KR101908772B1

KR101908772B1 - Anti-diabetic composition of mulberry twig hot water extract and oxyresveratrol as an efficient component and preparation method of the same

Info

Publication number: KR101908772B1
Application number: KR1020160031775A
Authority: KR
Inventors: 최상원; 김은정; 안은영; 전영희
Original assignee: 대구가톨릭대학교산학협력단
Priority date: 2016-03-17
Filing date: 2016-03-17
Publication date: 2018-10-16
Also published as: KR20170108222A; WO2017159912A1

Abstract

The present invention relates to an antidiabetic composition containing a topical hydrothermal extract and an ORT as an upper index component. When administered to Streptozotocin-induced diabetic rats, it inhibits the activity of intestinal disaccharide degrading enzyme compared to the hydrothermal extract of the alfalfa extract to lower blood glucose and improve plasma lipid Which can be used as a diabetic prophylactic and therapeutic functional medicinal drug and food composition containing the topical hydrothermal extract and the upper index component ORT by confirming the blood glucose lowering effect by increasing glucose uptake into liver tissue and increasing glycogen synthesis It has excellent effect.

Description

[0001] The present invention relates to an antidiabetic composition comprising an active ingredient of a topical hot-water extract and oxyresveratrol, and an anti-diabetic composition of mulberry twig hot water extract and oxyresveratrol as an active ingredient,

The present invention relates to an antidiabetic functional composition containing an active ingredient of a topical hot-water extract and oxyresveratrol, and a method of preparing the same. More particularly, the present invention relates to a composition for preventing and treating diabetes by administering the topical hydrothermal extract and oxyresveratrol in a streptozotocin- .

Diabetes mellitus is increasing worldwide, and the incidence of metabolic diseases and cancer is gradually increasing in Korea due to recent westernized diet and lack of exercise. According to the statistics of death statistics conducted by the National Statistical Office (NSO) in 2013, diabetes mellitus is ranked sixth and fourth respectively in men and women, and the combined deaths from complications are expected to account for a larger percentage of all deaths. According to the National Health and Nutrition Examination conducted by the CDC in 2012, the prevalence of diabetes has increased by 2.4% over the past decade. Guariguata and others are expected to increase the number of diabetics in Korea by 8.9% in 2013 and by 2.5% in 2035 to 11.4% in 2013. Diabetes has already become a serious social and health problem in our society.

Diabetes is a type of metabolic disorder caused by insufficiency of absolute or relative insulin, which is divided into type 1 diabetes and type 2 diabetes depending on the cause of the disease. Type 1 diabetes is a disease caused by lack of insulin itself and can be divided into immune mediated and idiopathic. When the pancreatic beta cells are destroyed due to autoimmune reactions, it is called immune-mediated and corresponds to about 5-10% of total diabetes. Diabetes mellitus is one of the idiopathic types of type 1 diabetes. Type 1 diabetes is essential for the treatment of insulin and oral hypoglycemic agents can be taken as needed. Type 2 diabetes is a disease caused by increased resistance to insulin receptors or a decrease in insulin sensitivity, which is a genetic factor, but is associated with many environmental factors such as age, increased obesity, and reduced physical activity.

Diabetes is a problem due to complications rather than the disease itself. Acute complications of diabetes include hyperglycemia, coma due to hyperglycemia, ketone acidosis due to accumulation of ketone acid, misuse of insulin and hypoglycemic agents, This hypoglycemia is too low. Chronic complications include diabetic retinopathy, nephropathy, neuropathy, and foot disease due to poor blood circulation due to hyperglycemia.

In addition, diabetes is a metabolic disorder that increases the risk of cardiovascular complications, as it causes abnormalities in lipid metabolism as well as glucose metabolism related disorders caused by inability to use the sugar properly. The types of cardiovascular complications associated with diabetes include hypertension, coronary artery disease, stroke, peripheral arterial disease, cardiomyopathy, and heart failure.

