KR100921909B1 - Composition of healthy food comprising butanol solvent extract of Codonopsis lanceolata Benth et Hook having hematopoietic stem cell proliferation activity - Google Patents

Abstract

PURPOSE: A health food composition containing the butanol fraction of Condonopsis lanceolata is provided to ensure immunological enhancement and anticancer activity. CONSTITUTION: A method for preparing a health food composition containing the butanol fraction of Condonopsis lanceolata comprises the following steps of: extracting Condonopsis lanceolata roots in 5-10 times the amount of hot water and suspending the Condonopsis lanceolata extract in 5-10 times the amount of water; and fraction-extracting it in 5-10 times the amount of hexane, 5-10 times the amount of ethylacetate, and 5-10 times the amount of butanol in sequential order.

Description

Composition of healthy food comprising butanol solvent extract of Codonopsis lanceolata Benth et Hook having hematopoietic stem cell proliferation activity}

The present invention relates to a butanol solvent fraction of the hot water extract of the Korean special species Codonopsis lanceolata Benth et Hook, which exhibits immuno-enhancing and anticancer activity, which is a granulocyte macrophage colony-stimulating factor, GM-CSF ') increases secretion, tumor necrosis factor (TNF) -α production, and nitric oxide (NO) production to increase immunosuppression or cancer cell attack, thus preventing immunostimulants or cancer. And to provide a therapeutic agent or a pharmaceutical composition or functional food useful for improving the same.

Cancer treatment can be broadly classified into methods of directly killing cancer cells, such as surgery, administration of anticancer drugs, and irradiation, and methods of activating and treating in vivo immune functions that remove cancer cells, such as immunotherapy.

Conventionally, the former method of direct killing is used, but the latter uses the latter immunotherapy and combination therapy. In immunotherapy, as the mechanism of action of immune function is known recently, many attempts have been made to use immunomodulators. The immunomodulatory substance enhances the body's defense against disease factors by non-specifically stimulating the immune cells in vivo to enhance the biological immune function. Examples of such nonspecific immunomodulators include killed bacteria, chemical syntheses, biologics (cytokines or hormones), and researches are being conducted to obtain anticancer effects by enhancing biological immune function by administering them.

However, most of these nonspecific immunomodulators show many limitations in actual clinical applications due to toxicity or side effects. In particular, as the in vivo factors (cytokines) related to the immune response is revealed, studies are actively underway to mass-produce these factors and use them for cancer treatment. In other words, to achieve the anticancer effect of the immune stimulant including interleukin-2, tumor necrosis factor (TNF), etc., but this situation is also very limited due to severe toxicity.

On the other hand, hematopoietic function is a function of generating various blood cells including immune cells from hematopoietic stem cells of bone marrow, and is closely related to the immune activity of the living body. In particular, recently, as cytokines are known to act at each stage of differentiation from hematopoietic stem cells to blood cells, studies to actively use hematopoietic cell proliferation inducers such as GM-CSF have been actively conducted. However, these factors also require a large amount to be administered to the human body in order to be effective, in which case most of them are severely toxic, so they are used only for some limited purposes.

In recent years, immune-stimulating or anti-cancer effects are obtained by factors related to immune response and hematopoietic function, namely, immune stimulants including GM-CSF, hematopoietic cell proliferation inducers such as tumor necrosis factor (TNF-α), and hormones. However, there are also very limited attempts due to toxicity.

In addition, nitric oxide (NO) is a very unstable and highly reactive substance that functions in vivo. Enzymes for synthesizing nitric oxide are largely divided into constitutive NO synthase (cNOS) and inducible NO synthase (iNOS). The former is a protein that already exists in the cell and is activated by a stimulus, and the latter is synthesized into a new protein in response to the stimulus. Nitric oxide is known to produce a large amount of nitrates (NO ³ ) in test animals administered with the endotoxin lipopolysaccharide (LPS), and then macrophages are exposed to interferon-γ (IFN-γ) and LPS. It is activated to produce nitrite (NO ² ) and nitrate, which proved to be derived from nitric oxide produced in macrophages. It has also been found that these nitric oxides are important mediators of the antimicrobial and anticancer effects of macrophages. Thus, investigating nitric oxide production in macrophages is a good indicator for studying anticancer effects, so if an effective drug promotes nitric oxide production in macrophages, the anticancer effect of the drug can be inferred.

