KR101329727B1 - Composition comprising the extract of Loranthus yadoriki SIEB. having monoamine oxidase-inhibiting activity - Google Patents

Abstract

The present invention relates to a composition for the prevention and treatment of MAO-related depression, stress relief and fatigue recovery comprising an extract of Loranthus yadoriki SIEB. Having monoamine oxidase (MAO) inhibitory activity as an active ingredient. The oak mistletoe extract of the present invention strongly inhibited MAO enzyme activity in in vitro experiments, oral administration of oak mistletoe extract increased MAO-A activity after exercise of animals and lowered MAO-B enzyme activity to normal levels. By recovering, and also lowering the activity of DBH and increasing immune function, oak mistletoe extract of the present invention is a drug for depression associated with MAO enzymes, sports functionality for relieving stress after exercise, fatigue recovery and athletic performance It can be used as a food and beverage and dietary supplement.

Description

Composition containing oak mistletoe extract having monoamine oxidase inhibitory activity {Composition comprising the extract of Loranthus yadoriki SIEB. having monoamine oxidase-inhibiting activity}

The present invention relates to a composition for the prevention and treatment of depression containing oak mistletoe extract as an active ingredient.

1 Hajto, T., Berki, T., Boldizsar, F., and Nemeth, P. Galactoside-specific plant lectin, Viscum album agglutinin-Ⅰ induces enhanced proloferation and apoptosis of murine thymocytes in vivo . Immunol . Lett. 86 (1): 23-27 (2003).

2. Siegle, I., Friz, P., McClellan, M., Gutzeit, S., and Murdter, TE Combined cytotoxic action of Viscum album agglutinin-Ⅰ and anticancer agent against human A549 lung cancer cells. Anticancer Res . 21 (4A): 2687-2691 (2001).

3. Kim, YK, Kim, YS, Choi, SU, and Ryu, SY Isolation of flavonol from Loranthus tanakae and cytotoxic effect of them on human tumer cell lines. Arch . Pharm . Res . 27 (1): 44-47 (2004).

[4] 4. Karagoz, A., Onay, E., Arda, N., and Kuru, A. Antiviral potency of mistletoe ( Viscum album ssp. album ) extracts against human parainfluenza virus type 2 in Vero cells. Phytother . Res . 17 (5) : 560-562 (2003).

5. Fernandez, T., cerda, ZP, Aulicino, P., Caldas, LE, Wagner, M., Ricco, R., Hajos, S., Gurni, A., and Alvarez, E. Immunobiological features of the galactoside lectin L-Lc isolated from the Argentine mistletoe Ligaria cuneifolia . J. Ethnopharmacol . 85 (1) : 81-92 (2003).

6. Boneberg, EM and Hartung, T. Mistletoe lectin-I increases tumor necrosis factor-alpha release in lipopolysaccharide-stimulated whole blood via inhibition of interleukin-10 production. J. Pharmacol . Exp . Ther . 298 (3) : 996-1000 (2001).

7. Tabiasco, J., Pont, F., Forrnie, JJ, and Vercellone, A. Mistletoe viscotoxins increase natural killer cell-mediated cytotoxicity. Eur . J. Biochem . 269 (10) : 2591-2600 (2002).

8. Mengs, U., Gothel, D., and Leng-Peschlow, E. Mistletoe extracts standardized to mistletoe lectins in oncology: review on current status of preclinical research. Anticancer Res . 22 (3) : 1399-1407 (2002).

[Document 9] 9. Chang Bok Lee. Korean Plant Book, 783. Hyangmunsa, Seoul, South Korea (1999).

10. Hwang, K.H., Ma, J.Y. and Kim, I.R .: The studies on the theory of KIMI by the activity of monoamine oxidase. Kor. J. Herbology 14: 1-14 (1999)

11. Hwang, KH, Kim, IR and Han, YN Effects of cold and hot drugs on the activity of monoamine oxidase. Kor . J. Pharmacogn . 30: 145-150 (1999).

12. Hwang, K. H. Development of new food materials to improve athletic performance and improve stress. Report of research results on the development of native plants using technology of the Ministry of Science and Technology. (2003).

13. Creveling, C. R., Daly, J. W., Witkop, B. and Udenfriend, S. 1962. Substrates and Inhibitors of Doipamine-β-Hydroxylase. Biochemica et Biophysica Acta. 64: 125.

According to the World Health Organization (WHO), depression is the fourth leading cause of discomfort and death worldwide. Although the levels of a key protein called MAO-A (monoamine oxidase A) are elevated in the brains of depressed patients, most antidepressants do not affect MAO-A. Lowering elevated levels of MAO-A is necessary to fully treat depression and prevent relapse. In this study, MAO-A, an enzyme that degrades dietary monoamine, and dopamine beta, an enzyme that degrades dopamine to norepinephrine, as well as MAO-A, which uses serotonin as a substrate from native plants, -Research on the inhibitory activity of dopamine β-hydroxylase (DBH) and its active ingredients to improve the depression without side effects and to develop functional foods that can supplement the action of antidepressant drugs currently used. Korean Mistletoe ( Viscum album L. var. coloratum Ohwi) are anticancer ^1-3), antihypertensive, antibacterial, and the pharmacological effects have been reported, such as anti-viral, cardiac ^{effects. 4, 5).}

In particular, mistletoe has been widely recognized as an anticancer activity in Europe such as Germany for a long time, and is widely used as a treatment and treatment aid for various malignant tumors. ^6-8) the current viscotoxin belonging is well known.

