protein 70 was significantly increased.

As has been said, heat shock protein 70 plays a role in the organization and maintenance of myofibrils in response to various stresses (for example, oxidative stress, mechanical stress, etc.), and thus plays an essential role in muscle contraction function, . Thus, a natural substance that can induce the expression of heat shock protein 70 but has no side effects is required for the treatment of atrophy.

Korean Patent Laid-Open Publication No. 2012-0097705 discloses that the expression of heat shock proteins having cytoprotective function is inhibited by Celastrol (CEL), a quinone methide triterpene compound derived from a plant of Celastraceae To promote muscle protein synthesis and inhibit protein degradation factors, thereby exhibiting an anti-atrophic effect against atrophy.

Korean Patent Publication No. 1,349,361 and Korean Laid-Open Patent Publication No. 2014-0000767 each inhibit myocardial damage, necrosis and apoptosis caused by oxidative stress by the extracts of spine herb extracts and chicory extract, respectively, resulting in prevention of muscle atrophy , Treated, or ameliorated.

Muscle dystrophy, which is associated with degenerative muscular dystrophy as well as physical factors such as weightlessness and nerve damage, is a rare disease in which there are about 30,000 patients in the US and Europe. There is no. Natural extracts are ideal as a drug for lifelong ingestion at the time when no clear treatment for these atrophy patients has been developed. In addition, as the aging and space age, which are based on state-of-the-art technologies, has come, the problem of gradual atrophy has gradually become a problem. Therefore, the pharmaceutical composition according to the present invention is expected to be usefully used for rehabilitation of patients having muscle atrophy and physical disability, and the identification of effective ingredient materials through natural products without side effects has not only high value in research, I think it will help.

Evening primrose is a two-year-old herbaceous plant belonging to the plumidaceae family. It is a naturalized plant native to South America Chile and widely distributed in Central America, Europe and Asia. In Korea, it is distributed throughout the country. It grows mainly in water, roadside, and winter . Evening primrose flowers are bloomed in July, yellow bellworms are run one on each side of the leaf, 2 to 3 cm in diameter, and bloom in the evening and drown in the morning. Four calyxes are combined, two are combined, flowers are bloomed backward, petals are ending with four, the stamens are eight, the pistil is one, and the stigma is divided into four. Ovary is conical and has hair. The fruit is oval, long elliptical, 2.5 cm long, split into four pieces, and seeds emerged. Seeds have several corner angles and when wet they produce mucus. The young leaves eat cows but do not eat the grown leaves.

The roots of evening primrose in one room are used as medicinal medicine called monthly primrose. It is high in fever with cold, when it is in sore throat, it is put into water and dalyeoseo, and seed is used as hypertonemia. Generally, it has antipyretic and anti-inflammatory effect, and it is known to have a therapeutic effect on cold, sore throat, bronchitis and dermatitis. Evening primrose oil has been traditionally used as an indigenous medicine for the inflammation and rash by Indians. It has been passed on to asthma as a pandemic for white people. It has been called "King's panacea" and has been selected as a national medicine. Recently, evening primrose oil has been recognized as a medicine and health food because it is found that the unsaturated fatty acid called gamma linolenic acid, which is abundantly contained in evening primrose oil, becomes the mother of physiologically active substance called prostaglandin. Prostaglandins play a wide role in various physiological functions such as expansion and contraction of blood vessels in the human body, contraction and relaxation of bronchial muscles, inhibition of gastric secretion, uterine muscle contraction, excretion of water, induction of platelet aggregation or inhibition. Evening primrose oil significantly reduces the amount of cholesterol to improve circulatory or heart disease, including myocardial infarction, arteriosclerosis, hypertension and angina. In addition, there are many reports that weight loss effect was increased by administration of evening primrose oil, and other excellent effects such as rheumatoid arthritis, menstrual irregularity, alcohol poisoning and diabetes have been recognized. In addition, it is effective for allergic skin diseases. Especially, in the case of atopic dermatitis child and atopic eczema patients, the symptom improvement effect in the ingestion group has been confirmed, and it is known that it is effective for prevention of skin aging and whitening, It is also widely used in beauty products such as cosmetics and soap.

