KR101705547B1 - Composition for antioxidant or anticancer or antidiabete or antimicrobial containing balloon flower extract - Google Patents

Abstract

The present invention relates to a pharmaceutical composition having an antioxidant, anti-cancer, anti-diabetic or antimicrobial activity comprising an extract of Platycodon grandiflorum as an active ingredient. The composition according to the present invention increases DPPH, nitrite or ABTS cation scavenging activity, It has anticancer activity in breast cancer cells, rectal cancer cells, lung cancer cells, hepatic cancer cells and cervical cancer cells. It has high antitumor activity in glucoses, and it has antimicrobial activity against many bacteria of bronchial diseases. And can be usefully used as a pharmaceutical composition having an activity. In addition, there is no cytotoxicity, toxicity to the drug, and side effects are avoided, so that they can be used safely even when taken for a long period of time and have a stable effect in the body.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition having antioxidant, anticancer, antidiabetic or antimicrobial activity, which comprises a bellflower extract as an active ingredient.

The present invention relates to a composition for antioxidant, anti-cancer, anti-diabetic or antimicrobial activity, which comprises a bellflower extract as an active ingredient.

Bellflower is a perennial herb that belongs to the longevity field and is native to Korea, China, and Japan. It is a medicinal herb that has been used for respiratory diseases such as drainage, germosis, bronchitis and asthma. In addition, there are various pharmacologic actions such that the number of herbal medicine using this drug is 273 in Donguibogam and 49 in amalgamation. The main pharmacological component of the phloem is triterpenoid saponin These ingredients include anticholinergic action against chlamydia, genomic action, central nervous system inhibition (sedation, analgesic, antipyretic effect), anti-inflammatory action against acute chronic inflammation, anti-ulcer and gastric secretion inhibition, anticholinergic action to lower blood pressure by expanding blood vessels, Hypoglycemic effect, and cholesterol metabolism improvement action.

In addition, bellflower extract inhibits fungal aprotoxin. Inulin fraction has strong antitumor activity against phagocytosis and solid and multiple cancers, and 40% bellflower extract has an alcohol absorption inhibitory action.

On the other hand, since plants containing bioactive useful physiologically active substances, including bellflower, are the primary producers and become food for all animals including humans, it is necessary to study the improvement and the increase in production.

As a result, plant cell and tissue cultivation techniques have been developed, and plant regeneration has been fully enabled from callus, which is a undifferentiated cell mass, to enable effective utilization of plant resources. As an example of the research result, plant growth regulators are used to promote plant growth And the plants organs and organs are grown with special in-cabin environmental conditions, so that continuous plant products can be obtained in the cabin without being adversely influenced by the outside. In addition, studies are underway to increase plants by producing polyploids.

On the other hand, in the developed conventional method, a method of inducing tetraploid plants is used in the colchicine method, but conventionally, there is a disadvantage in that the organic efficiency of the tetraploid is low by obtaining the plant by treating the seed with colchicine solution.

In addition, plant propagation propagation under a special environment of a cabin requires selective nutrients suitable for various cabin environments, and there is a problem that a proper medium is required depending on the type and characteristics of plants to be treated.

Accordingly, in the present invention, efforts have been made to find a method for maximizing the physiologically active substance of Platycodon grandiflorum, and a pharmaceutical composition having antioxidant, anti-cancer, anti-diabetic or antimicrobial activity was prepared using the extract thus prepared.

Korean Patent No. 1259421

Accordingly, an object of the present invention is to provide a pharmaceutical composition having antioxidant, anti-cancer, anti-diabetic or antimicrobial activity, which comprises as an active ingredient, a bellflower extract having various pharmacological effects without adverse effects on living bodies.

It is also an object of the present invention to provide a health functional food having antioxidant, anti-cancer, anti-diabetic or antimicrobial activity which comprises a bellflower extract as an active ingredient.

In order to achieve the object of the present invention as described above, the present invention provides a pharmaceutical composition having antioxidant, anti-cancer, anti-diabetic or antimicrobial activity, which comprises Platycodon grandiflorum extract as an active ingredient.

In one embodiment of the present invention, the bellflower may be diploid or tetraploid.

