KR20080022315A - Poncirus polyandra extracts having whitening activity and anti-inflammatory activity - Google Patents

Abstract

An extract of Poncirus polyandra is provided to whiten the skin by inhibiting activity of melanocyte and tyrosinase, inhibit oxidation by removing DPPH(1.1-diphenyl-2-picrylhydrazyl) radical and suppress inflammation without side effects, so that the extract is useful for skin whitening, and prevention and treatment of inflammatory disease. An extract of Poncirus polyandra having skin-whitening, anti-oxidative and anti-inflammatory effects is prepared by washing immature Poncirus polyandra with water, pulverizing the washed Poncirus polyandra, freeze-drying the pulverized Poncirus polyandra, dipping the pulverized product in 70% ethanol with agitation at room temperature for 2-3 days, filtering the extract of Poncirus polyandra, concentrating the filtered extract of Poncirus polyandra under reduced pressure, suspending the concentrates in distilled water, and extracting the suspension with hexane, ethylacetate and butanol sequentially. A composition for whitening the skin or preventing and treating inflammatory disease comprises the extract of Poncirus polyandra.

Description

PONCIRUS POLYANDRA EXTRACTS HAVING WHITENING ACTIVITY AND ANTI-INFLAMMATORY ACTIVITY < RTI ID = 0.0 >

Figure 1 shows the chemical structure of Poncirin < RTI ID = 0.0 >

FIG. 2 is a graph showing the results of HPLC analysis of the dragonfly extract of the present invention

A: immature, B: mature

3 is a graph showing the results of LC-M / M analysis of pyscholine contained in the dragonfly extract of the present invention

FIG. 4 is a graph showing the results of ¹ H-NMR analysis of ponsyrin contained in the dragonfly extract of the present invention

FIG. 5 is a graph showing the results of ¹³ C-NMR analysis of pysyline contained in the dragonfly extract of the present invention

6 is a graph showing the content of pysylin contained in the dragonfly extract of the present invention

A: immature, B: mature

7 is a graph showing the inhibitory activity on melanin synthesis of the dragonfly extract of the present invention

A: Suppression of proliferation of melan-A cells

B: Growth inhibition in RAW 264.7 cells

FIG. 8 is a graph showing the inhibitory effect of tyrosinase activity on the dragonfly extract of the present invention

A: ethanol extract, B: hexane fraction

9 is a graph showing the effect of inhibiting melanin synthesis on the dragonfly extract of the present invention

A: ethanol extract, B: hexane fraction

10 is a Western blot photograph showing the results of immunoblot analysis of tyrosinase, TRP-1, TRP-2, and β-actin according to the concentration of the dragonfly extract of the present invention

A: immature, B: mature

1: control group, 2: melasolve cells (30 μM), 3: 12.5 μg / ml

4: 25 占 퐂 / ml, 5: 50 占 퐂 / ml, 6: 100 占 퐂 / ml

11 is a photograph showing the results of RT-PCT analysis of tyrosinase, TRP-1, TRP-2 and Mitf mRNA gene expression according to the concentration of the dragon extract of the present invention

A: immature, B: mature

M: Size Marker (100 bp), C: control group, 1: melasolve cells (30 μM)

     2: 12.5 占 퐂 / ml, 3: 25 占 퐂 / ml, 4: 50 占 퐂 / ml, 5: 100 占 퐂 / ml

12 is a graph showing the anti-inflammatory activity against the dragonfly extract of the present invention and a Western blot photograph

A: Graph, B: Western Blot Photo

The present invention relates to a drip irrigation extract showing whitening activity and anti-inflammatory activity.

The skin color of a person is influenced by the color of skin melanin, hemoglobin, carotenoid, etc. produced by melanocytes, which are pigment cells present in the epidermis, and the thickness and reflectivity of the skin.

Melanin pigment, which is the most important factor for determining skin color, is derived from tyrosinase (Tyrosinase), an enzyme present in melanocytes, which is a type of amino acid normally present in human body, And finally produces a dark brown polymer melanin through a complicated oxidation process.

Synthesis of melanin is facilitated by factors such as ultraviolet light exposure, melanoma, hyperpigmentation disease, and the like.

Recently, molecular biologic approaches have been attempted with the reports that the expression and synthesis of tyrosinase, TRP-1, and TRP-2 involved in melanin synthesis are directly involved in melanin synthesis.

On the other hand, when the inflammatory reaction is infected with tissue (cell) damage or external infectious agents (bacteria, fungi, viruses, various kinds of allergens), various inflammatory mediators and immune cells in the local blood vessels and body fluids are involved, Inflammatory mediator secretion, body fluid infiltration, cell migration, tissue destruction, and other external symptoms such as erythema, edema, fever, and pain.

In normal cases, the inflammatory reaction removes the external infectious agent and regenerates the damaged tissue to regenerate the organism's function. However, if the antigen is not removed or the internal substance causes the inflammatory reaction to occur excessively or continuously, Some lead to diseases such as cancer.

