KR100768084B1 - Euphorbia jolkini extracts having physiological activity - Google Patents

Abstract

A composition comprising an extract of Euphorbia jolkini having excellent physiological activity is provided to secure excellent anti-oxidizing, anti-inflammatory and antibacterial activities. A composition comprises an extract of Euphorbia jolkini. The extract is obtained by crushing Euphorbia jolkini dried in the shade using a mill, precipitating the obtained fine powder in ethanol and extracting it using ultrasonic wave three times per one hour, concentrating recovered supernatant of the extract under reduced pressure, suspending the concentrate in water, and extracting the suspension with hexane, dichloromethane, ethylacetate and butanol in sequence.

Description

[0001] The present invention relates to an EUPHORBIA JOLKINI EXTRACTS HAVING PHYSIOLOGICAL ACTIVITY

1 is a graph showing the content of polyphenol compounds in the cancer antiproto extract of the present invention

2 is a graph showing the antioxidant activity of the cancer antiproto extract of the present invention

A: DPPH radical scavenging activity, B: Xanthine oxidase inhibitory activity

C: Peroxide scavenging activity

Fig. 3 is a graph showing the anti-inflammatory activity of the cancer antiproto extract of the present invention

Fig. 4 is a photograph showing the antimicrobial activity of Gram positive bacilli of the cancer antiproduct extract of the present invention

A: Staphylococcus aureus , B: Bacillus cereus

1: hexane fraction, 2: dichloromethane fraction,

3: ethyl acetate fraction 4: butanol fraction

5: Water fraction, C: Control group, E: Ethanol extract

5 is a photograph showing the antimicrobial activity of Gram negative bacteria of the cancer antiproto extract of the present invention

A: Pseudomonas aeruginsosa , B: Vibrio parahaemolyticus

1: hexane fraction, 2: dichloromethane fraction,

3: ethyl acetate fraction 4: butanol fraction

5: Water fraction, C: Control group, E: Ethanol extract

FIG. 6 is a graph showing changes in the concentration of Bacillus sp. cereus strain Growth curve

A: ethanol extract, B: hexane fraction, C: dichloromethane fraction

D: ethyl acetate fraction, E: butanol fraction, F: water fraction

FIG. 7 is a graph showing the effect of the concentration of Listeria monocytogenes Growth curve

A: ethanol extract, B: hexane fraction, C: dichloromethane fraction

D: ethyl acetate fraction, E: butanol fraction, F: water fraction

FIG. 8 is a graph showing the concentration of Staphylococcus aureus strain Growth curve

A: ethanol extract, B: hexane fraction, C: dichloromethane fraction

D: ethyl acetate fraction, E: butanol fraction, F: water fraction

Fig. 9 is a graph showing the effect of the concentration of Escherichia < RTI ID = 0.0 > coli Growth curve

A: ethanol extract, B: hexane fraction, C: dichloromethane fraction

D: ethyl acetate fraction, E: butanol fraction, F: water fraction

The present invention relates to a cancer heavy pole extract having physiological activity.

Recently, there has been a growing interest in physiologically active substances, which are secondary metabolites of natural plants, by extracting specific components from medicinal plants.

Among the secondary metabolites of natural plants, the phenolic compounds in plants have a phenolic hydroxyl group, so they have a property of binding to macromolecules such as proteins and have antioxidant activity, antimicrobial activity and oxidative (1997), which has been reported to be effective in the prevention of diseases such as heart disease and cancer. (1976). The flavonoids, a class of semiessential food components, and their role in human nutrition World Rev. Nutr. Diet., 24. 117-120).

Meanwhile, the Euphorbia Recently, it has been reported that Chinese pekinensis has been applied to the treatment of psychiatric diseases such as schizophrenia in China. However, almost no studies have been reported on the ingredients, but most studies on euphane triterpenoids triterpenoid, euphol, and the like.

Korean Patent Laid-Open Publication No. 1998-027092 (a composition for skin whitening containing a plant extract in a major pole) contains a compound selected from the group consisting of 1, 2, 3, 4 and 5 selected from the group consisting of consumed candles, recited poles, cancer pollen, The present invention relates to a composition exhibiting a skin whitening effect, which is characterized by containing an extract from a plant belonging to the genus Major.

