KR20180004076A - Ultrasonic extract of Lespedeza cuneate and method for extracting the same - Google Patents

Abstract

The present invention relates to an ultrasonic extract of an anchovy and an extract thereof, and more particularly, to an anchovy extract having antioxidant, anti-inflammatory and anticancer effects using an ultrasonic extraction method and a method for effectively producing the same. The method for preparing the non-squeezed ultrasound extract according to the present invention can effectively extract the active ingredient contained in the syrup by treating the ultrasound under specific conditions after adding the solvent to the syrup, Has excellent antioxidative, anti-inflammatory and anti-cancer activities. Therefore, the non-squeezable ultrasound extract prepared by the method of the present invention can be used for various industrial fields such as medicines, foods, cosmetics and the like with various functionalities. Particularly, since it is a natural medicinal plant and can be edible, the composition of the present invention containing an extract derived therefrom as an active ingredient has safety advantages even for long-term use.

Description

[0001] Ultrasonic Extract of Anchovy and Its Extraction Method [

The present invention relates to an ultrasonic extract of an anchovy and an extract thereof, and more particularly, to an anchovy extract having antioxidant, anti-inflammatory and anticancer effects using an ultrasonic extraction method and a method for effectively producing the same.

It is a perennial herbaceous or supernatural herb that grows in mountains and fields around the country. The growing environment grows wherever it is sunny. The key is about 1m, the leaves are shifted, there is no hairs on the surface of the leaves, there are fine hairs on the back, the length of the leaves is 1 ~ 2㎝, and the width is 0.2 ~ 0.4㎝. The stalk is climbing thinly and has many fine hairs. The flower is shorter than the leaf, attached to the leaf axilla with a butterfly shape, and bloomed in white, with a purple stripe in the center. The fruit is dark brown and runs in October and contains one seed with red spots on the yellowish green background. Outposts containing roots are used for medicinal purposes. As a generic name, it is called a "yamantan", and it has become more common to drink alcohol recently.

On the other hand, the flavonoid is derived from the flavus which means yellow in Greek, and refers to a plant pigment having flavone as a basic structure. Vitamin P (permeable vitamin) or vitamin C2 (vitamin C as a synergist) is also called. It is relatively small in animals, and is contained in many kinds of plants, such as leaves, leaves, In addition, it has antibacterial antiviral anti-allergic and anti-inflammatory activity, and it is reported that there is almost no toxicity. In addition, as the fact that it inhibits in vivo oxidative action is known, there is a growing interest in the development and application of flavonoid-based materials.

The substances such as phenolic compounds, flavonoids and tocopherols of the syrup can inhibit the action of reactive oxygen species and prevent adult diseases such as cancer, cardiovascular diseases, and contribute to delay and prevention of aging. Many hydroxyl groups (-OH) present in polyphenols have the property of easily binding to various compounds and thus have excellent antioxidative, anti-cancer and anti-inflammatory effects, and flavonoids are known to have high antioxidant ability by effectively removing active oxygen species Like polyphenols, it is known to have antiviral, anti-inflammatory and anti-cancer effects. Therefore, there is a need for research on a method for efficiently producing such an insecticidal extract.

In general, the active ingredient in the syrup can be extracted using a solvent such as water or ethanol. When extracting with water, high-temperature and long-time extraction conditions are required, and the components contained in the syrup can be decomposed. It is disadvantageous in that an insecticidal extract having low physiological activity is obtained due to the evaporation phenomenon. On the other hand, in the case of ethanol extraction, it can be extracted by lowering the extraction temperature and the extraction time. However, since the organic solvent is used, the cost is increased and there is a problem such as environmental pollution.

The inventors of the present invention have found that when an ultrasonic treatment is carried out under a specific condition after adding ethanol to a non-syrup, the antioxidative, anti-inflammatory and anti-cancer The present inventors have completed the present invention by confirming that it is possible to produce an insecticidal extract having excellent activity.

Korean Patent Publication No. 10-2008-0079134

Accordingly, it is an object of the present invention to provide a pharmaceutical composition which can prevent or treat diseases such as inflammation disease and cancer by using an aspirate ultrasonic extract.

It is another object of the present invention to provide a food composition capable of preventing or ameliorating diseases such as inflammation disease and cancer by using an aspirate ultrasonic extract.

It is still another object of the present invention to provide a health functional food which can prevent or ameliorate diseases such as inflammation disease and cancer by using an aspirate ultrasonic extract.

It is still another object of the present invention to provide a cosmetic composition which can improve skin through antioxidant or anti-inflammatory function using non-sour ultrasound extract.

It is still another object of the present invention to provide a method for producing an aqueous non-squeezable extract capable of effectively and rapidly extracting an active ingredient of an insecticide.

Yet another object of the present invention is to provide an ultrasonic extract prepared by the above method.

In order to achieve the above-mentioned object of the present invention,

There is provided a pharmaceutical composition for preventing or treating an inflammatory disease or cancer disease comprising an aspirate ultrasonic extract as an active ingredient.

In one embodiment of the present invention, the non-squeaky ultrasound extract may be an extract obtained by adding ethanol to a syrup followed by treatment with ultrasound.

In one embodiment of the present invention, the cancer may be colon cancer or lung cancer.

In addition, the present invention provides a food composition for preventing or ameliorating an inflammatory disease or cancer disease comprising an aspirate ultrasonic extract as an active ingredient.