In recent years, in order to prevent and treat various diseases such as inflammatory diseases as described above, researches for using natural substances that are not artificial substances in various fields such as substances, functional foods, and functional cosmetics have been actively conducted.

On the other hand, Poncirus polyandra It is a genus, such as P. trifoliata , whose origin is known as a Chinese male, and is known as a dragon (dragon) because it has the characteristic that it grows like a dragon.

In the 1970s, it was introduced by Dr. Vitas of the University of California that there was a possibility of being selected as a low-end (small-sized) passage.

P. trifoliata , which is contained in genus such as P. polyandra , has been used as a cleansing agent and a digestive circulation agent since it was first known as a fungus.

In the United States, Bitters et al. (1979) studies on Varensiia orange have been widely used in studies to make small citrus trees after proven tormenting phenomena. In Japan, since 1980, But it did not reach to practical use.

From the mid-1980s, the researchers began to study the possibility of using the high-grade citrus citrus citrus citrus fruit as the main ingredient of the "Qingdao Wenzhou" citrus fruits in Japan. .

Since the late 1990s, Jeju has become interested in low-key lines, and has been conducting full-scale research on dykes since 1999.

The dripstone is a deciduous tree like a tiger. It is medium to slightly weak and straight. The height of the tree is more than 2m. The leaf is small and bent with the leaves of the leaves. The branches, thorns and roots are bent.

Fruits are spherical, about 30 g, seeds contain about 20 per fruit, and have resistance similar to tangerine for trita virus (CTV).

Drosophila is similar to Tazza in its initial growth until the end of August after sowing.

As a result of the study on the dylan tree in Japan, it was observed that the size of the 4th and 5th year trees after the first year students "Pyeongdokjo" and "Hinoaaebono" And it is reported that the tendency is larger in "wind demodulation".

Korean Patent Registration No. 10-0526992 entitled " Composition for treatment of tangerine extract fermented product and Helicobacter pylori infection disease "discloses a composition for treating diseases caused by infection of Helicobacter pylori with effective components extracted fermented product extracted from tangerine The present invention relates to a therapeutic composition.

Korean Patent Laid-Open Publication No. 10-2001-0018805 entitled " Antiinflammatory and anti-irritant cosmetic composition containing tannin extracts "includes 0.001 to 20.0 wt% of tannin extracts in a cosmetic composition for anti- Are disclosed.

However, research on the physiological activity of dyrrhythmia has not been conducted so far, so it is necessary to study various physiological activities of dyrrhion.

It is an object of the present invention to provide a drip irrigation extract showing whitening activity and anti-inflammatory activity.

Further, the present invention provides a whitening cosmetic composition comprising a dyer extract as an active ingredient, and an object of the present invention is to provide an antioxidant composition and a composition for prevention and treatment of inflammatory diseases.

The present invention relates to a drip irrigation extract showing whitening activity and anti-inflammatory activity.

The scarlet dragon extract of the present invention can be used as a pest- polyandra immature fruits were pulverized by a wet pulverizer, the pulverized material was lyophilized to prepare a powder, the powder was immersed in 70% ethanol, stirred for 2 to 3 days at room temperature and leached, The filtrate was concentrated under reduced pressure. The concentrate was suspended in distilled water, and the suspension was successively extracted with hexane, ethyl acetate and butanol. The residue was purified by filtration through a filter, and the leaching and filtration process was repeated 2 to 3 times. .

The dyed dragon ( P. polyandra ) It is a genus, such as P. trifoliata , whose origin is known as a Chinese male, and is known as a dragon (dragon) because it has the characteristic that it grows like a dragon.

Drake is a deciduous tree like a tiger. It is medium to slightly weak and straight. Tree height is more than 2m. Leaves are small and bent with exuberant leaves. Branches, thorns and roots are bent. Fruits are spherical 30 g, and the seed contains about 20 seeds per one fruit, and has a resistance similar to the tangerine for the trita virus (CTV).

The inventors of the present invention found that a drip irrigation extract was prepared using the above-described dripsticks, and then the ethyl acetate fraction was selected and analyzed to find that 98% poncirin was contained therein ~ 5).

Poncirin is a substance with a chemical structure of C ₂₈ H ₃₄ O ₁₄ and a molecular weight of 594, which is contained in an extract of fruits and has been reported to have an inhibitory activity against Helicobacter pylori.

In addition, quantitative comparison of poncirin content in the ethyl acetate fraction of the matured and immature strains of the present invention by HPLC was confirmed to be 3.4 times as much as that of mature in the immature strains (Fig. 6).

The inventors of the present invention have studied and experimented on various physiological activities using the above-mentioned drip irrigation extract. As a result, it has been found that the drip irrigation extract of the present invention is excellent in whitening activity and has antioxidative activity and anti-inflammatory activity .

