KR20100029369A - Method for preparing green tea saponin, 21-o-angeloyltheasapogenol e3 - Google Patents

Abstract

PURPOSE: A method for preparing a green tea saponin 21-O-angeloyltheasapogenol E3 is provided to massively produce 21-O-angeloyltheasapogenol E3 through hydrolysis using microorganism. CONSTITUTION: A method for preparing 21-O-angeloyltheasapogenol E3 comprises: a step of collecting extract containing saponin from plant seeds using eater or organic solvent; and a step of hydrolyzing the extract using acid, base, enzyme, or microorganism producing the enzyme to isolate the 21-O-angeloyltheasapogenol E3 of chemical formula 1. The plant seed is green tea seed. The acid is hydrochloric acid, sulfuric acid, nitric acid, or ethanol, methanol, or butanol thereof. The base is sodium hydroxide, or potassium hydroxide, or base, ethanol, methanol, or butanol thereof. The enzyme is glucosidase, arabinosidase, rhamnosidase, xylosidase, cellulose, hesperidinase, naringinase, [glucuronidase, pectinase, galactosidase, or amyloglucosidase.

Description

Method for preparing green tea saponin 21-O-angeloylthiasapogenol E3 {Method for preparing green tea saponin, 21-O-angeloyltheasapogenol E3}

The present invention relates to a method for producing a green tea saponin derivative 21-O-angeloyltheasapogenol E3 represented by the following formula (1) as an oleanan-based triterpenoid derivative.

Green tea is the oldest drink in the world, and as the interest in green tea has recently increased, studies on the composition of tea and its pharmacological effects have been conducted. Green tea is higher in threamine and polyphenols than other foods. The functional ingredients of green tea are catechins based on flavan-3-ol, which belongs to polyphenols in the multileaf, and are (+)-catechin and (-)-epicatechin. (epicatechin), (-)-epigallocatechin-3-gallate, (-)-gallocatechin and the like are known to be the main ingredients. In particular, polyphenols contained in green tea have been reported to lower blood cholesterol, antioxidant, anticancer, detoxification, antibacterial, caries prevention, anti-aging, whitening and fragrance. In addition, polyphenols contained in green tea have been reported to prevent obesity and increase the resistance of capillaries by preventing gout peroxide, delaying aging and inhibiting the production of neutral lipids.

However, most of the green teas having such various efficacy use leaves, and green tea seeds containing similar potent substances have not been specifically used for cultivation purposes.

Accordingly, the present inventors have found a large amount of aglycone-type green tea saponin 21-O-angeloyltheasapogenol E3 having excellent physiological activity from green tea, in particular, green tea seeds which are not used for special purposes. As a result of studying the production method, the present invention has been completed.

Accordingly, an object of the present invention is to provide a method for producing aglycone type green tea saponin 21-O-angeloyltheasapogenol E3, in particular, hydrolyzing green tea seed extract. The purpose of the present invention is to provide a method for producing 21-O-angeloyltheasapogenol E3 and isolating it.

In order to achieve the above object, the present invention is a first step of obtaining an extract containing saponin from water or organic solvent from the plant seed; And a second step of separating 21-O-angeloyltheasapogenol E3 by hydrolyzing the extract with an acid, a base, an enzyme, or a microorganism producing the enzyme. 21-O-angeloyltheasapogenol E3 Provided is a method for preparing -O-angeloylthiasapogenol E3.

Using the method for preparing 21-O-angeloyltheasapogenol E3 of the present invention, after obtaining saponin extract from green tea seed, the microorganism producing the acid, base, enzyme or the enzyme Through the hydrolysis method, 21-O-angeloyltheasapogenol E3, one of the main bioactive substances, can be produced in large quantities.

The method for preparing 21-O-angeloyltheasapogenol E3 represented by the following Chemical Formula 1 according to the present invention is to obtain a crude saponin extract from plant seeds using water or an organic solvent. Stage 1; And a second step of separating 21-O-angeloyltheasapogenol E3 by hydrolyzing the extract with an acid, a base, an enzyme, or a microorganism producing the enzyme. It is done.

