KR100553578B1 - Puerarin derivatives with high solubility and preparation method for the same - Google Patents

Abstract

The present invention relates to furan derivatives and methods for their preparation. In particular, the present invention provides a furin derivative having excellent water solubility and stability by preparing glucose and maltose by transferring glycosylated glycosides to glycosides in a sugar transfer reaction of maltogenic amylase.

Furarin derivatives, sugar transition, furarin, maltogenic amylase

Description

Purinine derivatives having a high water solubility and a method for preparing the same {PUERARIN DERIVATIVES WITH HIGH SOLUBILITY AND PREPARATION METHOD FOR THE SAME}

Figure 1 shows the results of thin layer chromatography analysis of the reaction product prepared by the transfer to the purine by maltogenic amylase.

Figure 2 shows the high performance liquid chromatogram of the reaction product prepared by transferring sugar to furin using maltogenic amylase.

FIG. 3 is an LC / MASS spectrum of α-1,6-glucosylfuranine prepared and purified according to the present invention.

4 is an LC / MASS spectrum of α-1,6-maltosyl furanine of the present invention.

Fig. 5 is the ¹³ C-NMR spectrum of α-1,6-glucosyl furarin of the present invention.

Figure 6 is a ¹³ C-NMR spectrum of the α-1,6-maltosyl furanine of the present invention.

[TECHNICAL FIELD OF THE INVENTION]

The present invention relates to a furanine derivative having a high water solubility and a method for preparing the same, and more specifically, α-1,6-malto prepared by transferring glucose or maltose to furanin by a sugar transfer reaction of maltogenic amylase. Oligosaccharides furarin.

[Private Technology]

Pueraria lobata , which belongs to the legumes, is used for medicinal purposes in Korea, China and Japan. Brown root is widely used for sweating, urticaria treatment, branching and antipyretic in oriental medicine. Brown root has been used as a medicinal or sulfurous plant in Korea, and in recent years, the amount of use as a health food in addition to medicinal. The active principal components of cartilage are known as isoflavone-based components, such as purine, dydazine, and genistin, and these are plant hormones, phytoestrogen, which are in the spotlight for treating breast cancer and osteoporosis.

On the other hand, furraline has a unique structure different from that of ordinary isoflavones. In other words, Dydzin and Genistin have glycosides with glycosides between aglycone and oxygen, but furanin has direct carbon-carbon bonds with glycosides and aglycones. .

Recently, many studies have been conducted on the physiological activity of furin, and it has been reported that the root extract has a high affinity for estrogen receptor β (ER β) and shows strong anti-breast cancer activity. In addition, purine has an antioxidant effect and is involved in various physiological effects such as cancer cell growth or bone metabolism by being involved in signaling within cells.

Due to the pharmacological effects of the above-mentioned purine, research is being conducted to utilize it as a health supplement. However, in the case of extracting and purifying the furanin to be commercialized, there is a problem that the furanin is low in water solubility insoluble in water. Therefore, there is a need for a study on how to improve the water solubility of furarin.

In order to solve the problems of the prior art, an object of the present invention is to provide a purine derivative having excellent water solubility.

It is another object of the present invention to provide a method for producing a purine derivative having excellent water solubility.

In order to achieve the above object, the present invention provides a purine derivative represented by Formula 1:

(Formula 1)

In Formula 1, R is α-1,6-maltosyl or α-1,6-glucosyl.

In another aspect, the present invention provides a method for producing a purine derivative comprising the transfer of glucose and maltose to glycosides furarin or derivatives thereof by a sugar transfer reaction of maltogenic amylase.

Hereinafter, the present invention will be described in detail.

The inventors of the present invention have completed the present invention by recognizing that the water solubility is remarkably increased when the sugar transition to the furanine or a derivative thereof is conducted while researching to develop a high water solubility derivative.

FIELD OF THE INVENTION The present invention relates to furarine or derivatives thereof, which have been sugars with glucose and maltose.

The purine derivatives of the present invention have remarkable water solubility, such as about 100 times higher water solubility than purine. Therefore, the furanine can be used in the state of sugar transition can be commercialized in various forms.

In another aspect, the present invention relates to a method for producing a purine derivative using a sugar transfer enzyme, wherein the method for preparing a purine derivative comprises transferring glucose and maltose to glycosides furarin or derivatives thereof by a sugar transfer reaction of maltogenic amylase. It provides a manufacturing method.

More specifically, furanine and maltotriose are added to the sugar transfer enzyme in a ratio of 1 to 1 by 10, and mixed and reacted in a buffer of pH 5.5 to 6.5. The buffer may be sodium citrate, and the reaction may be performed at 45 to 65 ° C. for 5 hours to 30 hours. After the reaction, the reaction solution is filtered, and the purine derivative is separated by chromatography.

