KR101898345B1 - The composition comprising the extracts of Ramaria formosa(Fr.) Quel for prevention of skin whitening as an active ingredient - Google Patents

Abstract

The present invention relates to a skin whitening cosmetic composition containing, as an active ingredient, Ramarin C (ramarin C), a compound of Ramaria formosa (Fr.) Quel extract and a fraction thereof, a labdane diterpene compound More particularly, the present invention relates to a cosmetic composition for whitening comprising ramarin C extracted from red mulberry fruiting body as an active ingredient which inhibits melanin formation and exhibits excellent whitening effect.
The cosmetic composition according to the present invention contains an extract of Ramaria formosa (Fr.) Quel and L-marine C as active ingredients, and the effect of inhibiting tarosinase , MITF expression and melanin synthesis is remarkably superior to that of arbutin Bar, skin whitening cosmetic material.

Description

[0001] The present invention relates to a cosmetic composition for whitening comprising an extract of red mulberry extract as an active ingredient,

The present invention relates to a skin whitening cosmetic composition containing as an active ingredient an extract of Ramaria formosa (Fr.) Quel or a fraction thereof, LARMARIN C, and more particularly to a cosmetic composition for skin whitening comprising Tarrosinase , MITF expression, The present invention relates to a cosmetic composition for whitening which exhibits an excellent whitening effect by inhibiting melanin synthesis.

In general, skin is the largest organ in the human body, and it plays important physiological functions such as secretion, respiration, and synthesis as well as physical and chemical protection. Melanin occurs in the melanosome in the melanocyte present in the basal layer of the mammalian skin and has a crucial effect on the color of skin, hair, and eyes.

Also, Melanin is synthesized as a defense to protect the body. Melanin is deposited as pigment on skin surface due to aging and immune degradation. Excessive melanin deposits, especially spiny, freckles, and black spots, such as causing cosmetic problems. Melanin synthesis is regulated by ultraviolet light, cytokine, growth factor, and hormone, and is a complex process involving many factors. It is synthesized through several intracellular signal transduction mechanisms including cyclic adenosine monophosphate (cAMP) / protein kinase A (PKA) pathway. This is because when the skin is exposed to UV light, it activates the downstream signaling substance PKA by amplifying the cAMP signal of the melanocyte, activates the intracellular cAMP response element binding (CREB) protein and activates the microphthalmia-associated transcription factor (MITF) Increases expression.

Melanin functions to protect the skin, but most whitening cosmetics have the effect of inhibiting melanin production, because freckles and stains caused by the deposition of melanin due to adverse effects adversely affect the skin.

On the other hand, the mushrooms are a mushroom belonging to the family Aphyllophorales, Ramariaceae and Ramaria, which are a kind of mushroom species, and a total of 7 species are reported including red mushroom in Korea. Among them, The red mushroom is the largest mushroom grown in the country and is known as a mushroom which is easy to obtain (Korean mushroom, Korea Arboretum, 2013. 386-393)

Ramaria fomosa is known to be tasty and sweet, and various biological activities are known. For example, antioxidant activity (Ramesh CH, Pharmacognosy Res. 2: 107-112, 2010), anticancer activity by polysaccharides (Yoo IS, Kor. J. Mycol., 10: 165-171, 1982) (Ginterova A, Folia Microbiol., 26: 133-136, 1981), the human HIV-1 reverse transcriptase inhibitory action (Zhang R, J. Basic Microbiol., 55: 269-275, (KC, Chem. Pharm. Bull., 63: 554-557, 2015 and Lee IS, Biosci. Biotech. Biochem., 79: 1921-1925, 2015). Other Japanese behold patent (Japanese Patent No. 2909825 No. 1999) Hagi mushrooms (Ramaria fumigata ) has been disclosed as a cosmetic composition composition having inhibitory activity against tiotropin , but it has a different scientific name from that of Ramaria formosa proposed in the present invention, and a novel laminarin C compound isolated from red tiger mushroom has no patent It is not mentioned. Therefore, a document that red mushroom extract and its fraction, L-marine C, are related to skin whitening efficacy is reported for the first time in the present invention.