[Formula 1]

In the first step, the plant seed is characterized in that the green tea seed.

As the organic solvent, at least one organic solvent selected from the group consisting of ethanol, methanol, butanol, ether, ethyl acetate and chloroform or a mixture of these and water, preferably 50% ethanol, may be used.

In the second step, the acid may be used at least one acid selected from the group consisting of hydrochloric acid, sulfuric acid and nitric acid, or a mixed solvent of these acids with at least one alcohol selected from the group consisting of ethanol, methanol and butanol. At this time, the concentration of acid that can be used is 0.1N ~ 2N, the alcohol content of the mixed solvent is 10 ~ 50%. In addition, when hydrolysis is performed using an acid, the reaction temperature is preferably 50 to 100 ° C., and the reaction time is 0.5 to 8 hours.

The base may be one or more bases selected from the group consisting of sodium hydroxide and potassium hydroxide, or a mixed solvent of these bases with one or more alcohols selected from the group consisting of ethanol, methanol, butanol. At this time, the concentration of base that can be used is 0.1N ~ 2N, the alcohol content of the mixed solvent is 10 ~ 50%. In addition, when hydrolysis is performed using a base, the reaction temperature is preferably 50 to 100 ° C., and the reaction time is 0.5 to 24 hours.

The enzyme or microorganism producing the enzyme is an enzyme that breaks down the sugar bonds of green tea saponin contained in the extract or an enzyme that breaks down the sugar bond, and removes the sugar portion of green tea saponin to remove 21-O-angelo. It is characterized by producing ylthiasapogenol (21-O-angeloyltheasapogenol) E3.

In addition, the enzyme is more preferably glucosidase, arabinosidase, rhamnosidase, xylosidase, cellulase, hesperidinase ), Naringinase, glucuronidase, pectinase, pectinase, galactosidase and amyloglucosidase. It is characterized by using.

In addition, the microorganisms producing the enzyme are aspergillus genus, Bacillus genus, penicillium genus, rhizopus genus, rhizomucor genus, talaomyces (talaromyces), bifidobacterium (genus), mortierella (mortierella), genus Cryptococcus (cryptococcus) and microbacterium (microbacterium), characterized by using one or more selected from the group consisting of do.

Hereinafter, the present invention will be described in detail.

In the first step, in order to obtain a crude saponin extract using water or an organic solvent from a plant, preferably green tea seed, about 1 to 6 times, preferably about 3 times, water or organic solvent is added to the plant. After extracting and degreasing with stirring at 1 to 5 times at room temperature, and then about 1 to 8 times, preferably about 4 times of water or an organic solvent to the degreased plants, and extracted under reflux 1 to 5 times, 10 to 20 After immersion at 1 ° C. for 3 days, the residue and the filtrate were separated through filtration and centrifugation, and the extract obtained by concentrating the separated filtrate under reduced pressure was suspended in water, and then the pigment was removed using ether or the like. The aqueous layer was extracted 1 to 5 times using an organic solvent, and the obtained organic solvent layer was concentrated under reduced pressure to obtain an organic solvent extract. After dissolving it in a small amount of methanol, a large amount of ethyl acetate was added. And by drying the resulting precipitate, it is possible to afford the crude saponin extract of the present invention.

