KR101407150B1 - A composition and functional food comprising an extracts of Rosa rugosa preventing or treating a physical stress-involved disease - Google Patents

Abstract

The present invention relates to a composition containing a stem and a leaf extract, which comprises a powder or an extract obtained by extracting and concentrating the stem and leaf of a corresponding stem and leaves with various solvents, and suppressing stress- The present invention relates to a pharmaceutical composition and a health functional food for preventing or ameliorating sleep deprivation, wherein the stem and leaf extract according to the present invention has an effect of increasing the survival rate, reducing the cortisol content, reducing the cortisol content, We confirmed the excellence.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a composition for the treatment and prevention of mobility-related physical stress-related diseases,

The composition containing the extract of the present invention as an active ingredient is excellent for suppressing, preventing and improving physical stress-related diseases because it is excellent in resistance against physical stress.

Literature 1 Eriksen G et al., Lancet., 352 (9130), 759-62, 1998

[Literature 2] Ji LL., Free Radic Biol Med., 18 (6), 1079-86, 1995

[Literature 3] Khansari, D. N. et. al., Immunol., Today 11, 170-175

[Document 4] Kim, K.J., et. al., Seoul International Sport Science Congress, Proceedings, II, 817-825, 1994

[Literature 5] Pedersen, B. K., & Ullum, H., Med. Sci., Sports Exerc., 26 (2), 140-146

[Literature 6] Nieman, D. C. et. al., Int. J. Sports Med., 15, 199-206, 1994

[Literature 7] Hoffman-Goetz, L, & Pederson, B. k., Immunology Today., 15, 382-387, 1994

[Document 8] Clarkson, PM Food Sci . Nutr ., 35, 131-141 , 1995

[Document 9] Sen, C. K., J. APPl. Physiol., 79, 675-686, 1995

[Literature 10] McEwen B. S., Brain Res., Dec 15, 886 (1-2), 172-189, 2000

[Document 11] Cheng HY. et al., J Neurosci., 26 (50), 12984-12995, 2006

[Document 12] Fulkerson WJ, Tang BY. J Endor., 1979; 81: 135-141

[Literature 13] Fell LR, Proc Aust Anim Prod., 16: 203-206, 1986

[Literature 14] Lee HJ., Korean J Biotechnol Bioeng ., 19 (1), 67-71, 2004

[Literature 15] Choi YS., Korean J Biotechnol Bioeng ., 8 (3), 224-229, 1993

[Document 16] Murasc, T. et. al., Am . Physiol . Regul . Integr . Comp Physiol ., 288, R708-R715, 2005

[Literature 17] Singh, A. et. al., J. Med . Food ., 5 , 211-220, 2002

[Literature 18] Bonen, A. et. al., Med . Sci . Sports Exerc ., 32 , 778-789, 2000

[Literature 19] Keuchi, M. et al. et. al., J. Nutr . Sci . Vitaminol ., 51, 40-44, 2005

[Document 20] Kim, KM, J Nutr ., 128, 1978-1983, 2001

[Document 21] You.Y.H., Biosci. Bio technol. Biochem., 70 (10), 2532-2535, 2006

[Literature 22] Matsumoto K. et al., J Appl Physiol., 81, 1843-1849, 1996

[Literature 23] Jung. KA., Journal of Ethnopharmacology., 93, 75-81, 2004

[Literature 24] Packer L. Oxidant, antioxidant nutrients and the athlete. J Sport Sci, 15: 353-63, 1997

[25] Qiumei Youa., Et al., Toxicology and Applied Pharmacol., 230 (1), 1-8, 2008

The present invention relates to a composition for treating and preventing a physical stress-related disease containing an extract of Alaska pollack as an active ingredient.

In recent years, exercise has been expanding from competitive sports to prevent disease and promote health. Regular and severe exercise provides a health effect that reduces the risk of cardiovascular disease, cancer, osteoporosis, and obesity. (Eriksen G et al., Lancet., 352 (9130), 759-62, 1998), which is known to reduce the risk of prolonged human life and premature death. However, increased oxygen consumption during exercise and increased production of reactive oxygen species (ROS) have been reported to cause oxidative stress acceleration and cell and tissue damage in endogenous oxygen consumption exercise (Ji LL, (Kim et al., 1998; Nieman, et al., 1998). In the present study, 1994; Pedersen, & Ullum, 1994). (Hoffman-Goetz, L, & Pederson, B. K., Immunology Today, 15, 382-387, 1985). It is known that rapid high-intensity exercise can cause a greater change in circulating hormone levels than mental stress 1994).

