KR100260612B1 - A process for preparing alpha-glycosidase inhibitors from silkworm urine - Google Patents

Abstract

PURPOSE: An α-glycosidase inhibition material from silkworm urine is provided, which shows nearly same α-glycosidase inhibition effect as the existing diabetes medicine, the Acarbose, thereby contributing to increase of farm income and enhancement of national health. CONSTITUTION: A process for the preparation of α-glycosidase inhibition material from silkworm urine comprises the steps of: filtering the silkworm urine, freeze-drying to get powder, and adding methanol(alcohol-precipitation); processing anion-exchange column chromatography with the methanol-soluble part to get active part; separating into methanol-soluble part and insoluble part to remove inorganic material in silkworm urine; adding water and acetonitrile to methanol-soluble part to separate into acetonitrile layer and water layer, and concentrating the water layer and separating into A, B and C fractions(1-deoxynojirimycin, fagomine, 1,4-dideoxy-1,4-imino-D-araviniton showing excellent α-glycosidase inhibition effect).

Description

Method for preparing α-glycosidase inhibitor from silkworm urine

The present invention relates to a method for producing α-glycosidase inhibitor from silkworm urine. More specifically, the present invention is freeze-dried silkworm urine, alcohol precipitation and a series of ion exchange column chromatography, 1-deoxynojirimycin which are excellent substances for inhibiting α- glycosidase (deoxynojirimycin) , A method for preparing fagomine and 1,4-didioxy-1,4-imino-di-arabinitol (1,4-dideoxy-1,4-imino-D-aravinitol) will be.

Insulin secretion in healthy people is characterized by the fact that insulin is released into the blood at a rapid rate during glucose loading, and then insulin is released gradually over an hour, while promoting insulin or insulin secretion administered for the treatment of diabetics. Drugs have limitations in mimicking the normal insulin secretory reaction, and thus, side effects such as hypoglycemia and hyperinsulinemia are often encountered.

As a way to solve such discrepancies between elevated blood sugar and insulin content after eating, numerous methods have been tried, including the adjustment of drug administration time, the development of new drug routes and the development of new formulations to promote drug absorption. It has been, but not yet satisfactory.

Meanwhile, Jekins recommends a “lente carbohydrate concept,” which includes foods that contain a lot of dietary fiber, such as pectin, or contain as much carbohydrates as possible in the form of foods, such as starch. It has been suggested that this may partially solve the post-prandial hyperglycemic problem (Jenkins, D., Lente carbohydrate: A new approach to the diary management of diabetes, Diabetes Care, 5: 634 (1982)). However, such a regime is virtually impossible without becoming a herbivorous. In addition, during the development of various methods to solve the post-prandial hyperglycemic problem, a method of using an α-glycosidase inhibitor, which has an activity of delaying glucose absorption in the small intestine, has been devised. The drug was proved through. As such, administration of the α-glycosidase inhibitor in the small intestine can control blood sugar levels that rise rapidly after meals, and can solve a large increase in blood sugar levels and decreases that are commonly seen in diabetics. The release of can also be suppressed, which may help prevent the development of other circulatory complications caused by hyperinsulinemia.

On the other hand, the present inventors already have a significant inhibitory effect on the blood sugar rise after the silkworm-related substances, which, like acarbose (acarbose), which is an existing diabetic drug, inhibits α-glycosidase, an enzyme involved in carbohydrate digestion in the small intestine. (Jeong-sun Lee et al., Journal of Pharmacy, 39 (4): 367-372 (1995); Chung Sung-hyun et al., Kyung Hee Pharmacy Thesis, 24: 95-100 (1996)). However, it is not known which component of the silkworm acts as an inhibitor of α-glycosidase.

Accordingly, the present inventors have made diligent efforts to prepare α-glycosidase inhibitors from silkworm urine. As a result, 1-dioxynojirimycin, pagomin and 1,4-didie are excellent substances for inhibiting α-glycosidase. It was confirmed that oxy-1,4-imino-D-arabinol could be separated, and the present invention was completed.

After all, it is an object of the present invention to provide a method for producing a series of α-glycosidase inhibitors from silkworm urine.

1 is a graph showing substrate specificity of α-glycosidase inhibitory activity in vitro of Amberlite activity fractions.

2 is a graph showing blood glucose levels of α-glycosidase inhibitory activity according to the dose of the Amberlite active fraction.

3 is a graph showing the degree of α-glycosidase inhibitory activity according to the substrate of the Amberlite active fraction.

4 is a graph showing the degree of α- glycosidase inhibitory activity over time of the Amberlite activity fraction.