In general, insulin preparations are used for the treatment of type 1 diabetes, and oral hypoglycemic agents for type 2 diabetes. The oral hypoglycemic agents include sulfonylureas, which stimulate insulin secretion and improve insulin resistance, dipeptidyl peptidase-4 (DPP-4) inhibitors, which increase insulin secretion and inhibit glucagon secretion, Biguanide to improve resistance, thiazolidinedione (TZD) system to reduce insulin resistance in peripheral tissues and liver, and α-glucosidase inhibitor to delay glucose uptake. Oral hypoglycemic agents are not prescribed according to the diabetes classification because they are diverse according to their functional groups. In many cases, insulin preparations and oral hypoglycemic agents or oral hypoglycemic agents are used together according to the period and progress of the diabetes . There are many reasons for the development of diabetes, so many studies are underway to develop new drugs based on each etiology. In addition, since diabetes is a problem caused by complications, the development of drugs that can improve the complications is an important part of the treatment of diabetes.

However, the drugs developed so far have been reported to cause side effects such as hypoglycemia, loss of insulin secretion, gastrointestinal disorders, renal toxicity, diarrhea and stomach pains, heart failure, anemia, and liver failure. In addition, some medications may be prohibited due to serious hepatotoxicity and cardiovascular side effects. Some of the new drugs known to have hypoglycemic effects are difficult to use in clinical practice due to their high production costs, so it is necessary to develop new drugs or health functional foods derived from natural products that help patients with diabetes either alone or in combination with drugs.

Saccharin products are products related to mulberry, silkworm, oedi, top leaf, top, and bark. It has been known that latent products are effective for various diseases including diabetes. It is said that Dongyubogam is effective for diabetes, and the upper limb is said to be effective against wind and diuresis, and the upper limb is effective for swelling. In addition, it is said that it is effective for diabetes in the main stem, mulberry, mulberry, obi, and dusk in the main stem.

The antidiabetic effect of the latent products in the animal models studied showed that the silkworm decreased serum glucose concentration and inhibited glucosidase in streptozotocin (STZ) -induced diabetic rats, and decreased the activity of the disaccharide degrading enzyme in the proximal part of the small intestine . In the STZ-induced diabetic rats, serum levels of triglyceride (TG), total-cholesterol (T-CHO), low-density lipoprotein cholesterol (LDL-CHO) and thiobarbituric acid reactive substance (TBARS). On the leaf of mulberry leaf, reduction of fasting blood sugar and glycated hemoglobin concentration and reduction of disaccharide degrading enzyme activity of small intestine were reported in STZ induced diabetic rats. In addition, there was a decrease in TG concentration, T-CHO concentration, low density lipoprotein concentration, and TBARS content and there was an increase in high density lipoprotein cholesterol (HDL-CHO) concentration.

In addition, studies on antidiabetic efficacy of topical and topical antioxidants reported above have shown that the topical water extract significantly inhibited the α-glucosidase activity in the small intestine and significantly lowered fasting glucose in the alloxan-induced diabetic rats, glucosidase has been reported to be as potent inhibitor of acarbose as an antidiabetic drug. Recently, our researchers have found that the extract of the upper part of the mulberry tree significantly inhibits the activity of α-glucosidase from other sites. The antidiabetic efficacy of these extracts has been reported to be due to the suppression of oxidative stress or inflammatory response in the pancreas, and the upper limb extracts have been shown to increase the body weight and insulin levels of streptozotocin (STZ) induced diabetic rats , Which has been shown to be as efficacious as metformin, a well known type 2 diabetes drug.

Recently, oxyreservatrol derivatives (glycosides mulberroside A and oxyresveratrol, resveratrol, and 2-arylbenzofuran derivatives) have been identified in the upper limbs, and oxyreservatrol is a major physiologically active substance in the upper extremities. Antimelanin, and antioxidant components. And mulberoside A, which is also reported to be a major physiologically active substance in alfalfa, has recently been reported to have antidiabetic efficacy in alloxan-induced diabetic rat models. Thus, the antidiabetic effect of topical water extract and the main ingredient mulberroside A has been reported. However, almost all studies on the antidiabetic effect in the type 2 diabetic model were conducted. There have been no reports on the antidiabetic efficacy and mechanism of oxyresveratrol in the type 1 diabetes model.

The prior art related to the present invention is disclosed in Korean Patent No. 10-1438543, but it relates to a method for producing an extract of mulberry tree containing anti-inflammatory, anti-aging functional oxiresveratrol, trans-resveratrol and morasin. However, to date, there has been no known invention relating to an antidiabetic functional composition of a topical hot-water extract and oxyresveratrol (hereinafter referred to as ORT).

Accordingly, an object of the present invention is to provide a composition containing an upper surface hot-water extract and an index component ORT, which are effective for diabetes, as an active ingredient, and a method for producing the same.

