KR20030019727A - Extracts of prosomillet inhibiting the hepatic glucose-6-phosphatase and composition comprising them as active materials - Google Patents

Abstract

PURPOSE: A composition containing a Panicum miliaceum L. extract exhibiting excellent hypoglycemic activity by inhibiting synthesis of new sugar and decomposition of glycogen is provided which can be effectively used for prevention and improvement of diabetic. CONSTITUTION: The composition containing a Panicum miliaceum L. extract as a main component lowers a blood sugar level by lowering glucose-6-phosphatase activity of liver cells. A methanol extract and n-hexane extract of Panicum miliaceum are added to a microsome pellet dispersion solution of a rat to investigate G-6-Pase activity inhibition, a test diet containing the methanol extract of Panicum miliaceum and a contrast diet are supplied to the rat, thereafter G-6-Pase activity and sugar change are measured using collected blood and liver tissue thereof.

Description

Extract of millet extract having a hypoglycemic effect and a blood glucose lowering composition containing the same as an active ingredient {Extracts of prosomillet inhibiting the hepatic glucose-6-phosphatase and composition comprising them as active materials}

The present invention relates to a natural extract for reducing blood sugar and a composition for lowering blood sugar containing the same as an active ingredient. More specifically, the present invention is a natural extract that reduces blood glucose by inhibiting glucose-6-phosphatase (G-6-Pase) activity of liver tissue, which is extracted from millet and contains it as an active ingredient. It relates to a composition to be.

There are two major mechanisms of action in liver tissue that play a central role in glycemic control: neosynthesis and glycogen breakdown (Nordie, RC, Trends Biochem. Sci., 10, 70-75 (1985)). Glucose-6-phosphatase (G-6-Pase) catalyzes the reaction of hydrolysis of glucose-6-phosphate to glucose and phosphate ions. This is necessary at the end of both sugar synthesis and glycogen breakdown. It has been reported that G-6-Pase activity greatly affects excessive glucose production in diabetic liver tissue (Jakobssen, SV, Dallner, G., Biochim. Biophys.Acta 165, 380-392 (1968)). . Therefore, inhibition of G-6-Pase activity of liver tissues inhibits glucose production, which is helpful in treating diabetes susceptible to hyperglycemia. G-6-P hydrolysis in the liver includes at least six distinct components, including the catalytic unit D-glucose-6-phosphate phosphohydrolase. Is needed. The other five components are stabilizing proteins, four types of transporters, one for G-6-P (T1), one for phosphate (T2α, T2β), and one for glucose (T3). G-6-Pase activity is not only insulin (Gardner, LB, Liu, Z., Barrett, EJ, Diabetes, 42, 1614-1620 (1993)) but also proline (Bode, AM, Foster, JD, Nordlie). , RC, J. Biol. Chem., 267, 2860-2863 (1992)), α-ketoglutarate (Minassian. C., Ajzannay, A., Riou.JP, Mithieux, G. , J. Biol., Chem., 269, 16585-16588 (1994)), unsaturated fatty acids (Daniele, N., Bordet, J.-C., Mithieux, G., J. Nutr., 127, 2289-2292 (1997)), and metabolism of long-chain fatty acyl CoA (Mithieux, G., Zitoun, C., Eur. J. Biochem., 235, 799-803 (1996)). It has also been reported to be controlled by water. In addition, chlorogenic acid and 2-hydroxy-5-nitrobenzaldehyde inhibited G-6-Pase activity in naturally occurring substances (Schindler). , PW, Below, P., Hemmerle, H., Berger, H.-J., Arion, WJ, Efendic, S., Diabetologia 37 (Suppl. 1), A134 (1994); Arion, WJ, Canfield, WK , Ramos, FC, Schindler, PW, Burger, H.-J., Hemmerle, H., Schubert, G., Below, P. and Herling, AW, Arch.Biochem. Biophys. 339, 315-322 (1997) ). In addition, researchers at the pharmaceutical company have synthesized several derivatives with higher inhibitory activity than chlorogenic acid, including some that have hypoglycemic effects in vivo (Arion, WJ, Canfield, WK, Ramos, FC, Su, ML, Burger, H.-J., Hemmerle, H., Schubert, G., Below, P. and Herling, AW, Arch.Biochem.Biophys. 351, 279-285 (1998); Herling, AW, Burger, H.-J Schwab, D., Hemerle, H., Below, P. and Schubert, G., Am. J. Physiol. 274, G1087-G1093 (1998); Parker JC, VanVolkenburg, MA, Levy, CB, Martin, Burk, SH, Kwon, Y., Giragossian, C., Gant, TG, Carpiro, PA, McPherson, K., Vestergaard, P. and Treadway, JL, Diabetes 47, 1630-1636 (1998)).

