WO2017159912A1 - Antidiabetic functional composition containing hot water extract of ramulus mori and oxyresveratrol as active ingredients and method for producing thereof - Google Patents

Antidiabetic functional composition containing hot water extract of ramulus mori and oxyresveratrol as active ingredients and method for producing thereof Download PDF

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WO2017159912A1
WO2017159912A1 PCT/KR2016/003209 KR2016003209W WO2017159912A1 WO 2017159912 A1 WO2017159912 A1 WO 2017159912A1 KR 2016003209 W KR2016003209 W KR 2016003209W WO 2017159912 A1 WO2017159912 A1 WO 2017159912A1
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hot water
group
ort
extract
ethanol
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French (fr)
Korean (ko)
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최상원
김은정
안은영
전영희
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대구가톨릭대학교산학협력단
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/60—Moraceae (Mulberry family), e.g. breadfruit or fig
    • A61K36/605—Morus (mulberry)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate

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  • the present invention relates to an antidiabetic functional composition containing a hot water extract and oxyresveratrol as an active ingredient, and more particularly, to a composition for preventing and treating diabetes by administering the upper extremity hot water extract and oxyresveratrol in Streptozotocin-induced diabetic rats. It is about.
  • Diabetes is on the rise all over the world, and in Korea, the incidence of metabolic diseases and cancer is gradually increasing due to westernized diet and lack of exercise. According to the 2013 Cause of Death Statistics conducted by the National Statistical Office, deaths due to diabetes rank 6th and 4th, respectively, and the deaths from complications are expected to account for a greater proportion of all deaths. In addition, according to the National Health and Nutrition Survey conducted by the Centers for Disease Control and Prevention in 2012, the prevalence of diabetes has increased by 2.4% over the last decade. Guariguata et al. Estimated that diabetes mellitus in Korea is expected to increase by 2.5% to 8.9% of Korea's total population in 2013 and 11.4% by 2035. Diabetes has already become a serious social and health problem in our society.
  • Diabetes is a metabolic disease caused by an absolute or relative lack of insulin and is divided into type 1 diabetes and type 2 diabetes depending on the cause of the onset.
  • Type 1 diabetes is a disease caused by the lack of insulin itself and can be divided into immunomediated and idiopathic. The destruction of pancreatic beta cells due to autoimmune reactions is called immunomediation, which accounts for 5-10% of all diabetes. Diabetes of unknown type 1 diabetes is idiopathic.
  • Type 1 diabetes requires the treatment of insulin and can be taken with oral hypoglycemic agents as needed.
  • Type 2 diabetes is a disease caused by increased resistance to insulin receptors or decreased insulin sensitivity. There are genetic factors, but it is combined with many environmental factors such as age, obesity, and decreased physical activity.
  • Diabetes is more problematic due to complications than the disease itself.
  • Acute complications of diabetes include hyperglycemic coma that leads to coma due to hyperglycemia, ketoacidosis due to overaccumulation of ketone acid, and blood sugar due to incorrect use of insulin or hypoglycemic agents.
  • the hypoglycemia that is too low can be mentioned largely.
  • Chronic complications include diabetic retinopathy, nephropathy, neuropathy, and foot problems caused by poor blood circulation due to hyperglycemia.
  • diabetes is a metabolic disease that increases the risk of cardiovascular complications because it causes abnormalities in lipid metabolism as well as glucose metabolism-related disorders caused by inadequate use of sugar.
  • Cardiovascular complications related to diabetes include hypertension, coronary artery disease, stroke, peripheral artery disease, cardiomyopathy, and heart failure.
  • oral hypoglycemic agents are used for type 2 diabetes.
  • Types of oral hypoglycemic agents include sulfonylureas, which stimulate insulin secretion and improve insulin resistance, dipeptidyl peptidase-4 (DPP-4) inhibitors that increase insulin secretion and inhibit glucagon secretion, and inhibit hepatic angiogenesis. Biguanides improve resistance, thiazolidinedione (TZD) decreases insulin resistance in peripheral tissues and liver, and ⁇ -glucosidase inhibitors that delay glucose absorption.
  • DPP-4 dipeptidyl peptidase-4
  • Biguanides improve resistance
  • thiazolidinedione (TZD) decreases insulin resistance in peripheral tissues and liver
  • ⁇ -glucosidase inhibitors that delay glucose absorption.
  • Oral hypoglycemic agents vary depending on the mechanism of action, so they are not determined according to the type of diabetes, but insulin and oral hypoglycemic agents, or oral hypoglycemic agents, are often used in combination with diabetes mellitus. .
  • the reasons for the development of diabetes vary, and many studies are being conducted to develop new drugs based on the etiology of each.
  • the development of drugs that can improve the complications is an important part of diabetes treatment.
  • the latent product refers to products related to mulberry trees, and includes silkworms, mulberry, upper leaves, upper limbs, and lettuce.
  • Latent products have long been known to be effective in various diseases including diabetes. It is recorded that the auditory effect is effective for diabetes, and the upper limb is effective for wind and diuresis, and the upper lobe is effective for body swelling.
  • the herbaceous wood has been reported to have an effect on diabetes in the upper lobe, upper and lower skin, mulberry, and Jambun, and the upper leaf, upper limb and Jambun are also reported to be effective in wind.
  • the leaves of the mulberry which have been reported to reduce fasting glucose and glycated hemoglobin concentrations and decreased intestinal disaccharide enzyme activity in STZ-induced diabetic rats.
  • TG concentration, T-CHO concentration, low density lipoprotein concentration and TBARS content were decreased, and high density lipoprotein cholesterol (HDL-CHO) concentration was reported.
  • oxyresveratrol derivatives (glycosides mulberroside A and aglycone oxyresveratrol), resveratrol, and 2-arylbenzofuran derivatives have recently been identified from the upper limbs. It has been reported as an anti-melanin, and an antioxidant component.
  • mulberroside A which is also present in the upper limbs, has been reported to have anti-diabetic effects in alloxan-induced diabetic rats.
  • the antidiabetic effect of the water extract of the upper limbs and mulberroside A the main component, has been reported, but most of the studies have revealed the antidiabetic effect in the type 2 diabetes model. There have been no studies on the antidiabetic efficacy and mechanism of oxyresveratrol in water extract and type 1 diabetes in type 1 diabetes model.
  • the present invention comprises the steps of extracting 200 g of dried upper limbs and 100 g of baekryepi and 1L of ionized water, most preferably in an ultrasonic extractor for 2 hours; Concentrating the extracted upper limb and lettuce extract under reduced pressure under reduced pressure to obtain an extract of the upper limb and lettuce extract;
  • the upper extremity hot water extract has an excellent effect of providing a pharmaceutical and food composition with superior antidiabetic efficacy when compared with the extract of hot water extract.
  • Figure 1 is a graph showing the HPLC results of the upper liquor hot water extract, baekryepi hot water extract and ORT.
  • Figure 2 is a schematic diagram showing the separation and purification method of the two functional indicators (ORT & ORTG) from the upper liquor ethanol extract according to the present invention.
  • Figure 3 is a schematic diagram showing the MT and MRB administration after the STZ administration to the induction of diabetes mellitus according to the present invention.
  • Figure 4 is a schematic diagram showing the ORTG, ORT and Met administration after STZ administration to experimental animals for diabetes induction according to the present invention.
  • Figure 5 is a graph showing the fasting blood glucose measurement of the diabetes-induced experimental animal according to the present invention.
  • Figure 6 is a graph showing the measurement of plasma glucose and insulin content of the diabetes-induced experimental animals according to the present invention.
  • Figure 7 is a graph showing the change in lipid metabolism according to the administration of hot water extracts to the diabetes-induced experimental animals according to the present invention.
  • FIG. 8 is a graph showing triglyceride content after administration of hot water extracts of the upper limbs to a diabetic-induced experimental animal according to the present invention.
  • FIG. 9 is a graph showing the measured value of free fatty acid content after administration of hot water extracts of the upper limbs to the diabetic-induced experimental animal according to the present invention.
  • FIG. 10 is a graph showing changes in triglyceride and lipid metabolism in liver tissues after administration of hot water extracts of the upper limbs to a diabetic-induced experimental animal according to the present invention.
  • Figure 11 is a graph showing the results of measuring the disaccharide enzyme activity of the small intestine mucosa after administration of hot water extracts to the diabetes-induced experimental animals according to the present invention.
  • FIG. 12 is a graph showing the measured blood fasting glucose levels after administration of ORT, which is an indicator of the upper limbs, in the diabetes-induced experimental animal according to the present invention.
  • Figure 13 is a graph showing the measured value of plasma glucose and insulin content after ORT administration to the diabetic animal-derived experimental animal according to the present invention.
  • Figure 14 is a graph showing the change in lipid metabolism after administration of ORT to the diabetes-induced experimental animals according to the present invention.
  • 15 is a graph showing the measured value of free fatty acid content in plasma after ORT administration to a diabetic animal-derived experimental animal according to the present invention.
  • Figure 16 is a graph showing the disaccharide enzyme activity of the small intestine mucosa after the ORT administration to the diabetic induced animal according to the present invention.
  • Figure 17 is a graph showing the measurement value of liver tissue GLUT2 mRNA expression after ORT administration to the diabetes-induced experimental animals according to the present invention.