In the present invention, 1 L of ionized water is added to 200 g of dried top and 100 g of corn syrup, and extracted with an ultrasonic extractor for 2 hours most preferably; Extracting the upper and upper bark extracts obtained in the above step under reduced pressure to obtain a topical extract and a hydrolyzate extract, respectively;

Ultrasonically extracting the upper limb with 80% ethanol aqueous solution twice, followed by filtration and concentration under reduced pressure; The 80% ethanol extract which is concentrated under reduced pressure in the above step is further dissolved in 80% ethanol, and then fractionated with hexane to concentrate the defatted ethanol extract obtained under reduced pressure; Oxyresveratrol (ORT) in the EtOAC fraction and oxyresveratrol 3 ', 4-O-β-naphthol fraction in the n-BuOH fraction were successively fractionated with CH2Cl2, EtOAC and n-BuOH solvents, The antidiabetic function of oxyresveratrol obtained by isolating and purifying D-diglucopyranoside (ORTG), respectively, was verified and evaluated in streptozotocin (STZ) -induced diabetic rats.

There is an excellent effect that it is possible to provide a diabetic preventive and improved functional medicine and a food composition containing the topical hot water extract and ORT extracted and extracted from mulberry according to the present invention as an active ingredient.

According to the present invention, the topical hot-water extract has an excellent effect of providing pharmaceuticals and food compositions having superior anti-diabetic efficacy as compared with the hydrothermal extract of perianth gland.

FIG. 1 is a graph showing the HPLC results of the hot-water extract of the upper body, the hot-water extract of the perianth and the ORT.
FIG. 2 is a schematic diagram showing a method for separating and purifying two functional index components (ORT & ORTG) from a topical ethanol extract according to the present invention.
FIG. 3 is a schematic diagram showing administration of MT and MRB after administration of STZ to an experimental animal to induce diabetes mellitus according to the present invention. FIG.
FIG. 4 is a schematic diagram showing administration of ORTG, ORT and Met after STZ administration to experimental animals for diabetes induction according to the present invention. FIG.
FIG. 5 is a graph showing fasting blood glucose measurement values of an experimental animal induced by diabetes mellitus according to the present invention. FIG.
FIG. 6 is a graph showing measured values of plasma glucose and insulin levels in an experimental animal induced by diabetes mellitus according to the present invention. FIG.
FIG. 7 is a graph showing the amount of lipid metabolism change according to administration of topical hot-water extract to diabetic-induced experimental animals according to the present invention.
FIG. 8 is a graph showing triglyceride content after administration of topical hydrothermal extract to diabetic test animals according to the present invention. FIG.
FIG. 9 is a graph showing measured values of free fatty acid after administering the hot-water extract of the upper extremities to the diabetic-induced experimental animals according to the present invention.
FIG. 10 is a graph showing changes in triglyceride and lipid metabolism in liver tissues after administration of topical hydrothermal extract to diabetic experimental animals according to the present invention. FIG.
11 is a graph showing the results of measuring the activity of the disaccharide-degrading enzyme in the small intestine mucosa after administering the hot-water extract of the upper extremity to the diabetic-induced experimental animal according to the present invention.
FIG. 12 is a graph showing blood glucose fasting value measured after administration of ORT, which is the upper index component, to the diabetic test animal according to the present invention.
FIG. 13 is a graph showing measured values of plasma glucose and insulin levels after administration of ORT to an experimental animal that induces diabetes according to the present invention. FIG.
FIG. 14 is a graph showing changes in lipid metabolism after administration of ORT to a diabetic-induced experimental animal according to the present invention.
FIG. 15 is a graph showing measured values of free fatty acid in plasma after administration of ORT to diabetic test animals according to the present invention. FIG.
FIG. 16 is a graph showing the activity of the disaccharide degrading enzyme of the small intestine mucosa after administration of ORT to diabetic test animals according to the present invention.
FIG. 17 is a graph showing measured values of liver tissue GLUT2 mRNA expression after administration of ORT to diabetic test animals according to the present invention. FIG.
18 is a graph showing measured values of hepatic glycogen content after administration of ORT to diabetic-induced experimental animals according to the present invention.
FIG. 19 is a schematic diagram showing the effect of inhibiting diabetes in the case of administration of topical hot-water extract and ORT according to the present invention. FIG.

The present invention relates to a medicament for preventing and treating diabetes and a food composition containing an upper body hot water extract and an ORT as an active ingredient.

