KR100642744B1 - Tsunokaori skin extracts having physological activity and foods using that - Google Patents

Abstract

A peel extract of Citrus tree "Tsunokaori" having excellent physiological activity such as antioxidant activity, anti-aging activity, antibacterial activity, etc. is provided. A food is manufactured by using the peel extract of Citrus tree "Tsunokaori". The peel extract of Citrus tree "Tsunokaori" is obtained by the steps of: separating the peel of Tsunokaori from the flesh; freeze drying the separated peel; grinding the freeze dried peel; soaking the ground peel in 70% ethanol; removing residues from an ethanol precipitate using a filter and recovering the filtrate; concentrating the filtrate under reduced pressure; and drying the concentrate under vacuum and freeze drying residual moisture. For an example, the food such as a drink composition is obtained by mixing 100ml Tsunokaori peel extract, 0.3g citric acid, 0.1g sodium citrate, 0.1ml orange extract, 700ml purified water and 12ml liquid fructose and agitating with 200ml purified water.

Description

Physiologically active fruit extract and food using it {TSUNOKAORI SKIN EXTRACTS HAVING PHYSOLOGICAL ACTIVITY AND FOODS USING THAT}

1 is a manufacturing process diagram of the true-flavored skin extract of the present invention

2 is a standard graph for flavonoid component determination

Figure 3 is a graph showing the effect of citrus peel extract on HepG2 cells (MTT reductive activity)

A: Graph showing the results measured after 1 day treatment with H ₂ O ₂ (5 mM)

B: Graph showing the results measured after 4 days treatment with H ₂ O ₂ (5 mM)

  I1: Serious peel extract K1: Axilla extract

  M1: Wenzhou Immature and Skin Extract

Figure 4 is a graph showing the effect of citrus peel extract on HepG2 cells (LDH dissociation activity)

  I1: Serious peel extract K1: Axilla extract

  M1: Wenzhou Immature and Skin Extract

5 is a photograph showing the effect of citrus peel extract on the viability of HepG2 cells against oxidative stress

  I1: Serious peel extract K1: Axilla extract

  M1: Wenzhou Immature and Skin Extract

Figure 6 is a graph showing the results of ROS MTT measurement in HepG2 cells for citrus fruit extract

  I1: Serious peel extract K1: Axilla extract

  M1: Wenzhou Immature and Skin Extract

7 is an experimental result showing the degree of inhibition of iNOS, COX-2, ρ-ERK1 / 2 activity in RAW cells for the hot persimmon persimmon extract (H1)

A: Western blot showing the degree of inhibition of iNOS, COX-2 and ρ-ERK1 / 2 activity in RAW264.7 cells

B: graph showing NO production

8 is an experimental result showing the degree of inhibition of iNOS, COX-2, ρ-ERK1 / 2 activity in RAW cells for citrus peel extract

A: Western blot showing the degree of inhibition of iNOS, COX-2 and ρ-ERK1 / 2 activity in RAW264.7 cells

B: graph showing NO production

  I1: Serious peel extract K1: Axilla extract

  M1: Wenzhou Immature and Skin Extract

The present invention relates to a true flavoured skin extract and a food using the same.

Citrus fruits are the most produced fruit in Korea (Ministry of Agriculture, Fruit and Vegetable Production by Year, 2002), and mainly contain various compounds such as flavonoids, vitamins, citric acid and carotenoids.

Citrus fruits are known to have various pharmacological activities. Especially, flavonoids, one of the main components of citrus peels, are known to have an effect on cardiovascular disease and anti-cancer, antioxidant, anti-inflammatory, antiviral and capillary potentiating effects. have.

Citrus peels contain four times more vitamin C and more than eight times more vitamin C than the flesh (Recommended Dietary Allowances for Koreans, 7 th Rewision, p316). 317, 2000).

In recent years, consumer preferences for citrus fruits have been diversified and advanced (Jeju citrus experiment station: Research paper of citrus corp for 1998, Jeju citrus grower's agr. Coper., Jeju, p. 46-50; Shewfelt, RL 1999 What is quality, Postharvest Biol, Technol, 3: 197-200) Producers are also diversifying their varieties.

As a result, the area of cultivation of citrus fruits such as dogs, localization, jinjihyang, crisps, and Wenzhou immature is gradually increasing (Jeju provincial government: 2000 Annual report of citrus marketing, Jeju provincial government, Jeju, Bul. 5-14) .