  • Figure 18 is a graph showing the measurement value of liver tissue glycogen content after ORT administration to a diabetic-induced experimental animal according to the present invention.
  • Figure 19 is a schematic diagram showing the effect of inhibiting diabetes appeared when the hot water extract and ORT administration in accordance with the present invention.
  • the present invention relates to a pharmaceutical and food composition for preventing and treating diabetes containing the upper limb hydrothermal extract and ORT as an active ingredient.
  • the upper limb hydrothermal extract has a content of mulberroside A and ORT 2 to 3 times higher than that of the hot birch extract.
  • the upper limb hydrothermal extract has a higher ORT content than the hot water extract, but the content of ORTG is very low [upper lichen hydrothermal extract: ORTG (2.45 ⁇ 0.06%), ORT (0.92 ⁇ 0.02%); Lepidoptera hot water extract: ORTG (19.71 ⁇ 2.80%), ORT (0.04 ⁇ 0.01%)]
  • the present invention relates to a pharmaceutical composition and a food composition characterized by superior antidiabetic effect when the upper limb hydrothermal extract and ORT administered as an index component thereof are higher than hot water extract in diabetic-induced mice using STZ.
  • the upper limb extract of the present invention may be extracted by a hydrophilic organic solvent such as water, ethanol, methanol, or a mixed solvent thereof, most preferably a hot water extract extracted using hot water.
  • a hydrophilic organic solvent such as water, ethanol, methanol, or a mixed solvent thereof, most preferably a hot water extract extracted using hot water.
  • ORT of the present invention is most preferably separated by EtOAC when separated from the hot water extract.
  • the food composition according to the present invention provides a food composition, characterized in that the formulation of any one of beverage, gum, tea, and wire.
  • composition according to the present invention is a pharmaceutical composition characterized in that it has any one formulation selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, emulsions, syrups, non-aqueous solvents, suspensions and lyophilized preparations. To provide.
  • 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 limbs and limbs of the present invention are usually commercially available.
  • the numerical value of the upper limb hydrothermal extract or ORT content is limited to 1% by weight, but may be added up to 0.5 to 2.5% by weight.
  • the upper limbs were dried for one week after harvesting from mulberry trees without leaves in spring and then dried in a hot air dryer (COBP-15S, Sjunheung dryer, Boryyeong, Korea) below 50 ° C.
  • COBP-15S hot air dryer
  • the upper limbs were extracted twice with an 80% ethanol solution using an ultrasonic extractor, and filtered and concentrated under reduced pressure.
  • the degreased ethanol extract concentrated in the above step was dissolved in water, and then fractionated sequentially using CH 2 Cl 2, EtOAC, and n-BuOH solvent.
  • the upper limbs and epithelium hot water extracts (1 g) were diluted with 100 mL of distilled water, filtered through a 0.45 ⁇ m filter, and then injected into 10 ⁇ l of HPLC, where HPLC analysis was performed using Waters e2690.5 HPLC high performance liquid chromatography, column. ; YMC-Pack Pro C18, Solvent A (0.05% H3PO4 in H2O), Solvent B (CH3CN), flow rate; It was performed at 0.8 mL / min conditions.
  • the two functional indicators ORT and ORTG of the upper limbs and epithelium hot water extracts measured by HPLC were analyzed in comparison with the retention time of the standard, and the contents of the two extracts were calculated from the calibration curves of the two standard.
  • the experimental animals used for the antidiabetic effect of the upper limbs and epithelial hot water extract were male and 6-week-old ICR mice weighing 29 ⁇ 0.2 g and fed solid feed for 1 week and adapted to the experimental environment.
  • the experimental animals were 10 normal controls (NC), 10 diabees control (DC), 10 upper limbs (Mulberry twig (MT)) administered with the upper limbs hot water extract, and an epithelial diabetic group treated with hot water extract. (Mulberry root bark, MRB) was divided into 10 numbers.
  • the normal control group and the diabetic control group were treated with sterile saline, the upper limb diabetic group was administered with a concentration of 5 g / kg of the upper extremity hot water extract, and the epithelial diabetic group was administered with an epithelial hot water extract at 600 mg / kg.
  • the experimental animals used for the antidiabetic effect of the upper limb markers were male and 6 week old ICR mice weighing 33 ⁇ 0.3 g.
  • the experimental animals were 7 normal controls (NC) administered sterile saline, 7 diabetic controls (DC) administered sterile saline, 10 diabetic groups administered ORTG at a concentration of 600 mg / kg,
  • NC normal controls
  • DC diabetic controls
  • the experiment was divided into 10 diabetic groups administered ORT at a concentration of 600 mg / kg and 10 diabetic groups (Metformin, Met) administered at a concentration of 600 mg / kg.
  • STZ was dissolved in 0.1M citrate buffer (pH 4.5) and injected into the abdominal cavity at a constant time for 5 days at a dose of 50 mg / kg / day.
  • blood was collected from the vein of the vein to check the blood glucose to confirm the induction of diabetes.
  • the test animal was confirmed to be induced diabetes orally oral administration of upper extremity hot water extract 5g / kg to the upper extremity diabetic group (MT) for 15 days from the start of the experiment 16 days or 6g / kg oral administration of hot water extract to the epithelial diabetic group (MRB) (Fig. 3).
  • animals were sacrificed and blood, liver, soleus muscle, epididymal fat and small intestine were collected and used for sample analysis.
  • Example 2 In order to confirm the antidiabetic effect of the upper extremity indicator component, the experimental animals of Example 2 were intraperitoneally injected with STZ at a dose of 40 mg / kg / day for 5 days. Confirmed. Experimental animals confirming the blood glucose level of the above step was orally administered with ORTG, ORT and Met at a concentration of 0.6 g / kg, respectively, for 18 days after the start of the experiment (Fig. 4). Animals were sacrificed after sample administration and blood, liver, pancreas and small intestine were collected and used for sample analysis.
  • body weight was reduced in three groups of DC, MT, and MRB which induced diabetes compared to NC group.
  • the animals were fasted for 12 hours 10 days after the administration of the test sample, and fasting blood glucose was measured before the test sample was administered the next morning.
  • Glucose was measured using a blood glucose monitoring kit (ARRKRAY) with a small amount of blood taken from the vein.
  • the blood glucose measured in plasma after sacrifice of experimental animals was increased in the DC group and the MT group in the MT group compared to the NC group, but there was no significant difference in the MRB group.
  • Plasma insulin concentration showed a significant decrease only in the MT group compared to the NC group.
  • Plasma lipid concentration was measured using the plasma obtained in Example 5 to measure blood glucose and blood insulin concentrations following the administration of the upper limbs and epithelium hot water extract. Asan kit was used for glucose measurement. Plasma insulin was measured using a mouse Insulin ELISA kit (U-type, Shibayagi Co.).
  • the total cholesterol (T-CHO) concentrations of the MT and MRB groups were reduced by 72% and 73%, respectively, and the HDL-cholesterol (HDL-CHO) concentrations were not different between the experimental groups.
  • the HDL-CHO / T-CHO ratio was 80% lower in the DC group than the NC group, and 135% and 130% in the MT and MRB groups, respectively.
  • MT group showed lower T-CHO concentration and HDL-CHO / T-CHO ratio compared to DC group in MRB group.
  • triglyceride (TG) content in plasma was reduced in the DC group compared to the NC group and increased in the MT group compared to the DC group.
  • TG, T-CHO and FFA concentrations were measured using liver tissue obtained by the method of Example 5 to determine TG and T-CHO concentrations in the liver following administration of the upper limbs and epithelial hot water extract.
  • the liver TG concentration was measured to be 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 normal level by 44%.
  • the liver T-CHO concentrations were 2.6 in the NC group, 2.9 in the DC group, 2.5 in the MT group, and 1.9 mg / g in the MRB group.
  • the DC group increased compared to the NC group and the MT and MRB groups decreased compared to the DC group. There was no significant difference.
  • the FFA content was decreased in the DC, MT and MRB groups compared to the NC group, and there was no difference between the three groups.
  • MT group had no effect on liver FFA level compared to DC group, but decreased TG concentration to normal level and decreased T-CHO level to improve liver lipid level.
  • Lactase, sucrase and maltase activity of small intestinal mucosa using the small intestine obtained in Example 5 to determine whether the hypoglycemic effect of MT group according to the administration of the upper limb and epithelium hot water extract was different was measured by Dahlqvist's method.
  • 0.1 mL of diluted enzyme samples (lactase: 1x, sucrase: 8x, maltase: 10x), substrate solution 0.056M disaccahride solution and 0.1mL 0.1M sodium maltase buffer (pH 6.0) were added to the test tube and mixed well. After reacting in a water bath for 1 hour, 0.8 mL of distilled water was added, soaked in water for 2 minutes, and the absorbance was measured at 420 nm.
  • the lactase activity of proximal was 70% decreased in the MT group compared to the DC group.
  • Sucrase activity was increased by 120% in DC group and 67% and 75% in MT and MRB groups, respectively.
  • Maltase activity was increased by 138% in DC group and 77% in MT group.
  • the MRB group was 70% less than the DC group.
  • lactase and sucrase activity was decreased in DC, MT and MRB groups compared to NC group.