Accordingly, the inventors of the present invention have found that when a substance having skin whitening activity is studied, a novel compound, Ramarin C, in the extract of Ramaria formosa or a fraction thereof, effectively inhibits the expression of tyrosinase, MITF and melanin synthesis And that it is not cytotoxic, it can be effectively used as a cosmetic composition for skin whitening, thereby completing the present invention.

KR 10-0530669 KR 10-1006341 KR 10-1511279 KR 10-0602684 KR 10-0888742

It is an object of the present invention to provide a skin whitening cosmetic composition containing Ramarin C as an active ingredient in a Ramaria formosa extract or a fraction thereof.

In order to achieve the above object, the present invention provides a composition comprising Ramaria formosa extract or Ramaria formosa (Fr.) Quel or Ramarin C (ramarin C) represented by the following formula as an active ingredient A cosmetic composition for whitening is provided.

The content of the active ingredient is 0.0001 to 10% by weight based on the total weight of the whitening cosmetic composition.

The cosmetic composition is characterized by inhibiting melanin synthesis.

The present invention relates to a skin whitening cosmetic composition containing an extract of Ramaria formosa (Fr.) Quel as an active ingredient, a red foliar extract or a fraction thereof, and a novel compound, L-marine C, The effect of inhibiting the expression of tyrosinase enzyme is excellent, and thus the composition can be effectively used as a skin whitening cosmetic composition.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram of a method for separating laminarin C from red mushroom.
2 shows ¹ H-NMR (500 MHz, in CDCl ₃ ) spectrum of laminarin C. Fig.
3 shows the ¹³ C-NMR (125 MHz, in CDCl ₃ ) spectrum of laminarin C. Fig.
Fig. 4 shows the effect of suppressing the expression of tyrosinase of the red mulberry extract ( Ramaria formosa (Fr.) Quel).
Fig. 5 shows the effect of suppressing the expression of MITF of the red mulberry extract ( Ramaria formosa (Fr.) Quel).

Hereinafter, the present invention will be described in detail. In the following description of the present invention, detailed description of known functions and configurations incorporated herein will be omitted when it may make the subject matter of the present invention rather unclear.

The present invention provides a whitening cosmetic composition comprising, as an active ingredient, Ramaria formosa (Fr.) Quel extract or its fraction, ramarin C.

Larmarine C is a compound represented by the following general formula (1), and only the human neutrophil elastase effect is known by the present inventors. However, the whitening effect has not been clarified yet. For this reason, LaMarine C was isolated from red fungus fruiting bodies for the first time by the present inventors in the natural world, and the physicochemical properties, chemical structure and inhibitory effect on human neutrophil elastase were found (Lee et al ., Bioscience, Biotechnology, In a subsequent study, Lamarin C identified tyrosinase and MITF expression in the course of identifying various biological activities of laminarin C (Fig. 1). And shows excellent whitening effect, thus completing the present invention.

Tyrosinase, tyrosinase related protein-1 (TRP 1) and tyrosinase related protein-2 (TRP-2) are known as major enzymes of melanin biosynthesis. Melanin synthesis is regulated by melanocyte-specific enzymes tyrosinase and tyrosinase-related proteins (TRP-1, TRP-2) Two types of melanin are produced: pheomelanin, a yellow to red pigment, and eumelanin, a black to brown color. Tyrosinase required for melanin synthesis converts tyrosine to 3,4-dihydroxyphenyl-alanine (DOPA), facilitates its oxidation to DOPA quinone, oxidizes 5,6-dihydroxyindole (DHI) to indole-5,6-quinone . Among these, tyrosinase acts as an essential enzyme for the production of eumelanin and pheomelanin, but TRP-1 and TRP-2 are known to act only on the production of eumelanin. In addition, microphthalmia-associated transcription factor (MITF), well known as a regulator of melanin synthesis, is a transcription factor with helix-loop-helixleucine zipper structure and is known to regulate the process of blackening, cell proliferation and survival through melanin synthesis . Since MITF is well known to bind to M-box sequences of tyrosinase and TRPs and regulate the expression of enzymes involved in melanin synthesis, it is considered to be a key index for developing skin-lightening agents that control melanin synthesis.

