KR101315942B1 - Preparing method of astragalin - Google Patents

Abstract

The present invention relates to a method for preparing astragalin represented by the following formula (1). More specifically, the present invention relates to a method for producing astragalin, characterized in that astragalin is isolated by selectively removing sugars using an enzyme from a plant extract containing camphorol glycosides. Using this method of the present invention, it is possible to produce a large amount of astragalin, a major bioactive substance from plants.

Astragaline, Camperol Glycoside, Camellia Seed, Selective Sugar Removal, Enzyme, Green Tea Seed, Green Tea Leaf

Description

Preparation method of astragalin {Preparing method of astragalin}

The present invention relates to a method for preparing astragalin represented by Formula 1 using an enzymatic reaction.

[Formula 1]

In most plants, including green tea, many substances are present in glycosides. Although chemical methods are used to remove sugars bound to glycoside materials, particularly when chemicals are used, it is possible to produce aglycone-type substances, but it is difficult to selectively remove sugars at specific positions. However, by selectively removing sugars from glycosides using enzymes with specific activities, not only various kinds of substances can be made, but also various substances can be prepared.

Astragalin, a form of flavonoids, combined with glucose (glucose) in camphorol (kaempferol), is one of flavonols and is distributed on the leaves and flowers of plants of various species. It is known to be excellent, and the application to various fields, including an atopy therapeutic agent, is seeking. However, currently used astragalin is mostly in the form of extracts, and since the actual amount of astragalin is very small, it is difficult to express the actual efficacy of the substance. In addition, although it is reported that persimmon or bokbunja extract contains a lot of astragalin, the content is only a few tens of ppm, and separation and purification to produce a high content of astragalin also has problems in terms of economics. There is no research on the mass production of this product.

Accordingly, the present inventors have effectively treated enzymes with glycosides, such as camelliaside A and camelliaside B, which are present in large amounts in green tea and the like, and selectively remove sugars, thereby reducing glucose to camphorol. As a result of studying a method for mass production of astragaline, which is a form of), the present invention was completed.

Accordingly, the present invention provides a method for producing astragalin, and a camphorol glycoside, such as camelliaside A and camelliaside B, which are contained in a large amount in plants, particularly green tea seeds or green tea leaves ( The purpose of the present invention is to provide a method for effectively separating and preparing astragalin by deriving optimal enzymatic reaction conditions to selectively remove sugars from glycosides.

In order to achieve the above object, the present invention provides a method for producing astragaline represented by the formula (1), the enzyme capable of selective sugar removal from Camelliaside A (camelliaside A) or Camelliaside B (camelliaside B) It provides a method for producing astragalin characterized in that to obtain astragalin.

[Formula 1]

Method for producing astragalin according to the present invention is to obtain a plant extract containing camelliaside A (camelliaside A) or camelliaside B (camelliaside B) using water or an organic solvent from the plant, enzymes capable of selective sugar removal By using to remove the sugar (sugar) bound to the extract is characterized in that the separation of astragalin (astragalin).

As the organic solvent, one or more organic solvents selected from the group consisting of ethanol, methanol, butanol, ether, ethyl acetate and chloroform or a mixture of these and water, preferably 80% ethanol can be used.

In addition, the enzyme is an enzyme that can be obtained from various microbial species as an enzyme that breaks down sugar bonds. This enzyme can be used commercially or prepared and used as needed. This enzyme is particularly useful for the astragalin glycosides, preferably the galactopyranose and rhamnopyranose groups of camelliaside A, the xylopyranose group of cameliaside B and rhamnopyranose ( rhamnopyranose) is characterized in that the enzyme having the activity to selectively remove the group.

In addition, the enzyme is preferably rhamnosidase, galactosidase, pectinase, xylosidase, hesperidinase, naringinase ( naringinase), cellulase (cellulase) is characterized in that at least one member selected from the group consisting of.

In addition, the enzyme is more preferably rhamnosidase, galactosidase, pectinase, xylosidase, hesperidinase, naringinase (naringinase), cellulase (cellulase) is characterized in that the combination consisting of two or more selected from the group consisting of.

Hereinafter, the present invention will be described in detail.