However, there have been no reported cases of using evening primrose extracts for use in the prevention, treatment, or rehabilitation of muscle atrophy. Accordingly, it is an object of the present invention to provide a novel use for preventing, treating, or ameliorating atrophy of the evening primrose oil extract by no-load, fine gravity, or nerve damage and muscle nonuse.

In order to achieve the above object, the inventors of the present invention have conducted intensive studies, and have found that even-evening primrose extracts exhibit significantly inhibited thermal shock proteins in animal experiments such as cell experiments conducted under microgravity assuming cosmic state and animal models The present invention can be utilized as a main component of a pharmaceutical composition for improving or treating muscular atrophy caused by microgravity or nerve damage and a food for improvement, by confirming that the evening primrose oil extract greatly contributes to prevention or treatment of muscle atrophy. .

Korean Patent Publication No. 2012-0097705 Korean Patent Publication No. 1,349,361 Korean Patent Laid-Open Publication No. 2014-0000767

Booth FW. (1982). &Quot; Effect of limb immobilization on skeletal muscle ", J. Appl Physiol Respir Environ Exerc Physiol., 52 (5): 1113-8. Iain W. McKinnell & Michael A. Rudnick (2004). &Quot; Molecular Mechanisms of Muscle Atrophy ", Cell, 19 (7): 907-910. Arya R1, Mallik M, Lakhotia SC. (2007). &Quot; Heat shock genes - integrating cell survival and death ", J Biosci., 32 (3): 595-610. Fitts RH., Riley DR., Widrick JJ. (2001). &Quot; Functional and structural adaptations of skeletal muscle to microgravity ", J Exp Biol., 204 (Pt 18): 3201-8. Servais S., Letexier D., Favier R., Duchamp C., Desplanches D. (2007). "Prevention of unloading-induced atrophy by vitamin E supplementation: links between oxidative stress and soleus muscle proteolysis", Free Radic Biol Med., 42 (5): 627-35. Selsby JT. & Dodd SL. (2005). &Quot; Heat treatment reduces oxidative stress and protects muscle mass during immobilization ", Am J Physiol Regul Integr Comp Phys Physiol., 289 (1): R134-9.

It is an object of the present invention to provide a method for preventing or preventing the loss of muscle tissue or atrophy of muscles caused by microgravity or nerve damage caused by not using muscles by promoting the expression of heat shock proteins of evening primrose ( Oenothera odorata ) To find new materials for treatment or improvement and to provide new uses thereof.

In order to achieve the above object, the present invention provides a method of inhibiting the damage and necrosis of myocardial cells through the antioxidant activity of Oenothera odorata extract and increasing the expression of heat shock protein (HSP70) Treatment, or amelioration of atrophy, and a health functional food composition.

Oenothera odorata extract inhibits myocyte injury and necrosis, and exhibits antioxidative effect through increased expression of heat shock protein with cytoprotective function. Therefore, it is possible to prevent, treat or ameliorate muscle atrophy caused by microgravity or nerve damage of evening primrose extract And a health functional food composition.

FIG. 1 is a graph showing cytotoxicity observed when evening primrose extracts were treated at 0, 5, 10, 25, 50, and 100 μg / ml and then cultured in muscle cells.
FIG. 2 is a graph showing the effect of evening primrose extract to prevent and recover necrosis of muscle cells in a concentration-dependent manner after inducing muscle cell necrosis by exposure to microgravity with a clinostat.
FIG. 3 shows the effect of microgravity using a clinostat on the expression of heat shock protein 70 in muscle cells and the effect of evening primrose oil extract on it.
FIG. 4 shows the effect of evening primrose oil extract on the muscle atrophy using bio-micro-CT in a small animal model that artificially induces atrophy by total neuronectomy.
FIG. 5 is a graph showing the effect of preventing evening primrose when the evening primrose oil extract was treated in a small animal model that artificially induces atrophy by artificial neurotomy.
FIG. 6 shows the effect of evening primrose oil extract on the expression of heat shock protein 70 in a small animal model that artificially induced muscle atrophy by total neuronectomy.