In the second step, the saponin crude extract is hydrolyzed using an acid, a base, an enzyme, or a microorganism producing the enzyme to prepare 21-O-angeloyltheasapogenol E3. At this time, when using an acid, a saponin crude extract is added with a concentration of 0.1N to 2N, preferably 1N, or a mixed solvent of acid and alcohol (preferably 50% ethanol mixed solvent), and then 50 to 100. The hydrolysis is carried out by heating to reflux for 1 hour in a water bath at 80 ° C., preferably at 80 ° C. In addition, when the base is used, the plant extract is dissolved, and then a base of 0.1N to 2N concentration, preferably 1N concentration, or a mixed solvent of a base and an alcohol (preferably 50% butanol mixed solvent) is added to 50 to 100 ° C. Hydrolysis is preferably carried out by heating to reflux for 12 hours in a 100 ° C. water bath. In addition, when the enzyme is used, the plant extract is dissolved in an acid buffer solution of 5 to 20 times, preferably about 10 times, and then the enzyme is added, about 40 to 55 hours in a water bath of about 37 ° C., preferably about 48 hours. While stirring for an hour, the substrate was confirmed by thin layer chromatography, and when the substrate was completely lost, it was hydrolyzed by heating in hot water (80-100 ° C.) for 5-15 minutes to terminate the reaction. When using microorganisms, plant extracts are dissolved in 5 to 10 times, preferably about 10 times, ionized water, sterilized at 121 ° C. for 30 minutes, cooled to 30 ° C., and then 5 to 10% After incubation at 30 ° C. for 2 to 5 days, preferably 5 days, the substrate was confirmed by thin layer chromatography to confirm the scavenging rate of the substrate. When the substrate was completely lost, the precipitate was recovered by centrifugation at 5,000 to 10,000 rpm. After washing three times with distilled water and centrifuged to obtain a precipitate.

After hydrolysis using an acid, a base, an enzyme, or a microorganism producing the enzyme as described above, the reaction solution was concentrated under reduced pressure to remove the solvent, and stirred 1 to 5 times by adding alcohol to the residue, followed by precipitation. The salts were removed through filtration, and the filtrate was concentrated under reduced pressure to give a crude product. The crude product was separated by silica gel column chromatography (chloroform: methanol = 8: 1-4: 1) to obtain 21-O-. Angeloylthiasapogenol (21-O-angeloyltheasapogenol) E3 can be obtained.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples, but the present invention is not limited only to these examples.

Example 1 Preparation of Green Tea Seed Extract

6 kg of hexane was added to 2 kg of green tea seeds, stirred and extracted three times at room temperature, and then degreased. Then, 4 l of 50% ethanol was added to 1 kg of degreased green tea seeds, and refluxed three times. . Thereafter, the residue and the filtrate were separated through filter cloth filtration and centrifugation, and the extract obtained by concentrating the separated filtrate under reduced pressure was suspended in water, and then extracted five times with 1 L of ether to remove the pigment, and the aqueous layer was 1-. Extracted three times with 500 ml of butanol. The total 1-butanol layer thus obtained was concentrated under reduced pressure to obtain 1-butanol extract, which was dissolved in a small amount of methanol and then added to a large amount of ethyl acetate, and the resulting precipitate was dried to obtain 300 g of green tea seed extract. .

Example 2 Preparation of 21-O-angeloylthiasapogenol (21-O-angeloyltheasapogenol) E3 by Acid Hydrolysis Method

20 g (v / w) of 1 N HCl-50% methanol solution (v / v) was added to 10 g of the green tea seed extract obtained in Example 1, and heated to reflux for 8 hours in an 80 ° C. water bath. Sugars bound to seed crude saponins were hydrolyzed. The reaction solution was concentrated under reduced pressure to remove the solvent, and the residue was stirred by adding ethanol (200 mL) to the residue (three times). The precipitated salts were removed by filtration, and the filtrate was concentrated under reduced pressure to give a crude product. The crude product obtained was then separated by silica gel column chromatography (chloroform: methanol = 7: 1-3: 1) to give 0.55 g of 21-O-angeloyltheasapogenol E3.

Example 3 Preparation of 21-O-angeloylthiasapogenol (21-O-angeloyltheasapogenol) E3 by Base Hydrolysis Method

10 g of the green tea seed extract obtained in Example 1 was dissolved in dry pyridine (500 ml), and sodium methoxide (powder, 10 g) was added thereto, and the mixture was refluxed for 8 hours on an oil bath. After the sugars bound to saponin were hydrolyzed, the reaction solution was concentrated under reduced pressure to remove the solvent, and ethanol (200 mL) was added to the residue, followed by stirring (three times). The precipitated salts were removed by filtration. The filtrate was concentrated under reduced pressure to give a crude product, and the obtained crude product was separated by silica gel column chromatography (chloroform-methanol = 8: 1 to 4: 1) to obtain 21-O-angeloylthiasapogenol (21 0.35 g of -O-angeloyltheasapogenol) E3 was obtained.