  • lactase, sucrase and maltase activities were decreased in DC, MT and MRB groups compared to NC group.
  • the animals fasted 12 hours after 19 days of administration of the test sample, and fasting blood glucose was measured before administration of the test sample the next morning.
  • Glucose was measured using a blood glucose monitoring kit (ARRKRAY) with a small amount of blood taken from the vein.
  • fasting blood glucose was increased by 232% in the DC group compared to 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.
  • the blood glucose measured in plasma after sacrifice of experimental animals was increased in the DC group compared to the NC group and decreased by 43% in the ORT group compared to the DC group. There was no significant difference between ORTG group and Met group. Plasma insulin concentrations were significantly decreased in the DC, ORT, ORTG, and Met groups that induced diabetes compared to the NC group, and there was no difference between the four groups. Therefore, it was shown that the change in blood glucose level of each group was not due to the change in insulin concentration.
  • Plasma lipid concentration was measured by using the plasma obtained in Example 5 to measure blood lipid concentrations according to the administration of the upper limb index component, and a glucose measuring solution (Asan kit) was used to measure plasma glucose content. Plasma insulin was measured using a mouse Insulin ELISA kit (U-type, Shibayagi Co.).
  • the plasma TG concentration and the GDL-CHO concentration did not differ between the experimental groups.
  • T-CHO concentration was decreased in ORTG, ORT and Met groups compared to DC group, and there was no difference between NC and DC groups.
  • the HDL-CHO / T-CHO ratio was significantly increased in the Met group compared to the other experimental groups.
  • 0.1 mL of diluted enzyme samples (lactase: 1x, sucrase: 8x, maltase: 10x), substrate solution 0.056M disaccahride solution and 0.1mL 0.1M sodium maltase buffer (pH 6.0) were added to the test tube and mixed well. After reacting in a water bath for 1 hour, 0.8 mL of distilled water was added, soaked in water for 2 minutes, and the absorbance was measured at 420 nm. Since the disaccharide dehydrogenase activity effect was shown only in the proximal part in the results of Experimental Example 5, only the proximal part was measured for disaccharide dehydrogenase activity.
  • mRNA expression level of glucose transporter glucose transporter 2 was measured using liver tissue obtained by the method of Example 5.
  • liver tissue was homogenized in a mortar containing liquid nitrogen and 1 mL of RNAiso Plus (TaKaRa) was added per 0.1 g of colon tissue. The supernatant was taken and chloroform 200uL was added and centrifuged at 4 ° C. and 10,000 rpm for 5 minutes. After isopropanol 500uL was added to the supernatant obtained by centrifugation, RNA pellet was obtained by centrifugation at 4 ° C and 14,000 rpm for 20 minutes. The RNA pellet obtained in the above step was washed with 75% ethanol, centrifuged at 4 ° C. and 9,500 rpm for 5 minutes and then air-dryed and dissolved in diethyl pyrocarbonate (DEPC) -treated water for analysis.
  • DEPC diethyl pyrocarbonate
  • upper limb hydrothermal extracts inhibited the activity of small intestinal disaccharides, lowering blood glucose, reducing plasma T-CHO levels, and reducing liver tissue TG levels, resulting in lipid-improving effects and superior efficacy compared to epithelial hot water extracts.
  • the antidiabetic effect of the upper limb markers confirmed the hypoglycemic effect of ORT, the major substance in the upper limb.
  • the mechanism of ORT increased the expression of GLUT2 mRNA in liver tissue, which increased glucose inflow into liver tissue and the synthesis of glycogen. It has been shown to increase blood sugar levels. It is also effective in improving lipids by reducing plasma T-CHO concentration (Fig. 19).
  • the present invention is a very useful invention for the health food and pharmaceutical industry because it has an excellent effect of providing a composition for the prevention and treatment of diabetes containing the upper limbs hot water extract and the upper limb index ORT.

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Abstract

The present invention relates to an antidiabetic composition containing a hot water extract of Ramulus Mori and ORT, which is an indicator component of Ramulus Mori. When administered to Streptozotocin-induced diabetic rats, it has been confirmed that the antidiabetic composition of the present invention is more excellent in inhibiting the activity of small intestine disaccharidase to lower blood glucose and improving plasma lipid, compared to a hot water extract of Mori Cortex Radicis, and that the antidiabetic composition of the present invention has a hypoglycemic effect by increasing glucose uptake into liver tissues and increasing glycogen synthesis. Therefore, the antidiabetic composition of the present invention has an excellent effect that can be used as a diabetic preventive and therapeutic functional medicine and a food composition containing a hot water extract of Ramulus Mori and ORT, which is an indicator component of Ramulus Mori.

Description

상지 열수추출물과 OXYRESVERATROL을 유효성분으로 함유하는 항당뇨 기능성 조성물 및 그 제조방법Antidiabetic functional composition containing the upper limbs hot water extract and OXYRESVERATROL as an active ingredient, and a method of manufacturing the same
본 발명은 상지 열수추출물 및 oxyresveratrol을 유효성분으로 함유하는 항당뇨 기능성 조성물 및 그 제조방법에 관한 것으로 더욱 상세하게는 Streptozotocin 유도 당뇨쥐에서 상기 상지 열수추출물 및 oxyresveratrol을 투여함으로써 당뇨 예방 및 치료용 조성물에 관한 것이다. The present invention relates to an antidiabetic functional composition containing a hot water extract and oxyresveratrol as an active ingredient, and more particularly, to a composition for preventing and treating diabetes by administering the upper extremity hot water extract and oxyresveratrol in Streptozotocin-induced diabetic rats. It is about.
당뇨병은 전 세계적으로 증가하는 추세에 있으며, 우리나라에서도 최근 서구화된 식생활과 운동부족으로 인하여 대사성 질환과 암 등의 발병률이 점차 증가하고 있다. 2013년 통계청에서 실시한 사망원인통계에 따르면 당뇨병으로 인한 사망이 남녀 각각 6위와 4위로 높은 순위를 차지하고 있으며, 합병증으로 인한 사망까지 합하면 전체 사망 원인 중 더 큰 비율을 차지할 것으로 예상된다. 또한 2012년에 질병관리본부에서 실시한 국민건강영양조사에 따르면 당뇨병 유병률은 최근 10년간 2.4% 증가하였다. Guariguata 등은 우리나라에서 당뇨병 유병인이 2013년에는 우리나라 전체 인구의 8.9%이며, 2035년도에는 11.4%로 2.5% 증가할 것으로 예상됨으로서 당뇨는 이미 우리 사회에 심각한 사회, 보건 문제가 되었다.Diabetes is on the rise all over the world, and in Korea, the incidence of metabolic diseases and cancer is gradually increasing due to westernized diet and lack of exercise. According to the 2013 Cause of Death Statistics conducted by the National Statistical Office, deaths due to diabetes rank 6th and 4th, respectively, and the deaths from complications are expected to account for a greater proportion of all deaths. In addition, according to the National Health and Nutrition Survey conducted by the Centers for Disease Control and Prevention in 2012, the prevalence of diabetes has increased by 2.4% over the last decade. Guariguata et al. Estimated that diabetes mellitus in Korea is expected to increase by 2.5% to 8.9% of Korea's total population in 2013 and 11.4% by 2035. Diabetes has already become a serious social and health problem in our society.
당뇨병은 인슐린이 절대적 또는 상대적으로 부족하여 생기는 대사성 질환의 일종으로 발병원인에 따라 1형 당뇨와 2형 당뇨로 나뉜다. 1형 당뇨는 인슐린 자체가 부족하여 생기는 병으로 면역매개성과 특발성으로 나눌 수 있다. 자가면역반응으로 인하여 췌장 베타세포가 파괴되어 일어나는 경우를 면역매개성이라고 하며, 전체 당뇨병의 5-10% 정도에 해당한다. 1형 당뇨 중 원인이 밝혀지지 않은 당뇨는 특발성에 속한다. 1형 당뇨는 인슐린의 치료가 필수적이며 필요에 따라 경구혈당강하제를 같이 복용할 수 있다. 2형 당뇨는 인슐린 수용체에 저항성이 증가되거나 인슐린 민감도가 떨어져서 생기는 병으로 유전적 요인도 있지만 연령증가, 비만도 증가, 육체활동 감소 등 많은 환경적 요인과 합쳐져 발병이 된다.Diabetes is a metabolic disease caused by an absolute or relative lack of insulin and is divided into type 1 diabetes and type 2 diabetes depending on the cause of the onset. Type 1 diabetes is a disease caused by the lack of insulin itself and can be divided into immunomediated and idiopathic. The destruction of pancreatic beta cells due to autoimmune reactions is called immunomediation, which accounts for 5-10% of all diabetes. Diabetes of unknown type 1 diabetes is idiopathic. Type 1 diabetes requires the treatment of insulin and can be taken with oral hypoglycemic agents as needed. Type 2 diabetes is a disease caused by increased resistance to insulin receptors or decreased insulin sensitivity. There are genetic factors, but it is combined with many environmental factors such as age, obesity, and decreased physical activity.
