KR100563232B1 - Method for Preparing Salicin Analogous, the Salicin Analogous therefrom and Blood Coagulation Control Composition Containing the Salicin Analogous - Google Patents

Abstract

The present invention relates to a method for preparing a salicylic analog, a salicylic analog prepared therefrom and an anticoagulant composition containing the same, comprising the steps of: separating and purifying dextran sucrase from a strain of Leuconostoc mesenteroides species; The glucose of the salicylic acid, salicylic acid was added to the solution containing 0.01-2 M of sugar by adding the dextran sucrase and then reacting it with salicylic acid, salicylic alcohol and / or phenol in a reactor at 15-50 ° C. Transitioning to alcohol and / or phenol to produce salicylic analogs; And separating and purifying the salicylic analog, a method for preparing a salicylic analog, a salicylic analog prepared from the method, and a composition for controlling coagulation containing the salicylic analog are provided.

According to the present invention, dextran sucrase can be used to enzymatically synthesize salicylic analogues, and some of the synthesized salicylic analogs exhibit better blood coagulation inhibitory effects than aspirin, and some have higher blood coagulation than salicylic acid or water. It has a promoting effect and can be usefully used as a composition for controlling blood coagulation.

Dextran sucrose, sucrose, aspirin, receptor response, blood coagulation

Description

Method for preparing salicin analogs, and salicylic analogs prepared therefrom and blood coagulation control compositions containing the same {Method for Preparing Salicin Analogous, the Salicin Analogous therefrom and Blood Coagulation Control Composition Containing the Salicin Analogous}

1 is a TLC result of analyzing the distribution of the reaction product when the reaction with the addition of salicylic or salicylic alcohol solution and dextran sucrase in the sucrose solution according to the present invention. The products indicated by the arrows are the products newly synthesized by the receptor reaction.

Mn, maltodextrin standard mixture; IMn, isomaltodextrin standard mixture; S, salin

(a)

Lane 1, 0.3M sucrose + buffer + L. mesenteroides NRRL B-1299 dextran sucrase;

Lane 2, 0.3M sucrose + salicylic acid + L. mesenteroides NRRL B-1299 dextran sucrase;

Lane 3, 0.3 M sucrose + buffer + L. mesenteroides NRRL B-1299C dextran sucrose;

Lane 4, 0.3 M sucrose + salicylic acid + L. mesenteroides NRRL B-1299C dextran sucrase;

Lane 5, 0.3M sucrose + buffer + L. mesenteroides NRRL B-512 dextran sucrase;

Lane 6, 0.3 M sucrose + salicylic acid + L. mesenteroides NRRL B-512 dextran sucrase;

Lane 7, 0.3M sucrose + buffer + E. coli KCTC 0859BP dextran sucrase;

Lane 8, 0.3M sucrose + salicyline + E. coli KCTC 0859BP dextran sucrase;

Lane 9, 0.3M sucrose + buffer + L. mesenteroides NRRL B-1355 dextran sucrase;

Lane 10, 0.3 M sucrose + salicylic acid + L. mesenteroides NRRL B-1355 dextran sucrase;

Lane 11, 0.3 M sucrose + buffer + L. mesenteroides NRRL B-742 dextran sucrase;

Lane 12, 0.3M sucrose + salicylic acid + L. mesenteroides NRRL B-742 dextran sucrase;

(b)

Lane 1, 0.3M sucrose + buffer + L. mesenteroides NRRL B-1299C dextransukrase;

Lane 2, 0.3 M sucrose + salicylic alcohol + L. mesenteroides NRRL B-1299C dextran sucrase;

P1-P14 and PA1-PA8 are newly synthesized receptor products with the transfer of glucose to salicylic acid and salicylic alcohol.

Figure 2 is a TLC result confirming the synthesized receptor product after reacting sugar and dextran sucrose using phenol.