The present invention also provides a health functional food for preventing or ameliorating an inflammatory disease or cancer disease comprising an aspirate ultrasonic extract as an active ingredient.

In one embodiment of the present invention, the food is selected from the group consisting of beverage, meat, chocolate, foods, confectionery, pizza, ram noodles, gums, candy, ice cream, alcoholic beverages, .

In addition, the present invention provides a cosmetic composition having antioxidant or anti-inflammatory activity comprising an aspirate ultrasonic extract as an active ingredient.

In one embodiment of the present invention, the cosmetic composition may be at least one selected from the group consisting of a soft lotion, a gel, a water-soluble liquid, a milk lotion, a nutritional cream, a massage cream, an essence, an oil in water emulsion, , Oil dispersions in aqueous phase using microspheres, ionic lipid vesicles, nonionic lipid vesicles, ointments, cleansing foams, cleansing waters, packs, body oils, oil-in-oil makeup bases, watershed makeup bases, foundations, skins And may be one type of formulation selected from the group consisting of a cover, a lipstick, a lip gloss, a face powder, a two-way cake, an eye shadow, a mascara, a cheek color and an eyebrow pencil.

Further, the present invention provides a method for producing a microcapsule comprising the steps of: a) treating ultrasonic waves after adding a solvent to a syringe; And b) centrifuging the solution after the step a), and filtering the separated supernatant.

In one embodiment of the present invention, the solvent may be ethanol.

In one embodiment of the present invention, the step (a) may be performed by immersing a syringe in an aqueous solution of 20 to 60 w / v% ethanol and treating the syringe at 20 to 30 ° C for 1 to 24 hours.

In one embodiment of the present invention, in the step b), the solution having been subjected to the step a) is centrifuged at a temperature of 0 to 10 ° C at a speed of 3000 to 5000 rpm for 5 to 30 minutes, Lt; RTI ID = 0.0 > 10 < / RTI >

In one embodiment of the present invention, after the step b), c) may further comprise pulverizing the filtered extract.

The In one embodiment, the frequency of the ultrasonic waves in the ultrasonic processing may be 20 to 30 kHz.

In addition, the present invention provides an ultrasonic extract obtained by the above method.

The method for preparing the non-squeezed ultrasound extract according to the present invention can effectively extract the active ingredient contained in the syrup by treating the ultrasound under specific conditions after adding the solvent to the syrup, Has excellent antioxidative, anti-inflammatory and anti-cancer activities. Therefore, the non-squeezable ultrasound extract prepared by the method of the present invention can be used for various industrial fields such as medicines, foods, cosmetics and the like with various functionalities. Particularly, since it is a natural medicinal plant and can be edible, the composition of the present invention containing an extract derived therefrom as an active ingredient has safety advantages even for long-term use.

FIG. 1 is a graph showing the results of measurement of the total flavonoid content of the anthracnose extract according to the extraction conditions.
FIG. 2 is a graph showing the DPPH radical scavenging activity of the extract of the anthilliae according to the extraction conditions (IQ: isoquercetin-treated group, R: treated group).
Fig. 3 is a graph showing the results of measurement of the inhibitory effect on the NO production of the extract of Asuncio according to the extraction conditions (P3: isoquercitrin-treated group).
FIG. 4 shows the results of measuring the inhibitory effect of NF-κB promoter binding activity on HUVEC cells with increased oxidative stress by TNF-α, respectively (P3: isoquercitrin-treated group) .
FIG. 5 relates to the results of principal components analysis of yield, antioxidant properties, anti-inflammatory activity, anti-arteriosclerosis activity, and anticancer activity according to the extraction conditions of anchovy.

The present invention provides a composition comprising an ashless ultrasonic extract as an active ingredient.

The composition of the present invention can be used for various purposes and applications requiring antioxidant, anti-inflammatory or anti-cancer activity. Specifically, the composition of the present invention can impart antioxidative activity to articles applied in various industrial fields such as medicines, cosmetics, It can be used as a functional material, and can also be used as a medicine preservative, a cosmetic preservative, a food preservative, a pharmaceutical additive, a cosmetic additive, a food additive, and a feed additive.

Hereinafter, the present invention has been described in detail in the form of medicines, foods, and cosmetics to which the composition of the present invention can be applied.

The composition of the present invention may be a pharmaceutical composition for the prevention or treatment of an inflammatory disease or cancer disease comprising an aspirate ultrasonic extract as an active ingredient.

The pharmaceutical composition of the present invention can be prepared by using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the above-mentioned active ingredients. Examples of the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, A lubricant or a flavoring agent can be used.

The pharmaceutical composition may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the above-described active ingredients for administration.

The pharmaceutical composition may be in the form of granules, powders, tablets, coated tablets, capsules, suppositories, liquids, syrups, juices, suspensions, emulsions, drops or injectable solutions. For example, for formulation into tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Also, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included as a mixture. Suitable binders include, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, natural and synthetic gums such as corn sweeteners, acacia, tracker candles or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum and the like. Acceptable pharmaceutical carriers for compositions that are formulated into a liquid solution include sterile water and sterile water suitable for the living body such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.

In one embodiment of the present invention, the non-sour ultrasound extract of the present invention may be contained in an amount of 0.00001 to 99% by weight based on the total weight of the composition.

In another embodiment of the present invention, the non-syringe ultrasonic extract of the present invention may be contained at a concentration of 0.1 to 1000 μg / ml on the composition.

