KR20170114773A - Anti-inflammatory composition comprising broccoli extracts or cosmetic composition for alleviating skin irritation - Google Patents

Abstract

The present invention provides an anti-inflammatory composition comprising a broccoli extract.
The composition containing the broccoli extract of the present invention shows high antioxidative, antibacterial and anti-inflammatory activities, and thus can be effectively used as a composition for anti-inflammation or a cosmetic composition for alleviating skin irritation.

Description

TECHNICAL FIELD [0001] The present invention relates to an anti-inflammatory composition containing a broccoli extract or a cosmetic composition for alleviating skin irritation,

The present invention relates to an antiinflammatory composition comprising a broccoli extract, and more particularly to a cosmetic composition for relieving skin inflammation comprising a broccoli extract in which a large amount of physiologically active substances such as glucuraphenin and sulforapan appear.

At present, cosmetics for skin moisturizing and moisturizing in domestic are not satisfactory because they have considerable limitations on their functionalities and effective ingredient delivery. Multifunctional cosmeceutical [compound word of cosmetic and pharmaceutical] equipped with all the effects of penetrating deep into the skin and promoting tissue regeneration.

Therefore, in order to demonstrate the performance of the product through the efficacy test for mitigating skin inflammation and inhibiting the inflammation, we tried to develop a differentiated product that is excellent in skin disease and inflammation relief by using the local super food product.

Broccoli (Brassica oleracea) is a variant of cabbage belonging to the Cabbage, a plant belonging to the cruciferous family, and is widely consumed as a salad or food additive in Western Europe, the United States and Far East Asian countries. In addition, broccoli, which is an abundant source of various vitamins and antioxidants, contains a secondary metabolite called glucosinolate (GSL) (Free Radical Research, 1996, 25 (1), 75-86; Food Science and Technology, 2009, 42, 1561-1572).

Glucoraphanin, which is contained in broccoli, acts on the digestive enzymes to form sulfuropha- ine, which is an anticancer substance (Proc. Natl. Acad Sci USA, 1997, 94, 10367-10372) (Proc. Natl. Acad Sci. USA, 2002, 99, 7610-7615) have been reported to specifically kill Helicobacter bacteria that cause gastric cancer.

Korean Patent Laid-Open No. 10-2012-0036516 (Apr. 18, 2012) discloses a cosmetic composition for skin whitening containing a broccoli seed extract, and Korean Patent No. 10-1429946 2014.08.07) discloses a natural soap and cosmetic composition containing broccoli extract and a method for producing the same.

Water is used as one of the most important components in cosmetic compositions, and water used in cosmetics is purified water purified by distillation, ion exchange, and ultrafiltration. Recently, however, consumers' awareness of the function and importance of water has greatly increased, and research on water that can replace purified water with the longing of modern people for uncontaminated pure nature has been actively conducted. For example, Korean Patent Laid-Open No. 10-1995-0023399 (August 18, 1995) discloses a cosmetic composition containing hot spring water, and 10-1995-0028760 (November 22, 1995) discloses hot spring water- and germanium- A cosmetic composition containing ground water is disclosed in Patent Literature 10-2001-0038510 (May 05, 2001), a preparation method of cosmetic composition containing hot spring water is disclosed in Patent Literature 10-2003-0093617 (December 11, 2003) 10-2006-0034739 (Apr. 25, 2006) discloses a cosmetic composition containing deep-seated hot spring water, and attempts have been actively made to use hot spring water, ground water, deep seawater, etc. as cosmetic base materials. It has a wealth of advantages.

Mineral water, on the other hand, is also called mineral water because it contains minute amounts of minerals such as calcium, magnesium and potassium. It is a spring water that springs from the ground and contains a large amount of gaseous or solid matter. In the source of water, at least 25 ℃ is called hot spring, and below is called cold spring. Mineral water usually refers to the latter. These mineral waters are made by using natural mineral springs and by removing chlorine from tap water and adding appropriate salts. They are used for drinking water, carbonated water, and the like. Natural mineral waters are often drunk as an enteral agent.

Recently, many cosmetic products using natural products have been developed to reduce skin irritation caused by various chemical substances and the like. In addition to low adverse effects on skin, natural materials have recently become increasingly appreciated as a cosmetic raw material as consumers' response to cosmetics using natural materials has increased.