The inhibitory activity of melanocyte melanocyte proliferation and growth was remarkable in the dillanol ethanol extract of the present invention when administered at 25 μg / ml or more. The concentration of 12.5 μg / The proliferation tended to be gradually suppressed, and the proliferation was significantly inhibited in a concentration-dependent manner as the treatment concentration was continuously increased (Fig. 7).

In addition, the tyrosinase activity of the dithionol ethanol extract of the present invention was tested and showed a tyrosinase inhibitory activity of 75% when treated with 30 uM of melazove.

In other words, when the ethanol extracts were treated at 12.5, 25 and 50 μg / ml, the inhibition rates were 44%, 62% and 77%, respectively. As the treatment concentration increased, the tyrosinase activity tended to be suppressed (Fig. 8).

These results suggest that the ethanol extract may inhibit the activity of tyrosinase and prevent the process of melanin synthesis.

The ethanol extract showed higher tyrosinase inhibitory activity than the respective fractions (water, hexane, ethyl acetate, and butanol) (FIG. 8A), and the hexane fraction showed the highest tyrosinase inhibitory activity among the fractions (FIG. 8B).

In addition, as a result of the experiment for inhibiting melanin synthesis, it was shown that the dosage of the ethanol extract of the present invention tended to inhibit melanin production with increasing treatment concentration, and it was statistically significant, (Fig. 9).

(IC ₅₀ ) was 25 / / ml, which showed a high whitening effect (Fig. 9A) and a high inhibitory effect (IC ₅₀ ) of 10 ㎍ / ㎖ in the hexane fraction (Fig. 9B ).

In addition, the effects of the present invention on the expression of tyrosinase, TRP-1, TRP-2, and MITF protein were examined. As a result, the expression pattern of tyrosinase protein was decreased And the expression of tyrosinase and TRP-2 protein was inhibited in the immature stage.

The expression of tyrosinase and TRP-1 protein was decreased in the ethanol extract, and the expression of tyrosinase protein was inhibited in a concentration-dependent manner in the hexane fraction and the ethyl acetate fraction (FIG. 10).

In addition, the effects of the extract of the present invention on the expression of tyrosinase, TRP-1, TRP-2 and MITF mRNA showed that TRP-2 mRNA expression . The expression of tyrosinase hk TRP-2 mRNA was inhibited by dillong maturation and ethanol extracts. Tyrosinase and ethylacetate fractions inhibited the expression of TRP-2 mRNA in hexane fraction and ethyl acetate fraction, respectively. (Fig. 11).

Thus, the dragonfly extract of the present invention suppresses tyrosinase activity, suppresses melanin synthesis, inhibits tyrosinase, TRP-1, TRP-2 and MITF protein expression, inhibits tyrosinase, TRP- 1, TRP-2, and MITF mRNA, and thus it was confirmed that the whitening activity was excellent.

In addition, the DPPH radical scavenging activity of the extract of the present invention was good, and the superoxide radical scavenging activity of ethanol extract of the present invention was IC ₅₀ > 500 ㎍, but the activity of xanthine oxidase inhibition was higher in hexane fraction and ethyl acetate fraction (IC ₅₀ 122 ㎍ / ㎖) (Table 3).

Therefore, it was confirmed that the extract of the dragonfly of the present invention has an antioxidative activity.

Further, the anti-inflammatory activity of the extract of the present invention was measured, and it was found that the NO produced by LPS was strongly inhibited (IC ₅₀ ≪ 150ug) (Fig. 12A).

In addition, it was confirmed that the inhibitory rate of iNOS expression was about 50% at 25 μg / ml of the dragon extract of the present invention, and 90% or more when treated with 100 μg / ml. Cox-2 expression was also suppressed at 100 μg / Up to 60%.

Therefore, it was confirmed that the dragonfly extract of the present invention is excellent in anti-inflammatory activity (FIG. 12).

Meanwhile, an antioxidant composition containing the dragon extract of the present invention as an active ingredient, a composition for the prevention and treatment of inflammatory diseases, and a composition having a whitening activity can be prepared.

In addition, the extract of the present invention can be widely used as a food additive, a beverage composition, an animal feed additive, a fish food feed additive, and the like.

Hereinafter, 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 the dragonfly extract of the present invention

P. polyandra was prepared by the Jeju Agricultural Research Institute in October 2005 and May 2006 for immature fruit.

1 kg of diller immature fruit was washed, pulverized with a wet grinder, lyophilized and powdered to prepare a dillon immature fruit powder.

This diller immature fruit powder was put into 70% ethanol and stirred for 3 days at room temperature of 25 ° C with stirring.

After leaching, the residue was removed by filtration through a filter to prepare a primary filtrate.

This primary filtrate was again added to 70% ethanol, and the mixture was stirred at room temperature for 2 days at 25 ° C, and then filtered through a filter to remove the residue to prepare a secondary filtrate.