The glycotransferase is a conventional maltogenic amylase enzyme, which may be isolated and purified from a native prokaryotic or eukaryotic organism, and may be prepared by artificially expressing a gene thereof by genetic recombination technology. For example, in the present invention, a maltogenic amylase obtained by transforming and expressing and purified a maltogenic amylase gene cloned from Bacillus stearothermophilus into E. coli MC1061.

Purine derivatives prepared according to the method can be identified by thin layer chromatography, nuclear magnetic resonance spectra, molecular weight measurement and high performance liquid chromatography.

In the present invention, in order to confirm the preparation of the purine derivatives, thin layer chromatography was performed using, for example, a reaction product prepared by reacting maltotriose and purine under maltogenic amylase as a sample. As a result, as can be seen in the development of the reaction product of lane 2 in Figure 1, it was confirmed that glucose or maltose was transferred to furarine, and the sugar transition to α-1,6 binding in nuclear magnetic resonance spectra.

Then, the reaction product was separated and purified by high performance liquid chromatography, respectively, and the molecular weight thereof was analyzed. The reaction products were shown in FIG. 3, the glucose peak was measured at 578 from the ion peak appearing at m / z 579.5 (M + H] ⁺ ), and m / z 741 (as shown in FIG. 4). It was maltosyl furanine whose molecular weight was measured at 740 from the ion peak shown by [M + H] ⁺ ), m / z 763 ([M + Na] ⁺ ), m / z 779 ([M + K] ⁺ ).

Maltooligosaccharide furanine of the present invention is about 100 times superior to furanin in water solubility.

Hereinafter, examples of the present invention will be described. The following examples are only for illustrating the present invention, but the protection scope of the present invention is not limited to the following examples.

Example 1: Preparation of Purine Derivatives

Purarin was extracted from methanol using methanol, and maltotriose was purchased from Sigma Chemical Co., St. Louis, Mo. USA.

Maltogenic amylase was prepared by transforming the maltogenic amylase gene cloned from Bacillus stearothermophilus into Escherichia coli MC1061, expressing the maltogenic amylase therefrom, and then purifying by column chromatography (Cha et al . , 1998, Eur. J. Biochem. 253, 251-262).

Maltogenic amylase 1 U / mg G3, 0.25% furarine and 1% maltotriose were mixed in sodium citrate buffer (pH 6, Sigma) and reacted at 55 ° C. for 20 hours. After the reaction, the reaction solution was boiled for 5 minutes and filtered using Whatman filter paper, and the filtrate was separated on a C ₁₈ column (Sep-Pak Plus C ₁₈ , 30 mm X 25 mm, VyDAC), followed by high performance circulation. The reaction product was separated and purified through a gel permeation column (JAIGEL-W251, 40mm x 500mm, JAI) using liquid chromatography.

Example 2 Analysis of Reaction Products Using Thin Film Chromatography

Thin film chromatography was performed to analyze the product of the reaction solution obtained in Example 1. After completion of the reaction, each sample was loaded in a 1 µl portion of Whatman K5F TLC plate (20 cm x 20 cm), and developed on a developing solution containing 5: 3: 1 of n-butanol: acetic acid: distilled water. After development, the plate was dried and checked at 254 nm UV, and in a coloring solution in which 0.3 (w / v)% N- (1-naphthyl) -ethylenediamine and 5 (v / v)% sulfuric acid were dissolved in methanol. After immersion, color development was performed for 10 minutes in an oven at 110 ° C.

Figure 1 shows the results of thin layer chromatography analysis of the reaction product prepared by the transfer to the purine by maltogenic amylase. In Figure 1, "Pu" means furanin, "G1-Pu" means glucosyl furanin, "G2-Pu" means maltosyl furanin. Lane 1 is the development of furin, lane 2 is the development of the reaction product prepared in Example 1.

In Fig. 1, glucose and maltose were transferred to furarin by maltogenic amylase, respectively, to confirm that α-1,6-maltosyl furarine and α-1,6-glucosyl furaline were produced.

Example 3 Isolation of α-1,6-Glucosyl Furan and α-1,6-Maltosyl Furanine Using High Performance Liquid Chromatography

The reaction product isolated and purified in Example 1 was analyzed using High Performance Liquid Chromatography (HPLC). The reaction product was diluted with ultrapure water, filtered through a 0.45 µm thin film filter paper, and then 20 µl was injected into the column, and the solvent was flowed at a flow rate of 1.2 ml / min. HPLC analysis was performed using Waters (USA) 600E gradient pump, UV detector and C ₁₈ column, solvents were A solution (water / formic acid, 100 / 0.1 volume ratio), B solution (methanol / water / formic acid, 50 /50/0.1 volume ratio) was used in combination. The detection wavelength was set to 265 nm.