Examples of the inflammation-related diseases in which the pharmaceutical composition of the present invention can exhibit a therapeutic effect include rhinitis, asthma, acute pain, chronic pain, periodontitis, gingivitis, inflammatory bowel disease, gout, myocardial infarction, arteriosclerosis, congestive heart failure, hypertension But are not limited to, angina pectoris, gastric ulcer, Alzheimer's disease, cerebral infarction, Down's syndrome, multiple sclerosis, obesity, diabetes, dementia, depression, schizophrenia, tuberculosis, sleep disorders, sepsis, burn or pancreatitis.

Examples of the cancer that can be treated by the pharmaceutical composition of the present invention include gastric cancer, liver cancer, colon cancer, lung cancer, breast cancer, uterine cancer, esophageal cancer, pancreatic cancer, skin cancer and blood cancer. .

The present invention also provides a method of preventing or treating an inflammatory or cancer-related disease, comprising the step of administering the pharmaceutical composition to a suspected individual of an inflammatory or cancer-related disease.

In the present invention, the suspected individual of the inflammatory or cancer-related diseases refers to all animals including humans who have developed or are capable of developing inflammation or cancer-related diseases. The pharmaceutical composition comprising the non- By administering to a suspected individual with an associated disease, the individual can be treated efficiently. Inflammation or cancer-related diseases are as described above.

The term "administering" as used herein means introducing the pharmaceutical composition of the present invention into a subject suspected of having an inflammatory or cancer-related disease by any appropriate method, and the administration route may be an oral or non- May be administered via various routes of administration.

The method of treatment of the present invention may comprise administering a pharmaceutically effective amount of a pharmaceutical composition comprising an insecticidal ultrasound extract.

The term " pharmaceutically effective amount " as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and the effective dose level will vary depending on the species and severity, age, The type of disease, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The composition of the present invention may be administered as an individual therapeutic agent or in combination with another therapeutic agent, and may be administered sequentially or simultaneously with a conventional therapeutic agent. And can be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art. The preferred dosage of the composition of the present invention will depend on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration, and the period of time, and the appropriate total daily dose can be determined by treatment within the proper medical judgment.

The composition of the present invention may also be a food composition. The food composition may contain various flavors or natural carbohydrates as an additional component, such as an ordinary food composition, in addition to an active ingredient, an insecticidal ultrasound extract.

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 polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. The above-described flavors can be advantageously used as natural flavorings (tau martin), stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.).

The food composition of the present invention can be formulated in the same manner as the above pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolates, foods, confectionery, pizza, ram noodles, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes, .

In addition, the above food composition may contain various additives such as various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring agents and intermediates such as cheese and chocolate, Acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the food composition of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks.

The non-squeezable ultrasound extract, which is an active ingredient of the present invention, is a substance extracted from a natural medicinal plant. Since it has almost no side effects such as chemicals, it can be safely used for long-term administration for antioxidant, anti-inflammation and anticancer functions.

The present invention may also be a health functional food for preventing or ameliorating an inflammatory disease or a cancerous disease comprising an aspirate ultrasonic extract as an active ingredient.

The health functional food of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, rings and the like for the purpose of preventing or improving inflammatory diseases or cancer diseases.

In the present invention, the term "health functional food" refers to a food prepared and processed by using raw materials or ingredients having useful functions in accordance with the Act on Health Functional Foods, and the nutrients can be regulated on the structure and function of the human body, ≪ / RTI > is intended to be taken for the purpose of obtaining a beneficial effect for health use such as action.

The health functional foods of the present invention may contain conventional food additives and, unless otherwise specified, whether or not they are suitable as food additives are classified according to the General Rules for Food Additives approved by the Food and Drug Administration, Standards and standards.

Examples of the food items included in the above food additives include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as persimmon extract, licorice extract, crystalline cellulose, high color pigment and guar gum; L-glutamic acid sodium preparations, noodle-added alkalis, preservative preparations, tar coloring preparations and the like.

For example, the health functional food in the form of tablets may be prepared by granulating a mixture obtained by mixing the active ingredient of the present invention with an excipient, a binder, a disintegrant and other additives, granulating the mixture in a conventional manner, The mixture can be directly compression molded. In addition, the health functional food of the tablet form may contain a mating agent or the like if necessary.

The hard capsule of the capsule form of the health functional food may be prepared by filling a normal hard capsule with a mixture of the non-sour lysozyme extract as an active ingredient of the present invention with an additive such as an excipient and the soft capsule may contain the above extract as an excipient And mixing the mixture with an additive into a capsule base such as gelatin. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative and the like, if necessary.

The ring-shaped health functional food can be prepared by molding a mixture of an active ingredient of the present invention and an excipient, a binder, a disintegrant, and the like in accordance with a conventionally known method, and if necessary, Or it may be coated with a material such as starch, talc.

The granular health functional food may be prepared by granulating a mixture of an active ingredient of the present invention and an excipient, a binder, a disintegrant, and the like in a granular form by a conventionally known method, and if necessary, adding a flavoring agent, And the like.

As shown in the following examples, the health functional food containing the non-squeezable ultrasound extract of the present invention as an active ingredient has been experimentally proved to have excellent antioxidative, anti-inflammatory and anti-cancer effects and is effective in preventing or ameliorating inflammation-related diseases and cancer.

The health functional food may be a beverage, a meat, a chocolate, a food, a confectionery, a pizza, a ramen, a noodle, a gum, a candy, an ice cream, an alcoholic beverage, a vitamin complex and a health supplement food.