Example 4 Preparation of 21-O-angeloylthiasapogenol (21-O-angeloyltheasapogenol) E3 by enzymatic digestion

10 g of the green tea seed extract obtained in Example 1 was dissolved in 100 ml of 0.1 M acetic acid buffer solution (pH 4.5), and 2.5 g of enzyme (0.5 g of hesperidinase, 0.5 g of naringinase, cellulase) was added thereto. 0.5 g, β-glucuronidase 0.2 g, β-galactosidase 0.5 g, amyloglucosidase 0.3 g; manufactured by Sigma) was added and thin layer chromatography was stirred for 48 hours in a 37 ° C water bath. After checking periodically, the green tea saponin disappeared and heated in hot water (80-100 ° C.) for 10 minutes to complete the reaction. The reaction solution was concentrated under reduced pressure to remove the solvent, and ethanol (200 mL) was added to the residue. After stirring (three times), the precipitate was removed by filtration, and the filtrate was concentrated under reduced pressure to give a crude product. The obtained crude product was separated by silica gel column chromatography (chloroform: methanol = 8: 1-4: 1) to obtain 1.02 g of 21-O-angeloyltheasapogenol E3.

Example 5 21-O-angeloyltheasapogenol E3 Preparation Using Microorganisms

10 g of the green tea leaf extract obtained in Example 2 was dissolved in 100 ml of ionized water, sterilized at 121 ° C. for 30 minutes, cooled to 30 ° C., and then pre-cultured Aspergillus niger KCCM 11885 in liquid quantity. 5-10% of the inoculum was incubated at 30 ° C. for 5 days, followed by thin layer chromatography to confirm the elimination of the substrate to confirm that the substrate was completely lost. The precipitate was recovered by centrifuging the culture at 5,000 to 10,000 rpm. Washed three times with distilled water and then centrifuged to obtain a precipitate. Ethanol (200 mL) was added to the precipitate, followed by stirring (three times). The precipitate was removed by filtration, and the filtrate was concentrated under reduced pressure to give a crude product. The obtained crude product was separated by silica gel column chromatography (chloroform: methanol = 8: 1-4: 1) to obtain 0.72 g of 21-O-angeloyltheasapogenol E3.

Experimental Example 1 Identification of 21-O-angeloyltheasapogenol E3

The products prepared in Examples 2 to 5 exhibited the following characteristics and were identified as 21-O-angeloyltheasapogenol E3 (Varian Gemini 2000 300MHz, Varian).

Physical and chemical properties of 21-O-angeloylthiasapogenol E3

Appearance: White fine crystal

Molecular Weight: 586.7

carbon δH δC dept One 1.12, 1.75 (2H) 39.71 CH ₂ 2 1.70 (2H) 27.15 CH ₂ 3 3.78 (1 H, t) 72.97 CH 4 - 56.92 C 5 1.34 (1 H) 48.67 CH 6 0.91 (2H, m) 21.86 CH ₂ 7 1.67 (2 H) 33.46 CH ₂ 8 - 41.43 C 9 1.80 (1 H) 48.11 CH 10 - 37.02 C 11 1.95 (2 H) 21.74 CH ₂ 12 5.35 (1H, brs) 124.57 CH 13 - 143.79 C 14 - 42.70 C 15 1.36, 1.79 (2H) 34.73 CH ₂ 16 4.14 (1H, brs) 68.89 CH 17 - 48.63 C 18 2.59 (1 H, t) 41.15 CH 19 1.12, 2.47 (1H each, dd-like) 48.47 CH ₂ 20 - 36.73 C 21 5.58 (1H, d, 10.1 Hz) 82.47 CH 22 3.99 (1H, d, 10.1 Hz) 74.11 CH 23 9.31 (1 H, s) 208.73 CH 24 1.13 (3H, s) 9.56 CH ₃ 25 1.12 (3H, s) 16.45 CH ₃ 26 0.95 (3H, s) 17.49 CH ₃ 27 1.48 (3H, s) 27.74 CH ₃ 28 3.20, 3.33 (1H each, dd, 10.3 Hz) 66.57 CH ₂ 29 0.85 (3H, s) 30.03 CH ₃ 30 1.13 (3H, s) 20.44 CH ₃ 21-O-Ang One' - 170.57 C 2' - 130.07 C 3 ' 6.07 (1H, dq like) 138.07 CH 4' 1.97 (3H, doublet, 8.63 Hz) 16.15 CH ₃ 5 ' 1.92 (3 H, s) 21.12 CH ₃