In one embodiment of the present invention, the extract may be water, an organic solvent or a mixed solvent extract thereof.

In one embodiment of the present invention, the extract may be lyophilized, naturally dried, hot air dried or microwave dried.

In one embodiment of the present invention, the extract may be one that increases DPPH, nitrite or ABTS cation scavenging activity.

In one embodiment of the present invention, the composition may further comprise at least one additive selected from pale blue cabbage, corn oil and citron.

In one embodiment of the present invention, the extract may have rectal cancer or cervical cancer cell killing activity.

In one embodiment of the present invention, the extract is selected from the group consisting of Corynebacterium diphtheriae ) KCCM 40413, Klebsiella pneumoniae subsp . pneumoniae ) KCCM 41285, Streptococcus pyogenes KCCM 11817. In addition,

Further, the present invention provides a health functional food having antioxidant, anti-cancer, anti-diabetic or antimicrobial activity, which comprises a bellflower extract as an active ingredient.

The present invention relates to a pharmaceutical composition having an antioxidant, anti-cancer, anti-diabetic or antimicrobial activity comprising an extract of Platycodon grandiflorum as an active ingredient. The composition according to the present invention increases DPPH, nitrite or ABTS cation scavenging activity, It has anticancer activity in breast cancer cells, rectal cancer cells, lung cancer cells, hepatic cancer cells and cervical cancer cells. It has high antitumor activity in glucoses, and it has antimicrobial activity against many bacteria of bronchial diseases. And can be usefully used as a pharmaceutical composition having an activity. In addition, there is no cytotoxicity, toxicity to the drug, and side effects are avoided, so that they can be used safely even when taken for a long period of time and have a stable effect in the body.

FIG. 1 shows the results of measurement of SOD enzyme activity of the diploid and tetraploid strained extracts (FD: freeze drying, ID: natural drying, HD: hot air drying, MD: microwave drying).
FIG. 2 shows the results of measurement of CAT activity by the drying method of diploid and tetraploid strained extract (FD: freeze drying, ID: natural drying, HD: hot air drying, MD: microwave drying).
FIG. 3 shows the results of measurement of APX activity of the diploid and tetraploid strained extracts (FD: freeze drying, ID: natural drying, HD: hot air drying, MD: microwave drying).
FIG. 4 shows the result of measurement of POD activity of the diploid and tetraploid strained extracts (FD: freeze drying, ID: natural drying, HD: hot air drying, MD: microwave drying).
Figure 5 is a graph showing the effect of the Corynebacterium diphtheriae ) KCCM 40413 (A: diploid, B: tetrad).
FIG. 6 is a graph showing the activity of Klebsiella pneumoniae subsp . pneumoniae ) KCCM 41285 (A: diploid, B: tetrad).
Figure 7 is a graph showing the effect of Streptococcus pyogenes ) KCCM 11817 (A: diploid, B: tetrad).

The present invention is characterized by providing a pharmaceutical composition having antioxidant, anti-cancer, anti-diabetic or antimicrobial activity, which comprises Platycodon grandiflorum extract as an active ingredient.

'Doraji' is called Gyungyang in oriental medicine, but its quality is slightly warm and poisonous and its taste is spicy. In one room, Gyung-kyung refers to a disease of the bronchi and lungs of people who are not fully decayed or discarded. It is mainly due to saponin, which is the main component of the bellflower, and it can be used for the treatment of diabetes, anti-inflammation, anti-ulcer, promotion of corticosteroid secretion, airway mucus secretion , Saliva secretion and promote the action of the pharmacological effects are known to enhance nutritional power.

Therefore, the present inventors have found that the effect of the physiologically active substance is better in the quadriceps than in the diploid bellflower when trying to find a method of maximizing the physiologically active substance of the bellflower, .

According to one embodiment of the present invention, the bellflower extract is divided into a diploid and a tetraploid, and the method of freeze drying (FD), indoor drying (ID), hot air drying (HD), microwave drying (MD) , DPPH radical scavenging activity was higher than that of diploid bellflower, and the degree of DPPH radical scavenging activity was higher than that of diploid bellflower. The degree of DPPH scavenging activity was higher than that of freeze dried sample, (See Table 2).