However, there is no active research on cancer antagonism, and research on the pharmacological activity of cancer antagonists is still lacking.

An object of the present invention is to provide a cancer antipark extract having excellent antioxidant activity, anti-inflammatory activity and antibacterial activity.

It is also an object of the present invention to provide a composition for prevention and improvement of diseases, which comprises a cancer polymolecular extract having excellent physiological activity as an active ingredient.

The present invention relates to a cancer heavy pole extract having physiological activity.

Euphorbia jolkini of the present invention is shaken and pulverized into fine powder, which is then immersed in 80% ethanol, and the ethanol precipitate is extracted three times for 1 hour each time by using ultrasonic waves. Then, The solution is recovered and concentrated under reduced pressure. The concentrate is suspended in water, and the suspension is successively extracted with hexane, dichloromethane, ethyl acetate and butanol to prepare a cancer antipyretic extract.

Euphorbia jolkini , a main material of the present invention, is a perennial herbaceous plant in the main pole and is native to rocky areas of Jeju and southern provinces. It is used for ornamental and medicinal purposes. It is a toxic plant used as a medicinal agent for poisoning, bloating, diuretic, sweating, edema, swine, toothache, diabetes, gonorrhea and the like.

The present invention aims at preparing an extract having a large physiological activity using a cancer counter electrode and finding a possibility as a composition for prevention and improvement of a disease using the cancer repellent extract having the physiological activity as an active ingredient.

As a result of analyzing the polyphenol content of the cancer antiprotoin extract of the present invention by fractionating the cancer counterpart with four solvents having different polarities (ethanol, hexane, dichloromethane, ethyl acetate, and butanol), the ethanol extract and all fractions (Fig. 1). The highest content was found in the ethyl acetate fraction (Fig. 1).

This is inferred that the cancer polymorphic extract of the present invention contains many phenolic compounds having various physiological activities.

In addition, the present invention measured the antioxidative activity of DPPH radical scavenging activity, xanthine oxidase inhibiting activity, and peroxide radical scavenging activity of the cancer antagonist extract.

As a result, the anti-oxidant activity of the cancer polymorphic extract of the present invention was found to be high, and it was confirmed that the ethyl acetate fraction showed the highest antioxidative activity (Table 1, Fig. 2).

In addition, LDH cytotoxicity and inhibition of NO production were measured.

As a result, it was confirmed that the cancer antiproduct extract of the present invention had an excellent anti-inflammatory activity, and the production of NO was remarkably inhibited especially in hexane, dichloromethane and ethyl acetate fractions. In the ethyl acetate fraction, (Fig. 3).

These results suggest that the inhibitory effect of NO in the ethyl acetate fraction is due to the toxic effect.

In order to search for antimicrobial activity against Gram-positive bacteria and Gram-negative bacteria in food harmful microorganisms of the present invention, the degree of antimicrobial activity was measured by measuring the size of the growth inhibition circle, and the degree of inhibition of microbial growth and microorganisms And the minimum inhibitory concentration was measured.

As a result, as the concentration of the cancer polymorphous extract of the present invention was increased, the size of growth inhibitory rings exhibiting antimicrobial activity was increased (Table 3, Table 4). As a result of measuring the minimum concentration inhibiting the growth of food harmful microorganisms, Ethanol extract, ethyl acetate extract and butanol fraction showed inhibitory effect on growth at 100 ppm concentration, and 250 ppm of water fraction and 500 ppm of dichloromethane fraction showed the minimum growth inhibitory effect (Table 5).

Therefore, it was confirmed that the anticancer activity of the present invention was superior to the anticancer activity of the present invention, and the ethyl acetate fraction and the butanol fraction showed higher antimicrobial activity (FIGS. 4 to 9).

On the other hand, the cancer polymolecular extract having the physiological activity of the present invention can be utilized variously as a food additive, a feed additive, a beverage composition, and the like, and can be utilized as a composition for prevention and improvement of various diseases.

In addition, a composition having antioxidant activity, anti-inflammatory activity and antimicrobial activity, which comprises the cancer polymorph extract of the present invention as an active ingredient, can be prepared.

Hereinafter, the cancer antipyretic extract having the physiological activity of the present invention will be described in detail with reference to Examples and Experimental Examples, but the scope of the present invention is not limited thereto.