The secondary filtrate was concentrated at 40 ° C using a rotary evaporator to obtain 330.6 g of an ethanol extract.

This ethanol extract was suspended in 1 L of distilled water.

The suspension was extracted twice with hexane to obtain a hexane layer and an aqueous layer, and the hexane layer was concentrated under reduced pressure to obtain 13.2 g of hexane fraction.

The water layer was extracted twice with ethyl acetate to obtain an ethyl acetate layer and an aqueous layer. The ethyl acetate layer was concentrated under reduced pressure to obtain 18.4 g of ethyl acetate fraction.

Again, the water layer was extracted twice with butanol to obtain a butanol layer and a water layer. The butanol layer was concentrated under reduced pressure to obtain 39.6 g of a butanol fraction. The water layer was concentrated under reduced pressure to obtain 28.8 g of a water fraction.

<Experimental Example 1> Analysis of the extract of the dragonfly extract of the present invention

Ethyl acetate fractions were prepared from the drip irrigation extract prepared by the method of Example 1 of the present invention.

1 g of the prepared ethyl acetate fraction was dissolved in 10 ml of methanol for HPLC and passed through a 0.45 μm sample filter, which was separated using a stable high-performance liquid chromatography.

High-performance liquid chromatography was performed using a Waters Co model DeltaPrep equipped with a Waters Co SunfirePrep (5 μm, 19 × 150 mm), acetonitrile and distilled water 30:70, and the mobile phase solvent was removed by flowing at a flow rate of 5 ml / min.

Using a Waters dual λ Absorbance Detector, the compounds were separated using an ultraviolet absorbance pattern of the material and an absorbance at a wavelength (254 nm), resulting in 34 mg of a compound in 20 minutes (FIG. 2) .

5 mg of the separated compound was dissolved in 5 mL of methanol for HPLC, and the mass was measured using a LC-Mass-Mass stabilized at a concentration of 1 mg / mL passed through a 0.2 μm sample filter.

As a result of a negative analysis, a molecular weight of 594 was obtained (FIG. 4).

12 mg of pure purified compound was completely dried and dissolved in CD ₃ OD (0.6 ml), injected into a 5 mm NMR tube, and analyzed with a Bruker (Germany) model (AVANCE-500 MHz) ¹ H-NMR, ¹³ C-NMR, Dept 45, Dept 90, Dept 135, COZY, HSQC, TOCSY and HMBC were analyzed by NMR.

As a result of the measurement, the physicochemical properties of the ethyl acetate fraction of the present invention are as follows.

(1) Chemical name: poncirin (purity 98%)

(2) Molecular formula: C ₂₈ H ₃₄ O ₁₄

(3) Molecular weight: 594 (ESI-MS, m / z 593 [M-H])

(4) Chemical structure: see FIG. 1, Table 1

(5) Spectral analysis of ¹ H and ¹³ C-NMR: See FIGS. 4 and 5

(6) Solubility: Soluble in methanol, ethanol, butanol, ethyl acetate, insoluble in chloroform,

(7) Comparisons of poncirine contents in Bryong maturation and Wakamisu

The quantitative analysis of pysilein content in the skipjack matured and mature mature skeletal muscles by HPLC was 3.4 times higher than that of mature mature skeleton (Fig. 7).

<Table 1> Structural analysis of pysyline in the ethyl acetate fraction

Carbon number Chemical shifts (ppm) δC δH 2 78.31 5.57 (1H, dd, J = 2.2,9.1 Hz ) 3 42.05 2.50 (1H, d, J = 2.2 Hz, cis), 2.80 (1H, d, J = 9.1 Hz, trans) 4 197.12 5 162.88 12.07 (1H, s, OH) 6 96.33 6.09 (1H, d, J = 2.1 Hz ) 7 164.85 8 95.13 6.13 (1H, d, J = 2.2 Hz ) 9 162.65 10 103.29 OCH ₃ 3.77 (3 H, s) One' 30.35 2' 128.42 7.45 (2H, d, J = 8.7 Hz ) 3 ' 113.90 6.97 (2H, d, J = 8.7 Hz ) 4' 159.50 5 ' 113.90 6.97 (2H, d, J = 8.7 Hz ) 6 ' 128.42 7.45 (2H, d, J = 8.7 Hz ) One〃 100.35 5.10 (1H, d, J = 8.0 Hz ) 2〃 77.10 3 76.08 4〃 69.58 5 76.87 6 60.41 One'" 97.43 4.55 (1H, s) 2'" 70.44 3 '' ' 70.34 4'" 71.79 5 '' ' 68.23 6 '' ' 17.97

<Experimental Example 2> Measurement of the whitening activity of the extract of the present invention

1. Melan-a cell culture

Melan-a cell lines were cultured in DMEM medium supplemented with 10% FBS (folate serum), 100 nM TPA (12-O-tetradecarnoylphobol-13-acetate) and 50 μg / ml streptomycin and penicillin.