Among them, Tsunokaori is one of the citrus varieties made by crossing the Citrus sinensis and the Citrus unshiu varieties and the endemic species produced only in Jeju Island.

The fruit size is 160 ~ 200g, which is smaller than Cheongju Orange, and the fruit shape is closer to Wenzhou Citrus, and the skin color is darker than Cheongju Orange, and the sugar content is higher than Cheongdog Orange and the fruit flesh is soft and juicy. .

Recently, with the development of domestic fruit drinks, there has been interest in the food use of citrus peels emitted as by-products in the manufacturing process.

However, there is no report on the development of functional foods using physiologically active skin extracts, and the acquisition of new materials is not only the development of functional foods but also its application to the pharmaceutical industry. It is true.

Korean Patent Application Publication No. 1999-0080214 (Health Improvement Functional Drink Containing Citrus Peel Extract) discloses a functional drink for health improvement manufactured using a citrus peel extract or a bioflavonoid derived from citrus peel. .

In Korean Patent Application Publication No. 10-2004-0069587 (biological manufacturing process of active ingredient from citrus gourd), dietary fiber material and functional sweetener in citrus gourd are produced biotechnologically, using continuous process and solvent recovery process. Therefore, it is disclosed that the pollution prevention process through the feed through the fermentation of the remaining residue to prevent the consumption and spillage of the solvent.

Korean Patent Laid-Open Publication No. 1999-0080216 (Enhanced Tangerine Tea) discloses a high value-added functional tangerine tea manufactured using solids generated after the juice of citrus juice, which is currently used as compost, waste or livestock feed. .

However, as described above, the research on the true flavour-derived skin extract with excellent physiological function activity has not been made yet compared to the conventional citrus extract.

It is an object of the present invention to provide a true skin extract having a physiological activity excellent in antioxidant activity, anti-aging activity, anti-inflammatory activity.

In addition, an object of the present invention is to provide a beverage composition, food additives using the extract of the true flavor fragrance.

The present invention relates to a true flavoured skin extract and a food using the same.

The true-flavored rind extract of the present invention is a first step of separating the rinds and the rinds of the true-oriented, a second process of lyophilizing the separated rind, and a third process of grinding the lyophilized rind of the lyophilized rind into a fine powder. , The fourth step of depositing the fine powder of the third step in 70% ethanol, the fifth step of recovering the filtrate after removing the residue from the ethanol deposits of the fourth step using a filter, the second step of recovering the filtrate under reduced pressure It consists of preparing a true-flavored skin extract with physiological activity through 6 steps.

In addition, after the true-flavored skin extract of the present invention as it is or after vacuum drying, the residual moisture is lyophilized to prepare a powder to prepare a food additive using the true-flavored skin extract.

In addition, the beverage extract containing the true extract of the extract of the present invention is prepared in a conventional manner by diluting or diluting the extract from the extract of the present invention.

In addition, after the true-orientated skin extract of the present invention as it is or after vacuum drying, residual moisture is lyophilized to prepare a powder to provide a disease prevention composition using the true-oriented skin extract.

The true peel was extracted with 70% ethanol and concentrated to analyze the flavonoid content.

In addition, citrus fruit extracts were measured for physiological activities such as hydrogen donating ability and cytotoxicity by DPPH.

As a result, among the various citrus fruit extracts, the flavonoid content of the true fruit extract was the highest (Table 3).

By treating a citrus peel extract for a type of HepG2 cells for liver cell line of human were investigated for cell viability, the results oriented peel extract true of a number of citrus peel extract in a concentration dependent manner with the cell death phenomena induced by H ₂ O ₂ Inhibition showed a high cytoprotective effect (FIG. 3).

In addition, the activity of lactate dehydrogenase (LDH) was examined to determine the cytotoxicity of the citrus peel extract. Experimental results showed that the cytotoxicity increased by H ₂ O ₂ was generally reduced (FIGS. 4, 5). HepG2 hepatocellular carcinoma cell lines showed significant changes in ROS scavenging activity in HepG2 cells after treatment with Hydrogen peroxide. Significant antioxidant activity was shown (FIG. 6).

