KR20060131360A - Seed Extract of Hwangchil Tree with High Physiological Activity - Google Patents

Abstract

The present invention relates to a seed extract of Hwangchil-tree having excellent physiological activity.

The seed extract of the Hwangchil tree excellent in physiological activity of the present invention, after drying the seeds of the Hwangchil tree, grind it with a grinder to prepare a fine powder, and deposit this Hwangchil tree seed powder in 70% ethanol, using ultrasonic waves 1 After extracting three times each time, the supernatant of the ethanol extract was recovered and concentrated under reduced pressure, and the concentrate was suspended in water, and then extracted sequentially with hexane, ethyl acetate and butanol to obtain respective fractions.

According to the present invention, a seed extract of Hwangchil tree having excellent physiological activity such as antioxidant activity, anticancer activity, and antibacterial activity is provided, and can be used in various fields such as functional health supplements, food additives, beverage compositions, feed additives of livestock and fish, etc. A seed extract of Hwangchil-tree having excellent physiological activity is provided.

Description

Seed Extract of Hwangchil Tree with High Physiological Activity {THE FRUITS EXTRACTS OF DENDROPANAX MORBIFERA LEVEILLE HAVING THE EXCELLENT BIOLOGICAL ACTIVITY}

1 is a manufacturing process of the Hwangchil wood seed extract of the present invention

Figure 2 is a graph showing the DPPH radical scavenging activity for the Hwangchil tree seed extract of the present invention

A: experimental group (M-ethanol extract, a-hexane fraction, b-ethyl acetate fraction,

                 c-butanol fraction, d-water layer)

B: control group (BHA, trolox, ascorbic acid)

Figure 3 is a graph showing the superoxide scavenging activity and xanthine oxidase radical inhibitory activity against the Hwangchil tree seed extract of the present invention

A: Ethanol extract B: Hexane fraction C: Ethyl acetate fraction

D: Butanol fraction E: Water layer F: Control group (allopurinol)

Figure 4 is an electrophoresis picture for confirming the DNA fragmentation phenomenon for the HL-60 cell line and Jurkat cell line of the Hwangchil tree seed extract of the present invention

Figure 5 is a flow cytometry graph and pictures of the cell nuclei for HL-60 cells for morphological measurements of apoptosis

A: untreated HL-60 cells

B: HL-60 Cells Treated with Ethyl Acetate Fraction from Hwangchil Seeds

Figure 6 is a flow cytometry graph and pictures of the cell nucleus for Jurkat cells for the morphological measurement of apoptosis

A: Untreated Jurkat Cells

B: Jurkat Cells Treated with Ethyl Acetate Fraction from Hwangchil Seeds

Figure 7 is an immunoassay photograph of PARP, pro caspase-3, β-actin in HL-60 cells and Jurkat cells treated with ethyl acetate fraction of the yellow lacquer seed of the present invention

The present invention relates to a seed extract of Hwangchil-tree having excellent physiological activity.

Hwangchil- tree ( Dendropanax morbifera Le'veille ') is a plant belonging to the family Araliaceae, and it is economically valuable as Korea's own traditional paints. It is an evergreen broad-leaved arborescent

It is a species that does not lose leaves even in winter. When the bark is damaged, yellow resin solution comes out. This is called Hwangchil (黃 漆). Hwangchil has been used as a rare paint that emits the golden color of emperor's armor, helmets, and other metal ornaments since the Three Kingdoms. Hwangchil wood is said to be effective and harmless in eliminating heat burns, cures and burns, and leprosy. .

  Hwangchil's components consist of 66.7% of non-volatile components, 10.8% of aromatic components, 8.1% of moisture, and 14.4% of solid components, which are the paint components that form the golden coating. The aromatic component consists mainly of sesquiterpenes, β-cubebene, γ-selinene, and δ-cadinene, which are known to exhibit sedative and tonic effects on the nervous system. .

In addition, the sap of sakil wood sap has the effect of clearing the head and relaxing the mind and body, and it has a fascinating and fascinating scent.It is used as a perfume for women. It is said that it has an excellent effect on the treatment of inflammation.

In Korean Registered Patent Publication No. 10-0318019 (hwangchil-tree extract having anticancer activity), the hwangchil-tree or its sap is extracted with a lower alcohol, and again fractionated sequentially using hexane or the like to show the anti-cancer and antioxidant activity. Is open to the public.