In addition, the composition of the present invention may be a cosmetic composition in addition to a pharmaceutical composition or a food composition.

Since the non-sour ultrasound extract of the present invention is excellent in antioxidative and anti-inflammatory activities, the composition comprising the non-sour organic extract as an active ingredient is useful as an effective ingredient for preventing damage to human cells and promoting aging, Skin protection and skin treatment, so that it can be effectively used as a cosmetic composition.

When the composition of the present invention is prepared with a cosmetic composition, the composition of the present invention may contain not only the above-mentioned non-aqueous ultrasonic extract but also components commonly used in cosmetic compositions, such as antioxidants, stabilizers, , Customary adjuvants such as pigments and flavoring agents, and carriers.

In addition, the composition of the present invention may be mixed with an antioxidant that has been used in the past so long as it does not impair the skin protecting effect by reacting with the non-sour ultrasound extract, in addition to the above-described non-sour ultrasound extract.

Examples of products to which the cosmetic composition of the present invention can be added include cosmetics such as astringent lotion, softening longevity lotion, nutrition lotion, various creams, essences, packs, foundation and the like, cleansing, cleanser, soap, , And shampoo.

As a specific formulation of the cosmetic composition of the present invention, there may be mentioned a skin lotion, a skin softener, a skin toner, an astringent, a lotion, a milk lotion, a moisturizing lotion, a nutrition lotion, a massage cream, It includes formulations such as soap, shampoo, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, latex, lipstick, makeup base, foundation, press powder, loose powder, eye shadow.

The present invention relates to a method for producing a microcapsule comprising the steps of: a) treating ultrasonics after addition of a solvent to a syringe; And b) centrifuging the solution obtained in the step a), and filtering the separated supernatant, characterized in that it provides a method for producing an ultrasonic extract.

The step (a) of the present invention is a step of adding ultrasonic waves after adding the solvent to the syringe.

The solvent may be any pharmaceutically acceptable organic solvent, and water or an organic solvent may be used. Examples of the solvent include, but are not limited to, purified water, methanol, ethanol, Alcohol having 1 to 4 carbon atoms, acetone, ether, benzene, chloroform, ethyl acetate (including ethyl acetate) containing propanol, isopropanol, butanol, ), Methylene chloride, hexane, and cyclohexane may be used alone or in combination. It is preferable to use ethanol (alcohol), more preferably 20 to 60 w / v% ethanol.

In one embodiment of the present invention, the step a) may be a step of immersing a syringe in an aqueous solution of 20 to 60 w / v% ethanol and treating the ultrasonic wave at 20 to 30 ° C for 1 to 24 hours.

In another embodiment of the present invention, the frequency of the ultrasonic wave in the ultrasonic processing may be 20 to 30 kHz.

In the step b) of the present invention, the solution having been subjected to the step a) is centrifuged and the separated supernatant is filtered. Specifically, the solution having been subjected to the step a) is filtered at a temperature of 0 to 10 ° C at a rate of 3000 to 5000 rpm For 5 to 30 minutes, and filtering the separated supernatant through a membrane having a pore size of 0.2 to 10 mu m.

The method for producing the non-squeezed ultrasonic extract of the present invention may further include the step of c) pulverizing the filtered extract after the step b).

That is, the primary extract extracted through the step b) of the present invention can be prepared into a powder state by an additional process such as vacuum distillation, freeze-drying, or spray drying. Further, the primary extract can be further purified by using various chromatographies such as silica gel column chromatography, thin layer chromatography, high performance liquid chromatography and the like, You can get it.

Therefore, in the present invention, the non-squeezing ultrasonic wave extract is a concept including all the extracts, fractions and tablets obtained in each step of extraction, fractionation or purification through the steps a), b) and c), diluted solution thereof, to be.

In addition, the present invention provides a non-squeezable ultrasound extract prepared by the above-described method.

The extract of the present invention is more effective in extracting the extract than the extract obtained by using ethanol alone, and further exhibiting excellent antioxidative, anti-inflammatory and anti-cancer activities.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.

≪ Example 1 >

Preparation of an Asiatic Extract

≪ 1-1 > Preparation of Unsulfurized Powder

The bisuri (nomenclature used in this experiment: Lespedeza cuneata , domestic) were directly sampled and dried. All samples were milled to 0.6 mm size with a pin mill and stored frozen.

<1-2> Preparation of Anthracnose Extract Using Ethanol Solvent Extraction

Ethanol (60%) of 100 times (w / v) was added to the non-syrupy dry powder prepared in Example <1-1>, and the mixture was stirred at 25 ° C for 24 hours for extraction. The thus prepared non-viable ethanol extract was used as a comparative control sample in the following experiment.

<1-3> Preparation of anisakii extract using ultrasonic extraction

The ultrasonic extraction method used in the present invention was to extract ultrasonic waves by generating ultrasonic waves at 28 kHz and amplitude 80% using an ultrasonic wave extractor. To maintain the temperature constant, a circular water bath (Circulator Water Bath) Respectively.

The dried non-syrupy powder prepared in Example <1-1> was immersed in an aqueous solution of 0, 20, 40 and 60 w / v% ethanol, and the extraction time was 1, 2, 3 , 6, 9, 12 and 24 hours, respectively. The extract was centrifuged (4000 rpm, 20 minutes) at 4 ° C, and the separated supernatant was filtered through a membrane having a pore size of 0.22 μm and then concentrated under reduced pressure (Evaporator, Tokyo rikakikai co.Ltd., Japan) Ultrasonic ultrasound extracts were prepared and used as analytical samples in the following experiments.