Mn, maltodextrin standard mixture

IMn, isomaltodextrin standard mixture;

Lane 1, 0.2 M sucrose + phenol + L. mesenteroides NRRL B-1299C dextran sucrase;

Lane 2, 0.2M sucrose + buffer + L. mesenteroides NRRL B-1299C dextransukrase;

Lane 3, phenyl-glucose;

Lane 4, phenyl--glucose;

Lane 5, salicyline;

Lane 6, salicylic alcohol;

P15; Phenyl α-glucoside, P16; Phenyl α-Isomaltoside

Figure 3 is the result of measuring the molecular weight after the purified purified salicylic analogs.

Figure 4 shows the NMR-spectrum structure analysis of salicylic alcohol isomaltotrioside in the synthesized salicy analog.

Figure 5a shows the chemical formula of the synthesized salicylic analogs.

Figure 5b shows the chemical structure of the salicylic analogs synthesized according to the present invention divided into three types.

The present invention relates to a method for preparing salicylic analogs, and more particularly, to a method for enzymatically synthesizing salicylic analogs using dextran sucrase, a salicylic analog prepared therefrom, and a composition for controlling blood coagulation containing the same. .

Aspirin (acetylsalicylic acid) has been shown to be effective in reducing the risk of cardiovascular disease, analgesic, anti-inflammatory and anticoagulant since its use as a medicine in 1899. Aspirin belongs to the compound family of salicylate substances and is converted to salicylic acid in the body. Salicycin has a structure similar to aspirin and when converted into salicylic acid, it has a powerful effect as an anti-inflammatory and analgesic agent. Currently salicylic acid (C1 ₃ H ₁₈ O ₇ ) is obtained from the bark of willow and poplar trees.

There are several difficulties in chemically preparing chemicals such as salicylic or salicylic derivatives. For example, chemically binding sugars to chemicals requires a difficult and difficult process to protect all chemical groups that can react outside of the desired sites, and then to deprotection after the reaction. In particular, it is very difficult to selectively attach two or more sugars to a particular site. For this reason, as a method for preparing salicylic acid or salicylic derivatives, a study in which a salicylic alcohol was put into culture using plant cells and converted to isosalcin in 31.1% yield after 2 days of incubation (Syahrani, A et al., 1998, Glycosylation of salicylic alcohol by cell suspension cultures of Solanum Iaciniatum, Journal of Asian Natural Products Research 1: 111-117) and salicylic alcohol using different plant cells to produce salicylic acid and isosalisine ( H. Mizukami et al., 1983, Glucosylation of salicylic alcohol in cultured plant cells, Phytochemistry, 22, 679-680).

However, these methods produce products by converting the glycosyl groups of the substrates that have been directly incubated with plant cells, or by combining one glycosyl group with a substrate that has been added to the glycosyl groups produced during metabolism during cell growth. As a method for producing isosalcin, a longer time is required than an enzymatic method as proposed in the present invention, and in particular, the synthesis result of two or more glucosyl groups obtained in the present method is not shown. In other words, no enzymatic synthesis of salicylic analogs has been proposed.

On the other hand, enzymatic synthesis has been studied a lot, for example, to prepare a material such as oligosaccharide synthesis.