The present invention relates to a pharmaceutical composition for preventing or treating muscular atrophy caused by microgravity or nerve damage comprising an extract of Oenothera odorata as an active ingredient.

In one embodiment of the present invention, the evening primrose oil extract may be any one selected from the group consisting of flowers, leaves, branches, roots, stems, fruits, shells and mixtures thereof of evening primrose, and more particularly, Roots can be used.

In one embodiment according to the present invention, the evening primrose extract may be extracted with any one solvent selected from the group consisting of water, an organic solvent and a mixed solvent thereof.

In one embodiment of the present invention, the organic solvent is a non-polar solvent such as ether, chloroform, benzene, hexane, or dichloromethane, or a mixture of water and a polar solvent such as alcohol, ethyl acetate or acetone having 1 to 5 carbon atoms, have.

In one embodiment according to the present invention, the alcohol may be selected from methanol, ethanol, propanol, butanol, isopropanol and the like, and more specifically, it may be methanol or ethanol.

In one embodiment according to the present invention, the alcohol may be methanol or ethanol, and the evening primrose extract may be an ethanol extract of evening primrose root.

In one embodiment of the present invention, the Evening Primrose Oil Extract comprises fractions obtained by further fractionating the first extract extracted using the above-mentioned extraction solvent using an extraction solvent having a different polarity. For example, the Evening Primrose Oil Extract may be a fraction obtained by extracting with an alcohol having 1 to 5 carbon atoms and then fractionating again with a solvent having a different polarity such as ether, benzene or hexane.

In one embodiment of the present invention, at least two solvents may be used in the fractionation, and each solvent extract may be used sequentially or in combination, depending on the polarity of the solvent, but is not limited thereto.

In one embodiment of the present invention, the produced extract or the fraction obtained by performing the fractionation process may be subjected to filtration, concentration or drying to remove the solvent, followed by filtration, concentration and drying .

 In one embodiment of the present invention, the filtration can be performed using a filter paper or a vacuum filter, and the concentration can be reduced by using a vacuum concentrator, for example, a rotary evaporator. Can be performed.

In one embodiment according to the present invention, the pharmaceutical composition is almost toxic and harmless to the human body because it is a component separated from a natural product extract or a derivative thereof as an active ingredient.

In one embodiment according to the present invention, the evening primrose oil extract of the pharmaceutical composition is present in an amount of 0.001 to 30% by weight, more specifically 0.01 to 20% by weight, and more particularly 0.1 to 10% by weight of the total composition .

In one embodiment according to the present invention, evening primrose extract may be included in the pharmaceutical composition at a concentration of 0.001 to 10 mg / kg.

In one embodiment according to the present invention, the pharmaceutical composition may be administered in an amount of 0.01 to 500 mg / kg, more specifically 0.1 to 200 mg / kg, More specifically from 0.1 to 100 mg / kg, more particularly from 0.1 to 20 mg / kg, more particularly from 1 to 10 mg / kg, and the dosage form unit dosage form May be administered several times at predetermined time intervals as needed.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier and a vehicle. Particularly preferred are ion exchange resins, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as various phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids ), Water, salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride and zinc salts), colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose based substrate, polyethylene glycol, sodium carboxy Methylcellulose, polyarylate, wax or wool, and the like.

The composition comprising a pharmaceutically acceptable carrier and vehicle can be of various oral or parenteral formulations. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used.

Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like. Such solid preparations can be prepared by mixing the pharmaceutical composition of the present invention with at least one excipient such as starch, calcium carbonate, Sucrose or lactose, calcium carbonate, 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 include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The pharmaceutical composition is selected from the group consisting of tablets, pills, powders, granules, coated tablets, capsules, suspensions, solutions, emulsions, syrups, juices, sterile aqueous solutions, non-aqueous solutions, injections, lyophilized preparations and suppositories Any one of the formulations can be used.