당뇨병은 질병 그 자체보다 합병증으로 인해 문제가 되는데, 당뇨의 급성 합병증으로는 고혈당으로 인해 혼수상태에 빠지게 되는 고혈당성 혼수, 케톤산이 과 축적되어 생기는 케톤산혈증, 인슐린이나 혈당강하제의 잘못된 사용으로 인해 혈당이 너무 낮아지는 저혈당을 크게 들 수 있다. 만성 합병증으로는 고혈당으로 인해 혈액 순환이 잘 되지 않아 생기는 당뇨병성 망막증, 신증, 신경병증, 족부질환 등이 있다. Diabetes is more problematic due to complications than the disease itself.Acute complications of diabetes include hyperglycemic coma that leads to coma due to hyperglycemia, ketoacidosis due to overaccumulation of ketone acid, and blood sugar due to incorrect use of insulin or hypoglycemic agents. The hypoglycemia that is too low can be mentioned largely. Chronic complications include diabetic retinopathy, nephropathy, neuropathy, and foot problems caused by poor blood circulation due to hyperglycemia.
또한 당뇨병은 당을 제대로 이용하지 못해 일어나는 당 대사 관련 장애뿐만 아니라 지질대사에도 이상을 초래하기 때문에 당뇨병은 심혈관계 합병증의 위험 또한 증가시키는 대사성 질환이다. 당뇨병과 관계된 심혈관계 합병증의 종류로는 고혈압과 관상동맥질환, 뇌졸중, 말초동맥질환, 심근증, 심부전 등이 있다.In addition, diabetes is a metabolic disease that increases the risk of cardiovascular complications because it causes abnormalities in lipid metabolism as well as glucose metabolism-related disorders caused by inadequate use of sugar. Cardiovascular complications related to diabetes include hypertension, coronary artery disease, stroke, peripheral artery disease, cardiomyopathy, and heart failure.
일반적으로 1형 당뇨의 치료에는 인슐린 제제를 투여하며, 2형 당뇨에는 경구혈당강하제를 사용한다. 경구혈당강하제의 종류로는 인슐린의 분비를 자극하며 인슐린 저항성을 개선하는 sulfonylurea계, 인슐린 분비를 증가시키며 글루카곤 분비를 억제하는 dipeptidyl peptidase-4(DPP-4) inhibitor계, 간의 당 신생을 억제하며 인슐린 저항성을 개선하는 biguanide계, 말초 조직과 간에서 인슐린 저항성을 감소시키는 thiazolidinedione(TZD)계, 포도당 흡수를 지연시키는 α-glucosidase 저해제 등이 있다. 경구혈당강하제는 작용기전에 따라 종류가 다양하기 때문에 당뇨의 분류에 따라 정해져 있는 것이 아니라 당뇨병이 걸린 시기와 진행 정도에 따라 인슐린제제와 경구혈당강하제를, 또는 경구혈당강하제끼리 병용해서 사용하는 경우가 많다. 당뇨병이 발병하는 이유는 다양하므로 각각의 병인에 근거한 새로운 약물의 개발을 위해 많은 연구가 진행되고 있다. 또한 당뇨병은 합병증으로 인한 문제가 크기 때문에, 그 합병증을 개선시킬 수 있는 약물의 개발은 당뇨병 치료 중 중요한 부분이다.In general, insulin treatment is administered for the treatment of type 1 diabetes, and oral hypoglycemic agents are used for type 2 diabetes. Types of oral hypoglycemic agents include sulfonylureas, which stimulate insulin secretion and improve insulin resistance, dipeptidyl peptidase-4 (DPP-4) inhibitors that increase insulin secretion and inhibit glucagon secretion, and inhibit hepatic angiogenesis. Biguanides improve resistance, thiazolidinedione (TZD) decreases insulin resistance in peripheral tissues and liver, and α-glucosidase inhibitors that delay glucose absorption. Oral hypoglycemic agents vary depending on the mechanism of action, so they are not determined according to the type of diabetes, but insulin and oral hypoglycemic agents, or oral hypoglycemic agents, are often used in combination with diabetes mellitus. . The reasons for the development of diabetes vary, and many studies are being conducted to develop new drugs based on the etiology of each. In addition, since diabetes is a problem due to complications, the development of drugs that can improve the complications is an important part of diabetes treatment.
그러나 지금까지 개발된 약물들은 저혈당증, 인슐린 분비능 상실, 위장장애, 신장독성, 설사와 배탈, 심부전, 빈혈, 간부전 등을 유발하는 부작용이 보고되어 왔다. 또한 일부 약물은 심각한 간독성 및 심혈관계 부작용으로 사용이 금지된 경우도 있다. 혈당강하작용이 있다고 알려진 새로운 물질 중 몇몇 약제는 고가의 생산비 등으로 인해 현재 임상에서의 이용이 어려우므로, 단독 혹은 약물을 도와 당뇨의 증상을 낮추는 천연물 유래 신약 혹은 건강기능성 식품의 개발이 필요하다.However, the drugs developed so far have been reported side effects such as hypoglycemia, loss of insulin secretion, gastrointestinal disorders, kidney toxicity, diarrhea and upset stomach, heart failure, anemia, liver failure. In addition, some drugs are banned due to severe hepatotoxicity and cardiovascular side effects. Some new drugs known to have hypoglycemic effects are currently difficult to use in clinical trials due to high production costs. Therefore, it is necessary to develop new drugs or health foods derived from natural products that lower the symptoms of diabetes alone or by helping drugs.
잠상산물은 뽕나무와 관련된 산물을 말하며 누에, 오디, 상엽, 상지, 상백피 등이 있다. 잠상산물은 예로부터 당뇨를 비롯한 여러 가지 질병에 효과가 있다고 알려져 왔다. 동의보감에 오디가 당뇨에 효과가 있다고 기록되어있으며, 상지는 풍과 이뇨에, 상엽은 몸이 붓는 증세에 효과가 있다고 기록되어있다. 또한 본초강목에서는 상엽, 상백피, 오디, 잠분에서 당뇨병에 효과가 있다고 기록되어 있으며 상엽, 상지, 잠분은 또한 풍에 효과가 있다고 기록되어 있다.The latent product refers to products related to mulberry trees, and includes silkworms, mulberry, upper leaves, upper limbs, and lettuce. Latent products have long been known to be effective in various diseases including diabetes. It is recorded that the auditory effect is effective for diabetes, and the upper limb is effective for wind and diuresis, and the upper lobe is effective for body swelling. In addition, the herbaceous wood has been reported to have an effect on diabetes in the upper lobe, upper and lower skin, mulberry, and Jambun, and the upper leaf, upper limb and Jambun are also reported to be effective in wind.
지금까지 연구된 동물모델에서 잠상산물의 항당뇨 효과를 보면 누에는 streptozotocin(STZ) 유도 당뇨쥐에서 혈청 포도당 농도를 낮추고 -glucosidase 억제 효과를 나타냈으며, 소장 proximal 부분의 이당류 분해효소 활성 감소 효과를 나타내었다고 보고되었다. 오디는 STZ 유발 당뇨쥐에서 혈당을 낮추며 혈청 중성지방 (triglyceride, TG) 농도, 총 콜레스테롤 (total-cholesterol, T-CHO) 농도, 저밀도지단백질 (low density lipoprotein cholesterol, LDL-CHO) 농도, thiobarbituric acid reactive substance(TBARS) 함량을 낮춘다고 보고되었다. 뽕나무의 잎인 상엽은 STZ 유발 당뇨쥐에서 공복혈당과 당화헤모글로빈 농도의 감소 및 소장의 이당류분해효소 활성 감소가 보고되었다. 또한 TG 농도와 T-CHO 농도, 저밀도지단백질 농도, TBARS 함량이 감소하였으며 고밀도지단백질 (high density lipoprotein cholesterol, HDL-CHO) 농도가 증가하였다는 보고가 있었다.The antidiabetic effects of latent products in the animal models studied so far showed lower glucose concentration and -glucosidase inhibitory effect in streptozotocin (STZ) -induced diabetic rats, and reduced disaccharide degrading enzyme activity in the small intestine proximal. Was reported. Audi lowers blood sugar levels in STZ-induced diabetic rats with serum triglyceride (TG) levels, total cholesterol (total-cholesterol (T-CHO)) levels, low density lipoprotein cholesterol (LDL-CHO) levels, and thiobarbituric acid reactive It has been reported to lower the substance (TBARS) content. The leaves of the mulberry, which have been reported to reduce fasting glucose and glycated hemoglobin concentrations and decreased intestinal disaccharide enzyme activity in STZ-induced diabetic rats. In addition, TG concentration, T-CHO concentration, low density lipoprotein concentration and TBARS content were decreased, and high density lipoprotein cholesterol (HDL-CHO) concentration was reported.
