KR20110018178A - Compound isolated from the bark of phellodendron amurense, isolating method of thereof and application - Google Patents

Abstract

PURPOSE: A compound isolated from Phellodendron amurense bark is provided to ensure antioxidation and whitening and to prevent senile pigmentation. CONSTITUTION: A compound isolated from Phellodendron amurense bark is selected from structural formulas 1-5. A method for isolating Phellodendron amurense bark comprises: a step of extracting Phellodendron amurense bark using distilled water and concentrating to obtain the concentrate of Phellodendron amurense bark; a step of fractioning the concentrate of Phellodendron amurense bark with a water soluble part; a step of performing column chromatography of the concentrate to obtain lysate of PAW-1 to PAW-3; and a step of performing column chromatography of PAW-1 to PAW-3 using a solvent of MeOH-H_2O(1:1, 1:3, v/v) to obtain protocatechuic acid of structural formula 2 from PAW-3-2, vanillic acid of structural formula 3 from PAW-3-31, or ferulic acid of structural formula 1 from PAW-3-32.

Description

Compound isolated from bark of bark, its isolation method and use of the compound {Compound isolated from the Bark of Phellodendron amurense, isolating method of approximately and application}

The present invention relates to a compound separated from the bark of bark, a method for separating the same, and the use of the compound. More specifically, the compound of the following structural formulas (1) to (5) separated from the bark of bark; It relates to the use of the compound.

.....구조식(1)

..... Structure (1)

.....구조식(2)

..... Structure (2)

.....구조식(3)

..... Structure (3)

.....구조식(4)

..... Structure (4)

.....구조식(5)

..... Structure (5)

Phellodendron amurense is a deciduous arboreous tree of Rutaceae, reaching 10m in height, bark deeply divided into gray or greyish brown, and cork layer well developed. Conical inflorescences 6-8 ㎝ long in early summer, several small flowers are running, the fruit is ball-shaped and ripened into a core of about 1 ㎝ in diameter and contains 5 cores and 5 seeds. The bark dried flat by removing the cork layer was used as a dry agent as a yellow wall, and the inner skin of the bark was mainly used for dyeing (Kim Tae-wook, 1996). The hairs on the back of the leaves have villus ( P. molle Nak.), 3 ~ 5 pairs of lobule ( P. insulare Nak.), Thin cork layer and less bristles It is called P. sachalinense Sarg.

The wood of yellow wall trees was also planted as a building, furniture, utensils, boxes, etc., and the seeds were bitter, used as medicinal and insecticides, and distributed in Korea, northeastern China, and Japan (Lee Chang-bok, 1993). . The main component of the pharmacological action of the bark of the yellow bark is the berberine, which is a vegetable base.

As a study on the yellow bark tree, Lee Jong-gu et al. (2007) reported that melanin biosynthesis inhibitors were isolated from Hwangbaek (黄柏) and measured melanin production inhibitory activity in the B-16 melanoma cell line (Lee Jong-gu, Ji-young Choi, Jun-seok Oh, Hee-wook Jung , Choi Eun-hyang, Hee-sang Lee, Jung-a Kim, Tae-soo Jang, Jong-geun Son, Seung-ho Lee 2007. Isolation of Melanin Biosynthesis Inhibitors from Hwangbaek. Ikuta et al. (1998) also reported the isolation of six alkaloid compounds in addition to the new compound, 7,8-dehydrorutaecarpine, from the bark of the yellow bark (Ikuta, A., T. Nakamura, and H. Urabe. 1998. Indolopyridoquinazoline, Furoquinoline and Canthinone Type Alkaloids from Phellodendron Amurense Callus Tissues.Phytochemistry 48 (2): 28-291.). Lee et al. (2006) reported that yellow bark showed the highest GST activity of 174.8% in a study examining the properties of glutathione S-transferase (GST) active compounds in medicinal plants (Lee, JH, DH Lee, HE Yu, JH Kim and JS Lee. 2006.Isolation and characterization of a novel glutathione S-transferase-activating peptide from the oriental medicinal plant Phellodendron amurense.PEPTIDES 27: 2069-2074.), And Kishi et al. (1992) Two new compounds, kihadalactone A and B, along with seven tirucallanes triterpenoids were isolated (Kishi, K., K. Yoshikawa and S. Arihara. 1992. Limonoids and protolimonoids from the fruits of Phellodendron amurense . Phytochemistry 31 (4) : 1335-1338.) Ida et al. (1994) reported the isolation of glycosides of (±) -5,5'-dimethoxylariciresinol, 2 ( p -hydroxy-phenyl) -ethanol, and N -methylhigenamine, including nine phenolic compounds from the bark of a yellow bark. Ida, Y., Y. Satoh, M. Ohtsuka, M. Nagsao and J. Shoji. 1994. Phenolic constituents of Phellodendron amurense bark.Phytochemistry 35 (1): 209-215.), And Kim and Park (2007) Kim, H. and S. Park. 2007. Effects of introduced chemical groups on the dyeing of cotton fabric by applying functional groups having anionic properties and plasticity to cotton fiber dyeability of cotton fabrics with Phellodendron amurense Rupr .. Dyes and Pigments 75: 351-355.

Yellow wall has been used for herbal medicine and folk medicine since ancient times, and many researches have been carried out on yellow wall trees containing yellow pigments. It is not done.

Recently, interest in well-being has increased due to rising awareness of health due to rising incomes and living standards, and research on natural materials has also increased as interest in natural products that are beneficial and harmless to humans has also increased. It is very active. Therefore, the present invention intends to establish the basic data by separating the extract components of the bark of bark tree and various physiological activity tests, and also to apply the functional cosmetic additives as natural pigments and / or functional materials based on such data.