The Encyclopedia of plant mistletoe are classified into five kinds ^9). This study aims to identify 39 species of mistletoe collected from various regions, hosts, genders, directions, etc. according to the growth conditions of mistletoe. The content of the indicator components was very different according to the species of mistletoe in this process. Mistletoe has been used for ornamental and medicinal purposes since ancient times. Recently, studies on the anticancer and anti-tumor immunomodulatory activity of mistletoe have been actively conducted, but there is no research on antidepressant activity.

Therefore, the present inventors focused on the fact that mistletoe has different active ingredient contents and oil species according to the species and searched for various activities. The present invention was completed by confirming that yadoriki SIEB.) exhibited inhibitory activity against the MAO-A and MAO-B enzymes, separating the active ingredient to reveal the structure, and revealing the mechanism of inhibiting the enzyme activity of the active ingredient.

Accordingly, it is an object of the present invention to provide a composition for the prevention and treatment of monoamine oxidase inhibitors and monoamine oxidase antidepressants comprising oak mistletoe extracts exhibiting monoamine oxidase (MAO) inhibitory activity as active ingredients.

It is also an object of the present invention to provide a composition, sports food and beverage and health functional food for antidepression, fatigue, stress relief and athletic performance.

In order to achieve the above object, the present invention is oak mistletoe ( Loranthus It provides a monoamine oxidase (MAO) inhibitor containing yadoriki SIEB.) extract.

The present invention also provides a pharmaceutical composition for the prevention and treatment of MAO enzyme-related depression containing oak mistletoe extract having a monoamine oxidase inhibitory activity.

The present invention also provides a health functional food for antidepression, fatigue, stress relief and athletic performance containing oak mistletoe extract having a monoamine oxidase inhibitory activity.

The extracts as defined herein include crude extracts, polar solvent-soluble extracts and / or non-polar solvent-soluble extracts.

The crude extract is an extract available in water, spirits, lower alcohols having 1 to 4 carbon atoms or a mixed solvent thereof, preferably a mixed solvent of water and methanol, more preferably 30 to 90% mixed solvent of water and methanol, including purified water. It includes.

The nonpolar solvent-soluble extract includes an extract which is soluble in a non-polar solvent such as ethyl acetate, chloroform, hexane or dichloromethane, preferably ethyl acetate.

The polar solvent-soluble extract includes water, alcohol, lower alcohols having 1 to 4 carbon atoms, or a mixed solvent thereof, preferably an alcohol soluble in butanol.

The method of separating the extract of the present invention is as follows.

Hereinafter, the present invention will be described in detail.

The extract of the present invention, after crushing the dried oak mistletoe with a grinder, about 1 to 20 times, preferably about 1 to 15 times the water, alcohol, lower alcohol of 1 to 4 carbon atoms, acetone , Chloroform, methylene chloride, hexane or a mixed solvent thereof, preferably water and methanol mixed solvent, more preferably water and methanol 30 to 90% mixed solvent, 12 hours to 1 week, preferably 24 hours to For 72 hours, extraction methods such as cold sediment extraction, hot water extraction, ultrasonic extraction, reflux cooling extraction, etc., preferably at 10 ° C. to 100 ° C., preferably at 30 ° C. to 70 ° C., to obtain an extract by performing a hot water extraction method. Stage 1; The crude extract of the present invention can be obtained through the production process of the second step in which the extract obtained above is filtered with a filter cloth and filtered to obtain a filtrate.

The polar solvent or nonpolar solvent soluble extract of the present invention may be obtained by adding water to the crude extract obtained above and then performing a conventional fractionation process using n-hexane, methylene chloride, ethyl acetate, and butanol, and then eluting with n-hexane, A non-polar solvent-soluble fraction extracted in a non-polar solvent such as ethyl acetate; And polar solvent soluble extract fractions soluble in polar solvents such as butanol, water and the like.

The present invention provides a pharmaceutical composition for the prevention and treatment of monoamine oxidase-related depression containing the oak mistletoe extract obtained in the production method as an active ingredient.

The present invention provides a monoamine oxidase inhibitor containing the oak mistletoe extract obtained in the extraction process as an active ingredient.

The present invention also provides an antidepressant composition containing the oak mistletoe extract obtained in the extraction process as an active ingredient.

In addition, the present invention provides a composition for improving athletic performance and fatigue comprising oak mistletoe extracts obtained in the extraction process.

The composition for the prevention and treatment of monoamine oxidase-related depression of the present invention and the composition for relieving stress of the body, restoring fatigue, and improving athletic performance include the oak mistletoe extract in an amount of 0.5 to 50% by weight based on the total weight of the composition. .