한편, 지금까지 보고된 상지 및 상백피의 항당뇨 효능에 관한 연구를 보면 먼저 상지 물추출물은 소장의 α-glucosidase 활성을 크게 억제하였으며, alloxan 유발 당뇨쥐에서 공복혈당을 크게 낮추었는 데 그 효과는 α-glucosidase 강력저해제로서 항당뇨약으로 잘 알려진 acarbose 만큼의 효능이 있는 것으로 보고된 바가 있다. 그리고 최근 본 연구진의 연구에 의하면 뽕나무 부위 중 상지 물추출물이 다른 부위보다 α-glucosidase 활성을 크게 억제하는 것으로 확인된 바가 있다. 이러한 상지 물추출물의 항당뇨 효능은 추출물에 존재하는 다당류가 췌장에서의 산화적스트레스나 염증 반응을 억제함으로써 나타나는 것으로 보고된 바가 있으며, 그리고 상지추출물은 streptozotocin(STZ) 유발 당뇨쥐의 체중 및 인슐린 증가를 유도함으로써 제2형 당뇨치료제로 잘 알려진 metformin 만큼의 효능을 나타냄을 확인한 바가 있다.On the other hand, studies on the antidiabetic effect of the upper limbs and epithelial epithelium reported so far showed that the upper limb extract significantly inhibited α-glucosidase activity of the small intestine and significantly lowered fasting blood glucose in alloxan-induced diabetic rats. As a glucosidase inhibitor, it has been reported to be as effective as acarbose, an antidiabetic drug. Recently, according to the research of the researchers, it was confirmed that the water extract of the upper limbs of the mulberry tree inhibits α-glucosidase activity more than other sites. The antidiabetic effect of these water extracts has been reported to be due to the inhibition of oxidative stress or inflammatory responses in the pancreas by the polysaccharides present in the extract, and the extracts from the streptozotocin (STZ) -induced diabetic rats. By inducing it has been confirmed to show the efficacy as metformin well known as a type 2 diabetes treatment.
한편, 최근 상지로부터 옥시레스베라트롤 유도체(배당체인 mulberroside A와 아글리콘인 oxyresveratrol), 레스베라트롤, 및 2-arylbenzofuran 유도체가 확인된 바가 있으며, 이 중 옥시레스베라트롤은 상지의 주된 생리활성물질로서 지금까지 항염증, 항멜라닌, 및 항산화성분으로 보고된 바가 있다. 그리고 상지에도 존재하며, 특히 상백피의 주된 생리활성물질로 보고된 mulberroside A 성분은 최근 alloxan 유발 당뇨쥐 모델에서 항당뇨 효능이 보고된 바가 있다. 이와같이 지금까지 상지 물추출물 및 주된 성분인 mulberroside A의 항당뇨 작용이 보고된 바가 있으나 거의 대부분 제2형 당뇨모델에서의 항당뇨 효능을 밝힌 연구이었으며, 본 연구에서와 같이 STZ을 여러번 처리하여 유발된 제1형 당뇨모델에서의 상지 물추출물 및 oxyresveratrol의 항당뇨 효능과 그 기전에 관한 연구보고는 아직 없다.On the other hand, oxyresveratrol derivatives (glycosides mulberroside A and aglycone oxyresveratrol), resveratrol, and 2-arylbenzofuran derivatives have recently been identified from the upper limbs. It has been reported as an anti-melanin, and an antioxidant component. In addition, mulberroside A, which is also present in the upper limbs, has been reported to have anti-diabetic effects in alloxan-induced diabetic rats. As described above, the antidiabetic effect of the water extract of the upper limbs and mulberroside A, the main component, has been reported, but most of the studies have revealed the antidiabetic effect in the type 2 diabetes model. There have been no studies on the antidiabetic efficacy and mechanism of oxyresveratrol in water extract and type 1 diabetes in type 1 diabetes model.
본 발명과 관련된 선행문헌으로는 대한민국 등록특허 제10-1438543호에 공지되어 있으나 이는 항염증, 항노화 기능성 옥시레스베라트롤, 트란스-레스베라트롤 및 모라신이 함유된 뽕나무 가지추출물의 제조방법에 관한 것이다. 그러나 현재까지 상지 열수추출물 및 oxyresveratrol(이하 ORT라 한다)의 항당뇨 기능성 조성물에 관련된 발명은 공지된 바 없다.Prior art related to the present invention is known in the Republic of Korea Patent No. 10-1438543, but it relates to a method for producing a mulberry branch extract containing anti-inflammatory, anti-aging functional oxyresveratrol, trans- resveratrol and Moracin. However, there is no known invention related to the antidiabetic functional composition of the upper limb hydrothermal extract and oxyresveratrol (hereinafter referred to as ORT).
따라서 본 발명의 목적은 당뇨병에 효과가 있는 상지 열수추출물 및 지표성분 ORT를 유효성분으로 함유하는 조성물 및 그 제조 방법을 제공하는 데 있다.Accordingly, it is an object of the present invention to provide a composition containing the upper limb hydrothermal extract and the indicator component ORT as an effective ingredient and a method for producing the same which are effective for diabetes.
본 발명은 건조한 상지 200g 및 상백피 100g에 이온수 1L를 가하여 초음파추출기에서 가장 바람직하게는 2시간 동안 추출하는 단계와; 상기 단계에서 추출한 상지 및 상백피 추출액을 감압 농축하여 각각 상지 및 상백피 열수 추출물을 얻는 단계와; The present invention comprises the steps of extracting 200 g of dried upper limbs and 100 g of baekryepi and 1L of ionized water, most preferably in an ultrasonic extractor for 2 hours; Concentrating the extracted upper limb and lettuce extract under reduced pressure under reduced pressure to obtain an extract of the upper limb and lettuce extract;
상지를 80% 에탄올수용액으로 2회 초음파추출한 후 여과 및 감압농축하는 단계와; 상기 단계에서 감압농축한 80% 에탄올추출물을 다시 80% 에탄올에 녹인 후 헥산으로 분획하여 얻은 탈지 에탄올추출물을 감압농축하는 단계와; 상기 단계에서 감압농축한 탈지 에탄올추출물에 가수한 다음 CH2Cl2, EtOAC 및 n-BuOH 용매로 순차적으로 분획하여 EtOAC 분획에서 oxyresveratrol(ORT)을, n-BuOH 분획에서 oxyresveratrol 3‘,4-O-β-D-diglucopyranoside(ORTG)를 각각 분리 및 정제하는 단계를 통해 얻은 oxyresveratrol의 항당뇨 기능성을 streptozotocin(STZ) 유도 당뇨쥐에서 검증하고 이를 평가함으로써 달성하였다.Ultrasonically extracting the upper limb twice with an aqueous 80% ethanol solution, and then filtration and concentration under reduced pressure; Concentrating the degreasing ethanol extract obtained by dissolving the 80% ethanol extract concentrated under reduced pressure in 80% ethanol again and then fractionated with hexane; In the above step, the ethanol extract was concentrated under reduced pressure and hydrolyzed, and then sequentially fractionated with CH2Cl2, EtOAC and n-BuOH solvent, oxyresveratrol (ORT) in the EtOAC fraction, and oxyresveratrol 3 ', 4-O-β- in the n-BuOH fraction. The antidiabetic function of oxyresveratrol obtained by separating and purifying D-diglucopyranoside (ORTG), respectively, was achieved by evaluating and evaluating it in streptozotocin (STZ) -induced diabetic rats.
본 발명에 따른 뽕나무로부터 채취 및 추출한 상지 열수추출물 및 ORT를 유효성분으로 함유하는 당뇨병 예방 및 개선 기능성 의약품 및 식품 조성물을 제공할 수 있는 뛰어난 효과가 있다.There is an excellent effect that can provide a diabetes prevention and improvement functional medicines and food compositions containing the upper limbs hot water extract and ORT extracted from the mulberry according to the present invention as an active ingredient.
본 발명에 따르면 상기 상지 열수추출물은 상백피 열수추출물과 비교했을 때 항당뇨 효능이 더 우수한 의약품 및 식품 조성물을 제공할 수 있는 뛰어난 효과가 있다.According to the present invention, the upper extremity hot water extract has an excellent effect of providing a pharmaceutical and food composition with superior antidiabetic efficacy when compared with the extract of hot water extract.
도1은 상지 열수추출물, 상백피 열수추출물 및 ORT의 HPLC 결과를 나타낸 그래프이다.Figure 1 is a graph showing the HPLC results of the upper liquor hot water extract, baekryepi hot water extract and ORT.
도2는 본 발명에 따른 상지 에탄올추출물로부터 두 가지 기능성 지표성분(ORT & ORTG)의 분리 및 정제방법을 나타낸 모식도이다.Figure 2 is a schematic diagram showing the separation and purification method of the two functional indicators (ORT & ORTG) from the upper liquor ethanol extract according to the present invention.
도3은 본 발명에 따른 당뇨 유발을 위해 실험동물에 STZ 투여 후 MT 및 MRB 투여를 나타낸 모식도이다.Figure 3 is a schematic diagram showing the MT and MRB administration after the STZ administration to the induction of diabetes mellitus according to the present invention.
도4는 본 발명에 따른 당뇨 유발을 위해 실험동물에 STZ 투여 후 ORTG, ORT 및 Met 투여를 나타낸 모식도이다.Figure 4 is a schematic diagram showing the ORTG, ORT and Met administration after STZ administration to experimental animals for diabetes induction according to the present invention.
도5는 본 발명에 따른 당뇨 유발 실험동물의 공복혈당 측정값을 나타낸 그래프이다.Figure 5 is a graph showing the fasting blood glucose measurement of the diabetes-induced experimental animal according to the present invention.
도6은 본 발명에 따른 당뇨 유발 실험동물의 혈장 포도당 및 인슐린 함량의 측정값을 나타낸 그래프이다.Figure 6 is a graph showing the measurement of plasma glucose and insulin content of the diabetes-induced experimental animals according to the present invention.