Cereals are among the plant foods that contribute the most to human nutrition. These grains are known to contain many polyphenol compounds in addition to fibrin, which is important for hypoglycemic action (Salunkhe. D, K., Jadhav, SJ, Kadam, SS and Chavan, JK, Crit. Rev. Food.Sci Nutr . 17, 277-305 (1982); Yuganari, T., Tan, BK, Teh, M. and Das, NP, Lipids 27, 181-186 (1992)).

Millet ( panicum milaceum ) is a perennial herb that grows to 50 ~ 120㎝ in height and has been cultivated in mountainous region for a long time. However, little research has been conducted on components other than fibrin so far.

In view of the above, the present inventors have attempted to search for G-6-Pase activity inhibitors in the extracts of millet, followed by millet 80% methanol crude extract, secondary extracts and column choromatgraphy. section G-6 in which the use of the purified fractions after a review of G-6-Pase activity inhibition of microsomal (microsome) between the rat in in vitro millet powder and millet methanol crude extract ingestion to rats after blood sugar and in vivo The present invention was completed by investigating changes in -Pase activity.

Accordingly, an object of the present invention is to provide a millet extract that lowers blood glucose by lowering the G-6-Pase activity extracted from the millet. Another object of the present invention is to provide a composition for reducing blood glucose, which contains the extract of millet as an active ingredient to lower the blood sugar separated from the millet.

The object of the present invention is to add millet methanol extract and n-hexane extract to the microsome pellet dispersion solution of rats to investigate the G-6-Pase activity and to supply an experimental diet containing millet methanol extract and powder Blood and liver tissues of rats were reared to obtain G-6-Pase activity and blood glucose.

Hereinafter, the configuration of the present invention.

Figure 1a is a graph showing the G-6-Pase activity inhibitory effect of rat liver microsomes by millet methanol extract.

Figure 1b is a graph showing the inhibitory effect of G-6-Pase activity of the second solvent extract of millet methanol extract.

Figure 2 shows the TLC results of the millet hexane extract.

Figure 3a is a graph showing the inhibitory effect of G-6-Pase activity of the TLC fractions 1 to 7 samples of the millet hexane extract.

Figure 3b is a graph showing the inhibitory effect of G-6-Pase activity according to the dose of TLC fractions 3 to 7 samples of millet hexane extract.

Figure 4 is a graph comparing the inhibitory effect of G-6-Pase activity of TLC fraction No. 3 sample of the millet hexane extract and a combination of HNB and PLP.

The present invention comprises the steps of preparing a methanol extract and n-hexane extract each; Preparing microsomal pellets by collecting liver tissue from rats; Measuring the G-6-Pase activity inhibition after reacting the millet methanol extract and n-hexane extract in a microsomal pellet dispersion solution of the rat; Preparing an experimental diet containing millet methanol extract and powder and a control diet not containing the millet component; Supplying the diet prepared in the step to the rats to breed and then collecting blood and liver tissue; The blood and liver tissue collected in the above step consists of measuring G-6-Pase activity and blood glucose change.

Hereinafter, the specific method of the present invention will be described step by step with reference to Examples, but the scope of the present invention is not limited only to these Examples.

Example 1 Preparation of Millet Extract with Methanol and n-hexane

The millet grown in Chungbuk area was washed with water, dried, and ground to about 40 mesh. Methanol extract was prepared by extracting twice by adding 80% methanol, which is five times the length of millet powder, and refluxing at 90 ° C. for 3 hours. The methane wool extract was filtered and concentrated under reduced pressure at 40 ° C or less.

The methanol extract (15.6 g) was dispersed in 10% methanol, followed by stepwise volume of n-hexane (2 L × 2), chloroform (2 L × 2), ethyl acetate (2 L × 2) and butanol (2 L × 2). Extraction was carried out with a solvent of to obtain respective secondary solvent fractions, which were dried in vacuo.