The effects of NO formation and expression of iNOS, COX-2 production and activity inhibition from RAW 264.7 cells, a murine macrophage cell line of citrus peel extracts, were determined in a concentration-dependent manner. It was confirmed that the expression of the protein is significantly inhibited, the extract of true-oriented skin extract was confirmed that the protein expression of COX-2 was significantly inhibited as in the result of inhibiting the protein expression of iNOS (Fig. 7, 8).

In addition, we observed the effect of LRK (100 ng / mL) on the inhibition of ERK1 / 2 protein phosphorylation, which is induced by LPS (100 ng / mL) in RAW264.7 cells. It could be confirmed (Fig. 7,8).

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples for the physiologically active fruit extract and food using the same, but these do not limit the scope of the present invention.

Example 1 Preparation of True-orientated Peel Extract Having Physiological Activity

Jinji-hyang was harvested on April 2004 in farms in Seogwipo, Jeju.

2 kg of the prepared jinjihyang was separated into pulp and skin.

500 g of the separated rind were lyophilized.

150 g of lyophilized true peel was ground to a fine powder.

100 g of the ready-made true skin powder was immersed in 1 L of 70% ethanol.

After removing the residue from the ethanol deposits using a filter, 900 ml of the filtrate was recovered.

The recovered filtrate was concentrated under reduced pressure to prepare 300 ml of the extract of true-flavored skin of the present invention.

<Comparative Example 1> Preparation of elbow skin extract

A skin extract was prepared in the same manner as in Example 1 of the present invention, but was prepared using Citrus hassku Hort. Instead of true incense .

Comparative Example 2 Wenzhou Immature and Skin Extract Preparation

It was prepared in the pericarp extract in the same manner as in Example 1 of the present invention, instead of a directed Ontario immature and; was prepared using the (Citrus unshiu Unripe Citrus).

<Comparative Example 3> Wenzhou citrus fruit extract preparation

A skin extract was prepared in the same manner as in Example 1 of the present invention, but was prepared using Citrus unshiu instead of true flavor .

<Example 2> Preparation of food additives using the true-flavored skin extract of the present invention

The true extract of perilla extract prepared in Example 1 of the present invention was prepared.

100 ml of the prepared skin extract was vacuum-dried and lyophilized to remove residual moisture, and then powdered to prepare 5 g of a fruit extract, a food additive.

Example 3 Preparation of Beverage Composition Using Ginseng Extract Perfume Extract of the Present Invention

The true extract of perilla extract prepared in Example 1 of the present invention was prepared.

0.3 g of citric acid, 0.1 g of sodium citrate, 0.1 ml of orange essence, 700 ml of purified water were added to 100 ml of the prepared extract, and the sugar content was adjusted using 12 ml of liquid fructose, followed by stirring with 200 ml of the remaining purified water. A beverage composition was prepared using the extract of the true fragrance skin extract of the present invention.

Experimental Example 1 Analysis of Flavonoid Content in True Perfume Extract of the Present Invention

1. Sample Preparation

10 ml of true-flavored skin extract prepared by Example 1, 10 ml of fresh skin extract prepared by Comparative Example 1, 10 ml of Wenzhou immature and skin extract prepared by Comparative Example 2, Wenzhou prepared by Comparative Example 3 10 ml of citrus peel extract was prepared.

Each solution was filtered through a 0.45 μm filter (MFS, MFS-25, CA syringe filter, 25 mm), respectively.

The filtered filtrate was used as a sample for flavonoids analysis.

2. HPLC Assay

Standard reagents used for HPLC (Knauer) analysis were rutin hydrate (Sigma Co.), ferulic acid (Sigma Co.), naringin (Sigma Co.), hesperidin (Sigma Co.), naringenin (Sigma Co.), hesperitin (Sigma Co.) was used.

The mobile phase was prepared by adding 5 ml of acetic acid to acetonitrile and 1 L of ultrapure distilled water.

Analytical conditions for analyzing flavonoid components from the composition of the mobile phase and HPLC are as follows (Table 1, Table 2, Figure 2).