In one embodiment according to the present invention, the pharmaceutical composition may be administered to mammals such as rats, mice, livestock, humans, and the like in a variety of routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection.

The dosage of the pharmaceutical composition according to the present invention may vary depending on the purpose of administration, the type of disease, the severity of the disease, the kind and amount of the active ingredient and other ingredients contained in the composition, the type of the formulation and the age, weight, And the diet, the time of administration, the route of administration and the rate of administration of the composition, the duration of the treatment, the drugs used concurrently, and the like.

The present invention relates to a health functional food composition for prevention or amelioration of muscle atrophy caused by microgravity or nerve damage comprising an extract of evening primrose ( Oenothera odorata ) as an active ingredient.

In one embodiment according to the present invention, the evening primrose may be any one selected from the group consisting of flowers, leaves, branches, roots, stalks, fruits, husks, and mixtures thereof of evening primrose, Evening primrose root can be used.

In one embodiment according to the present invention, the evening primrose extract may be extracted with any one solvent selected from the group consisting of water, an organic solvent and a mixed solvent thereof.

In one embodiment of the present invention, the organic solvent is a non-polar solvent such as ether, chloroform, benzene, hexane, or dichloromethane, or a mixture of water and a polar solvent such as alcohol, ethyl acetate or acetone having 1 to 5 carbon atoms, have. In one embodiment according to the present invention, the alcohol may be selected from methanol, ethanol, propanol, butanol, isopropanol and the like, and more specifically, it may be methanol or ethanol.

In one embodiment according to the present invention, the alcohol may be methanol or ethanol, and the evening primrose extract may be an ethanol extract of evening primrose root.

In one embodiment of the present invention, the Evening Primrose Oil Extract comprises fractions obtained by further fractionating the first extract extracted using the above-mentioned extraction solvent using an extraction solvent having a different polarity. For example, the Evening Primrose Oil Extract may be a fraction obtained by extracting with an alcohol having 1 to 5 carbon atoms and then fractionating again with a solvent having a different polarity such as ether, benzene or hexane.

In one embodiment of the present invention, at least two solvents may be used in the fractionation, and each solvent extract may be used sequentially or in combination, depending on the polarity of the solvent, but is not limited thereto.

In one embodiment of the present invention, the produced extract or the fraction obtained by performing the fractionation process may be subjected to filtration, concentration or drying to remove the solvent, followed by filtration, concentration and drying .

In one embodiment of the present invention, the filtration can be performed using a filter paper or a vacuum filter, and the concentration can be reduced by using a vacuum concentrator, for example, a rotary evaporator. Can be performed.

The health functional food composition may be a health functional food composition for prevention or amelioration of muscle atrophy, which is contained in space food for suppressing muscle aging due to cosmic life or for improving exercise function.

The health functional food may be commercialized in the form of tablets, capsules, pills, powders, granules, liquid beverages, concentrates and the like.

In the case of health functional foods in the form of granules, tablets or capsules, it is usually 0.0001 to 100% by weight, more particularly 0.01 to 80% by weight, more particularly 0.1 to 50% by weight, more particularly 1 to 20% Evening primrose oil extracts may be added, but are not limited thereto.

In another embodiment according to the present invention, the Evening Primrose Oil Extract can be commercialized as a food. For example, the food may include beverages, meats, chocolates, cookies, pizza. Examples of the food include all kinds of foods in a conventional sense, such as noodles, noodles, gum, ice cream, vitamin complex, alcoholic beverage, dairy product, juice, ring, granule, tea tea bag and stick.

The food typically contains from 0.0001 to 100% by weight, more specifically from 0.001 to 80% by weight, more specifically from 0.01 to 60% by weight, more specifically from 0.1 to 50% by weight, more particularly from 1 to 20% But the present invention is not limited thereto and may be added within a range that does not impair the inherent taste of the food.

Various additives such as flavors or natural carbohydrates can be used as the food-aid additive of the present invention. The natural carbohydrates are sugar saccharides such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like.

Hereinafter, the present invention will be described in more detail with reference to the following Experimental Examples and Preparation Examples. It should be understood, however, that the same is by way of illustration and example only and is not intended to limit the scope of the present invention.