It is an object of the present invention to provide a compound, a method for separating the same, and the use of the compound, which has been isolated from the bark of bark.

An object of the present invention is to provide a compound of the following structural formulas (1) to (5) separated from yellow bark bark, a separation method thereof, and a use of the compound.

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..... Structure (1)

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..... Structure (2)

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..... Structure (3)

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..... Structure (4)

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..... Structure (5)

Compounds of the above formulas (1) to (5) separated from the bark of the bark by the present invention can be used as a material of cosmetics because there is an effect such as antioxidant effect, whitening effect.

Compounds of the above formulas (1) to (5) isolated from the bark of the bark by the present invention is toxic to skin cancer cells in addition to the antioxidant effect, whitening effect, and may exhibit an effect on blotch or senile pigmentation It can be judged that it can serve as a functional additive, such as cosmetics, especially aging cosmetics.

The present invention represents the compounds of the following structural formulas (1) to (5) isolated from the bark of yellow bark.

The present invention represents any one compound selected from the following structural formulas (1) to (5) isolated from bark of bark.

.....구조식(1)

..... Structure (1)

.....구조식(2)

..... Structure (2)

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..... Structure (3)

.....구조식(4)

..... Structure (4)

.....구조식(5)

..... Structure (5)

In the compounds of the formulas (1) to (5), the compound of formula (1) represents ferulic acid, the compound of formula (2) represents protocatechuic acid, The compound of (3) represents vanillic acid, protocatechuic acid vanillic acid, the compound of formula (4) represents berberin, and the compound of formula (5) represents limonin.

The present invention shows a method for separating the compounds of the formulas (1) to (5) from the bark of bark.

Separation method of the compounds of the formulas (1) to (3) from the bark of bark of the present invention comprises the steps of extracting the bark of bark with distilled water and concentrating to obtain a bark of bark concentrate; Fractionating the yellow bark concentrate into a water soluble portion; Performing column chromatography on the water soluble portion of the bark concentrate of the yellow bark to obtain a separation of three portions of PAW-1 to PAW-3 in a separation order; And PAW-3 of the three isolates was subjected to continuous column chromatography using MeOH-H ₂ O (1: 1, 1: 3, v / v) as an eluting solvent to obtain PAW-3-2. Protocatechuic acid of (2) is obtained, vanillic acid of the following structural formula (3) is obtained from PAW-3-31, or ferrule of structural formula (1) is obtained from PAW-3-32. You can get ferulic acid.

.....구조식(1)

..... Structure (1)

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..... Structure (2)

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..... Structure (3)

Upon separation of the compounds of the above formulas (1) to (3) from the bark of the present invention, the bark concentrate is cold water extracted with 4.5 liter (L) of distilled water per 1 kg of the bark of the bark and the cold water. The yellow bark concentrate can be obtained by concentrating the extract to 0.8 to 1.2 liter (l), preferably 1 liter (l). At this time, the bark of the yellow bark may be cut or crushed by a cutter or a grinder to cut and / or crushed to a size of 5 ~ 10mm. The cut and / or crushed yellow bark bark may be placed in distilled water for cold water extraction at 20 to 26 ° C. for 40 to 80 hours, preferably at 25 to 26 ° C. for 70 to 75 hours.

In the above column chromatography may be used to fill the Sephadex LH-20 (Sephadex LH-20, Merck) with a filling material.

In the column chromatography, an aqueous methanol solution (MeOH: H ₂ O = 3: 1, v / v) may be used as the elution solvent.

In the column chromatography, Sephadex LH-20 (Mephadex LH-20, Merck) may be used as a filling material, and an aqueous methanol solution (MeOH: H ₂ O = 3: 1, v / v) may be used as the elution solvent. .

Separation method of the compound of formula (4) from the yellow bark bark of the present invention comprises the steps of extracting the yellow bark bark with distilled water and concentrating to obtain a yellow bark bark concentrate; Fractionating the yellow bark concentrate into a water soluble portion; Dilute hydrochloric acid is added to the water soluble portion of the bark concentrate of the yellow bark, and then reacted in a constant temperature water bath. The resulting precipitate is purified by recrystallization to obtain berberin of the formula (4).

.....구조식(4)

..... Structure (4)

When separating the compound of formula (4) from the yellow bark bark of the present invention, the yellow bark bark concentrate is extracted with 4.5 liters (L) of distilled water with respect to 1 kg of yellow bark bark and the cold water extract content is 0.8 Concentrate to ˜1.2 liter (L), preferably 1 liter (L) to obtain a yellow bark concentrate. At this time, the bark of the yellow bark may be cut or crushed by a cutter or a grinder to cut and / or crushed to a size of 5 ~ 10mm. The cut and / or crushed yellow bark bark may be placed in distilled water for cold water extraction at 20 to 26 ° C. for 40 to 80 hours, preferably at 25 to 26 ° C. for 70 to 75 hours.

When dilute hydrochloric acid is added to the water soluble part of the yellow bark bark concentrate and reacted above, 8-12 ml g of dilute hydrochloric acid is added to 200 ml of the water soluble part, and for 2 to 3 hours, preferably 10 ml g of dilute hydrochloric acid is used. Can be added and reacted for 2.5 hours.

When dilute hydrochloric acid is added to the water soluble portion of the bark bark concentrate, the dilute hydrochloric acid may be diluted hydrochloric acid at a concentration of 1 to 5%, preferably dilute hydrochloric acid at a concentration of 3%.