As described above, while improving the anti-cancer effect and immune function and hematopoietic function through the activation of immune function, it is urgently necessary to secure a material that is safe for chemotherapy, and GM-CSF, TNF-α, iNOS It is expected that the superior safety of anti-cancer or immune-enhancing substances will be produced with excellent safety.

Therefore, in recent years, many researches have been conducted on the development of natural physiologically active substances having no side effects from natural products, and the physiologically active substances, which are effective for bioregulation and biological defense, have been actively searched for industrialization as health supplement foods or therapeutic agents. Is reaching.

On the other hand, Codonopsis lanceolata Benth et Hook is a perennial vine plant belonging to Campahutaceae, which has wild wild squirrel and artificial cultivated wild squirrel, which is superior to wild squirrel in taste and aroma. There is no objective data.

For studies on immunopotentiation or anticancer activity against Codonopsis lanceolata Benth et Hook, Korean Patent Laid-Open Publication No. 2003-57317 discloses the proliferation of lymphocytes and regulation of NO in macrophages, iNOS gene expression and cytokine expression. It can be said that it can contribute to the enhancement of nourishment and tonic effect through the regulation of Korean Patent Laid-Open Publication No. 2008-12812 describes that hot water extract and ethanol extract of Deodeok have cancer cell proliferation inhibitory effect. No. 5,000, which contains a purified functional milk composition which improves immune activity, contains prickly pear extract, deodeok extract, mushroom mushroom extract, and matsutake mushroom extract. Soyangin's immune enhancing composition containing the extract as an active ingredient is described.

As described above, scientific studies of specific pharmacological actions on hematopoietic stem cell proliferation such as extracts related to Deodeok and increased GM-CSF secretion in Korea have not been conducted so far.

Therefore, while the present inventors are studying natural products having hematopoietic stem cell proliferation and immunomodulatory activity, butanol solvent fraction of deodeok root hot-water extract shows hematopoietic stem cell proliferation activity and has been proven to be safe, and thus has no side effects, so as to improve immunotherapy or chemotherapy. The present invention has been completed by revealing the effectiveness.

The present inventors found that the butanol solvent fraction of the decocted rooted hot water extract regulates hematopoietic stem cell proliferation effect and immune response and has immuno-enhancing or anti-cancer activity.

Accordingly, it is an object of the present invention to provide a butanol solvent fraction of deoderm root hot water extract having immuno-enhancing or anticancer activity.

It is another object of the present invention to provide a pharmaceutical or functional health food composition for preventing or treating a disease related to these butanol solvent fractions of deodeok root hot water extract providing tumor necrosis factor-α and iNOS expression.

The present invention provides a butanol solvent fraction of deodeok root hot water extract having immuno-enhancing or anticancer activity.

Deodeok root extract of the present invention comprises an extract available in polar solvent, preferably hot water or 70% ethanol selected from water, lower alcohols having 1 to 4 carbon atoms and mixed solvents thereof.

Deodeok root extract of the present invention, dried and powdered deodeok root is about 1 to 20 times, preferably about 5 to 10 times the volume of water, lower alcohol having 1 to 4 carbon atoms or about 1: 0.1 thereof To 1:10, preferably a mixed solvent having a mixing ratio of 1: 0.2 to 1: 3, hot water extraction and reflux for about 1 hour to 2 days, preferably 2 hours to 1 day at an extraction temperature of 20 to 100 ° C. After extracting by repeated extraction 1 to 5 times, preferably 2 to 3 times by an extraction method such as cold extraction, ultrasonic extraction, and then filtered under reduced pressure and the filtered extract was mixed with a rotary vacuum concentrator 20 to 100 ℃, Preferably, the solvent is removed by concentration under reduced pressure at 50 to 70 ° C. to obtain a crude extract that is a soluble extract according to water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof.

In addition, the non-polar solvent soluble extract may be prepared by suspending the crude extract by 5 to 20 times the weight of distilled water, and then adding about 1 to 10 times, preferably about 5 to 10 times, the volume of n-hexane to the suspension. Fractions, preferably 2 to 4 times, are separated into n-hexane soluble fraction and water soluble fraction, and ethyl acetate in about 1 to 10 times volume is added to the water soluble fraction and ethyl acetate soluble fraction and water soluble fraction are added. To the water-soluble fraction, and to the water-soluble fraction, by adding about 1 to 10 times the volume of butanol to separate the butanol soluble fraction, and to separate the water-soluble fraction of about 1 to 10 times the volume of chloroform soluble fraction and the water-soluble fraction, respectively The solvent fractions were concentrated under reduced pressure with a rotary vacuum concentrator to remove the solvents to obtain n-hexane, ethyl acetate, butanol, chloroform and water fractions. Can.