≪ Example 1 > Preparation of cancer antiproton extract of the present invention

Euphorbia jolkini , which is growing on the coast of Jeju Island, was collected around April 2006 Prepared.

The prepared cancer counterparts were shredded and crushed by a crusher. The cancer counterpart fine particles were immersed in 80% ethanol and extracted three times for 1 hour using ultrasonic waves.

Thereafter, the supernatant was recovered and concentrated under reduced pressure to obtain an ethanol extract.

The concentrated ethanol extract was suspended in water and extracted successively with hexane (1 L x 3), dichloromethane (1 L x 3), ethyl acetate (1 L x 3) and butanol (1 L x 3) .

<Experimental Example 1> Analysis of polyphenol content of the cancer antiproto extract of the present invention

Phenolic materials are one of the secondary metabolites widely distributed in plants and have various structures and molecular weights.

Because they have a phenolic hydroxyl group, they have a property of binding to proteins and other macromolecules, and they also have a physiological activity function such as an antioxidant effect.

The total polyphenol content of the cancer pollen extract of the present invention was measured and compared using a standard curve of tannic acid.

The content of the polyphenol compound was measured by slightly modifying the Folin-Denis method (Gutfinger, T .: Polyphenols in olive olis, J. Am. Oil Chem. Soc., 58, 966-968 (1981).

After 0.2 ml of Folin-ciocalteu's phenol reagent (Sigma) was added thereto, the mixture was well mixed, and then the mixture was incubated at room temperature for 3 minutes .

After 3 minutes, 0.4 ml of 2 M Na ₂ CO ₃ solution was added and mixed. Distilled water was added to make 4 ml, and the solution was allowed to stand at room temperature for 1 hour. Absorbance of the supernatant was measured at 725 nm.

A standard curve using tannic acid (Sigma) was prepared by dissolving 1 mg of tannic acid in 1 ml of a 50% methanol solution, taking the final concentration of the solution to 0, 32.5, 75, 125, 250 and 500 占 퐂 / nm and absorbance was measured.

As a result, total polyphenol contents were 16.20% in ethanol extract, 1.26% in nucleic acid fraction, 4.81% in dichloromethane fraction, 54.20% in ethyl acetate fraction, 17.64% in butanol fraction and 3.0% in residue fraction The fraction with the highest polyphenol content tended to be 3.4 times higher than the crude extract in ethyl acetate fraction (Fig. 1).

As a result, it was found that the cancer antipole extract of the present invention contains a large number of phenolic compounds.

<Experimental Example 2> Antioxidant activity test for the cancer antiproto extract of the present invention

The cancer pole extract prepared in Example 1 of the present invention was prepared and used in the experiment.

As a control group, ascorbic acid, butylated hydroxy anisole (BHA), and trolox were used.

1. Detection of antioxidant activity by DPPH radical scavenging activity

The most characteristic mechanism of antioxidants is to react with free radicals, and the free radical scavenging function is used as a measure to inhibit the antioxidant effect and the aging in the human body by donating electrons to free radicals.

DPPH is a stable free radical and is reduced by sulfur amino acids such as cysteine, glutathione, ascorbic acid, aromatic amine (ρ-phenylenediamine, ρ-aminophenol) (Blois, MS 1958. Antioxidant determination by the use of a stable free radical, Nature 26: 1199-1200).

Through the DPPH radical scavenging activity experiment, it is possible that the separation of active substances that inhibit the toxic action of active oxygen in the human body may be an antioxidant.

Antioxidants are radical scavengers that inhibit rancidity by destroying non-radical compounds by acting as a donor of hydrogen or an electron to peroxy radicals of various free radicals or oils, and other functional scavengers Removing agent, releasing peroxide and ascending agent.

Synthetic antioxidants such as butylated hydroxy toluene (BHA) and butylated hydroxy anisole (BHT), which have been widely used for decades, have excellent antioxidant power. However, due to their safety concerns, (1996), Antioxidative effects of extracts from ganoderma, Korean Journal of Food Science and Technology, 28: 1157-1163).

The electron donating ability was determined according to the DPPH free radical scavenging method by the Blosis method (Blois, M. S. 1958. Antioxidant determination by the use of a stable free radical., Nature 26: 1199-1200).