According to the present invention, the content of mulberoside A and ORT is 2 to 3 times higher than that of the hydrothermal extract of Manganese bark extract.

According to the present invention, the topical hot water extract has a higher ORT content than that of the hydrothermal extract, and the content of ORTG is very low (upper water hot water extract: ORTG (2.45 ± 0.06%), ORT (0.92 ± 0.02%); Water extract of Alaska pollack: ORTG (19.71 ± 2.80%), ORT (0.04 ± 0.01%)

According to the present invention, the present invention relates to a pharmaceutical composition and a food composition, which are superior to the hydrothermal extract of Panax ginseng extract in diabetic rats using STZ, and have superior antidiabetic effect upon administration of ORT, an indicator component thereof.

The topical extract of the present invention may be one extracted with a hydrophilic organic solvent such as water, ethanol or methanol or a mixed solvent thereof, and most preferably is a hot-water extract obtained by using hot water.

In addition, the ORT of the present invention is most preferably separated into EtOAC when it is separated from the topical hot water extract.

The food composition according to the present invention is a food composition characterized by being one of beverage, gum, tea, and sunscreen.

The composition according to the present invention may have any one of formulations selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, emulsions, syrups, non-aqueous solutions, suspensions and freeze- .

The compositions of the present invention may be prepared in the form of pharmaceutical compositions and may further comprise one or more non-toxic and pharmaceutically acceptable carriers, excipients, adjuvants or diluents or other active ingredients.

The upper and upper alfalfa of the present invention are usually commercially available.

The present invention will be described in more detail with reference to Examples, Experimental Examples and Preparation Examples. However, the following Examples and Experiments are only for illustrative purposes and are not intended to limit the scope of the present invention.

In Production Examples 1 to 4 of the present invention, the value of the content of the topical hydrothermal extract or ORT is most preferably limited to 1 wt%, but it may be added in an amount of 0.5 to 2.5 wt%.

Production Example 1 Beverage containing topical hot water extract or ORT ingredient Content (% by weight) honey 0.3 Dioctanoic acid amide 0.0003 Nicotinic acid amide 0.0003 Sodium riboflavin hydrochloride 0.0001 Pyridoxine hydrochloride 0.0001 Inositol 0.001 Ortho acid 0.002 water 98.6962 Topical hot water extract or ORT One Sum 100

Production Example 2 Gum containing topical hydrothermal extract or ORT ingredient Content (% by weight) Gum base 20 Sugar 75 Spices 2 water 2 Topical hot water extract or ORT One Sum 100

Production Example 3 Tea containing topical hot water extract or ORT ingredient Content (% by weight) Functional glucose 96.8 Citric acid 0.05 Vitamin c 0.15 Xylitol One HPMC One Topical hot water extract or ORT One Sum 100

Production Example 4 Extreme hot-water extract or ORT-containing wire ingredient Content (% by weight) Brown rice 30 Yulmu 20 barley 20 Glutinous rice 7 Perilla 7 Black beans 7 Black sesame 7 wisdom 0.5 Rehab 0.5 Topical hot water extract or ORT One Sum 100

Example 1. Extraction, isolation and purification of ORTG and ORT from upper limbs

The upper limbs were harvested from mulberry leaves that did not grow in spring, shade for a week, and then dried in 50 ° C or less hot air dryer (COBP-15S, Sjunheung dryer, Boryyeong, Korea). To separate the two functional index components from the dried upper extremities, the upper extremities were extracted twice with an 80% aqueous ethanol solution using an ultrasonic wave extractor, and then filtered and concentrated under reduced pressure. The depilated concentrated ethanol extract of the above step was dissolved in water and then fractionated sequentially using CH2Cl2, EtOAC and n-BuOH solvents. Oxyresveratrol (ORT) from the EtOAC fraction and oxyresveratrol 3 ', 4-O- beta -D-diglucopyranoside (ORTG) from the n-BuOH fraction were separately purified (FIG.

Experimental Example 1 HPLC analysis of surface component ORT of hot-water extract of upper and lower alfalfa

For the HPLC analysis of the ORT and ORTG of the surface components of the upper and upper alfalfa hot water extracts extracted by the method of Example 1 above, analysis was performed using Merck HPLC grade (Darmastadt, Germany).