Dilute hydrochloric acid is added to the water soluble portion of the bark concentrate of the yellow bark, and the temperature of the constant temperature bath can be reacted by maintaining the temperature in the constant temperature bath at 35 to 45 ° C.

When dilute hydrochloric acid is added to the water soluble part of the bark concentrate of the yellow bark, the temperature of the constant temperature bath can be reacted while maintaining the temperature in the constant temperature bath.

Separation method of the compound of formula (4) from the yellow bark bark of the present invention comprises the steps of extracting the yellow bark bark with distilled water and concentrating to obtain a yellow bark bark concentrate; Fractionating the yellow bark concentrate with an ethyl acetate soluble portion; The precipitate produced by concentrating the ethyl acetate soluble part of the yellow bark bark concentrate may be purified by recrystallization to obtain limonin of the following structural formula (5).

.....구조식(5)

..... Structure (5)

Upon separation of the compound of formula (5) from the yellow bark bark of the present invention, the yellow bark bark concentrate is extracted with 4.5 liter (L) of distilled water with respect to 1 kg of yellow bark bark and the cold water extract content is 0.8 Concentrate to ˜1.2 liter (L), preferably 1 liter (L) to obtain a bark concentrate. At this time, the bark of the yellow bark may be cut or crushed by a cutter or a grinder to cut and / or crushed to a size of 5 ~ 10mm. The cut and / or crushed yellow bark bark may be placed in distilled water for cold water extraction at 20 to 26 ° C. for 40 to 80 hours, preferably at 25 to 26 ° C. for 70 to 75 hours.

The present invention includes cosmetics containing any one or more compounds selected from the group of the compounds of the formulas (1) to (5) isolated from the bark of the yellow bark.

The present invention includes cosmetics containing any one or more compounds selected from the group of the compounds of the formulas (1) to (5) separated from the bark of bark by the above-mentioned method.

The cosmetic may be a cosmetic having a whitening effect.

In the above cosmetics may be a cosmetic for the elderly having the function of removing blotch.

In the above cosmetics may be a cosmetic for the elderly having the function of removing the aging pigmentation.

In the above cosmetics may be a cosmetic for the elderly having the function of removing the blotch and / or aging pigmentation.

The compound isolated from the bark of the present invention, the separation method thereof was investigated under various conditions, in order to achieve the object of the present invention, the compound separated from the bark of bark by the conditions mentioned above, the separation method thereof It is desirable to provide.

Hereinafter, the present invention will be described in detail with reference to Examples and Test Examples. However, these are intended to explain the present invention in more detail, and the scope of the present invention is not limited thereto.

≪ Example 1 >

2kg of yellow bark bark was crushed into 7mm size and added to 9 liters of distilled water at room temperature, followed by cold water extraction at 25 ° C. for 72 hours (3 days), and the cold water extract was concentrated to 1 L. Tree bark extract was fractionated into hexane soluble part, ethyl acetate soluble part and water soluble part using a separatory funnel.

Sephadex LH-20 (Merck) was used as a filler for the water soluble part (95.8 g) of the bark of the bark obtained above, and MeOH-H ₂ O (3: 1, v / v) was used as the elution solvent. Column chromatography was performed to separate the three parts according to the separation order, which was designated as PAW. PAW-3 was subjected to continuous column chromatography with MeOH-H ₂ O (1: 1, 1: 3, v / v) to protocatechuic acid of the following formula (2) in PAW 3-2 21 mg), and PAW-3-31 obtained the following formula (3) to obtain Valinic acid (Vanillic acid, 27 mg), and PAW-3-32 represented the following formula (1) by phenolic acid (Ferulic acid, 58 mg). )

In the above, the compounds of the formulas (1) to (3) isolated from the bark of the bark were identified by thin layer chromatography using Cellulose F (20 × 20 cm, Merck) TLC plate. The developing solvent used t -butanol: acetic acid: H ₂ O (3: 1: 1, v / v, TBA (solvent A)) and 6% HOAc (solvent B) and UV lamp (Beckman Du-70) It was observed at 254nm.

.....구조식(1)

..... Structure (1)

.....구조식(2)

..... Structure (2)

.....구조식(3)

..... Structure (3)

Structural analysis of the isolated compounds of the formulas (1) to (3) isolated from the bark of the yellow bark was performed using Bruker Advance DPX 400 MHz spectrometor (Germany) for ¹ H--NMR (400MHz), ¹³ C- NMR (100MHz) was analyzed, DEPT, COSY, HMBC, etc., 2D-NMR were analyzed, and CD ₃ OD and DMSO were used as analytical solvent. Mass spectrometry measured the molecular weight using EI-MS and showed the result of these measurement in following (A)-(C).

(A) Ferulic acid (125.3 mg), a compound of formula (1)

EI-MS m / z: 194 ([M] ⁺ , Base ion), 179, 133, 105, 77.

¹ H-NMR (400 MHz, δ , CD ₃ OD): 3.88 (3H, s , H-OCH ₃ ), 6.31 (1H, d , J = 15.76 Hz, H-8), 6.81 (1H, d , J = 8.11 kPa, H-5), 6.86 (1H, d , J = 8.46 kPa, H-5 '), 7.06 (1H, dd , J = 1.90 kPa and J = 8.20 kPa, H-6), 7.17 (1H , d , J = 1.89 μs, H-2), 7.59 (1H, d , J = 15.89 μs, H-7).

¹³ C-NMR (100 MHz, δ, CD ₃ OD): 56.84 (C-OCH ₃ ), 112.07 (C-2), 116.35 (C-8), 116.87 (C-5), 124.40 (C-6) , 128.21 (C-1), 147.32 (C-7), 149.76 (C-3), 150.89 (C-4), 171.45 (C-9).