The composition comprising the oak mistletoe extract of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

The composition comprising the extract of the present invention may further comprise a suitable carrier, excipient and diluent according to conventional methods.

Carriers, excipients and diluents that may be included in the composition comprising the extract of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium Phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

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, Can be used.

The amount of the extract of the present invention may vary depending on the age, sex, and weight of the patient, but may be administered once to several times in an amount of 0.1 to 1000 mg / kg. The dose of the extract may also be increased or decreased depending on the route of administration, degree of disease, sex, weight, age, and the like. Thus, the dosage amounts are not intended to limit the scope of the invention in any manner.

The composition comprising the oak mistletoe extract of the present invention can be used in a variety of drugs, foods and beverages for stress relief, fatigue recovery and athletic performance of the body. Examples of the food to which the oak mistletoe extract can be added include various foods, sports foods and beverages, beverages, gums, teas, vitamin complexes, and health functional foods.

Oak mistletoe extract of the present invention is a drug that can be used with confidence even for long-term use for the purpose of prevention because there is little toxicity and side effects.

The extract of the present invention can be added to food or beverages for the purpose of improving athletic performance and fatigue recovery. At this time, the amount of the extract in the food or beverage is generally the health food composition of the present invention can be added to 0.01 to 30% by weight of the total food weight, the health beverage composition is 0.02 to 30 g based on 100 ml, preferably Can be added in a ratio of 0.3 to 1 g.

In another aspect, the present invention provides an anti-fatigue, anti-stress and exercise performance functional drinks containing oak mistletoe extract as an active ingredient.

In another aspect, the present invention provides a health functional food for antidepression, fatigue, stress relief and athletic performance containing oak mistletoe extract as an active ingredient.

The health functional food includes a functional beverage, sports food and beverage, candy, various foods, beverages, gums, tea, vitamin complexes, or health supplement foods, the functional beverage of the present invention contains the oak mistletoe extract has proven stability By preparing a beverage, it is possible to provide a functional beverage that is safe and easy to take, yet has effects such as fatigue recovery, stress relief, and athletic performance.

The functional beverage of the present invention is preferably characterized in that it comprises 0.1 to 10% by weight of oak mistletoe extract, 10 to 25% by weight of the usual beverage additives such as sweeteners and acidulants, and the remaining weight% of water. Typical beverage additives in the functional beverage of the present invention may include liquid fructose, sugar, maltodextrin, glucose, citric acid, nicotinic acid amide, pantothenic acid, sodium benzoate, flavoring agents, flavoring agents and the like.

In addition, the health beverage composition of the present invention, except for containing the extract as an essential ingredient in the indicated ratio, there is no particular limitation on the liquid component and may contain various flavors or natural carbohydrates, etc. as additional ingredients, like ordinary drinks. . Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of said natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and salts thereof. , Organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation 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.

The present invention confirms that oak mistletoe extract exhibits inhibitory activity against the MAO-A and MAO-B enzymes, and isolates the active ingredient to reveal the structure and reveals the mechanism of inhibiting the enzyme activity of the active ingredient, thereby inhibiting monoamine oxidase inhibitors. It can be used as a health functional food as well as a functional food and drink for sports, which is effective for antidepressant or depression medicine and fatigue, stress relief and exercise function improvement.

1 is a diagram showing the extraction and preparation work of oak mistletoe extract for the detection of MAO inhibitory activity,
Figure 2 is a diagram showing the change in the MAO enzyme activity in the body of rats ingested oak mistletoe extract,
Figure 3 is a diagram showing the changes in CD4 + and CD8 + cell expression of rats fed the oak mistletoe extract,
Figure 4 is a diagram showing the change in the ratio of CD4 + / CD8 + cells of rats ingested oak mistletoe extract,
5 is a diagram showing the effect of increasing the secretion capacity of IL-4, IFN-γ cells in the spleen of rats ingested oak mistletoe extract.

Hereinafter, the present invention will be described in detail by reference examples, examples and experimental examples.

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

Example 1. Extract preparation and screening in vitro activity of oak mistletoe

1-1. Materials and reagents

The oak mistletoe used in the experiment was supplied from Korea National Arboretum and used for measuring enzyme activity, serotonin (H-9523, Sigma), benzylamine (B-5136, Sigma), ion exchange resin Amberlite CG-50 (CG50-500G, Sigma) Sigma Co., Ltd. was used as a solvent for HPLC, and Merk Co., Ltd. was used as a solvent for HPLC, and a domestic special reagent was used as a solvent for column chromatography and sampling.

1-2. Isolation of Oak Mistletoe Extract

773 g of oak mistletoe dry powder was supplied from Korea National Arboretum (www.forest.go.kr) and extracted with 70% MeOH 2L for 7 days at room temperature. The extract was combined three times, and then concentrated under reduced pressure under reflux cooling at 50 ° C. in a water bath. The oak mistletoe extract of 218.5 g of methanol (hereinafter referred to as "LYS") was obtained (yield 28.27%) (see Fig. 1).