The concentration of each sample dissolved in methanol was dispensed into a 96-well plate in an amount of 100 μl. An equal amount of 0.4 mM DPPH solution was added thereto, and the mixture was allowed to stand at room temperature for 10 minutes and absorbance was measured at 517 nm.

The DPPH radical scavenging activity was calculated from the following equation and expressed as the concentration of the sample (IC ₅₀ ) when the absorbance of DPPH decreased by 50%. Each sample was repeated three times and the average value was obtained.

DPPH radical scavenging activity (%) = (A _control - A _sample ) / A _control × 100

A _sample = Absorbance of the reaction solution to which the sample is added

A _control = Absorbance of the reaction solution to which methanol was added instead of the sample

DPPH (1,1-diphenyl-2-picryl-hydrazyl), the antioxidative activity of the cancer antipark extract of the present invention was found to be significantly higher than that of other solvent fractions in crude extract, ethyl acetate and butanol fractions Radical scavenging activity (Fig. 2).

Showed the highest of the radical scavenging activity in the ethyl acetate fraction, IC ₅₀ values of the ethyl acetate fraction of the DPPH free-radical scavenging activity indicated the highest was found to be 8.38 ㎍ / ㎖ (Table 1).

This value can be regarded as an activity which is not inferior to ascorbic acid, butylated anisole (BHA), and trolox used as a control group.

2. xanthine oxidase inhibitory activity experiment

Xanthine oxidase is produced from xanthine dehydrogenase in an oxidative environment.

Xanthine oxidase oxidizes hypoxanthine to ultimately produce uric acid and oxygen, and oxygen free radicals and hydrogen peroxide are generated from this oxygen.

Since accumulation of uric acid is known to cause hyperuricemia and gout, inhibitors of uric acid formation may be useful as therapeutic agents for these diseases.

In addition, the generation of oxygen free radicals generated by xanthine oxidase in are known to cause damage to the cells (Cheng, ZJ, SC Kuo, SC Chan, Ko FN, and Teng CM. 1998. Antioxidant properties of butein isolated from Dalbergia odorifera. Biochim . Biophys Acta, 1392:. 291-299 ).

Thus, substances capable of cleaving the free radicals of the oxygen free radicals will also be useful for preventing oxidative damage.

Gout occurs when the uric acid level rises, causing them to become adhered to the joints as crystals, causing inflammation and, in severe cases, complications such as kidney and heart.

Xanthine oxidase inhibitors have been used for treating urethral acidosis causing gout, kidney disease, ischemia, and cardiomyopathy. Allopurinol and alloxanthine are known to be used in gout treatment. have.

Thus, in this experiment, the xanthine oxidase inhibitory activity of the cancer polymorphic extract was examined.

Uric acid production by xanthine / xanthine oxidase was measured by increased absorbance at 290 nm.

The reaction solution was prepared in 100 μl of 200 mM phosphate buffer (pH 7.5) containing 0.5 mM xanthine and 1 mM EDTA at various concentrations of each sample, and the production of uric acid was performed by adding 50 mU / ml xanthine oxidase Respectively.

The xanthine oxidase inhibitory activity is expressed as the concentration of the sample (IC ₅₀ ) when the absorbance of the resulting uric acid is reduced by 50%.

As a result of the inhibition of xanthine oxidase activity by the concentration of the cancer polymorphic extract of the present invention, the highest inhibition of xanthine oxidase activity was shown in the ethyl acetate fraction (FIG. 2).

The IC ₅₀ value of the ethyl acetate fraction was 466.01 ㎍ / ㎖ (Table 1).

3. Superoxide scavenging activity experiment

About 0.4 to 4% of the total oxygen consumed during the course of normal oxidative phosphorylation is converted to free radical peroxides and the resulting peroxides are converted to other reactive oxygen species (ROS), either directly or indirectly It is known to cause cell damage.

Normally, peroxides are rapidly converted to hydrogen peroxide by superoxide dismutase (SOD) by the endogenous antioxidant defense mechanism.

However, if this endogenous antioxidant defense system fails to maintain the intracellular oxidation-reduction balance, oxidative stress will result, and this oxidative stress will play an important role in directly damaging intracellular macromolecules or causing cell damage .

