KR101894156B1 - Method for producing codonopsis lanceolata extract and uses thereof - Google Patents

Abstract

The present invention relates to the use of Rootgrass extract and its solvent fractions and their preparation, and the yield and efficacy of the extract and its solvent fractions are optimized by specifying the conditions of the production process.

Description

METHOD FOR PRODUCING CODONOPSIS LANCEOLATA EXTRACT AND USES THEREOF FIELD OF THE INVENTION [0001]

The present invention relates to rosemary extract and its preparation and to various uses of rosemary extract and its solvent fractions.

Bronchial asthma is the most common allergic respiratory disease, and allergens that are inhaled by the airways secrete several kinds of interleukines from various immune cells in vivo. By these mast cells, Leukotrienes, prostaglandins, and histamines, which are primarily inflammatory reactions that cause acute bronchoconstriction, followed by secondary bronchoconstriction. Disodium cromoglycate, cromolyn sodium, nedcromil sodium, ketotifen, and the like have been used to treat asthma. In recent years, Development of medicines using inhibitors or metabolic inhibitors of inflammatory mediators focusing on various inflammatory reactions involved in bronchial relaxants and asthma is also under way.

Most of the expectorants have been used as medicinal medicines or experience prescriptions by using plants, and recently, cysteine derivatives such as carbocysteine and N-acetylcysteine have been used. However, since these drugs do not have completely satisfactory effects, and they are toxic and chemically unstable, there has been a demand for development of an antipyretic agent having low toxicity, no side effects, and excellent dandruff effect.

Obesity is one of the most prevalent diseases in which the pattern of disease is rapidly changing to a developed country type as the hygiene environment is improved due to the improvement of the living standard and the average life span is extended due to the westernization of the diet. Thus, adult diseases have emerged as the biggest medical problem today, and the obesity, which is a major cause of these adult diseases, is increasing rapidly. Obesity refers to the phenomenon that extra calories are accumulated in the body in the form of fat by consuming excessive calories compared to the calories consumed. Obesity is thought to be caused by various causes such as genetic influences, environmental influences due to westernized diet, and psychological effects due to stress. However, the precise cause and mechanism of obesity has not been established yet. However, since obesity can act not only as a problem of obesity itself, but also as a cause of diseases such as cardiovascular disease or diabetes (Manson et al., New England J. Med., 333, pp677-685, 1995; JAMA, 282, pp1523-1529, 1999). There is a growing interest in the treatment of obesity worldwide.

In addition, studies on the regulation of the human immune system against the natural extract have been actively conducted because the immune system abnormality breaks health and causes various diseases. Therefore, the development of immunomodulators for safe natural products that can minimize side effects can be a very important material for the development of food, cosmetics and pharmaceuticals. For example, mulberry bark and shiitake mushrooms are used as immunostimulants, Starfish and Ganoderma mushrooms as anti-cancer adjuvants, and licorice, licorice glycyrrhizin, sea cucumber, Research is underway on the differentiation materials. The development of immunomodulatory agents using these natural materials has attracted many people's interest and interest because they can safely treat cancer and chronic inflammatory diseases without side effects.

Korean Patent No. 10-1290749

Accordingly, the present inventors have completed the present invention by optimizing the extracting conditions of Root Root Extract or its solvent fraction to improve the content of active ingredients of Root Root Extract and improve the efficacy.

Accordingly, it is an object of the present invention to provide a method for producing rosacea extract.

It is another object of the present invention to provide a process for the preparation of rosemary solvent fractions from Root Root Extract.

Another object of the present invention is to provide a composition for prevention or treatment of asthma, which comprises the extract of Rootgrass or a solvent fraction thereof as an active ingredient.

It is another object of the present invention to provide an antioxidative composition comprising Rootgrass extract or a solvent fraction thereof as an active ingredient.

It is another object of the present invention to provide an anticancer composition comprising a Rootgrass extract or a solvent fraction thereof as an active ingredient.

Another object of the present invention is to provide an anti-obesity composition comprising the roots extract or a solvent fraction thereof as an active ingredient.

In order to accomplish the above object, the present invention provides a method for manufacturing a semiconductor device, Lyophilizing the cut dough; And extracting the lyophilized yolk with 30% alcohol alcohol.

The present invention provides a process for preparing a rosemary extract fraction comprising fractionating the rosemary extract according to the present invention into a solvent selected from the group consisting of n-hexene, methyl chloro rode, ethyl acetate, butyl alcohol and distilled water.

The present invention provides a composition for prevention or treatment of asthma, comprising a rosemary extract or a solvent fraction thereof as an active ingredient.