&Lt; 1-4 > Extraction yield (%)

The extraction yields (%) of each of the non-sour extracts prepared in Examples 1-2 and 1-3 were determined after the lyophilization (weight of the dried extract) / (raw material weight ) × 100. As a result, as shown in Table 1, extraction yield of 13.5% was found in the conventional ethanol 60% solvent extraction, whereas extraction yield was 11.5 ~ 19% in the ultrasonic extraction according to the extraction conditions.

Extraction Yield (%) of Extracts from Extracts by Extraction Conditions and Extraction Conditions Extraction method Temperature
(° C) time
(hr) ethanol
(w / v%) Sample Name Extraction yield
(%) Ethanol solvent 25 24 60 L0 13.5 ultrasonic wave 25 One 0 L1 13.5 20 L2 15.5 40 L3 17 60 L4 15 2 0 L5 11.5 20 L6 18 40 L7 16 60 L8 13.5 3 0 L9 14.5 20 L10 16.5 40 L11 16.5 60 L12 12.5 6 0 L13 13 20 L14 14.5 40 L15 16 60 L16 15 9 0 L17 14 20 L18 16 40 L19 17.5 60 L20 16 12 0 L21 15.5 20 L22 17.5 40 L23 18.5 60 L24 19 24 0 L25 14 20 L26 18 40 L27 17.5 60 L28 17.5

< Experimental Example  1>

The total flavonoid content

In this experiment, the content of total flavonoids contained in each of the non-sour extracts prepared in Examples <1-2> and <1-3> was measured.

The total flavonoid content was determined according to the Folin-Denis method (Swain T & Hills WE 1959, J Sci Food Agric 10: 63-68). The phenolic constituents of Prunus domestica I-the quantitative analysis of phenolic constituents.

Briefly, 0.1 mL of Folin-Ciocalteus phenol reagent and 1.4 mL of distilled water were added to 0.2 mL of the sample, and then 0.3 mL of 20% sodium carbonate was added thereto. The mixture was allowed to stand in a dark place for 20 minutes, and then the absorbance was measured at 765 nm using a microplate spectrophotometer , Bioteck, VT, USA) were measured. As a standard substance, the content was indicated by using a routine.

As a result, as shown in Fig. 1 and Table 2, L2 (202.36 uM routine / 2 mg), L3 (232.7 uM routine / 2 mg), L6 (198.83 uM routine / 2 mg), L8 (248.5 uM routine / 2 mg), L10 (203.58 uM routine / 2 mg), and L24 (262.4 uM routine / 2 mg). On the other hand, L8 and L24 showed the highest total flavonoid contents under ultrasonic extraction conditions (p <0.05). The optimum extraction conditions of total flavonoid extracts were 60% ethanol solvent, 60% solvent extraction method than ultrasonic extraction method.

The total flavonoid content Extraction method Temperature
(° C) time
(hr) ethanol
(w / v%) Sample Name Total flavonoid content
(%) Ethanol solvent 25 24 60 L0 224.79 ultrasonic wave 25 One 0 L1 166.61 20 L2 202.36 40 L3 232.67 60 L4 204.48 2 0 L5 145.90 20 L6 198.83 40 L7 211.15 60 L8 248.53 3 0 L9 99.33 20 L10 203.58 40 L11 223.37 60 L12 180.85 6 0 L13 171.86 20 L14 162.57 40 L15 200.65 60 L16 218.42 9 0 L17 150.65 20 L18 212.87 40 L19 211.76 60 L20 219.54 12 0 L21 189.64 20 L22 110.65 40 L23 184.69 60 L24 262.36 24 0 L25 105.49 20 L26 189.43 40 L27 218.22 60 L28 217.62

< Experimental Example  2>

DPPH Radical Scavenging Activity of Anthracnose Extracts by Extraction Conditions

In this experiment, DPPH radical scavenging activity was examined to evaluate the antioxidative activity of each of the non-sour extracts prepared in Examples <1-2> and <1-3>.

Methods for measuring DPPH radical scavenging activity are described in Llorach et al. (2002) method. Briefly, the reducing power of each sample against the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical was measured. The DPPH solution was made up to 10 μM using 80% methanol. The absorbance was measured at 517 nm using a spectrophotometer (Agilent 8453, Agilent Technologies, CA, USA) after reacting 0.1 ml of the sample and 2.9 ml of the DPPH solution. Ascorbic acid was used as a standard for the comparison of the activity of the sample. In the control blank, 0.1 mL of 50% methanol was added instead of the sample. The sample blank was measured by adding 2.9 mL of 50% methanol instead of the DPPH solution.

DPPH radical scavenging activity (%) = (Abs _Blank- Abs _Sample / Abs _Blank ) 100

As a result, as shown in Fig. 2 and Table 3, L0 using the conventional ethanol 60% extraction method was 41.4%, and L2 (43.5%), L3 (59.1%), L4 (51.4% L15 (49.7%), L15 (49.5%), L16 (49.3%), L7 (48.3%), L8 (68.8% ), L20 (54.2%), L23 (47.6%), L24 (65.7%) and L28 (48.3%), respectively. DPPH radical scavenging activity was significantly higher in L8, L24 and IQ (isoquercetin) (p <0.05). The higher the ethanol content of the extraction solvent, the higher the antioxidant activity. The optimal extraction conditions of the antioxidant extracts were more efficient than the ethanol extraction method by using the ultrasonic extraction method.