The composition of the present invention comprises the extract of the deodeur root, non-polar solvent soluble extract or polar solvent soluble extract obtained according to the production method.

The composition of the present invention comprises 0.1 to 50% by weight of the extract relative to the total weight of the composition. The composition comprising the extract of the deodeur root of the present invention may further comprise a suitable carrier, excipient or diluent according to conventional methods. Carriers, excipients and diluents that may be included in the compositions 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. Compositions comprising extracts according to the invention are each formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories or sterile injectable solutions according to conventional methods. Can be used. In detail, when formulated, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. which are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations include at least one excipient such as starch, calcium carbonate, sucrose, lactose, gelatin, and the like. Can be prepared by mixing. In addition to simple excipients, lubricants such as magnesium stearate, talc can also be used. Liquid preparations for oral use include suspensions, solvents, emulsions, and syrups, and include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Can be. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The extract of the present invention may vary depending on the age, sex, and weight of the patient, but in general, an amount of 0.01 to 500 mg / kg, preferably 0.1 to 100 mg / kg, may be administered once or several times a day. Can be. In addition, the dosage of the extract can be increased or decreased depending on the route of administration, the degree of disease, sex, weight, age and the like. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

The pharmaceutical composition of the present invention can be administered to various mammals such as rats, mice, livestock, humans, and the like. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injections. Deodeok plant extract of the present invention is a drug that can be used with confidence even for long-term administration for the purpose of prevention because there is little toxicity and side effects. The present invention also provides a dietary supplement for the prevention and amelioration of inflammation-related diseases, including extracts of deoderm root and food supplements.

The composition comprising the extract of the genus Roots of the present invention can be used in a variety of drugs, food and beverages for the prevention of dementia diseases. Foods to which the extract of the present invention may be added include various foods, for example, beverages, gums, teas, vitamin complexes, dietary supplements, and the like, which are pills, powders, granules, tablets, tablets, capsules or beverages. Available in form. At this time, the amount of the extract in the food or beverage, in the case of the health food composition of the present invention can generally be added to 0.01 to 15% by weight of the total food weight, 0.02 to 10g, based on 100ml for the health beverage composition Can be added at a ratio of 0.3 to 1 g. The health beverage composition of the present invention has no particular limitation on the liquid component except for containing the extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as additional ingredients, such as 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; Polysaccharides such as dextrin, cyclodextrin; Conventional sugars such as and the like and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (tauumatin, stevia extract, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of such natural carbohydrates 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, 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 its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like.

The compositions of the present invention may also contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected from the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

The present inventors measured the expression and production of GM-CSF, TNF-α, and inducible nitric oxide (iNO) on various solvent extracts of Deodeok root, and showed that the hot water extract of Deodeok root showed excellent activity. Butanol fraction of the hot water extract was found to show the highest immune or anticancer activity.

The present invention is described in more detail based on the following examples, but the present invention is not limited thereto.

Deodeok root extract of the present invention significantly increases the expression and production of GM-CSF, TNF-α, inducible nitric oxide (iNOS) and significantly proliferates at leukocyte levels reduced by cyclophosphamide By confirming, it can be effectively used as a pharmaceutical composition or health functional food for effective and safe immunity or cancer treatment.

Obtain 70% ethanol extract (EE, ethanol extract) and hot water extract (HWE, hot water extract) of the deodeok root in the same manner as in Example 1, 70% ethanol extract and hot water extract of the deodeok root in the same manner as in Example 2 For each of n-hexane, ethyl acetate, n-butanol, chloroform and water solvent fractions were obtained, the effects of these extracts and their fractions on GM-CSF secretion were measured. 2 is shown.

Table 1 shows the effects of the degroot root on GM-CSF secretion in RAW264.7 cells against 70% ethanol extract (100 μg / ml) and hot water extract (100 μg / ml) of the deodeur root of Example 1 of the present invention. The 70% ethanol extract showed a 27-fold increase in GM-CSF secretion, and the dried hot-water extract of Deodeok root showed 54-fold increase in GM-CSF secretion.