During exercise, the cell has an increase in oxygen uptake, an increase in intermediate metabolites during partial reduction of oxygen, an increase in oxygen radicals when epinephrine and other catecholamines are inactivated due to exercise, , A change from a weak free radical, superoxide, to a slightly stronger free radical, hydrodxyl radical, muscle damage from excessive exercise leads to lipid peroxidation in the cell membrane, and macrophages and white blood cells produce various free radicals in damaged tissues (Clarkson, PM Food Sci., Nutr ., 35, 131-141, 1995). In addition, the free radicals and various types of highly reactive oxygen species are produced. Thus, high-intensity aerobic exercise can increase reactive oxygen species to produce free radicals above the body's antioxidant defense system, induce lipid peroxidation of the body's molecules, and even cause muscle damage (Sen, CK, J. APPl Physiol., 79, 675-686, 1995).

The nervous system, the endocrine system, and the immune system interact throughout the mammalian physiological system. They are working to adapt to environmental changes. Stress is defined as 'a threat, real or implied, to the maintenance of a narrow range of vital homeostatic parameters necessary for survival' (McEwen BS, Brain Res. Dec 15, 886 (1-2), 172-189, 2000) do. In many studies in The vivo stress study is performed on experimental animals designed with neuropsychiatric disorder model, and the time limit, mental behavioral changes are studied by exposing stresser. It also causes physical stress by endurance exercise or improvisation exercise and studies exercise time, muscle strength, blood biomarker, and hormone content.

Cortisol is a glucocorticoid secreted from the adrenal cortex, including cortisol, corticosterone and cortisone, and their secretion and metabolism are well known. Most mammals contain cortisol, but rheumatoid arthritis is known to have similar concentrations of cortisol and corticosterone. The blood cortisol concentration, which is regulated by ACTH stimulation, is dependent on the amount released into the blood and the metabolic rate of cortisol. Serum cortisol levels are measured not only for the diagnosis of adrenal hyperplasia and adrenocortical insufficiency in animals and humans but also for measuring the degree of stress in animals and humans (Cheng HY et al., 2006 ; Fulkerson W. et al., 1979; Fell LR et al., 1986).

To investigate the molecular mechanisms of the active extracts, the enzymatic activities and hormone analyzes were carried out. In addition, we evaluated physiological activities of positive control and active extracts and tried to provide scientific evidence data to utilize active extracts as health - oriented foods and health functional foods.

Rosa rugosa Thunb.) is a deciduous shrub belonging to the Rosaceae. It has hairy hairs and grows well on the sandy beach. Chemical composition studies have been carried out on leaves, flowers, fruits, and underground parts of these plants. In Japan, flower coloring is used as a natural coloring agent, and in Korea, as a treatment for diabetes mellitus, Other physiologically active research has been reported, such as anti-inflammatory, antispasmodic activity, hypoglycemic activity, serum cholesterol lowering action, blood pressure lowering action, HIV protease inhibitory activity (Lee HJ.,. Korean J Biotechnol Bioeng, 19 (1) , 67-71, 2004). Recently, it has been shown that the methanol extract of roots from the roots significantly reduced the hepatic triglyceride when administered orally (Choi YS., Korean J Biotechnol Bioeng ., 8 (3), 224-229, 1993).

Increased global athletic ability in mice treated with green tea extract. Green tea contains rich polyphenols lays beneficial results for the health and sport shot performance as antioxidants (Murasc, T. et. Al. , Am .J. Physiol. Regul. Integr. Comp Physiol ., 288, R708-R715, 2005). Auxiliaries such as quercetin with antioxidant effects have enhanced global exercise performance (Singh, A. et al., J. Med . Food , 5, 211-220, 2002).

High intensity exercise produces lactic acid in excess of the amount that the tissue will remove lactic acid, which releases lactic acid into the blood and causes fatigue (Bonen, A. et al., Med . Sci . Sports Exerc ., 32 , 778-789, 2000). The amount of lactic acid in blood is a fat marker. Increasing the amount of lactic acid in the blood by continuous exercise can be inhibited by ingestion of nutritional supplements (Keuchi, M. et al., J. Nutr . Sci . Vitaminol ., 51, 40-44, 2005).

Nutritional supplements can protect the oxidative stress caused by exercise, which can reduce extreme fatigue due to continuous physical activity (Kim, KM, J Nutr ., 128, 1978-1983, 2001).

However, none of the above documents discloses or discloses an inhibitory activity against a physical stress-related disease containing an extract of R.

Accordingly, the present inventors have succeeded in preparing a corresponding extract and producing an in Oral administration in vivo the mouse and evaluate the limit swimming exercise capacity using the swimming tank, and exercising the limit swimming exercise time compared to sweetbrier extract administration group is the control group to measure the blood lactate amount increase after, and the amount of blood lactate by movement Compared with the control group. The results of this study were as follows. First, it was confirmed that Korean native extract has a marginal swimming ability and anti - fatigue effect and can be used as a raw material for health functional foods such as sports drinks. The present invention has been completed.

In order to solve the above-mentioned object, the present invention provides a pharmaceutical composition for treating and preventing physical stress-related diseases containing an extract of Rumonium japonica as an active ingredient.