According to one embodiment of the present invention, the bellflower extract is divided into a diploid and a tetraploid, and the method of freeze drying (FD), indoor drying (ID), hot air drying (HD), microwave drying (MD) The nitrite scavenging activity of Rhodophyllum japonica was measured at pH 1.2. Most of the extracts were able to decompose more than 60% of nitrite, % And 66.7%, respectively, which showed the highest nitrite scavenging activity. In diploid bellflower, it showed relatively higher elimination than quadruple bellflower, but not significant difference (see Table 4).

According to one embodiment of the present invention, the ABTS cation-scavenging activity of each of the tetraploids and tetraploids was found to increase proportionally with increasing concentration of the extract, It was found that the drying method had no significant effect on the change of the scavenging activity although the dredge showed relatively higher scavenging activity than the diploid doric (see Table 6).

According to one embodiment of the present invention, in order to investigate whether or not the platycodon grandiflorum extract has anticancer activity, MTT assay was applied to human cancer cells of various sites, and as a result, it was found to be more effective than tetrapod platycodon In MCF-7 cells and Calu-6 cells, HCT 116 cells and Hela cells, which are cervical cancer cells, showed relatively higher effects than other cancer cell lines. (See Table 12).

In addition, according to one embodiment of the present invention, in order to investigate whether or not an antidiabetic effect of Platycodon grandiflorum extract has been divided into a diploid and a tetrad, the endurance test according to the drying method was carried out. As a result, , And it was found that the resistance against frost-drying, hot-air drying and microwave drying was superior to that of natural drying (see Table 13).

Furthermore, the present inventors measured antimicrobial activity against three types of bronchial disease-causing bacteria in order to investigate whether or not they have antimicrobial activity on Platycodon grandiflorum extract. As a result, Quaternary Platycodon grandiflorum extract was relatively active compared to Diclofened Platycodon, In particular, Klebsiella pneumoniae subsp . pneumoniae ) (see Figs. 5 to 7).

Accordingly, the inventors of the present invention have found that the platycodon grandiflorum extract of the present invention can be used as a pharmaceutical composition having antioxidant, anti-cancer, anti-diabetic or antimicrobial activity.

The broodstock according to the present invention can be obtained by extracting and isolating from nature using an extraction and separation method known in the art, and the 'extract' defined in the present invention is extracted from bellflower using an appropriate solvent For example, hot water extracts of bellflower, polar solvent-soluble extracts, or non-polar solvent-soluble extracts.

As a suitable solvent for extracting the extract from the bellflower, any solvent acceptable in the art may be used, and water or an organic solvent may be used. Examples of the solvent include alcohols having 1 to 4 carbon atoms, acetone, ether, and the like, including purified water, methanol, ethanol, propanol, isopropanol, butanol, Various solvents such as benzene, chloroform, ethyl acetate, methylene chloride, hexane and cyclohexane may be used alone or in combination. But is not limited to.

As the extraction method, any one of the methods such as hot water extraction method, cold extraction method, reflux cooling extraction method, solvent extraction method, steam distillation method, ultrasonic extraction method, elution method and compression method can be selected and used. In addition, the desired extract may be further subjected to a conventional fractionation process or may be purified using a conventional purification method. There is no limitation on the production method of the Platycodon grandiflorum extract of the present invention, and any known method can be used.

For example, the Platycodon grandiflorum extract contained in the composition of the present invention can be prepared into a powdery state by an additional process such as vacuum distillation, freeze drying, spray drying, or the like, with the primary extract extracted by the hot water extraction or solvent extraction method described above . 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 bellflower extract is a concept including all extracts, fractions, and purified products obtained in each step of extraction, fractionation, or purification, and diluted solutions, concentrates, or dried products thereof.

The composition of the present invention containing such a bellflower extract as an active ingredient may be a pharmaceutical composition.

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 platycodon grandiflorum extract of the present invention may be contained at a concentration of 0.1 ug / ml to 500 ug / ml based on the total weight of the composition.

The composition of the present invention may also be a food composition. The food composition may contain, as an additional component, an active ingredient, a bellflower extract, as well as various flavors or natural carbohydrates, .