FIG. 5 is a graph showing the degree of α-glycosidase inhibitory activity in alloxane-induced hyperglycemic mice of the Amberlite activity fraction.

Hereinafter, the present invention will be described in more detail.

The inventors of the present invention, in order to isolate and manufacture the α-glycosidase inhibitor, first, silkworm urine was lyophilized, powdered, and methanol was added thereto. Subsequently, the methanol soluble portion was subjected to anion exchange column chromatography to obtain a fraction having α-glycosidase inhibitory activity. Methanol was added to separate the methanol soluble part and the insoluble part in order to remove the inorganic substances included in the electricity and the active fraction. Then, a small amount of water and acetonitrile were further added in the methanol soluble portion, and the mixture was partitioned into an acetonitrile layer and a water layer. A fraction of this fraction was concentrated under reduced pressure, and methanol was added to give compound I as white crystals. On the other hand, the methanol soluble portion was further concentrated under reduced pressure, subjected to cation / anion exchange chromatography, eluted with hydroxylamine to obtain an active fraction. The active fraction was then recrystallized from methanol to give compound II. Subsequently, ethanol was added to the C fraction eluted with hydroxylamine to divide the soluble part and the insoluble part, and the ethanol insoluble part was recrystallized with methanol to obtain Compound IV, and the ethanol soluble part was subjected to cation / anion exchange column chromatography, Elution with hydroxylamine gave compound V and compound VI.

On the other hand, in order to investigate the inhibitory activity of the silkworm urine fraction on glycosidase, the silkworm urine active fraction showed that α-glycoside was found in vitro and in vivo. It was confirmed that it has a substrate specificity for (glycosidic) binding and has the same α-glycosidase inhibitory activity as that of acarbose, a conventional diabetes treatment.

In addition, in order to investigate the glycosidase inhibitory activity of the finally prepared compounds I to VI, various glycosidase inhibitors were tested to show that compounds IV, V, and VI exhibit the highest α-glycosidase inhibitory activity. In order to identify the chemical structures of compounds IV, V, and VI, Rf values, melting points, molecular weights were examined and analyzed by NMR, FABMS and TLC. Oxynojirimycin, compound V was identified as pagomin, and compound VI as 1,4-didioxy-1,4-imino-D-arabinol.

Hereinafter, the present invention will be described in more detail with reference to the following examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1

[Preparation of α-glycosidase inhibitor from silk urine]

Silkworm urine was lyophilized to a powder and methanol was added thereto. Subsequently, 250 g of the silk soluble portion of the silkworm urine was subjected to anion exchange resin (Dowex 1 × 2-400) column chromatography (3 × 45 cm, Cl ⁻ form) to obtain 8.3 g of an active fraction. In order to remove the inorganic matter contained in the urine urine contained in the active fraction, methanol soluble part and insoluble part were divided. Subsequently, a small amount of water and acetonitrile were added to the methanol soluble portion, and the mixture was partitioned into an acetonitrile (0.5 g) layer and a water layer (3.6 g), and then the water layer was concentrated. , H ⁺ form). Then eluted with water to obtain A fraction in fractions 21-45 (fraction size: 10 ml, 320 mg) and B fractions in fractions 57-58 (13 mg), respectively, followed by 0.5 M hydroxylamine (NH ₄ OH). Elution was performed to obtain a C fraction having a fraction No. 98-100 (900 mg) (hereinafter, the three fractions of A, B, and C were combined to be called “amberlight active fraction”).

The A fraction was then concentrated under reduced pressure and methanol was added to give compound I (40 mg, yield: 0.01%) as white crystals. Meanwhile, the methanol soluble portion was further concentrated under reduced pressure, subjected to cation / anion exchange (Amberlite MB-1) column chromatography, and eluted with 0.1 M hydroxylamine to obtain an active fraction (fraction number 81-93, fraction size 2.5 ml). . The active fractions were then recrystallized from methanol to give compound II (16 mg, yield: 0.004%).

The B fraction was also washed with ethanol to give compound III (13 mg, yield 0.0036%).

On the other hand, ethanol was added to the C fraction and divided into an soluble part and an insoluble part. The insoluble part was recrystallized with methanol to obtain Compound IV (500 mg, yield: 0.13%), and the ethanol soluble part was subjected to cation / anion exchange (Amberlite MB-1) column chromatography and eluted with 0.1 M hydroxylamine. At this time, two active peaks were obtained, which were Compound V (13 mg, yield: 0.003%) and Compound VI (11 mg, yield: 0.002%), respectively.

Example 2

[Investigation of α-glycosidase inhibitory activity]

The α-glycosidase inhibitory activity in the process of separating the α-glycosidase inhibitory activity fraction from the silkworm urine of Example 1 was investigated by the following method.

