KR101316132B1 - A method for separating and purifying cosmetic effective compounds comprising p-coumaric acid from Sasa querlpaertensis Nakai - Google Patents

Abstract

The present invention relates to a method for producing Sasa querlpaertensis Nakai) relates to a method for separating and purifying a cosmetically active ingredient from the invention, and according to the separation and purification method of the present invention, it is possible to obtain an active ingredient in Jeju including p-coumaric acid in a yield more than twice as conventional.

Description

A method for separating and purifying cosmetic effective compounds comprising p-coumaric acid from Sasa querlpaertensis Nakai}

The present invention relates to a method for producing Sasa querlpaertensis Nakai) relates to a method for separating and purifying cosmetic active ingredients, including p-coumaric acid, and more specifically, alcohol extraction of Jeju chopsticks, followed by carbohydrate hydrolase and pectin hydrolase, followed by organic solvents. The present invention relates to a method for separating and purifying the cosmetically active ingredient of Jeju chopsticks, including p-coumaric acid, by fractionation, followed by separation and purification of the active ingredient by column chromatography.

Jeju jeretdae is one of the most abundant plant species in the forests of Jeju Island. Jeju choridae (rice family) is an edible bamboo leaf, dried leaves have been used in the manufacture of leaf tea for the treatment of diabetes and gastritis. Among the compound components constituting the plant, p-coumaric acid has already been identified.

Tyrosinase (EC 1.14.18.1) is a copper-containing oxygenase enzyme that is widely distributed in plants and animals. L-tyrosine is oxidized to 3,4-dihydroxyphenylalanine (DOPA) and then to tyrosinase enzyme to DOPA quinone, which is an important step in melanin biosynthesis. Tyrosinase not only causes animal melanination, but also causes plant browning. Thus, tyrosinase inhibitors are used not only for skin melanin inhibition in cosmetics but also for keeping fruits and vegetables fresh in the food industry.

Strong tyrosinase inhibitory activity was observed from alcohol extract of Jeju jeju bar. Three known compounds, namely Np-coumaroylserotonin (hereinafter referred to as 'Compound 3'), N-feruloylserotonin (hereinafter referred to as 'Compound 4'), from the bio-derived fraction of Jeju jeju leaves In addition to p-coumaric acid (hereinafter referred to as 'compound 5') two new phenyl propanoids, namely 3-Op-coumaryl-1- (4-hydroxy-3,5-dimethoxy-phenyl ) -1-propanone (hereinafter referred to as 'Compound 1') and 3-Op-coumaryl-1- (4-hydroxy-3,5-dimethoxyphenyl) -1-O-β-D- Glucopyranosylpropanol (hereinafter referred to as 'compound 2') has been separated. The five compounds are referred to herein as cosmetically active ingredients.

Republic of Korea Patent Application No. 2005-59961 'Jeju jeokdae leaf extract with whitening activity' is a extract obtained from the sake extract, jeju jeokdae leaves showing the antioxidant activity, anti-inflammatory activity, tyrosinase inhibitory activity, melanin formation inhibitory properties The extract is described.

Republic of Korea Patent Application No. 2007-134908 'method of purifying para-coumarin acid from jeju choridae and functional cosmetics and pharmaceutical compositions using the para-coumarin acid' describes a method of purifying p-coumaric acid from jeju chori .

However, the method used for extracting p-coumaric acid from Jeju jeokdae in the prior art is in need of improvement because it is not satisfactory in yield.

The inventors of the present invention seek to provide a method that can further increase the yield in the method for separating and purifying cosmetic active ingredients including p-coumaric acid from Jeju jeokdae.

In order to achieve the above object, according to the present invention, the step of alcohol extraction of jeju jeoldae, followed by treatment with carbohydrate hydrolase and pectin hydrolase, followed by fractionation by organic solvent, followed by cosmetic by column chromatography The present invention provides a method for separating and purifying a cosmetically active ingredient from Jeju jerk by separating and purifying the active ingredient.