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 food or food. 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 food composition may contain flavoring agents such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and thickening agents (such as cheese and chocolate), pectic 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 drinks 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 bellflower extract of the present invention is a natural substance and has little toxicity and side effects. Therefore, it can be safely used for long-term administration for antioxidant, anti-cancer, anti-diabetic and antimicrobial effects.

The health functional food of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and circles for the purpose of antioxidation, anti-cancer, anti-diabetic and antimicrobial effects.

In the present invention, the term " health functional food " refers to foods manufactured and processed using raw materials or ingredients having useful functions in accordance with Law No. 6727 on Health Functional Foods. Or to obtain a beneficial effect in health use such as physiological 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 items listed in the above-mentioned "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 can be obtained by granulating a mixture obtained by mixing the active ingredient of the present invention, the bellflower extract, with an excipient, a binder, a disintegrant and other additives in a usual manner, Alternatively, 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-type health functional food can be prepared by filling a common hard capsule with a mixture of an effective ingredient of the present invention, such as a bellflower extract, with an additive such as an excipient, and the soft capsule is prepared by adding a bellflower extract as an excipient And filling the mixture with 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 a borage extract, which is an effective ingredient of the present invention, an excipient, a binder, a disintegrant, and the like, by a conventionally known method and, if necessary, Or the surface may be coated with a material such as starch, talc.

The granular health functional food may be prepared by granulating the mixture of the active ingredient of the present invention, the granule extract, the excipient, the binder and the disintegrant in a granular form by a known method. If necessary, a flavoring agent, ≪ / RTI >

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.

Hereinafter, the present invention will be further described by way of 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 bellflower extract

In order to prepare a bellflower extract, the present inventors frozen and dried the bellflower, and extracted three times at a ratio of 50%: 50% of distilled water and alcohol at a room temperature. The extract was concentrated under reduced pressure, . A lyophilized powder 100g n - using a solvent such as hexane (n -hexane), methylene chloride (Methylene chloride), AcOEt (Ethyl acetate), butyl alcohol (Butyl alcohol), distilled water (DW) were sequentially extracted continuously After fractionation, the extract was completely concentrated with a vacuum concentrator, and the recovery of each fraction was examined by measuring the weight.

As a result, it was found that both of the diploid and tetraploid donor showed the highest recovery rate in water extraction and the yield was extremely small in the ethyl acetate layer (see Table 1).

Solvent extraction yield of Platycodon grandiflorum extract Solvents Yield  (g) Diploid ( Diploid ) Tetraploid ( Tetraploid ) n - hexane 2.31 2.64 Methylene chloride 2.44 3.04 Ethyl acetate 0.26 0.30 Butyl alcohol 9.97 10.05 D.W. ( distilled water ) 83.03 80.12

< Example  2>

Bellflower extract DPPH Radical Scatters  Measure

<2-1> Depending on the drying method Diploid , Tetraploid  Bellflower DPPH  Measurement of scavenging activity

The present inventors measured the DPPH radical scavenging ability using the method of Choi et al. (2003) in order to investigate whether or not the borax extract prepared by the method of Example 1 had antioxidative effect.

First, samples of various concentrations were dissolved in a methanol solvent, and 900 μL of DPPH solution (100 μM) and 100 μL of each sample were mixed and stirred. After incubation in the dark for 30 minutes, the absorbance of the mixture was measured at 517 nm. The hydrogen electron donating ability was calculated by repeating each experiment three times, and the absorbance of the control was calculated by the following equation.

An = (A ₀ -A) / A ₀ 100

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

A ₀ : Absorbance of DPPH solution without added sample

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

Platycodon grandiflorum extract of the present invention was divided into diploid and tetraploid and treated by various methods such as freeze drying (FD), indoor drying (ID), hot air drying (HD), microwave drying (MD) , DPPH radical scavenging activity was higher in the tetraploid morcellation than in the diploid bellflower. The degree of DPPH radical scavenging activity was higher in the freeze dried samples than in the other drying methods. The increase in the DPPH scavenging activity was dependent on the concentration (See Table 2).

<2-2> Selection of materials for functional enhancement DPPH  Measurement of scavenging activity

The present inventors measured the DPPH scavenging activity of various plant materials in order to further prepare antioxidant-enhanced extracts by adding other plant materials in addition to the platycodon grandiflorum extract of the present invention.