The present invention provides an antioxidative composition comprising a roots extract or a solvent fraction thereof as an active ingredient.

In one embodiment of the present invention, the solvent fraction may be an ethyl acetate fraction.

The present invention provides an anticancer composition comprising roots extract or a solvent fraction thereof as an active ingredient.

In one embodiment of the present invention, the solvent fraction may be an n-hexene fraction.

The present invention provides an anti-obesity composition comprising Root Root Extract or a solvent fraction thereof as an active ingredient.

In one embodiment of the present invention, the solvent fraction may be a methyl chloride fraction.

In one embodiment of the invention, the compositions may comprise the rosemary extract or a solvent fraction thereof in a concentration of 0.01 to 20 mg / mL. For example, the roeae extract may be contained at a concentration of 0.5 to 20 mg / mL, and the solvent fraction may be contained at a concentration of 0.01 to 10 mg / mL.

The present invention can be applied to a variety of therapeutic uses such as anti-asthma, antioxidation, anticancer, anti-obesity, etc., by optimizing the extraction conditions of Root Root Extract or its solvent fraction to improve the content of active ingredients of Root Root Extract and improve the yield and efficacy .

FIG. 1 is a view illustrating a manufacturing process of a roe deer extract according to an embodiment of the present invention.
FIG. 2 is a view illustrating a manufacturing process of a fragrance solvent fraction according to an embodiment of the present invention.
FIG. 3 is a view illustrating a manufacturing process of the fragrance solvent fraction according to an embodiment of the present invention.
FIG. 4 is a graph showing the growth of human T cell line of Rootgrass extract according to the storage period according to an embodiment of the present invention. FIG.
FIG. 5 is a graph showing the growth of human T cell line of roe deer extract according to the size of chopped roots in an embodiment of the present invention.
FIG. 6 is a graph of human T cell line growth results of Root Root Extract according to storage temperature in one embodiment of the present invention. FIG.
FIG. 7 is a graph showing the growth of human B cell line of Duckweed extract according to the storage period according to an embodiment of the present invention. FIG.
FIG. 8 is a graph showing the growth of human B cell line of D. melanogaster extract according to the size of Broodstock according to one embodiment of the present invention. FIG.
FIG. 9 is a graph showing the growth of human B cell line of Root seed extract according to storage temperature in one embodiment of the present invention. FIG.
FIG. 10 is a graph showing the SOD activity of Rootgrass extract according to the cutting size of roots in one embodiment of the present invention. FIG.
FIG. 11 is a graph showing SOD activity of Root Root Extract according to storage temperature in an embodiment of the present invention. FIG.
12 is a graph showing the SOD activity of the solvent fraction of dodder according to the fraction solvent in an embodiment of the present invention.
Figure 13 is a graph showing the CAT activity of the solvent fraction of dodder according to the fraction solvent in one embodiment of the invention.
Figure 14 is a graph showing APX activity of solvent fractions of rosemary according to fractional solvent in one embodiment of the invention.
15 is a graph showing the POD activity of the solvent fraction of Doduck according to the fraction solvent in an embodiment of the present invention.
FIG. 16 is a graph (a) showing the result of intracellular fat accumulation of the solvent fraction of dodder according to the fraction solvent, and an image (b) showing the result of cell staining according to an embodiment of the present invention.
FIG. 17 is a graph showing the results of measuring the expression level of IFN-y according to the treatment of roeae extract in one embodiment of the present invention.
18 is a graph showing the results of measurement of IL-4 expression level according to the treatment of roeae extract in one embodiment of the present invention.
FIG. 19 is a graph showing the measurement result of IL-5 expression level according to the treatment of roeae extract in one embodiment of the present invention. FIG.
20 is a graph showing the results of measurement of IL-13 expression level according to the treatment of roeae extract in one embodiment of the present invention.

The present invention provides a method for preparing rosemary extract, its solvent fraction, and its use for anti-asthma, antioxidant, anti-cancer and anti-obesity.

The present invention can produce a roasted extract having an improved yield and an increased content of active ingredients by optimizing various conditions of the roasted roots of the roasted roots.

The method for preparing rosemary extract according to the present invention is as shown in FIG. 1, and can be specifically prepared by the following steps:

1) washing the roots and cutting them to 1 to 10 mm, for example 5 mm;

2) washing the truncated trichomes again, freeze-drying and drying;

3) extracting the dried roots with 30% alcohol;

4) filtering and concentrating the roots extract and sterilization;

5) lyophilization of sterilized roots extract, if necessary.