The DPPH radical scavenging ability Extraction method Temperature
(° C) time
(hr) ethanol
(w / v%) Sample Name DPPH radical scavenging ability
(%) Ethanol solvent 25 24 60 L0 41.38 ultrasonic wave 25 One 0 L1 34.47 20 L2 43.54 40 L3 59.14 60 L4 51.45 2 0 L5 27.56 20 L6 49.68 40 L7 48.29 60 L8 68.77 3 0 L9 31.27 20 L10 45.53 40 L11 54.38 60 L12 48.25 6 0 L13 28.98 20 L14 45.66 40 L15 49.46 60 L16 49.20 9 0 L17 27.60 20 L18 39.05 40 L19 39.87 60 L20 54.21 12 0 L21 32.27 20 L22 29.03 40 L23 47.60 60 L24 65.66 24 0 L25 12.14 20 L26 28.47 40 L27 42.12 60 L28 48.34

< Experimental Example  3>

The inhibitory effect on the production of NO (nitrogen monoxide) by the extract condition

In this experiment, the inhibitory effect of NO (nitrogen monoxide) formation on the anti-inflammatory activity of each of the non-sour extracts prepared in Examples <1-2> and <1-3> was investigated.

<3-1> Cell culture

Mouse macrophage Raw 264.7 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin, 100 mg / mL streptomycin and Human Endothelial Growth Medium at 37 ° C in a 5% CO2 incubator . Cells were subcultured at 2-day intervals to maintain 70-80% confluency.

&Lt; 3-2 >

Raw 264.7 cells were adjusted to a cell number of 2 × 10 ⁷ cells / mL using 10% FBS DMEM medium and cultured in a 96-well plate. After 24 hours, the samples were treated for 1 hour before, and then cultured for another 24 hours in a medium containing LPS (1 μg / mL) and samples. The amount of NO produced was measured using the Griess reaction. 50 μl of the cell culture supernatant was mixed with 50 μl of Griess reagent [1% (w / v) sulfanilamide, 0.1% (w / v) naphtylethylenediamine in 2.5% (v / v) phosphoric acid] The absorbance was measured at 540 nm using a microplate reader and quantified as the value for the standard concentration curve obtained by stepwise dilution of sodium nitrite (NaNO ₂ ).

For reference, NO (nitrogen monoxide) is produced from L-arginine by NOS. When LPS (lipopolysaccharide), a cell wall component of gram-negative bacteria, binds to TLR (Toll like receptor) 4 of macrophages, it activates NF-κB transcription factor Thereby promoting protein expression of iNOS and eventually producing excess NO. It is known that NO produced is an active oxygen, which is involved in cytotoxicity and tissue damage, and plays an important role in inflammation induction. Therefore, a material inhibiting NO production may be useful as an anti-inflammatory functional material.

As a result, as shown in Fig. 3 and the following Table 4, the NO production amount was decreased in the extract of all conditions, as compared with the LPS group not containing the extract. In addition, extracts with similar inhibitory effect on NO formation were confirmed as compared with those treated with 20 μM of isoquercitrin, which is one of the representative functional materials contained in the non-sourdough. The highest inhibitory activity of L28 (24 h, 60% ethanol, ultrasonication) was found to be about 61% inhibitory.

Inhibition of NO production by extractive conditions Extraction method Temperature
(° C) time
(hr) ethanol
(w / v%) Sample Name Anti-inflammatory (NO production,% inhibition) Ethanol solvent 25 24 60 L0 54.89 ultrasonic wave 25 One 0 L1 37.34 20 L2 43.19 40 L3 45.42 60 L4 44.05 2 0 L5 39.23 20 L6 43.01 40 L7 46.46 60 L8 48.18 3 0 L9 40.43 20 L10 43.70 40 L11 43.53 60 L12 46.46 6 0 L13 36.65 20 L14 44.22 40 L15 49.72 60 L16 9.72 9 0 L17 38.20 20 L18 44.05 40 L19 51.27 60 L20 52.48 12 0 L21 38.89 20 L22 33.21 40 L23 41.47 60 L24 44.91 24 0 L25 38.54 20 L26 49.38 40 L27 53.85 60 L28 61.25

<Experimental Example 4>

Inhibitory effect of TNF-α on NF-κB promoter binding activity

In order to screen for the cardiovascular health-controlling efficacy of each of the non-sour extracts prepared in Examples <1-2> and <1-3> in this experiment, blood vessels of endothelial cells with increased oxidative stress by TNF- Was treated with an extract of Asisaceae having different extraction conditions to determine the inhibitory effect of NF-κB promoter binding activity.

Recently, interest in the inflammatory response in the blood vessels has been increasing as a cause of vascular disease. Inflammation is one of the in-vivo responses to various infectious agents such as external stimuli including tissue damage, and various immune cells undergo organic interactions. The induction of arteriosclerosis is caused by repeated physical or chemical stimulation of various risk factors and damage of vascular endothelial cells due to biological damage, and it is recognized that oxidative stress and inflammation play an important role in endothelial cell damage. Activated macrophages activate various nuclear cytokines and reactive oxygen species, activating nuclear factor-κB (NF-κB), which is a transcription factor of inflammatory response. As a result, inducible nitric oxide synthase (iNOS), cyclooxygenase- 2), resulting in inflammation. If this inflammatory reaction occurs continuously or excessively in the blood vessel, the initial response of atherosclerosis is activated.