Table 2 shows the n-hexane fraction (50 ㎍ / ㎖), ethyl acetate fraction (50 ㎍ / ㎖), n-butanol fraction (50 ㎍) for each of the extract 70% ethanol extract and hot water extract of Example 2 of the present invention / Ml), chloroform fraction (50 µg / ml), water fraction (50 µg / ml) as the effect of increasing the secretion of GM-CSF secretion in the n-butanol fraction (50 µg / ml) of the hot-water extracts of the Rhizome root The increase of GM-CSF secretion of the embryo (see Fig. 2) was shown, and the increase of GM-CSF secretion showed a concentration-dependent effect (Fig. 4).

 Table 1. Effect of Deok-root Root Water Extract (100 μg / mL) and 70% Ethanol Extract (100 μg / mL) on GM-CSF Secretion Treatment group GM-CSF (pg / ml) Normal 0.6 ± 0.2 LPS (Lipopolysaccharide) 50.8 ± 1.3 Hot Water Extract (HWE) 32.7 ± 3.2 70% Ethanol Extract (EE) 16.4 ± 1.6

  Table 2. Effect of Fraction (100 ㎍ / mL) of Solder Extract of Deodeok Root on GM-CSF Secretion Treatment group GM-CSF (pg / ml) Normal 0.6 ± 0.2 LPS 50.8 ± 1.3   70% Ethanol Extract Hexane fraction 6.4 ± 0.7 Chloroform fraction 17.4 ± 0.9 Ethyl acetate fraction 14.1 ± 1.2 Butanol fraction 12.8 ± 1.0 Distilled water fraction 13.5 ± 0.3   Hot Water Extract Hexane fraction 6.1 ± 2.2 Chloroform fraction 13.2 ± 1.2 Ethyl acetate fraction 3.4 ± 1.9 Butanol fraction 63.1 ± 1.4 Distilled water fraction 15.5 ± 2.6

Experimental Example 1: Experiment of Cell Viability of Deodeok Root Extract

       The effect on the cell viability of the deodeur root solvent extract prepared by the method of Example 1 was measured. As can be seen in Figures 1a, 1b fractions of the deodeur root solvent extract and hot water extract of Example 1 did not induce cytotoxicity even at the concentration of the test maximum concentration of 100 μg / ㎖.

Experimental Example 2: Formation Effect of Nitric Oxide (iNOS), a Cancer Attack Factor of Deodeok Root Extract

In the present invention, the results of the effect on the production of nitric oxide (iNOS), which is a cancer attack factor against the solvent fractions of deodeok root hot water extract and hot water extract, in particular, the butanol fraction of the deodorant root hot water extract showed the best effect (Fig. 3) Butanol fraction of the deodeok root hot water extract showed a concentration-dependent effect (Fig. 4).

Experimental Example 3: Effect of TNF-α As a Cancer Attack Factor of Deodeok Root Extract

In the present invention, the results of the effect on the production of TNF-α, which is a cancer attack factor against the solvent fractions of deodeok root hot water extract and hot water extract, in particular, the butanol fraction of deodeok root hot water extract showed the best effect (see Fig. 3).

Experimental Example 4: Effect of Deodeok Root Extract and Solvent Fractions on Increased Leukocyte Levels Reduced by Cyclophosphoramide

In the present invention, the effect on the leukocytes reduced by cyclophosphosamide on the 70% ethanol extract and hot water extract, and the solvent fraction fractions of Deodeok root was compared to the Deokroot root hot water extract rather than the 70% ethanol extract from Among them, the butanol fraction showed the best 3.5-fold leukocyte proliferation effect (Figs. 6a and 6b) among the solvent fractions of the dendrites root hot water extract.

  Table 3. Increased Leukocyte Levels Decreased by Cyclophosphoramide after Solvent Extract and Solvent Fraction (200 mg / kg / day / 2 Week Oral Administration) of Deodeok Root Treatment group Leukocyte count increase (multiple) Normal - 70% Ethanol Extract 1.8   70% Ethanol Extract Hexane fraction 1.2 Chloroform fraction 1.4 Ethyl acetate fraction 1.3 Butanol fraction 1.7 Distilled water fraction 1.1 Hot Water Extract 2.9   Hot Water Extract Hexane fraction 2.2 Chloroform fraction 3.0 Ethyl acetate fraction 2.6 Butanol fraction 3.5 Distilled water fraction 1.1

Experimental Example 5: Cytotoxicity Test of Cancer Cells by MTT Analysis of Deodeok Root Extract

The cytotoxic effect of the cancer cells by MTT analysis of the deodeur root solvent extract and fraction prepared by the method of Example 8 showed the butanol fraction of the solvent fraction of the deodorant root hot water extract showed the best anticancer effect.