[Example]

Example 1: Preparation of Evening Primrose Oil Extract

Evening primrose was cultivated by the Rural Development Administration (RDA) Good Agricultural Practice (GAP) and was harvested in Chungcheongbuk-do Voice (GPS: E 128 ° 62'N 36 ° 56 ') in 2009. Extraction of the sample was performed as follows. To 50 g of evening primrose root samples, 150 ml of 99% ethanol was added, and the mixture was reacted at 150 rpm for 48 hours, filtered and concentrated under reduced pressure to obtain lyophilized powder.

[Experimental Example] Physiological activity confirmation experiment

Experimental Example 1: Antioxidative effect of evening primrose oil extract on muscle cell culture system to prevent muscle atrophy

(1) Cell culture

Normal muscle cells (C2C12) were cultured in DMEM medium containing 5% fetal bovine serum (FBS). Namely, normal skeletal muscle cells were inoculated into 75 cm 2 plastic flasks (Falcon Co., England) with 4.4 μl / well of 10% FBS, 7.5% NaHCO ₃ 150 μg / ml, glutamine 58.4 μg / ml and antibiotics / antimycotics, in DMEM medium containing the ㎖ were cultured in 37 ℃, conditions of 5% CO _2. The cell line was maintained by secondary culture once every 2-3 days.

(2) Cell quantification

Cells were removed from the bottom of the flask by treatment with 0.25% trypsin / EDTA followed by washing with CMF-PBS (calcium magnesium free-phosphate buffered saline, pH 7.2) Neutralized with the culture medium, and centrifuged (1200 rpm, 3 min). The culture medium was added to the pellet of the remaining cells and repeatedly inhaled with a sterile pipette to make a single cell suspension. Trypan blue was mixed with the cell suspension at a ratio of 9: 1, were measured using a hemocytometer.

(3) Cytotoxicity measurement

Ez-Cytox kit was used to measure the toxicity of Evening Primrose Oil extract. That is, normal muscle cells (C2C12) were dispensed in a 96-well plate at 2 × 10 ⁴ cells / well. After culturing in an incubator at 37 ° C and 5% CO ₂ , the evening primrose extracts were added at various concentrations of 0, 5, 10, 25, 50, 100 ㎍ / ml and cultured for 24 hours. Cytotoxicity of Evening Primrose Oil (Evening Primrose) extracts was determined by using Ez-Cytox kit for normal muscle cells cultured for 24 hours.

The results of cytotoxicity measurement in cultured normal muscle cells supplemented with evening primrose extract are shown in FIG. The survival rate was similar to that of the non - supplemented group when the evening primrose extract was added. Therefore, it was found that the evening primrose extract used in this experiment did not show toxicity to cultured muscle cells.

(4) Effect of evening primrose extract on concentration of muscle cell necrosis induced by microgravity in clinostat

In order to observe the effect of the evening primrose extract on the muscle cells exposed to microgravity with Clinostat, muscle cells were first placed in 96-well plates at a density of 2 × 10 ⁴ cells / well and cultured in a CO ₂ incubator. Then, the evening primrose extracts were added at concentrations of 0, 25 and 50 μg / ml, respectively. The wells were filled and then cultured in clinostat for 24 hours under the condition of 5% CO ₂ . After incubation, 10 E of Ez-Cytox kit was added and the absorbance was measured after 1 hour to determine its effect.

As can be seen from FIG. 2, the concentration of evening primrose extract was increased from 0 / / ㎖ to 50 / / ㎖ by measuring the muscle cell survival rate of evening primrose extract against concentration of muscle cell necrosis induced by clinostat The survival rate of the cells was recovered. Thus, it was proved that the effect of micronutrient on muscle cell necrosis is increased in a concentration dependent manner on the evening primrose oil extract.