Korean Patent Publication No. 10-2012-0036516 (April 18, 2012) Korean Patent No. 10-1429946 (August 4, 2014) Korean Patent Publication No. 10-1995-0023399 (August 18, 1995) Korean Patent Publication No. 10-1995-0028760 (November 22, 1995) Korean Patent Laid-Open No. 10-2001-0038510 (May 15, 2001) Korean Patent Publication No. 10-2003-0093617 (December 11, 2003) Korean Patent Publication No. 10-2006-0034739 (April 25, 2006). Korean Patent Publication No. 10-2014-0132668 (Nov. International Patent No. 2012-033422 (2012.03.15)

Free Radical Research, 1996, 25 (1), 75-86 Food Science and Technology, 2009, 42, 1561-1572 Proc. Natl. Acad. Sci. USA, 1997, 94, 10367-10372 Proc. Natl. Acad. Sci. USA, 2002, 99, 7610-7615

The inventors of the present invention found that the content of glucosinolate-based substances and the anti-inflammatory effect were high in broccoli extracts, especially when investigating anti-inflammatory compositions, in particular cosmetic products for skin irritation. In particular, And exhibit antibacterial activity against bacteria. Accordingly, it is an object of the present invention to provide an anti-inflammatory composition containing a broccoli extract or a cosmetic composition for alleviating skin irritation.

According to one aspect of the present invention, there is provided an anti-inflammatory composition comprising a broccoli extract as an active ingredient.

In one embodiment, the broccoli extract may be prepared as a water extract, at room temperature or by heating extraction.

In one embodiment, the broccoli extract is a C ₁ to C ₃ lower alcohol solvent extract, which may be prepared by room temperature extraction or ultrasonic extraction.

In one embodiment, the anti-inflammation may be skin inflammation relief and the anti-inflammatory composition may be a cosmetic composition for alleviating skin irritation.

According to the present invention, the extract of Broccoli showed a high anti-inflammatory effect, and particularly, when 70% ethanol was extracted with a solvent, the anti-inflammatory effect was more excellent and the antibacterial activity against the skin uptake bacteria was confirmed. Therefore, the broccoli extract of the present invention can be effectively used as a composition for anti-inflammation or a composition for alleviating skin irritation.

1 is a graph showing the DPPH radical scavenging activity of broccoli extract (water C, ascorbic acid, WE-RT, water extract at room temperature, WE-100, water extract at 100 ° C, MWE-RT, Extract, MWE-100, mineral water extract at 100 ℃, EE, 70% ethanol extract at room temperature, USEE, ultrasonic extract of 70% ethanol).
2 is a graph showing the ABTS radical decolorizing effect of the broccoli extract.
Figure 3 shows the results of MTT analysis of survival rates of broccoli extracts treated with RAW264.7 cells at various concentrations (0, 5, 10, 50, 100, 500 and 1000 ug / mL) for 24 hours .
Figure 4 is a graph showing the effect of broccoli extract on NO production induced by LPS in Raw 264.7 macrophages.
FIG. 5 is a photograph showing the anti-inflammatory effect of a broccoli extract inhibiting the expression of COX-2 and iNOS protein induced by LPS in Raw 264.7 macrophages.

As used herein, "water" includes not only chemically pure water but also water including inorganic salts, minerals and various compounds, including, but not limited to, distilled water, purified water, mineral water, .

The present invention provides an anti-inflammatory composition comprising an extract of Broccoli as an active ingredient.

In one embodiment of the present invention, the broccoli extract is a water extract, which can be prepared by room temperature extraction or heat extraction

When the solvent is distilled water or mineral water, the solvent achieves the effect of increasing the content of the glucosinol-based substance, and the glucosylinol-based substance may be sulfolane or glucopraphone. The mineral water may be pure mineral water.

In one embodiment of the present invention, the broccoli extract is a C ₁ to C ₃ lower alcohol solvent extract, which can be prepared by room temperature extraction or ultrasonic extraction.

When the solvent is 70% ethanol, it achieves the effect of enhancing antioxidant, antibacterial activity and anti-inflammatory activity.

In one embodiment of the present invention, the anti-inflammation may be skin inflammation relief, and the anti-inflammatory composition may be a cosmetic composition for skin inflammation relief.