According to the present invention, the cosmetically active ingredient is 3-Op-coumaryl-1- (4-hydroxy-3,5-dimethoxy-phenyl) -1-propanone, 3-Op-coumaro. Yl-1- (4-hydroxy-3,5-dimethoxyphenyl) -1-O-β-D-glucopyranosylpropanol, Np-coumaroylserotonin, N-feruloylserotonin and p-coumar Provide a method of acid.

In addition, according to the present invention, the extraction step provides a method characterized in that the stirring is carried out for 3 days at room temperature using 10 to 20 times the alcohol based on the weight of the jeju choridae.

In addition, according to the present invention, the fractionation step provides a method characterized in that the fractionation using n-hexane, ethyl acetate, n-butanol.

According to the separation and purification method of the present invention, it is possible to obtain a cosmetically active ingredient including p-coumaric acid from Jeju jewdae more than twice as much as conventionally.

1 is jeju jeoldae ( Sasa querlpaertensis Nakai) is a view schematically showing a process for separating and purifying the cosmetic active ingredient.
Figure 2 is a graph showing the tyrosinase inhibitory activity of the compounds 1 to 5 separated and purified from jeju jeoldae.
FIG. 3 is a graph showing 50% inhibitory activity (IC ₅₀ ) of compounds 1-5 and arbutin against mushroom tyrosinase. FIG.

Jeju jejudae according to the present invention ( Sasa querlpaertensis Nakai) is a method for separating and purifying cosmetic active ingredients, including p -coumaric acid from alcohol, extracting the jeju jeoldae with alcohol, filtering the concentrate and concentrated under reduced pressure using a rotary concentrator, fractions of the extract with an organic solvent And concentrating under reduced pressure to obtain a fraction, and performing column chromatography on the fraction to separate and purify the cosmetically active ingredient.

In the extraction step, in order to extract the active ingredient from the jeju jeoldae crushed naturally dried jeju jeoldae and 10 to 20 times the alcohol by weight based on the extraction using a stirrer for 3 days at room temperature. The alcohol usable in the present invention is not particularly limited as long as it is an alcohol having 1 to 4 carbon atoms.

After the extraction step, the extracted liquid was filtered and by using the rotation and the filtrate concentrator (rotary evarporator) and concentrated under reduced pressure Jeju Sasa (Sasa querlpaertensis Nakai) alcohol extract is obtained.

In the hydrolase treatment step, carbohydrate hydrolase and pectin hydrolase are added to the alcohol extract obtained from the treatment. More specifically, the carbohydrate hydrolase may be 10% by weight biskozyme mixed enzyme, the pectin hydrolase may be 10% by weight pectinase, but is not limited thereto. The hydrolase treatment is preferably done at 40 to 70 ° C., more preferably at 40 to 50 ° C. and under stirring for about 12 hours.

Subsequently, the material obtained from the above is sequentially fractionated using n-hexane, ethyl acetate, n-butanol in the fractionation step, and n-hexane has a low polarity and serves to remove impurities such as lipids and chlorophyll, and ethyl acetate And n-butanol are effective for obtaining compounds having a polarity in the range similar to the active ingredient.

The fractions were each concentrated under reduced pressure to obtain a fraction, and then column chromatography (VLC) was performed using n-hexane, ethyl acetate and ethanol as mobile phases. VLC (vaccum liquid chromatography) is performed by filling a silica gel into a filtration flask and connecting an aspirator. In this case, the separation time can be minimized.

In order to further purify the fraction obtained by performing VLC (vaccum liquid chromatography), column chromatography was performed with n-hexane, ethyl acetate, chloroform and ethanol as mobile phases, and silica gel and Sephadex LH-20 as fixed phases.

Sephadex LH-20 has the property of separating compounds according to their size and polarity, making them ideal for the separation of cosmetically active ingredients.

N-hexane, ethyl acetate, ethyl acetate and ethanol are used as the mobile phase of the VLC (vaccum liquid chromatography). First, the eluate is obtained by increasing the amount of ethyl acetate using 100% n-hexane to 100% ethyl acetate using n-hexane and ethyl acetate. Likewise, the eluate is obtained by increasing the amount of ethanol to 100% ethyl acetate to 50% ethanol using ethyl acetate and ethanol as the mobile phase after 100% ethyl acetate. The desired compound 1 to compound 5 were obtained by washing the eluate.