When preparing the hexane extract treated with the millet powder from the beginning, 4kg millet powder was immersed in hexane, which is twice the millet capacity, for 1 week, and then reextracted twice in the same manner every three days. The extract was dried under vacuum at 30 ° C. The hexane extract (3.54 g) was dissolved in hexane and subjected to silicic acid column chromatography (3 cm × 50 cm, i.d) to obtain seven fractions; Fraction 1 (0.11 g), fraction 2 (1.20 g), fraction 3 (0.49 g), fraction 4 (0.45 g), fraction 5 (0.16 g), fraction 6 (0.41 g) and fraction 7 (0.14 g). The solvent was started with 1 L of a solution of hexane / ethylacetate ratio of 8: 1 and 500 mL of 4: 1, 1: 1dml mixed solution was added stepwise, and finally 500 mL of ethyl acetate was used. . The fraction was crystallized using the same solvent, and then the effect of inhibiting G-6-Pase activity was measured.

Example 2 Investigation of G-6-Pase Activity Inhibitory Effect of Millet Extract

Step 1: Collect Sample

Hepatic microsomes from overnight fasted rats Junk (Junker) and Story method (Ha, TY, Cho, IJ and Lee, SH, Korean J. Food Sci.Technol., 30, 224-229 (1998) )) Was used as a sample of the enzyme activity. The rats were lightly anesthetized with ether, and liver tissue was collected. 250 mM sucrose, 40 mM Tris-HCl, 2 mM EDTA, 5 mM dithiothreitol, 50 mM KCl, 50 mM KF It was homogenized in a cold buffer solution containing pH 7.4. The homogenate was centrifuged at 12,000 g at 4 ° C. for 10 minutes and the supernatant was further centrifuged at 100,000 g for 75 minutes to obtain microsomal pellets. The microsome pellet was dispersed in a small amount of buffer solution (40 mM Tris-HCl, 1 mM EDTA, 5 mM dithiothreitol, 50 mM KCl, 50 mM KF, pH 7.4) at -75 ° C until the enzyme activity was measured. Stored.

Second Step: Investigation of G-6-Pase Inhibitory Activity

G-6-Pase activity was measured by monitoring the release of phosphoric acid from G-6-P (Baginski, ES, Foa, OP, Zak, B., Bergmeyer, HU (ed) Vol II, 2nd ed. , Academic Press, Inc., New York and London (1974)). Microsomes (about 500 μg of protein content) were dispersed in 550 μl reaction mixture (3.2 mM histidine, 1 mM EDTA, 18 mM G-6-Pase) at pH 6.5 and reacted at 37 ° C. To determine the in vitro inhibitory effect of G-6-Pase activity, the samples were dissolved in 10-50 μl of DMSO and added to the culture mixture. The control group used pure DMSO and HNB (2-hydroxy-5-nitrobenzaldehyde) and PLP (pyridoxal-5-phosphate) (reversible inhibitor of G-6-Pase complex) in the same amount as the experimental group. Were 1.4 and 9 mM, respectively. After reacting for 30 minutes, free phosphoric acid was colored with ammonium molybdate and absorbance was measured at 660 nm.

Inhibition of liver tissue microsomal G-6-Pase by methanol extract of millet is shown in Figure 1a. When 50 μg of methanol extract was added to microsomes with 500 μg of protein, the inhibitory effect began to show. When 100 μg was added, 38.5% of the activity was inhibited. Did not do it.

Inhibition of G-6-Pase activity of the secondary solvent extracts of methanol extract is shown in FIG. Experimental results showed that the inhibition of hexane (ME-Hx) and chloroform (ME-Ch) fractions was large, whereas the inhibition of ethyl acetate (ME-EtOAc) or butanol (ME-Bu) fractions was almost increased even if the amount of addition was increased up to 600 ㎍ It did not appear. Therefore, it was confirmed that the substance having an inhibitory effect is a nonpolar substance, which is different from chlorogenic acid and its derivatives. Thus, the millet was extracted with hexane from the beginning to investigate the components.

The hexane crude extract of millet was collected in stages by fractionation of the fractions obtained by silicic scid chromatography, and the components were examined by TLC in FIG. 2.