The red scarlet mucilage extract of the present invention or fractions thereof, and the novel compound, L-marine C, can be formulated in the form of a cosmetic preparation, a soap or a skin external ointment. Cosmetics made from cosmetic compositions can be prepared in the form of conventional emulsions and solubilized formulations. Cosmetics of the emulsified form include nutritive lotion, cream, essence and the like, and the cosmetics of the solubilized form include the flexible lotion. In addition to cosmetics containing the Ramarin C compound of the present invention, it can also be formulated as a topical or systemic adjuvant which is conventionally used in the field of dermatology by containing a dermatologically acceptable medium or base.

Suitable cosmetic formulations include, for example, solutions, gels, solid or kneaded anhydrous products, emulsions obtained by dispersing the oil phase in water, suspensions, microemulsions, microcapsules, microgranules or ionic (liposomes) In the form of a foliar dispersant, in the form of a cream, a skin, a lotion, a powder, an ointment, a spray or a conceal stick. It can also be prepared in the form of a foam or an aerosol composition further containing a compressed propellant.

In addition, the cosmetic composition of the present invention may further comprise, in addition to the red mulberry extract or a fraction thereof, and the novel compound R-marine C, a lipid, an organic solvent, a solubilizing agent, a thickening agent and a gelling agent, a softening agent, an antioxidant, a suspending agent, A foaming agent, a fragrance, a surfactant, water, an ionic or nonionic emulsifier, a filler, a sequestering and chelating agent, a preservative, a vitamin, a barrier, a wetting agent, A lipophilic active agent, a lipid vesicle or any other ingredient conventionally used in cosmetics, as well as adjuvants commonly used in cosmetics or dermatology. And the above ingredients may be introduced in amounts commonly used in the dermatology field.

As a specific formulation of the cosmetic composition of the present invention, there may be mentioned a skin lotion, a skin softener, a skin toner, an astringent, a lotion, a milk lotion, a moisturizing lotion, a nutrition lotion, a massage cream, And includes formulations such as soap, shampoo, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, milky lotion, press powder, loose powder, eye shadow and the like.

The content of the whitening active ingredient contained in the cosmetic composition according to the present invention is not limited to 0.0001 to 10% by weight, 0.001 to 10% by weight, 0.01 to 10% by weight, 0.1 to 10% by weight, Lt; / RTI > If it is less than 0.0001% by weight, the whitening effect may be insignificant, and if it exceeds 10% by weight, the formulation stability may deteriorate.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.

However, the following examples and experimental examples are intended to illustrate the present invention in detail, and the content of the present invention is not limited by the examples and the experimental examples.

Red mushroom ( Ramaria  formosa ) Preparation of extract

The fruiting body of the red mulberry collected in Chungbuk Ramsan was shaded and pulverized. 8 kg of ethanol was added to 1 kg of the extract, and the mixture was extracted three times at room temperature for 7 days. Then, the mixture was filtered and concentrated to obtain 70 g of a red mulberry ethanol extract Respectively.

Red mushroom  From ethanol extracts Fraction  Produce

In order to prepare fractions from the red mulberry extract, 70 g of the ethanol extract prepared in Example 1 was suspended in 2 L of water, 2 L of n-hexane was added and the mixture was distributed to obtain nalum 3 -Hexane layer was separated, and the remaining ethyl acetate layer (ethylacetate, EtOAc) was added to the remaining water layer to separate the ethyl acetate layer 3 times. In this way, 8.0 g of the ethyl acetate fraction was obtained.

Red mushroom  From the extract ramarin  Separation of C

The process of isolating ramarin C from red fruiting body fruity body extract is shown schematically in FIG. In the middle of August 2014, 1.0 kg of Ramaria formosa fruiting body collected from the Ramsangsan area, Chungcheongbuk-do province was shredded, shredded, and repeatedly dipped in ethanol (8L) for 3 days at room temperature for 3 days. g. The ethanol extract (70 g) was suspended in 2 L of distilled water, and then fractionated sequentially using n-hexane and ethyl acetate to obtain ethyl acetate layer (8 g). The tyrosinase inhibitory activity of the obtained ethyl acetate layer was measured, and the fractions exhibiting strong activity were purified. 8 g of EtOAc layer was separated into four fractions (E1E4) by silica gel column chromatography (70230 mesh, 50 × 5.0 cm) under gradient solvent of MC: MeOH (10: The fraction E4 (380 mg) was subjected to YMC RP-18 column chromatography (50 × 3.5 cm) under gradient solvent conditions of MeOHH ₂ O (7: 3 → 9: 1, v / v) (E4-1, E4-2). The small fraction E4-2 was subjected to prep-HPLC [Gilson TRILUTION system; YMC-pack Pro C18 (250 x 10 mm, id) column; _{ACNH 2 O (70:30, v /} v); UV detection, 220 nm; flow rate 3.0 mL / min] to obtain ramarin C It was isolated _{(69 mg, t R = 30.2} min, 0.01% yield).

