KR20030017092A - Hypoglycemic effect of extracts from Paeoniae radix - Google Patents

Abstract

The present invention relates to a composition for preventing and treating diabetes, which contains an alcoholic extract of peony as an active ingredient. The extract of the peony active ingredient according to the present invention inhibits the absorption of glucose by inhibiting the activity of alpha-glucosidase, an enzyme that catalyzes the final stage of carbohydrate digestion in the intestine, thereby suppressing the rapid absorption of glucose after food intake. It has an inhibitory effect and can be effectively used as a hypoglycemic agent.

Description

Hypoglycemic effect of extracts from Paeoniae radix

The present invention relates to a peony extract having a hypoglycemic effect. More specifically, the present invention contains the extract obtained by extracting the peony with alcohol as an active ingredient, the extract of the active ingredient inhibits the glucose absorption of the human body by inhibiting alpha-glucosidase activity in the intestine A composition useful as a hypoglycemic agent.

Diabetes causes abnormal physiological metabolic control functions such as carbohydrates, protein, lipid and electrolyte metabolism due to hormone imbalances such as insulin, glucagon and glucocorti-coid. It is characterized by high blood sugar, diabetes and other characteristic symptoms, and if these diabetes symptoms continue to bring serious chronic complications, including blood circulation disorders. Diabetes mellitus is a major cause of autoimmune processes that destroy pancreas β-cells, leading to insulin-dependent diabetes, which causes insulin secretion, genetic causes, insulin resistance, and beta cell dysfunction. It is classified as insulin-independent diabetes mellitus due to its interaction. Because insulin-dependent diabetes is severely deficient in insulin, it is absolutely dependent on insulin to prevent ketosis and death. Insulin-independent diabetes, however, maintains the ability of the pancreas to secrete insulin, but it is also called adult diabetes because insulin secreted by various reasons, such as obesity, lack of exercise, stress, and aging, is the most common form of diabetes. It accounts for more than 90%.

There are about 250 million people with diabetes worldwide. The best goal of diabetes treatment is to bring the blood sugar of diabetics to normal blood sugar as much as possible. In practice, however, normalization of blood glucose is a difficult task and the control of postprandial hyperglycemia is even more difficult.

Currently, diabetics are using diet and medicine to control hyperglycemia.

The most effective medicines for postprandial hyperglycemia include insulin lispro, insulin analogues, and alpha-glucosidase inhibitors.

The enzyme alpha-glucosidase (EC 3.2.1.20) catalyzes the final step in the digestion of carbohydrates. Thus, alpha-glucosidase inhibitors can slow the digestion of carbohydrates to control postprandial hyperglycemia. Acarbose, miglitol, and boglibose are used as drugs as alpha-glucosidase inhibitors that inhibit post-prandial hyperglycemia by inhibiting carbohydrate digestive enzyme activity in diabetics. Side effects occur frequently depending on the user.

Therefore, it is necessary to develop a safe blood glucose lowering agent with less side effects to improve the quality of life of diabetics and their families by appropriately treating diabetes, which is increasing at home and abroad.

Therefore, the present inventors prepared extracts for dozens of herbal medicines for the purpose of developing post-prandial hypoglycemic drugs and measured the inhibitory ability of alpha-glucosidase, and the specific extract of peony was alpha-glucosidase ( By inhibiting alpha-glucosidase), the present invention was completed by identifying the hypoglycemic effect.

Peony ( Paeoniae radix ) is a plant belonging to the genus Peony, perennial and thick, but grows long, fusiform and long, about 30cm long. This plant is an alleviation, analgesic, astringent, relieving medicine that is effective for muscle spasms, headaches, abdominal pain, dysentery, bacterial infections, cold, and landscaping. Mainly acting in the liver, and softens the liver energy and blood replenishment function is irregular menstrual cycles and stress is an excellent effect when you are irritated.

Therefore, the inventors of the present invention extracted alpha-glucosidase obtained from the small intestinal mucosa of fractions obtained by extracting various extracts obtained by extracting the peony with various organic solvents using high performance liquid chromatography. (alpha-glucosidase) was found to be the first to complete the present invention was found to have the effect of inhibiting the blood glucose increase very strongly in the glucose-load experiments of animal experiments.