In order to evaluate this, 1 g of topical extract of the upper and lower alfalfa was diluted with 100 mL of distilled water, filtered with a 0.45 μm filter, and 10 μL was injected into the HPLC. The HPLC analysis was performed using a Waters e2690.5 HPLC high performance liquid chromatography ; YMC-Pack Pro C18, Solvent A (0.05% H3PO4 in H2O), Solvent B (CH3CN), flow rate; 0.8 mL / min. The two functional index components ORT and ORTG of the upper and upper alfalfa hot water extracts as measured by HPLC were compared with the retention time of the standard substances and the contents of the two extracts were calculated from the calibration curves of the two standard substances.

The results of the ORTG (1) and ORT (2) measurements of the two reference materials (A), topical hot water extract (B) and hydrocortisone hot water extract (C) are shown in FIG.

Example 2. Experimental animal

Anti-diabetic effect of topical extracts from the upper and lower alfalfa extracts Male and 6-week-old ICR mice weighing 29 ± 0.2 g were purchased from Coatec Biotech and fed solid feed for 1 week and adapted to the experimental environment. The experimental animals were divided into 10 normal control group (NC), 10 diabetes control group (DC), 10 water soluble Mulberry twig group (MT), and 10 bark diabetic diabetic group (Mulberry root bark, MRB). In the normal control and diabetic control groups, sterile saline was administered. In the upper diabetic group, topical hot water extract was administered at a concentration of 5 g / kg. In the diabetic group, bovine perianal hot water extract was administered at a concentration of 600 mg / kg.

The experimental animals used for the anti-diabetic effect of upper limb component were male, 6-week-old ICR mice weighing 33 ± 0.3 g. The animals were divided into three groups: normal control (NC) 7, diabetic control (DC) 7, ORTG 600 mg / kg, The ORT was divided into 10 diabetic groups administered at a concentration of 600 mg / kg and 10 diabetic groups (Metformin, Met) administered with 600 mg / kg of metformin.

Example 3. Diabetes induction

STZ was dissolved in 0.1 M citrate buffer (pH 4.5) and injected at a dose of 50 mg / kg / day for 5 days at a constant time to induce diabetes in the experimental animal of Example 2. [ After 14 days from the start of STZ administration, venous blood was collected from the venous blood to confirm blood glucose levels. In the experimental animals with diabetes mellitus induction, 5 g / kg of topical hydrothermal extract was orally administered to the upper diabetic group (MT) for 15 days starting from the 15th day after the start of the experiment, and 6 g / kg of hydrocortisone hydrothermal extract was administered to the bark diabetic group (MRB) (Fig. 3). Animals were sacrificed at the end of the sample administration period, and blood, liver, sputum, epididymal fat, and small intestine were collected and used for sample analysis.

Example 4. Topical index component Anti-diabetic effect

STZ was intraperitoneally injected into the experimental animals of Example 2 at a dose of 40 mg / kg / day for 5 days in order to confirm the anti-diabetic effect of the upper surface marker component. After 17 days from the start of STZ administration, Respectively. ORT, ORT and Met were orally administered at a concentration of 0.6 g / kg (FIG. 4) for 22 days from the 18th day after the start of the experiment. The animals were sacrificed and the blood, liver, pancreas and small intestine were collected and used for sample analysis.

Example 5. Sample collection

To analyze the samples of the experimental animals sacrificed in Example 3 and Example 4, the animals were anesthetized with CO ₂ for 12 hours before sacrifice, and blood was collected from the abdominal vena cava using a 1000 IU / mL heparin syringe. Blood was centrifuged at 13,200 rpm at 4 ° C, plasma was separated and stored at -80 ° C. After collection, the liver, the soleus, the epididymis and the pancreas were weighed, weighed and stored frozen at -80 ° C with liquid nitrogen. At the same time, the small intestine was scraped into the small intestine and rapidly frozen with liquid nitrogen and stored at -80 ° C.

Experimental Example 1 Weight change in the experiment of hot water extract of upper and lower alpaca

In the antidiabetic effect test of the upper and lower alfalfa hot water extracts, the initial body weights before the start of the experiment and the final body weights after the end of the experiment were measured and compared in the NC, DC, MT and MRB groups.

As shown in Table 5, the body weight of the DC, MT and MRB groups, which induced diabetes, was decreased compared to the NC group.