Table 1. Conditions for Flavonoid Analysis by HPLC

Instrument: Knauer HPLC system Column: Eurospher-100 C-18 (300 × 4.6 mm ID.5 μm) Detector wavelength: 254 nm Flow rate: 1.0 ml / min Injection volume: 20 μl Column Temp: 25 ℃

Table 2. Fluid Bed Conditions of Flavonoid Analysis by HPLC

Program order Time (min) Acetonitrile (%) Water (%) One 0 18 82 2 25 18 82 3 30 38 62 4 40 38 62 5 45 18 82 6 60 18 82

In the above method, the results of analyzing the flavonoid components content of the citrus fruit extracts are shown in Table 3.

Table 3. Flavonoid Components Content Analysis of Citrus Peel Extracts

Skin Extract Type Naringin (ml / ℓ) Hesperitin (ml / ℓ) Serious orientation (Example 1) 9.23 14.86 Elbow 7.83 - Wenzhou Immature 9.55 - Wenzhou Citrus 4.65 -

As described above, as a result of analyzing the content of flavonoid components of citrus fruit extracts, the contents were analyzed using Naringin and Hesperitin as a standard among various components reported to be contained in a large amount of citrus peels.

Among flavonoids, Naringin contained 4.65 ㎖ / ℓ in the currently produced hot jujube persimmon peel, while it was 9.23 ㎖ / ℓ in the true flavour peel, which was twice as high as that of the junju peel and hesperitin peel. A high content of 14.86 ml / l was shown.

<Experimental Example 2> Antioxidant Activity Test on True Perfume Extract of the Present Invention

The most characteristic mechanism of antioxidants is the reaction with free radicals. The free radical scavenging action is used as a measure of the antioxidant effect in plants and the aging of 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 amines (ρ-phenylenediamine, ρ-aminophenol), which are widely used for measuring the antioxidant activity of antioxidants (Blois, MS 1958). , Antioxidant determination by the use a stable free radical, Nature 26: 1199-1200).

Therefore, the antioxidative activity test was performed on the extract of the true-flavored skin of the present invention as follows.

1. Preparation of experimental materials

10 ml of true-flavored skin extract prepared by Example 1, 10 ml of fresh skin extract prepared by Comparative Example 1, 10 ml of Wenzhou immature and skin extract prepared by Comparative Example 2, Wenzhou prepared by Comparative Example 3 10 ml of citrus peel extract was prepared.

2. Measurement of electron donating ability

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

In other words, 100 μl of each concentration of the sample dissolved in methanol was dispensed in a 96 well plate, and 0.4 mM DPPH solution was added in the same amount, and the resultant was allowed to stand at room temperature for 10 minutes, and then the absorbance was measured at 517 nm. Butylated hydroxy anisole (BHA), and trolox were used.

DPPH free radical scavenging activity was calculated from the following equation, expressed as the concentration of the sample (IC ₅₀ ) which appears 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 _control is the absorbance of the reaction solution to which the sample is added.

* A _sample is the absorbance of the reaction solution with methanol added instead of the sample.

In the above method, the results of measuring the antioxidant activity of the citrus fruit extract sample by the free free radical scavenging activity of DPPH are shown in Table 4.

Table 4. Free free radical scavenging activity of DPPH in citrus peel extracts

DPPH scavenging ^a) IC ₅₀ (μl / ml) True Oral Skin Extract (Example 1) 104.4 ± 15.8 Crisp Skin Extract 609.8 ± 84.2 Wenzhou Immature and Skin Extract 132.2 ± 16.4 Wenzhou Citrus Skin Extract 215.2 ± 10.6 Trolox 11.2 ± 0.6 ^b) BHA 6.8 ± 0.0

* ^a) IC ₅₀ is the statistical value of the concentration of each sample that appears when the absorbance of DPPH is reduced by 50%.

^b) BHA is butylated hydroxy anisole.

As a result of measuring the antioxidant activity of citrus fruit extract samples by the free free radical scavenging activity of DPPH (1,1-dipheny1-2-picryl-hydrazyl) as described above, it was found that the radical scavenging activity was significantly higher in true-orientated, hot-rice immature and skin extracts. Among them, it showed the highest radical scavenging activity in true extract.

Experimental Example 3 Effect Test of True-Oriented Peel Extract of the Present Invention on HepG2 Cell Viability

In order to examine the effect of Jeju citrus fruit extracts on the viability of HepG2 cells, they were treated with HepG2 cell line, a kind of human hepatocellular carcinoma cell line.

In this experiment, the effect of Wenzhou persimmon rind extract was significantly lower than that of other citrus rinds.