Korean Patent Laid-Open Publication No. 2003-0079205 (hwangchil extract having hepatoprotective effect, hwangchil fraction and pharmaceutical composition containing them), extracts the resin of Hwangchil tree with an alcohol solvent, and the extract is extracted with hexane, chloroform, butanol, Discloses a yellow lacquer extract and a yellow lacquer fraction having a hepatocellular protective effect consisting of fractions obtained by successive repeated extractions as a solvent.

However, the extract using the conventional Hwangchil wood is obtained by extracting the bark or sap of the Hwangchil tree with alcohol, and relates to an extract showing anticancer activity, antioxidant activity and hepatocyte protective effect, cell renewal effect, etc. In addition, studies on the seeds of Hwangchil-tree and other effects of Hwangchil-tree seed extract have not been conducted.

In order to solve the above problems, an object of the present invention is to provide a seed extract of hwangchil wood with excellent physiological activities such as antioxidant activity, anticancer activity, antibacterial activity.

In addition, an object of the present invention is to provide a seed extract of Hwangchil-tree having excellent physiological activity that can be used in various fields such as functional health supplements, food additives, beverage compositions, feed additives of livestock and fish.

The present invention relates to a seed extract of Hwangchil-tree having excellent physiological activity.

The seed extract of the Hwangchil tree having excellent physiological activity according to the present invention is a first step of drying the seed of Hwangchil tree, and then grinding it with a grinder to prepare fine powder. It is composed of extracting three times by one hour using the ethanol extract.

In addition, the supernatant of the ethanol extract is recovered and concentrated under reduced pressure, and then the concentrate is suspended in water, and then sequentially extracted with hexane, ethyl acetate and butanol to obtain respective fractions.

The main material used in the present invention, Dendropanax morbifera Le'veille ', is a plant belonging to the family Araliaceae, which has economic value as a traditional paint unique to Korea, and the southwest coast of Jeollanam-do, including Wando, Haenam, and Bogildo. It is an evergreen broad-leaved arboreous tree native to Hallasan in Jeju Island

Hwangchil's components consist of 66.7% of non-volatile components, 10.8% of aromatics, 8.1% of moisture, and 14.4% of solids. The aromatic component consists mainly of β-cubebene, γ-selinene, and δ-cadinene of sesquiterpenes. These components are known to exhibit sedation and tonic effects on the nervous system. , Hwangchil sap is applied to clear the head and to relax the mind and body, and it has a fascinating and fascinating scent.It is used as a perfume for women. It is said that it has an excellent effect on treatment.

In the past, but mainly used as a paint, but the research on the sap of the hwangchil in recent years, the antioxidant and anti-cancer activity of the hwangchil sap is known, the situation is used as food, such as drinks.

However, the inventors of the present invention, the sap of the saplings of the hwangchil wood is extremely low, the effect is also somewhat reduced, the resin extract of the hwangchil wood has a problem that the effect is inferior, while the seeds of the hwangchil sap while studying at a different angle As well as more excellent antioxidant and anticancer activity, it was found to be excellent in physiological activity.

Therefore, with respect to the physiological activity of the seed extract of the Hwangchil-tree, more specifically, it is confirmed that the physiological activity is the greatest when extracted by using a solvent, and to use the physiologically excellent Hwangchil-tree seed extract in various ways. The study completed the present invention.

Seeds of Hwangchil-tree were extracted with 70% ethanol and partitioned sequentially into solvents of different polarity, hexane, ethyl acetate and butanol.

The ethanol extracts and the fractions of the Hilchi chinensis were measured by DPPH hydrogen donating ability, physiological activities such as superoxide scavenging activity, xanthine oxidase inhibitory effect, antibacterial activity and anticancer activity.

As a result, the ethyl acetate fraction showed the highest antioxidant activity among the fractions of Hwangchil-tree seeds, and showed the relatively high activity inhibition rate in the butanol fraction, and the results of experiments on the antimicrobial activity by applying it to various food rot bacteria and food poisoning bacteria It showed the highest antimicrobial activity in hexane fraction.