Group Initial BW (g) Final BW (g) NC 34.00 ± 0.28 35.10 ± 0.61 DC 34.87 + - 0.38 30.67 + - 0.84 MT 34.56 ± 0.32 30.42 + 0.78 MRB 36.06 ± 0.29 31.49 + - 0.45

Experimental Example 2 Measure blood sugar and plasma insulin content

In order to confirm the antidiabetic effect of the upper and lower alfalfa hot water extracts, the animals were fasted for 12 hours after the administration of the test sample 10 days later and the fasting blood glucose level was measured the morning before the test sample. Blood glucose was measured using a blood glucose monitoring kit (ARKRAY).

As a result, as shown in FIG. 5, fasting blood glucose level was increased in the DC group compared to the NC group after 10 days of oral administration, and decreased in the MT group compared to the DC group. There was no significant difference in MRB group compared to DC group.

As can be seen from FIG. 6, blood glucose levels in the plasma of the experimental animal sac were increased in the DC group compared to the NC group, and 44% in the MT group, but not significantly in the MRB group. Plasma insulin concentrations were significantly lower in the MT group than in the NC group.

Experimental Example 3. Blood lipid concentration measurement

Blood lipid concentration was measured using the plasma obtained in Example 5 to measure blood glucose and insulin concentration according to the administration of the upper and lower alfalfa hot-water extracts. Asan kit was used to measure plasma glucose content. Plasma insulin was measured using a mouse insulin ELISA kit (U-type, Shibayagi Co.).

As shown in FIG. 7, total cholesterol (T-CHO) concentration was 72% and 73% lower in the MT and MRB groups than in the DC group, and HDL-cholesterol (HDL-CHO) . The HDL-CHO / T-CHO ratio was 80% lower in the DC group than in the NC group and 135% and 130% higher in the MT group and the MRB group, respectively, as compared to the DC group. In conclusion, T-CHO concentration in MT group MRB group was decreased and HDL-CHO / T-CHO ratio was increased compared with DC group, thus confirming the effect of improving blood lipid concentration.

As shown in Fig. 8, triglyceride (TG) content in plasma decreased in the DC group compared to the NC group, and increased in the MT group compared to the DC group.

As can be seen from FIG. 9, plasma free fatty acid (FFA) levels were decreased in the DC, MT and MRB groups compared with the NC group, but there was no significant difference between the three groups.

Example 4. Liver tissue TG and T-CHO content

TG, T-CHO and FFA concentrations were measured using liver tissues obtained by the method of Example 5 to measure TG and T-CHO concentrations in the liver according to the administration of the topical and hydrocortisone extracts.

As shown in FIG. 10, the liver TG concentration was 9.5 in the NC group, 19.4 in the DC group, 8.5 in the MT group, and 22.5 mg / g in the MRB group, MT group decreased to 44% compared to DC group. The T-CHO concentration in the NC group was 2.6, the DC group was 2.9, the MT group was 2.5, and the MRB group was 1.9 mg / g, which was higher in the DC group than in the NC group and decreased in the MT group and the MRB group There was no significant difference. FFA content was decreased in DC group, MT group and MRB group compared with NC group, but there was no difference between the three groups. In conclusion, MT group had no effect on hepatic FFA concentration compared to DC group, but hepatic TG concentration was decreased to normal level and T-CHO level was decreased to improve liver lipid level.

Experimental Example 5 Disaccharide-degrading enzyme activity of small intestine mucosa

In order to determine whether the hypoglycemic effect of the MT group was superior to that of the control group, the lactose, sucrase and maltase activities of the small intestine mucosa were measured using the small intestine obtained in Example 5 Were measured by the Dahlqvist method.

In order to evaluate this, the pancreas of the small intestine obtained from the method of Example 5 was cut from 10 cm away from the appendage to the appendix, divided into three sections divided into proximal and middle distal portions and washed with physiological saline. The cells were washed with physiological saline, scraped with a microscopic glass on a cold plate, weighed, and homogenized with 4 times the weight of distilled water, homogenized at 4 ° C and centrifuged at 7000xg for 10 minutes. After centrifugation, the supernatant was taken and used as an enzyme activity measurement sample. 0.1 mL of the diluted enzyme sample (1: 1, sucrase: 8-fold, maltase: 10-fold), 0.1 mL of the substrate solution 0.056M disaccahride solution and 0.1M sodium maltase buffer (pH 6.0) After reacting in a water bath for 1 hour, 0.8 mL of distilled water was added, and the absorbance was measured at 420 nm after immersing in water for 2 minutes.