(5) Effects of microgravity using clinostat on the expression of heat shock protein 70 (HSP 70) in muscle cells and the effect of evening primrose oil extract

The expression of heat shock protein 70 was analyzed by western blotting. The muscle cells were lysed in lysis buffer [50 mM HEPES (pH 7.4), 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40, 20 mM beta-glycerophosphate, 0.2 mM Na _The cells were homogenized in ₃ VO ₄ , 50 mM NaF, protease inhibitor Complete Mini, phosphatase inhibitor cocktail to make lysates and then analyzed by Bradford assay Respectively. Then, 20 μg of the protein was electrophoresed on SDS-polyacrylamide gel (SDS-polyacrylamide gel). The developed gel was transferred to a nitrocellulose membrane and blocked with 5% skim milk for 1 hour. Then, the primary antibody of the protein was stuck at 4 ° C for 12 hours, and the secondary antibody conjugated with horseradish peroxidase (HRP) was incubated for 2 hours. Then, a color reaction was induced with an enhanced chemiluminescence (ECL) kit, and the band was identified after being sensitized to an X-ray film. Beta-actin was used as a standardization indicator and the amount of protein detected was measured with a densitometer (Gel Document, Bio-Rad.).

As shown in FIG. 3A, when the muscle cells were cultured in clinostat over time, the expression of heat shock protein 70 was inhibited at 24 hours. However, as shown in FIG. 3B, when 50 μg / ml of evening primrose extract was treated, the expression of heat shock protein 70 was significantly increased as compared with the control. It has been confirmed that the extract acts as a heat shock protein 70 inducer.

Experimental Example 2 Effect of Evening Primrose Oil Extract on Small Animal Model Induced Muscle Atrophy by Total Nerve Excision

Preparation of experimental animals

C57BL6 mice (male, 5 weeks old, weighing approximately 25 g) were pre-fed for one week and used for surgery. The sciatic nerve was excised to create an atrophic model in the hindlimb muscles. In the model of muscle atrophy induction, the evening primrose extract was inoculated into the hind leg muscles at different doses (0, 1, 2 mg / kg) per mouse for 5 times / 7 days.

(1) In a small animal model inducing an artificial muscle atrophy by total neuronectomy, CT Effect of evening primrose extract

Two weeks after the operation, CT images of the muscles were taken from the tibia of each mouse using micro-computed tomography (micro-CT) at a resolution of 35 μm (N = 5, 100 kV, 100 mA, 790 ms, and a rotation step of 1.2 [deg.]). The mice undergoing anesthesia underwent anesthesia. Three-dimensional models of tibia were reconstructed using CT-1.11 (CT-1.11, Bruker, Germany) to assess muscle mass.

As shown in FIG. 4, when comparing the biomicro-CT images of the mice inoculated into the hindlimb muscles in the amounts of 0, 1, and 2 mg / kg of the evening primrose extracts after the resection of the normal mice (control group) and the sciatic nerve, It was confirmed that the hindlimb muscles of the mice not receiving the evening primrose extract inoculated after the neurotomy compared with the hindlimb of the mouse were markedly inactivated, and it was confirmed that the induction of the atrophy of the muscle was further inhibited when the administration dose was increased to 0, 1 or 2 mg / kg.

(2) Effects of muscular atrophy induced by small nerve resection on small animals and the effect of evening primrose oil extract

The weight and volume of the gastrocnemius and the soleus muscle of the hind limb removed 2 weeks after surgery were measured by micro-CT for small animals and expressed as leg muscle ratios atrophied against the normal legs. As shown in FIG. 5A, it was confirmed that the muscle volume of the neurally-resected mice was reduced to almost half of that of the normal mice (control group). However, as shown in FIG. 5B, it was confirmed that when the evening primrose extract was inoculated intramuscularly, the loss of muscle volume decreased as the administration dose was increased to 0, 1, or 2 mg / kg.