In order to obtain a plant extract containing the camperol glycoside Camelliaside A or Camelliaside B from the plant using water or an organic solvent, about 1 to 6 times, preferably about 3 times, water or an organic solvent to the plant. Extracted, degreased by stirring with 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 refluxed 1 to 5 times, and then After immersion for 1 to 3 days at ~ 20 ℃, the residue and the filtrate is separated through filtration and centrifugation, and the extract obtained by concentrating the separated filtrate under reduced pressure in water, and then removed the pigment using ether, etc. After extraction of the aqueous layer with an organic solvent 1 to 5 times, the obtained organic solvent layer was concentrated under reduced pressure to obtain an organic solvent extract, which was dissolved in a small amount of methanol and the like, Dry the resulting precipitate by adding such as lactate, it is possible to obtain the plant extract of the present invention.

Astragalin is prepared using an enzyme from Camellia seeds A or B of the plant extract prepared in the same manner as above.

In order to increase the efficiency of the production of astragaline, the enzyme reaction time is preferably in the range of 10 to 60 hours, and particularly preferably in the range of 40 to 50 hours, and the reaction pH is in the range of 4.0 to 9.0, particularly, in the range of pH 5.0 to 6.5 range i is preferred. In addition, the concentration of the enzyme is suitably in the range of 0.1 to 10% relative to the substrate, in particular, 1% is preferred, the temperature of the reaction is preferably in the range of 30 to 60 ℃, particularly preferably 50 ℃. If the range is out of the above range, productivity decreases relative to the amount of enzyme used, or the enzyme reaction is delayed.

In the final step, the reaction solution is concentrated under reduced pressure to remove the solvent, the alcohol is added to the residue, stirred 1 to 5 times, the precipitated salts are removed by filtration, and the filtrate is concentrated under reduced pressure to give a crude product, The crude product obtained can be separated by silica gel column chromatography (methanol: chloroform: distilled water = 5: 4: 1) to give astragalin.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. However, the present invention is not limited only to these examples, and may be modified and modified without departing from the scope of the present invention.

In addition, since the activity of the enzyme on the substrate of naringinase and rhamnosidase in the present invention is similar, naringinase was used in the embodiment of the present invention.

Preparation Example 1 Preparation of Green Tea Seed Extract

6 kg of hexane was added to 2 kg of green tea seeds, extracted by stirring three times at room temperature, and then degreased. 4 kg of 80% methanol was added to 1 kg of degreased green tea seeds. 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, extracted five times with 1 L of ether to remove the pigment, and the aqueous layer was 1-butanol. Extracted three times with 500 ml. 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 to dry the resulting precipitate, thereby obtaining 250 g of green tea seed extract.

Preparation Example 2 Preparation of Green Tea Leaf Extract

6 kg of hexane was added to 2 kg of green tea leaves, stirred and extracted three times at room temperature, and then degreased. 4 L of 80% methanol was added to 1 kg of degreased green tea leaves, and the mixture was extracted under reflux 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, extracted five times with 1 L of ether to remove the pigment, and the aqueous layer was 1-butanol. Extracted three times with 500 ml. 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 150 g of green tea leaf extract.

Example 1 Screening of the Combination of Optimal Enzymes to Selectively Remove Sugars from Camellia Seed A

The enzymatic reaction was carried out at 50 ° C. for 40 hours by adding a combination water to the plant extract extracted in Preparation Example 1, and then adding a combination of enzymes as follows. After the reaction was centrifuged to determine the production rate of astragalin by high performance liquid chromatography, the effect of enzyme treatment was examined. In order to determine the optimal combination of enzymes the amount of green tea seed extract was adjusted to 100mg, pH 6.0 as a substrate and proceeded the enzyme reaction for each combination, the results are shown in Table 1.

Astragalin from Green Tea Seed Extract
Optimal combination for produce enzyme Reaction Rate (%) Naringinase 23 β-galactosidase
(β-galactosidase) 11 Pectinase 14 Cellulase 9 Naringinase + β-galactosidase 68 Pectinase + Naringinase 79 Cellulase + naringinase 70 Pectinase + Cellulase 75 β-galactosidase + pectinase 69 Cellulase + β-galactosidase 72 Enzyme substrate: Green tea seed extract 100 mg, pH 6.0, reaction time 40 hours

As shown in Table 1, it is possible to maximize the production rate of astragalin when a mixture of different enzymes is used rather than a single enzyme.

Example 2 Screening for the Combination of Optimal Enzymes to Selectively Remove Sugars from Camellia Seed B

The enzyme reaction was carried out at 50 ° C. for 40 hours by adding a combination of the following enzymes to the plant extract extracted in Preparation Example 1 and adding a combination of the following enzymes. After the reaction was centrifuged to determine the production rate of astragalin by high performance liquid chromatography, the effect of enzyme treatment was examined. To find the optimal combination of enzymes, the amount of green tea seed extract was adjusted to 100 mg, pH 6.0 as a substrate, and the results of the enzyme reaction for each combination are shown in Table 2 below.

