KR100396986B1 - A process for preparing α-glycosidase inhibitor derived from powdery silkworm - Google Patents

Abstract

The present invention provides a method for producing a blood glucose lowering substance excellent in inhibiting postprandial blood sugar increase from silkworm powder. According to the method for preparing a blood glucose lowering substance from the silkworm powder of the present invention, the silkworm powder is extracted with ethanol, filtered, and subsequently subjected to a series of ion exchange column chromatography to perform 1-dioxynojirimycin, an excellent substance for lowering blood glucose. (1-deoxynojirimycin) is isolated. The hypoglycemic substance isolated by the manufacturing method of the present invention was confirmed to have almost the same hypoglycemic activity as acarbose (acarbose), a conventional diabetes treatment. Therefore, it is expected that if the substance produced by the present invention is developed as a diabetes treatment agent, it will contribute to increase of income of farmers and improvement of national health.

Description

Method for preparing α-glucosidase inhibitor from silkworm powder {A process for preparing α-glycosidase inhibitor derived from powdery silkworm}

The present invention relates to a method for preparing α-glycosidase inhibitor from silkworm powder, and more specifically, 5 days and 3 days silkworm powder is lyophilized, 50% ethanol extraction and serial ion exchange column chromatography. The present invention relates to a method for preparing 1-deoxynojirimycin, which is an excellent substance for inhibiting α-glycosidase.

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 slowly released 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 of solving the mismatch between elevated blood sugar and insulin content after meals, numerous methods have been attempted, such as the adjustment of drug administration time, the development of a new drug route, and the development of new formulations to promote drug absorption. It has come but has not yet been satisfactory.

In addition, during the development of various methods to solve the post-prandial hyperglycemia 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. In this way, administration of an α-glycosidase inhibitor in the small intestine can control blood sugar levels that rise rapidly after meals, and can solve the large increase and decrease of blood sugar levels commonly seen in diabetic patients. It can also be suppressed and help prevent the development of other circulatory complications due to hyperinsulinemia.

On the other hand, the present inventors that silkworm powder has a significant inhibitory effect on postprandial blood sugar, and this is due to the inhibitory effect of α-glucosidase, an enzyme involved in carbohydrate digestion in the small intestine, like acarbose, a conventional diabetes treatment It has been revealed (Patent Registration No. 151731)

However, it is not known which component of the silkworm powder itself acts as an inhibitor of α-glycosidase.

Accordingly, the present inventors have made diligent efforts to prepare α-glycosidase inhibitors from silkworm powder. As a result, the present inventors have found that 1-dioxynozirimycin having excellent α-glycosidase inhibitory activity can be isolated alone, It was completed.

Accordingly, an object of the present invention is to provide a method for producing 1-dioxynojirimycin, which is an α-glycosidase inhibitor, from silkworm powder.

1 is a photograph of 1-deoxynojirimycin prepared by the method of the present invention.

Figure 2 is a graph showing the hypoglycemic effect in streptozotocin (STZ) induced hyperglycemic rats.

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

The present invention starts from the fact that 84% of diabetic patients in Korea are insulin-independent diabetes mellitus to separate blood glucose lowering substances from natural products with higher physiological activity than insulin therapy. Conventionally, a method for separating blood glucose lowering substances from such natural products, for example, 1-deoxy nozirimycin which is an α-glycosidase inhibitor from silkworm urine in Korean Patent Publication No. 1999-54967 (July 15, 1999) A method for simultaneously separating, pagomin and 1,4-didioxy-1,4-imino-D-arabinol is disclosed, which is a silkworm powder because silk urine is metabolized from silkworm Although the compound contained therein is simpler, each of the α-glycosidase inhibitors is separated in the form of a mixture rather than separately, so that the present inventors have found silkworm powder containing more compounds than silkworm urine. It was attempted to separate the α-glycosidase inhibitors alone, and therefore, it was successful to separate 1-dioxynozirimycin alone among the α-glycosidase inhibitors.