1. Sample Preparation

10 ml of true-flavored skin extract prepared by Example 1, 10 ml of fresh skin extract prepared by Comparative Example 1, 10 ml of Wenzhou immature and skin extract prepared by Comparative Example 2, Wenzhou prepared by Comparative Example 3 10 ml of citrus peel extract was prepared.

2. Cell Culture

In this experiment, HepG2 cells, liver cancer cell lines, were cultured in a serum-free midium for 24 hours, and then the prepared samples were prepared for 1 hour according to the concentration group (125, 250, 500, 1000, 2000 μl / ml). The cell viability was examined after pretreatment and then treatment with H ₂ O ₂ (5 mM) for 1 day and 4 days.

Cells were treated with 5% CO using Dulbecco's minimal essential medium (D-MEM) containing 100 U / ml penicillin, 100 μg / ml streptomycin and 10% fetal bovine serum. ₂ and 37 ℃ were incubated in the incubator, subculture was carried out once every 3 to 4 days.

3. MTT assay

MTT assaysms is a method of measuring the mitochondria activity of the cells and indirectly measuring the survival of the cells. After cell culture, the culture medium is removed and 200 μl of MTT reagent (Sigma) is mixed for 30 minutes to 1 hour at 37 ℃. After the reaction solution was removed, 200 μl of isopropanol was added to induce a color reaction, and the absorbance was measured at 570 nm.

4.LDH cytotoxicity detection

Lactate dehydrogenase (LDH) is a stable cytoplasmic enzyme present in most cells and is released into cell culture media when the plasma membrane is damaged.

Therefore, it is a simple and simple colorimetric assay to measure the LDH activity released by damaged cells.

In this experiment, LDH cytotocixity detection kit (Takara) was used to measure activity at 492 nm.

5. H33342 Dyeing

In order to examine cell survival or apoptosis, H33342 (Sigma), a fluorescent dye that specifically binds to intracellular DNA, was added to the cultured cells and incubated at 37 ° C for 30 minutes, followed by fluorescence with a CoolSNAP-Pro color digital camera. Observation was made under a microscope.

The degree of cell condensation and apoptotic body formation were observed and used as an indicator of cell survival or apoptosis.

6. Experimental Results

As a result of this experiment, the extract of true-orientated skin extract increased the viability of HepG2 cell line in a concentration-dependent manner (Fig. 3, 4).

In addition, as a result of investigating the dissociation activity of LDH to determine the cytotoxicity of the citrus fruit extract, the cytotoxicity increased by H ₂ O ₂ showed a tendency to decrease generally (FIG. 4).

As a result of staining the cells by H33342 staining method, each citrus peel extract showed a concentration-dependent inhibition of H ₂ O ₂ induced apoptosis. (FIG. 5).

In conclusion, the results of H33342 staining showed similar results to MTT assay, and especially for the extract of true-oriented skin extract, it could increase the cytoprotective effect by suppressing H ₂ O ₂ induced cell death. It is believed to be.

Experimental Example 4 ROS Measurement Experiment

In this experiment, the results of the wine extract were not significantly lower than those of other citrus fruit extracts.

1. Sample Preparation

10 ml of true-flavored skin extract prepared by Example 1, 10 ml of fresh skin extract prepared by Comparative Example 1, 10 ml of Wenzhou immature and skin extract prepared by Comparative Example 2, Wenzhou prepared by Comparative Example 3 10 ml of citrus peel extract was prepared.

2. Experimental method

Samples prepared in this experiment were treated according to various concentration groups (62.5, 125, 250, 500, 1000 μl / ml) to verify the antioxidant activity of the fractions.

First, HepG2 liver cancer cell line was cultured in nutrient deficient medium for 24 hours, and then each fraction was pretreated for 1 hour and then treated with H ₂ O ₂ (1 mM).

After 15 minutes of treatment with hydrogen peroxide, changes in the contents of intracellular reactive oxygen species (ROS) were measured.

Fluorescent marker H ₂ DCFDA (2'-7'-dichlorofluoresin diacetate) was used as an index of ROS as the intensity of fluorescence emitted in the presence of hydrogen peroxide in the cell.

2. Experimental Results

Citrus fruit extracts showed significant ROS scavenging activity in HepG2 cells, especially concentration-dependent antioxidant activity in true skin extracts (FIG. 6).