In addition, the cell growth inhibition of HL-60 cells and Jurkat cells of H. chinensis seed extract and solvent fractions was investigated using HL-60 cells and Jurkat cells, which were leukemia cell lines, and its proliferation inhibitory effect was apoptosis. As a result of the induction, the ethyl acetate fraction showed the highest inhibition rate of cell proliferation, and the DNA fragmentation caused by induction of apoptosis was also best observed.

In addition, when the ethyl acetate fraction was treated with 50 μg / ml, the cell size was reduced, the shape of the nucleus was irregular and partial nucleation was observed. Sub-G1-hypodiploid cells (sub-G1 hypodiploid cell) was confirmed to be increased.

In addition, the concentration of pro-caspase-3 protein was decreased depending on the concentration of ethyl acetate fraction and the fragments of PARP were observed at the concentration of decreasing the amount of pro-caspase-3 protein. That is, it was confirmed that the ethyl acetate fraction of the Hilchi chinensis seed extract showed inhibition of cell growth by induction of apoptosis.

On the other hand, using the hwangchil wood seed extract of the present invention can be used in various fields such as functional health supplements, food additives, beverage compositions, feed additives of livestock and fish.

Particularly, since ethyl acetate fraction in the fraction of the Hwangchil tree seed extract is excellent in antioxidant and anticancer activity, the ethyl acetate fraction is vacuum-dried and powdered, and then encapsulated in a conventional manner to provide functional health supplement food having antioxidant and anticancer activity. The butanol fraction of the Hilchi chinensis extract is excellent in antioxidant activity, so that the butanol fraction is vacuum-dried and powdered and then encapsulated in a conventional manner to produce a functional dietary supplement having antioxidant activity. You can do it.

In addition, since the hexane fraction of the Hwangchil tree seed extract has excellent antimicrobial activity, the hexane fraction may be dried by vacuum drying, powdered, and then encapsulated by a conventional method to prepare a functional health supplement having antimicrobial activity. have.

At this time, the functional health supplement using the seed extract of the hwangchil wood of the present invention is preferably ingested 5 ~ 50 mg / kg (body weight) on an adult basis.

In addition, the seed extract of the hwangchil wood of the present invention can be used as a beverage composition, food additives, feed additives, etc. At this time, when preparing a beverage composition, it is preferable to add the extract to be 0.5 to 50% of the total weight, food When prepared as an additive, it is preferable to use it as an additive to consume 5 to 50 mg / kg (body weight) on an adult's daily basis. In addition, when used as a feed additive, it is preferable to add so that 1 to 50% by weight of conventional livestock and fish feed.

Hereinafter, the seed extract of the Hwangchil tree having excellent physiological activity of the present invention will be described in detail through Examples and Experimental Examples. However, these do not limit the scope of the present invention.

<Example 1> Preparation of Hwangchil-tree seed extract excellent in physiological activity of the present invention

In October 2004, the seeds of Hwangchil-tree were collected and prepared in Uigu-ri, Namwon-eup, Namjeju-gun, Jeju Island.

1 kg of the prepared Hwangchil wood seeds were dried and then ground into a grinder to prepare a fine powder.

The prepared Hwangchil-tree seed powder was immersed in 70% ethanol and extracted three times for 1 hour using ultrasonic waves. The supernatant was recovered, concentrated under reduced pressure, and 68.1 g of ethanol extract was obtained.

Among them, 50 g of the ethanol extract of the seed was suspended in 1 L of distilled water, and then extracted with hexane (1 L x 3), ethyl acetate (1 L x 3) and butanol (1 L x 3), respectively, and the respective fractions were vacuum dried. 0.49 g of hexane fraction, 7.6 g of ethyl acetate fraction, 7.12 g of butanol fraction and 33.57 g of water layer were obtained.

Example 2 Preparation of Functional Health Supplement Using Hwangchil-Tak Seed Extract of the Present Invention 1

The ethyl acetate fraction of the Hilchi chinensis seed extract prepared by the method of Example 1 of the present invention was prepared.

The ethyl acetate fraction was dried in vacuo to produce a powder, and then encapsulated in a conventional manner to prepare a functional dietary supplement with excellent antioxidant and anticancer activity.

Example 3 Preparation of Functional Health Supplement Using Hwangchil-Tak Seed Extract of the Present Invention 2

A butanol fraction of the Hilchi chinensis seed extract prepared by the method of Example 1 of the present invention was prepared.