The compound of formula (1) is a cinnamic acid-based compound, showing a typical peak of the methoxyl group at 3.88 ppm in the ¹ H-NMR spectrum, and the doublet signal of 7.59 ppm and 6.31 ppm is H-7. The peaks for and H-8 are J = 15.89㎐ and J = 15.76 보, indicating that they are combined in a trans form. ^{In the 13} C-NMR spectrum, the signal of methoxyl group is shown at 56.84ppm, the signal of 171.45ppm is the peak of ketone group of C-9, and the signals of 147.32ppm and 116.35ppm are typical cinnamic acid, C-7 and C-8. It shows the peak of the double bond of the series compound. The signal of 116.87ppm represents C-5, which appears to be upfielded due to the OH group bonded to the adjacent carbon. The signal of 112.07ppm is the peak of C-2. When OH group is present in C-3, the signal is similar to C-5, but the methoxyl group is substituted. Gentry et al. (1998) showed that the C-5 of hycandinic acid esters-1 was 114.7ppm and C-2 was 109.4ppm, which was upfield about 5ppm, C-3 was 146.8ppm and C-4 was 148.1ppm. (Gentry, EJ, HB Jampani, A. KS, MD Morton, DV Velde, H. Telikepalli, and LA Mitscher. 1998. Antitubercular Natural Products: Berberine from the roots of commercial Hydrastis canadensis powder.Isolation of inactive 8 -oxotetrahydrothalifendine, canadine, β -hydrastine, and two new quinic acid esters, hycandinic acid esters-1 and -2.J. Nat. Prod. 61 (10): 1187-1193.). Therefore, it can be seen that the OH group of C-3 is substituted with a methoxyl group. Molecular weight analysis by EI-MS spectrum was consistent with the molecular weight 194 of this compound as [M] ⁺ m / z 194.

(B) Protocatechuic acid (186.7 mg), a compound of formula (2)

EI-MS m / z: 154 [M] ⁺ , 137 (Base ion), 109, 81, 57.

¹ H-NMR (400 MHz, δ , CD ₃ OD): 6.80 (1H, d , J = 7.61 ㎐ H-5), 7.42 (1H, d , J = 2.06 ㎐, H-6), 7.44 (1H, s , H-2).

¹³ C-NMR (100 MHz, δ , CD ₃ OD): 116.18 (C-2), 118.13 (C-5), 123.52 (C-1), 124.32 (C-6), 146.47 (C-3), 151.95 (C-4), 170.69 (C-7).

The compound of formula (2) is a derivative of pyrocatechol, a benzoic acid family, and has a ¹ H-NMR spectrum as a compound in which an OH group is bonded to C-3 and C-4 of benzoic acid. The peak of H-5 is shown as doublet at 6.80ppm, J = 7.61 보, it can be seen that ortho coupling with H-6, H-6 is doublet peak at 7.42ppm, H-2 is At 7.44 ppm it was attributed to a singlet peak. ^{In the 13} C-NMR spectrum, the signal of 170.69ppm shows typical peak of carboxyl group, and C-3 and C-4 with OH group show characteristic peaks at 146.47 ppm and 151.95 ppm, respectively. The above data is based on data of protocatechuic acid reported by Cha et al. (2000) (Cha, BC, SB Lee, TJ Rhim, and KH Lee. 2000. Constituents of Antioxidative Activity and Free Radical Scavenging Effect from Galla Rhois ( Rhus javanica Linne). Kor. J. Pharmacognosy 31 (2): 185-189.), And molecular weight analysis by EI-MS spectrum was consistent with the molecular weight 154 of this compound as [M] ⁺ m / z 154.

(C) Vanillic acid (56.4 mg), a compound of formula (3)

EI-MS m / z: 168 ([M] ⁺ , base ion), 153, 151, 125, 97.

¹ H-NMR (400 MHz, δ , CD ₃ OD): 3.89 (3H, s , H-OCH ₃ ), 6.84 (1H, d , J = 8.87 ㎐ H-5), 7.55 (1H, dd , J = 1.73 μs and J = 3.54 μs H-6), 7.57 (1H, d , J = 1.97 μs, H-2).

¹³ C-NMR (100 MHz, δ , CD ₃ OD): 56.79 (C-OCH ₃ ), 114.17 (C-2), 116.24 (C-5), 123.45 (C-1), 125.68 (C-6) , 149.06 (C-3), 153.07 (C-4), 170.44 (C-7).

The compound of formula (3) is a compound in which methoxy (methoxyl) group is bonded to C-3 of protocatechuic acid, which is the compound of formula (2), instead of OH group, and it is a methoxyl group at 3.89 ppm in ¹ H-NMR spectrum. The typical peak of is shown and the remaining hydrogen peaks are the same as the protocatechuic acid. ¹³ C-NMR spectrum also shows a peak of methoxyl group at 56.79ppm, and the methoxyl group is bonded to C-3, resulting in a difference of 2ppm downfield signal of adjacent C-4. The remaining carbon peaks show the same shape as the protocatechuic acid. The above data is from Choi et al. (2004) data on the compounds isolated from the inflorescences (Choi In-Ho, Hak-Joo Lee, Don-Ha Choi, Jae-In Park, and Tae-Ho Choi. 2004. Extracts from Magnolia sieboldii . 39.) and molecular weight analysis by EI-MS spectrum is [M] ⁺ m / z 168, consistent with the molecular weight of 168 of this compound.