Therefore, in this experiment, it was evaluated whether or not the extract of the present invention had cardiovascular health controlling effect through the inhibitory effect of NF-κB, which is a transcription factor of inflammatory reaction.

<4-1> Transformation and DNA extraction

MMP-1 plasmid DNA was incubated in competent cells at 42 ° C for 1 min. The reaction mixture was added to 0.4 ml of LB medium and incubated at 37 ° C for 1 hr with shaking at 250 rpm. The culture medium shaken in LB medium (ampicillin 100 μg / mL) prepared in advance was plated and cultured at 37 ° C. for 24 hours. The next day, 100 mL of liquid LB medium (ampicillin 100 μg / mL) was inoculated with single colony and cultured at 37 ° C. with shaking. When the cells were sufficiently grown, DNA was extracted using Plasmid midi kit (QIAGEN). DNA extraction was performed according to the test method (QIAZEN) provided by the reagent manufacturer.

<4-2> Transfection

293T cells were transfected with NF-κB-luciferase viral vector to obtain viral particles. 293T cells were plated in a 10 cm dish at 7 × 10 ⁵ cells / plate and cultured for 24 hours. After 24 hours of incubation, the cells were exchanged for antibiotic-free serum free medium for transfection, and 3 μg of each of MMP-1 plasmid DNA, psPAX2 and pMD2G DNA, 100 μl of Opti MEM mixed solution, 20 μl of lipofectamine and 100 μl of Opti MEM were prepared Respectively. The prepared DNA mixture and transfection reagent were mixed, vortexed, reacted at room temperature for 5 minutes, mixed with DNA-reagent mixture, and incubated for 24 hours.

<4-3> Infection

Human Umbilical Vein Endothelial Cell (HUVEC) was prepared to be 60% confluent. 5 mL of fresh medium and 5 mL of transfected cell medium were mixed and then filtered with 0.45 μM membrane filter. The prepared cell medium was removed, and the filtered medium containing 1 μl / mL of polybrene was added thereto, followed by incubation for 24 hours. Subsequently, selection was carried out until the uninfected cells were 100% killed by exchanging puromycin with 1 μg / mL of medium in control and infected cells. Nuclear factor-kappa B (NF-κB) luciferase-expressing HUVEC cell line was constructed by culturing the selected medium.

<4-4> Cardiovascular Health Regulation Screening

NF-κB luc-expressing HUVEC cells were counted in 96-well plates at 2 × 10 ⁴ cells / mL and cultured in a 5% CO ₂ , 37 ° C cell incubator for 24 hours. The cells were cultured for 24 hours in serum free medium. After 24 hours, the sample was treated with 100 μg / mL of Tumor Necrosis Factor-αlpha (TNF-α) for 1 hour. After 3 hours, the medium was removed and the cells were lysed with 100 μl of lysis buffer for 1 hour. 50 μl of luciferase reagent was added to 50 μl of the solution and measured using Spectramax (Molecular devices, Silicon Valley, CA, United States).

As a result, as shown in FIG. 4 and Table 5, when treated at a concentration of 100 μg / mL for each extraction condition, 64.8% in L0 (24 hours, 60% in ethanol, shaking), L20 (9 hours, 60% Ultrasound) showed the highest inhibitory effect (68.9%) and ultrasound was suppressed to similar level in shorter time. P3, which is isoquercitrin, was inhibited to 37.5%.

Inhibitory activity of NF-κB promoter binding activity of the ASSR Extraction method Temperature
(° C) time
(hr) ethanol
(w / v%) Sample Name Anti-atherosclerosis (NF-κB transactivity,% inhibition) Ethanol solvent 25 24 60 L0 64.86 ultrasonic wave 25 One 0 L1 61.00 20 L2 43.78 40 L3 55.01 60 L4 50.60 2 0 L5 53.68 20 L6 63.89 40 L7 38.44 60 L8 29.85 3 0 L9 29.64 20 L10 65.39 40 L11 61.00 60 L12 54.14 6 0 L13 56.93 20 L14 37.36 40 L15 42.24 60 L16 27.31 9 0 L17 23.32 20 L18 46.82 40 L19 36.73 60 L20 36.85 12 0 L21 52.73 20 L22 68.94 40 L23 45.21 60 L24 35.97 24 0 L25 68.01 20 L26 66.40 40 L27 -3.90 60 L28 54.29

< Experimental Example  5>

Anticancer efficacy of the extract of Asahiri extract according to extraction condition

In order to investigate the anticancer effects of each of the non-sour extracts prepared in Examples 1-2 and 1-3, human colon cancer cell lines and lung cancer cell lines were cultured in the same manner as in Example 1, After the treatment of the extracts, the degree of cancer cell death was measured.

In detail, human colon cancer cells (HT29, HCT116) and lung cancer cells (HCC827, HCC827 GR) were allowed to have a concentration of 4 x 10 ⁴ cells / mL and then cultured in a 96-well plate for 24 hours. The extracts were then treated at a concentration of 100 μg / mL and then cultured for another 24 hours. MTS ([3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- ) -2H-tetrazolium) and phenazine methosulfate (PMS) were added and the absorbance at 492 nm and 690 nm was measured after 1 hour. The killing effect of the extract on cancer cells was relative to 100% of the untreated group after subtracting the absorbance at 690 nm from the absorbance at 492 nm.