[Experimental Example 2] Confirmation of 21-O-angeloyltheasapogenol E3 Production after Hydrolysis (enzymatic degradation)

After the green tea seed extract was prepared by the method of Example 1, and then subjected to the enzymatic reaction by the method of Example 4, the change during the enzymatic reaction was measured by high performance liquid chromatography. The result of measuring the content of 21-O-angeloyltheasapogenol E3 after enzymatic reaction of green tea seed extract is shown in FIG. 1 and it was isolated and 21-O-angeloylthiasapogenol (21- The result of preparing O-angeloyltheasapogenol) E3 is shown in FIG. 2.

As a result, as shown in Figures 1 and 2, it was found that 21-O-angeloyltheasapogenol E3 was converted in the green tea seed saponin extract after the enzyme reaction.

Figure 1 shows the measurement of the production of 21-O-angeloylthiasapogenol E3 by hydrolyzing the green tea seed extract with an enzyme.

Figure 2 shows the measurement of the separation of 21-O-angeloylthiasapogenol E3 from the hydrolyzate of the green tea seed extract.

Claims (7)

A first step of obtaining an extract containing saponin from water or organic solvent from a plant seed; And The second step of separating the 21-O-angeloyltheasapogenol E3 represented by the following formula 1 by hydrolysis of the extract using an acid, a base, an enzyme or a microorganism producing the enzyme ; Method for producing a 21-O-angeloylthiasapogenol E3 comprising a. [Formula 1]

The method of claim 1, wherein the plant seed is a green tea seed. The method of claim 1, wherein the organic solvent is at least one organic solvent selected from the group consisting of ethanol, methanol, butanol, ether, ethyl acetate and chloroform or a mixture of water and 21-O-angeloylthiasapogenol How to make E3. The method of claim 1, wherein the acid is at least one acid selected from the group consisting of hydrochloric acid, sulfuric acid and nitric acid, or a mixed solvent of these acids with at least one alcohol selected from the group consisting of ethanol, methanol and butanol. Process for preparing O-angeloylthiasapogenol E3. The method of claim 1, wherein the base is at least one base selected from the group consisting of sodium hydroxide and potassium hydroxide, or a mixed solvent of these bases with at least one alcohol selected from the group consisting of ethanol, methanol, butanol Process for preparing 21-O-angeloylthiasapogenol E3. The enzyme of claim 1, wherein the enzyme is glucosidase, arabinosidase, rhamnosidase, xylosidase, cellulase, hesperidinase ), Naringinase, glucuronidase, pectinase, pectinase, galactosidase and amyloglucosidase. Characterized by a method for preparing 21-O-angeloylthiasapogenol E3. According to claim 1, wherein the microorganism producing the enzyme is genus Aspergillus, Bacillus, Bacillus, Penicillium, Rhizopus, Rhizomucor, One or more selected from the group consisting of genus talomyces, genus bifidobacterium, genus mortierella, genus cryptoococcus, and genus microbacterium Method for producing 21-O-angeloylthiasapogenol E3 according to the present invention.

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