The dodok extract prepared according to the above conditions has an increased content of active ingredients such as polyphenols and flavonoids. In addition, the expression level of human immune cells is increased, and DPPH radical scavenging activity, total ABTS cation scavenging ability, reducing power, Xanthine oxidase inhibiting activity, and SOD (Superoxide Dismutase) activity are improved and excellent antioxidative effect is exhibited.

In addition, the roots extract may be fractionated with a solvent selected from the group consisting of n-hexene, methyl chlo- roide, ethyl acetate, butyl alcohol and distilled water to produce rosemary solvent fractions.

The composition comprising the rosemary extract of the present invention or a solvent fraction thereof as an active ingredient can be used as a pharmaceutical composition for immunity enhancement, antioxidant, anti-asthmatic, anti-cancer and anti-obesity use, and a pharmacologically effective amount of rosemary extract or its solvent Fractions may be included alone or may include one or more pharmaceutically acceptable carriers, excipients or diluents.

The pharmaceutical composition of the present invention may employ an appropriate solvent fraction depending on the use, for example, the antioxidant composition may contain ethyl acetate fraction of rosemary, and the anti-cancer composition may include nodule n-hexane fraction , The anti-obesity composition may comprise the methyl chloride fraction of rosemary.

In the present invention, the term "immune enhancement" refers to a cell-growth promoting action of T cells and B cells, and may include immune cells other than the above-described immune cells.

In the present invention, anti-obesity refers to the ability of cells to inhibit α-amylase, α-glucose, pancreatic lipase inhibition and adipocyte differentiation.

In the present invention, 'obesity' means that the amount of fat in the body is excessively increased. Obesity is caused by an imbalance in the synthesis and degradation of fat in adipocytes. Obesity, which is caused by excess fat accumulation, increases the risk of developing chronic degenerative diseases and increases the risk of coronary artery disease, hypertension, stroke, hyperlipemia and diabetes .

In the present invention, " prevention " means all actions that inhibit asthenia, cancer, and obesity or delay progression by the administration of the composition of the present invention.

&Quot; Improvement " in the present invention refers to all actions in which administration of the composition of the present invention improves or alleviates symptoms of asthma, cancer and obesity.

In the present invention, " treatment " means, unless otherwise indicated, reversing, alleviating, inhibiting or preventing the disease or disorder to which the term applies, or one or more symptoms of the disease or disorder And the term " treatment " as used herein refers to an act of treating when " treating " is defined as described above.

The term " administration " as used herein is meant to provide any desired composition of the invention to a subject in any suitable manner.

The composition of the present invention may contain rosemary extract or its solvent fraction at a concentration of 0.01 to 20 mg / mL, for example, the rosemary extract may be contained at a concentration of 0.5 to 20 mg / mL, The fractions may be included at a concentration of 0.01 to 10 mg / mL

The "pharmaceutically effective amount" as used herein refers to an amount sufficient for the physiologically active component to be administered to an animal or a human to exhibit a desired physiological or pharmacological activity. However, the pharmaceutically effective amount may be appropriately changed depending on the severity of symptoms, the age, body weight, health condition, sex, administration route and treatment period of the patient.

The term "pharmaceutically acceptable" as used herein means physiologically acceptable and does not generally cause an allergic reaction such as gastrointestinal disorder, dizziness, or the like when administered to a human. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Further, it may further include a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and an antiseptic agent.

The compositions of the present invention may also be formulated using methods known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal, and may be formulated as a variety of compositions for oral or parenteral administration . ≪ / RTI > The formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatine capsules, sterile injectable solutions, sterile powders, ointments, lotions, creams,

The composition according to the present invention may be administered through various routes including oral, transdermal, subcutaneous, intravenous or muscular, and the dose of the active ingredient may be varied depending on various factors such as route of administration, age, sex, And the like. In addition, the composition of the present invention may be administered in combination with a known compound capable of raising the desired effect.

Furthermore, the composition according to the present invention can be used not only as a pharmaceutical composition as described above, but also as a health functional food. For example, it can be easily used as a raw material for food, an ingredient, a food additive, a functional food or a beverage.

The term " food " means a natural product or a processed product containing one or more nutrients, preferably a state of being able to be eaten directly through a certain degree of processing, , Food additives, functional foods and beverages.

Foods to which the food composition can be added include, for example, various foods, beverages, gums, tea, vitamin complexes, and functional foods. In addition, it is also possible to use special nutritive foods (eg crude oil, milk, infant food, etc.), meat products, fish products, tofu, jelly, noodles (eg ramen, (Such as fermented milk, cheese, etc.), processed foods, kimchi, pickled foods (various kinds of kimchi, etc.), sauces, confectionery (eg, snacks), candies, chocolate, gums, ice cream, But not limited to, beverages (e.g., fruit drinks, vegetable beverages, beverages, fermented beverages, etc.), natural seasonings (e.g., ramen soup, etc.). The food, beverage or food additive may be prepared by a conventional production method.