The butanol fraction was vacuum dried to prepare a powder, and then encapsulated in a conventional manner to prepare a functional health supplement with excellent antioxidant activity.

Example 4 Preparation of Functional Health Supplement Using Hwangchil Tree Seed Extract of the Present Invention 3

The hexane fraction of the Hilchi chinensis seed extract prepared by the method of Example 1 of the present invention was prepared.

The hexane fraction was vacuum dried to prepare a powder, and then capsule-treated in a conventional manner to prepare a functional health supplement with excellent antibacterial activity.

Example 5 Preparation of Food Additive Using Hwangchil Tree Seed Extract of the Present Invention

An ethanol extract of Hwangchil-seed seed prepared by the method of Example 1 of the present invention was prepared.

The ethanol extract was freeze-dried to prepare a powder to prepare a food additive using the Hwangchil tree seed extract of the present invention.

Example 6 Preparation of a Beverage Composition Using the Hwangchil Tree Seed Extract of the Present Invention

An ethanol extract of the Hwangchil-seed seeds prepared in Example 1 of the present invention was prepared.

1 g of purified water was added to 100 g of the prepared extract, and 10 g of sodium citrate, 40 g of citric acid, 5 kg of liquid fructose, and 6 kg of syrup were mixed to prepare a beverage composition using the extract of Hilchi chinensis of the present invention.

<Example 7> Preparation of feed additives using the Hwangchil wood seed extract of the present invention

An ethanol extract of Hwangchil-seed seed prepared by the method of Example 1 of the present invention was prepared.

The extract was lyophilized to prepare a powder, thereby preparing a feed additive for livestock and fish using the Hwangchil tree seed extract of the present invention.

Experimental Example 1 Antioxidant Activity Test of Hwangchil Tree Seed Extract of the Present Invention

1. Preparation of experimental materials

Each fraction prepared in Example 1 was prepared.

2. DPPH radical scavenging activity experiment

2-1. Experiment method

The most characteristic mechanism of antioxidants is the reaction with free radicals. Free radical scavenging action is used as a measure of antioxidant activity in plants and aging in the human body by donating electrons to free radicals.

DPPH is a stable free radical that is reduced and decolorized by sulfur-containing amino acids such as cysteine and glutathione, ascorbic acid, and aromatic amine (ρ-phenylenediamine, ρ-aminophenol). It is widely used for measuring antioxidant capacity.

Electron donating ability was measured according to the DPPH free radical scavenging method by the Blois method.

100 μl of the sample to be analyzed was dispensed into a 96 well plate, and 0.4 mM DPPH solution was added in the same amount, and left at room temperature for 10 minutes, and the absorbance was measured at 517 nm.

Butyl hydroxy anisole (BHA) and trolox were used as controls.

DPPH radical scavenging activity was calculated from the following equation, expressed as the concentration of the sample (IC ₅₀ ) appearing when the absorbance of DPPH was reduced by 50%, and each sample was repeated three times to obtain an average value.

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

* A _sample = absorbance of the reaction solution added

* A _control = absorbance of the reaction solution with methanol added instead of the sample

2-2. DPPH radical scavenging activity test results

Antifreeze activity of DPPH (1,1-diphenyl-2-picryl-hydrazyl) was measured by the antioxidant activity of Hwangchil-silk extract and each fraction, compared with other solvent fractions in 70% ethanol extract and butanol and ethyl acetate fractions. It exhibited relatively high radical scavenging activity, of which the highest radical scavenging activity was shown in the butanol fraction (FIG. 2).

3. Xanthine oxidase inhibition and superoxide scavenging activity test

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

Xanthine oxidase oxidizes hypoxanthine and finally produces uric acid oxygen, and oxygen free radicals and hydrogen peroxide groups are generated from this oxygen.

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

In addition, oxygen free radicals produced by xanthine oxidase cause cell damage). Thus, materials capable of eliminating free radicals in oxygen free groups will also be useful in the prevention of oxidative damage.

Gout occurs as the level of uric acid rises, and they form crystals and adsorb on the joints, causing inflammation, and in severe cases, it is known to cause complications in the kidneys and the heart.

Xanthine oxidase inhibitors have been used for the treatment of uric acid causing gout, kidney defects, ischemia and cardiomyopathy.Allopurinol and alloxanthine are used for the treatment of gout. Etc. are known.