As a result, DPPH scavenging activity was observed in various plant materials. However, DPPH scavenging activity was remarkably enhanced in the extracts of corn syrup and snakeskin, and this effect was increased in a concentration-dependent manner (see Table 3).

< Example  3>

Nitrite of bellflower extract Scatters  Measure

<3-1> Depending on the drying method Diploid , Tetraploid  Nitrite of bellflower Scatters  Measure

To measure the nitrite scavenging ability of Platycodon grandiflorum extract, 140 μl of a sample extract and 40 μl of 0.1 N HCl (pH 1.2) or 0.2 M citrate buffer (citrate buffer (pH 4.2, 6.0)) were added to 20 μl of 1 mM NaNO ₂ And the volume was adjusted to 200 μl. The reaction solution was reacted in a constant temperature water bath at 37 ° C for 1 hour, and then 1000 μl of 2% acetic acid and 1% of a Griess reagent (1% sulfanilic acid and 1% naphthylamine prepared by 30% acetic acid) , Prepared immediately before use), and the mixture was well mixed. The mixture was reacted at room temperature for 15 minutes with blocking of light, and the absorbance at 520 nm was measured to determine the nitrite scavenging ability as follows.

  N (%) = [1- (A-C) / B] 100

  N: nitrite scavenging ability

A: Add 1 mM nitrite to the sample after waiting 1 hour, and then add absorbance of 1 mM NaNO ₂ added after standing for 1 hour.

B: Absorbance of nitrite (absorbance of 1NaNO ₂ )

C: absorbance of control (control)

The Platycodon grandiflorum extract of the present invention was divided into diploid and tetraploid and treated with various methods such as freeze drying (FD), indoor drying (ID), hot air drying (HD), microwave drying (MD) When the reaction solution was pH 1.2, most of the extracts could decompose more than 60% of the nitrite. Among them, the highest degree of nitrite (69.8% and 66.7% Respectively. In diploid bellflower, it was relatively higher than quadruple bellflower, but it was not significantly different. At pH 4.2, most of the nitrite scavenging activity was more than 50%, but almost no activity was observed at pH 6.0.

Therefore, the nitrite scavenging activity of the extracts of each plant by drainage method and drying method was the highest at pH 1.2 and the activity gradually decreased or decreased with increasing pH.

<3-2> Nitrite of choice material for functional enhancement Scatters  Measure

The present inventors measured the nitrite scavenging ability by varying the pH concentration of various plant materials in order to further prepare an extract having improved antioxidative function by adding other plant materials in addition to the plant extract of the present invention.

As a result, most of the sample extracts could decompose more than 85% of the nitrite when the reaction solution was pH 1.2, and the nitrite scavenging activity was more than 10% at pH 4.2, but almost no activity at pH 6.0.

Therefore, the nitrite scavenging activity of the extracts was the highest at pH 1.2, and the activity was gradually decreased or lost as the pH was increased. The nitrite scavenging activity of the extracts of Sansui and Yeoju (See Table 5).

< Example  4>

Bellflower extract ABTS  Cation ( ABTS +) Measurement of scavenging activity

<4-1> Depending on the drying method Diploid , Tetraploid  Bellflower ABTS  Measurement of cation-scavenging activity

The present inventors measured ABTS cation scavenging activity of Platycodon grandiflorum extract by using 7.4 mM ABTS (2,2'-azinbis- (3-ethyl-benzothiazoline-6-sulfonic acid)

mM and potassium persulphate were mixed and reacted in a dark place for about 15 hours. Then, the mixture was diluted to a absorbance of 1.5 at 414 nm.

150 μl of each sample was added to 3 ml of the diluted solution. The sample was shaken for 10 seconds in a vortex mixer, left at room temperature for 90 minutes, and then absorbed at 414 nm. On the other hand, ascorbic acid was prepared at the same concentration as the sample and the absorbance was measured by the same method.

The cationic scavenging activity was expressed as AEAC (relative ascorbic acid equivalent antioxidant capacity), which indicates the ABTS cation elimination activity of the same concentration sample when the scavenging activity of ascorbic acid is 1.000.