HPLC analysis was performed using a Gilson TRILUTION system and an analytical column, YMC pack Pro C18 (250 × 10 mm, id), for the HPLC analysis of purified and purified ramarin C. The analytical conditions including the gradient conditions of the mobile phase are shown in Table 3 below and the purity of the two samples was measured by HPLC analysis. As a result, ramarin C was found to be 99.6% in purity.

HPLC analysis conditions HPLC system Gilson TRILUTION system Detector UV 220 nm Column YMC pack Pro C18 (250 x 10 mm, id) Column oven 40 ° C Flow rate 3.0 mL / min Injection volume 5 μ Mobile phase ACN-H ₂ O (70:30, v / v)

ramarin  Analysis of physical properties and chemical structure of C

As a result of UV spectrum, IR spectrum, Mass and NMR analysis of purely separated ramarin C from the above, the results of the following Tables 2, 3 and 4 show that ramarin C having the structure of formula 1 Structure.

Namely, ramarin C was separated in the form of a white powder, and a compound having a molecular formula of C ₂₁ H ₃₆ O ₄ from a molecular ion [M + Na] ⁺ peak of m / z 375.2505 obtained in a high-resolution mass spectrometer (HR ESI-MS) Respectively. The maximum absorption wavelength of 221.5 nm was observed in the UV spectrum and the presence of the α, β-unsaturated carbonyl group was observed from the strong absorption band observed at 1665 cm ¹ of the IR spectrum. ^The carbon signals corresponding to the two ternary olefinic carbons (delta _C 142.8 and 126.6) and the ester carbonyl group (delta _C 167.6) observed in the ¹³ C-NMR spectrum (FIG. 4) In the world. ¹ Analysis of the H-NMR spectrum (Fig. _{3) δ H 6.67 (1H,} dd, J = 12.6, 7.2 Hz) from one olefinic _{hydrogen, δ H 3.42 (1H, dd} , J = 11.7, 5.3 Hz) at One hydroxymethine hydrogen, a pair of hydroxymethylene hydrogen at δ _H 3.36 and 3.28 (2H, each d, J = 10.8 Hz), δ _H 1.76, 0.78, 0.65 (each 3H, s), three tertiary methyl groups, δ _H One secondary methyl group at 0.89 (3H, d, J = 6.3 Hz), and 隆_H The methyl group bonded to the ester group was observed at 3.64 (3H, s). In ¹³ C NMR spectral analysis involving the DEPT spectrum, four methyl, δ _C 7 methylene containing 63.0 oxymethylene group, δ _C Four methine containing 72.5 oxymethine groups, two quaternary carbons, two olefinic carbons (δ _C 142.8 and 126.6), two carbons corresponding to the methyl ester group (δ _C 167.6 and 51.5, respectively).

(Phytochemistry 20: 2255-2258, 1981), Zdero (Phytochemistry 29: 326-329, 1990), Ayafor (J. Nat. Prod., 57: 917-923, 1994) (Phytochemistry 57: 537-541, 2001), and Fujita (Chem. Pharm. Bull. 32: 2117-2125, 1984), Hegazy (J. Nat. Prod., 71: 1070-1073, Compared with the reported literature, this compound was identified as a labdane-type diterpene compound with a C ₆ -side chain at the C-9 position. In particular, the characteristic secondary methyl signal observed at δ _H 0.89 (d, J = 6.3 Hz) was compared with the labdane diterpene methyl labd-13 E -en-15-oate (Mebe, 2001) Lt; / RTI > ¹ Comparison of the H and ¹³ C-NMR ramarin C is methyl labd-13 E H-13 compared to _{-en-15-oate (δ H} 6.67) and _C-14 (δ C 126.6) were observed to migrate and C-13 (δ _C 142.8) was observed at high magnetic field. This indicates that ramarin C is a methyl ester group and C-15 is a methyl group, unlike methyl labd-13 E -en-15-oate. H-13 / C- 14, C-15, C-16 and H ₃ -15 / C-13, C-14 and C-16. Ramarin C was determined in methyl 3β, 18-dihydroxy-8 S -labd-13 E -en-16-oate as shown in formula (1) in the first discovered in the natural world by the present inventors from the above results the compounds.