Meanwhile, about 0.4 to 4% of the total oxygen consumed during the normal oxidative phosphorylation process is converted into free radical superoxide (· O ₂ ⁻ ), and the generated O ₂ ⁻ is converted into other reactive oxygen species, ROS) is known to cause cell damage directly or indirectly.

Normally, · O ₂ ^- is rapidly converted to hydrogen peroxide by superoxide dismutase (SOD) by endogenous antioxidant defense mechanisms.

However, if this endogenous antioxidant defense system has problems in maintaining intracellular redox balance, the result is oxidative stress, which plays an important role in directly damaging intracellular macromolecules or causing cell damage. Do it.

3-1. Experiment method

The production of uric acid by xanthine / xanthine oxidase was measured by increased absorbance at 290 nm and the amount of superoxide was determined by nitroblue tetrazolium (NBT). It measured by the reduction method.

The reaction solution was prepared in various concentrations of each sample, 0.5 mM xanthine and 1 mM EDTA in 100 µl of 200 mM phosphate buffer (pH 7.5), and 50 mU / ml xanthine oxidase was added to generate uric acid. Induced.

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

Xanthine oxidase inhibition and superoxide scavenging activity were expressed as the sample concentration (IC ₅₀ ) that appears when the absorbance of the produced uric acid and superoxide decreased by 50%, respectively.

3-2. Xanthine oxidase inhibition and superoxide scavenging activity test results

Xanthine oxidase inhibition and superoxide scavenging activity of the seed extract and fractions of the present invention are shown in FIG.

Ethyl acetate fraction showed the highest inhibition of xanthine oxidase activity and relatively high activity inhibition rate in butanol fraction.

In particular, the hexane fraction showed almost no xanthine oxidase inhibitory effect.

The IC ₅₀ value of the ethyl acetate fraction which showed the best inhibition of xanthine oxidase activity was 261 μg / ml, and the order of their activity for inhibition of xanthine oxidase activity was different from that of DPPH radical scavenging activity. Butanol> ethanol.

In addition, the ethyl acetate fraction showed the highest scavenging activity, and the butanol fraction showed the relatively high superoxide radical scavenging activity.

The IC ₅₀ value of the ethyl acetate fraction which showed the highest superoxide radical scavenging activity was 97.46 μg / ml, and the activities were similar in the order of ethyl acetate>butanol> ethanol.

Experimental Example 2 Antimicrobial Activity of Hwangchil Tree Seed Extract

1. Preparation of strains and media

Strains used in the antimicrobial experiment were Gram positive bacteria (Gram positive bacteria) and Gram negative bacteria (Gram negative bacteria) three species were purchased from KCTC (Table 1).

The growth medium of the bacteria was incubated for 24 hours in an incubator at 37 ℃ using nutrient agar (Difco. USA) and BHI (Difco. USA) for each strain.

Table 1 Strains Used in the Antimicrobial Activity Experiment

division Strains Gram positive bacteria staphylococcus aureus (KCTC 1927) Bacillus subtilis (KCTC 1023) Staphylococcus epidermidis (KCTC 1917) Streptococcus mutans (KCTC 3065) Gram negative bacteria Vibro parahaemolyticus (KCTC 2471) Escherichia coli (KCTC 1042) Salmonella typhimurium (KCTC 2514)

2. Antimicrobial Activity Test Method

Antimicrobial activity screening was done using the paper disc method.

Incubate the bacteria in a flat broth for 24 hours, smear the bacteria on nutrient agar medium, place sterilized paper discs on this medium to the number of samples, and adhere to the sample number. 50 µl inoculation at a concentration of 50 mg / ml was used to measure the diameter of the clear zone (mm) around the paper disk.

3. Antibacterial activity test result

The results of searching for antimicrobial activity by applying Hwangchilseol extract and fractions to various food rot and food poisoning bacteria by the paper disc method are shown in Table 2 below.