1-3. Fraction of Soluble Extract with Polar Solvent and Nonpolar Solvent

218.5 g of oak mistletoe methanol extract obtained by concentrating under reduced pressure in Example 1-2 was suspended in an appropriate amount of distilled water, and the extract obtained by solvent fractionation was concentrated under reduced pressure using a solvent such as Hexane, CHCl ₃ , EtOAc, BuOH, and the like. The remaining water layer was concentrated under reduced pressure to give Hexane fraction (hereinafter referred to as “LYS-HE”) (2.16 g, 0.98%), CHCl ₃ Fraction (hereinafter referred to as "LYS-CH") (8.09 g, 3.70%), EtOAc fraction (hereinafter referred to as "LYS-EA") (28.85 g, 13.20%), BuOH fraction (hereinafter referred to as "LYS-BU" (39.93g, 18.27%) and a water layer (hereinafter referred to as “LYS-WA”) (119.89g, 54.87%) were obtained (Table 1; see FIG. 1).

sample Dry weight
(g) yield
(%) IC ₅₀ values (mg / ml) MAO-A MAO-B DBH MeOH 218.5 28.27 0.5 0.95 0.3 Hexane 2.16 0.98 0.4 0.73 - CHCl ₃ 8.09 3.70 0.3 1.09 0.013 EtOAc 28.85 13.20 0.2 0.34 0.2 BuOH 39.93 18.27 0.2 0.12 0.065 Water 119.89 54.87 1.2 2.80 1.1

Experimental Example  One. MAO -A, MAO -B enzyme in vitro  Inhibitory activity search

In order to test the inhibitory activity against the MAO-A and MAO-B enzymes of the extracts obtained from the above examples, the experiments were performed as follows by applying the method disclosed in the literature (11).

1-4. From brain tissue MAO -A enzyme preparation

Rats were anesthetized with ether-doped anesthesia, opened, and blood was collected from the left ventricle. This was washed with 0.01 M phosphate buffered saline (PBS, pH 7.0) and 9 ml cold 0.25 M sucrose solution per 1 g of wet weight was added to homogenate with Turrax disperser for 1 minute. The homogenate was centrifuged at 700 × g for 20 minutes at 4 ° C., and the supernatant was taken and further centrifuged at 18,000 × g for 20 minutes. The supernatant was discarded and the pellet was suspended in 5 ml of PBS per 1 g of weight (see Table 2).

Reference Example  1. Experimental animals

Sprague Dawley male rats, 5 weeks old, were supplied by Biogenomes Co., Ltd., and were freely supplied with general solid feed and water in an animal breeding room where the temperature was controlled at 23 ± 3 ℃, humidity 50 ± 10%, and controlled for 12 hours. It was used for the experiment after adapting for ˜4 weeks.

Experimental Example  1.Oak Mistletoe In vitro  brain MAO -A inhibitory activity

In order to determine the brain MAO-A inhibitory activity of oak mistletoe extract, the following experiment was conducted using the method described in the literature (11).

1-1. Enzyme source  pharmacy

5 g of 0.25 M sucrose solution per 1 g of bovine adrenal wet weight was added and homogenated with a waring blender (IKA®T25, Basic) for 2 minutes and centrifuged at 700 x g for 10 minutes at 4 ° C. The supernatant was taken and centrifuged again at 10,000 × g for 20 minutes at 4 ° C., and the pellets were suspended in 2 ml of 0.25 M sucrose solution and frozen in a -15 ° C. refrigerator. When used, it was dissolved at room temperature and diluted 30 times with 0.25M sucrose solution to use as an enzyme source (see Table 3).

1-2. Brain tissue MAO -A enzyme activity measurement

0.5 ml of the prepared enzyme source was placed in a test tube, 0.5 ml of 1.0 mM serotonin solution was added as a substrate solution, and the mixture was incubated for 90 minutes in a 37.5 ° C incubator. The reaction was stopped by heating for 3 minutes on a 95 ° C. water bath, immediately centrifuged at 700 × g, and 1.0 mL of the supernatant was taken and poured into a previously prepared Amberlite CG-50 (H ⁺ form) column (0.6 × 4 cm). After sufficiently washing the resin with distilled water (40 ml or more), 3 ml of 4N acetic acid solution was poured into the resin, and the eluate was received in a test tube and the absorbance was measured at 277 nm. Separately, the correction group with the substrate solution at the reaction end point instead of the reaction start point was performed with the test group. Based on the control group of each experimental group, the change in enzyme activity according to the temperature change and drug administration was calculated according to the following Equation 1 (Equation 1; Table 4).

1-3. From liver tissue MAO -B enzyme preparation

Liver mitochondria fractions of rats were separated according to the conventional method and used as an enzyme source. In other words, rats anesthetized in Ether bottles were immediately subjected to blood collection in the left ventricle, and blood was collected. The livers were extracted, washed in 0.01M phosphate buffered saline (PBS, pH 7.0), and 0.25M sucrose per 1g of wet weight. 5 ml of the solution was added and homogenized with a Turrax disperser (IKA®T25, Basic) for 1 minute. This homogenate was immediately centrifuged at 700 x g for 20 minutes at 4 ° C. The supernatant was taken and again centrifuged at 18,000 × g for 20 minutes. The supernatant was discarded and the pellet was suspended and suspended in 5 ml of PBS (see Table 5).