<Experiment 5> Anti-inflammatory activity test for the true extract of skin extract of the present invention

LPS, also known as endotoxin, is present in the extracellular membrane of Gram-negative bacteria, and is mediated by inflammatory mediators such as TNF-α, IL-6, and IL-1β in macrophage or monoocytes such as RAW264.7. is known to increase pro-inflammatory cytokine

In addition, the formation of such inflammatory mediators is known to lead to a process of converting arachidonic acid to prostagladin and NO formation due to the activity of phospholipase A2.

NO is an inorganic free body produced by L-arginine by NO synthase (NOS) and is known to be involved in various biological processes such as immune response, cytotoxicity, neurotransmitter and vascular relaxation. Depending on the concentration, it is known to play an important role in maintaining cell function and to cause cytotoxicity.

NOS, which produces NO, is present in cells and is activated by macrophages or vascular endothelial cells independent of calcium and calmodulin-dependent constitutive NOS (cNOS) and calcium. There are two forms of inducible NOS (iNOS), which are forms induced by bacterial endotoxins or various cytokines such as lipopolysaccharide (LPS).

INOS expressed by LPS stimulation produces a large amount of NO, and cytotoxicity is known to be involved in inflammatory reactions, cell mutations and tumor development.

Increased expression of NO and iNOS has been reported in tissue damage associated with inflammatory responses.

In this study, we investigated the effects of LPS stimulation on NO formation, iNOS expression, COX-2 production, and activity inhibition of citrus rind extracts from murine macrophage RAW264.7 cells.

1. Sample Preparation

10 ml of true-flavored skin extract prepared by Example 1, 10 ml of fresh skin extract prepared by Comparative Example 1, 10 ml of Wenzhou immature and skin extract prepared by Comparative Example 2, Wenzhou prepared by Comparative Example 3 10 ml of citrus peel extract was prepared.

2. Cell Culture

RAW264.7 cells, a mouse macrophage line, were distributed from Korean Cell Line Bank (KCLB) using DMEM medium containing 100 units / ml penicillin-straptomycin and 10% fetal bovine serum (FBS). C, incubated in a 5% CO ₂ incubator, subculture was performed once every 3 to 4 days.

Lipopolysaccharide (LPS. E. coli serotype 0111: B4) was purchased from Sigma and used in the experiment.

3. Cytotoxicity Experiment

1) MTT ASSAY

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, and then samples were added for each concentration.

This was incubated for 24 hours, then 100 μg of MTT was added and further incubated for 3 hours.

After removing the medium, 150 μl of dimethylsulfoxide (DMSO, Sigma) was added to dissolve the formazan precipitate produced by the reduction of MTT, and then absorbance at 540 nm using a microplate reader. Measured.

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) LDH cytotoxicity detection

LDH is a stable cytoplasmic enzyme present in most cells and is released into cell culture media when the plasma membrane is damaged.

Therefore, it is a simple and simple colorimetric assay to measure the 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, and then the samples were added by concentration to measure the outflow of LDH after cell culture. The degree of damage was investigated.

Activity was measured at 492 nm using LDH cytotocixity detection kit (Takara).

4. NO production inhibition rate measurement

RAW264.7 cells were adjusted to 1.0 × 10 ⁵ cells / ml in DMEM medium with 10% FBS, inoculated into 48 well plates, and treated with test material and LPS (100 ng / ml) for 24 hours. Incubated.

The amount of NO produced was measured in the form of NO ₂ ⁻ present in the cell culture solution using Griess reagent.

Mix 100 μl of cell culture supernatant with 100 μl of Griess reagent [1% (w / v) sulfanilamide, 0.1% (w / v) naphylethylenediamine in 2.5% (v / v) phosphoric acid] for 10 minutes on 96 well plates. After absorbance was measured at 530 nm.

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

5. Western blot analysis

RAW264.7 cells (1.0 × 10 ⁵ cells / ml) were collected after treatment with citrus peel extracts by concentration.

After washing the cells with PBS (Phosphate Buffered Saline) 2-3 times, 1 ml of lysis buffer was added, lysis was carried out for 30 minutes to 1 hour, and centrifuged at 12,000 rpm for 20 minutes to remove cell membrane components.