The cosmetic composition for improving skin irritation according to the present invention further comprises a cosmetically acceptable carrier.

The carrier can be used without limitation as long as it is known in the technical field of the present invention, and can be appropriately selected and configured according to the desired formulation. For example, it is possible to use various additives such as a solvent, a purified water, a moisturizer, a pigment, an emulsifier, a chelating agent, a softener, a buffer, a wax, a paraffin, a starch, a talc, , But are not limited to, proteins, antioxidants, hydrocarbons, synthetic esters, fatty alcohols, glycerin, wetting agents, anti-settling agents, dispersing agents, gelling agents, solubilizing agents and emulsifying agents.

The cosmetic composition for improving skin irritation of the present invention can be provided as a formulation for improving skin troubles such as skin inflammation, itching and the like.

The formulation may be provided without limitation as long as it is known in the art. More specifically, the present invention relates to a cosmetic composition for ointment, cosmetic composition, cosmetic composition, cosmetic composition, cosmetic composition, cosmetic composition, cosmetic composition, But are not limited to, depilatories, packs, soaps, body lotions, body creams, body oils, body essences, emulsions, hair tonics, sprays, oil gels and the like.

Hereinafter, the present invention will be described in more detail with reference to examples and measurement examples. However, the following examples and measurement examples are provided for illustrating the present invention, and the scope of the present invention is not limited thereto.

EXAMPLES Extraction of Broccoli Extract and Evaluation of Its Activity

1. Extraction method

Broccoli was purchased directly from Cheongju, Chungcheongbuk - do and was used as a test material by removing the foreign materials, washing and freeze - drying. The extraction solvent was water, purified water, and 70% ethanol, and extracted by adding about 10 times of the solvent.

Extracts were extracted with water or purified water at room temperature. The extracts were extracted with stirring for 24 hours at room temperature, and the supernatant was separated. The same method was repeated for the precipitates, and the extracts were repeated three times. For 3 hours under reflux. The supernatant was separated, and the same procedure was repeated for the precipitate.

The extracts at room temperature using 70% ethanol were extracted with stirring for 24 hours at room temperature. The supernatant was separated and the same method was repeated for the precipitates three times repeatedly. The ultrasonication extract was treated with ultrasonic at 60 kHz After 6 hours of extraction, the supernatant was separated and the precipitate was further extracted twice at 60 ° C for 12 hours.

Each extract was filtered under reduced pressure, concentrated under reduced pressure using a vacuum concentrator (EYELA, Germany), and the concentrate was lyophilized and used for the test.

The water extract at room temperature was WE-RT, the water extract at 100 ° C was WE-100, the mineral water extract at room temperature was MWE-RT, the mineral water extract at 100 ° C was MWE-100, The extracts were labeled with EE and the ultrasound extract with 70% ethanol solvent was designated as USEE.

<Test Example>

1. Evaluation of physiologically active substance content of broccoli extract

The content of sulforaphane in broccoli is very different from one variety to the other, and it is produced by the action of myrosinase on glucuraphenin.

The content of glucoraphenin in broccoli extract was determined by HPLC (Dionex, Sunnyvale, CA, USA) and column (C18, inertsil ODS2, 4.6 x 250 mm, GS Science, Tokyo, Japan). The column was maintained at 35 ° C using a column oven, and the mobile phase was analyzed at 227 nm while flowing 1.0 mL / min. The mobile phase was eluted with a linear gradient of 1:99 (A: B, v / v) to 99: 1 using a 20% acetonitrile (solvent A) and deionized water (solvent B) , 99: 1 for 11 minutes, and 99: 1 to 1:99 for 3 minutes.

Analysis of the sulfolane content in the broccoli extract was carried out using HPLC and column equipped with SP930D dual pump and UV730 detector (NovaPak C18 60 Å 4 μm), and the absorbance was set at 254 nm. The solvent mobile phase begins with acetonitrile: distilled water as the gradient elution at an initial 20:80 (v / v) ratio and flows through the line to change to a 60:40 ratio for 10 minutes. , The initial state was maintained at 20:80 ratio from 16 minutes, and the flow rate was maintained at 1 mL / min. The analysis was carried out using a column oven at a temperature of 30 ° C, and 10 μL of the sample was injected.