Figure 2 shows the high performance liquid chromatogram of the reaction product prepared by transferring sugar to furin using maltogenic amylase. In FIG. 2, peak 1 is furarin, peak 2 is α-1,6-glucosyl furan, and peak 3 is α-1,6-maltosyl furanin.

Only fractions eluted with peak 2 of FIG. 2 were obtained as α-1,6-glucosyl furarin, and only fractions eluted with peak 3 were α-1,6-maltosyl furanine, respectively.

Example 4 Identification of α-1,6-Glucosyl Furan and α-1,6-maltosyl Furan

4-1. Molecular Weight

The molecular weights of α-1,6-glucosyl furanine and α-1,6-maltosyl furanine purified in Example 3 were measured.

LC / MS was used, the system used JMS-LC mate (JEOL, Japan), and the ion mode used APCI ⁺ . Experimental results are shown in FIGS. 3 and 4.

Figure 3 is an LC / MASS spectrum of α-1,6-glucosyl furarin of the present invention. The molecular weight of the sample was determined to be 578 from the ionic peak shown at 579.5 ([T1 + H] ⁺ ) as α-1,6-glucosylfuranine, confirming that it was glucoseofuran.

4 is an LC / MASS spectrum of α-1,6-maltosyl furanine of the present invention, wherein α-1,6-maltosyl furanine is 741 ([T2 + H] ⁺ ), 763 ([T2 + Na). ] ⁺ ) And the molecular weight of the sample was measured at 740 from the ion peak appearing at 779 ([T2 + K] ⁺ ) to verify that it is maltosyl furan.

4-2. ¹³ Structural Analysis of α-1,6-Glucosyl Furan and α-1,6-maltosyl Furan by C NMR

In order to confirm the structures of α-1,6-glucosyl and maltosyl furanine purified in Example 3, ¹³ C nuclear magnetic resonance analysis (NMR; JNM LA-400 FT-NMR spectrometer (JEOL, Japan)) Was performed.

Samples were prepared by dissolving α-1,6-glucosyl and maltosyl furanine purified in Example 3 in DMSO-d6, and the results are shown in FIGS. 5 and 6.

Example 5 Properties of α-1,6-Glucosyl Furan and α-1,6-maltosyl Furan

Each of α-1,6-glucosyl furanine and α-1,6-maltosyl furanine was added to distilled water, sonicated for maximum dissolution, and stirred at room temperature for 1 hour. Subsequently, the resultant was filtered with a 0.45 μm filtration membrane and quantified by high performance liquid chromatography.

The solubility test results of α-1,6-glucosyl furanine and α-1,6-maltosyl furanine are shown in Table 1 below. In Table 1, the solubility of furin was 1.77 x 10 ^-2 mol / L, which was hardly soluble in water, but the solubility of α-1,6-glucosylfuran was about 2.0. mol / L water, and the solubility of α-1,6-maltosyl furanine was 2.0 With mol / L water, the solubility of each of about 100-fold higher than that of furarin was increased. Therefore, when the sugar transfer to the furanrin can significantly increase the water solubility of the furanin.

compound Solubility (mol / L water) Control (Furarin) 1.77 x 10 ^-2 Glucosyl Furanine > 0.2 x 10 Maltosyl Furan > 0.2 x 10

As described above, the present invention can be produced in various forms by providing the α-1,6-glucosyl fururin and α-1,6-maltosyl fururin excellent in water solubility as compared to the fururin. .

Claims (5)

delete A method for preparing a purine derivative of Formula 1, comprising transferring glucose or maltose to glycosides furanine or a derivative thereof by a sugar transfer reaction of maltogenic amylase. (Formula 1)

R is α-1,6-maltosyl or α-1,6-glucosyl. The method of claim 2, wherein the maltogenic amylase is a recombinant protein prepared by transforming a maltogenic amylase derived from Bacillus stearothermophilus or a maltogenic amylase gene derived from Bacillus stearothermophilus . A method of increasing the water solubility of furarine comprising transferring glucose or maltose to glycosides furarine or derivatives thereof by a sugar transfer reaction of maltogenic amylase. The method of claim 4, wherein the maltogenic amylase is a recombinant protein prepared by transforming a maltogenic amylase derived from Bacillus stearothermophilus or a maltogenic amylase gene derived from Bacillus stearothermophilus .

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