As a result, as shown in Table 6, it was confirmed that the extract of the present invention had an anticancer effect on lung cancer cells and colon cancer cells. In particular, the anticancer activity of L2, L6 and L10 was excellent overall, and the anticancer activity against HCC 827 cell which is a lung cancer cell line was compared with that of corn ethanol extract prepared by the conventional ethanol extraction method for 3 hours or 12 hours by ultrasonication (L9, L10, L11, L21, L22, and L23), and L14 (6 hours, ethanol 20%, ultrasonication) showed a high killing effect on HCT 116 cells as typical cancer-resistant colon cancer cells. %, Respectively. On the other hand, when 20 μM of isoquercitrin known as the main functional material of the non-sushi was treated, the survival rate of the cancer cells was suppressed to 14.2%, and it was confirmed that the non-sushi extract of the present invention showed more excellent anti-cancer activity.

Effect of Extract of Anthracnose on Cancer Cell Death by Different Extraction Conditions Extraction method Temperature
(° C) time
(hr) ethanol
(w / v%) Sample Name Cancer cell killing efficacy HCC 827 HCC 827 GR HCT 116 HT 29 Ethanol solvent 25 24 60 L0 1.13 18.14 29.42 25.27 ultrasonic wave 25 One 0 L1 -5.53 11.95 23.02 10.88 20 L2 4.20 15.59 29.49 13.03 40 L3 4.20 19.50 31.37 24.48 60 L4 -1.87 19.85 30.34 21.57 2 0 L5 -12.13 19.69 32.05 20.96 20 L6 3.93 12.11 26.73 14.21 40 L7 5.33 -28.24 27.91 24.08 60 L8 9.40 16.67 24.13 24.54 3 0 L9 11.33 20.46 30.79 23.93 20 L10 14.67 21.90 27.13 21.30 40 L11 10.67 19.34 13.49 20.84 60 L12 2.27 10.99 14.63 7.97 6 0 L13 2.40 14.04 31.34 12.24 20 L14 3.20 22.36 36.88 13.97 40 L15 4.33 23.44 36.11 24.69 60 L16 4.73 23.71 25.19 19.57 9 0 L17 4.20 16.02 24.88 33.57 20 L18 4.67 20.54 25.40 -2.57 40 L19 5.07 25.15 19.37 24.42 60 L20 6.80 26.89 22.90 32.93 12 0 L21 13.67 25.45 25.00 33.26 20 L22 14.20 20.35 26.18 27.20 40 L23 19.53 24.37 19.43 26.99 60 L24 5.87 14.78 15.50 -3.51 24 0 L25 7.13 13.04 33.26 15.33 20 L26 2.20 12.03 29.89 -23.1 40 L27 11.73 25.84 29.30 22.81 60 L28 8.40 16.94 21.13 22.78

< Experimental Example  6>

Analysis of Principal Component of Extracts by Extraction Conditions

(DPPH), total flavonoid content (TF), anti-inflammatory (AI), anti-photoaging (AP), anti-aging activity The results of principal component analysis (PCA) of anti-atherosclerosis (AA), anticancer activity (HCC827; H8, HCC827GR; H8G, HCT116; HCT116; 116, HT29; 29) .

As a result, the first principal component (PC1) and the second principal component (PC2) accounted for 34.93% and 16.19%, respectively, accounting for 51.12% of the total variation. In the ultrasonic extraction condition, the samples located in the negative direction of the first main component were mainly 3 hours, 9 hours, 12 hours and 24 hours, and the solvent concentrations were 20%, 40% and 60% ethanol extracts. It was confirmed that the yield and anticancer activity of L10 (ultrasound-3 hours-20% EtOH) and L27 (ultrasonic-24 hours -40% EtOH) L3 (ultrasound-1 hour-60% EtOH), L16 (ultrasound-6 hours), and 60% ethanol extracts were used for the samples located in the negative direction of the second main component. -60%). The antioxidant properties and anti-arteriosclerotic activity of the extracts were found to be high.

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 (7)

Characterized in that the non-sour ultrasound extract is obtained by ultrasonic treatment at a frequency of 20 to 30 kHz at 20 to 30 ° C for 1 to 24 hours using 20 to 60 w / v% ethanol. doing,
A pharmaceutical composition for preventing or treating lung cancer. Characterized in that the non-sour ultrasound extract is obtained by ultrasonic treatment at a frequency of 20 to 30 kHz at 20 to 30 ° C for 1 to 24 hours using 20 to 60 w / v% ethanol. doing,
A food composition for preventing or ameliorating lung cancer. 3. The method of claim 2,
Wherein said food is selected from the group consisting of beverage, meat, chocolates, foods, confectionery, pizza, ram noodles, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes and health supplement foods. Food composition for improvement. a) immersing the syrup in an aqueous solution of 20 to 60 w / v% ethanol and sonicating at a frequency of 20 to 30 kHz at 20 to 30 ° C for 1 to 24 hours; And
b) centrifuging the ultrasound treatment solution after the step a), and filtering the separated supernatant. 5. The method of claim 4,
In the step b), the ultrasonic treatment solution after the step a) is centrifuged at a temperature of 0 to 10 ° C at a speed of 3000 to 5000 rpm for 5 to 30 minutes, and the separated supernatant is filtered with a membrane having a pore size of 0.2 to 10 μm Wherein the method comprises the steps of: 6. The method of claim 5,
Further comprising the step of pulverizing the filtered extract after step b). &Lt; Desc / Clms Page number 20 &gt; An unsulfurized ultrasonic extract having lung cancer inhibitory activity according to any one of claims 4 to 6.

Images