50 mM PIPES buffer (pH 6.8), 10 mM maltose, a small amount of α-glycosidase and the sample to be examined were added to make the total volume of the reaction solution 60 μl. Subsequently, the reaction was carried out at 37 ° C. for 30 minutes, 1 ml of TRINDER reagent was added, and then left at room temperature for 18 minutes, and the absorbance was measured at 505 nm to investigate α-glycosidase inhibitory activity.

Example 3

Investigation of In Vitro α-Glycosidase Inhibitory Activity of Amberlite Activity Fractions

2x10 ^-3 g of Amberlite active fraction obtained in Example 1, 50 mM PIPES buffer solution (pH 6.8), a small amount of α-glycosidase and artificial substrate p-nitrophenyl-α-D-glucopyranoside (p- nitrophenyl-α-D-glucopyranoside), p-nitrophenyl-β-D-glucopyranoside or p-nitrophenyl-β-D-galactopyranoside (p -nitrophenyl-β-D-galactopyranoside) was added to make the total volume of the reaction solution 0.5 ml, and the reaction solution was reacted at 37 ° C for 30 minutes. Subsequently, 1 ml of ethylenediamine was added to terminate the reaction, and the absorbance was measured at 400 nm. As a result, 82% for p-nitrophenyl-α-D-glucopyranoside, p-nitrophenyl-β-D-glucopyranoside and p-nitrophenyl-β-D-galactopyranoside, respectively. , 4%, 13% showed α-glycosidase inhibitory activity (see FIG. 1). In FIG. 1, PNPαGcl is p-nitrophenyl-α-D-glucopyranoside, PNPβGlc is p-nitrophenyl-β-D-glucopyranoside, PNPβGal is p-nitrophenyl-β-D-galactopy It shows the case of using the ranoside as a substrate, (▒) graph is the 2x10 ^-2 g of the Amberlite active fraction, (□) graph is 2x10 ^-3 g, (■) graph is the Amber 2 x 10 ^-4 g of light active fractions are shown.

On the other hand, the graph obtained in Example 1 shows that the Amberlite activity fraction is 2x10 ^-2 g, 2x10 ^-3 g or 2x10 ^-4 g 50 mM PIPES buffer solution (pH 6.8), natural substrate maltose, isomalt sugar 1mM and a small amount Glycosidase was added to bring the total volume of the reaction solution to 60 μl. Subsequently, the reaction was carried out at 37 DEG C for 30 minutes, 1 ml of a TRINDER reagent was added, and then left at room temperature for 18 minutes, and the absorbance was measured at 505 nm to investigate α-glycosidase inhibitory activity. As a result, all concentrations of the Amberlite active fractions showed α-glycosidase inhibitory action against natural substrate maltose and isomaltose (see FIG. 1). From this result, it was found that the silkworm urine active fraction selectively inhibited α-glycosidase which hydrolyzed α-glycosidic bond.

Example 4

[In vivo α-Glycosidase Inhibitory Activity of Amberlite Activity Fractions]

To investigate the degree of α-glycosidase inhibitory activity according to the dose of the Amberlite active fraction, the rats were fasted for 14 hours, and then the Amberlite active fractions 40, 60, 80 mg / kg and maltose obtained in Example 1 were fasted. 2 g / kg was orally administered together and blood glucose was measured. As shown in FIG. 2, the relative values of blood glucose levels based on blood glucose levels obtained by oral administration of maltose alone showed 46%, 52%, and 70% of significant dose-dependent increase in blood glucose, depending on the dose of Amberlite active fraction, respectively. Inhibitory effect was shown. In FIG. 2, 'V' indicates that oral administration of 0.9% sodium chloride, physiological saline instead of active fraction and maltose, oral administration of only 2g / kg of maltose, and '+ Aca' means 2g / kg of maltose. And oral administration of 50 mg / kg of acarbose, a control drug. In addition, in FIG. 2, '+ A', '+ B', and '+ c' are indicated by oral administration of 2 g / kg malt sugar and 40 mg / kg, 60 mg / kg, and 80 mg / kg of Amberlite active fraction. Each case is shown.

On the other hand, in order to investigate the degree of α-glycosidase inhibitory activity according to the substrate, after fasting normal mice for 14 hours, 60 mg / kg of the Amberlite active fraction obtained in Example 1 and sugars (maltose, lactose, sugar, starch) And glucose) 2 g / kg orally together and blood glucose was measured. As shown in FIG. 3, the relative value of blood glucose was measured based on the blood glucose level by oral administration of maltose alone. As a result, Amberlite active fraction showed 50% of maltose, 27% of lactose, 25% of sugar, and 42% of starch. It showed a synergistic inhibitory effect. In FIG. 3, the (▒) graph shows only 0.9% sodium chloride, which is a physiological saline solution, the (□) graph shows a sugar only, and the (그래프) graph shows a 50 mg / kg of sugar and acarbose together, ( ■) The graph shows the case where the saccharide and the Amberlite active fractions were administered together.