  Table 4. Effect of Fraction (100 ㎍ / ml) of Solder Extract of Deodeok Root on Cell Viability of Cancer Cells  Treatment group Cell survival rate (%) HT-29 C6 glioma Normal 100 ± 3 100 ± 5   70% Ethanol Extract Whole fraction 92 ± 3 90 ± 2 Hexane fraction 64 ± 5 65 ± 3 Chloroform fraction 83 ± 4 82 ± 3 Ethyl acetate fraction 71 ± 1 73 ± 2 Butanol fraction 68 ± 2 65 ± 4 Distilled water fraction 89 ± 4 91 ± 1   Hot Water Extract Whole fraction 45 ± 4 41 ± 1 Hexane fraction 60 ± 2 58 ± 3 Chloroform fraction 52 ± 3 50 ± 1 Ethyl acetate fraction 57 ± 1 56 ± 2 Butanol fraction 32 ± 3 30 ± 1 Distilled water fraction 86 ± 2 87 ± 4

Experimental Example 6. Oral Acute Toxicity in Rats

Acute toxicity test was performed using 6-week-old SPF SD rats as follows. The butanol fraction of the deodeur root hot water extract of the present invention was dissolved in two animals per group and administered orally at a dose of 1 g / kg / ml. After administration of the extract, mortality, clinical symptoms, and changes in body weight were observed. Hematological and hematological examinations were performed. Necropsy was performed to observe abdominal and thoracic organ abnormalities. As a result, no significant clinical symptoms or dead animals were noted in all animals treated with the test substance, and no toxic changes were observed in weight changes, blood tests, blood biochemical tests, and autopsy findings. Therefore, the butanol fraction of Deodeok root hot water extract according to the present invention did not show toxicity change up to 5,000 mg / kg in all rats, and the minimum lethal dose (LD50 value) of oral administration was determined to be more than 5,000 mg / kg.

As described above, butanol fraction of the deodeok root hot water extract according to the present invention is excellent in the ability to produce a variety of leukocytes, GM-CSF, TNF-α, iNOS and the like as a safety-enhanced material for immune enhancement and cancer prevention And it can be usefully used as a therapeutic drug or food additives.

Example 1 Preparation of Deodeok Root Extract

175 g of deodeok root was washed well to remove foreign substances, and then pulverized using a grinder. The extract was extracted three times with 1.5 L of 70% ethanol at 80 ° C. The extract was concentrated using a rotary vacuum evaporator, and then freeze-dried to 70%. 50 g of ethanol extracts were obtained. In addition, 175 g of Deodeok root was extracted by heating for 6 hours at 1.5 L of hot water heated to 95 ° C., and concentrated to obtain 34 g of hot water extract.

Example 2 Preparation of Deodeok Root Extract

50 ml of 70% ethanol extract of Example 1 was added with 500 ml of water and suspended, and 500 ml of n-hexane was added thereto to separate the n-hexane layer, which was then repeated twice. 4.5 g of fractions were obtained. 500 ml of ethyl acetate, 500 ml of n-butanol, and 500 ml of chloroform were fractionated in the same manner as described above, followed by concentration, followed by concentration of 7.0 g of ethyl acetate fraction, 19.0 g of butanol fraction, 13.0 g of chloroform fraction and water fraction. 23.0 g were obtained respectively.

In addition, 340 ml of water was added and suspended in 34 g of the hot water extract of Example 1, and then n-hexane was added to 340 ml of n-hexane to separate the n-hexane layer. 4.4 g was obtained. Subsequently, 340 ml of ethyl acetate, 340 ml of n-butanol, and 340 ml of chloroform were fractionated in the same manner as described above, followed by concentration. 14.0 g were obtained respectively.

<Example 3> Cytotoxicity test through MTT analysis of Deodeok root extract and fractions

Cytotoxicity experiments of the dendrites root solvent extracts and their solvent fractions were determined by MTT assay. Cytotoxicity test cultures were added 10 μl of MTT solution (10 mg / ml in phosphate buffer solution-saline, pH 7.4) 3 hours before the end of the culture test, and the cells were incubated until the end of the reaction. The culture was stopped by adding 15% sodium dodecyl sulfate to increase the solubility of formazan. Optical density (OD) at 570 nm was measured using a Spectramax 250 microplate reader.