The method for separating 1-deoxynojirimycin from silkworm powder according to the present invention

(1) 5 days 3 days freeze-dried silkworms were lyophilized to a powder state, and then 50% ethanol was added thereto to obtain an extract;

(2) eluting the extract with cation exchange column chromatography with 0.5N NH ₄ OH, and then concentrating the eluate to obtain a concentrate;

(3) dissolving the concentrate in water, then eluting with about 10 times the volume of anion resin by anion exchange column chromatography, and then concentrating the eluate to obtain a concentrate;

(4) dissolving the concentrate in water, followed by cation exchange column chromatography eluting with about 5 times the volume of cation resin to obtain five fractions, eluting with 0.5N NH ₄ OH to obtain one fraction;

(5) anion exchange column chromatography on a second of the six fractions to obtain a pure 1-dioxynojirimycin fraction; And

(6) methanol precipitation of the 1-dioxynojirimycin fraction to obtain 1-dioxynojirimycin crystals

Characterized in that made.

Each step will be described in detail as follows.

(1) 5 days 3 days freeze-dried silkworms were lyophilized to a powder state, and then 50% ethanol was added thereto to obtain an extract.

The extraction using 50% ethanol is because 50% ethanol can extract only alkaloids and amino acids including 1-deoxynojirimycin. The extraction method is extracted five times with ultrasonic waves for about 2 hours, and then filtered through celite.

(2) the extract was subjected to anion exchange column chromatography for 0.5N NH. ₄ Eluting with OH and then concentrating the eluate to obtain a concentrate.

Cation exchange column chromatography uses Amberlyst 15 resin (NH ₄ ⁺ type, 1.2 L) to allow the compounds contained in the extract to adhere onto the resin through ion exchange and then elute with 0.5 N NH ₄ OH (12 L). Concentrate and weigh. By this process, an anion compound adheres to resin.

(3) dissolving the concentrate in water, followed by anion exchange column chromatography to elute with about 10 times the volume of anion resin, and then concentrating the eluate to obtain a concentrate.

Anion exchange column chromatography uses Dowex 1 × 2 resin (OH ⁻ type). This step removes other amino acid components other than alkaloids.

(4) dissolving the concentrate in water, followed by cation exchange column chromatography eluting with about 5 times the volume of cation resin to obtain five fractions, eluting with 0.5N NH ₄ OH to obtain one fraction .

Cation exchange column chromatography uses Amberlyst 15 resin (NH ₄ ⁺ type, 2.7 × 85 cm).

(5) anion exchange column chromatography on a second of the six fractions to obtain a pure 1-dioxynojirimycin fraction.

Anion exchange column chromatography uses Dowex 1 × 2 resin (OH ⁻ type).

(6) the 1-dioxynojirimycin fraction is methanol precipitated to obtain 1-dioxynojirimycin crystals.

1-deoxynojirimycin obtained by the above method can be used as a dietary supplement, such as functional foods, as well as health care foods that can be easily taken by diabetics to control blood sugar, which can increase the consumption of biscuit products. It can contribute to the development of new uses of sheep products, to improve farm incomes and to improve public health.

Hereinafter, although an Example is given and this invention is demonstrated in detail, this invention is not limited only to these examples.

Example 1 Isolation of 1-dioxynojimycin from silkworm powder

After 5 days 3 days silkworms were lyophilized to a powder form of 1kg, 50% ethanol was added thereto. Amberlyst 15 after passing through the extract to (NH ₄ ⁺⁾ and, 0.5N NH ₄ OH and concentrated to a leak, Dowex 1X2 (OH ^-) passing the concentrate and the concentrated leakage to 50% ethanol and Amberlite CG-50 ( NH ₄ ⁺ ) was concentrated by distillation with H ₂ O and 0.5N NH ₄ OH, eluting Dowex 1 × 2 (OH ⁻ ) with H ₂ O. The fraction was concentrated under reduced pressure and methanol was added to give a white crystal of 3.0 g (yield 0.3). %) Was obtained.