Thus, in this experiment, peroxide scavenging activity of the cancer pollen extract was examined.

The amount of superoxide was measured by NBT (nitroblue tetrazolium) reduction method.

Superoxide scavenging activity was obtained by adding 0.5 mM NBT to the reaction solution.

The peroxide scavenging activity is expressed as the concentration of the sample (IC ₅₀ ) when the absorbance of the peroxide is reduced by 50%.

The results of superoxide radical scavenging activity are shown in Fig.

The ethyl acetate fraction and the butanol fraction showed the highest peroxidative radical scavenging activity and the IC ₅₀ value of the ethyl acetate fraction showing the highest peroxide radical scavenging activity was 11.39 ㎍ / ㎖ (Table 1).

Their activity showed a pattern similar to that of DPPH radical scavenging activity.

<Table 1> Antioxidant test results of the cancer antiproto extract of the present invention

process IC ₅₀ ([mu] g / ml) ^a) DPPH radical scavenging activity Xanthine oxidase inhibitory activity Peroxide radical scavenging activity Ethanol extract 22.22 ± 1.37 > 1000 26.11 + 1.41 Hexane fraction 266.83 + - 2.43 > 1000 > 1000 Dichloromethane fraction 71.93 + - 1.85 > 1000 117.48 ± 2.07 Ethyl acetate fraction 8.38 ± 0.92 466.01 + - 2.67 11.39 ± 1.06 Butanol fraction 12.90 ± 1.11 > 1000 19.701 + - 1.29 Water fraction 53.47 ± 1.72 > 1000 93.73 + 1.97 BHA ^{b )} 22.70 ± 0.61 NA ^{c )} NA ^{c )} Ascorbic acid 3.90 + - 3.22 - - Trolox 8.62 ± 2.20 288.60 ± 4.4 189.9 ± 2.03 Allopurinol NA ^{c )} 3.12 ± 0.17 22.65 + - 0.35

* a) The IC ₅₀ value is the concentration of the sample when the absorbance is reduced by 50%.

* b) BHA: Butylated hydroxy anisole

* c) NA: Not applicable

<Experimental Example 3> Anti-inflammatory activity test of the cancer antipode extract of the present invention

LPS, known as endotoxin, is present in the extracellular membrane of Gram-negative bacteria and is known to increase pro-inflammatory cytokines such as TNF-α, IL-6 and IL-1β in macrophages or monocytes such as RAW264.7.

In addition, the formation of such an inflammatory mediator leads to a process of converting arachidonic acid into prostagladin and a process of forming nitrogen oxide (NO) due to the activity of phospholipase A2.

Nitric oxide (NO) is an inorganic vitreous substance produced from L-arginine by nitric oxide synthase (NOS) and is known to be involved in various biological processes such as immune response, cytotoxicity, neurotransmitter system and vascular relaxation Depending on the concentration, it may play an important role in cell function maintenance and cytotoxicity.

The NOS that produces NO is a non-naturally occurring constitutive NOS (cNOS) that is present in cells and is dependent on calcium or calmodulin, and activates macrophages or vascular endothelial cells independent of calcium, (inducible NOS, iNOS), a form induced by various cytokines and endotoxins of bacteria such as lipopolysaccharide (LPS).

INOS expressed by LPS stimulation produces a large amount of NO, which is known to be involved in inflammatory reactions, cell mutations and tumorigenesis. It has been reported that the production of NO and iNOS is increased in tissue damage associated with inflammatory reaction.

Therefore, the present study confirmed the inhibitory effect of LPS stimulation from the murine macrophage cell line (RAW264.7) and the degree of toxicity thereof.

1. Cell culture

Mouse macrophage RAW264.7 cells were purchased from the Korean Cell Line Bank and contained 100 units / ml penicillin-streptomycin and 10% fetal bovine serum (FBS) DMEM medium at 37 ° C in a 5% CO ₂ incubator, and subculture was performed every 3 to 4 days.

Lipopolysaccharide (LPS, E. coli serotype 0111: B4) was purchased from Sigma and used for the experiments.

2. LDH cytotoxicity detection

Lactate dehydrogenase (LDH) is a stable cytoplasmic enzyme present in most cells. When the plasma membrane is damaged, it is released into cell culture fluids.