Further, the present invention provides a health functional food for improving and preventing physical stress-related diseases containing an extract of R. koreensis as an active ingredient.

Said adaptations as defined herein include stems, flowers, roots, outposts, etc., preferably stem or leaf parts.

The extract as defined herein is a polar solvent selected from water, alcohol, lower alcohols having 1 to 4 carbon atoms or a mixed solvent thereof, preferably water or water and a mixed solvent, more preferably water or 20 to 90% water And an extract that is soluble in the alcohol mixture.

The physical stress-related disease as defined herein refers to a disease caused by physical stress and refers to a disease caused by physical stress and includes fatigue, hypothermia, hypothermia, hypothyroidism, headache, insomnia, muscle pain, muscle stiffness, palpitations, chest pain, abdominal pain, , Cold sores, facial flushing, sweating, or hypoimmunities, in particular, fatigue, hypogonadism, hypothyroidism, or lethargy.

Hereinafter, the present invention will be described in detail.

The stem or flower of the present invention may be freeze-dried by freeze-drying the stem or leaf material such as flower, roots, and outposts, and then washed with water having a volume of about 1 to 100 times, preferably about 2 to 20 times the weight of the sample , A water, or a water-miscible solvent, more preferably water or a 20 to 90% water-miscible solvent and a mixed solvent of 20 to 120 ° C , Preferably 30 to 80 ° C for about 1 to 72 hours, preferably 2 to 12 hours, using an extraction method such as hot water extraction, cold extraction, reflux cooling extraction or ultrasonic extraction, preferably by hot water extraction After extraction, it is filtered under reduced pressure and concentrated to obtain corresponding extracts of the present invention.

The present invention also provides a pharmaceutical composition and a health functional food for the treatment and prevention of physical stress-related diseases containing the extract prepared from the above-mentioned method and the above-mentioned method as an active ingredient.

The extracts prepared from the above extracts were in In vivo , mice were orally administered, and the global swimming ability was assessed using a swimming bath, and the amount of lactic acid in the blood after exercise was measured. As a result, the swimming time of global swimming increased compared to the control group, And the amount of lactic acid was not increased. Therefore, it has been confirmed for the first time that the Korean native extract can be used as a raw material for manufacturing health functional foods such as sports drinks and the like, which has global swimming ability and anti-fatigue effect. .

The composition of the present invention contains the herbal extract in an amount of 0.01 to 99% by weight based on the total weight of the composition.

However, the composition is not limited thereto, and may vary depending on the condition of the patient, the type of disease, and the progress of the disease.

Compositions comprising the extract of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions.

The composition containing the extract according to the present invention may be formulated in the form of powders, granules, tablets, capsules, oral preparations such as suspensions, emulsions, syrups and aerosols, external preparations, suppositories and sterilized injection solutions, Examples of carriers, excipients and diluents that can be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Silicates, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate and mineral oil. 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, which may contain at least one excipient, such as starch, calcium carbonate, sucrose, Or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a suppository base, witepsol, macrogol, tween 61, cacao paper, laurin, glycerol gelatin and the like can be used.

The preferred dosage of the extract of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the administration route and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the extract is preferably administered at a dose of 0.01 mg / kg to 10 g / kg per day, preferably 1 mg / kg to 1 g / kg per day. The administration may be carried out once a day or divided into several doses. Therefore, the dose is not intended to limit the scope of the present invention in any aspect.

The composition of the present invention may be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, including, for example, oral and rectal or intravenous administration.

In addition, the present invention provides a health functional food for improving and preventing physical stress-related diseases containing an extract of Rumonium japonica as an active ingredient.

The health functional food containing the extract of the present invention can be used variously for medicines, foods and beverages for the improvement and prevention of physical stress related diseases. Examples of the foods to which the extract of the present invention can be added include various foods, beverages, gums, tea, vitamin complex, leach tea, health supplement foods and the like, and they are in the form of powder, granule, tablet, capsule or beverage Can be used.

Accordingly, the present invention also provides a food or food additive containing as an active ingredient an extract of the present invention having an effect of improving and preventing physical stress-related diseases.

The food forms to which the extract of the present invention can be added include various foods of candy, beverages, gums, tea, a vitamin complex, or foods that are health supplement foods.