The properties of the compound are as follows.

R _f : 0.42

m.p .: 200 ° C

Molecular Weight: 163

¹ H-NMR (500 MHz, D ₂ O): δ 2.52 (dd, 1H, J _{1ax, 1eq} = 12.1, J _{1ax, 2} = 10.9 Hz H _1ax ), 2.61 (ddd, 1H, J _{1ax, 1eq} = 12.1, J _{1eq, 2} = 5.1Hz, H _1eq ), 3.3 (t, 1H, J _3,4 = J _4,5 = 9.1Hz, H ₄ ), 3.38 (t, 1H, J _2,3 = J _3,4 = 9.1 Hz, H ₃ ), 3.56 (ddd, 1H, J _{1ax, 2} = 10.9, J _{1eq, 2} = 5.1, J _2,3 = 9.1 Hz, H ₂ ), 3.7 (dd, 1H, J _5,6ax = 6.2, J _{6ax, 6eq} = 11.3Hz, H _6ax ), 3.84 (dd, J _5,6eq = 2.9, J _{6ax, 6eq} = 11.3Hz, H _6eq )

¹³ C-NMR (125 MHz, D ₂ O): δ 51.3 (C ₁ ), 63.1 (C ₅ ), 64.7 (C ₆ ), 73.5 (C ₂ ), 74.1 (C ₄ ), 81.3 (C ₃ )

FABMS (NBA matrix) m / z 164 (M + 1): Anal (C ₆ H ₁₃ NO ₄ ) C, H, N

The properties of the compounds described above and the properties of 1-dioxynojirimycin described in the literature were in agreement, and R _f values were compared on TLC with the standard of 1-dioxynojirimycin obtained from Professor Asano (Hokuriku University, Japan). As a result, since they matched, the compound of the white crystal obtained by the method mentioned above was identified as 1-dioxy nozirimycin of the following general formula.

<Test Example 1>

(1) laboratory animals and animal experiments

SD rats (male, 160 ± 10 g) are being bred at Samsung Medical Center, Seoul, Korea.The animals are pre-divided for 2 weeks in the animal room.They are treated with diabetes by streptozotocin (STZ) 65mg / kg. After induction, blood glucose was measured for 3 days, and only rats having 350 mg / dL or more were used in the experiment. In the animal experiment, the experimental group was divided into four groups of six animals, and the experimental control group and the induced group (STZ) were fed with distilled water, and the 1-deoxynojiri of Example 1 was investigated to investigate the antidiabetic effect. The anti-diabetic effect was evaluated by analyzing the hypoglycemic effect of 12 mg of mycin (DNJ) by dissolving intraperitoneally with 40 mg of acarbose, a insulin-independent type II diabetes treatment, currently available as a hypoglycemic agent. All reagents used in this experiment are from Sigma, USA.

(2) Preparation of prepared food

The feed composition used in this experiment was composed of carbohydrate 59.0% (α-corn starch: 44.0% + sucrose 15.0%), protein 18.0% (sodium-free casein), lipid 15.0% (lard: 10.0% + corn oil: 5.0%), 1.0%, 3.5%, and 3.0% fiber, 0.3% DL-methionine and 0.2% choline chloride were added.

(3) Experiment Method

① Induction of experimental diabetes

STZ, which is widely used as a diabetic inducer, was dissolved in 0.1 M sodium citrate buffer (pH 4.3) to 65 mg / kg BW in SD rats and injected through the tail vein. Blood was collected from the tail vein of diabetes-induced rats after 3 days and analyzed for blood glucose levels. On the 4th day, the rats with the blood glucose level of 350 mg / dl or more were fed and fed the DNJ 20mg / kg of Example 1 to analyze the blood glucose level for 11 days. The blood glucose change was observed for 3 days without the administration of DNJ. The hypoglycemic effect was observed. As a control drug, 40 mg / kg of acarbose, a commercial diabetes treatment, was fed and analyzed for glucose content analysis.