Thus, it is a simple and simple colorimetric assay that measures LDH activity released by damaged cells.

In this experiment, RAW264.7 cells were added to each well of a 48-well plate at a concentration of 1.0 × 10 ⁵ cells / ml, and cultured for 24 hours. After the cell culture was completed, the LDH efflux was measured Were investigated.

Activity was measured at 492 nm using the LDH cytotoxicity detection kit (Promega).

3. Detection of NO production inhibition rate

RAW264.7 cells were adjusted to 1.0 × 10 ⁵ cells / ㎖ using DMEM medium supplemented with 10% FBS, and inoculated into 48 well plates. LPS (1 ㎍ / ㎖) Lt; / RTI &gt;

The amount of NO produced was measured in the form of NO ₂ ^- present in cell culture medium using Griess reagent.

100 μl of the cell culture supernatant was mixed with 100 μl of Griess reagent [1% (w / v) sulfanilamide, 0.1% (w / v) naphylethylenediamine in 2.5% (v / v) phosphoric acid] And the absorbance was measured at 530 nm.

The amount of NO produced was compared with sodium nitrite (NaNO ₂ ) as standard.

As a result, it was confirmed that NO was produced excessively in the LPS alone treatment group, and NO production was decreased in the test group treated with the cancer antiproduct extract. Especially, the amount of NO produced in the hexane, dichloromethane and ethyl acetate fractions (Fig. 3).

In addition, the ethanol extract of cancer antagonist showed little cytotoxicity in RAW264.7, but the cytotoxicity of ethyl acetate fraction was strong.

As a result, the inhibitory effect of NO on the ethyl acetate fraction was considered to be due to the toxic effect, and the butanol fraction was somewhat toxic (FIG. 3).

&Lt; Experimental Example 4 > Antibacterial activity of the cancer antiproto extract of the present invention

1. Preparation of used strain and medium

A total of 10 strains were used, including five gram-positive bacteria and five gram-negative bacteria, which are food-harmful microorganisms (Table 2).

Tryptic soybean agar (TSA), Tryptic soybean broth (TSB) and Brain heart infusion were purchased from Difco (USA) (Table 2).

<Table 2> List of strain and medium for antibacterial activity test

Strain name badge Temperature (℃)   Gram positive bacteria Bacillus cereus TSA / TSB 30 Listeria monocytogenes BHI 37 Staphylococcus aureus TSA / TSB 37 Staphylococcus epidermidis TSA / TSB 37 Streptococcus faecalis BHI 37   Gram-negative bacteria Escherichia coli TSA / TSB 37 Pseudomonas aeruginosa TSA / TSB 37 Salmonella enteritidis TSA / TSB 37 Salmonella  trphimurium TSA / TSB 37 Vibrio parahaemolyticus TSA / TSB + 3% NaCl 30

2. Antimicrobial activity measurement

In order to search for the antibacterial substance of the cancer antiproto extract of the present invention, in this experiment, a paper disk method (JH Bae. Effect of Artemisia capillaris extract on the growth of food-borne pathogens. Kor J Food Sci Technol. 36 (2): 147-153, 2003).

Bacteria cultured for 18 hours at the optimal culture temperature (Table 2) were analyzed with a spectrophotometer (Ultrospec 2100 pro, Amersham Biosciences, USA) using 10 ml of broth, , The absorbance was adjusted to an optical density value of 0.4 (620 nm), and the mixture was uniformly mixed in a petri dish in which the medium was divided according to a pour-plate, and then solidified at room temperature.

A sterilized paper disc was placed on the culture medium, and each sample was absorbed at 1000 ppm.

The control group was sprayed with 80% ethanol in the same manner as the experimental group.

After 24 hours of incubation at the optimal temperature condition, the size of the clear zone (mm) produced around the dest was measured and the degree of antimicrobial activity was compared and analyzed.

As a result, the antimicrobial activity of the ethanol extract and the fraction of the cancer antagonist against Gram-positive bacteria is shown in Table 3.

As the concentration of ethanol extract, ethyl acetate, butanol and water fraction increased, the size of inhibition zone, which showed antimicrobial activity, increased. In particular, in the case of ethyl acetate fraction, Staphylococcus aureus showed a growth zone of 20 mm at the concentration of 1000 ppm and a clear zone of 22 mm at the Bacillus cereus , showing excellent antibacterial activity as compared with the other fractions (FIG. 4).