In addition, to investigate the degree of α-glycosidase inhibitory activity of the Amberlite activity fraction over time, after fasting for 14 hours in normal rats, 60mg / kg of Amberlite active fraction obtained in Example 1 and 2g / kg malt per malt Were orally administered together and blood glucose was measured after 18, 35, 60, 90 and 120 minutes. As shown in FIG. 4, the relative values of blood glucose levels were examined based on blood glucose levels when oral administration of maltose alone showed 50% and 51% inhibition of blood glucose increase at 18 and 35 minutes after administration. In Figure 4 (◇) is administered only maltose, (○) is administered together with maltose and acarbose 50mg / kg, (▲) is administered together with maltose and Amberlite active fraction, (■) Are the cases where only 0.9% sodium chloride, the physiological saline, was administered.

Finally, to investigate the α-glycosidase inhibitory activity of the Amberlite active fraction in alloxan-induced hyperglycemic mice, fasting normal mice for 14 hours, and then intravenously administering 75 mg / kg alloxan to hyperglycemic mice. 48 hours later, blood glucose was measured, and mice having a blood glucose level of 250-400 mg / dl were selected. The selected high blood glucose mice were fasted for 16 hours, and 120 mg / kg of the Amberlite active fraction obtained in Example 1 and 2 g / kg of maltose were orally administered together, and blood glucose was measured 35 minutes later. As shown in FIG. 5, the relative values of blood glucose levels were investigated based on blood glucose levels by oral administration of maltose alone. As a result, the Amberlyte fraction showed an inhibitory effect on blood glucose increase of 61%. Inhibition of blood glucose increase by 68% at the dose of kg, there was no significant difference between the two groups. In FIG. 6, V denoted only when physiological saline was administered only 0.9% sodium chloride, maltose was administered only 2g / kg malt, and acarbose was administered when 2g / kg malt sugar and 100mg / kg acarbose were administered. The fraction represents the case where maltose and Amberlite active fraction were administered together.

Example 5

[Glycosidase Inhibitory Activity of Compound I-VI]

50 mM PIPES buffer (pH 6.8), 10 mM maltose, a small amount of glycosidase and the sample to be examined were added to make the total volume of the reaction solution 60 μl. Subsequently, the reaction was carried out at 37 ° C. for 30 minutes, 1 ml of TRINDER reagent was added thereto, and then left at room temperature for 18 minutes, and the absorbance was measured at 505 nm. The glycosidase inhibitory activity of each compound isolated in Example 1 was measured. Investigate. As shown in Table 1 below, Compounds IV and VI had the greatest inhibitory effects on mouse small intestine maltose-glucoamylase (IC ₅₀ : 0.2 μm, 2.38 μm). Compound V was relatively weak but had a selective inhibitory action on α-glycosidase. Compound I had a greater inhibitory effect on β-glycosidase than other compounds. Compound II had no inhibitory effect at less than 100 μm compared to several glycosidases, and Compound I-VI had no hepatic vesicle α-glycosidase, mouse liver lysosomal α-mannosidase, β-mannosidase. It did not inhibit for.

Asano et al. Reported that 1-dioxynozirimycin had potent inhibitory effects on rat liver vesicles glucosidase II and lysosomal glycosidase (IC ₅₀ : 4.6 μm, 0.4 μm). The results of this experiment were different. In addition, 1,4-didioxy-1,4-imino-di-aravinitol is also weak against rat liver lysosomal α-mannosidase. Reported that there is an inhibitory activity (IC ₅₀ : 110㎛), but no inhibitory activity was observed in this experiment.

TABLE 1

*: Not less than the IC ₅₀ has 1000㎛ reports that the inhibitory activity against glycosidase, expressed NI la. Here, IC ₅₀ means the concentration of the enzyme activity inhibitor necessary to reduce 50% of the enzyme activity.