The term " functional food " or " health functional food " refers to a food group imparted with added value to function or express the function of the food to a specific purpose by physical, biochemical or biotechnological techniques, Defensive rhythm control, prevention and restoration of disease, and the like, and it can be a health functional food. The functional food may include a food-acceptable food-aid additive, and may further comprise suitable carriers, excipients and diluents conventionally used in the production of functional foods.

The health supplements may be in the form of powders, granules, tablets, capsules or drinks, although not limited thereto.

Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described in detail below. Hereinafter, the present invention will be described in detail with reference to examples. However, these examples are for illustrating the present invention specifically, and the scope of the present invention is not limited to these examples.

Example 1: Preparation of roots extract

Root cane extract was prepared according to the process shown in Fig. Specifically, the roots grown in Jeju Island were washed, cut and lyophilized. The lyophilized cuts were then washed and then extracted with alcohol, filtered, concentrated and sterilized. If necessary, it can be stored by freeze drying.

In order to minimize the loss of active ingredients, to minimize component loss, and to increase the yield, we optimized the conditions for the preparation of roe deer extract by varying the roasted condition, storage temperature, alcohol concentration, and so on.

<Experimental Example 1> Evaluation of yield according to raw material cutting conditions and storage temperature

In case of extracting the dried powder of Doduck, the active ingredient dissolves relatively much, but since it is not easy to filtrate with a specific viscous substance, it is necessary to cut to the optimum size to maximize the efficiency. Therefore, samples of 100 g of the lyophilized powder were extracted with 30% alcohol by varying the size in five steps, and the yield was investigated to establish the optimal truncation conditions. The results are shown in Tables 1 and 2.

As can be seen from Tables 1 and 2, the yield of the extract according to the pulverization size was the highest in the powder phase, but the size of the pulverization process was selected to be about 5 mm in consideration of the efficiency of the filtration after the extraction. The yields of freeze - dried powder extracts were not different by storage temperature.

<Experimental Example 2> Changes in major components and physiological activity according to storage period, storage temperature, and crushing size

Lancemaside A, a major potent agent of Dodeca

The changes of lancemaside A component by storage temperature, storage period and crushing size were measured by HPLC. The HPLC conditions are shown in Table 3, and the measurement results are shown in Tables 4 to 6. The lancemaside A component was detected at 38.6 min.

As can be seen from Tables 4 to 6, changes in the lancemaside A component according to the storage temperature, storage period (2 months), and crushing size (5 levels) showed that the longer the storage period or the higher the storage temperature, And the lancemaside A component was found to be higher in the degree of crushing.

Total polyphenol and flavonoid content determination

- Total polyphenol content measurement

The total polyphenol content of Duckweed extract was analyzed by measuring the absorbance at 725 nm using a spectrophotometer. The extract was dissolved in a constant concentration and 0.25 ml of each test tube was mixed with 0.5 ml of a 10-fold diluted Folin &Ciocalteu's phenol reagent. The mixture was left at room temperature for 5 minutes. After that, 0.5 ml of 7.5% sodium carbonate (Na ₂ CO ₃ ) was added, and the mixture was allowed to stand at 30 ° C for 90 minutes, and the absorbance was measured at 725 nm using a spectrophotometer. To obtain the total polyphenol content, a calibration curve was prepared using chlorogenic acid as a reference material, and the total polyphenol content of dodec was determined and quantified.

- Determination of total flavonoid content

The total flavonoid content was determined by dissolving Dodok extract at a concentration of 1 mg / mL using 80% methanol, adding 10 mL of diethylene glycol and 0.1 mL of 1N NaOH, reacting at 37 ° C for 1 hour, and measuring the absorbance at 420 nm Respectively. Naringin was used as a reference material.

The results of the polyphenol and flavonoid content measurements are shown in Tables 7 to 9. Data were expressed as mean ± SE of three experiments.

The total polyphenol content and flavonoid content of the extracts of Dulcidae, which had different alcoholic alcohol contents, were higher than those of 30% EtOH extracts. This result is considered to be meaningful in relation to the selection of 30% EtOH as a sample extraction solvent for the standardization of the material in this study. In addition, it was found that the content of the fine particles increased with the change of the content by the pulverization size. However, depending on storage temperature, there was almost no difference in total polyphenol content and flavonoid content.