Hereinafter, the present invention will be described in detail.

In the present invention, the extract was extracted with hexane, ethyl acetate, butanol, and water, and the extract was concentrated using a concentrator, and then fractionated using a high-performance liquid chromatography having a silica column. To obtain fractions.

As a result of measuring the inhibitory activity of the fractionated components in vitro on the alpha-glucosidase activity of rat small intestine, the compounds of the peony fractions showed inhibitory activity against alpha-glucosidase. In animal experiments, fractions of the peony extract also inhibited the activity of alpha-glucosidase in the small intestine, thereby lowering the glucose concentration in the blood by inhibiting intestinal degradation of the disaccharide maltose.

Thus, peony alcohol extracts slow down the metabolism of glucose by inhibiting alpha-glucosidase in the small intestine, thereby lowering the concentration of glucose in the blood. Therefore, the composition containing the active ingredient of these plants exhibits an effect of inhibiting excessive synergistic effect of glucose appearing after carbohydrate intake, showing an excellent effect as a hypoglycemic agent.

The present invention is explained in more detail by the following examples.

The following examples illustrate the present invention in detail and are not intended to limit the scope of the present invention.

Example 1

Preparation of Extract

Peony (production area: Uiseong, Gyeongbuk, Salesman: Yeongju Nonghyup) was purchased from Nonghyup Hana Club, Yangjae-dong, Seoul, as a dry herb, and then used as an extracting material.

Peony dry medicinal herbs and methanol (methanol) was mixed at a ratio of 1: 3 (W / V), and then extracted by cooling for 7 days, filtered through a filter paper filter, and the filtrate was concentrated at 40 ℃ with a vacuum concentrator.

The concentrate was re-dissolved in 70% methanol, and then extracted sequentially using four solvents (hexane, ethyl acetate, butanol, and water) according to the polarity, and concentrated at 40 ° C. using a vacuum condenser, and the water layer was freeze-dried. Lyophilization was used to extract the sample.

Example 2

Preparation of Fractions

Hexane, ethyl acetate and butanol solvent extracts were injected into the silica column (22 × 250 mm, DAISO) of high performance liquid chromatography (AKTA explorer 10) by injecting 77 mg of cyclohexane (cyclohexane). ), Butanol, isopropanol, and methanol (methanol) were taken in a test tube with 17 ml, and 36 fractions were obtained by concentration under reduced pressure using a speed vac and used as a fraction sample. .

The water extract was injected into a prep column (Prepak C ₁₈ cartridge, 25 × 100 mm, Waters) of High Speed Liquid Chromatography (Waters Delta 600), and the test tube was changed while changing the concentration of water and methanol as mobile phase solvents. After receiving 10 ml of 30 fractions were obtained by concentration under reduced pressure using a concentrator (speed vac) was used as a fraction sample. Table 1 shows the fractionation conditions of the high performance liquid chromatography for the preparation of the fractions of the organic solvent extracts.

Example 3

Screening of Alpha-glucosidase Inhibitory Activity from Rat Small Intestine

(3-1) Preparation of alpha-glucosidase coenzyme solution and glucose reagent

Intestinal epithelial layer of male Sprague-Dawley rats fasted for 16 hours was taken, suspended in 0.1 M phosphate buffer (pH 7.0), pulverized three times by ultrasound for 15 seconds, and then at 4 ℃ and 10,000 rpm. The supernatant was used as a crude enzyme solution by centrifugation for 30 minutes.

Glucose reagent to be used for the determination of alpha-glucosidase enzyme activity is 125 ㎍ / ml O -phenylenediamine, 5 units / ml peroxidase and glucose oxidase (glucose oxidase) 0.96 units / ml was used.