Example 6

[Inhibitory Activity of Compound I-IV on Rat Small Intestine α-glycosidase]

Inhibition of glycosidase activity was measured in the same manner as in Example 6. As shown in Table 2 below, Compound I showed selective inhibitory action against maltase among various α-glycosidases (IC ₅₀ : 4 μm), sucrase, isomaltase, The inhibitory activity was weak against lactase, and there was no inhibitory activity against trehalase and cellubioase. Compounds II and III showed no inhibitory effect on rat small intestinal glycosidase at less than 1000 μm, but also at high concentrations. Compound IV had a potent inhibitory action on α-glycosidase, maltase, sucrase, and isomaltase, except for weak or no inhibitory activity on trehalase and cerubiase. Compound V had an inhibitory effect on α-glycosidase, Compound VI had a selective inhibitory effect on maltase among α-glycosidase, and compounds IV and VI had potent α-glycosidase inhibitory activity but substrate Selectivity was found to be inferior.

TABLE 2

*: Not less than the IC ₅₀ has 100㎛ reports that the inhibitory activity against glycosidase, expressed NI la. Here, IC ₅₀ means the concentration of the enzyme activity inhibitor necessary to reduce 50% of the enzyme activity.

Example 7

[Chemical structure determination of compound IV, V, VI]

As a result of Example 5 and Example 6, the chemical structure was determined as follows for the compounds IV, V, VI confirmed to have the best α- glycosidase inhibitory activity.

(1) Compound IV: White needle crystal, Rf = 0.42, Melting point 200 ° C, Molecular weight 163

Rf value shows the distance which the sample moved from the origin on TLC as a relative ratio with respect to the distance which the developing solvent finally moved. On the other hand, the melting point was determined by measuring the temperature at the time of melting the crystal using a melting point measuring instrument (Mel-temp II, Laboratory Devices, U. S. A.).

Elemental analysis of NMR (Bruker, AMX 500, Germany), FABMS (VG Analytical, VG, 70-USEQ, U. S. A.) of Compound IV is as follows:

Since the NMR and FABMS data and the data of 1-deoxynojirimycin described in the literature were in agreement, and Rf values were consistent with the 1-deoxynojirimycin standard and TLC, Compound IV was 1-di. It was identified as oxynojirimycin.

(2) Compound V: White needle crystal, Rf = 0.28, melting point 186 ° C, molecular weight 147, well soluble in water but insoluble in methanol.

The measurement method of Rf value and melting point is the same as the measurement method of compound IV mentioned above.

The NMR and FABMS elemental analysis of Compound V is as follows:

The compound N was identified as pagomin because the NMR and FABMS data and the pagomine data described in the literature were in agreement, and the Rfcl was in agreement with the standard.

(3) Compound VI: White acicular crystal, Rf = 0.72, Melting point 115 ° C, Molecular weight 133

The measurement method of Rfcl and melting point is the same as the measurement method of compound IV mentioned above.

The NMR and FABMS elemental analysis of Compound VI is as follows.

The above-mentioned NMR and FABMS data and the 1,4-didioxy-1,4-imino-di-arabinitol (1,4-dideoxy-1,4-imino-D-aravinitol) described in the literature match Compound VI was identified as 1,4-didioxy-1,4-imino-di-arabinitol because the standard and Rf value were identical.

As described and demonstrated in detail above, the present invention provides a method for producing an α-glycosidase inhibitor from silkworm urine. The method for separating and producing the α-glycosidase inhibitor of the present invention is a method for separating a substance having excellent α-glycosidase inhibitory activity from silkworm urine which has been completely discarded before. It was confirmed that it had the same alpha-glycosidase inhibitory activity as the therapeutic agent acarbose. Therefore, when the substance produced by the method of the present invention is developed as an antidiabetic agent, it is expected to contribute to increase of income of farmers and improvement of national health.

Claims (4)

(i) lyophilizing silkworm urine and precipitating with methanol; (ii) anion exchange column chromatography on methanol solubilizer obtained from above; (iii) eluting the active fraction obtained in the previous step with methanolamine, acetonitrile extraction and cation exchange column chromatography eluting with hydroxylamine; And, (iv) ethanol precipitation of the hydroxylamine elution fraction in the foregoing, ethanol amorphous portion recrystallized from methanol to separate 1-deoxynojirimycin, and ethanol soluble portion was cationic / anion exchange column chromatography. To separate fagomine and 1,4-didioxy-1,4-imino-D-arabinitol (1,4-dideoxy-1,4-imino-D-aravinitol) Simultaneous preparation of 1-dioxy nozirimycin, pagomin and 1,4-didioxy-1,4-imino-D-arabinol which are α-glycosidase inhibitors from silkworm urine. The method of claim 1, wherein the anion exchange column chromatography uses Dowex 1 × 2-400 resin. The process of claim 1 wherein the cation exchange column chromatography uses Amberlite IRC-50 resin. The method of claim 1, wherein the cation / anion exchange column chromatography uses Amberlite MB-1 resin.

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