RAEAC = Caa × △ As

              Aaa Cs

ΔAaa: Absorbance change when ascorbic acid is added, Caa: Concentration of ascorbic acid

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

The ABTS cation-scavenging activity of the tetraploid bellflower showed a tendency to increase proportionally with the increase of the concentration of the extract. In general, the tetraploid bellflower exhibited a relatively higher (See Table 6). However, it was found that the drying method had little effect on the change of the scavenging activity (see Table 6).

<4-2> Selection of materials for functional enhancement ABTS  Measurement of cation-scavenging activity

The present inventors measured the ABTS cation scavenging activity by varying the concentration of various plant materials in order to produce an extract having improved antioxidative function by adding other plant materials in addition to the plant extract of the present invention.

As a result, ABTS cation-scavenging activity was present in various plant materials, but ABTS cation-scavenging activity was particularly remarkable in corn oil, snakeskin and citrus (pea or leaf) extracts, and this effect was increased in a concentration-dependent manner (See Table 7).

The DPPH scavenging ability, nitrite scavenging ability and ABTS scavenging ability of the eight plants selected as the functional reinforcing materials were found to be higher than those of other plants. Therefore, these plants are suitable as the materials other than the bellflower.

< Example  5>

Determination of Antioxidant Enzyme Activity by Drying Method of Bellflower Extract

<5-1> SOD  ( SuperOxide Dismutase ) Enzyme activity measurement

To 0.5 g of the sample was added an extraction buffer [100 mM K-PO ₄ (pH 7.5), 100 mM EDTA, 1% PVP, 100 mM PMSF] and centrifuged at 15,000 g for 20 min. The supernatant was used for antioxidant activity assay. Ascorbate peroxidase (APX) was prepared by adding 10 mM of the above composition to an extraction buffer. Protein quantification was performed using Bradford (1976) method using BSA as a standard and SOD enzyme activity assay was 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 mixture 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 )

As a result, the highest activity was shown to be 90% in the freeze-dried and naturally dried quadriporadia, while the lowest activity was found in microwave dried dodecahontrate, which was the lowest in 47.5% (See Fig. 1).

<5-2> CAT ( Catalase ) Active measurement

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.

As a result, the activity of tetraploid bellflower was higher in diploids than in all other drying conditions. The drying method showed relatively high activity in freeze drying and natural drying (see FIG. 2).

<5-3> APX  ( Ascorbate Peroxidase ) Active measurement

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

As a result, although slightly higher activity was observed in lyophilized bellflower, there was no significant difference between drainage and drying method (see FIG. 3).

<5-4> POX  ( Peroxidase ) Active measurement

POX activity was measured by the method of Egley et al. (1983), and the reaction solution had 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 change at 470 nm for 2 minutes using a spectrophotometer by mixing the sample into the reaction solution.

As a result, both diploid and tetraploid gypsophila showed relatively high activity in freeze drying and natural drying, but did not show any significant difference between the palatability (see FIG. 4).

< Example  6>

Bellflower extract By solvent fraction  Antioxidant enzyme activity measurement

The SOD and CAT activities of the extracts were determined by ethyl acetate fraction and methylene chloride fraction, APX activity by Butyl alcohol fraction and water fraction, In the fraction, POD activity showed relatively high activity in methylene chloride fraction and water fraction. Overall, it was found that the quadriceps dandelion was more active than the dodecane bellflower (see Tables 8-11).

< Example  7>

Analysis of Anticancer Activity of Platycodon grandiflorum Extract

The present inventors prepared tetraploid and tetraploid Dorrhiza to investigate the anticancer activity of Platycodon grandiflorum extract and analyzed MTT (3- (4,5-dimethyl-thiazol-2-yl) -2,5- diphenyltetrazoliumbromide) Respectively.

Seven types of cells, including SNU-601, SNU-1066, MCF-7 breast cancer cell, HCT-116 rectal cancer cell, Calu-6 lung cancer cell, Hep 3B hepatocarcinoma cell, Hela cell cervical cancer cell, Well plate, and cultured in a 5% CO ₂ incubator at 37 ° C for 24 hours.