Example 4 GM-CSF Production Measurement of Deodeok Root Extract and Fractions

Deodeok root solvent extract and fractions were diluted to 100 ㎍ / ㎖ with DMSO solvent and treated in RAW264.7 cells, and then cultured for 24 hours, and then the supernatant was separated by centrifugation of the culture supernatant and ELISA to generate GM-CSF of the supernatant Measured by the method (Endogen, Cambridge, MA).

Example 5 Production or Expression Measurement of TNF-α

Soldered root extract and fractions were diluted 100 mg / ml with DMSO solvent, pretreated in RAW264.7 cells, incubated for 24 hours, and then cultured for 24 hours, the supernatant was separated and the supernatant was separated and TNF-α production of the supernatant was ELISA. Measured by the method (Endogen, Cambridge, MA).

Example 6 Production Measurement of Nitric Oxide (iNOS)

37 ° C. in RPMI medium containing 10% FBS, penicillin (100 unit / ml), streptomycin sulfate (100 μg / ml) in macrophage RAW264.7 (1 × 10 ⁶ cells / ml) in 96-well plate, Cultured in an environment of 5% carbon dioxide, 100 ㎍ / ㎖ of each hot water extract and 50 ㎍ / ㎖ fraction of the hot water extract was pretreated for 30 minutes, and then incubated for 24 hours by treatment with LPS (1 ㎍ / ㎖). 100 μl of cultured cell medium was added to Gries [1% (w / v) sulfanylamide in the same amount dissolved in reagent 5% (v / v), phosphoric acid and 0.1% (w / v) naphthylethylenediamine-hydrochloric acid]. After mixing to 100 μl, the cells were incubated for 10 minutes at room temperature, and then absorbance was measured at 550 nm using a microplate reader (Power Wave 340, Bio-Tek, USA). Fresh medium was used as untreated group in all experiments. The amount of nitric oxide in the sample was calculated from the sodium nitrite standard curve prepared in the medium.

Example 7 Hematopoietic Stem Cell Production Measurement

Male BALB / c mice (approximately 25 g) were subjected to oral administration of deodeur root solvent extracts and their solvent fractions (100 mg / kg) at 10 AM for one week using sonde for two weeks, followed by oral administration on the last 7 days. Post cyclophosphamide (200 mg / kg) was injected intravenously to induce a decrease in hematopoietic stem cells. After collecting blood from the tail blood vessels of these mice, 95 μl of Truk's solution and 5 μl of collected blood were mixed to measure the number of white blood cells.

Example 8 Cytotoxicity in Cancer Cells by MTT Analysis

Cytotoxicity experiments in cancer cells of the deodeok-derived solvent extracts were determined by MTT assay. Colorectal cancer cells HT-29 (5 × 10 ⁵ cells / ml) in 96-well plate were 37, 5% in RPMI medium containing 10% FBS, penicillin (100 unit / ml) and streptomycin sulfate (100 / ml). The glioma cells C6 glioma (5 × 10 ⁵ cells / ml) were cultured in a carbon dioxide environment and 37% in DMEM medium containing 5% FBS, penicillin (100 unit / ml) and streptomycin sulfate (100 μg / ml). C was incubated in an environment of 5% carbon dioxide. Each solvent fraction (100 mg / ml) was treated and incubated for 72 hours. Cytotoxicity test cultures in cancer cells were added 10 ul of MTT solution (10 mg / ml in phosphate buffer solution-saline, pH 7.4) 3 hours before the end of the culture experiment, the cells were continued incubation until the end of the reaction. The culture was stopped by adding 15% sodium dodecyl sulfate to increase the solubility of formazan. Optical density (OD) at 570 nm was measured using a Spectramax 250 microplate reader.

Example 9 Preparation of Pharmaceuticals

9-1. Manufacture of powder

Butanol fraction of Deodeok root hot water extract of Example 2. 300 mg

Lactose ... ... 100 mg

Talc ........................ ... 10 mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

9-2. Manufacture of tablets

Butanol fraction of Deodeok root hot water extract of Example 2. 300 mg

Corn Starch ... Mg

Lactose ... ... 100 mg

Magnesium Stearate ............................................... 2 mg

After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.