<Table 2> Antimicrobial Activity Test Results for Seed Extracts of Hwangchil Tree

Bacteria Growth inhibition ring (mm)  Medium (℃) ethanol Hexane Ethyl acetate Butanol Water layer Staphylococcus aureus (KCTC 1927) 11 10 8 10 - NA (37) Bacillus subtilis (KCTC 1023) 12 12 9 12 14 NA (30) Staphylococcus epidermidis (KCTC 1917) 11 10 9 10 - NA (37) Streptococcus. mutans (KCTC 3065) - 12 - - - BHI (37) Vibro parahaemolyticus (KCTC 2471) - - - - - NA (37) Escherichia coli (KCTC 1042) - - - - - NA (37) Salmonella typhimurium (KCTC 2514) - - - - - NA (30)

As shown in Table 2, four Gram (+) bacteria showed the most antimicrobial activity, in particular against the strain of Bacillus subtilis ( Bacillus subtilis ) was the most antimicrobial activity, crude extract 70% ethanol The highest antimicrobial activity was found in the and hexane fractions.

However, antimicrobial activity against Gram (-) bacteria was not so high.

In general, the ethyl acetate layer showed high antibacterial activity, but the antibacterial activity was low, while the hexane layer showed the highest antibacterial activity.

Experimental Example 3 Anticancer Activity Test of Hwangchil Tree Seed Extract

1. Cell Culture

HL-60 and Jurkat cell lines derived from leukemia patients were distributed from Korea Cell Line Bank (KCLB) and 100 units / ml penicillin-streptomycin (GIBCO) and 10% fetal bovine serum (FBS, RPMI 1640 medium (GIBCO) containing GIBCO) was incubated in a 37 ° C., 5% CO 2 incubator, and passages were performed once every 3 to 4 days.

2. Measurement of cell proliferation inhibitory effect

2-1. How to measure

Proliferation inhibition of HL-60 and Jurkat cells by treatment of each sample was measured by cell metabolism activity.

The effect on the growth of HL-60 and Jurkat cells was measured by the absorbance of formazan produced by the reduction of MTT using MTT, one of tetrazolium salts.

Cells (2.5 × 105 / ml) were placed in each well of a 96 well plate and samples were added at different concentrations. After incubating for 4 days, 100 μg of 3- (4,5-dimethylthiazol) -2,5-diphenyltetrazolium bromide (MTT, Sigma) was added and further incubated for 4 hours.

The plate was centrifuged at 1,000 rpm for 10 minutes, the medium was carefully removed, and 150 μl of dimethylsulfoxide (dimethylsulfoxide, DMSO, Sigma) was added to dissolve the formazan precipitate produced by the reduction of MTT. Absorbance was measured at 540 nm using a microplate reader (BIO-TEK INSTRUMENIS. INC).

The average absorbance values for each sample group were obtained, and growth inhibition was examined by comparing with the absorbance values of the control group.

2-2. Result of growth inhibition effect of cells

Table 3 shows the results of experiments on cell growth inhibition of the Hwangchil-tree seed extract and fractions of the present invention.

<Table 3> Result of cell growth inhibitory effect

Cell line  Concentration (μg / ml) Cell growth inhibition rate (%) ethanol Hexane Ethyl-acetate Butanol Water layer  HL-60 12.5 21.2 ± 5.552 16.23 ± 4.449 13.56 ± 1.125 2.6 ± 7.79 5.73 ± 1.343 25 27.73 ± 7.696 89.18 ± 0.567 53.59 ± 3.615 4.43 ± 3.357 6.89 ± 3.426 50 32.02 ± 5.16 92.13 ± 0.915 57.15 ± 1.785 13.4 ± 7.693 9.86 ± 6.636 100 47.79 ± 3.022 93.75 ± 0.09 72.05 ± 1.982 35.7 ± 4.196 6.95 ± 8.119  Jurkat 12.5 21.16 ± 6.831 28.09 ± 5.875 16.45 ± 5.56 23.58 ± 2.38 8.17 ± 4.732 25 31.69 ± 4.922 89.81 ± 0.464 59.56 ± 4.459 29.24 ± 3.437 7.08 ± 1.787 50 47.5 ± 5.09 91.89 ± 0.846 76.71 ± 2.652 31.48 ± 8.691 8.48 ± 2.122 100 53.79 ± 3.179 92.63 ± 0.248 78.77 ± 3.277 47.03 ± 6.898 22.81 ± 4.917

As shown in Table 3 above, the treatment of Hwangchil-tree seed extracts and fractions showed concentration-dependent cell growth inhibition in all treatments, but in the case of hexane fraction, more than 12.5 μg / ml as shown in Table 3 below. At the time of treatment, it is considered to have cytotoxicity as it causes rapid cell necrosis in both HL-60 and Jurkat cells.