<Example 2>

2 kg of yellow bark bark was crushed to 7 mm size and added to 9 liter (ℓ) of distilled water at room temperature, followed by cold water extraction at 25 ° C. for 72 hours (3 days), and the cold water extract was concentrated to 1 L. Tree bark extract was fractionated into hexane soluble part, ethyl acetate soluble part and water soluble part using a separatory funnel.

To the water soluble part (95.8 g) of the yellow bark bark obtained above, 10 ml of 3% dilute hydrochloric acid was added to 200 ml of the water soluble part, and then reacted for 2.5 hours in a 40 ° C. constant temperature water bath. Was purified by recrystallization to obtain berberin (1.25 g) of the following structural formula (4).

The compound of Structural Formula (4) isolated from the bark of bark was identified by thin layer chromatography using Cellulose F (20 × 20 cm, Merck) TLC plate. The developing solvent used t -butanol: acetic acid: H ₂ O (3: 1: 1, v / v, TBA (solvent A)) and 6% HOAc (solvent B) and UV lamp (Beckman Du-70) It was observed at 254nm.

.....구조식(4)

..... Structure (4)

Structural analysis of the isolated compound of Structural Formula (4) isolated from the bark of bark was carried out using ¹ H--NMR (400 MHz) and ¹³ C-NMR (100 MHz) using Bruker Advance DPX 400 MHz spectrometor (Germany). 2D-NMR, DEPT, COZY, HMBC, etc. were analyzed, and CD ₃ OD and DMSO were used as analytical solvent. Mass spectrometry measured molecular weight using TOF MS. The measurement results of Compound (4) are shown below.

Berberine of formula (4) (2.7 g)

TOF-MS m / z: [M] ⁺ 336.

¹ H-NMR (400 MHz, δ , CD ₃ OD): δ 9.76 (1H, s , H-8), 8.67 (1H, s , H-13), 8.10 (1H, d , J = 9.2 ㎐, H -11), 7.99 (1H, d , J = 9.2 ㎐, H-12), 7.63 (1H, s , H-1), 6.95 (1H, s , H-4), 6.10 (2H, s , OCH ₂ O), 4.93 (2H, t , J = 6.4 ㎐, H-6), 4.20 (3H, s , C-9-OCH ₃ ), 4.10 (3H, s , C-10-OCH ₃ ), 3.27 (2H , m , H-5).

¹³ C-NMR (100 MHz, δ , CD ₃ OD): δ 152.2 (C-10), 152.1 (C-3), 150.0 (C-2), 146.5 (C-8), 145.8 (C-9) , 139.7 (C-13a), 135.2 (C-12a), 132.0 (C-4a), 128.2 (C-11), 124.6 (C-12), 123.4 (C-1a), 121.9 (C-8a), 121.6 (C-13), 109.5 (C-4), 106.6 (C-1), 103.8 (C-OCH ₂ O), 62.7 (C-9-OCH ₃ ), 57.8 (C-10-OCH ₃ ), 57.3 (C-6), 28.3 (C-5).

The compound of formula (4) showed 9.76 ppm singlet peak attributable to the signal of H-8 in the ¹ H-NMR spectrum downfield to the low field due to the influence of adjacent nitrogen, and 8.67 ppm singlet peak showed the same phenol. It was attributed to the signal of H-13 in the ring. Peaks attributed to signals H-11 and 12 appear as doublets of J = 9.2 Hz at 8.10 and 7.99 ppm, and signlets at 7.63 and 6.95 ppm were attributed to signals H-1 and 4. In addition, a singlet signal characteristic of methylene dioxy hydrogen was observed at 6.10 ppm, and the peaks of 4.20 and 4.10 ppm were attributed to signals of two methoxyl groups bonded to C-9 and 10. The 4.93ppm triplet signal was downfielded under the influence of nitrogen as the H-6 peak, and the 3.27ppm multiplet peak was attributed to the H-5 signal. The above results were consistent with the NMR spectra of berberine isolated by Ju Hye-kyeong (2000) (Shu Hye-kyung, Hwang Bang-yeon, Ahn Byung-tae, Kim Mi-jung, Choi Woo-ho, Cho Bong-jin, Roh Jae-sub, Lee Kyoung-soon, 2000. ): 300-305.).

^{In the 13} C-NMR spectrum, the signal of 103.8 ppm due to methylene dioxy carbon, typical of berberine, and the signal of 62.7 and 57.8 ppm due to methoxyl carbon were identified. The signals of 152.2 and 145.8 ppm were the signals of C-10 and 9 combined with methoxyl groups, and the signals of 146.5, 139.7 and 57.3 ppm were downfielded by the effects of nitrogen. In addition, the signals of 152.1 and 150.0 ppm are signals of C-3 and 2 downfield combined with methylene dioxy, and signals of 106.6, 109.5, 121.6, 124.6 and 128.2 ppm are methine carbons of C-1, 4, 13, 12, It was attributed to the peak of 11. In addition, the 28.3 ppm signal was the peak for C-5, methylene carbon, and the signals of 123.4, 132.0, 121.9, and 135.2 ppm were the peaks of C-1a, 4a, 8a, and 12a, quaternary carbons originating from the phenol ring. . The results were consistent with berberine reported by Lee et al. (1997) isolated from barberry (Hyang Lee, Jong-Won Kim. 1997. Studies on the components of Berberis amurensis Ruprecht. Journal of Pharmacognosy 28 (4): 257-263 .). TOF-MS was m / z [M] ⁺ 336, which exactly matches the molecular weight of this compound, followed by berberine structure.