1-4. Liver tissue MAO Determination of -B

Enzyme activity was measured according to the method of McEwen et al. That is, 0.5 ml of 4.0 mM benzylamine-HCl solution was added to the test tube with 0.5 ml of enzyme source and substrate solution, and the culture was continued for 90 minutes in a 37.5 ° C. thermostat. To stop the reaction, 0.2 ml of 60% perchloric acid was added thereto, and 4 ml of cyclohexane was added thereto, followed by shaking, followed by centrifugation at 700 × g for 20 minutes to obtain a cyclohexane layer. The absorbance of this cyclohexane layer was measured at 242 nm. Separately, the calibration group was run with the test group as in MAO-A. Based on the control group of each experimental group, the change in enzyme activity according to temperature change and drug administration was calculated according to the following Equation 2 (Table 6; Equation 2).

As a result of the experiment, the methanol extract of oak mistletoe and each solvent fraction were prepared using 1 mg / mL solution using distilled water as a stock solution, and diluted to 1/2 and 1/10 solutions of MAO-A and The inhibitory activity against MAO-B was searched to obtain the results of Table 1 above.

Methanol extract of oak mistletoe was found to significantly inhibit the enzymatic activity of MAO-A and MAO-B in vitro. The IC ₅₀ values for MAO-A and MAO-B of methanol extracts were 0.5 mg and 0.95 mg, respectively. Methanol extract of oak was found to show relatively strong inhibitory activity against MAO-A activity and the activity of each solvent fraction was compared with IC ₅₀ value. The IC ₅₀ value of the methanol extract was 0.5 mg / ml, the IC ₅₀ value of the hexane fraction was 0.4 mg / ml, the IC ₅₀ value of the chloroform fraction was 0.3 mg / ml, and the IC ₅₀ value of the ethylacetate fraction was 0.2 mg / ml, buthanol. The IC ₅₀ value of the fraction was also 0.2 mg / ml, and the remaining water layer showed weak activity at 1.2 mg / ml. The MAO-B inhibitory activity of the oak mistletoe methanol extract was also shown to be relatively strong, and the activity of each solvent fraction was compared with an IC ₅₀ value. IC ₅₀ values of the methanol extract of the stock solution was 0.95mg / ml IC ₅₀ values of the hexane fraction was 0.73mg / ml IC ₅₀ value of the chloroform fraction is 1.09mg / ml, IC ₅₀ value of the ethylacetate fraction is 0.34mg / ml, buthanol The IC ₅₀ value of the fraction was found to be the strongest, indicating 0.12 mg / ml, and the remaining water layer showed weak activity at 2.80 mg / ml.

This extract was used as a sample of MAO-A, MAO-B inhibitory activity screening and the results are summarized in Table 1 above.

Experimental Example  2. DBH  Inhibitory Activity Assay

In order to measure the DBH inhibitory activity of oak mistletoe extract, it was experimented as follows using Crevelling et al. (12).

2-1. Enzyme source  pharmacy

5 g of 0.25 M sucrose solution per 1 g of bovine adrenal wet weight was added and homogenated with a waring blender (IKA? T25, Basic) for 2 minutes and centrifuged at 700 × g for 10 minutes at 4 ° C. The supernatant was taken and centrifuged again at 10,000 × g for 20 minutes at 4 ° C., and the pellets were suspended in 2 ml of 0.25 M sucrose solution and frozen in a -15 ° C. refrigerator. When used, it was dissolved at room temperature and diluted 30 times with 0.25M sucrose solution to use as an enzyme source (see Table 3).

2-2. Preparation of Reaction Aid

Sodium fumarate, N-ethylmaleimide, iproniazid phosphate and ascorbic acid were 0.06, 0.06, 0.006 and 0.06M, respectively, and the solution dissolved in distilled water was used as a reaction aid.

2-3. DBH  Inhibitory Activity Assay

0.3 ml of enzyme source, 1.0 ml of sample solution, 0.2 ml of 0.4% catalase, 0.5 ml of 1M acetic acid buffer (pH 5.0), and 0.5 ml of reaction aid were added and preincubated at 37 ° C. for 15 minutes. After adding 0.5 ml of 0.12M tyramine and HC1 and incubating for 90 minutes, 0.4 ml of 3M trichloroacetic acid was added to stop the enzyme reaction. Immediately after the reaction was stopped, the mixture was centrifuged at 700 × g for 10 minutes, and 3.0 ml of the supernatant was poured into a Dowex 50W × 8 (H ⁺ form, 200-400 mesh) column (0.8 × 3 cm) and washed with 30 ml of distilled water. 3 ml of 4N NH ₄ OH was added thereto, and the eluted eluate was separately received in a test tube and 0.2 ml of 4% NaIO ₄ solution was added thereto. After 10 minutes, 0.2 ml of 20% Na ₂ S ₂ O ₅ solution was added thereto, and the UV absorbance was measured at 330 nm. A control group containing the same amount of distilled water instead of the test solution and a blank test group in which the substrate solution was added at the reaction end point instead of the reaction start point, and a sample correction group in which the same amount of distilled water was added instead of the substrate solution were performed. Each group was calculated by performing duplicates, and the enzyme inhibition rate of the sample solution was calculated according to Equation 3 below (Equation 3; Table 7; see FIG. 2).