Baek et al. (Synthesis of new oligosaccharides using glucansucurases, et al., Journal of the Korean Society for Industrial Microbiology, 26, 179-186, 1998) show that various dextran sucrases and receptors include maltose, genthiobioses and lactose. In this study, the effects of enzyme and receptor concentration on the product of the receptor have been studied. Lee et al., (Jin Ha, et al., Deformation and characterization of flulan using dextran sucrase, Journal of the Korean Society for Industrial Microbiology, 26, 264-268) , 1998), using polysaccharides such as flulan as receptors, Kim et al., Modification of Acetobacter xylinum bacterial cellulose using dextransucrase and alternansucrase, J. Microbial Biotechnol., 9, 704-708, 1999) studied the production of cellulose, cellulose composed of cellulose and cellotriose as receptors. Also recently, Yoon and Robyt, Synthesis of acarbose analogues by transglycosylation reactions of Leuconostoc mesenteroides B-512 FMC and B-742CB dextransucrases, Carbohydr. Res., 337, 2427-2435, 2003) synthesized a receptor product that significantly increased the activity inhibitory activity of amylase, a starch degrading enzyme, using acarbose as a receptor. Park et al., Enzymatic synthesis of fructosyl oligosaccharides by levansucrase from Microbacterium aevaniformans , Enzyme Microbial Technol., 32, 820-827, 2003) is a sugar-glucose receptor that uses melibanase as a receptor using levansukraase, one of the flactosyl-transferases. Receptor products transfected were synthesized.

Korean Patent Application No. 1998-24355 discloses dextran sucrase obtained from a mutant strain of Leuconostoc mesenteroides using sugar as a substrate and maltose, genthiobiose, raffinose or lactose as receptors. A method of producing novel oligosaccharides is disclosed.

However, studies have been conducted to prepare various novel substances through receptor reactions through enzymatic synthesis methods. However, the method for enzymatically synthesizing salicylic analogs using the dextran sucrase proposed by the present invention has not been carried out until now. It has not been presented or performed.

The present invention has succeeded in synthesizing salicylic analogs by using salicylic acid, salicylic alcohol and / or phenol as receptors and by transferring glucose to said receptors using dextran sucralase, and in particular, according to the structure of the salicylic analogs thus obtained, It was found that blood coagulation properties and blood coagulation promoting properties are excellent.

It is therefore an object of the present invention to provide a method for enzymatically synthesizing salicylic analogs using dextran sucrase.

Another object of the present invention is to provide a salicylic analog prepared from the above method.

Still another object of the present invention is to provide a composition for controlling blood coagulation containing the salicylic analog.

According to the first aspect of the present invention,

Separating and purifying dextran sucrase from Leuconostoc mesenteroides species strain;

Add glucose dextran sucrase to a solution containing 0.01-2 M of sugar and react it with salicylic acid, salicylic alcohol and / or phenol in a reactor at 15-50 ° C. Transitioning to alcohol and / or phenol to produce salicylic analogs; And

Isolating and purifying the salicylic analog;

Provided is a method for preparing salicylic analogs, including.

According to a second aspect of the present invention, there is provided a novel salicylic analog prepared by the above method.

According to a third aspect of the present invention, there is provided a composition for controlling blood coagulation, which contains 0.01-2% by weight of salicylic alcohol isomaltoside or salicylic glucoside (isosalcin) and has anticoagulant properties.

According to a fourth aspect of the present invention, there is provided a composition for controlling blood coagulation, which contains 0.01-2% by weight of salicylic alcohol isomaltotrioside and has a blood coagulation promoting property.

Hereinafter, the present invention will be described in detail.

The inventors have discovered a method for synthesizing salicylic analogs by transferring glucose of sugar to the receptor using phenol, salicylic and / or salicylic alcohol as receptors and dextransucrase, and moreover some salicylic analogs thus produced. According to the structure, it was found to exhibit improved anticoagulant properties or blood coagulation properties.

Dextran sucase is an enzyme that synthesizes oligosaccharides by transferring glucose units of sugar to added carbohydrates when other carbohydrates other than sugar are added to the enzyme reaction solution together with dextran sucase. The added carbohydrate is called the receptor and this reaction is called the receptor reaction. Depending on the type of enzyme, the binding structure of the glucose transferred to the receptor and the reaction efficiency of the carbohydrate, which is a receptor, are different. The concentration of the enzyme and the receptor is different depending on the concentration of the biosynthetic receptor product.

In the present invention, dextran sucrase produced from a specific microbial strain was used to make salicylic analogs by enzymatic method, and anticoagulation properties and blood coagulation promoting properties were confirmed according to the structure of the synthetic product.