The culture conditions were maintained at 10% CO ₂ , 37 ° C.

2. MTT assay

The MTT method was used to measure inhibition and growth of the cell growth of the dragon extract of the present invention.

15 [mu] l of MTT (5 [mu] g / ml) was added to each well and incubated in a 5% CO ₂ incubator at 37 [deg.] C for 4 hours.

After incubation, the solution was removed and 150 ㎕ of Formazan precipitate formed with DMSO was dissolved and added to 490 nm using an ELISA reader to measure the optical density.

The inhibitory concentration (IC ₅₀ ) was calculated as the concentration of extract added corresponding to 50% of the value obtained by removing the MTT untreated control OD value from the OD values of the untreated group.

As a result, as shown in FIG. 8, the cell proliferation by the ethanol extract of the present invention was slightly lower than that of the control group at a concentration as low as 12.5 占 퐂 / ml, but did not show a significant change.

However, in the hexane fraction, the cell proliferation was gradually inhibited from the concentration of 12.5 ㎍ / ㎖ as compared with the control, and the proliferation was significantly inhibited in a dose dependent manner as the treatment concentration was continuously increased.

In conclusion, the ethanol extract inhibited the proliferation and growth of Melan-a melanocyte cells at a concentration of 25 ㎍ / ㎖ or higher.

In addition, the ethanol extract of the present invention had no cytotoxicity to RAW 264.7 murine macrophage cells up to 25-200 / / ml (FIG. 6B).

3. Experiments to inhibit tyrosinase

Tyrosinase is a metalloprotein that contains a pair of copper ions at the active site and plays an important role in melanin production. Tyrosinase, which oxidizes tyrosine in melanomas to form dopa, It acts as tyrosine hydroxylase and dopa oxidase and is known to act as a major enzyme in the synthesis of melanin.

In addition, the tyrosinase related proteins TRP1 and TRP2, together with tyrosinase, are important proteins for melanin synthesis.

TRP1 promotes melanin synthesis by converting from DHICA to indole-5,6-quinone-2-carboxylic acid.

TRP-2 is a protein involved in the synthesis of melarin, such as TRP-1 and tyrosinase, by converting DOPA chrome to DHICA.

In order to investigate the effect of the extract of the present invention on the melanin synthesis, the final product of tyrosine metabolism, the extracts of melanin were applied to melan-a cells at various concentrations ranging from 12 to 50 μg / And cultured cells were harvested.

That is, the Melan-A cell line was cultured in a 6-well plate (5 × 10 ⁴ cells / ml) for 24 hours and then the extract was added at a concentration of 50-100 μg / ml per well and cultured for 72 hours.

Cells were harvested and the number of cells was measured.

The amount of melanin was measured by adding 200 μl of 1N NaOH to 4 x 10 ⁵ cells, reacting at 95 ° C for 10 minutes, and measuring at 490 nm.

Lysis buffer (50 mM phosphate buffer, pH 6.8, 1% triton X-100, 2 mM MSF) was added to 5 x 105 cells and lysed by ultrasonic method.

The mixture was centrifuged at 15,000 rpm for 20 minutes, and the supernatant was used as an assay for tyrosinase activity.

0.3 ml of the sample was added to 0.3 ml of L-tyrosine (1.5 mM) and 30 μM of L-dopa (0.06 mM), and the mixture was reacted at 37 ° C for 30 minutes.

The activity of tyrosinase was measured at 475 nm.

As a result, it showed a thiocyanate inhibitory activity of 75% when treated with melazove 30 uM (Pacific).

The extracts of Trichoderma viride showed inhibition rates of 44%, 62% and 77% at 12.5, 25 and 50 ㎍ / ㎖, respectively.

As shown in FIG. 8, tyrosinase activity tended to be suppressed with increasing treatment concentration and it was statistically significant.

These results suggest that the dragonfly extract of the present invention may inhibit the activity of tyrosinase and prevent the progress of melanin synthesis.

The ethanol extract showed higher tyrosinase inhibitory activity than the respective fractions (water, hexane, ethyl acetate, butanol) (Fig. 8A).

Furthermore, the hexane fraction is showed the above four kinds of fractions highest tee tyrosinase inhibition effect in the resulting 5% respectively process the 2.5, 5, 10 ㎍ / ㎖ , 22%, inhibition of the 50% effect, IC ₅₀ is 10 [mu] g / ml (Fig. 8B).

In particular, the whitening effect of melazove (30 uM), which is a commercially available whitening compound, was overcome.

At this time, treatment with more than 10 ㎍ / ㎖ of hexane fraction showed cytotoxicity when treated with 25 ㎍ / ㎖ of MTT and showed whitening effect within the range of no cytotoxicity.