On the other hand, ethanol extract, ethyl acetate fraction, and buntanol fractions were found to have prominent cell proliferation inhibitory effects in both HL-60 and Jurkat cells.

Among them, the IC ₅₀ value of the ethyl acetate fraction with the highest inhibitory effect was 23.38 μg / ml for HL-60 cells and 17.28 μg / ml for Jurkat.

3. Experiment to Induce Apoptosis

3-1. Analysis for DNA fragmentation

Each sample was treated with cells (2.5 × 105 / ml) and then incubated for 48 hours.

After the cells were collected, DNA was isolated using a reagent extraction kit (Promega Wizard® Genomic DNA Purification Kit).

The isolated DNA was electrophoresed for 60 minutes (100 V) on a 1.5% agarose gel, stained with ethidium bromide, and DNA fragmentation was observed under an UV transilluminator.

3-2. Morphological changes in the nucleus

In order to observe the change in the nucleus, one of the morphological characteristics of apoptosis, DNA was stained using DAPI, a fluorescent dye that binds specifically to DNA, and observed with a fluorescence microscope.

That is, each sample was added to the cells (2.5 × 105 / ml), and then incubated for 48 hours.

Cells were collected and washed twice with PBS (phosphate-buffered saline) solution, and then 1 ml of 4% paraformaldehyde was added and fixed at room temperature for 1 hour. After fixing the cells, washed twice with PBS and 2.5 μg / ml DAPI (4,6-diamidino-2-phenylindole, Sigma) solution was added and stained for 30 minutes under room temperature.

After washing twice with PBS again, morphological changes of nuclei were observed under fluorescence microscopy.

3-3. Cell cycle analysis

Each sample was treated with cells (2.5 × 10 5 / ml) and then incubated for 48 hours, after which the cells were harvested and washed with PBS.

The washed cells were fixed with 70% ethanol at -20 ° C. for 30 minutes, washed with PBS, treated with RNase A, and then stained with propidium iodide (PI) (Sigma), Cell cycles were analyzed with an analyzer (Flow Cytometer, FACS Calibur. BD Bioscience. USA).

3-4. Western blot analysis

Samples were treated with HL-60 and Jurkat cells (3.5 × 105 / ml) and incubated for 48 hours. After washing the cells 2-3 times with PBS (Phosphate Buffered Saline), 500 μl of lysis buffer was added and lysed for 1 hour, followed by centrifugation at 14,000 rpm for 20 minutes to remove cell membrane components. .

Protein concentration was quantified using the Bradford method.

40 μg of lysate was denatured by 10% mini gel SDS-PAGE (Poly Acrylamide Gel Electrophoresis) and transferred to PVDF membrane (BIO-RAD) at 200 mA for 2 hours.

The blocking of membranes was performed for 1 hour at room temperature in TTBS (TBS + 0.1% Tween 20) solution containing 5% skim milk.

The primary antibody for detecting PARP and Pro-caspase-3 protein amount was anti-PARP (Santa-Cruz), anti-Pro-caspase-3 (Santa-Cruz), and diluted 1,000-fold in TTBS solution at room temperature. Reaction was carried out for 2 hours. As a secondary antibody, anti-rabbit IgG (Amersham Co.) conjugated with HRP (Horse Radish Peroxidase) was diluted 1: 5000, and reacted overnight at 4 ° C.

The cell membrane was washed three times with TTBS, and then reacted with ECL substrate (Amersham Co.) for 1 to 3 minutes, and then was exposed to X-ray film.

4. Experimental Results of Induction of Apoptosis

DNA fragmentation phenomenon to investigate whether cell proliferation inhibitory effect is induced by apoptosis with ethanol extract and ethyl acetate fraction which were the most effective among Hwangchilsil extract and fractions in HL-60 cells and Jurkat cells. Was observed by electrophoresis.

When treated with HL-60 and Jurkat cells at a concentration of 50 μg / ml for 48 hours, DNA fragmentation was best observed in HL-60 and Jurkat cells when the ethyl acetate fractions were treated (FIG. 4).