2-4. Sample  50% enzyme inhibition concentration IC ₅₀ Output of

By diluting the sample solution step by step, the enzyme inhibition rate was calculated, and the enzyme inhibition rate for the concentration of the sample solution was plotted on a logit log paper to obtain 50% enzyme inhibition concentration.

As a result of the experiment, 1 mg / mL solution was prepared using methanol extract of oak mistletoe and each solvent fraction using distilled water to prepare a stock solution. The inhibitory activity against β-hydroxylase was searched to obtain the results of Table 1 above.

It was confirmed that oak mistletoe showed strong DBH inhibitory activity of methanol extract. IC ₅₀ values of the crude methanol extract was 0.3mg / ml hexane fraction did not exhibit activity had IC ₅₀ values of the chloroform fraction showed up most strongly to 0.013mg / ml, IC ₅₀ values of ethylacetate fraction 0.2mg / ml In addition, the IC ₅₀ value of the buthanol fraction was also strongly expressed as 0.065mg / ml, and weakly at 1.1mg / ml in the remaining water layer.

From the above results, the antidepressant efficacy of oak mistletoe may be due to the inhibitory activity against MAO-A and MAO-B by the components coexisting in the ethyl acetate and butanol fractions and the DBH inhibitory activity present in the chloroform fraction. Could. Based on the results, anti-depressant efficacy components were determined using MAO inhibitory activity as guide assay for ethyl acetate and butanol fractions of oak mistletoe.

Experimental Example  3. The body MAO  Enzyme activity ( in vivo ) Measure

In order to measure mouse MAO by oral administration of oak mistletoe extract, the following experiment was conducted using the method described in the literature (11).

3-1. Preparation of Samples for Oral Administration

1L of 70% EtOH was added to 325.4 g of oak mistletoe dry powder, and the mixture was extracted by heating three times for 6 hours under reflux cooling at 100 ° C. water bath. After cooling to room temperature, the filtrate was concentrated under reduced pressure in a 40 ° C. water bath to remove EtOH, and an extract 76.56 g (23.5%) was obtained. This extract was stored in a -80 ° C. freezer and dissolved in distilled water during the experiment.

3-2. Oral administration of sample

1 g of oak mistletoe extract obtained in 3-1 was dissolved in 10 mL of distilled water, and diluted appropriately to prepare 16, 32, and 64 mg / mL. The solution was orally administered to the animal fasted from the afternoon of the previous day at 1 mL each morning. The positive control group and the blank test group were orally administered 40 ml of iproniazid and 1 mL of distilled water under the same conditions. This amount is the dry weight of the sample at 0.8-3.2 g / kg body weight, reflecting the IC ₅₀ value obtained in the in vitro experiments and is the amount equivalent to the literature value for the daily human dose in terms of animal dose.

40 oak male 5 week-old ICR (CrljOri: CD1) mice with a weight of about 30 g were distributed from Orient Co., Ltd. for 7 days while feeding indefinitely with water and feed. Mistletoe extract was divided into low-dose, medium-dose, and high-dose groups so that the daily intake was 0.8, 1.6, 3.2g / kg, and orally administered once a day for 7 days, dissected, blood was drawn from the left ventricle, and brain and liver were extracted. Activity changes of brain MAO-A and liver MAO-B were measured. On the other hand, the test group of animals treated with distilled water instead of the drug and the positive control group receiving iproniazid 40μmol were measured separately as a control group for the change of enzyme activity by the drug.

3-3. Experiment result

As a result of the experiment, as shown in FIG. 2, when compared with the enzyme activity of the control group administered tap water instead of oak mistletoe extract, MAO-A showed a slight decrease in the enzyme activity by oak mistletoe extract and the MAO-B enzyme. The activity tended to increase strongly. These results were found to affect the enzyme activity in the same trend as the positive control group using Ipronizid (I-7627, Sigma), which is used as a drug for the treatment of depression in clinical trials. Was significantly larger than the control drug and it was confirmed that the change in enzyme activity was significantly increased (see FIG. 2).

3-4. Statistical processing

All experimental results are expressed as mean ± SEM. Two-way ANOVA was performed to verify the significance of each group, and statistically significant difference was set at p <0.05.

Experimental Example  4. Immunological Analysis

In order to determine the immunological analysis of oak mistletoe extract, the following experiment was conducted using the method described in the literature (12).