The content of glucoraphenin in broccoli extract was found to be 795 mg / kg when broccoli was extracted at room temperature in the purified water. The content of sulforaphane was determined by extracting with water at room temperature 63.6 mg / kg, respectively (Table 1).

Extraction process Content (mg / kg) Glucoraphnin Sulforaphane WE-RT 486 63.6 WE-100 416 55.3 MWE-RT 795 40.0 MWE-100 273 0.29 EE 500 5.99 USEE 412 0.882

2. Evaluation of Antioxidative Activity of Broccoli Extract

2.1. Electron donating ability measurement

The electron donating ability is an index of antioxidant activity against phenolic acid, flavonoid and other phenolic substances. The electron donating ability of such a substance is higher when the reducing power is higher, and the electron donating ability is mainly expressed by the fruit, stem, flower, , And roots.

The scavenging activity of DPPH radical was measured as follows. 3 mL of 60 μM DPPH (1,1-diphenyl-2-picryl hydrazyl) was added to 0.5 mL of each sample, followed by stirring. After reacting for 15 minutes, the absorbance was confirmed at 517 nm and the electron donating ability was calculated according to the following equation (1).

As a result of measuring the electron donating ability of the broccoli extract, as shown in FIG. 1, the electron donating ability tended to increase in all the extracts in a concentration-dependent manner. Especially, the ethanol extract (EE) showed a marked increase in antioxidant activity with increasing concentration of extract and a high electron donating ability of 98.18% at 10,000 ug / mL.

2.2. ABTS activity measurement

For the analysis using ABTS radical cation decolorization, 5 mL of 7 mM ABTS and 88 μL of 140 mM K ₂ S ₂ O ₈ were mixed and reacted for 14-16 hours under dark conditions. ethanol) and 1:88 (ABTS and K ₂ S ₂ O ₈ : anhydrous ethanol) to prepare an ABTS solution. Absorbance was measured at 734 nm and ABTS solution with an absorbance value of about 0.7 was used as a control. 50 μL of Broccoli extract sample solution and 1 mL of ABTS solution were stirred for 30 seconds, incubated for 2.5 minutes, and the inhibition rate was calculated by measuring the absorbance at 734 nm.

ABTS reacts with free radicals such as hyroxyl, peroxyl, alkoxyl and inorganic radicals to form stable ABTS + type cation radicals, and the resulting ABTS + forms hydrophilic and hydrophobic The antioxidant capacity of the substance can be used for the measurement. The ABTS radical scavenging activity of broccoli extract was measured by ABTS radical scavenging activity. The ABTS radical scavenging activity tended to increase with increasing concentration in all extracts. Ethanol extract (EE) was the highest at 95.93% Indicating a radical scavenging ability (Fig. 2).

2.3. Measurement of FRAP activity

The ferric reducing antioxidant power assay (FRAP) is a process that converts Fe (III) -TPTZ (ferric tripyridyltriazine) complex to Fe (II) -TPTZ (ferrous tripyridyltrianzine) by reducing agent at acidic pH. It is a method to measure the total antioxidant capacity in a sample.

Acetate buffer (pH 3.6, 23 mM) was prepared by using C ₂ H ₃ NaO ₂ and acetic acid (C ₂ H ₄ O ₂ ), and 40 mM HCl and TPTZ (2,4,6-tripyridyl -s-triazine) was used to make a 10 mM TPTZ solution. As a reaction solution for this test, acetic acid buffer (pH 3.6, 23 mM), 10 mM TPTZ (2,4,6-tripyridyl-s-triazine) and 20 mM FeCl ₃ .6H ₂ O were mixed in a ratio of 10: , And stored at 37 캜 until measurement. Broccoli extract (2 μL) and coloring reagent (198 μL) were treated in a 96-well microtiter plate (well volume: 200 μL), reacted for about 30 minutes under dark condition, and absorbance was measured at 590 nm .

In order to compare the antioxidative activities of broccoli extracts, FRAP values were determined by substituting FeSO _{4 on the} calibration curves. All of the extracts showed a tendency to increase in the concentration-dependent reducing power and the ethanol extracts showed the highest values (Table 2). All values were measured in triplicate and expressed as mean ± standard deviation.