Protein concentration was quantified using the Bio-Rad Protein Assay Kit by standardizing BSA (Bovine serum albumin).

30 to 50 ㎍ of lysate was denatured by 8-12% mini gel SDS-PAGE (Poly Acrylamide Gel Electrophoresis), and transferred to PVDF membrane (BIO-RAD) at 200 mA for 2 hours.

Membrane blocking was performed for 2 hours at room temperature in TTBS (TBS + 0.1% Tween 20) solution containing 5% skim milk.

Anti-mouse iNOS (1: 1000) (CALBIOCHEM) was used as an antibody to examine the expression level of iNOS, and anti-mouse COX-2 (1: 1000) was used as an antibody to examine the expression level of COX-2. As an antibody for examining the expression level of ERK1 / 2, the antibody was diluted in anti-mouse ERK1 / 2 (1: 1000) (Cell Signaling) TTBS solution, reacted at room temperature for 2 hours, and washed three times with TTBS. It was.

As a secondary antibody, anti-mouse or anti-rabbit IgG (Amersham Co.) conjugated with HRP (Horse Radish Peroxidase) was diluted to 1: 5000, reacted at room temperature for 30 minutes, washed three times with TTBS, and then ECL. After reacting with the substrate (Amersham Co.) for 1 to 3 minutes, the X-ray film was photosensitive.

6. Experimental Results

As a result of the anti-inflammatory activity of the citrus fruit extract of the present invention, the cytotoxicity in the RAW264.7 of the citrus fruit extract showed little cytotoxicity at the concentration used in the experiment.

Accordingly, the NO production inhibitory effect was measured in the form of NO ₂ ⁻ present in the cell culture by treating at a concentration that does not exhibit cytotoxicity.

As a result, it was confirmed that the excessive generation of NO in the LPS-only treatment group, NO production was reduced in a concentration-dependent manner in the test plot treated with citrus fruit extract at the same time, especially the true-flavored extract (IC ₅₀ = 139) At low concentrations at μl / ml), NO production was significantly inhibited (FIG. 8 -B).

On the other hand, in the case of Wenzhou persimmon rind extract used as a control, the IC ₅₀ was 527.6 μl / ml, and the NO activity inhibition rate was not very high. It was shown (Fig. 7,8).

LPS (100 ng / ml) was used to induce the production of iNOS and COX-2 in RAW264.7 cells. by The degree of inhibition was determined by Western blot.

As a result, the citrus fruit extract significantly inhibited iNOS expression in a concentration-dependent manner, and it was confirmed that COX-2 expression also inhibited in true-orientated and crust extracts as well as the result of protein expression of iNOS (FIG. 7,8). .

However, it was confirmed that the protein extract inhibition of iNOS was not good in the case of Wenzhou persimmon rind extract (FIG. 7,8).

In addition, true and biphasic rind extract showed the effect of inhibiting the phosphorylation of ERK1 / 2 protein, an intranuclear transcription factor induced by LPS (100 ng / ml) in RAW264.7 cells. In particular, it showed a concentration-dependent phosphorylation inhibitory effect in the extract of the true-flavored skin (Fig. 7,8).

According to the present invention, there is provided a true skin extract having an physiological activity excellent in antioxidant activity, anti-aging activity, anti-inflammatory activity and the like.

In addition, the present invention provides a beverage composition, food additives, and the like using the true extract skin extract.

Claims (4)

In skin extract, 1st process for separating the flesh and skin of Tsunokaori, A second step of lyophilizing the separated skin, A third process of grinding the lyophilized true-flavor peel into a fine powder, A fourth step of depositing the fine powder of the third step in 70% ethanol, A fifth step of recovering the filtrate after removing the residue from the ethanol deposit of the fourth step by using a filter; Manufactured by the sixth step of concentrating the recovered filtrate under reduced pressure, Seriously scented rind extract with physiological activity. After the vacuum-derived extract of claim 1 for vacuum extraction, the remaining moisture is lyophilized to prepare a powder, Seriously scented rind extract with physiological activity. In the true-flavored skin extract of claim 1, Characterized in that it is used in one selected from food additives, beverage composition, Seriously scented rind extract with physiological activity. In the true-flavored skin extract of claim 2, Characterized in that it is used in one selected from food additives, beverage composition, Seriously scented rind extract with physiological activity.

Images