도7은 본 발명에 따른 당뇨 유발 실험동물에 상지 열수추출물 투여에 따른 지질대사 변화량을 나타낸 그래프이다.Figure 7 is a graph showing the change in lipid metabolism according to the administration of hot water extracts to the diabetes-induced experimental animals according to the present invention.
도8은 본 발명에 따른 당뇨 유발 실험동물에 상지 열수추출물 투여 후 triglyceride 함량을 나타낸 그래프이다.8 is a graph showing triglyceride content after administration of hot water extracts of the upper limbs to a diabetic-induced experimental animal according to the present invention.
도9는 본 발명에 따른 당뇨 유발 실험동물에 상지 열수추출물 투여 후 free fatty acid 함량의 측정값을 그래프이다.9 is a graph showing the measured value of free fatty acid content after administration of hot water extracts of the upper limbs to the diabetic-induced experimental animal according to the present invention.
도10은 본 발명에 따른 당뇨 유발 실험동물에 상지 열수추출물 투여 후 간 조직에서 triglyceride 및 지질대사 변화량을 나타낸 그래프이다.10 is a graph showing changes in triglyceride and lipid metabolism in liver tissues after administration of hot water extracts of the upper limbs to a diabetic-induced experimental animal according to the present invention.
도11은 본 발명에 따른 당뇨 유발 실험동물에 상지 열수추출물 투여 후 소장 점막의 이당류분해효소 활성을 측정한 결과를 나타낸 그래프이다.Figure 11 is a graph showing the results of measuring the disaccharide enzyme activity of the small intestine mucosa after administration of hot water extracts to the diabetes-induced experimental animals according to the present invention.
도12는 본 발명에 따른 당뇨 유발 실험동물에 상지 지표성분인 ORT 투여 후 혈중 공복혈당 측정값을 나타낸 그래프이다.12 is a graph showing the measured blood fasting glucose levels after administration of ORT, which is an indicator of the upper limbs, in the diabetes-induced experimental animal according to the present invention.
도13은 본 발명에 따른 당뇨 유발 실험동물에 ORT 투여 후 혈장 포도당 및 인슐린 함량의 측정값을 나타낸 그래프이다.Figure 13 is a graph showing the measured value of plasma glucose and insulin content after ORT administration to the diabetic animal-derived experimental animal according to the present invention.
도14는 본 발명에 따른 당뇨 유발 실험동물에 ORT 투여 후 지질대사 변화량을 나타낸 그래프이다.Figure 14 is a graph showing the change in lipid metabolism after administration of ORT to the diabetes-induced experimental animals according to the present invention.
도15는 본 발명에 따른 당뇨 유발 실험동물에 ORT 투여 후 혈장에서 free fatty acid 함량의 측정값을 나타낸 그래프이다.15 is a graph showing the measured value of free fatty acid content in plasma after ORT administration to a diabetic animal-derived experimental animal according to the present invention.
도16은 본 발명에 따른 당뇨 유발 실험동물에 ORT 투여 후 소장 점막의 이당류분해효소 활성을 나타낸 그래프이다.Figure 16 is a graph showing the disaccharide enzyme activity of the small intestine mucosa after the ORT administration to the diabetic induced animal according to the present invention.
도17은 본 발명에 따른 당뇨 유발 실험동물에 ORT 투여 후 간 조직 GLUT2 mRNA 발현량의 측정값을 나타낸 그래프이다.Figure 17 is a graph showing the measurement value of liver tissue GLUT2 mRNA expression after ORT administration to the diabetes-induced experimental animals according to the present invention.
도18은 본 발명에 따른 당뇨 유발 실험동물에 ORT 투여 후 간 조직 글리코겐 함량의 측정값을 나타낸 그래프이다.Figure 18 is a graph showing the measurement value of liver tissue glycogen content after ORT administration to a diabetic-induced experimental animal according to the present invention.
도19는 본 발명에 따라 상지 열수추출물 및 ORT 투여 시 나타나는 당뇨 억제 효과를 나타낸 모식도이다.Figure 19 is a schematic diagram showing the effect of inhibiting diabetes appeared when the hot water extract and ORT administration in accordance with the present invention.
본 발명은 상지 열수추출물 및 ORT를 유효성분으로 함유하는 당뇨병 예방 및 치료용 의약품 및 식품 조성물에 관한 것이다.The present invention relates to a pharmaceutical and food composition for preventing and treating diabetes containing the upper limb hydrothermal extract and ORT as an active ingredient.
본 발명에 따르면 상기 상지 열수추출물은 상백피 열수추출물보다 mulberroside A 및 ORT의 함량이 2~3배 높다.According to the present invention, the upper limb hydrothermal extract has a content of mulberroside A and ORT 2 to 3 times higher than that of the hot birch extract.
본 발명에 따르면 상기 상지 열수추출물은 상백피 열수추출물보다 ORT 함량은 높은 반면, ORTG의 함량은 매우 낮다[상지 열수추출물: ORTG(2.45±0.06%), ORT(0.92±0.02%); 상백피 열수추출물: ORTG(19.71±2.80%), ORT(0.04±0.01%)]According to the present invention, the upper limb hydrothermal extract has a higher ORT content than the hot water extract, but the content of ORTG is very low [upper lichen hydrothermal extract: ORTG (2.45 ± 0.06%), ORT (0.92 ± 0.02%); Lepidoptera hot water extract: ORTG (19.71 ± 2.80%), ORT (0.04 ± 0.01%)]
본 발명에 따르면 STZ를 이용한 당뇨 유발 마우스에서 상백피 열수추출물보다 상지 열수추출물 및 그 지표성분인 ORT 투여시 항당뇨 효과가 더 뛰어난 것이 특징인 의약품 및 식품 조성물에 관한 것이다. The present invention relates to a pharmaceutical composition and a food composition characterized by superior antidiabetic effect when the upper limb hydrothermal extract and ORT administered as an index component thereof are higher than hot water extract in diabetic-induced mice using STZ.
본 발명의 상기 상지 추출물은 물, 에탄올, 메탄올과 같은 친수성 유기용매 또는 이들의 혼합 용매에 의해 추출된 것일 수 있으며, 가장 바람직하게는 열수를 이용하여 추출된 열수추출물이다.The upper limb extract of the present invention may be extracted by a hydrophilic organic solvent such as water, ethanol, methanol, or a mixed solvent thereof, most preferably a hot water extract extracted using hot water.
또, 본 발명의 상기 ORT는 상지 열수추출물로부터 분리시 가장 바람직하게는 EtOAC로 분리하는 것이다.In addition, the ORT of the present invention is most preferably separated by EtOAC when separated from the hot water extract.
본 발명에 따른 상기 식품 조성물은 음료, 껌, 차 및 선식 중 어느 하나의 제형인 것이 특징인 식품조성물을 제공한다.The food composition according to the present invention provides a food composition, characterized in that the formulation of any one of beverage, gum, tea, and wire.
본 발명에 따른 상기 조성물은 정제, 환제, 산제, 과립제, 캅셀제, 현탁제, 유제, 시럽제, 비수성용제, 현탁제 및 동결건조제제로 이루어진 군으로부터 선택되는 어느 하나의 제형을 가지는 것이 특징인 약학적 조성물을 제공한다.The composition according to the present invention is a pharmaceutical composition characterized in that it has any one formulation selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, emulsions, syrups, non-aqueous solvents, suspensions and lyophilized preparations. To provide.
본 발명의 조성물은 약제학적 조성물 형태로 제조할 수 있으며, 하나 또는 그 이상의 무독성 및 약제학적으로 허용 가능한 담체, 부형제, 보조제 또는 희석제 또는 다른 활성성분을 추가로 더 포함할 수 있다.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.
본 발명의 상기 상지 및 상백피는 통상 시중에서 구입할 수 있다. The upper limbs and limbs of the present invention are usually commercially available.
본 발명을 실시예, 실험예 및 제조예에 따라 더욱 상세하게 설명한다. 그러나 하기 실시예 및 실험예는 본 발명을 예시하기 위한 것에 불과하므로 본 발명의 권리범위를 한정하는 것으로 의도되지는 않는다.The present invention will be described in more detail according to Examples, Experimental Examples and Preparation Examples. However, the following examples and experimental examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention.
본 발명의 제조예1 내지 제조예4에는 가장 바람직하게는 상지 열수추출물 또는 ORT 함유량의 수치를 1중량%로 한정하였으나 0.5~2.5중량%까지 첨가하여도 좋다.In Production Examples 1 to 4 of the present invention, most preferably, the numerical value of the upper limb hydrothermal extract or ORT content is limited to 1% by weight, but may be added up to 0.5 to 2.5% by weight.