<Example 3>

2kg of yellow bark bark was crushed into 7mm size and added to 9 liters of distilled water at room temperature, followed by cold water extraction at 25 ° C. for 72 hours (3 days), and the cold water extract was concentrated to 1 L. Tree bark extract was fractionated into a hexane soluble part, an ethyl acetate soluble part and a water soluble part using a fraction funnel.

The precipitate produced in the process of concentrating the ethyl acetate soluble part (5.6 g) obtained above was purified by recrystallization to obtain limonin (limonin, 2.45 g) of the following structural formula (5).

The compound of formula (5) isolated from the bark of the yellow bark was identified by thin layer chromatography using Cellulose F (20 × 20 cm, Merck) TLC plate. The developing solvent used t -butanol: acetic acid: H ₂ O (3: 1: 1, v / v, TBA (solvent A)) and 6% HOAc (solvent B) and UV lamp (Beckman Du-70) It was observed at 254nm.

.....구조식(5)

..... Structure (5)

Structural analysis of the isolated compound of formula (5) isolated from the bark of bark was carried out using ¹ H--NMR (400 MHz) and ¹³ C-NMR (100 MHz) using Bruker Advance DPX 400 MHz spectrometor (Germany). 2D-NMR, DEPT, COZY, HMBC, etc. were analyzed, and CD ₃ OD and DMSO were used as analytical solvent. Mass spectrometry measured molecular weight using TOF MS. The measurement results of Compound (5) are shown below.

Limonin of formula (5) (2.4g)

TOF-MS m / z: [M] ⁺ 470.

¹ H-NMR (400 MHz, δ , DMSO- d ₆ ): δ 7.72 (1H, s , H-21), 7.66 (1H, s , H-23), 6.50 (1H, s , H-22), 5.47 (1H, s , H-17), 4.92 (1H, d , J = 13.00 μs, H-19e), 4.48 (1H, d , J = 13.50 μs, H-19a), 4.11 (1H, s , H -15), 4.10 (1H, s , H-1), 3.12 (1H, dd , J = 15.50 μs and 4.0 μs, H-2e), 2.77 (1H, dd , J = 15.5 μs and 3.3 μs, H- 6e), 2.64 (1H, dd , J = 16.5 ㎐ and 4.0 ㎐, H-2a), 2.57 (1H, dd , J = 12.5 ㎐ and 3.2 ㎐, H-9), 2.46 (1H, dd , J = 15.5 ㎐ and 3.2 ㎐, H-6a), 2.27 (1H, dd , J = 14.5 ㎐ and 3.2 ㎐, H-5), 1.72 (2H, m , H-11), 1.25 (2H, m , H-12) , 1.19 (3H, s , H-28), 1.11 (3H, s , H-29), 1.02 (3H, s , H-18), 1.00 (3H, s , H-30).

¹³ C-NMR (100 MHz, δ , DMSO- d ₆ ): δ 208.0 (C-7), 170.2 (C-3), 167.3 (C-16), 143.3 (C-23), 141.7 (C-21 ), 120.2 (C-20), 110.2 (C-22), 79.5 (C-4), 78.4 (C-1), 77.4 (C-17), 66.7 (C-14), 64.7 (C-19) , 57.9 (C-5), 53.7 (C-15), 50.2 (C-8), 46.4 (C-9), 45.2 (C-10), 37.6 (C-13), 36.1 (C-6), 35.6 (C-2), 29.7 (C-12), 29.1 (C-30), 21.4 (C-29), 19.6 (C-28), 17.5 (C-11), 17.0 (C-18).

The compound of formula (5) is a signal of the typical H-21, 23, 22 that appears when the singlet of 7.72, 7.66, 6.50 ppm in the ¹ H-NMR spectrum is combined with C-17 of the limonoid compound and C-20 of the furan ring These were similar to the furan rings of linonoid compounds reported by Bennett et al. (1994) (Bennett, RD, M. Miyake, Y. Ozaki and S. Hasegawa. 1991. Limonoid glucosides in Citrus aurantium . Phytochemistry 30 (11): 3803- 3805.). The doublet peaks of 4.92 and 4.48 ppm attributable to the H-19 signal were J = 13 Hz, indicating that C-3 and C-19 are limonoid compounds in the form of lactone linkage. It was confirmed that H-1 and H-15 appeared as almost overlapping signals at 4.10 and 4.11 ppm. And 1.00ppm, 1.02ppm, 1.11ppm, 4 of the signal 1.19ppm was attributed to the hydrogen peak of methyl group bonded to C-4, 8, 13, respectively, ¹³ C-NMR spectrum at 29.1ppm, 17.0ppm, Four signals of 21.4ppm and 19.6ppm are shown, and Ozaki et al. (1991) compared with limonin 17- β- D-glucopyranoside isolated from Tetradium rutaecarpa .

^{In the 13} C-NMR spectrum, peaks of 208.0, 170.2, and 167.3 ppm were attributed to signals of ketones of C-7, 3, and 16, and peaks occurring at 143.3, 141.7, 120.2, and 110.2 ppm were derived from the C-23 furan ring. , 21, 20, 22 were attributed to the signal. On the other hand, C-1 and 4 were shown at 78.4 and 79.5 ppm, indicating that they are in cyclic ether form. If C-4 shows a signal near 72 ppm, it does not form a ring with C-1. C-1 and tetracycline can be attributed to the cyclic ether (Bennett et al ., 1991). In addition, the signals of C-17, 14, 19, and 15 affected by adjacent oxygen are downfielded at 77.4, 66.7, 64.7, and 53.7 ppm, respectively, and the peaks of 35.6, 36.1, 17.5, and 29.7 ppm are m-carbon, C-. 2, 6, 11, 12 belonged to the signal. In addition, it was confirmed that the signal for methine carbon, C-5 and 9, was shown at 57.9ppm and 46.4ppm, which is consistent with the compounds isolated by Lee Jong-gu et al. (2007). , Hee-Sang Lee, Jung-A Kim, Tae-Soo Jang, Jong-Geun Son, Seung-Ho Lee 2007. Isolation of Melanin Biosynthesis Inhibitors from Hwangbaek.