The glycosyltransferase used is an enzyme that synthesizes dextran from sugar with dextransucrase (EC 2.4.1.5), and the reaction mechanism of the dextran sucrase sugar is as follows.

n sucrose (glucose) n-m-w + n-m lactose +

                       m Leucrose + w glucose

(Where n, m, w are the moles of each compound, n = m + w)

The main products of this enzymatic reaction are high molecular weight dextran and monosaccharide lactose of about 10 ⁷ ~ 10 ⁸ Da, and by-products of glucose and lucrose (5-O-α-D-glucopyranosyl-D-fructopyranose) Is produced.

Dextran sucrase used in the present invention may be obtained from a strain of Leuconostoc mesenteroides or Streptococcus sp. Separation of dextran sucrose from the microorganism is, for example, inoculated with LB medium containing a yeast extract, peptone, K ₂ HPO ₄ and sucrose and incubated in the culture medium, and then only the cells in the culture medium. By separating and purifying dextran sucrose from it. Isolation and purification of the dextran sucrase can be done by general methods known in the art.

Preferred strains of luconosestock mesentroid species for obtaining dextran sucrase include Leuconostoc mesenteroides NRRL B-1299, Leuconostoc mesenteroides NRRL B-1299C, and luconosestock messen. Leuconostoc mesenteroies NRRL B-1355, Leuconostoc mesenteroides NRRL B-742, Leuconostoc mesenteroides NRRL B-512 (ATCC 10830) or mutants thereof. In addition, the dextran sucrase used in the present invention is derived from Streptococcus strains such as Streptococcus mutans , Streptococcus sobrinus , Streptococcus salivarius , or the like. You can get it.

When the dextran sucrase is added to a solution containing 0.01-2 M of sugar and reacted with salicylic acid, salicylic alcohol and / or phenol, the sugar is decomposed so that the glucose of sugar is salicylic, salicylic alcohol and / or Or transferred to phenol to synthesize various salicylic analogs. At this time, the dextran sucrase is preferably added so that the reactor final active concentration is 0.03-3.3U / ml, and the salicycin, salicylic alcohol and / or phenol is each so that the reactor final active concentration is 0.05-2.0M It is preferable to add. In this case, the reaction is generally performed for 1-18 hours at all the sugars, preferably at 15-50 ° C, more preferably at 28-37 ° C, until all the sugars are decomposed.

When salicycin is used in the reaction, salicylic alcohol isomaltoside, salicylic alcohol nigeroside, salicylic alcohol isomaltrioside, salicylic alcohol nigerosyl glucoside, salicylic isomaltotetroside, Salicylic alcohol isomaltetotroside, salicylic alcohol nigerosyl isomaltoside, salicylic isomaltopentoside, salicylic alcohol isomaltopentoside, salicylic alcohol isomaltohexaside and / or salicylic alcohol isomaltopento Sides and the like can be created.

When salicylic alcohol is used in the reaction, salicylic analogs such as salicylic glucoside (isosalcin), salicylic isomaltoside, salicylic alcohol isomaltoside, salicylic isomaltotroside, salicylic isomaltopentoside, Salicylic alcohol isomaltopentoside and / or salicylic alcohol isomaltohexoside may be produced, and when phenol is used in the reaction, the salicylic analogs are phenyl alpha-glucopyranose and / or phenyl isomaltoxide; Can be generated.

The salicylic analogs synthesized according to the present invention can be divided into three types as shown in Chemical Formulas 1, 2, and 3 as a structure in which glucose is increased one by one to a free hydroxyl group of phenol, salicylic alcohol, and salicylic acid.