Physicochemical properties of Ramarin C compound 6 Appearance White powder [?] ²⁵ _D +17? (C = 0.1, CHCl 3) Molecular formula C ₂₁ H ₃₆ O ₄ Molecular weight 352 HR ESI-MS m / z 375.2505 [M + Na] < ⁺ > UV (MeOH) [lambda] _max nm 221.5 IR (KBr) v _max cm ^-1 3420, 2995, 1680, 1635, 1230, 1020 Solubility CHCl _3, DMSO, Pyridine

¹ H NMR (500 MHz) and ¹³ C NMR (125 MHz) analysis of Ramarin C Carbon ¹³ C ¹ H (m, J < / RTI > in Hz) One 36.0 t 1.55 m, 1.42 m 2 29.5 t 1.41 m 3 72.5 d 3.42 dd (11.7, 5.4) 4 45.2 s 5 43.1 d 1.89 m 6 33.8 t 1.05 m 7 27.1 t 1.23 m, 1.20 m 8 35.4 d 1.95 m 9 50.2 d 1.47 m 10 48.4 s 11 34.5 t 1.82 m, 1.65 m 12 24.8 t 2.05 m 13 142.8 d 6.67 dd (12.6, 7.2) 14 126.6 s 15 12.1 q 1.76 s 16 167.6 s 17 18.3 q 0.89 d (6.3) 18 63.0 t 3.36 d (10.8), 3.28 d (10.8) 19 16.8 q 0.65 s 20 15.0 q 0.78 s OCH ₃ 51.5 q 3.64 s

The present invention also provides a compound represented by the following formula, an optical isomer thereof, or a pharmaceutically acceptable salt thereof:

The compound of the above formula is preferably isolated from red mulberry ( Ramaria formosa (Fr.) Quel).

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.

[Experimental Example 1]

B16F10  mouse Melanoma  Toxicity to cells

1-1. Toxicity to B16F10 mouse melanoma cells

To confirm the whitening effect of ramarin C in new whitening material, it is first necessary to confirm cytotoxicity of ramarin C. Therefore, we used the cytotoxicity assay (NRU assay) to measure the cytotoxic effect. B16F10 melanoma cells derived from mice, which were distributed at the BRC, were used. B16F10 cells cultured in DMEM supplemented with 10% FBS and 5% antibiotic-antimyotic at 37 ° C and 5% CO ₂ were inoculated in a 96-well plate at 5 × 10 ⁴ cells / well each and cultured for 24 hours. After replacing with the same medium, ramarin C was diluted with the medium by concentration and cultured in a 5% CO ₂ incubator at 37 ° C for 72 hours. Cells were cultured in 200 μl of 5 mg / ml neutral red solution, DMEM supplemented with 10% FBS and 5% antibiotic-antimyotic, and cultured for 2 hours. 1% CaCl 2 and 1% formaldehyde solution were added, and the mixture was stirred for 1 minute and then absorbance was measured at 570 nm using a microplate reader. Cell viability was calculated as follows.

×100 Cell survival rate (%) =

× 100

As shown in Table 5, when ramarin C treatment was 100%, it was 101.56 ± 5.44% at 1 μg / ml of ramarin, 96.25 ± 2.94% at 5 μg / ml and 92.63% The cell viability was 80.75 ± 3.63% at 50 μg / ml, 61.55 ± 0.75% at 100 μg / ml and ramarin C was more than 80% at the concentration of 50 μg / ml or less. Based on the above cytotoxicity test results, the experiment was continued by setting the concentration that does not show cytotoxicity.