② Measurement of blood glucose

The glucose content in serum isolated from blood collected from the tail vein was measured by a kit reagent (Sigma, Co., USA) by enzyme method. First, 5 μl of glucose standard solution (400 mg / dl) was added as a serum and a standard solution, and 1.0 mL of a color developing reagent was added thereto, followed by well mixing and reaction at 37 ° C. for 15 minutes. The absorbance was measured at 505 nm with a blank containing the color reagent in distilled water to calculate the glucose content according to the following equation.

Glucose content (mg / dl serum) = (OD _sample / OD _{standard solution} ) × 400 ^*

(Wherein * The concentration of glucose standard solution.)

The results are shown in Table 1 and FIG. 2.

Comparison of hypoglycemic effect according to days of acarbose and DNJ administration (%) Dosing Day One 2 3 4 5 6 7 8 9 10 11 Akabozu 11.6 11.1 26.2 27.8 35.2 41.5 57.6 60.4 61.6 59.4 54.8 DNJ 22.7 12.4 16.1 47.1 49.9 60.0 66.3 64.0 60.9 69.6 51.6

From Table 1, the control drug acarbose showed an excellent hypoglycemic effect of 57.6% from the 7th day of administration, but DNJ showed an effect of 47.1% from the 4th day of administration, and continued to have an excellent hypoglycemic effect during the administration period. Able to know.

In addition, as can be seen from Figure 2, the blood glucose of the STZ-induced hyperglycemic rat group maintained more than 450mg / dl throughout the experimental period, but the blood glucose content of the DNJ-administered group isolated from silkworm powder was 47.1 to 47.1 compared to the control group. The hypoglycemic effect of day 11 was 51.6%, and the blood glucose gradually increased to the blood glucose of STZ-induced group. The Acarbose-administered group, which was administered as a control drug, showed a hypoglycemic effect of more than 50% from 7 days of administration and a good hypoglycemic effect throughout the administration period. In the three days of discontinuation to investigate the DNJ and Acarbose drug mechanisms, blood glucose levels are elevated, suggesting that these drugs may be used as treatments for insulin-independent forms.

In conclusion, DNJ isolated from silkworm is DNJ 20mg which has anti-diabetic effect, considering that it is ineffective against anti-diabetes of diabetes drug Acarbose. Development of antidiabetic agents would be desirable.

As described above, the present invention is a method for separating and producing 1-dioxynojirimycin, which is an α-glucosidase inhibitor, from a silkworm powder. Since it has been confirmed to have almost the same hypoglycemic activity as acarbose, it is expected that if it is developed as a diabetes treatment agent, it will contribute to increase of income of farmers and improvement of national health.

Claims (3)

(1) 5 days 3 days freeze-dried silkworms were lyophilized to a powder state, and then 50% ethanol was added thereto to obtain an extract; (2) eluting the extract with cation exchange column chromatography with 0.5N NH ₄ OH, and then concentrating the eluate to obtain a concentrate; (3) dissolving the concentrate in water, then eluting with about 10 times the volume of anion resin by anion exchange column chromatography, and then concentrating the eluate to obtain a concentrate; (4) dissolving the concentrate in water, followed by cation exchange column chromatography eluting with about 5 times the volume of cation resin to obtain five fractions, eluting with 0.5N NH ₄ OH to obtain one fraction; (5) anion exchange column chromatography on a second of the six fractions to obtain a pure 1-dioxynojirimycin fraction; And (6) methanol precipitation of the 1-dioxynojirimycin fraction to obtain 1-dioxynojirimycin crystals A method for producing 1-dioxy nozirimycin which is an α-glucosidase inhibitor from silkworm powder, characterized in that consisting of. The method of claim 1, wherein the cation exchange column chromatography uses Amberlyst 15 resin (NH ₄ ⁺ type) or Amberlite CG-50 resin (NH ₄ ⁺ type). The method of claim 1, wherein the anion exchange column chromatography uses Dowex 1 × 2 resin (OH ⁻ type).