[Formula 1]

(Wherein n is 0-8)

[Formula 2]

(In general formula, m is 1-8)

[Formula 3]

(Wherein l is 0-4)

As type A, preferably salicylic isomaltopentoside, salicylic isomaltoside or salicylic isomaltotetroside may be produced, and as type B, preferably salicylic alcohol isomaltside, salicylic alcohol iso Maltotrioside, salicylic alcohol isomaltotetroside, salicylic alcohol isomaltopentoside or salicylic alcohol isomaltohexoside may be produced, and as type C, preferably salicylic alcohol nigeroside, salicylate Salicylic analogs of real alcohol nigerosyl glucoside or salicylic alcohol nigerosyl isomaltoside can be produced.

Among these salicylic analogs, salicylic alcohol isomaltside and salicylic glucoside (isosalcin) represented by the following formulas (4) and (5), respectively, have excellent properties in inhibiting blood coagulation and may be useful for anticoagulants. When the salicylic alcohol isomaltoside and salicylic glucoside are used for the anticoagulant, 0.01-2% by weight based on the total weight of the composition is preferred.

[Formula 4]

[Formula 5]

In addition, salicylic alcohol isomaltotriosides represented by the following formula (6) among the salicylic analogs are excellent in blood coagulation promoting effect, it is considered that they can be usefully used as a coagulation accelerator. When the salicylic alcohol isomaltotrioside is used for the coagulation accelerator, 0.01-2% by weight is preferred based on the total weight of the composition.

[Formula 6]

Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples, but the present invention is not limited thereto.

Example 1 (Preparation of Dextran Sucrase Enzyme Liquid)

Preparation Example 1

E. coli DH5α / pFMCM (KCTC 0859BP) was inoculated in LB liquid medium containing ampicillin (50 μg / ml) (tryptone 1%, yeast extract 0.5%, NaCl 0.5%) and incubated at 37 ° C. Only the cells were isolated from the culture medium to prepare a crude enzyme solution, which was used as the dextran sucrase enzyme solution.

Preparation Example 2

L. mesenteroides NRRL B-512 was inoculated in LB medium containing 5 g of yeast extract, 5 g of peptone, 5 g of K ₂ HPO ₄ and 20 g of sucrose in 1 L of water and incubated at 28 ° C. Was prepared and purified dextran sucrose from this enzyme solution. Purification of enzyme was previously equilibrated with DEAE-Sepharose (2.5x35cm) with 20mM sodium phosphate buffer (pH6.8) and then co-enzyme solution was applied to remove unbound protein with three times the buffer of the column, The adsorbed enzyme was separated by NaCl (0-1M) solution prepared with the same buffer. The active protein fractions were collected, dialyzed with the same buffer solution, and then applied to the column three times to separate and purify the dextran sucrase activity.

Preparation Example 3

L. mesenteroides NRRL B-1299, B-1299C, B-742, and B-1355 were inoculated in LB medium containing 5 g of yeast extract, 5 g of peptone, 5 g of K ₂ HPO ₄ and 20 g of sucrose in 1 L of water, and cultured at 28. After that, only the cells were separated from the culture medium to prepare a crude enzyme solution, and dextran sucrase was purified from the enzyme solution. Purification of the enzyme was carried out in the same manner as in Preparation Example 2.

Example 2 (confirmation of oligosaccharide production reaction and composition of reaction products)

1) Oligosaccharide Production Reaction

In this example, 0.3M sugar solution was prepared to produce oligosaccharides, and the final sugar concentration of the enzyme reactor was adjusted to 0.16 by mixing the dextran sucralase enzyme obtained in Example 1 with 0.05M salicylic acid or 0.05M salicylic alcohol. The reaction was carried out at 28 ° C. as M. Phenol was added to the enzyme reactor so that the final concentration was 0.08 M, and the final activity of the enzyme was 1 U / ml. One unit of enzyme is expressed as the number of moles of lactose liberated from sugar per ml of enzyme per minute. The reaction was carried out at 28 until all of the sugar in the reactor was consumed (about 12 hours). Production of oligosaccharides and confirmation of complete use of sugar were carried out with 1 μl of the reaction solution, which was deposited on a Merck K6F TLC plate and prepared with nitromethane: normalpropyl alcohol: water = 2: 5: 1.5 or acetonitrile: water = 85: 15. The developed carbohydrates were separated by using a coloring reagent containing 0.5% (w / v) α-naphthol and 5% (v / v) sulfuric acid on a high pressure thin layer chromatograph (TLC) plate. Analyzed. Quantitative analysis of each carbohydrate was performed with the NIH Image Program using a Macintosh (Power PC; 7100/80) computer.