표 1
제조예1. 상지 열수추출물 또는 ORT를 함유하는 음료
성분 함량(중량%)
꿀 0.3
치옥토산아미드 0.0003
니코틴산아미드 0.0003
염산리보플라빈나트륨 0.0001
염산피리독신 0.0001
이노시톨 0.001
오르트산 0.002
물 98.6962
상지 열수추출물 또는 ORT 1
합계 100
Table 1
Preparation Example 1. Beverage containing upper limb hot water extract or ORT
ingredient Content (% by weight)
honey 0.3
Chioctosanamide 0.0003
Nicotinic acid amide 0.0003
Riboflavin Sodium Hydrochloride 0.0001
Pyridoxine hydrochloride 0.0001
Inositol 0.001
Ortsan 0.002
water 98.6962
Upper limb hydrothermal extract or ORT One
Sum
100
표 2
제조예2. 상지 열수추출물 또는 ORT를 함유하는 껌
성분 함량(중량%)
껌 베이스 20
설탕 75
향료 2
물 2
상지 열수추출물 또는 ORT 1
합계 100
TABLE 2
Preparation Example 2. Gum containing upper limb hydrothermal extract or ORT
ingredient Content (% by weight)
Gum base 20
Sugar 75
Spices 2
water 2
Upper limb hydrothermal extract or ORT One
Sum
100
표 3
제조예3. 상지 열수추출물 또는 ORT를 함유하는 차
성분 함량(중량%)
함수포도당 96.8
구연산 0.05
비타민c 0.15
자일리톨 1
HPMC 1
상지 열수추출물 또는 ORT 1
합계 100
TABLE 3
Preparation Example 3. Tea containing upper limb hydrothermal extract or ORT
ingredient Content (% by weight)
Functional glucose 96.8
Citric acid 0.05
Vitamin c 0.15
Xylitol One
HPMC One
Upper limb hydrothermal extract or ORT One
Sum
100
표 4
제조예4. 상지 열수추출물 또는 ORT를 함유하는 선식
성분 함량(중량%)
현미 30
율무 20
보리 20
찹쌀 7
들깨 7
검정콩 7
검정깨 7
영지 0.5
지황 0.5
상지 열수추출물 또는 ORT 1
합계 100
Table 4
Preparation Example 4. Wire type containing upper limb hot water extract or ORT
ingredient Content (% by weight)
Brown rice 30
Rule 20
barley 20
Glutinous rice 7
Perilla 7
Black Beans 7
Black sesame 7
wisdom 0.5
Yellow 0.5
Upper limb hydrothermal extract or ORT One
Sum
100
실시예1. 상지로부터 ORTG 및 ORT의 추출, 분리 및 정제Example 1 Extraction, Separation and Purification of ORTG and ORT from Upper Limbs
상지는 봄에 잎이 내돋지 않은 뽕나무로부터 채취 후 1주일간 음건한 다음 50℃ 이하 열풍건조기(COBP-15S, Sjunheung dryer, Boryyeong, Korea)에서 건조한 것을 공시재료로 사용하였다. 상기 건조한 상지로부터 두 가지 기능성 지표성분의 분리를 위해 상지를 초음파 추출기를 이용하여 80% 에탄올수용액으로 두번 추출하여 여과 및 감압농축 하였다. 상기 단계의 감압농축한 탈지 에탄올추출물을 물에 녹인 다음 CH2Cl2, EtOAC 및 n-BuOH 용매를 이용하여 순차적으로 분획하였다. EtOAC 분획으로부터 oxyresveratrol(ORT)을, n-BuOH 분획으로부터는 oxyresveratrol 3‘,4-O-β-D-diglucopyranoside(ORTG)를 각각 분리하여 정제하였다(도2).The upper limbs were dried for one week after harvesting from mulberry trees without leaves in spring and then dried in a hot air dryer (COBP-15S, Sjunheung dryer, Boryyeong, Korea) below 50 ° C. In order to separate the two functional indicators from the dried upper limbs, the upper limbs were extracted twice with an 80% ethanol solution using an ultrasonic extractor, and filtered and concentrated under reduced pressure. The degreased ethanol extract concentrated in the above step was dissolved in water, and then fractionated sequentially using CH 2 Cl 2, EtOAC, and n-BuOH solvent. Oxyresveratrol (ORT) from the EtOAC fraction and oxyresveratrol 3 ', 4-O-β-D-diglucopyranoside (ORTG) from the n-BuOH fraction was isolated and purified (Fig. 2).
실험예1. 상지 및 상백피 열수추출물의 지표성분 ORT의 HPLC 분석Experimental Example 1. HPLC analysis of the index component ORT of the upper limbs and epidermis hydrothermal extract
상기 실시예1의 방법으로 추출한 상지 및 상백피 열수추출물의 지표성분 ORT 및 ORTG의 HPLC 분석을 위해 Merck HPLC grade(Darmastadt, Germany)를 이용하여 분석하였다. Merck HPLC grade (Darmastadt, Germany) was analyzed for HPLC analysis of the index components ORT and ORTG of the upper limbs and epithelium hot water extract extracted by the method of Example 1.
이를 평가하기 위해 상지 및 상백피 열수추출물(1 g)을 증류수 100mL로 희석하고 0.45㎛ 필터로 여과 후 HPLC에 10㎕ 주입하여 정량분석하였으며, 이때 HPLC 분석은 Waters e2690.5 HPLC 고속액체크로마토그래피, 칼럼; YMC-Pack Pro C18, 용매A(0.05% H3PO4 in H2O), 용매B(CH3CN), 유속; 0.8mL/min 조건으로 수행하였다. 상기 HPLC로 측정한 상지 및 상백피 열수추출물의 두 가지 기능성 지표성분 ORT 및 ORTG는 표준물질의 보지시간(retention time)과 비교 분석하였으며, 두 추출물의 함량은 2개의 표준물질의 검량곡선으로부터 산출하였다.To evaluate this, the upper limbs and epithelium hot water extracts (1 g) were diluted with 100 mL of distilled water, filtered through a 0.45 μm filter, and then injected into 10 μl of HPLC, where HPLC analysis was performed using Waters e2690.5 HPLC high performance liquid chromatography, column. ; YMC-Pack Pro C18, Solvent A (0.05% H3PO4 in H2O), Solvent B (CH3CN), flow rate; It was performed at 0.8 mL / min conditions. The two functional indicators ORT and ORTG of the upper limbs and epithelium hot water extracts measured by HPLC were analyzed in comparison with the retention time of the standard, and the contents of the two extracts were calculated from the calibration curves of the two standard.
두 가지 표준물질(A), 상지 열수추출물(B) 및 상백피 열수추출물(C)의 ORTG(1) 및 ORT(2)의 측정 결과는 도1에 나타냈다.The measurement results of ORTG (1) and ORT (2) of two standard materials (A), upper limb hydrothermal extract (B) and epidermis hydrothermal extract (C) are shown in FIG.
실시예2. 실험동물Example 2. Laboratory animals
상지 및 상백피 열수추출물의 항당뇨 효과 실험에 사용된 실험동물은 체중 29±0.2g의 수컷, 6주령 ICR 마우스를 코아텍 바이오에서 구입하여 1주 동안 고형사료를 급여하며 실험환경에 적응시켰다. 실험동물은 정상대조군(Normal control, NC) 10수, 당뇨대조군(diabetes control, DC) 10수, 상지 열수추출물 투여한 상지당뇨군(Mulberry twig, MT) 10수 및 상백피 열수추출물 투여한 상백피당뇨군(Mulberry root bark, MRB) 10수로 분류하였다. 상기 정상대조군 및 당뇨대조군은 멸균식염수를 투여하였고 상지당뇨군은 상지 열수추출물을 5g/kg의 농도로 투여하였고 상백피당뇨군은 상백피 열수추출물을 600mg/kg 농도로 투여하였다.The experimental animals used for the antidiabetic effect of the upper limbs and epithelial hot water extract were male and 6-week-old ICR mice weighing 29 ± 0.2 g and fed solid feed for 1 week and adapted to the experimental environment. The experimental animals were 10 normal controls (NC), 10 diabees control (DC), 10 upper limbs (Mulberry twig (MT)) administered with the upper limbs hot water extract, and an epithelial diabetic group treated with hot water extract. (Mulberry root bark, MRB) was divided into 10 numbers. The normal control group and the diabetic control group were treated with sterile saline, the upper limb diabetic group was administered with a concentration of 5 g / kg of the upper extremity hot water extract, and the epithelial diabetic group was administered with an epithelial hot water extract at 600 mg / kg.
상지 지표성분의 항당뇨 효과 실험에 사용된 실험동물은 체중 33±0.3g의 수컷, 6주령 ICR 마우스를 사용하였다. 실험동물은 멸균식염수를 투여한 정상대조군(Normal control, NC) 7수, 멸균식염수를 투여한 당뇨대조군(Diabetes control, DC) 7수, ORTG를 600mg/kg의 농도로 투여한 당뇨군 10수, ORT을 600mg/kg의 농도로 투여한 당뇨군 10수 및 metformin을 600mg/kg의 농도로 투여한 당뇨군(Metformin, Met) 10수로 나뉘어 실험을 하였다.The experimental animals used for the antidiabetic effect of the upper limb markers were male and 6 week old ICR mice weighing 33 ± 0.3 g. The experimental animals were 7 normal controls (NC) administered sterile saline, 7 diabetic controls (DC) administered sterile saline, 10 diabetic groups administered ORTG at a concentration of 600 mg / kg, The experiment was divided into 10 diabetic groups administered ORT at a concentration of 600 mg / kg and 10 diabetic groups (Metformin, Met) administered at a concentration of 600 mg / kg.