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

Devices and Materials

All solvents used for extraction and column chromatography were for reagents and used without further purification. Melting points were measured on a Fisher Scientific 2555 melting point apparatus and were not calibrated. Optical rotation was measured on a Jasco P-1030 automatic digital polarimeter. UV spectra were recorded with a Biochrom Libra S22 UV-Visible spectrometer. IR spectra were obtained using a Shimadzu FT-IR 8400S spectrometer. ¹ H (400 MHz) and ¹³ C (100.60 MHz) NMR spectra were recorded on a JEOL, JNM-LA400 instrument where chemical shift values are reported in ppm (δ) relative to the solvent used. 2D NMR spectra were recorded on the same instrument using field gradient FG2 (inverse) probes. FABMS were obtained using an m-nitrobenzyl alcohol matrix on JEOL, JMS-700 spectrometer. Vacuum liquid chromatography (VLC) and column chromatography (CC) were carried out on Merck silica gel 60H (15 μm) and silica gel (0.063-0.2 mm), respectively. Silica gel 60 F ₂₅₄ coated aluminum plates for thin layer chromatography (TLC) were from Merck. Sephadex LH-20 (25-100 μm) for gel filtration chromatography (GFC) was from Fluka. L-tyrosine was purchased from Sigma Chemical Co. Arbutin was manufactured by Bioland Ltd. of Korea.

The stems and leaves of Jeju Loot were collected from Halla Mountain in Jeju Island in February 2007. Evidence specimen (J-101) has been deposited in Cheju National University's Chemistry and Natural Products Laboratory.

Example 1: From Jeju Journey p Separation and Purification of Cosmetic Active Ingredients, Including Coumaric Acid

670.6 g of Jeju dried leaves, which were naturally dried and crushed, were extracted with 90% ethanol solution while stirring at room temperature for 3 days. The extracted sample was filtered with a vacuum filter, and the filtrate was concentrated under reduced pressure with a rotary evaporator at 40 ° C to obtain 55.8 g of an extract.

10% by weight of pectin hydrolase (Pectinex, Novozymes, Denmark) and carbohydrate hydrolase (Viscozyme L, Novozymes, Denmark) were added to the obtained extract, and hydrolyzed with stirring at 40 to 50 ° C for 12 hours, followed by cooling. .

The hydrolyzate was suspended in 10 mL to 15 g in 1000 mL of distilled water, and fractionated with n-hexane, ethyl acetate and n-butanol.

Ethyl acetate fractions having excellent tyrosinase inhibitory activity in the fractions were used for separation and purification. 4.89 g of ethyl acetate fraction was adsorbed onto a pure silica gel column, and then sequentially purified by eluent n-hexane-ethyl acetate (0 to 100%), followed by ethyl acetate-ethanol (0 to 50%) using VLC (vacuum liquid chromatography). Fractionated. 200 mL each of the 26 fractions was obtained. Among them, 55-60% ethyl acetate / n-hexane fraction (185.9 mg) was adsorbed onto a Sephadex LH-20 column, and compound 1 (8.7 mg) was obtained using chloroform / ethanol as eluent, and the subfraction was chloroform / ethanol. Compound 5 (45.7 mg) was obtained using a pure silica gel column using (95: 5 v / v) as eluent. Additional compound 5 (30 mg), compound 3 using a normal phase silica gel column using chloroform / ethanol (90:10 v / v) combined 80-100% ethyl acetate / n-hexane fraction (471.8 mg) in VLC fractions (7.6 mg) and compound 4 (18.9 mg) were obtained. Finally, 2-7% ethanol / ethyl acetate fractions (1.10 g) were combined, gel filtration chromatographed using chloroform / ethanol (95: 5 v / v) as eluent, and finally chloroform / ethanol (90:10 v). / v) was used as the eluent to give a pure silica gel column to give compound 2 (55.6 mg). The precipitated fraction (250.8 mg) in 10% ethanol / ethyl acetate was purified by washing with chloroform to give compound 2 (162.8 mg).