2) Check the composition of the reaction product

The composition of the produced product was also confirmed using HPLC. In order to purify the reaction, Bio Gel P-2 and Superdex G-10 columns were prepared, samples were applied, eluted with water, and the degree of sugar separation was confirmed using TLC. The separated sugars were concentrated in vacuo and the monosaccharides were removed using yeast beads, and the degree of removal was confirmed using TLC. The composition and distribution of the synthetic product were confirmed using TLC, MALDI-TOF MS (Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometer) and NMR-spectrum, and the receptor reaction results and structures are shown in Figs. It was.

Eight or more receptor reaction products, including salicylic alcohol isomaltoside (P1 in FIG. 4), from the reaction of dextransucrase with salicycin from L. mesenteroides NRRL B-1299 (A-lane 2 in FIG. 1). Was synthesized, and nine or more receptor reaction products including salicyl isomaltoside were synthesized from the reaction of dextransucrase and salicylic acid obtained from the mutant L. mesenteroides NRRL B-1299C (lane 4 in Fig. 1). It was confirmed. In addition, it was confirmed that three receptor reaction products were synthesized from the reaction of dextran sucrase and salicylic acid obtained from L. mesenteroides NRRL B-512 (a-lane 6 in FIG. 1) (see Table 1). 7 Receptor reactions including salicylic isomaltoside (P1 in FIG. 4) also from the reaction of dextransukrases and salicylates from E. coli DH5α / pFMCM (KCTC 0859BP) (salin 8 in FIG. 1). It was confirmed that the product was synthesized, and 12 receptor reaction products were identified from the reaction of dextransucrase and salicylic acid obtained from L. mesenteroides NRRL B-1355 (A-lane 10 in FIG. 1), and L. mesenteroides NRRL B- Five receptor reaction products were identified from the reaction of dextransucrase and salicylic acid from 742 (a-lane 12 in FIG. 1), and the structure and amount of the product differed depending on the enzyme used in the synthesis. It was confirmed that eight receptor reaction products were synthesized from the reaction of dextran sucrase and salicylic alcohol obtained from L. mesenteroides NRRL B-1299 (b-lane 2 in FIG. 1).

The composition of the salicylic analog product produced is summarized in Table 1.

Dextransukrases of E. coli DH5α / pFMCM (KCTC 0859BP), L. mesenteroides NRRL B-512, B-1299, B-1299C, B-742, B-1355 commonly have salicylic alcohol isomaltsides ( A, P1) of FIG. 1, and a product in which glucose was combined with one (P4), two (P9), three (P12), four (P13), and five (P14), respectively. The product that combines one more glucose (P3) and two (P5, P6), three (P7, P8, P10) and four (P11) more glucose is determined to be linked to different structures depending on the enzyme From this, it can be seen that the structure of the synthesized product varies depending on the type of enzyme. In the case of using salicylic alcohol from b of FIG. 1, isosalicylic acid (Salicyl glucoside, PA1), glucose-salicylic isomaltoside (PA2), and salicylic alcohol glucoside-glued salicylic alcohol Eight receptor reaction products were synthesized including isomaltoside (PA2).

Meanwhile, the result of confirming the receptor reaction product by TLC after reacting sugar with dextran sucrase using phenol is shown in FIG. 2. Phenol α-glucose (P15) and phenyl α-isomaltose (P16), in which one and two glucoses were bound to phenol, were synthesized from the reaction of dextran sucrase and phenol obtained from L. mesenteroides NRRL B-1299. .