4. Melanin synthesis inhibitory effect experiment

In order to examine the effect of the extract of the present invention on melanin synthesis in vitro, the extracts of melanin were treated at various concentrations ranging from 25 to 50 μg / ml in melan-a cells and cultured for 7 days. Melanin production was measured.

The amount of melanin was modified by the method of Hosoi et al.

Cells were cultured, washed twice with PBS, and centrifuged to make cell precipitate.

200 ㎕ of 1 N NaOH solution was added, dissolved at 80 캜 for 1 hour, and absorbance was measured at 490 nm.

The amount of melanin in the experimental group was calculated as a percentage of the amount of melanin in the control group.

As a result, it was shown that the dose of the dragon extract of the present invention tended to be inhibited by melanin production as the treatment concentration was increased, and it was statistically significant (FIG. 9).

Ethanol extract inhibited melanin synthesis in a concentration dependent manner. Ethanol extract showed a high whitening effect (IC ₅₀ ) of 25 ㎍ / ㎖ (FIG. 9A) and inhibition rate (IC ₅₀ ) of hexane fraction of 10 ㎍ / Ml, showing a high whitening effect (Fig. 9B).

5. Analysis of effects on the expression of tyrosinase, TRP-1, TRP-2 and MITF protein

In order to examine the effect of the dragonfly extract of the present invention on melanin synthesis in vitro, the extracts of the dragonflies were treated at various concentrations ranging from 12.5 to 100 ㎍ / ㎖ in melan-a cells, cultured for 7 days, The expression patterns of tyrosinase, TRP-1, and TRP-2 proteins involved in the expression of these proteins were analyzed using western blotting.

As a result, as shown in FIG. 10, the expression of tyrosinase and TRP-1 protein in the ethanol extract of the present invention decreased sharply.

Tyrosinease is the main protein in the synthesis of melanin as an enzyme that acts on DOPA conversion from L-tyrosine to DOPA quinone conversion in DOPA and on indole-5,6-quinone-2-carboxylic acid conversion from DHICA in melanin synthesis.

In addition, TRP-1 is an enzyme that acts on the conversion of indole-5,6-quinone-2-carboxylic acid in DHICA and is involved in the synthesis of melanin together with tyrosinase.

Therefore, the treatment of tyrosinase and TRP-1 in the treatment of 25 μg / ml of diller immature and ethanol extract of the present invention shows a remarkable inhibitory effect.

In the diller immature and ethanol extracts of the present invention, the expression pattern of tyrosinase and TRP-1 protein was decreased in a concentration-dependent manner and was independent of the inhibition of TRP-2 protein expression (FIG. 10A).

However, in the hexane fraction, the expression pattern of tyrosinase and TRP-1 protein was somewhat less inhibitory than the ethanol extract (Fig. 10B).

6. Analysis of effects on the expression of tyrosinase, TRP-1, TRP-2 and MITF mRNA

In order to examine the effect of the dragonfly extract of the present invention on melanin synthesis in vitro, the extracts of the dragonflies were applied to melan-a cells at various concentrations ranging from 12.5 to 100 μg / ml, and the cells were cultured for 7 days. Immunoblot analysis and RT-PCR were performed to analyze the expression patterns of involved tyrosinase, TRP-1, TRP-2 and MITF mRNA.

For immunoblot analysis, the cell lysate was electrophoresed on an SDS-PAGE gel and transferred to a nitrocellulose membrane.

The NC (nitrocellulose) membrane was blocked with a blocking buffer containing 5% skim milk and reacted in the primary antibody solution for 1 hour at room temperature.

The cells were washed three to four times in TTBS buffer, reacted with a secondary antibody solution with HRP (horseradish peroxidase), reacted with ECL (Enhanced Chemiluminescence) substrate solution for 1 minute, and exposed to X-ray film for development.

CDNA was synthesized using the Improm-II ^™ cDNA kit (promega). The cDNA was amplified from the cDNA using whitening-related (Tyrosinase, TRP-1, TRP-2) and β- Actin primers, followed by tyrosinase, TRP-1, TRP-2, and Mitf mRNA expression were observed.

RT-PCR was performed using Trizol reagent (Invitrogen, Life Technologies). Total RNA was isolated according to the method provided by the kit and RNA was quantitated at 260 nm absorbance.

The cDNA was made up to a total volume of 5 μl with 1 μg of total RNA, 0.5 μg / μl of Oligo (dT) ₁₅ , and Nuclease-Free Water, heated at 70 ° C for 5 minutes and immediately left on ice for 5 minutes.

cDNA synthesis was performed using a previously prepared reverse transcription reaction mixture (Nuclease-Free Water, imProm-II ^™ 5X reaction buffer, 10 mM dNTP (final concentration 0.5 mM), recombinant RAasin ^R ribonuclease inhibitor, imProm-II ^TM reverse transcriptase] was synthesized as a mixture after 5 min at 25 ℃ in 42 ℃ 60 minutes.