As shown in FIG. 11, proximal lactase activity was reduced by 70% in MT group compared to DC group. The sucrase activity was increased 120% in the DC group compared to the NC group and 67% and 75% in the MT and MRB groups, respectively, compared to the DC group. Maltase activity was increased by 138% in the DC group and by 77% in the MT group compared with the NC group. MRB group was decreased by 70% compared with DC group. The lactase and sucrase activities of the middle part were not significantly different among the groups. Maltase activity was decreased in DC, MT and MRB groups compared with NC group. The lactase, sucrase, and maltase activities in the distal part of the rats were decreased in the DC, MT, and MRB groups compared to the NC group.

Experimental Example 6. Weight change in experimental animals

In the antidiabetic effect test of the upper limb surface, the initial body weight of the experimental animals in the NC, DC, ORTG, ORT and Met groups was measured and the final weight was measured and compared.

As shown in Table 6, there was no significant difference between the experimental groups.

Group Initial BW (g) Final BW (g) NC 33.24 34.78 DC 33.73 32.33 ORTG 33.43 33.19 ORT 33.92 31.66 Met 33.09 32.84

Experimental Example 7. Measure blood sugar and plasma insulin content

In order to confirm the antidiabetic effect of the upper limb component, the animals were fasted for 12 hours after the administration of the test sample for 19 days and the fasting blood glucose level was measured the morning before the test sample. Blood glucose was measured using a blood glucose monitoring kit (ARKRAY).

As a result, as shown in FIG. 12, the fasting blood glucose level was 232% higher in the DC group than in the NC group after 19 days of oral administration, and decreased in the ORTG group, the ORT group and the Met group compared to the DC group.

Also, as shown in FIG. 13, blood glucose levels in the plasma after the sacrifice of the experimental animals were increased in the DC group compared to the NC group and 43% lower in the ORT group than in the DC group. There was no significant difference between ORTG and Met groups. Plasma insulin concentrations were significantly lower in the DC, ORT, ORTG and Met groups than in the NC group, and there was no difference between the two groups. Therefore, changes in blood glucose concentration in each group were not due to changes in insulin concentration.

Experimental Example 8. Long-term weighing

The weight of the sample obtained by the method of Example 5 was measured in the anti-diabetic test of the upper index component.

As shown in Table 7, the liver and pancreas weights did not show any significant difference between the experimental groups.

Group Liver (g) Pancreas (g) NC 21.5 ± 0.53 5.7 ± 0.34 DC 23.0 ± 0.93 6.2 ± 0.52 ORTG 21.7 ± 1.27 6.0 ± 0.44 ORT 20.5 ± 1.37 5.9 ± 0.38 Met 23.4 ± 0.68 6.8 ± 0.26

Experimental Example 9 Blood lipid concentration measurement

In order to measure the blood lipid concentration according to administration of the upper surface index component, blood lipid concentration was measured using the plasma obtained in Example 5, and Asan kit for measuring glucose content was used for measurement of plasma glucose content. Plasma insulin was measured using a mouse insulin ELISA kit (U-type, Shibayagi Co.).

As shown in FIG. 14, there was no difference in plasma TG concentration and GDL-CHO concentration between the experimental groups. T-CHO concentration was decreased in ORTG group, ORT group and Met group compared to DC group, but there was no difference between NC group and DC group. The HDL-CHO / T-CHO ratio was significantly increased in the Met group compared to the other experimental groups.

As can be seen in FIG. 15, plasma free fatty acid (FFA) levels were decreased in DC, ORTG, ORT and Met groups compared with NC group, but there was no significant difference between the groups.

Experimental Example 10. Disaccharide-degrading enzyme activity of small intestine mucosa

The lactase, sucrase and maltase activities of the small intestine mucosa were measured by the method of Dahlqvist using the small intestine obtained in Example 5 to confirm whether there was a difference in the glucose uptake effect due to the topical index component administration Respectively.