The extract of the present invention can be added to food or beverage for the purpose of suppressing stress and preventing it. At this time, the amount of the extract in the food or beverage is generally 0.01 to 15% by weight of the total food weight of the health food composition of the present invention, and the health beverage composition is 0.02 to 10 g based on 100 ml, Can be added at a ratio of 0.3 to 1 g.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient, such as ordinary beverages, in addition to containing a mixture of the above extract as an essential ingredient in the indicated ratios, have. Examples of the above-mentioned natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like Sugar, and sugar alcohols such as xylitol, sorbitol, and erythritol. As natural flavors other than those described above, natural flavors (such as tau martin, stevia extract (e.g., rebaudioside A, glycyrrhizin)) and synthetic flavors (saccharin, aspartame, etc.) have. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above-mentioned composition, the composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and intermediates (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the compositions of the present invention may contain flesh for the production of natural fruit juices and fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

Targeting sweetbrier extract of the present invention in In vivo , the mice were orally administered, and the global swimming ability was evaluated using a swimming bath, and the amount of lactic acid in the blood after exercise was measured. As a result, the exercise time of marginal swimming increased compared to the control group, And the amount of the increase is not increased. It was confirmed for the first time that the Korean native extract has a global swimming ability and anti - fatigue effect and can be used as a raw material for manufacturing health functional foods such as sports drinks. The present invention provides a pharmaceutical composition or health functional food useful for inhibiting, treating, preventing and improving physical stress-related diseases by confirming that the compound is useful for treatment.

FIG. 1 is a graph showing the effect of the sample of the present invention and taurine on the global exercise stress (administered with distilled water or taurine (500 mg / kg body weight) or RRW extract (1 g / kg body weight / day) And the swimming time was measured at 7.5 L / min. The individual values were expressed as mean (mean ± SE), and the ^* mark on the top of the bar was significantly different from the control ( p <0.05 )
FIG. 2 is a graph showing the effect of the sample of the present invention and taurine on blood lactate level after endurance exercise (administration of distilled water or taurine (500 mg / kg body weight) or RR extract (1 g / kg body weight / day) and the level of lactate in the blood was measured after swimming. The individual values were expressed as the mean value (mean ± SE) in 6 mice in each group, and the ^* mark on the bar was significantly different from the control ( p < 0.05 )},
FIG. 3 is a graph showing the effect of the present invention sample and taurine on the intramuscular glycogen concentration after endurance exercise. The mice were treated with a vehicle preincubation agent, taurine (500 mg / kg body weight) or RRW extract (lg / kg body weight administration of the / day), and muscle glycogen is measured after incision, and each value is expressed in numerical average (mean ± SE) from 6 mice of each group, and the bar (bar) ^* mark in the top is significantly in the control group ( P < 0.05 )),
FIG. 4 is a graph showing the effect of the present invention sample and taurine on the MDA level of an experimental mouse (a mouse pre-exercise agent, taurine (500 mg / kg body weight) or a RRW extract sample ) for administering and, MDA level is measured after incision, and each value is expressed in numerical average (mean ± SE) from 6 mice of each group and ^* mark is different significantly in the control group (p <0.05)} ,
FIG. 5 is a graph showing the effect of the present invention sample and taurine on blood cortisol levels in experimental mice (mice were administered with vehicle excipient, taurine (500 mg / kg body weight) or RRW extract (lg / kg body weight / administration of the day), and serum cortisol levels are measured after incision, and each value is expressed in numerical average (mean ± SE) from 6 mice of each group and ^* mark being different significantly in the control group (p <0.05 )}.

Hereinafter, the present invention will be described in detail with reference to the following Reference Examples and Experimental Examples.

However, the following Reference Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Reference Examples and Experimental Examples.

Example  1. Preparation of Applied Stems and Leaf Extracts

Water was added to 14 kg of dry petroleum distillate (140 kg), and the mixture was refluxed at 100 ° C for 5 hours into an extractor (COSMOS 660, Kyesho Machinery Industries) After filtration using a filter equipped with a mesh filter mesh, the supernatant was depressurized and concentrated and then lyophilized to prepare an extract. 900 g of the extract was obtained and used as a sample for the following experiment. The RRW extract was used as a sample in this study. The extract was dissolved in distilled water and used in the experiment.

Reference example  1. Preparation of experimental material

Taurine was used as a positive control with proven activity. There is evidence that administration of taurine reduces oxidative stress induced by exercise (Dawson et al. 2004, Zhang et al. 2004) and has the effect of inhibiting oxidative stress associated with GSH oxidation during muscle contraction and exercise . The taurine used in this study was purchased from JIANGSU YUANYANG PHARMACEUTICAL CO., And was orally administered at 500 mg / kg body weight / day.

Reference example  2. Experimental animals

ICR 4 weeks old male mice with SPF (Specific pathogen free) were purchased and used in the experiments (Orient Bio Inc., Gyeonggi Province). The animals were housed in a polycarbonate cage (278 × 420 × 200 mm) in a hood equipped with a constant temperature and humidity system. The animals were housed at room temperature 22 ± 2 ℃, air velocity 13 ~ 18cm / sec, ventilation frequency 10 ~ / h, air pressure difference 2 ~ 10 mmH ₂ O, dark cycle 12 hours; 7: 00 ~ 19: 00, and illumination was 150 ~ 300 Lux. The body weight was measured twice a week to observe the appearance on a regular basis, and the bedding was changed periodically to maintain cleanliness. The diet was fully fed with AIN-76.