TOF-MS was m / z [M] ⁺ 470, which was exactly the same as the molecular weight of 470. As a result, compound 5 was identified as limonin.

Test Example 1 Antioxidant Activity Test (DPPH radical scavenging method)

Antioxidant activity using DPPH radical scavenging ability of the compounds of the structural formulas (1) to (5) obtained in Examples 1 to 3 was tested.

Antioxidant activity test using DPPH radical scavenging activity was put into 100 well by diluting the concentration of the compounds of the structural formulas (1) to (5) obtained in Examples 1 to 3 to 500 ppm each as a sample in a 96 well microplate 100 μl of 0.2 mM DPPH solution was added and reacted at room temperature for 30 minutes, and the absorbance was measured at 517 nm using a microplate reader.

The control group (no additive) was measured in the same manner by adding EtOH instead of the sample, and experimented with EtOH instead of 0.2mM DPPH solution to correct the color of the sample.

In Figures 1 to 3 of the compounds of the formula (1) to the formula (5) isolated from the bark of the bark in the protocatechuic acid, ferulic aicd, vanillic aicd more than 90% higher activity than the control Berberine showed about 75% and limonin showed no activity. At this time, the concentrations of the compounds of the structural formulas (1) to (5) isolated from the bark of the yellow bark in Examples 1 to 3 were measured to be somewhat high as 100 ppm, but the standard concentrations of the materials used in cosmetics were 1000 to 50000 ppm. Considering that it was possible to obtain a result that can be sufficiently used as a cosmetic additive material.

Test Example 2 Whitening Activity Test (Tyrosinase Activity Inhibition Test)

Diluting the compounds of the formulas (1) to (5) obtained in Examples 1 to 3 to a concentration of 1 mg / ㎖ each 0.1M PBS (pH 6.5) solution and tyrosine (1.5nM in 0.1M PBS) was added to the mixture to make a mixture, mushroom tyrosinase (1000 units in 0.05M PBS) was added, and the reaction solution was finally put into a 96 well plate to 200µl. After incubating the reaction solution at 37 ° C. for 10 minutes, the amount of dopachrome produced was measured at 490 nm using an ELISA.

As shown in FIG. 2, the compounds of the above structural formulas (1) to (5) isolated from the bark of the bark in Examples 1 to 3 were slightly weaker in whitening activity than the arbutin, which is a control group, but limonin. Except for all the compounds showed the inhibitory ability before and after 50%. Therefore, in Examples 1 to 3, the compounds of the structural formulas (1) to (5) isolated from the bark of the yellow bark could be confirmed that it can be used as a whitening-related cosmetic additive material.

Test Example 3 Cytotoxicity Test by MTT Assay

(1) disclosure cells

For cytotoxicity test, macrophage Raw 264.7 cell (Murine macrophage cell line) and fibroblast L929 cell (Mouse Fibroblast) were purchased from Korean Cell Line Research Foundation.

Macrophages are known to act as mediators of specific immune responses by phagocytosis by phagocytosis by phagocytosis and phagocytosis of foreign bodies, as well as being involved in immunity.

(2) Cell culture

All test cells were treated with a 37 ° C., 5% CO ₂ incubator using Dulbecco's modified Eagle's medium (DMEM) mixed with Gibco's fetal bovine serum (FBS) with 10%, 100 U / ml penicillin and 100 μg / ml streptomycin. incubator for 72 hours. The cell culture flasks were passaged so that the cell density was about 2 to 3 × 10 ⁶ / ml, and the cells were maintained under 5% CO ₂ conditions.

(3) Cytotoxicity test

In order to measure the cell viability, the cells to be tested were replaced with serum-free medium in which macrophages (Raw 264.7 cells) and fibroblasts (L929 cells), which were cultured to a density of 70% or more, were tested for each concentration. Was incubated for 48 hours. MTT (2 mg / ml in PBS) reagent was changed to a blue formazan product by the reaction of live mitochondrial dehydrogenases, and 20 μl of each well was incubated for 4 hours, and then the supernatant was removed. After washing with PBS and DMSO (dimethyl sulfoxide) was added to completely dissolve the formazan product, the absorbance was measured at 540nm with a microplate reader.

As shown in Figure 3 to Example 1 to Example 3 showed a cell viability of more than 80% of the macrophage (Raw 264.7 cells) in a sample of the compounds of the formula (1) to the formula (5) isolated from the bark of bark In Examples 1 to 3, the compounds of the formulas (1) to (5) isolated from the bark of bark were found to be cytotoxic.

In FIG. 3, the control is a control group without adding the sample to be measured during the cytotoxicity test, and the percentage is set as 100% as the activity.

Test Example 4 Cytotoxicity Test on Skin Cancer Cell (B16F10)

Cytotoxicity experiments on skin cancer cells (B16F10) were carried out on the compounds of the structural formulas (1) to (5) isolated from the bark of the bark in Examples 1 to 3.

At this time, the cytotoxicity test for skin cancer cells (B16F10) was carried out by applying the experimental method of Yoo Seon-sun et al. (2004) (Yoo Ji-sun, Kim An-geun. 2004. Combination administration of Flavonoid Myricetin with Vitamine C and Vitamine E in B16F10 cells Korean Journal of Pharmacognosy 35 (4): 357-363.) The results are shown in FIG.