(3) Effect of muscular dystrophy induced by neurotomy on the expression of heat shock protein 70 in small animals and the effect of evening primrose oil extract

The expression of heat shock protein 70 was analyzed by western blotting in the gastrocnemius of the hind limb removed 2 weeks after the operation. The muscle cells were lysed in lysis buffer [50 mM HEPES (pH 7.4), 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40, 20 mM beta-glycerophosphate, 0.2 mM Na _After homogenization by homogenization in ₃ VO ₄ , 50 mM NaF, protease inhibitor Complete Mini, phosphatase inhibitor cocktail) to make lysate, Bradford assay. Then, 40 μg of the protein was electrophoresed on SDS-polyacrylamide gel (SDS-polyacrylamide gel). The developed gel was transferred to a nitrocellulose membrane and blocked with 5% skim milk for 1 hour. Then, the primary antibody of the protein was stuck at 4 ° C for 12 hours, and the secondary antibody conjugated with horseradish peroxidase (HRP) was incubated for 2 hours. Then, a color reaction was induced with an enhanced chemiluminescence (ECL) kit, and the band was identified after being sensitized to an X-ray film. Beta-actin was used as a standardization indicator and the amount of protein detected was measured with a densitometer (Gel Document, Bio-Rad.).

As shown in FIG. 6, it was confirmed that the expression of heat shock protein 70 was suppressed in the animal model inducing the atrophy. However, the expression of heat shock protein 70 was markedly increased in the group inoculated with evening primrose extracts compared to the group not inoculated with evening primrose extract.

Claims (14)

A pharmaceutical composition for preventing or treating muscle atrophy caused by micro-gravity under muscular atrophy or nerve damage comprising Oenothera odorata extract as an active ingredient.
The method according to claim 1,
Wherein the evening primrose oil extract is any one selected from the group consisting of flowers, leaves, branches, roots, fruits, husks, and mixtures thereof of evening primrose. Or < / RTI >
The method according to claim 1,
Wherein the evening primrose oil extract is a root extract of evening primrose oil, and the evening primrose oil extract is a root extract of evening primrose.
The method according to claim 1,
Wherein the evening primrose oil extract is extracted with any one solvent selected from the group consisting of water, an organic solvent, and a mixed solvent thereof, wherein the evening primrose oil extract is extracted with a solvent selected from the group consisting of water, an organic solvent and a mixed solvent thereof.
5. The method of claim 4,
Wherein the organic solvent is methanol or ethanol, wherein the organic solvent is methanol or ethanol.
The method according to claim 1,
Wherein the dose of the evening primrose oil extract is 0.001 to 10 mg / kg. ≪ RTI ID = 0.0 > 25. < / RTI >
Mixing the evening primrose oil ( Oenothera odorata ) with any one solvent selected from the group consisting of water, an organic solvent and a mixed solvent thereof; And
≪ / RTI >
A method for preparing a pharmaceutical composition for preventing or treating muscle atrophy caused by micro-gravity muscle atrophy or nerve damage.
A health functional food composition for improving muscle atrophy caused by microgravity muscle atrophy or nerve damage comprising Oenothera odorata extract as an active ingredient.
9. The method of claim 8,
Wherein the evening primrose oil extract is any one selected from the group consisting of flowers, leaves, branches, roots, fruits, husks, and mixtures thereof of evening primrose. Or improving health functional food composition.
9. The method of claim 8,
Wherein the evening primrose oil extract is a root extract of evening primrose, and the evening primrose oil extract is a root extract of evening primrose oil.
9. The method of claim 8,
Wherein the evening primrose oil extract is extracted with any one solvent selected from the group consisting of water, an organic solvent and a mixed solvent thereof, wherein the evening primrose oil extract is extracted with a solvent selected from the group consisting of water, an organic solvent and a mixed solvent thereof.
12. The method of claim 11,
Wherein the organic solvent is methanol or ethanol, wherein the organic solvent is methanol or ethanol.
9. The method of claim 8,
Wherein the health functional food composition is contained in space food for suppressing muscle aging due to cosmic life or for improving exercise function, wherein the health functional food composition is for prevention or amelioration of muscle atrophy caused by micro-gravity under muscular atrophy or nerve damage.
9. The method of claim 8,
Wherein the evening primrose oil extract is contained in an amount of 0.0001 to 100% by weight in the health functional food, wherein the evening primrose oil extract is contained in the health functional food in an amount of 0.0001 to 100% by weight.

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