Experimental Example  1. Limit swimming activity evaluation

(Yoshi et al., Biosci. Bio-Technol., 70 (10), 1985), the following experiment was carried out by applying the method described in the literature in order to confirm marginal swimming activity of the extract obtained from the above- 2532-2535, 2006).

In the previous study, the swimming pool of the swimming movement was 90 × 45 × 45 cm and the water was filled up to 35 cm and the water temperature was maintained at 34 ℃. The surface flow rate was maintained at a flow rate of 7.5 L / min by using a pump with a voltage regulator and a water flowmeter (type F45500, Blue White Co, Westminster, Calif., USA) Limit swimming time was measured by the time the mouse was no longer able to swim, that is, the point at which the mouse did not come up to the water surface until 7 seconds had elapsed (Matsumoto K. et al., J Appl Physiol, 81, 1843-1849, 1996).

A total of 18 mice were divided into 6 control groups (control group) so that the mean marginal swimming time was the same after measuring the marginal swimming time of 2 times at a flow rate of 7 L / min after one week preliminary feeding period. , Taurine (taurine), and carbohydrate extract (RRW). In the control group, 500 mg / kg body weight / day was given for distilled water, taurine group, and 2 weeks for the RRW group without training at a concentration of 1 g / kg body weight / day. Limit swimming time was evaluated by fasting 3 hours before marginal swimming exercise after 2 weeks and oral administration of samples 2 hours before experiment.

The marginal swimming time of the mice was performed to evaluate whether the extract had a physical stress inhibitory activity, and the results are shown in Fig. The initial swimming time of each treatment group was divided into 25.0 minutes so as not to show difference. After 2 weeks of oral administration, the control group showed 35.5 minutes, taurine group had 40.7 minutes, and RRW group had 50.1 minutes. In the marginal exercise group, RRW was 1.4 times higher than control group . In the second group, the control group was 34.3 minutes, the taurine group was 53.0 minutes, and the RRW group was 45.8 minutes. In the taurine group, the marginal swimming time was 1.6 times and the RRW group was 1.3 times. There was no statistically significant difference between the taurine group and the RRW group. Limit swimming activity was increased by RRW administration and similar to that of taurine, a positive control.

Experimental Example  2. Blood lactate measurement

In order to confirm the effect of the extract obtained from the above Example on the blood lactate level after exercise, an experiment was conducted as described below (You.YH et al., Biosci. Bio technol. , 70 (10), 2532-2535, 2006).

After swimming (15 minutes of exercise), blood was taken from the venous blood of the tail of the experimental animals and lactic acid and blood glucose levels were measured. Blood lactate was measured using a test strip (ARKRAY, Kyoto, Japan).

The results of evaluating the amount of blood lactate after a certain period of swimming exercise are shown in FIG. DL in the control group, 2.9 mg / dL in the taurine group and 3.0 mg / dL in the RRW group. The taurine group and the RRW group showed a statistically significant decrease by 1.5 times compared to the control group. Since accumulation of lactic acid means fatigue, physical stress by swimming exercise was suppressed by taurine and RRW compared to the control group.

Experimental Example  3. Measurement of glycogen level in tissues

In order to confirm the effect on the amount of glycogen in post-exercise tissue of the extract obtained from the above example, the following experiment was carried out by applying the method described in the literature (Jung KA, Journal of Ethnopharmacology., 93, 75- 81, 2004).

The animals were sacrificed by oral administration of a sample (1 g / kg body weight / day) and taurine (500 mg / kg body weight / day). Muscle tissue (mid-light) was harvested and stored at -70 ° C until analysis. The medium was extracted with 30% KOH (10 times) and lysed at 100 ° C for 30 minutes. The samples were then transferred to a glass tube (100 μL). To prepare a glycogen standard curve, make a glycogen solution with the concentrations of 0, 100, 250, 500 and 1000 μg / mL together with the sample, and transfer the solution to 100 μL. To each well, 400 μL of distilled water was added to each well and 0.2% anthrone solution was added to sulfuric acid. After mixing, the absorbance was measured at 620 nm by cooling on ice for 15 minutes, and the absorbance value was converted to mg glycogen / g tissue by substituting the absorbance value into the standard curve.

The results of measurement of muscle glycogen content in each experimental group are shown in FIG. Glycogen content in the control group was 0.3 mg / g tissue, 0.4 mg / g tissue in the taurine group and 0.4 mg / g tissue in the RRW group. and 1.5 times higher than control group, respectively. There was no significant difference between the taurine group and the RRW group. It was confirmed that taurine and RRW administration showed the effect of suppressing stress due to marginal swimming movement, indicating the effect of reducing glycogen in muscle.