4 shows the cell viability of skin cancer cells (B16F10) with respect to the compounds of the structural formulas (1) to (5) isolated from the bark of bark in Examples 1 to 3, and from the yellow bark of the present invention The isolated compounds of the formulas (1) to (5) showed skin cancer cell (B16F10) toxicity, and especially berberine and limonin showed less than 60% cytotoxicity. Therefore, the compounds of the above formulas (1) to (5) isolated from the bark of the bark of the present invention, which are toxic to skin cancer cells, may be effective in blotting and / or senile pigmentation. Results that can be used as a functional additive, such as aging cosmetics.

In Figure 4, the control (control) is a group without addition of the sample to be measured during the cytotoxicity test to set this to 100% to indicate the percentage (%) for that activity.

As described above, although described with reference to a preferred embodiment of the present invention, those skilled in the art will be variously modified and modified within the scope of the present invention without departing from the spirit and scope of the invention described in the claims below. It will be appreciated that it can be changed.

The compounds of the formulas (1) to (5) separated from the bark of the bark by the present invention have antioxidant and whitening effects and can be used as natural materials for cosmetics. In particular, the compounds of the structural formulas (1) to (5) isolated from the bark of the bark of the present invention have toxicity to skin cancer cells in addition to the antioxidant and whitening effects, and thus may have an effect on blotch or senile pigmentation. It is judged that it can be used as a material of functional additives such as aging cosmetics.

1 is a graph showing the results of the sulfate activity test of the compounds of the formulas (1) to (5) isolated from the bark of the bark in Examples 1 to 3 (com.1 in Figure 1 is a structural formula (1 ), Com. 2 means a compound of formula (2), com. 3 means a compound of formula (3), com. 4 means a compound of formula (4), com .5 means a compound of formula (5)).

Figure 2 is a graph showing the test results of the whitening activity of the compounds of the formulas (1) to (5) isolated from the bark of the bark in Examples 1 to 3 (com.1 in Figure 2 is the formula (1) Com. 2 means the compound of formula (2), com. 3 means the compound of formula (3), com. 4 means the compound of formula (4), and com. 5 means a compound of formula (5)).

Figure 3 is a graph showing the cell survival test results for the macrophage (Raw 264.7 cells) of the compounds of the formulas (1) to (5) isolated from the bark of the bark in Example 1 to Example 3 (Fig. 3 Where com.1 means the compound of formula (1), com.2 means the compound of formula (2), com.3 means the compound of formula (3), and com.4 means formula (4) ) And com. 5 means the compound of formula (5).

Figure 4 is a graph showing the test results of the survival test results of the skin cancer cells (B16F10) of the compounds of the formulas (1) to (5) isolated from the bark of the bark in Example 1 to Example 3 (in Figure 4 com.1 means the compound of formula (1), com.2 means the compound of formula (2), com.3 means the compound of formula (3), com.4 means formula (4) Com.5 means a compound of formula (5)).

Claims (7)

The compound of any one selected from the following structural formulas (1) to (5) isolated from the bark of the yellow bark.

..... Structure (1)

..... Structure (2)

..... Structure (3)

..... Structure (4)

..... Structure (5) Extracting yellow bark bark with distilled water and concentrating to obtain a yellow bark bark concentrate; Fractionating the yellow bark concentrate into a water soluble portion; Performing column chromatography on the water soluble portion of the bark concentrate of the yellow bark to obtain a separation of three portions of PAW-1 to PAW-3 in a separation order; PAW-3 of the three isolates was subjected to continuous column chromatography using MeOH-H ₂ O (1: 1, 1: 3, v / v) as an eluting solvent, to obtain the following structural formula from PAW-3-2. Obtain protocatechuic acid of 2), vanillic acid of the following structural formula (3) in PAW-3-31 or ferulic acid of the structural formula (1) in PAW-3-32 (Ferulic acid), characterized in that the separation of the compound from the bark of bark.

..... Structure (1)

..... Structure (2)

..... Structure (3) Extracting yellow bark bark with distilled water and concentrating to obtain a yellow bark bark concentrate; Fractionating the yellow bark concentrate into a water soluble portion; Dilute hydrochloric acid is added to the water soluble part of the yellow bark bark concentrate, followed by reaction in a constant temperature water bath, and the resulting precipitate is purified by recrystallization to obtain berberin of the following structural formula (4). Method for separating compounds from

..... Structure (4) Extracting yellow bark bark with distilled water and concentrating to obtain a yellow bark bark concentrate; Fractionating the yellow bark concentrate with an ethyl acetate soluble portion; Separation of the ethyl acetate soluble portion of the bark bark concentrate is purified by recrystallization to obtain a limonin of the following structural formula (5) to separate the compound from the bark bark bark.

..... Structure (5) The cold water extract according to any one of claims 2, 3, or 4, wherein 1 liter of bark bark is extracted with 4.5 liter (L) of distilled water and the content of the cold water extract is 0.8-1.2 liter (L). Separating the compound from the bark bark, characterized in that concentrated to obtain a yellow bark concentrate. From at least one compound selected from the group of compounds of formulas (1) to (5) isolated from the bark of bark of claim 1 or from the bark of bark by the method of any one of claims 2 to 4. Cosmetics containing any one or more compounds selected from the group of the compounds of formulas (1) to (5) isolated. The cosmetic according to claim 6, wherein the cosmetic is an elderly cosmetic having a function of removing blotch or senile pigmentation.

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