Example 2 Identification of Structural Formula of the Compound Purified from Jeju Sterilizer

Compound structural formulas were identified using HRFAB mass spectrum, IR spectrum and NMR spectrum of the compound obtained in Example 1.

The [M + Na] ⁺ peak in the HRFAB mass spectrum of Compound 1 obtained as a yellow solid showed that the mass peak was m / z 395.1104, consistent with the molecular formula C ₂₀ H ₂₀ O ₇ . IR spectra showed absorption in hydroxy (3320 cm ⁻¹ ) and carbonyl (1658 cm ⁻¹ ) groups. The ¹ H and ¹³ C NMR spectra of compound 1 show that they correspond to the signal of the 3-hydroxy-1- (4-hydroxy-3,5-dimethoxy-phenyl) -1-propanone nucleus. . The ¹ H NMR spectrum of Compound 1 is obtained by two equivalent methoxy group signals at δ 3.90 (s, 6H), aliphatic methylene at δ 3.39 (t, J = 6.4 Hz) and oxymethylene at δ 4.55 (t, J = 6.4 Hz) And peaks of two aromatic methines equivalent at δ 7.34 (s). Ortho-coupled doublet pairs at δ 7.41 and δ 6.78 (2H, J = 8.5 Hz) indicate the presence of p -substituted aromatic rings and signal at δ 7.56 and δ 6.28 (d, J = 16.1 Hz) Indicates that the double bond form is present as a trans. The ¹³ C NMR spectrum of Compound 1 showed 20 signals including 9 carbons corresponding to the p -coumaroyl residue. Other 11 carbon signal are two equal-methoxy-carbon, two equal methine at δ 107.4 appearing in δ 57.0, δ 38.2, two methylene at δ 61.6, a carbonyl group, and the δ 149.2 (two equal at δ 198.3 Carbon), δ 129.1, and δ 142.9. The order of each carbon was confirmed by DEPT measurement and the hydrogen-carbon interaction was confirmed through the HMQC spectrum. Also identified the location of the hydrogen through the ¹ H- ¹ H COSY.

The position of the group was confirmed by long-range heteronuclear correlation using HMBC. Singlet signals of δ 3.90 representing two methoxy groups were located at C-3 ′ and C-5 ′, respectively. This shows a ³ J correlation with the quaternary carbon of δ 149.2 where oxygen is located in the HMBC spectrum. HMBC correlation of oxymethylene hydrogen ( δ H 4.55, H-9) with p -coumaric carbonyl carbon ( δ c 169.3, C-9 ′) was confirmed to be esterified at the C-9 ′ position. Based on this data, it was confirmed that compound 1 is a novel compound 3-Op-coumaryl-1- (4-hydroxy-3,5-dimethoxyphenyl) -1-propanone.

Compound 2 was obtained in the form of a yellowish syrup. This compound is consistent with the molecular formula C ₂₆ H ₃₂ O ₁₂ showing that the molecular ion [M + H] ⁺ is m / z 537.1928 in the HRFAB mass spectrum. A fragment ion peak of m / z 357 appears, because the hexose residue is missing. Compound 2 has a structural formula similar to Compound 1 except that there is no carbonyl signal and there are additional signals corresponding to oxymethine and sugar residues. 6 sp ³ carbon signals combined with oxygen are present at δ 62.2-104.3 and hydrogen signals are present at δ 5.20 and 3.93-4.49. Since the hydrogens H-2 '', H-3 '', and H-3 'of the sugars exhibit axial-axial coupling constants, it was found that the sugars attached were glucose. In addition, the observed chemical shift values of C-1 ''-C-6 '' suggest that glucose. The large binding constant ( J = 7.8 Hz) of the annomeric doublet of δ 5.20 (H-1 ″) represents the β -glucopyranose residue of compound 2. The location of β -glucopyranose residues in Compound 2 was confirmed by HMBC measurements. Anomer hydrogen of glucose ( δ _H 5.20) shows a ³ J correlation with C-7 ′ ( δ _c 79.6) of an aglycone residue, and oxymethine hydrogen ( δ _H 5.46) is part of the sugar C-1 ′ '( δ _c 104.3) and ³ J correlation was shown, so the location per C-7 was confirmed. ¹ H and ¹³ C chemical shift values were clearly determined based on HMQC, HMBC, and ¹ H- ¹ H COSY. Thus compound 2 was identified as a novel compound 3- O - p -coumaryl-1- (4-hydroxy-3,5-dimethoxyphenyl) -1- O - β -glucopyranosylpropanol.