In addition, morphological observation of the cell nuclei treated with the extract (50 μg / ml) obtained from the ethyl acetate fraction was performed. As a result, the shape of the nuclei of normal cells was normal round shape, but in the cells treated with ethyl acetate extract, the size of the cells was reduced, and the shape of the nucleus was irregular and partial nucleation of the nucleus could be observed (FIG. 5, 6).

The mechanism of apoptosis has not been clearly demonstrated, but calcium levels in the cytoplasm are increased to activate calcium-dependent endonuclease, resulting in DNA fragmentation in the nucleus, and transglutaminase to be activated. It is known to form apoptotic bodies as cross-linking of proteins in the cytoplasm occurs, cytoplasmic enrichment and sap flows out of the cell.

In addition, whether the growth of Sub-G1 hypodiploid cells increased by induction of apoptosis by ethyl acetate fraction of H. chinensis seed extract was analyzed by flow cytometry by processing PI, which is a fluorescence-binding substance. Treatment with ethyl acetate fraction at a concentration of 50 μg / ml and after 48 hours of incubation, it was confirmed that the sub-G1 hypodiploid cells are increased (Fig. 5, 6).

Caspase is a protease that has all of cysteine at its active site. So far, 14 kinds are known, and most of them are found to play an important role in apoptosis.

Therefore, after treatment for 48 hours of ethyl acetate fraction by concentration, protein was isolated and the expression level of pro-caspase-3, one of the effector caspases, was investigated.

In addition, PARP, a protein involved in damaged DNA repair and reported to be cleaved by caspase during apoptosis, was analyzed under the same conditions.

As a result, as shown in Figure 7, it was confirmed that the concentration of pro-caspase-3 (37 kDa) protein was decreased in a concentration-dependent manner by treating the ethyl acetate solvent fraction, and also the amount of pro-caspase-3 protein. At this decreasing concentration, cleaved fragments of PARP could be observed.

According to the present invention, a seed extract of Hilchi chinensis with excellent physiological activities such as antioxidant activity, anticancer activity and antibacterial activity is provided.

In addition, the present invention provides a seed extract of the Hwangchil wood with excellent physiological activity that can be used in various fields such as functional health food, food additives, beverage composition, feed additives of livestock and fish.

Claims (5)

After drying the seeds of Hwangchil-tree, grinding them with a grinder to prepare fine powder, and then dipping this Hwangchil-tree seed powder into 70% ethanol, extracting 3 times by 1 hour using ultrasonic waves to prepare an ethanol extract, A hexane fraction obtained by recovering the supernatant of ethanol extract, concentrating under reduced pressure, and then suspending the concentrate in water and extracting with hexane. Hexane fraction of yellow chile seed with excellent antibacterial activity. After drying the seeds of Hwangchil-tree, grinding them with a grinder to prepare fine powder, and then dipping this Hwangchil-tree seed powder into 70% ethanol, extracting 3 times by 1 hour using ultrasonic waves to prepare an ethanol extract, The supernatant of the ethanol extract was recovered, concentrated under reduced pressure, the concentrate was suspended in water, extracted with hexane, and the obtained water layer was extracted with ethyl acetate. Ethyl acetate fraction of Hwangchil-seed with excellent antioxidant and anticancer activity. After drying the seeds of Hwangchil-tree, grinding them with a grinder to prepare fine powder, and then dipping this Hwangchil-tree seed powder into 70% ethanol, extracting 3 times by 1 hour using ultrasonic waves to prepare an ethanol extract, The supernatant of the extract was recovered, concentrated under reduced pressure, and then the concentrate was suspended in water and extracted sequentially using hexane and ethyl acetate. Then, the obtained water layer was extracted with butanol as a butanol fraction. Butanol fraction of yellow chile seed with excellent antioxidant activity. As an ethanol extract manufactured by drying the seeds of Hwangchil-tree, grinding them with a grinder to prepare fine powder, and then dipping the Hwangchil-tree seed powder into 70% ethanol and extracting it three times for 1 hour using ultrasonic waves. Characterized in having antioxidant activity, antibacterial activity, anticancer activity, Seed extract of Hwangchil-tree having excellent physiological activity. The method according to any one of claims 1 to 4, Characterized in that it is manufactured from one selected from functional health food, food additives, beverage composition, feed additives, Seed extract of Hwangchil-tree having excellent physiological activity.

Images