Extraction method FRAP value (FeSO ₄ eq. UM) 25 50 100 500 1000 2500 5000 10000 WE-RT 7.3
± 2.1 5.2
± 7.8 13.5
± 0.4 42.8
± 1.7 72.2
± 0.6 298.8
± 2.7 428.5
± 2.6 536.5
± 6.5 WE-100 7.2
± 1.7 7.2
± 1.5 9.5
± 1.3 32.0
± 0.2 55.5
± 0.5 282.7
± 17.7 371.5
± 7.1 472.8
± 4.5 MWE-RT 5.6
± 0.8 6.3
± 0.6 8.7
± 0.9 29.7
± 0.2 49.2
± 1.2 283.4
± 20.0 390.2
± 3.8 456.3
± 2.4 MWE-100 8.7
± 0.6 12.0
± 0.6 18.4
± 1.3 57.4
± 1.3 102.4
± 2.8 36.6
± 8.4 555.6
± 10.5 720.1
± 6.0 EE 17.6
± 1.9 22.6
± 0.9 32.3
± 1.9 114.8
± 5.2 204.8
± 1.9 602.3
± 42.9 908.7
± 18.0 1235.6
± 56.6 USEE 10.8
± 0.6 11.3
± 0.5 14.1
± 2.6 41.6
± 1.2 76.5
± 1.3 307.6
± 7.1 469.0
± 10.7 732.3
± 1.8

3. Antimicrobial activity measurement

3.1. Test strains and medium conditions

Staphylococcus epidermidis KCTC 1917 (S. epidermidis), Staphylococcus aureus KCTC 1621 (S. aureus), Escherichia coli KCTC 1039 (E. coli), Malassezia furfur KCTC 7743 (M. furfur) and Propionibacteri acnes KCTC 3314 (P. acnes)] was used at the Korea Research Institute of Bioscience and Biotechnology, and another strain [Pityrosporum ovale KCCM 11894 (P. ovale)] was purchased from the Korean Microorganism Conservation Center. S. epidermidis, S. aureus and E. coli were grown in 37 ° C nutrient broth, M. furfur at 30 ° C LNA broth, P. ovale at 30 ° C pityrosporum broth, P. acnes Were cultured in anaerobic conditions (GasPack EZ Anaerobic Container System, BD, USA) in 37 ° C Reinforce Clostridial broth.

3.2. Antimicrobial activity measurement using paper disk

The antimicrobial activity was measured by a paper disc method. First, platelets of S. epidermidis, S. aureus, E. coli, and inflammation related bacteria M. furfur, P. ovale, and P. acnes cultured on a plate medium were plated in a volume of 10 mL in a liquid medium And incubated for 18-24 hours. Thereafter, 0.1 mL of the bacterial solution was inoculated into 10 mL of the liquid medium, and the cells were cultured for 3 to 6 hours. The cells were uniformly plated on a sterilized cotton swab, and the cells were inoculated at a density of about 10 ⁷ cells per plate. Sterile filter paper disks (Tokyo, 8 mm, Japan) were placed on the solid plate media and 0.05 mL samples per disc were absorbed by concentration (1, 5, 10 and 20 mg / mL). After incubation at 37 ° C for 18-24 hours, the diameter of the growth zone (clear zone, mm) around the disc was measured.

As shown in Table 3, the ethanol extracts showed higher antimicrobial activity than the water and the purified water. Especially, the ethanol extract of broccoli showed high antimicrobial activity against all the species.

4. Anti-inflammatory activity measurement

4.1. Cell viability measurement

Cell viability was determined by the reduction method using MTT solution [3- (4,5-dimethylthiazole-2-yl) -2,5-diphenyl-tetrazolium bromide]. RAW264.7 cells (5 × 10 ⁴ cells / well) were cultured in 96-well microplates overnight in 100 μL DMEM medium (Dulbecco's Modified Eagle Medium) , 50, 100, 500 and 1000 ug / ml) for 24 hours. 50 μL of 5 mg / mL MTT solution was added to each well and cultured for 4 hours. The culture was then removed and 100 uL of DMSO solution was added to completely dissolve the violet formazan crystals. The degree of color development was measured by absorbance at 550 nm using an ELISA reader. The cell viability was expressed by comparing the absorbance of the sample solution with that of the sample solution.

The cell viability of each of the extracts of broccoli was measured by concentration. As a result, it did not affect cell growth to 1,000 ppm in all the extracts (FIG. 3).