The TLC fractions obtained in Example 1 were used to investigate the inhibitory effect of G-6-Pase activity using fractions of samples 7 to 7 (FIG. 3A). The inhibitory effect of G-6-Pase activity of the TLC fractions 3 to 7 obtained in Example 1 was investigated. The inhibitory effect between the five crystalline samples did not differ significantly and was dose dependent (FIG. 3B). G-6-Pase activity was reduced by about 70% in each crystal sample of 40 μg / ml and was further reduced by increasing the amount of sample to 200 μg / ml. At this time, the content of liver tissue protein was approximately 1 mg / ml. HNB and PLP are reversible competitive inhibitors for T1 and T3, components of the G-6-Pase complex. HNB inhibited G-6-Pase activity by 36% at 1.4 mM, similar to the effect of 40 μg / ml Fraction 3 crystals on enzymatic activity (FIG. 4). PLP inhibited the enzyme activity by 9 mM at 70%. Inhibition by HNB and PLP did not change with the addition of fraction 3 crystals to the same reaction solution.

Most G-6-Pase inhibitors, such as chlorogenic acid and its derivatives and 3-mercaptopicolinic acid, are glucose-6-phosphate translocases of the G-6-Pase multienzyme complex. It is reported that the unit T1 is suppressed. These inhibitors, due to their fat soluble nature, inhibit the substances of millet more easily pass through the plasma plasma membrane and interfere with the hydrolysis of G-6-P. As a result, the active substance of the field differs from HNB and PLP, which are known to act on the T1 and T3 components of the G-6-Pase complex, to prevent reversible inhibition of these HNB and PLP.

Example 3: millet extract in vivo Inhibitory Activity of G-6-Pase Activity in

Step 1: prepare an experimental diet

An experimental diet containing millet methanol extract (0.3%, w / w) and powder (30%, w / w) was prepared and consumed by white rats. The diet is basically 20% casein, 15% starch, 45% sugar, 10% lard, 5% cellulose, 3.5% mineral mix, 1% vitamin mix, Three dietary formulas containing 0.3% DL-methionine and 0.2% choline (0.2% choline) and the control diet containing two kinds of millet components and the control formula not containing (Table 1) were prepared. Millet powder diet contains casein 16.6%, starch 0%, sugar 42.2%, lard 9.3%, cellulose 4.5, taking into account the nutritional content of millet Only minerals and vitamin mixes, DL-methionine and choline, were used in the same amounts as the other diets.

Components of Experimental Diet (g / ~ 1000 g) Control millet 0.3% Methanol Extract 30% powder Casein ^a 200 200 166 DL-methionine ^b 3 3 3 Starch ^c 150 150 0 Sugar ^c 450 450 372 Cellulose ^b 50 50 45 Lard ^c 100 100 93 Mineral mix ^a 35 35 35 Vitamin mix ^a 10 10 10 Choline ^b 2 2 2 Millet ^d Methanol extract 3 powder 300

a: Teklad Diet Diets (Madison, Wisconsin, USA)

b: Sigma-Aldrich Chemical (St. Louis, Missouri)

c: purchased from local market

d: Grown in Chungbuk, Korea

Step 2: G-6-Pase activity in the extract of millet in vivo inhibitory potency irradiation

Three groups of rats were given each diet prepared in step 1, reared for 4 weeks, and fasted for 16 hours before sacrifice. Blood was taken from the abdominal aorta and centrifuged in a heparin-treated test tube to obtain plasma and stored at −50 ° C. until analysis of blood glucose by enzyme method (Shinyang Chemical, Seoul). The microsomes of liver tissue were prepared by the method described above, and no solvent extract of millet was added again when measuring G-6-Pase activity.

As a result, the activity of millet ingredient intake group was not different from the control group. However, blood glucose levels were significantly lower in the millet powder diet group than in the control group, and lower in the methanol extract (0.3%, w / w) group (Table 2).

Inhibition of G-6-Pase Activity and Glucose Levels in Liver Tissues of Millet Diet Control 0.3% millet methanol extract 30% millet powder G-6-Pase Activity (Pi μg / minmg Protein) 13.79 ± 2.33 12.75 ± 1.24 13.64 ± 3.93 Blood glucose level (mg / 100 mL) 209.7 ± 5.5 191.4 ± 6.7 180.6 ± 9.0

As described above, the millet extract of the present invention has an excellent effect of lowering blood glucose by inhibiting neo-glucose synthesis and glycogen degradation by lowering glucose-6-phosphatase activity of liver tissues, thus preventing diabetes and It is a very useful invention in the health food industry for improvement.

Claims (2)

Millet extract characterized by lowering blood glucose by lowering glucose-6-phosphatase activity. The composition for reducing blood sugar, which contains the extract of millet 1 as an active ingredient.