9-3. Manufacture of capsule

Butanol fraction of Deodeok root water extract of Example 2 .......... 300 mg

Corn starch ........................... 100 mg

Lactose ... ... 100 mg

Magnesium Stearate ... ............ 2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

9-4. Preparation of Injectables

Butanol fraction of Deodeok root hot water extract of Example 2. 300 mg

Sterile Distilled Water for Injection ...

pH Adjuster ... Quantity

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

9-5. Preparation of liquid

Butanol fraction of Deodeok root hot water extract of Example 2 ...... 1 g

Isomerization ............................................... .. 10 g

Mannitol ... ... 5 g

Purified water ................................................. .... appropriate

According to the conventional method of preparing a liquid solution, each component is added to the purified water to dissolve, the lemon row is added appropriately, the above components are mixed, purified water is added, the whole is adjusted to 100 ml by the addition of purified water, and then filled in a brown bottle. The solution is prepared by sterilization.

Example 10 Preparation Example of Health Food

10-1. Manufacture of health food

Butanol fraction of Deodeok root hot water extract of Example 2 ....... 1000 mg

Vitamin Blend ...............

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

Vitamin E .............. ................... 1.0 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

Nicotinamide ............................................... 1.7 mg

Folic Acid ... ... 50 μg

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

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

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

Zinc Oxide ... 0.82 mg

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

Potassium monophosphate ......................................................... 15 Mg

Dibasic Calcium Phosphate ... Mg

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

Calcium Carbonate ... Mg

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

The composition ratio of the above-mentioned vitamin and mineral mixtures is composed of relatively suitable ingredients suitable for health foods in the preferred formulation example, but the formulation ratio may be arbitrarily modified. The granules may be prepared and used for preparing a health food composition according to a conventional method.

10-2. Manufacture of healthy drinks

Butanol Fraction of Deodeok Root Water Extract of Example 2 ......... 1000 mg

Citric Acid ... 100 mg

Oligosaccharides ... g

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

Taurine ... 1 g

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

After mixing the above components according to a conventional healthy beverage manufacturing method, and then stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed and sterilized and then refrigerated and stored in the present invention For the preparation of healthy beverage compositions.

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

1 is an MTT analysis result for confirming the cytotoxicity of the deodeok root-derived solvent extract.

Figure 2 shows the effect of the hot water extract and its solvent fractions of the deodeok root on GM-CSF expression.

Figure 3 shows the effect of the hot water extract and its solvent fractions of the deodeok root on GM-CSF expression.

Figure 4 shows the concentration-dependent effect of the butanol fraction of deodeok root hot water extract on the expression of GM-CSF, proliferation, cytokines and nitric oxide synthase.

Figure 5 shows the effect of the hot water extract and 70% ethanol extract of the deodeok root on the leukocytes reduced by cyclophosphamide.

Figure 6a shows the effect on the leukocytes reduced by cyclophosphosamide of the solvent fraction of deodeur root 70% ethanol.

Figure 6b shows the effect on the leukocytes reduced by cyclophosphosamide of the solvent fraction of the deodeur root hot water extract.

Claims (11)

delete delete 5 to 10 times (v / w) of hexane, suspended in 5 to 10 times (v / w) of water in the Deodeok root concentrate obtained by extracting deodeok roots with 5 to 10 times (v / w) of hot water. It is prepared by fractional extraction in the order of 5 ~ 10 times (v / w) ethyl acetate and 5 ~ 10 times (v / w) butanol. Among them, the extract contains butanol fraction of deodeok root hot water extract as an active ingredient. Pharmaceutic composition. The method of claim 3, wherein The immuno-enhancer is a pharmaceutical composition for immuno-enhancing, characterized in that the GM-CSF (granulocyte macrophage colony-stimulating factor) secretion is increased through. The method of claim 3, wherein The immune enhancing pharmaceutical composition for immuno-enhancing, characterized in that the tumor necrosis factor (TNF) -α production through increased production. The method of claim 3, wherein The immuno-enhancing pharmaceutical composition, characterized in that through the production of nitric oxide (iNO) increase. delete 5 to 10 times (v / w) of hexane, suspended in 5 to 10 times (v / w) of water in the Deodeok root concentrate obtained by extracting deodeok roots with 5 to 10 times (v / w) of hot water. It is prepared by fractional extraction in the order of 5 ~ 10 times (v / w) ethyl acetate and 5 ~ 10 times (v / w) butanol. Among them, the extract contains butanol fraction of deodeok root hot water extract as an active ingredient. Health functional food. delete delete delete

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