Experimental Example  4. Antioxidant system  Active measurement

In order to examine the antistress mechanism at the molecular level of the extract obtained from the above examples, antioxidant activity was measured to confirm the effect on enzyme activities such as CAT, SOD, GPx, GR, GSH and MDA, (Packer L. Oxidant, antioxidant nutrients and the athlete J Sport Sci, 15: 353-63, 1997).

4.1 Experimental Process

Excessive exercise produces various reactive oxygen species (ROS) in the human body. These reactive oxygen species are cleared by the action of various antioxidant systems in the human body, but in the presence of free radicals, this can act as a cause to lower exercise capacity. Therefore, in this experiment, we evaluated the antioxidant enzyme activity through sacrifice after marginal swimming exercise to evaluate the exercise capacity and exercise stress protection mechanism by suppressing oxidative stress. Respectively.

Antioxidant activity of muscles was evaluated by using gastrocnemius tissue stored at -70 ° C. D-PBS equivalent to 10 times the liver weight was homogenized, centrifuged at 13,000 rpm for 30 minutes, and the supernatant was diluted. Muscles were lysed with gastrocnemius muscle in PBS containing 1.13% KOH, centrifuged at 13,000 rpm for 30 minutes, and the supernatant was diluted. Prior to the measurement of antioxidant activity, Bradford assay was used to quantify protein using bovine serum albumin (BSA) as a standard curve. The activity and content of CAT, SOD, GPx, GR, GSH and MDA were analyzed by the same treatment as the method of extract administration.

4.2 Superoxide dismutase  ( SOD )

Superoxide dismutase activity was analyzed by spectrophotometric method. A 96-well plate was prepared, and 20 samples were dispensed into 2 wells of the sample and blank, and ddw 20 was dispensed into blank 1 and blank 3 wells. Dissolve 20 wells of 0.75 mM WST-1 in the wells, mix 20 and 20 wells in blank 2 and 3, add 20 xanthine oxidase solution to sample and blank 1, Min and absorbance was measured at 250 nm. The measured absorbance was superoxide anion radical inhibition rate (%) = [Abs. blank 1 - Abs. blank 3) - (Abs. sample - Abs blank 2) / (Abs. blank 1- Abs. blank 3) × 100], and the SOD enzyme activity was calculated as 0, The protein was converted to U / mg protein using a standard curve drawn at 3.125, 6.25, 12.5, 25, 50 U / ml.

SOD is an antioxidant enzyme that plays a role of dismutting superoxide anion radical, which is a highly reactive and toxic free radical, into hydrogen peroxide. SOD activity is shown in Table 1. In muscle tissue, SOD activity was 3.2, 4.4 and 3.9 U / mg protein, respectively, and the activities of taurine and RRW groups were about 1.4 times and 1.2 times higher than control group, respectively. As a result of SOD activity evaluation, taurine and RRW groups showed high activity of SOD related to global exercise stress protection in both tissues.

4.3 Catalase  ( CAT )

The catalase activity of converting hydrogen peroxide into water and oxygen was analyzed by spectrophotometric method such as Aebi. 96 well plates were prepared, and 20 mM H ₂ O ₂ solution 290 was added to the tissue fluid 10, and the change in absorbance was measured at 240 nm for 3 minutes using a microplate reader kinetic method. The catalase enzyme activity was converted to U / mg protein.

CAT has the effect of reducing the hydrogen peroxide to water in the peroxisome of most tissues and defending against tissue damage due to the increase of hydrogen peroxide. It has a high Km value compared to GPx and is an enzyme which acts mainly when hydrogen peroxide concentration is high. . The activity of CAT is shown in Fig. In muscle tissue, activity was 7.1, 8.1 and 7.5 U / mg protein, respectively. The CAT activities of liver and muscle were evaluated by the taurine and RRW groups.

4.4. Glutathione peroxidase  ( GPx )

Glutathione peroxidase is also an enzyme that plays an important role in reducing hydrogen peroxide to water. To measure the enzyme, 3.5 mM glutathione reduced form 120, 0.5 U / mL glutathione reductase was added to the sample 20 to be measured, and then 2.5 mM NADPH was added. Finally, 30 mM tert-butyl hydroperoxide was added to the reaction mixture, and the absorbance was measured at 340 nm for 1 minute at 4 minutes.

GPX protects oxidative stress by acting on low concentration of hydrogen peroxide. It is an antioxidative enzyme containing Se. It catalyzes the reaction of reducing GSH with oxidized GSSG, water and other peroxides by using NADP as an electron acceptor in the body Which prevents peroxidative damage and inhibits oxidative stress. GPx activity is shown in Table 1. The activity of the RRW group was 0.11, 0.12, and 0.13 U / mg protein, respectively, and the activity of the RRW group was statistically significantly higher than that of the control group. As a result of evaluating the GPx activity of liver and muscle, it was confirmed that GPx activity of taurine and RRW group which are involved in global exercise stress protection is high in both tissues.