Antimicrobial activity against Gram-negative bacteria As shown in Table 4, the antimicrobial activity of all the strains was excellent in both the antiprotozoal extract and the fraction, and the ethyl acetate fraction was particularly effective against Pseudomonas aeruginosa and Vibrio parahaemolyticus showed the highest antimicrobial activity with a clear zone of growth of 22 mm at 1000 ppm treatment (Fig. 5).

On the other hand, the hexane fraction showed no antimicrobial activity in all strains, and the dichloromethane fraction showed low antimicrobial activity at a concentration of 1000 ppm.

Taken together, these results indicate that the ethyl acetate fraction of the cancer antagonist has broad antibacterial activity against Gram positive bacteria and Gram negative bacteria.

<Table 3> Antimicrobial Activity Tests of Gram-positive Bacillus subtilis Extract

 Strain name Diameter of growth inhibition clear zone (mm) Concentration (ppm) Ethanol extract Hexane fraction Dichloromethane fraction Ethyl acetate fraction Butanol fraction Water fraction B. cereus 100 11 - - 13 11 - 250 15 - - 18 15 10 500 15 - - 20 16 11 1000 18 - 11 22 18 12 L. monocytogenes 100 10 - - 12 11 - 250 11 - - 13 12 10 500 12 - - 15 13 11 1000 15 - 11 18 15 12 S. aureus 100 11 - - 12 12 - 250 15 - - 18 16 10 500 15 - - 19 17 11 1000 19 - 11 20 18 13 S. epidermidis 100 11 - - 13 12 - 250 16 - - 17 13 10 500 17 - 9 18 14 11 1000 20 - 11 20 18 13 S. faecalis 100 11 - - 12 11 - 250 13 - - 15 13 10 500 14 - 9 16 14 11 1000 16 - 11 19 19 12

<Table 4> Antimicrobial Activity of Gram Negative Bacteria Extract

 Strain name Diameter of growth inhibition clear zone (mm) Concentration (ppm) Ethanol extract Hexane fraction Dichloromethane fraction Ethyl acetate fraction Butanol fraction Water fraction E. coli 100 11 - - 14 11 - 250 14 - - 17 13 10 500 14 - - 18 15 12 1000 18 - 11 20 18 13 P. aeruginosa 100 11 - - 14 11 - 250 14 - - 18 15 11 500 15 - 9 20 16 12 1000 18 - 11 22 18 14 S. enteritidis 100 11 - - 12 11 - 250 14 - - 17 15 11 500 16 - 10 18 16 12 1000 18 - 11 21 18 13 S. trphimurium 100 11 - - 14 11 - 250 14 - - 17 16 10 500 14 - - 18 17 11 1000 16 - 11 20 18 12 V. parahaemolyticus 100 11 - - 13 12 - 250 15 - - 18 15 10 500 17 - 9 20 16 11 1000 19 - 11 22 18 13

3. Microbial growth inhibition curve by concentration

The female counterpart fractions were sterilized with a membrane filter, and each fraction was added to the liquid medium at 100 ppm, 250 ppm, 500 ppm, and 1000 ppm concentrations.

100 쨉 l of the cultured bacterial culture was inoculated into 10 ml of fresh medium until the OD value reached 0.4 and cultured for 72 hours in an optimal temperature condition (Table 2) in a shaking incubator. The degree of proliferation was measured at 650 nm through a microplate reader (BIO-TEK INSTRUMEN INC, USA).

As a result, the food microorganisms Bacillus cereus strain carcinoma counter extract of the present invention (Bacillus cereus , Listeria monocytogenes ), Staphylococcus aureus ) And Escherichia coli ) on the growth characteristics of four strains.

Bacillus cereus ( Bacillus cereus ) As shown in FIG. 6, the growth inhibition effect was shown in all the fractions. Especially, in the ethyl acetate fraction, the growth of the bacteria was rapidly increased until 6 hours, and then the growth was suppressed.

Listeria monocytogenes ) Degree As shown in Fig. 7, it was observed that as the concentration was decreased in the ethyl acetate fraction, the butanol fraction and the water fraction, the growth of the microorganism was decreased, which shows a remarkable growth inhibition effect.