The cultured cells were treated with 10 μl of each sample to a concentration of 125, 250, 500, 1000, 2000, and 4000 μg / ml, and cultured for 24 hours. 10 μl of 0.2 mg / ml MTT solution was added to each well, For 4 hours. After the incubation, the supernatant was removed, and 100 μl of DMSO was added to each well. The mixture was reacted at room temperature for 10 minutes, and the absorbance was measured at 540 nm using a microplate reader. The cytotoxicity was measured by the absorbance of the sample As a percentage.

The cytotoxicity of each sample extract by MTT method to human cancer cell lines was found to be better than that of diploid bellflower in tetrads. In particular, HCT 116 cells and Hela Cells, which are rectal cancer cells and Hela Cells, showed relatively high toxicity, while MCF-7 and Calu-6 Cells showed slightly lower toxicity than the other cancer cell lines (See Table 12).

< Example  8>

Bellflower extract Antidiabetic activity  analysis

In addition, the present inventors conducted an endurance test to see whether or not the bellflower extract had an anti-diabetic effect.

The glucose tolerance test was carried out by measuring the glucose content. The inventors used the BCS glucoose kit to analyze the effect of each sample extract on the glucoside content. 10 μl of each plant extract was added to 1.5 ml of the enzyme solution dissolved in the buffer solution, mixed thoroughly, heated at 37 ° C for 5 minutes, and absorbance was measured at a wavelength of 505 nm within 40 minutes by blank control. And then the glucose content was measured. At this time, 20 ml of the standard solution (glucose 200 mg / dl) was added to 1.5 ml of the enzyme solution as a standard solution.

Glucose content (mg / dl) = (OD _sample / OD _standard ) 200 mg / dl

As a result of the endurance test by the above method, the glucose tolerance in the platycodon was somewhat good but there was no significant difference, and it was confirmed that the platycodon was excellent in endurance. In particular, it was found that the resistance to frost was superior in freeze drying, hot air drying and microwave drying than in natural drying (see Table 13).

< Example  9>

Analysis of Antimicrobial Activity of Platycodon grandiflorum Extract

The present inventors further investigated the antimicrobial activity of Platycodon grandiflorum extract by using a diploid and tetraploid platycodextrose extract as a sample and using a liquid culture medium for bronchial disease (1% meat extract, 1% tryptone, 0.5% NaCl, 0.2% yeast extract 0.3%, pepton 0.1%, pH 7.3). Bacterial culture medium was modified with blood agar base (Difco 0045). Antimicrobial activity assay was performed by disk agar plate diffusion method.

That is, a soft agar in which test microorganisms were cultured at a concentration of 10 ⁷ CFU / mL in a medium suitable for each test microorganism was dispensed into a petri dish, and a paper disc was placed on the petri dish. 40 μL of the sample extract was dispensed And the formation of inhibitory rings was observed. When the inhibitory ring was generated within 24 hours, the antimicrobial activity was judged to be positive, and the diameter of the inhibitory ring was measured and compared. Table 14 below shows the three kinds of strains and the culture conditions used in the platycodon microbial activity test of the present invention.

As a result of antimicrobial activity against three kinds of bronchial disease - causing bacteria, the tetraploid phytophagous extract was relatively more active than the diploid bellflower, and Klebsiella pneumoniae subsp. pneumoniae ) (see Figs. 5 to 7).

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 (9)

Wherein the extract comprises at least one selected from the group consisting of Corynebacterium diphtheriae KCCM 40413, Klebsiella spp., Klebsiella sp. A pharmaceutical composition for antibiosis having antimicrobial activity inhibiting at least one strain selected from the group consisting of Klebsiella pneumoniae subsp. Pneumoniae KCCM 41285 and Streptococcus pyogenes KCCM 11817. delete The method according to claim 1,
Wherein the extract is water, an organic solvent, or a mixed solvent thereof. delete delete delete delete delete Wherein the extract comprises at least one selected from the group consisting of Corynebacterium diphtheriae KCCM 40413, Klebsiella spp., Klebsiella sp. A health functional food having antimicrobial activity inhibiting at least one strain selected from the group consisting of Klebsiella pneumoniae subsp. Pneumoniae KCCM 41285 and Streptococcus pyogenes KCCM 11817.

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