(3-2) Screening of alpha-glucosidase inhibitory activity

The crude enzyme solution, 0.1 M phosphate buffer solution (pH 7.0), 2 mM maltose and sample solution were added to 20 ㎍ / ml of the final concentration and incubated at 37 ° C for 40 minutes, followed by a constant temperature bath (80 ° C, 3 minutes). The enzyme was inactivated at. Transfer 100 μl of the reaction solution to a new 96 well plate, add the same amount of glucose reagent, incubate for 30 minutes at 37 ° C., develop color, and add 3 N hydrochloric acid (HCl) to measure absorbance at 490 nm. It was. The degree of inhibitory activity was determined relative to the percentage of inhibition compared to the positive control.

(3-3) Alpha-glucosidase Inhibitory Effect of Peony Fraction

Although 36 fractions of hexane extract and 30 water extracts of Peony did not show the effect of inhibiting alpha-glucosidase activity, fractions 10-19 of ethyl acetate extract showed very strong inhibitory activity. It was. And 12, 22, 23 fractions of butanol extract also showed a strong inhibitory activity for alpha-glucosidase (alpha-glucosidase) activity (Fig. 1).

Example 4

Hypoglycemic Action of Peony Fractions in Vivo

(4-1) Maltose loading test method

As the experimental animals used in this experiment, male mice weighing 25 ± 5 g of the ICR system purchased from the Hallym Laboratory Animal Research Institute were used. The animals were fasted for 16 hours before the experiment, and divided into control and experimental groups, and each group was tested with six animals. Glucobay 100 (Biel Co., Ltd.) and rat small intestine used as a mechanism to investigate the effect of alpha-glucosidase inhibitors that delay carbohydrate absorption to prevent hyperglycemia after meals Peptides effective for screening of alpha-glucosidase inhibitory activity of maltose after 30 minutes after oral administration of fractions 10-19 of ethylacetate extract, positive control and experimental group (Glucobay 100, Peony) Blood glucose was measured 30 minutes after oral administration of 2 g / kg to each fraction). Blood glucose measurement was performed using a blood glucose meter (SUPER GLUCOCARD ^TM II, KDK Co., Ltd.) from blood collected from the orbital vein of the mouse.

(4-2) Hypoglycemic Effects of Fractions upon Maltose Administration

The blood glucose level of the first group orally administered only 5% dimethyl sulfoxide (solvent) was 95 mg / dl, indicating a normal blood glucose level. Therefore, it can be seen that the 5% dimethyl sulfoxide used as the solvent does not show an effect on this experiment. In the second group orally administered maltose, maltose is broken down into glucose by alpha-glucosidase in the small intestine and absorbed into the blood, resulting in high blood sugar levels of 187 mg / dl. Showed. However, glucoby 100 and peony fraction administered with maltose showed a significantly lowering effect on glucose-inhibiting alpha-glucosidase activity (Fig. 2).

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1 is a graph of the in vitro alpha-glucosidase inhibitory effect of the peony organic solvent fraction of the present invention.

Figure 2 is a graph of in vivo blood glucose lowering of the peony fraction of the present invention.

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Claims (4)

The peony dry medicinal herbs and methanol were mixed at a ratio of 1: 3 (W / V), and then extracted by cooling for 7 days, filtered through a filter paper filter, and the filtrate was concentrated at 40 ° C. using a vacuum condenser, and the concentrate was concentrated. Extract with ethyl acetate and butanol. 77 mg of ethyl acetate and butanol extracts were injected into a silica column of a high performance liquid chromatography, and eluted at a rate of 8 ml per minute while varying the concentrations of the mobile phase solvents cyclohexane, butanol, isopropanol and methanol. Alpha-glucosidase of Peony alcohol extract, characterized in that the ethyl acetate fraction of the 36 fractions received 17 ml in the test tube, fractions 10-19 and butanol fractions 12, 22, 23 were prepared. Extract exhibiting hypoglycemic effect, containing an active ingredient showing alpha-glucosidase) inhibitory activity A hypoglycemic agent comprising an extract obtained according to claim 1 which is subsequently lyophilized and obtained in powder form. Blood glucose lowering agent comprising the extract of claim 1 as an active ingredient and a pharmaceutically acceptable carrier Health supplement food for hypoglycemic, characterized in that the extract of claim 1 as an active ingredient