From the above, compounds 1 and 2 were novel compounds, and their physical properties were confirmed as follows.

Compound 1: yellow syrup; UV (MeOH) λ _max 297 nm; IR ν _max 3323, 1658, 1643, 1448, 1115, 1022, 650 cm ⁻¹ ; See Table 1 for ¹ H and ¹³ C NMR; HRFABMS m / z 395.1104 ([M + Na] ⁺ , calcd for C ₂₀ H ₂₀ O ₇ Na: 395.1107)

Compound 2: yellow amorphous solid; Mp 180-182 ° C. [α] _D ²⁵ -30.75 (c 0.16, MeOH); UV (MeOH) λ _max 312 nm; IR ν _max 3320, 1643, 1448, 1415, 1115, 1022, 850 cm ⁻¹ ; See Table 2 for ¹ H and ¹³ C NMR; HRFABMS m / z 537.1927 ([M + Na] ⁺ , calcd for C ₂₆ H ₃₃ O ₁₂ : 537.1972)

The remaining compounds 3, 4, and 5 were each identified to be N - p -coumarylserotonin, N -ferulonylserrulnin, and p -coumaric acid.

Example 3: From Jeju Journey p -Tyrosinase Inhibition Experiment of Cosmetic Active Ingredients Including Coumaric Acid

The anti-tyrosinase activity of compounds 1 to 5 was investigated using L-tyrosine as the substrate. Test solutions of each compound were prepared at various concentrations ( 5.0-100 μg / mL) and the inhibitory effect on mushroom tyrosinase was tested. Arbutin was used as a control to measure the increase in absorbance at 480 nm due to the formation of dopachrome with a spectrophotometer. The results are shown in Fig. Among the compounds tested, serotonin derivatives, Compound 3 (IC ₅₀ 0.027 mM) and Compound 4 (IC ₅₀ 0.026 mM), showed higher inhibitory activity than Arbutin (IC ₅₀ 0.048 mM). In addition, novel compounds Compound 1 (IC ₅₀ 0.055 mM) and Compound 2 (IC ₅₀ 0.053 mM) also exhibited strong inhibitory activity, whereas p -coumaric acid (Compound 5, IC ₅₀ 0.12 mM) showed relatively weak activity. Inhibitory efficacy (IC ₅₀ ) on compounds 1-5 and arbutin was compared in FIG. 3.

Comparative Example 1: Comparison of Product Yields by Enzyme Treatment

Compounds 1 to 5 were obtained from the jeju ridae in the same manner as in Example 1 except that the pectin hydrolase and carbohydrate hydrolase treatment steps were not performed. The yields of Compound 1, Compound 2, Compound 3, Compound 4, and Compound 5 were 4.3 mg, 130.6 mg, 6.7 mg, 20.1 mg, and 150.7 mg, respectively. It was then found that the separation and purification process according to the present invention can produce more than twice as much p-coumaric acid product than the conventional process.

According to the present invention, when the active ingredient including p-coumaric acid is separated and purified from jeju jeoldae, it is possible to obtain the active ingredient in a yield more than twice as high as conventional, which is advantageous in terms of industrial use.

Claims (4)

delete delete Extracting jeju jeoldae with an organic solvent; Then fractionating with an organic solvent; Claims [1] A method for separating and purifying a cosmetically active ingredient from Jeju jerk by the method comprising the step of separating and purifying the cosmetically active ingredient by column chromatography.
The organic solvent extraction step was carried out under stirring for 3 days at room temperature using 10 to 20 times the ethanol on the basis of the dry weight of jeju jeoldae, carbohydrate hydrolase against the total weight of the ethanol extract in jeju jeol obtained above And adding 10 wt% of each pectin hydrolase to the organic solvent fractionation step immediately after hydrolysis while stirring at 40-50 ° C. for 12 hours. delete

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