Cytotoxicity of Ramarin C on B16F10 cells sample density % of CTL Control - 100.00 + - 4.28 Blank - 103.49 + 9.59 Ramarin C 1 μg / ml 101.56 + - 5.44 5 μg / ml 96.25 + 2.94 10 μg / ml 92.63 + - 7.96 50 μg / ml 81.75 ± 3.63 100 μg / ml 61.55 + 0.75

[Experimental Example 2]

Melanin production measurement results

B16F10 cells cultured in DMEM supplemented with 10% FBS and 5% antibiotic-antimyotic in a 5% CO ₂ incubator at 37 ° C were inoculated in a 6-well plate at 5 × 10 ⁴ cells / well each and cultured for 24 hours. Ramarin C was prepared at a concentration of 200 nM in a medium containing α-MSH and cultured for 72 hours. Arbutin was used as a positive control. After the culture medium was removed, the cells were washed with 1 × PBS and treated with trypsin to recover the cells. The recovered cells were centrifuged at 1,000 rpm for 5 minutes and the supernatant was removed. The recovered pellet was dried at 70 ° C for 1 hour, and 400 μl of 1 M NaOH solution containing 10% DMSO was added to dissolve the melanin, and the absorbance was measured at 490 nm with a microplate reader. As a result, the cells treated with ramarin C at concentrations of 6.25, 12.5, 25.0, and 50.0 μg / ml were collected and the amount of melanin was measured as shown in Table 6. As a result, as shown in Table 2, the highest effective concentration was 50 μg / Melanin synthesis was inhibited. Arbutin used as a positive control was inhibited by about 40% at 500 μg / ml concentration. Therefore, ramarin C inhibits melanin synthesis similar to arbutin at a significantly lower concentration than arbutin.

Melanin production inhibition rate of B16F10 cells of Ramarin C process density melanin synthesis rate (%) α-MSH - 100.00 ± 2.25 + 184.25 + - 8.62 arbutin 500 μg / ml 141.61 + - 3.34 ramarin C 6.25 μg / ml 180.35 + - 3.21 12.5 μg / ml 162.79 + - 8.67 25.0 μg / ml 150.65 + 2.53 50.0 μg / ml 135.65 + 2.55

[Experimental Example 3]

Tyrosinase  Protein expression inhibitory activity

B16F10 cells cultured in DMEM supplemented with 10% FBS and 5% antibiotic-antimyotic in a 5% CO ₂ incubator at 37 ° C were inoculated in a 6-well plate at 5 × 10 ⁴ cells / well each and cultured for 24 hours. The red mushroom extract was prepared at a concentration of 200 nM of α-MSH and cultured for 72 hours. Cells were lysed with Pro-Prep protein extraction solution and centrifuged. The resulting supernatant was electrophoresed using 10% SDS-PAGE and transferred to a PVDF membrane. After blocking with tris buffer containing 5% skim milk for 1 hour, the cells were reacted with tyrosinase and microphthalmia-associated transcription factor (MITF), respectively. As a result, as shown in Fig. 4, the red mulberry extract inhibited the expression of tyrosinase protein in a concentration-dependent manner. The expression of tyrosinase protein was inhibited at 120, 102, 87, and 73%, respectively, when treated with 6.25, 12.5, 25.0, and 50.0 μg / ml of red mulberry extract.

[Experimental Example 4]

MITF  Protein expression inhibitory activity

Meanwhile, FIG. 3 shows the effect of red mulberry extract on the expression of MITF (microphthalmia-associated transcription factor), which is a transcription factor of tyrosinase, which is most important for melanin biosynthesis. As a result, MITF protein expression inhibition rates were 122, 103, 85, and 75%, respectively, when MITF protein extracts were treated at 6.25, 12.5, 25.0 and 50.0 μg / Was similar to that of tyrosinase. From the above results, it can be concluded that red mulberry extract inhibits the expression of tyrosinase and inhibits melanin production by suppressing the expression of MITF.

Claims (3)

A cosmetic composition for whitening comprising Ramarin C (ramarin C) represented by the following formula as an active ingredient, which fraction is obtained by shredding Ramaria formosa (Fr.) Quel by shade, pulverizing and soaking in ethanol.

The method according to claim 1,
Wherein the content of the active ingredient is 0.0001 to 10% by weight based on the total weight of the whitening cosmetic composition.
The method according to claim 1,
Wherein the cosmetic composition inhibits melanin synthesis.

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