In contrast, the hexane fraction and the dichloromethane fraction showed inhibition of growth, but no significant difference was observed between the concentrations.

Staphylococcus As shown in FIG. 8, the growth inhibition effect of aureus was observed in the ethyl acetate fraction after 6 hours, but no significant inhibitory effect was observed in the ethanol extract and other fractions.

Escherichia As shown in FIG. 9, E. coli showed growth inhibition effect in all the fractions. In particular, the ethyl acetate fraction and butanol fraction showed remarkable growth inhibition effect after 6 hours.

4. Measurement of minimum inhibitory concentration of microorganisms

It was confirmed that the cancer antiproduct extract and the fraction of the solvent had excellent antimicrobial activity by the paper disc method. Therefore, the minimum inhibitory concentration of the cancer antiproduct extract and the fraction thereof inhibiting the growth of the food harmful microorganism I measured it.

The minimum inhibitory concentration (MIC) of each strain was determined by adding 100 ppm, 250 ppm, 500 ppm and 1000 ppm concentrations of each fraction to a liquid medium, diluting the culture with 100 times of the culture medium until the OD value reached 0.4 After inoculation and incubation for 24 hours, the degree of propagation of the bacterial culture was measured at 650 nm through a microplate reader (BIO-TEK INSTRUMEN INC, USA).

As a result, as shown in Table 5, the growth inhibition effect of ethanol extract, ethyl acetate fraction and butanol fraction was observed at the concentration of 100 ppm in all 10 strains, while the water fraction and the dichloromethane fraction were 250 ppm and 500 ppm, respectively. Respectively.

<Table 5> Results of Minimum Concentration Measurement to Prevent Growth in Food-Harmful Strain

 Strain name Minimum inhibitory concentration (ppm) Ethanol extract Hexane fraction Dichloromethane fraction Ethyl acetate fraction Butanol fraction Water fraction B. cereus 100 <M <0 - 1000 < M < 500 100 <M <0 100 <M <0 250 < M < 100 L. monocytogenes 100 <M <0 - 1000 < M < 500 100 <M <0 100 <M <0 250 < M < 100 S. aureus 100 <M <0 - 1000 < M < 500 100 <M <0 100 <M <0 250 < M < 100 S. epidermidis 100 <M <0 - 500 <M <250 100 <M <0 100 <M <0 250 < M < 100 S. faecalis 100 <M <0 - 1000 < M < 500 100 <M <0 100 <M <0 250 < M < 100 E. coli 100 <M <0 - 1000 < M < 500 100 <M <0 100 <M <0 250 < M < 100 P. aeruginosa 100 <M <0 - 500 <M <250 100 <M <0 100 <M <0 250 < M < 100 S. enteritidis 100 <M <0 - 1000 < M < 500 100 <M <0 100 <M <0 250 < M < 100 S. trphimurium 100 <M <0 - 1000 < M < 500 100 <M <0 100 <M <0 250 < M < 100 V. parahaemolyticus 100 <M <0 - 1000 < M < 500 100 <M <0 100 <M <0 250 < M < 100

The present invention provides a cancer antipark extract having excellent physiological activities such as antioxidative activity, anti-inflammatory activity and antibacterial activity.

In addition, there is provided a composition for preventing and / or ameliorating diseases, which comprises, as an active ingredient, a cancer heavy pole extract having excellent physiological activity.

Claims (4)

delete An antioxidative composition comprising, as an active ingredient, at least one selected from the group consisting of an ethanol extract and fractions prepared by extracting cancerous counterparts with ethanol and sequentially fractionating the fractions with hexane, dichloromethane, ethyl acetate and butanol. For the improvement and prevention of an inflammatory disease comprising, as an active ingredient, at least one selected from the group consisting of an ethanol extract and fractions prepared by extracting cancerous antigens with ethanol and sequentially fractionating the fractions with hexane, dichloromethane, ethyl acetate and butanol Composition. An antimicrobial composition comprising at least one selected from the group consisting of an ethanol extract and fractions prepared by extracting cancerous antigens with ethanol and sequentially fractionating the fractions with hexane, dichloromethane, ethyl acetate and butanol.

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