Then, the reaction was stopped by heating at 70 ° C for 15 minutes, and PCR was performed.

The primers shown in Table 2 below were used. PCR was carried out for 25 cycles in total with denaturation (1 minute at 94 ° C), annealing (1 minute at 52 ° C), and extension (72 ° C for 1 minute).

The PCR product was confirmed by electrophoresis on agarose gel of 1 to 1.5% depending on the size of the product.

Table 2: Primer sequences used for RT-PCR analysis in melan-a melanocytes

gene Primer base sequence SEQ ID NO: Short size Reference Tyrosinase F 5'-GGCCAGCTTTCAGGCAGAGGT-3 ' One  510 Jung et al . (2001) R 5'-TGGTGCTTCATGGGCAAAATC-3 ' 2 TRP-1 F 5'-GCTGCAGGAGCCTTCTTTCTC-3 ' 3 290 Jung et al . (2001) R 5'-AAGACGCTGCACTGCTGGTCT-3 ' 4 TRP-2 F 5'-GGATGACCGTGAGCAATGGCC-3 ' 5 1200 Jung et al . (2001) R 5'-CGGTTGTGACCAATGGGTGGC-3 6 Mitf F 5'-CCCGTCTCTGGAAACTTGATCG-3 ' 7 410 Kim et al . (2003) R 5'-CTGTACTCTGAGCAGCAGGTG-3 ' 8 β-actin F 5'-TGGAATCCTGTGGCATCCATGAAA C-3 ' 9  350 Jung et al . (2001) R 5'-TAAAACGCAGCTCAGTAACAGTCC G-3 10

As a result, as shown in FIG. 11, the expression of TRP-2 mRNA was inhibited when treated with dillong immature and ethanol extracts, and the expression of tyrosinased hk TRP-2 mRNA was inhibited when dripstone maturation and ethanol extracts were treated Respectively.

In addition, tyrosinase and hexane fraction inhibited the expression of TRP-2 mRNA in the dillon immature and ethyl acetate fractions.

The extract of the present invention suppresses the expression of tyrosinase, TRP-1, and TRP-2 mRNA, which are involved in melanogenesis, as compared with the suppression of protein expression, It may also be involved in post-transcriptional modification.

<Experimental Example 3> The antioxidant activity test for the extract of the dragon of the present invention

1. DPPH radical scavenging activity experiment

The antioxidant activity of the extract of the present invention was analyzed using the DPPH method.

That is, 0.5 ml of a 0.2 mM DHHP solution and 1 ml of a red blood cell extract sample were mixed, and the OD was measured at 517 nm after incubation at room temperature for 10 minutes.

The antioxidative effect of the sample is indicated by the reducing power of each sample, which is expressed as scaenging activity SC ₅₀ .

The electron donating ability was calculated as a percentage difference between the absorbance of the sample added and the non - added sample.

As a result, the dragonfly extract of the present invention showed much better inhibitory activity than Trolox, BHA, and ascorbic acid (< 100 ug) commonly used (Table 3).

2. Experiments on xanthine oxidase and peroxide radical scavenging activity

Xanthine / xanthine oxidase was prepared by adding 100 μl of the reaction buffer (200 mM sodium phophate buffer, pH 7.5, 0.5 mM xanthin, 1 mM EDTA) to a 96-well plate, .

The reaction was initiated by the addition of 100 μl xanthine-oxidase solution (200 mM sodium phsphate pH 7.5, 1 mM EDTA), followed by 30 min of reaction at 37 ° C., followed by addition of 20 μl of hydrochloric acid HCl) was added to the reaction.

Well mixed at room temperature for 10 minutes and then measured at 290 nm.

Superoxide readical scavenging activity was added to the reaction solution (200 mM sodium phophate buffer, pH 7.5, 0.5 mM xanthin (Sigma X0626), 1 mM EDTA, 0.5 mM NBT (Sigma N5514) at various concentrations.

100 μl of xanthine oxidase (50 mU / ml, Sigma X1875) was added and reacted at 37 ° C for 30 minutes and then measured at 550 nM.

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

The xanthine oxidase inhibitory activity and the superoxide scavenging activity are expressed as the concentration of the sample (IC ₅₀ ) when the absorbance of the uric acid and the superoxide is respectively decreased by 50% .

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

(%) = (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

As a result, the superoxide radical scavenging activity of the ethanol extract of the present invention showed an IC ₅₀ > 500 ㎍. However, xanthine oxidase inhibitory activity showed a high uric acid inhibitory activity in hexane fraction and ethyl acetate (IC ₅₀ 122 ㎍ / ㎖) fraction (Table 3).