4-1. Immune Cell Assay and Results

The spleen was placed in 1.5 ml of eppendorff tube at 2 × 10 ⁶ / ml under RPMI 1640 medium to measure the fraction of CD4 + and CD8 + cells, and then 500 μl of PBS was added and centrifuged at 5,000 rpm for 1 minute. . The filtrate was removed and reacted after adding PE (phycoerythrine; sc-13573 PE; sc-18860 PE) anti-CD4 (sc-13573, Santacruz Biotec, USA) and Biotin anti-CD8 (sc-18860, Santacruz Biotec, USA). . On the other hand, after binding to FITC (fluorescine isothiocyanate; sc-13573 L,; sc-18860 FITC, Santacruz Biotec, USA), it was reacted with streptavidin-FITC or streptavidin-PE as a secondary antibody. For the reaction, the expression level of each immune cell was measured by fractionation using FACScan flow cytometry (Quanta SC, Beckton Coulter Flow cytometer, USA).

As a result of the experiment, It has been reported that the CD4 + T-cell population in the blood decreases significantly during depression or exercise stress. As the intensity of the exercise increased, it gradually decreased, and then gradually increased again during the recovery period to the peak ^{12 )} . In the experimental group administered oak mistletoe, it was confirmed that the CD4 + T-cell population was decreased compared to the control group administered with tap water. In the positive control group administered with the positive control drug, the CD4 + T-cell population was found to be larger than the control group. This result is an experimental result measured in the splenocytes and is estimated to be different from the expression of immune cells in the blood (see FIG. 3). Unlike CD4 + T-cells, the blood CD8 + T-cell population is known to be less sensitive to physical stress from depression and exercise. In contrast to the CD4 + T-cells, the CD8 + T-cell population of the control group treated with tap water maintained significantly higher levels than the other two groups. In addition, the positive control group administered with the positive control drug was found to have a lower CD8 + T-cell population than the control group. In the experimental group administered oak mistletoe, the CD8 + T-cell population was found to be decreased than the control group, and the reduction rate was higher than that of the positive control group (see FIG. 3). Blood CD4 + T-cell and CD8 + T-cell populations are normally 65% and 35%. Changes in the ratios of CD4 + / CD8 + peripheral T-cells are usually used as an indicator of clinically abnormal immune function. It is also known that this value is usually kept lower than that of normal athletes who are engaged in intense training. The normal CD4 + / CD8 + ratio is usually about 2, but lowers to 1.5 for athletes and long-term trainees. This value is lowered by physical stress, including depression and exercise, and is known to return to normal after a recovery period after exercise. In rats, the ratio of CD4 + / CD8 + in rats is about 1.0 and in mice about 2.5. In this experiment, the CD4 + / CD8 + ratio of the control group administered with tap water was measured to be 1.34. On the other hand, the experimental group administered oak mistletoe extract was found to be significantly increased compared to the control group to 2.12, and the positive control group administered the positive control drug was confirmed to be increased to 1.54 (see Fig. 4). In the experimental group administered oak mistletoe, it was found that the CD4 + / CD8 + ratio was increased more than the positive control group administered with the control drug.

4-2. Cytokine  Assays and Results

IL-4 and IFN-γ secretion ability was recovered 72 hours after incubation of splenocytes (2 × 10 ⁶ / ml) in an incubator at 37 ℃, kit for measuring IL-4 and IFN-γ (K0331144; K0331138, Koma Biotec, Korea).

As a result, IL-4 is produced by CD4 + helper T cells and stimulates B cells and macrophages, which are involved in the production of IgE, an important mediator of allergic reactions. (cytokine). The cell population of IL-4 was measured in splenocytes of mice fed oak mistletoe. Compared to the control group administered with tap water, the experimental group administered oak mistletoe extract was found to increase in a concentration-dependent manner. The positive control group also showed a significant increase compared to the control group, indicating that it was more effective in producing IL-4 cells than the positive control drug. In particular, all three groups intake of oak mistletoe extract showed a significant increase, especially in the high intake group was found to show a significant increase (see Figure 5). INF-γ is also an immunoregulatory cytokine produced by CD4 + helper T cells and CD8 + T cells. The population of IFN-γ was measured in splenocytes of mice fed oak mistletoe. Compared to the control group administered with tap water, the experimental group administered oak mistletoe extract was found to increase in a concentration-dependent manner. The positive control group also showed a significant increase compared to the control group, indicating that it was more effective in producing IFN-γ cells than the positive control drug. In particular, all three groups intake of oak mistletoe extract showed a significant increase, especially in the high intake group was found to show a significant increase (see Figure 5). This result suggests that the intake of oak mistletoe extract can prevent the deterioration of immune function and increase the immune function due to depression, and the mechanism of CD4 + / CD8 + ratio and IL-4, IFN-γ secretion. It was confirmed that according to the effect.

Examples of the formulation of the composition are described below, but are not intended to limit the present invention but to explain in detail only.

Formulation example  1. Preparation of tablets

LYS Extract ......................................... 200 mg

Lactose 100 mg

Starch ......................................... 100 mg

Magnesium Stearate ...............