4.5. Glutathione  ( GSH )

Glutathione is a tetrameric enzyme with a molecular weight of 95 kDa in humans and plays an important role in the host antioxidant defense system. This enzyme acts primarily to protect the hydrogen peroxide from CAT and to remove hydrogen peroxide It is involved in toxicity. The enzyme activity was determined by modifying the method of kerboom and measuring the degree of production of yellow 5-thio-2-mitrobensoic acid for 5 minutes at 412 nm for 1 minute.

Reduced GSH is a tripeptide consisting of glutamic acid, cysteine and glycine. It is a GSH with reducing ability due to SH ^- group of cystein. It is easily oxidized by oxidative stress to form hexapeptide The GSH content of the oxidized GSSG is shown in Table 1. The content of GSH in the muscle tissue was 19.8, 23.2, and 20.1 umole / mg protein, respectively, and the GSH content in the taurine group was statistically higher than that in the control group. The GSH activity in the muscle tissue of the RRW group was higher than that of GSH activity in the marginal swimming stress protection.

4.7. Glutathione reductase  ( GR )

The activity of GR catalyzed the reduction of GSSG in the presence of NADPH, causing NADPH to oxidize to NADP ^+, where the absorbance reduction was measured at 340 nm.

GR plays a role in rapidly converting GSSG produced by in vivo oxidative stress into GSH, and is known as an enzyme that maintains the redox potential of the antioxidant system. The GR activity is shown in Table 1. In liver tissues, the activity was 2.3, 2.2 and 2.0 U / mg protein, respectively. Their activity in muscle tissue was 0.2, 0.2 and 0.2 U / mg protein, respectively. There was no statistically significant difference between the control and the treatment groups.

Effect on Muscle Antioxidant Activity of Experimental Mice Group SOD CAT GPx GSH U / mg protein U / mg protein U / mg protein nmoles / g tissue Control 7.09 + 0.16b 3.22 ± 0.11 b 0.11 0.001 0.20 + 0.01a Taurine 8.07 ± 0.51ab 4.42 + 0.14 a 0.12 ± 0.01ab 0.19 0.02a RRW 7.46 ± 0.15ab 3.89 ± 0.16a 0.13 + 0.01a 0.20 + 0.02a Mice were given either vehicle or Taurine (500 mg / kg body weight) or RRW extracts (l g / kg body weight / day) before exhaustive exercise. The antioxidant activity was measured after dissecting. Data express the mean ± S.E. of 6 mice in each group. * Significantly different from control, p < 0.05

4.8. Malondialdehyde  ( MDA )

The content of lipid peroxide in the tissue fluid was assessed using malondialdehyde (MDA), a thiobarbitric acid substance, as a reference material, by using thiobarbitric acid substance (TBARS) produced by the reaction of lipid peroxide with thiobarbitric acid. Add 500 uL of 15% TCA to 10-fold diluted tissue fluid to precipitate proteins for 10 minutes. After centrifugation at 4,000 rpm for 10 minutes, 500 μL of the supernatant was transferred to a new 1.5 mL tube, and then 0.375% TBA was added to 500 μL. This mixture was boiled at 100 ° C for 30 minutes and cooled on ice for 10 minutes. After centrifugation at 4 ° C and 3000 rpm for 10 minutes, absorbance of the supernatant was measured at 535 nm. TBARS was calculated using the MDA standard curve and expressed as mM / mg protein or nM / mg protein.

MDA is an indicator of the level of lipid peroxidation in tissues by measuring the content of MDA, a lipid peroxide. Lipid peroxidation is caused by increased production of free radicals due to oxidative damage in the body and decreased ability of antioxidant defense. The degree of lipid peroxidation was measured by colorimetric determination of lipid peroxides bound to thibarbituric acid by the amount of MDA. The MDA content of the muscle tissue of each group is shown in Fig. The content of MDA in muscle tissue was 14.13, 7.47 and 7.13 nmole / mg protein, respectively, and the contents of taurine and RRW groups were significantly decreased by about 1.9 and 2.0 times, respectively. As a result of MDA content in muscle, it was confirmed that Taurine and RRW inhibited lipid peroxidation.

Experimental Example  5. Stress Blood Hormone cortisol  Content evaluation

In order to examine the molecular stress-induced antisstress mechanism of the extract obtained from the above examples, the following experiment was conducted by applying the method described in the literature (Qiumei Youa., Et , al., Toxicology and Applied Pharmacol., 230 (1), 1-8, 2008).

The blood obtained at the time of dissection was centrifuged at 3000 rpm for 10 minutes, and only the serum was separated and stored at 70 ° C. For the analysis, thawed serum was diluted 10 times with distilled water, and 25 μL of the cortisol standard solution and 25 μL of the cortisol standard conjugate were dispensed into each well and incubated at room temperature for 1 hour. Subsequently, the remaining material was removed and washed three times with washing buffer. After washing, 100 uL of TMB substrate was added to each well and incubated for 15 minutes. After stopping the reaction with 50 uL of stop solution, absorbance was measured at 450 nm within 20 min. The absorbance of the solution was then calibrated to the standard curve obtained from the kit (Calbio tec) and the absorbance was calculated as ng / mL.