Optimal Combination for Astragalin Production from Green Tea Seed Extract enzyme Reaction Rate (%) Naringinase 29 β-xylosidase 27 Pectinase 16 Cellulase 9 Naringinase + β-xylosidase 72 Pectinase + Naringinase 69 Cellulase + naringinase 63 Pectinase + Cellulase 70 β-xylosidase + pectinase 78 Cellulase + β-xylosidase 67 Enzyme substrate: Green tea seed extract 100 mg, pH 6.0, reaction time 40 hours

When producing astragalin from green tea seed extract, especially Camelliaside B, as shown in Table 2, it is best to combine different enzymes rather than single enzymes to maximize manufacturing efficiency. Can be.

Example 3 Change in Production Rate of Astragalin with Reaction Time

 In the green tea seed extract extracted in Preparation Example 1, naringinase and pectinase, naringinase and β-xylosidase, β-xylosidase, pectinase and cellulase Each treatment was adjusted to pH 6.0 to vary the reaction time and then reacted at 50 ° C. Thereafter, the activity of the enzyme was stopped by heating, and the production rate of astragalin was confirmed by high performance liquid chromatography, and the results are shown in Table 3 below.

Production rate of astragalin according to reaction time (%) reaction
time β-xylosidase Pectinase Naringinase Naringinase + pectinase Naringinase + β-xylosidase Pectinase + Cellulase 5 hours 3 5 7 14 21 17 10 hours 10 11 15 38 33 37 20 hours 14 15 22 75 69 72 30 hours 20 17 27 87 86 88 40 hours 27 18 29 93 92 95 50 hours 29 22 30 95 96 96 60 hours 30 23 31 95 96 96 70 hours 28 21 27 95 94 93 80 hours 28 21 19 85 87 84

After 10 hours of enzymatic reaction, the production rate of astragalin became 30% or more, and after 40 hours, the production rate of 90% or more was observed. After 50 hours, the amount of substrate added is exhausted and the reaction is terminated. The production rate is maintained at a constant level, but after 60 hours, the enzyme is denatured and the production rate begins to decrease. Therefore, while the production rate of astragalin increases within the range of 10 hours to 60 hours, it can be seen that the productivity is lowered compared to the amount of enzyme used after 60 hours.

 Example 4 Change in Production Rate of Astragalin with Reaction pH

In the green tea seed extract extracted in Preparation Example 1, naringinase and pectinase, naringinase and β-xylosidase, β-xylosidase, pectinase and cellulase. After each treatment was changed to pH conditions and reacted at 50 ℃ for 40 hours. After that, the activity of the enzyme was stopped by heating, and the production rate of astragalin was confirmed by high performance liquid chromatography, and the results are shown in Table 4 below.

Production rate of astragalin according to pH (%) pH β-xylosidase Pectinase Naringinase Naringinase + pectinase Naringinase + β-xylosidase Pectinase + Cellulase 3.0 3 4 11 53 58 57 4.0 5 6 15 72 73 74 5.0 15 17 23 86 87 89 6.0 26 21 29 96 97 98 7.0 29 23 28 97 95 95 8.0 29 23 28 98 95 95 9.0 20 19 17 94 94 75 10.0 4 5 13 69 72 72

According to the results, the production rate was 70% or more in the range of pH 4.0-9.0, and particularly, the production rate was higher than 85% in the range of pH 5.0-6.5. When pH 9.0 elapses, degeneration occurs in enzymes and the like, and the production rate begins to decrease, indicating that productivity is decreased compared to the amount of enzyme used.

Example 5 Optimal Concentration of Enzyme for Hydrolysis of Green Tea Seed Extract

After treatment with naringinase (naringinase) and pectinase (pectinase) on the green tea seed extract extracted in Preparation Example 1, the pH was adjusted to 6.0, and the reaction was performed at 50 ° C. for 40 hours. After that, the activity of the enzyme was stopped by heating, and as a result of confirming the production rate of astragalin by high performance liquid chromatography, it was as shown in Table 5 below.