< Experimental Example  3> Validation of physiological activity according to storage period, storage temperature and grinding size

Verification of the enhancement effect of immune cell growth

The immune function enhancing effect was verified using T cell and B cell (RPMI 8226, KCLB No.10155), which are human immune cells. The cells were cultured in RPMI 1640 medium supplemented with 10% FBS at 5% CO 2 at 37 ° C. After the cells were adjusted to a concentration of 2.5104 cells in a 24-well plate, the cells were filtered with a 0.2 μm filter Samples (0.5 μg / μL) were added to each well for 24 hours and then re-cultured for 8 days. Cells were counted using a hemocytometer in each well while culturing for 8 days. Immunoreactivity was measured according to cell growth and number of cells compared with control without sample extracts. The results are shown in FIGS. 4 to 9. As a result of the growth promotion experiments of T cells and B cells, which are human immune cells of D. japonicus extract according to storage period, storage temperature and crushing size, And there was no significant difference between storage period, storage temperature and crushing size.

DPPH  radical Scatters

The antioxidant activity was measured by hydrogen electron donating ability by the method of Choi et al. (2003). Several concentrations of the sample were dissolved in methanol (or DMSO) solvent, and 900 μL of DPPH solution (100 μM) and 100 μL of each sample were mixed and stirred. This mixture was reacted in a dark place for 30 minutes and absorbance was measured at 517 nm. The hydrogen electron donating ability was evaluated by repeating each experiment three times, and the degree of decrease in absorbance of the control was calculated by the following equation, and the results are shown in Tables 10 to 12. Data were expressed as mean ± SE of three experiments.

An = (A ₀ -A) / A ₀ 100

An: Antioxidant activity against DPPH radical scavenging activity (%)

A0: Absorbance of DPPH solution without added sample

A: The absorbance of DPPH in the reaction solution and the absorbance of the sample

As can be seen from Tables 10 to 12, the DPPH scavenging activity of each of 6 concentrations such as 0.5, 1, 2.5, 5, 10, and 20 mg / , The activity was relatively higher as the storage period was shorter or the storage temperature was lower and the crushed particles were finer. Overall, the increase in DPPH scavenging activity showed a concentration-dependent tendency that was proportional to the concentration.

gun ABTS  Cation ( ABTS +) Scatters

A solution of 7.4 mM ABTS (2,2'-azinbis- (3-ethyl-benzothiazoline-6-sulfonic acid) and 2.6 mM potassium persulphate was mixed and incubated in a dark place for about 15 hours. 1.5, 150 μl of each sample was added to each diluted solution, and the mixture was shaken for 10 seconds with a vortex mixer, allowed to stand at room temperature for 90 minutes, and the absorbance was measured at 414 nm. And the absorbance was measured by the same method. The cationic scavenging activity was expressed as AEAC (relative ascorbic acid equivalent antioxidant capacity), which was calculated as 1.000 when the scavenging activity of ascorbic acid was 1.000. Which was calculated by the following equation.

ΔAaa: change in absorbance when ascorbic acid is added, Caa: concentration of ascorbic acid

ΔAs: Absorbance change when sample is loaded, Cs: Sample concentration

The results are shown in Tables 13 to 15, and the data were expressed as the mean value ± SE of three experiments.

The ABTS cation - scavenging activity was increased in proportion to the concentration of the extracts by storage period, storage temperature, and crushing size. There was no significant difference in storage time and storage temperature, but relatively high scavenging activity was observed at the finer particle size near the powder phase in the pulverization size.

Measuring reducing power

The reducing power was measured by modifying Oyaizu 's method. After mixing 30 μL / mL of 30% ethanol extract (10 μg / mL) with 500 μL of 0.2 M sodium phosphate buffer (pH 6.6) and 500 μL of 1% potassium ferricyanide at 50 ° C for 20 minutes, 10% trichloroacetic acid acid 2.5 mL was added. The reaction solution was centrifuged at 650 rpm for 10 minutes, and 500 μL of the supernatant was added to 500 μL of distilled water and 100 μL of 1% ferric chloride. The absorbance of the reaction solution was measured at 700 nm. The results are shown in Tables 16 to 18, and the data were expressed as the mean value ± SE of three experiments.

Experimental results of reducing power according to storage period, storage temperature, and crushing size showed that the activity increased with increasing concentration and was dependent on concentration. Overall, the reducing power of the pulverized particles was relatively higher than that of the fine pulverized samples. However, there was no significant difference between the storage period and storage temperature.