The results of analysis of the content of cortisol, which is an index of stress, on the serum of the positive control group and the extract administration group are shown in FIG. Values were 520.8, 358.1, and 332.2 ng / mL from the control group. The content of cortisol in the taurine and RRW groups was 1.6 times and 1.5 times lower than that of the control group. Serum levels of cortisol were lower in the taurine and RRW groups than in the control group.

In conclusion, the Korean native extract has an effect on global exercise efficacy and inhibits oxidative stress induced by marginal exercise stress, that is, it is judged to be a functional material effective in suppressing physical stress. It can be very useful as a functional material by performing additional studies.

Hereinafter, formulation examples of the composition containing the extract of the present invention will be described, but the present invention is not intended to be limited thereto but will be specifically described.

Formulation example  One. Sanje  Produce

RRW ------------------------------------ 200 mg

Lactose ----------------------------------- 100 mg

Talc ------------------------------------ 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

Formulation example  2. Preparation of tablets

RRW ------------------------------------ 200 mg

Corn starch ----------------------------- 100 mg

Lactose ----------------------------------- 100 mg

Magnesium stearate ---------------------- 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

Formulation example  3. Preparation of capsules

RR20 ----------------------------------- 200 mg

Crystalline cellulose ------------------------ 3 mg

Lactose ------------------------------ 14.8 mg

Magnesium stearate 0.2 mg &lt; RTI ID = 0.0 &gt;

The above components are mixed in accordance with a conventional method for producing a capsule, and filled in a gelatin capsule to prepare a capsule.

Formulation example  4. Preparation of injections

RR80 ----------------------------------- 200 mg

Mannitol --------------------------------- 180 mg

Sterile sterile distilled water for injection -------------------- 2974 mg

Na ₂ HPO _{4 ,} 12H ₂ O - 26 mg

(2 ml) per ampoule in accordance with the usual injection method.

Formulation example  5. Liquid  Produce

RRW ------------------------------------ 200 mg

Isolation Party --------------------------------- 10 g

Mannitol ------------------------------------ 5 g

Purified water -----------------------------------

Each component was added and dissolved in purified water according to the usual liquid preparation method, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was added with purified water to adjust the total volume to 100 ml, And sterilized to prepare a liquid preparation.

Formulation example  6. Manufacture of health food

RR20 ----------------------------------- 1000 mg

Vitamin mixture -----------------------------

Vitamin A Acetate ---------------------- 70 ㎍

Vitamin E -------------------------------- 1.0 mg

Vitamin B1 ------------------------------ 0.13 mg

Vitamin B2 ------------------------------ 0.15 mg

Vitamin B6 ------------------------------- 0.5 mg

Vitamin B12 ------------------------------ 0.2 g

Vitamin C --------------------------------- 10 mg

Biotin ----------------------------------- 10 [mu] g

Nicotinic acid amide 1.7 mg

Folic acid ------------------------------------- 50 μg

Calcium pantothenate --------------------------- 0.5 mg

Inorganic mixture -----------------------------

Ferrous sulfate ------------------------------ 1.75 mg

Zinc oxide ------------------------------- 0.82 mg

Magnesium carbonate --------------------------- 25.3 mg

Potassium monophosphate ------------------------------ 15 mg

Secondary calcium phosphate ------------------------------ 55 mg

Potassium citrate ------------------------------- 90 mg

Calcium carbonate -------------------------------- 100 mg

Magnesium Chloride --------------------------- 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

Formulation example  7. Manufacture of health drinks

RR80 ----------------------------------- 1000 mg

Citric acid --------------------------------- 1000 mg

Oligosaccharides --------------------------------- 100 g

Applicable concentrate ------------------------------- 2 g

Taurine ------------------------------------- 1 g

Add purified water - 900 ml total

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 ° C for about 1 hour. The resulting solution was filtered to obtain a sterilized 2-liter container, which was sealed and sterilized, It is used in the production of the health beverage composition of the invention.

Although the composition ratio is a mixture of the components suitable for the preferred beverage as a preferred embodiment, the blending ratio may be arbitrarily varied according to the regional and national preferences such as the demand level, the demanding country, and the intended use.

Claims (7)

A pharmaceutical composition for the treatment and prevention of fatigue, dysesthesia, hypothyroidism, or lethargy, comprising a water extract of a corresponding stem or a leaf as an active ingredient. delete delete delete A health functional food for improving and preventing fatigue, dysesthesia, hypogonadism, or lethargy which contains water extract of the corresponding stem or leaf as an active ingredient. delete delete

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