In order to evaluate this, the pancreas of the small intestine obtained by the method of Example 5 was cut 10 cm away from the small intestine to just before the cecum, and then divided into three into proximal and middle distal portions. After washing, the small intestine was washed with cold physiological saline, scraped with a microscopic glass on a cold plate, weighed, and homogenized with a homogenizer with 4 times the weight of distilled water measured at 4 ℃, 7000xg at 10 Minute centrifugation. After centrifugation, the supernatant was taken and used as an enzyme activity measurement sample. 0.1 mL of the diluted enzyme sample (1: 1, sucrase: 8-fold, maltase: 10-fold), 0.1 mL of the substrate solution 0.056M disaccahride solution and 0.1M sodium maltase buffer (pH 6.0) After reacting in a water bath for 1 hour, 0.8 mL of distilled water was added, and the absorbance was measured at 420 nm after immersing in water for 2 minutes. In the result of Experimental Example 5, only the activity of the disulfide cleavage enzyme was shown in the proximal portion, so that the activity of the disulfide cleavage enzyme was measured only in the proximal portion.

As shown in FIG. 16, activity of lactase, sucrase and maltase in the proximal portion was increased in the DC, ORTG, ORT, and Met groups compared to the NC group, and there was no significant difference between the four groups .

Experimental Example 11. Confirmation of GLUT2 mRNA expression level in liver tissue

The glucose transporter 2 (GLUT2) mRNA expression level of glucose transporter, glucose transporter 2 (GLUT2), was measured using liver tissue obtained by the method of Example 5 to ascertain whether glucose uptake by liver was increased.

To evaluate this, liver tissue was homogenized in a mortar bowl containing liquid nitrogen and 1 mL of RNAiso Plus (TaKaRa) was added per 0.1 g of colonic tissue. The supernatant was taken, and 200 μL of chloroform was added thereto, followed by centrifugation at 4 ° C. and 10,000 rpm for 5 minutes. The supernatant obtained by centrifugation was added with 500 uL of isopropanol, and centrifuged at 4 ° C and 14,000 rpm for 20 minutes to obtain an RNA pellet. The RNA pellet obtained in the above step was washed with 75% ethanol and centrifuged at 9,500 rpm for 5 minutes at 4 ° C. After air-drying, the RNA pellet was dissolved in diethyl pyrocarbonate (DEPC) -treated water.

As shown in FIG. 17, the amount of mRNA expression in the liver was decreased in the DC group compared to the NC group, but the ORTG group was increased compared to the DC group, but there was no significant difference. However, ORT group and Met group were significantly increased compared to DC group.

Experimental Example 12. Determination of liver glycoconut content

Since the difference in the expression level of GLUT2 mRNA was confirmed in Experimental Example 11, the amount of glucose introduced into the liver tissue according to administration of the epithelial component was considered to be different, and the change in the amount of glycogen, .

As shown in FIG. 18, the glycogen content of the liver was decreased in the DC group compared to the NC group, but the ORTG group and the ORT group were increased, but the difference was not significantly different between the DC group and the Met group.

In conclusion, the topical hydrothermal extract inhibited the activity of small intestinal disaccharide degrading enzymes, lowering blood glucose level, lowering plasma T-CHO concentration, reducing liver tissue TG concentration, and showing lipid-lowering effect and superior efficacy compared with hydrocortisone hydrothermal extract . In the antidiabetic effect of the upper limb component, the effect of ORT on blood glucose lowering, which is the main substance of the upper limb, was confirmed. As a result, the ORT increased the amount of GLUT2 mRNA expression in the liver tissue to increase glucose uptake into liver tissues and glycogen synthesis To lower the blood sugar level. In addition, the plasma T-CHO concentration is decreased to improve the lipid composition (FIG. 19).

The present invention is an extremely useful invention in the health food and medicine industry because it has an excellent effect of providing a composition for preventing and treating diabetes, which comprises a topical hot-water extract and an ORT as a top surface indicator component.

Claims

Ultrasonic wave on the extracted upper extremities was added aqueous ethanol and then with hexane fraction was concentrated under reduced pressure, hydrolysis in the skim ethanol extract, CH ₂ Cl _2, EtOAC and the n-BuOH fraction by sequentially separated from the n-BuOH fraction by purification oxyresveratrol 3,4- O-β-D-diglucopyranoside (ORTG).

The food composition according to claim 1, wherein the food composition is any one selected from beverage, gum, tea, and wire.

The pharmaceutical composition according to claim 2, wherein the pharmaceutical composition has any one of formulations selected from the group consisting of tablet, pill, powder, granule, capsule, suspension, emulsion, syrup, non-aqueous solution, suspension and freeze- &Lt; / RTI >

3. The pharmaceutical composition according to claim 2, wherein the composition further comprises a pharmaceutically acceptable carrier, excipient or diluent.