The result of measuring the molecular weight of the synthesized salicylic analogs after pure purification is shown in FIG. 3, and NMR-spectral analysis was also performed by purifying salicylic alcohol isomaltotrioside. The results are shown in FIG. In addition, using the MALDI-TOF MS (Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometer) and the NMR spectrum results confirmed the structure and the results are shown in Figure 5a. As a result, it was confirmed that the size of the form in which the glucose group was increased one more time in salicylic acid (molecular weight 286.3), accordingly, glucose (1, P1, PA3), 2 (P4), 3 (P9, PA5), 4 Products (P12, PA7), five (P13, PA8) and six (P14) more confirmed the combined product. Thus, products of the combined structure are synthesized by increasing glucose one by one to the free hydroxyl groups of phenol, salicylic alcohol, and salicylic acid, and the structure is mainly composed of three types, for example, salicylic β-isomaltooligosaccharide (Fig. A type 5b; n is 0 or more; has a structure such as PA2, PA4, PA6, P7, P11) and the other is for example salicylic alcohol β-isomaltooligosaccharide (type B of FIG. 5B; m is 0 Above; have a structure such as P1, P4, P9, P12, P13, P14, PA3, PA5, PA7), and another form a branched bond, for example salicylic alcohol nigerosyl isomaltooligosaccharide (Fig. C type of 5b; l is divided into the same structure as 0 or more; P3, P5, P12).

Example 3 (Coagulation inhibitory effect of salicylic analogs)

The blood coagulation inhibitory effect of the synthesized salicylic analogs was investigated using whole blood isolated from rabbits. Synthetic salicylic analogs were added to the blood at 1% (w / v) and measured at the time of blood coagulation while reacting at 37 ° C. In addition, the same experiment was performed using aspirin and salicycin as a comparison group and pure blood as a control group, and the time of blood coagulation was compared. The results are shown in Table 2 below.

Inhibitory Effect of Salicylic Analogs on Blood Coagulation Type of sample added to the blood Pure blood aspirin Salinity Salicylic Alcohol Salicylic Glucopyranoside Salicylic alcohol isomaltoside Coagulation 10 minutes + * + + － ** － － 20 minutes + + + + － － / + ***

* +: Blood clots

**-: No blood clot

***-/ +: blood does not coagulate and begins to coagulate

As a result of the test, the addition of salicylic glucoside and salicylic alcohol isomaltoside in the synthesized salicylic analogs was superior to the reaction of adding aspirin and salicylic acid to the coagulation delaying effect.

Example 4 (blood coagulation promoting effect of the salicylic analogs)

The blood coagulation promoting effect of the synthesized salicylic analogs was investigated using a commercial blood coagulation test diagnostic reagent (Pacific Hemostasis Thromboplastin-DS). 20 μl of the synthesized salicylate analog was added to 25 μl of thromboplastin, 480 μl of water, and 100 μl of plasma to react at 37 ° C., and the time of solidification was measured while measuring absorbance at 600 nm. In addition, the same experiment was performed using aspirin and salicycin as a comparison group and water as a control group, and the time of blood coagulation was compared. Among the salicylic analogs, especially salicylic alcohol isomaltotrioside showed a blood coagulation promoting effect, the results are shown in Table 3 below.

Blood clotting-promoting effect of salicylic analogs Sample type added water Salinity Salicylic alcohol isomaltotrioside Time taken for blood coagulation to progress by 50% 220 seconds 290 seconds 90 sec

According to the present invention, dextran sucrase can be used to enzymatically synthesize salicylic analogs. Some of the synthesized salicylic analogs can be effectively used as anticoagulant compositions because they exhibit superior blood coagulation inhibitory effects than aspirin. In addition, according to the structure of the analogues exhibits a higher blood coagulation promoting effect than salicylic acid or water can be usefully used as a composition for the coagulation promoter.