The tablets were prepared by mixing the above components and tableting according to a conventional method for producing tablets.

Formulation example  2. Preparation of Capsule

LYS-EA Extract ........................ 100 mg

Lactose ...... 50 mg

Starch ............................ 50 mg

Talc ........................................ 2 mg

Magnesium Stearate ...............

The capsules were prepared by mixing the above components and filling the gelatin capsules according to a conventional method for preparing capsules.

Formulation example  3. Liquid  Produce

LYS Extract ................................... 1000 mg

Sugar ......................................... 20 g

Isomerized sugar ......................................... 20 g

Lemon scent .....................

Purified water was added to adjust the total volume to 100 ml.

The above-mentioned components were mixed according to a conventional method for preparing a liquid solution, filled into a 100 ml brown bottle, and sterilized to prepare a liquid solution.

In addition, sports functional drinks, health functional foods and health functional drinks were prepared in the following manner.

Formulation example  4. sports drinks

LYS Extract ..................................... 1000 mg

Citric acid ........... 0.375 g

Vitamin C ..................... 0.075 g

Sodium Chloride ............... 0.125 g

Potassium chloride ..................................... 0.125 g

Calcium Lactate ......................................... 0.3 g

Magnesium Carbonate ......................................... 5 mg

Sodium Glutamate ......................................... 0.015 g

Spices ............................

Purified water was added to adjust the total volume to 250 ml.

The above ingredients were mixed according to a conventional beverage production method and filled into 250 ml of cans to prepare sports drinks.

Formulation example  5. Preparation of dietary supplements

LYS-EA Extract ........................ 1000 mg

Vitamin Blend ......... 20 g

Vitamin A Acetate ............... 70 μg

Vitamin E ..................................... 1 mg

Vitamin B1 ................................. 0.13 mg

Vitamin B2 ................................. 0.15 mg

Vitamin B6 ......................................... 0.5 mg

Vitamin B12 ................................. 0.2 μg

Vitamin C ......................................... 10 mg

Biotin .......................... 10 μg

Nicotinic Acid Amide ...................................... 1.7 mg

Folic acid ......................................... 50 μg

Calcium Pantothenate ......................................... 0.5 mg

Mineral mixture ......................

Ferrous Sulfate ................................. 1.75 mg

Zinc Oxide ......................................... 0.82mg

Magnesium Carbonate ............... 25.3 mg

Potassium monophosphate ......................................... 15 mg

Dicalcium Phosphate ......................................... 55 mg

Potassium Citrate ..................... 90 mg

Calcium Carbonate ......................... 100 mg

Magnesium Chloride .............. 24.8 mg

Although the composition ratio of the vitamin and mineral mixture is a composition suitable for a relatively healthy food in a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health functional food production method. The granules may be prepared and used for preparing the nutraceutical composition according to a conventional method.

Formulation example  6. Health functional drink  Produce

LYS Extract ................................... 1000 mg

Citric Acid ..................... 100 mg

Oligosaccharide ......................................... 100 g

Plum concentrate ..................................... 2 g

Taurine ......................................... 1 g

Purified water is added to the whole ............. 900 ml

After mixing the above components in accordance with a conventional healthy beverage production method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and then refrigerated and stored in the present invention It is used to prepare a health functional beverage composition.

Although the composition ratio is a composition suitable for a preferred beverage in a preferred embodiment, the compounding ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and use purpose.

Claims (8)

delete After crushing the oak mistletoe ( Loranthus yadoriki SIEB.) With a grinder, 1 to 20 times the water and methanol mixed solvent of the pulverized product was added for 12 hours to 1 week, the extract was carried out by performing a hot water extraction method at 10 ℃ to 100 ℃ A first step of obtaining; Crude extract soluble in a mixed solvent of water and methanol obtained through the second step of producing a filtrate by filtration of the extract obtained by the above filter and filter filtration; After adding water to the crude extract obtained above, a non-polar solvent soluble extract fraction soluble in ethyl acetate, chloroform, or hexane obtained by performing a fractionation process using ethyl acetate, chloroform, hexane and butanol; Or a pharmaceutical composition for preventing and treating depression related to monoamine oxidase (MAO) enzyme containing a polar solvent soluble extract fraction soluble in butanol as an active ingredient. delete delete delete delete After crushing the oak mistletoe ( Loranthus yadoriki SIEB.) With a grinder, 1 to 20 times the water and methanol mixed solvent of the pulverized product was added for 12 hours to 1 week, the extract was carried out by performing a hot water extraction method at 10 ℃ to 100 ℃ A first step of obtaining; Crude extract soluble in a mixed solvent of water and methanol obtained through the second step of producing a filtrate by filtration of the extract obtained by the above filter and filter filtration; After adding water to the crude extract obtained above, a non-polar solvent soluble extract fraction soluble in ethyl acetate, chloroform, or hexane obtained by performing a fractionation process using ethyl acetate, chloroform, or hexane and butanol; Or health functional food for the prevention and improvement of depression containing a polar solvent soluble extract fractions soluble in butanol as an active ingredient. delete

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