4.2. NO inhibitory activity measurement

NO (Nitric oxide) measurement was performed by measuring the amount of NO from the supernatant of the cell culture as nitrite and nitrate. A safe nitrate reduction form of grease (griess reagent, Sigma, USA) was used compared to nitrite. When confluence was 80% in ⁶ -well plates (2 × 10 ⁶ cells / well), the cells were washed twice with PBS and then cultured for 12 hours or longer using serum-free medium. After the samples were treated at different concentrations, they were cultured for 1 hour, treated with 1 μg / mL of LPS (lipopolysaccharide), and incubated for 24 hours. The supernatant of the culture was taken and reacted with a grease reagent, and the absorbance at 540 nm was measured with an ELISA reader. The NO production rate was expressed as a percentage.

In the body inflammation process, inflammatory factors such as excessive NO (nitric oxide) and prostaglandin E2 are formed by induction NO synthesis and cyclooxygenase-2. Among these, NO has various physiological functions such as defense function in the body, signal transduction function, and vasodilation. However, overexpression of NO leads to inflammation, tissue damage, genetic mutations, and nerve damage. In order to measure the inhibition of NO production in RAW 264.7 cells, broccoli extract was treated with LPS, a NO inducer, at a concentration of 1,000 ug / mL or less, which is less cytotoxic. The amount of NO production inhibition produced by LPS was measured (Fig. 4). At the concentration of 1,000 ug / mL, the ethanol extract (EE) showed about 50% inhibition rate. All values were measured in triplicate and expressed as mean ± standard deviation.

4.3. iNOs, COX-2 activity measurement

To measure iNOS and COX-2 protein activity, macrophage Raw 264.7 cells were added at 2 × 10 ⁴ cells / mL per well and cultured for 24 hours. The broccoli extract was treated with 150 ppm of the extract and cultured for 1 hour, followed by treatment with 1 μg / mL of LPS for 24 hours.

After incubation, the cells were washed 2-3 times with PBS, and 1 mL of lysis buffer (50 mM Tris-HCl pH 7.5, 50 mM NaCl, 1% Triton X-100, 1M DTT) The supernatant was centrifuged at 13,000 rpm for 10 minutes at 4 ° C, and the supernatant was used as the total protein extract.

Protein concentrations were quantified by standardizing bovine serum albumin (BSA) using a kit (Bio-Rad Protein Assay Kit). 20 uL of protein was denatured by 10% SDS-PAGE and transferred for 2 hours at 100 mA using PVDF (polyvinylidene difluoride) membrane and transfer buffer.

After the protein was transferred, the membrane was blocked with blocking buffer (5% skim milk in TBST (Tris-Buffered Saline and Tween 20)). The cells were washed three times with TBST at intervals of 10 minutes and cultured overnight at 4 ° C with the primary antibody [iNOS (BD Biosience 1: 1000), COX-2 (cayman 1: 1000)] diluted. And incubated for 2 hours at room temperature with each secondary antibody (mouse anti-rabbit IgG HRP, bovine anti-goat IgG HRP (santacruz 1: 1000)).

After washing three times, the cells were reacted with ECL (Millipore) solution in a dark room for 2 minutes, and the thickness of the bands exposed to Kodac was compared to confirm the presence or absence of protein expression.

As described above, RAW264.7 cells were stimulated with LPS to induce inflammatory responses, and broccoli extracts were treated to control the expression of inflammatory proteins iNOS and COX-2. As a result, And ultrasound ethanol extracts (Fig. 5). This test was repeated three times.

Claims (7)

A composition for antiinflammation comprising an extract of Broccoli as an active ingredient.
The anti-inflammatory composition according to claim 1, wherein the broccoli extract is a water extract.
The anti-inflammatory composition according to claim 2, wherein the broccoli extract is prepared by room temperature extraction or heat extraction.
The anti-inflammatory composition according to claim 1, wherein the broccoli extract is a C ₁ to C ₃ lower alcohol extract.
The anti-inflammatory composition according to claim 4, wherein the broccoli extract is prepared by room temperature extraction or ultrasonic extraction.
5. The composition according to claim 4, wherein the anti-inflammation is skin inflammation relief.
The anti-inflammatory composition according to claim 4, wherein the anti-inflammatory composition is a cosmetic composition for relieving skin irritation.

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