Xanthine oxidase  Measurement of inhibitory activity

The xanthine oxidase inhibitory activity of Duckweed extract according to storage time, storage temperature and crushing size was measured by Noro (1983) and Stirpe (1969). That is, 0.2 mL of a substrate solution in which 2 mM xanthine was dissolved was added to 0.1 mL of each sample solution and 0.6 mL of a 0.1 M potassium phosphate buffer (pH 7.5) solution, 0.1 mL of xanthine oxidase (0.2 U / mL) After reacting for 15 minutes, 1 mL of 1N HCl was added to terminate the reaction, and the amount of uric acid produced in the reaction solution was measured by UV / VIS spectrophotometer at the absorbance at 292 nm. At this time, 0.1 mL of distilled water was added to the control instead of the sample, and 1 mL of 20% trichloroacetic acid (TCA) was added to stop the reaction. The reaction was stopped and the protein was removed by filtration. The xanthine oxidase inhibitory activity was calculated as the absorbance reduction rate of the sample solution added group and the no-added group, ie, the percentage of uric acid produced in the reaction solution.

The results are shown in Tables 19 to 21, and data were expressed as the mean value ± SE of three experiments.

The activity of xanthine oxidase inhibitor was increased with storage period or storage temperature, and the activity of xanthine oxidase inhibitory activity was higher in powder with the finest size.

SOD (Super Oxide Dismutase) activity analysis

SOD enzyme activity assays were performed using an assay kit (Sigma, 19160). That is, the photochemical NBT method was used to test the ability of SOD enzyme to inhibit the reduction of NBT (Nitroblue tetrazolium). The reaction solution was adjusted to 50 mM carbonic buffer (pH 10.2), 0.1 mM EDTA, 0.1 mM Xanthine and 0.025 mM NBT, and the inhibition of NBT reduction was measured at 450 nm. Each sample was repeated 3 times, and SOD enzyme activity was calculated by the following equation.

SOD activity (NBT reduction inhibition rate,%) = {[(A _blank1 -A _blank3 ) - (A _sample -A _blank2 )] / (A _blank1 -A _blank3 )

The activity of SOD was not significantly different depending on the storage temperature.

< Example  2> Deutsche solvent Fraction  Produce

Hexane (nH), methylene chloride (MC), ethylene acetate (EA), and butyl alcohol (BA) were obtained by the process as shown in FIG. 3 using a 30% alcohol extract obtained from the step ) And distilled water (DW).

After cultivated in Jeju area, it was washed and lyophilized, and then it was extracted three times with 30% alcohol at room temperature and concentrated under reduced pressure. Fifty grams of the lyophilized powder obtained by concentrating the extract was subjected to fractionation three times for each solvent such as n-hexane, methylene chloride, ethyl acetate, butyl alcohol and DW (distilled water), and the extract was completely concentrated by using a vacuum concentrator The recovery rate of each fraction was investigated by weighing. As a result, the highest recovery was obtained in water extraction, and the yield was very slight in the ethyl acetate layer. That is, the extraction yield was 1.21 g of the normal nucleic acid soluble portion, 0.25 g of the methylene chloride soluble portion, 0.12 g of the ethyl acetate soluble portion, 2.38 g of the butyl alcohol soluble portion and 37.5 g of the water soluble portion.

< Experimental Example  4> dodok solvent Fraction  Antioxidant enzyme activity analysis

0.5 g of the dodecylsorbate fraction according to Example 2 was homogenized with 2 ml of Extract Buffer [100 mM K-PO4 buffer (pH 7.5), 100 mM EDTA, 1% PVP, 100 mM PMSF] and centrifuged at 15.000 g for 20 minutes The supernatant was then used to measure antioxidant activity. For ascorbate peroxidase (APX), 10 mM was added to the above extraction solution in extraction buffer. Protein quantification was performed according to the Bradford (1976) method using BSA as a standard.

SOD ( SuperOxide Dismutase )

SOD enzyme activity assays were performed using an assay kit (Sigma, 19160). That is, the photochemical NBT method was used to test the ability of SOD enzyme to inhibit the reduction of NBT (Nitroblue tetrazolium). The reaction solution was adjusted to 50 mM carbonic buffer (pH 10.2), 0.1 mM EDTA, 0.1 mM Xanthine and 0.025 mM NBT, and the inhibition of NBT reduction was measured at 450 nm. Each sample was repeated 3 times, and SOD enzyme activity was calculated by the following equation.

SOD activity (NBT reduction inhibition rate,%) = {[(A _blank1 -A _blank3 ) - (A _sample -A _blank2 )] / (A _blank1 -A _blank3 )

CAT ( Catalase )

CAT activity was measured by the method of Aebi et al. (1984). The reaction solution was 50 mM potassium phosphate buffer (pH 7.0) and 10 mM H 2 O 2. CAT activity was measured by adding the extract to the reaction solution and then changing the absorbance at 240 mM for 2 minutes.