<Table 3> Antioxidant Activity Test Results of the Drake Extract of the Present Invention

process IC ₅₀ ([mu] g / ml) ^a) DPPH radical scavenging activity Peroxide radical scavenging activity Xanthine oxidase inhibitory activity Ethanol extract 1119.2 ± 27.3 1460.0 ± 46.2 286.0 ± Water fraction 1314.7 ± 36.8 > 2000 > 2000 Butanol fraction 710.8 + - 23.5 700.8 ± 32.5 126.4 ± 24.6 Hexane fraction 1618.0 ± 14.1 540.2 ± 20.3 226.6 + - 67.9 Ethyl acetate fraction > 2000 1135.9 ± 39.1 - Trolox 8.0 ± 1.6 59.5 ± 15.5 144.7 ± 0.6 BHA ^{b )} 7.3 ± 0.1 NA ^{c )} NA ^{c )} Allopurinol - 1.2 ± 0.8 - Ascorbic acid 15.2 ± 0.2 8.8 ± 0.4 7.8 ± 1.9

* 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 is Not available method.

<Experimental Example 4> Measurement of anti-inflammatory activity against the extract of dragon dragon of the present invention

Anti - inflammatory activity was measured by RAW 264.7 cells, macrophage cell line, and the effect of LPS - induced nitric oxide production inhibiting effect of the extract of the present invention was measured.

Raw 264.7 cells were cultured in a 48-well plate (1.7 × 10 ⁵ cells / ml) for 24 hours and then treated with the dithionol ethanol extract of the present invention (50-100 μg / ml) and LPS (1 μg / Lt; / RTI &gt;

100 μl of the culture was transferred to a 96-well plate, and the same amount of griess reagent was added thereto. The reaction was allowed to proceed at room temperature for 10 minutes and then quantified at 540 nm.

And quantified by using sodium nitrate as a standard substance.

As a result, the drosophila ethanol extract of the present invention strongly suppressed NO produced by LPS ((IC ₅₀ &Lt; 150ug) (Fig. 13A).

In addition, anti-inflammatory activity of LPS-induced inhibition of iNOS and Cox-2 protein expression was analyzed by western blotting.

As a result, inhibition of iNOS expression was 50% inhibited by 25 μg / ml of the dyestuff ethanol extract of the present invention, 90% inhibited by 100 μg / ml (FIG. 12A), and Cox-2 expression (FIG. 12B) showed a suppressive effect up to 60% when treated with 100 μg / ml, indicating a high anti-inflammatory effect.

According to the present invention, a drip irrigation extract showing whitening activity, antioxidative activity, and anti-inflammatory activity is provided.

Further, the present invention provides a whitening composition comprising an extract of a dragonfly as an active ingredient, and provides an antioxidant composition and a composition for the prevention and treatment of inflammatory diseases.

<110> Jeju-Hi Tech Industry Development Institute <120> PONCIRUS POLYANDRA EXTRACTS HAVING WHITENING ACTIVITY AND          ANTI-INFLAMMATORY ACTIVITY <160> 10 <170> Kopatentin 1.71 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> B16 Murin Melanoma cells <400> 1 ggccagcttt caggcagagg t 21 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> B16 Murin Melanoma cells <400> 2 tggtgcttca tgggcaaaat c 21 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> B16 Murin Melanoma cells <400> 3 gctgcaggag ccttctttct c 21 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> B16 Murin Melanoma cells <400> 4 aagacgctgc actgctggtc t 21 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> B16 Murin Melanoma cells <400> 5 ggatgaccgt gagcaatggc c 21 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> B16 Murin Melanoma cells <400> 6 cggttgtgac caatgggtgg c 21 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> B16 Murin Melanoma cells <400> 7 cccgtctctg gaaacttgat cg 22 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> B16 Murin Melanoma cells <400> 8 ctgtactctg agcagcaggt g 21 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> B16 Murin Melanoma cells <400> 9 tggaatcctg tggcatccat gaaac 25 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> B16 Murin Melanoma cells <400> 10 taaaacgcag ctcagtaaca gtccg 25  

Claims (4)

In the extract of a plant of the genus Tangifera, Poncirus polyandra immature fruits were pulverized by a wet pulverizer, the pulverized material was lyophilized to prepare a powder, the powder was immersed in 70% ethanol, stirred for 2 to 3 days at room temperature and leached, The filtrate was concentrated under reduced pressure. The concentrate was suspended in distilled water, and the suspension was successively extracted with hexane, ethyl acetate and butanol. The residue was purified by filtration through a filter, and the leaching and filtration process was repeated 2 to 3 times. As a result, An antioxidant activity, and an anti-inflammatory activity. Dyrrhoid extract showing physiological activity. A whitening composition comprising the dyer extract of claim 1 as an active ingredient. Claims [1] A pharmaceutical composition comprising the dragon extract of claim 1 as an active ingredient, A composition for the prevention and treatment of inflammatory diseases. An antioxidant composition comprising the dragon extract of claim 1 as an active ingredient.

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