Claims (17)

Dextran sucrase was added to a solution containing 0.01-2 M of sugar and then reacted with salicylic, salicylic alcohol and / or phenol in a reactor at 15-50 ° C. And / or transition to phenol to produce salicylic analogs; And Isolating and purifying the salicylic analog; Method for producing salicylic analogs comprising a. The method of claim 1, wherein the dextran sucrase azepin Lu Pocono stock mesen steroid (Leuconostoc mesenteroides). Strain or Streptococcus (Streptococcus) raised analogues which is characterized that obtained from the sp method. The method of claim 2, wherein the Leuconostoc mesenteroides strain is Leuconostoc mesenteroides NRRL B-1299, Leuconostoc mesenteroides NRRL B-1299C, Luconostock Leuconostoc mesenteroies NRRL B-1355, Leuconostoc mesenteroides NRRL B-742, Leuconostoc mesenteroides NRRL B-512 (ATCC 10830) or mutants thereof Method of preparing salicylic analogs. The method of claim 1, wherein the dextran sucrase is added so that the reactor final active concentration is 0.03-3.3 U / ml. 2. The method of claim 1, wherein the salicylic acid, salicylic alcohol and / or phenol are each added such that the reactor final active concentration is 0.05-2.0 M. The method of claim 1, wherein the reaction is performed at 28-37 ° C. for 1-18 hours. The method of claim 1, wherein when salicylic acid is used in the reaction, salicylic alcohol isomaltoside, salicylic alcohol nigeroside, salicylic alcohol isomaltotriside, salicylic alcohol nigerosyl glucoside, salicylic Isomaltotetroside, salicylic alcohol isomaltotetroside, salicylic alcohol nigerosyl isomaltoside, salicylic isomalpentoside, salicylic alcohol isomaltopentoside, salicylic alcohol isomaltohexoside and / or Salicylic alcohol isomaltopentoside is produced. The method of claim 1, wherein when salicylic alcohol is used in the reaction, salicylic glucoside (isosalcin), salicylic isomaltoside, salicylic alcohol isomaltoside, salicylic isomaltotetroside, A method for preparing salicylic analogs, characterized in that salicylic isomaltopentoside, salicylic alcohol isomaltopentoside and / or salicylic alcohol isomaltohexoside are produced. The method of claim 1, wherein when phenol is used in the reaction, phenyl alpha-glucopyranose and / or phenyl isomaltside are produced as salicylic analogs. The salicylic analogs represented by the following formula (1). [Formula 1]

(In the formula, n is 0-8.) The salicylic analogs represented by the following formula (2). [Formula 2]

(In general formula, m is 1-8) The salicylic analogs represented by the following formula (3). [Formula 3]

(In the formula, l is 0-4) 10. The salicylic analogue according to claim 9, wherein the salicylic analog is salicylic isomaltopentoside, salicylic isomaltoside or salicylic isomaltotetroside. The method of claim 10, wherein the salicylic acid analog is salicylic alcohol isomaltoside, Salicylic analogs characterized by salicylic alcohol isomaltotrioside, salicylic alcohol isomaltotetroside, salicylic alcohol isomaltopentoside or salicylic alcohol isomaltohexoside. The salicylic analogue according to claim 11, wherein the salicylic analog is salicylic alcohol nigeroside, salicylic alcohol nigerosyl glucoside or salicylic alcohol nigerosyl isomaltoside. Salicylic alcohol isomaltoside represented by the following formula (4), or [Formula 4]

Salicylic Glucoside (Isosalcin) represented by Formula 5 [Formula 5]

Containing 0.01-2% by weight, the composition for controlling blood coagulation having anticoagulant properties. Salicylic alcohol isomaltotrioside represented by the following formula (6) [Formula 6]

Containing 0.01-2% by weight, the composition for controlling blood coagulation having blood clotting promoting properties.

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