APX  ( Ascorbate Peroxidase )

The activity of APX was measured by the Nacano and Asada (1981) method and the degree of oxidation of ascorbate was measured at 290 nm for 2 minutes. The reaction solution of APX was a 100 mM potassium phosphate buffer (pH 7.5) supplemented with 0.5 mM ascorbate and 0.2 mM H ₂ O ₂ .

POD ( Peroxidase )

POD activity was measured by the method of Egley et al. (1983). The reaction solution was adjusted to a final concentration of 40 mM K-PO ₄ buffer (pH 6.9), 1.5 mM guaiacol, and 6.5 mM H ₂ O ₂ . The POD activity was measured by measuring the absorbance at 470 nm for 2 minutes using a spectrophotometer.

The measurement results are shown in Figs. 12 to 15. Fig. Antioxidant enzyme activities of the extracts of Duckweed were highest in the ethyl acetate fraction as a whole, and relatively low in the n-hexane fraction and the DW fraction.

< Experimental Example  5> Human cancer cell  Doduk solvent Fraction  Analysis of antitumor activity

(MTT) (3- (4,5-dimethyl-thiazol-2-yl) -2,5-diphenyltetrazoliumbromide) assay was performed to measure the anticancer effect of the extracts of Solanum tuberosum Respectively. HeLa, breast cancer cell, MCF-7, lung cancer cell Calu-6 cell line were plated on a 96-well plate and incubated for 24 hours at 37 ° C in a 5% CO2 incubator. The cultured cells were treated with 10 μl of each sample to a concentration of 50, 100, 200, 400, 800 μg / ml, and cultured for 24 hours. 10 μl of 0.2 mg / ml MTT solution was added to each well, Lt; / RTI &gt; After the incubation, the supernatant was removed and 100 μl of DMSO was added to each well. After incubation at room temperature for 10 minutes, the absorbance was measured at 540 nm using a microplate reader. The cytotoxicity was expressed as a percentage of the absorbance of the control . The results are shown in Table 22, and the data were expressed as mean ± SE of three experiments.

As a result of examining the cytotoxicity of dodeca extract fraction by MTT method to human cancer cell line, the DW fraction showed slightly lower toxicity to human cancer cell line even at a high concentration of 1000 ug / mL, and n-Hexane, MethyleneChloride, Ethyl Acetate And n-BuOH fractions showed relatively high toxicity in both HeLa, MCF-7 and Calu-6 cell lines. Especially, n-Hexane fraction showed high cytotoxicity.

< Experimental Example  6 > 3T3-L1 cells Anti-obesity  Effect measurement

3T3-L1 cells were cultured in KCLB No. 10092.1 from Korean Cell Line Bank. Cells were cultured in DMEM containing 10% FBS in a 5% CO ₂ , 37 ° C incubator in a 75T-flask. After 3 to 4 days, the cells were treated with 0.05% Trypsin / 0.53 mM EDTA to separate the cells. The cells were collected at a centrifuge (2000 rpm, 2 min), and the cell density was diluted to 1 × 10 ⁴ cells / ml. and cultured in 1 mL increments. The cells were changed into new DMEM culture medium containing 10% FBS once every 2 days. After 3 to 4 days, the cells were fused. After 2 days, DMEM culture medium was supplemented with 5 mg / mL insulin, 0.25 mM dexamethasone and 0.5 mM IBMX Differentiation was induced. After 2 days, the cells were replaced with 5 mg / mL insulin alone and changed into new culture once every 2 days (Mac Dougald OA, Hwang CS, Fan H., Lane MD 1995). On day 6, cells were cultured in the absence of insulin, and after 7 days of incubation, the samples were treated with Oil red O staining 24 hours later.

Cytotoxicity measurement

MTT (methylthiazolyldiphenyltetrazolium bromide) assay for measuring cell viability was performed according to Sladowski et al. The cultured 3T3-L1 cells were plated at 1 × 10 ⁵ cells / well on a 96-well plate and cultured for 16-18 hours. The diluted sample extracts were treated for 24 hours. MTT solution dissolved at 5mg / ml was added to 10uL / 990ul medium and incubated at 37 ° C for 3 hours in 5% CO ₂ . After the culture was completed, the culture medium was removed, and the mixture was added to 200 μL / well of DMSO. The mixture was stirred for 20 minutes, and the absorbance at 595 nm was measured with an ELISA reader. The results are shown in Table 23. Data were presented as the mean ± SE of three experiments, and the same letters in the same column were not significantly different at the p <0.05 level by the Duncan's multiple range test.

The cell viability of 3T3-L1 cells at various concentrations of Dulbecco's soluble fraction was decreased in a concentration-dependent manner, especially in the butyl alcohol fraction, which was relatively more cytotoxic than the other fractions.