Optimum Concentration of Enzyme for Hydrolysis of Green Tea Seed Extract enzyme density(%) 0.1 One 2 4 6 8 10 12 Naringinase 16 29 31 35 35 29 28 15 Naringinase + pectinase 41 69 75 83 85 78 65 39 Enzyme substrate: Green tea seed extract 100 mg, pH 6.0, reaction time 40 hours

In Table 5, when the enzyme concentration is 0.1 to 6%, the production rate of astragalin is gradually increased, and when it is 1%, the production increase rate is particularly higher. In the case of 6-10%, the production rate is somewhat reduced, but it can be seen that the production rate of astragalin is more than 65%, and in the case of more than 10%, the production rate of astragalin is drastically lowered, indicating that productivity is lowered compared to the amount of enzyme used. Can be.

 Example 6 Change in Production Rate of Astragalin with Reaction Temperature

In the green tea seed extract extracted in Preparation Example 1, naringinase and pectinase, naringinase and β-xylosidase, β-xylosidase, pectinase and cellulase. After each treatment to change the reaction temperature, the pH is adjusted to 6.0 and reacted for 40 hours. After that, the activity of the enzyme was stopped by heating, and the production rate of astragalin was confirmed by high performance liquid chromatography. As shown in Table 6 below.

Production rate of astragalin according to enzyme temperature (%) reaction
Temperature β-xylosidase Pectinase Naringianaze Naringinase + pectinase Naringinase + β-xylosidase Pectinase + Cellulase 20 ℃ 8 8 9 21 22 25 25 ℃ 12 14 13 31 35 37 30 ℃ 18 17 17 75 78 76 35 ℃ 23 22 29 79 80 81 40 ℃ 25 25 29 87 88 88 45 ° C 26 27 30 91 90 92 50 ℃ 28 29 32 93 94 95 55 ° C 28 29 31 90 91 91 60 ° C 27 28 27 87 90 83 65 ℃ 17 16 15 71 68 61 70 ℃ 17 16 14 53 54 49

According to the above results, the production rate of astragalin is in the range of 30 to 60 ℃ 75% or more, in particular, it can be confirmed that the production rate is close to 95% at 50 ℃.

Experimental Example 1 Identification of Astragalin

The products prepared in Examples 1 to 6 exhibited the following properties and were identified as astragalin (Varian Gemini 2000 300MHz, Varian).

<Physicochemical Properties of Astragaline>

Appearance: Light yellow fine crystal

Positive FAB-MS: 449 [M + H] +

¹ H NMR: 3.20 (1H, ddd, H5 '′), 3.29 (1H, t, H4 ′ ′), 3.35 (1H, t, H3 ′ ′), 3.43 (1H, dd, H2 ′ ′), 3.58 ( 1H, dd, H6'′b), 3.69 (1H, dd, H6′′a), 5.24 (1H, d, H1 ′ ′), 6.20 (1H, H6), 6.39 (1H, d, H8), 6.88 (2H, d, H3 ', 5'), 8.05 (2H, d, H2 ', 6')

¹³ C-NMR: 62.61, 71.43, 73.10, 75.72, 78.43, 94.83, 99.91, 104.13, 105.72, 116.13, 122.81, 132.30, 135.53, 158.55, 159.11, 161.53, 163.02, 165.91, 179.52

As described in detail above, according to the method for preparing astragalin according to the present invention, after separating camelliaside A or camelliaside B from plants, especially green tea seeds or green tea leaves, By supplementing the disadvantages of the chemical treatment method by using an enzyme suitable for the substrate, it is possible to effectively produce astragalin through selective sugar removal from camelliaside A or camelliaside B.

Claims (10)

In the method for producing astragaline represented by the formula (1), Obtaining a plant extract containing camelliaside A or camelliaside B from the plant using water or an organic solvent; And Separating the astragalin using an enzyme capable of selective sugar removal, The enzyme is a combination of one or more selected from galactosidase, pectinase, cellulase and xylosidase and naringinase, The reaction time of the enzyme is 10 to 60 hours, The concentration of the enzyme is 0.1 to 10% relative to the substrate, The reaction pH of the enzyme is 4.0 to 9.0, The reaction temperature of the enzyme is a method for producing astragalin, characterized in that 30 to 60 ℃. [Formula 1]

delete The method of claim 1, wherein the astragalin selectively removes the sugars of the galactopyranose group and the rhamnopyranose group of camellia seed A, or selects the sugars of the xylopyranose group and the rhamnopyranose group from the camellia seed B. Astragalin production method characterized in that obtained by removing. delete delete delete delete delete delete The method of claim 1, wherein the plant extract containing camelliaside A or camelliaside B is green tea seed or green tea leaf extract.