Oil red O dyeing

The cell culture medium was discarded and 50 μL of 10% formaldehyde was added to each well to fix the cells, followed by incubation at 4 ° C. for 1 hour. After that, the formaldehyde was discarded and washed three times with PBS. Then, a solution of 0.25 g of Oil red O staining solution (Oil red O dissolved in 50 mL of isopropyl alcohol was mixed with distilled water at a ratio of 3: 2, filtered through a 0.45 μm filter Solution) was added to each well, and the plate was stained again at room temperature for 1 hour and then washed three times with PBS. The stained cells were observed under a microscope. After observation, the stained dye in the adipocyte was extracted with isopropyl alcohol (500 μL) per well, and the absorbance was measured at 520 nm by a spectrophotometer. The results are shown in FIG. In FIG. 16, the fat accumulation content was expressed as a percentage of control (Con), and fixed and stained cells were scanned for microspore captured images. The same letter in the same column means no significant difference at the p <0.05 level by Duncan's multiple range test. The bars in the graph indicate standard errors.

The oil red O staining of 3T3-L1 cells treated with 50 ㎍ / mL of solvent fraction showed fatty accumulation and adipocyte differentiation, and methyl chloride fraction showed relatively high effect.

< Experimental Example  7> Anti-asthma  Related Immune Indicator In Vitro Experiment

In order to test the anti - asthma - related immunoregulatory ability of the extracts, we examined the inflammatory factors. (TNF-α) and interferon gamma (IFN-) secretion levels of IL-4, IL-4, IL-5, After the induction of LPS inflammation, the extracts were treated with Immunocytochemistry (ICC).

The A549 cell line was inoculated at 1 × 10 ⁵ / well in RPMI 1640 medium containing 10% FBS and 1% penicillin and incubated for 18 hours. Then, LPS was treated with 150 μg / mL of rosemary extract, 250 μg / mL of rosemary extract, incubated for 24 hours, and the degree of inflammatory factor secretion was analyzed under the following conditions.

   IL-4 - Santa Cruz, 1: 100

   IL-5 - BioLegend, 1: 100

   IL-13 - Santa Cruz, 1: 100

   TNF-α-MYBioSource, 1: 100

   IFN- &lt; / RTI &gt; - Santa Cruz, 1: 100

The results of the analysis are shown in Figs.

For Thl -related cytokines, IFN- increased IFN- expression compared to CON-treated groups in LPS strain, and decreased IFN- (Fig. 17). For Th1-related cytokines, IL-4 increased the expression of IL-4 compared to the CON group in the LPS strain, and decreased in the Dactex extract-treated group (FIG. 18) In comparison, the expression level of IL-5 was increased, and it was decreased in the case of extract (Fig. 19). IL-13 increased the expression level of IL-13 in the LPS oil tool compared to the CON group, and decreased in the Duckex extract-treated group (Fig. 20).

As a result, the expression of Th1- & Th2-related cytokines was inhibited. Therefore, it was suggested that Thodacin was associated with Th1 and Th2 because Thodacin decreased Th1 and Th2 related factors (IFN-, IL-4, IL-5, IL-13)

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (10)

Washing the dodecae and cutting to 1 to 10 mm (step 1);
Lyophilizing the cut dough (step 2);
Extracting the lyophilized dodec with 30% alcohol (step 3); And
From the group consisting of cervical cancer, lung cancer, and breast cancer, which comprises the fractions of niger-hexane produced by fractionating the extract of step 1 with n-hexane and separating into n-hexane fraction and water layer (step 4) A pharmaceutical composition for the prevention or treatment of cancer selected.
Washing the dodecae and cutting to 1 to 10 mm (step 1);
Lyophilizing the cut dough (step 2);
Extracting the lyophilized dodec with 30% alcohol (step 3); And
From the group consisting of cervical cancer, lung cancer, and breast cancer, which comprises the fractions of niger-hexane produced by fractionating the extract of step 1 with n-hexane and separating into n-hexane fraction and water layer (step 4) A health functional food composition for preventing or ameliorating cancer selected.
Washing the dodecae and cutting to 1 to 10 mm (step 1);
Lyophilizing the cut dough (step 2);
Extracting the lyophilized dodec with 30% alcohol (step 3); And
From the group consisting of cervical cancer, lung cancer, and breast cancer, which comprises the fractions of niger-hexane produced by fractionating the extract of step 1 with n-hexane and separating into n-hexane fraction and water layer (step 4) A health food composition for preventing or ameliorating cancer selected. delete delete delete delete delete delete delete

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