실시예3. 당뇨유발Example 3. Diabetes
상기 실시예2의 실험동물의 당뇨를 유발시키기 위해 STZ를 0.1M citrate buffer(pH 4.5)에 녹여 50mg/kg/day의 용량으로 복강에 5일간 일정한 시간에 주사하였다. STZ 투여 시작일로부터 14일 후 미정맥에서 채혈하여 혈당을 확인하여 당뇨 유발을 확인하였다. 상기 당뇨 유발이 확인된 실험동물에 실험 시작 15일 후부터 16일 동안 상지 당뇨군(MT)에 상지 열수추출물을 5g/kg 경구 투여하였고 상백피 당뇨군(MRB)에 상백피 열수추출물을 6g/kg 경구 투여하였다(도3). 시료 투여기간이 지난 후 동물을 희생하여 혈액, 간, 가자미근, 부고환지방 및 소장을 채취하여 시료분석에 사용하였다.In order to induce diabetes in the experimental animal of Example 2, STZ was dissolved in 0.1M citrate buffer (pH 4.5) and injected into the abdominal cavity at a constant time for 5 days at a dose of 50 mg / kg / day. After 14 days from the start of STZ administration, blood was collected from the vein of the vein to check the blood glucose to confirm the induction of diabetes. The test animal was confirmed to be induced diabetes orally oral administration of upper extremity hot water extract 5g / kg to the upper extremity diabetic group (MT) for 15 days from the start of the experiment 16 days or 6g / kg oral administration of hot water extract to the epithelial diabetic group (MRB) (Fig. 3). After the sample administration period, animals were sacrificed and blood, liver, soleus muscle, epididymal fat and small intestine were collected and used for sample analysis.
실시예4. 상지 지표성분 항당뇨 효과Example 4. Upper limb index component antidiabetic effect
상지 지표성분의 항당뇨 효과를 확인하기 위해 상기 실시예2의 실험동물에 STZ를 40mg/kg/day의 용량으로 5일간 복강주사 하였으며, STZ 투여 시작일로부터 17일 후 미정맥에서 채혈하여 혈당 상승을 확인하였다. 상기 단계의 혈당 상승을 확인한 실험동물을 실험 시작 18일 후부터 22일간 ORTG, ORT 및 Met을 각각 0.6g/kg의 농도로 경구 투여하였다(도4). 시료 투여 후 동물을 희생시켜 혈액, 간, 췌장 및 소장을 채취하여 시료분석에 사용하였다.In order to confirm the antidiabetic effect of the upper extremity indicator component, the experimental animals of Example 2 were intraperitoneally injected with STZ at a dose of 40 mg / kg / day for 5 days. Confirmed. Experimental animals confirming the blood glucose level of the above step was orally administered with ORTG, ORT and Met at a concentration of 0.6 g / kg, respectively, for 18 days after the start of the experiment (Fig. 4). Animals were sacrificed after sample administration and blood, liver, pancreas and small intestine were collected and used for sample analysis.
실시예5. 시료 수집Example 5. Sample collection
상기 실시예3 및 실시예4에서 희생시킨 실험동물의 시료를 분석하기 위해 희생 전 12시간 절식 후 CO2로 마취한 실험동물을 복부 대정맥에서 헤파린 1000IU/mL 처리된 주사기를 사용하여 채혈하였다. 혈액은 4℃에서 13,200rpm으로 원심분리하여 혈장을 분리한 후 -80℃에 보관하였다. 채혈 후 간, 가자미근, 부고환지방 및 췌장을 적출하여 무게 측정 후 액체질소로 급속동결하여 -80℃에서 보관하였고 동시에 적출한 소장은 소장상피를 긁어 액체질소로 급속동결하여 -80℃에서 보관하였다.In order to analyze the samples of the experimental animals sacrificed in Examples 3 and 4, experimental animals anesthetized with CO 2 after 12 hours of fasting before sacrifice were collected using a heparin 1000IU / mL syringe in the abdominal vena cava. Blood was centrifuged at 13,200 rpm at 4 ° C to separate plasma and stored at -80 ° C. After blood collection, liver, soleus muscle, epididymal fat and pancreas were extracted, weighed and then rapidly frozen with liquid nitrogen and stored at -80 ° C.
실험예1. 상지 및 상백피 열수추출물 실험에서의 체중변화Experimental Example 1. Body Weight Changes in the Upper and Upper Epidermal Hot Water Extract Experiments
상지 및 상백피 열수추출물의 항당뇨 효과 실험에서 NC, DC, MT 및 MRB 그룹의 실험동물의 실험 시작 전 초기체중과 실험 종료 후 마지막 체중을 측정하여 비교하였다.In the antidiabetic effect test of upper and upper epithelial hot water extracts, the initial body weight and the final body weight after the end of the experiment in the NC, DC, MT and MRB group were measured.
실험 결과, 하기 표5에서 알 수 있듯이 NC군에 비하여 당뇨를 유발한 DC, MT 및 MRB 세군에서 체중이 감소하였다.As a result, as shown in Table 5, body weight was reduced in three groups of DC, MT, and MRB which induced diabetes compared to NC group.
표 5
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
Table 5
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
실험예2. 혈당 및 혈장 인슐린 함량 측정Experimental Example 2. Blood glucose and plasma insulin content determination
상지 및 상백피 열수추출물의 항당뇨 효과를 확인하기 위해 실험동물에 실험 시료 투여 10일 후에 12시간 절식시키고 다음날 오전에 실험 시료 투여 전 공복혈당을 측정하였다. 혈당 측정은 미정맥으로부터 소량의 혈액을 채취하여 혈당계(blood glucose monitoring kit, ARKRAY)를 이용하여 측정하였다. In order to confirm the antidiabetic effect of the upper limbs and epithelial hot water extracts, the animals were fasted for 12 hours 10 days after the administration of the test sample, and fasting blood glucose was measured before the test sample was administered the next morning. Glucose was measured using a blood glucose monitoring kit (ARRKRAY) with a small amount of blood taken from the vein.
실험 결과, 도5에서 알 수 있듯이 경구투여 10일 경과 후 공복혈당은 NC군에 비해 DC군에서 증가하였고, DC군에 비해 MT군에서 감소하였다. DC군에 비해 MRB군은 유의적인 차이는 없었다.As can be seen from Figure 5, 10 days after oral administration fasting blood glucose was increased in the DC group compared to the NC group, and decreased in the MT group compared to the DC group. There was no significant difference in MRB group compared to DC group.
또 도6에서 알 수 있듯이 실험동물 희생 후 혈장에서 측정한 혈당은 NC군에 비해 DC군이 증가하였고 DC군에 비해 MT군이 44% 감소하였으나 MRB군에서는 유의적인 차이는 없었다. 혈장 인슐린 농도는 NC군에 비해 MT군만 유의적인 감소를 나타냈다.As shown in FIG. 6, the blood glucose measured in plasma after sacrifice of experimental animals was increased in the DC group and the MT group in the MT group compared to the NC group, but there was no significant difference in the MRB group. Plasma insulin concentration showed a significant decrease only in the MT group compared to the NC group.
실험예3. 혈중지질 농도 측정Experimental Example 3. Blood lipid concentration measurement
상지 및 상백피 열수추출물 투여에 따른 혈당과 혈중 인슐린 농도를 측정하기 위해 상기 실시예5에서 얻은 혈장을 이용하여 혈중지질 농도를 측정하였다. 혈장 포도당 함량 측정을 위해 포도당 측정용 시액(Asan kit)을 사용하였다. 혈장 인슐린은 mouse Insulin ELISA kit(U-type, Shibayagi Co.)를 사용하여 측정하였다.Plasma lipid concentration was measured using the plasma obtained in Example 5 to measure blood glucose and blood insulin concentrations following the administration of the upper limbs and epithelium hot water extract. Asan kit was used for glucose measurement. Plasma insulin was measured using a mouse Insulin ELISA kit (U-type, Shibayagi Co.).
실험 결과, 도7에서 알 수 있듯이 Total cholesterol(T-CHO) 농도는 DC군에 비해 MT 및 MRB군이 각각 72%, 73% 감소하였고 HDL-cholesterol(HDL-CHO) 농도는 실험군간의 차이는 없었다. HDL-CHO/T-CHO 비율은 NC군에 비해 DC군이 80% 감소하였고 DC군과 비교했을 때 MT군과 MRB군이 각각 135%, 130% 증가하였다. 결론적으로 MT군가 MRB군에서 DC군과 비교했을 때 T-CHO 농도가 감소하였고 HDL-CHO/T-CHO 비율이 증가하여 혈중 지질 농도 개선의 효과를 확인하였다.As shown in FIG. 7, the total cholesterol (T-CHO) concentrations of the MT and MRB groups were reduced by 72% and 73%, respectively, and the HDL-cholesterol (HDL-CHO) concentrations were not different between the experimental groups. . The HDL-CHO / T-CHO ratio was 80% lower in the DC group than the NC group, and 135% and 130% in the MT and MRB groups, respectively. In conclusion, MT group showed lower T-CHO concentration and HDL-CHO / T-CHO ratio compared to DC group in MRB group.
또 도8에서 알 수 있듯이 혈장에서 triglyceride(TG) 함량은 NC군에 비해 DC군에서 감소하였으며 DC군에 비해 MT군에서 증가하였다.In addition, as can be seen in Figure 8 triglyceride (TG) content in plasma was reduced in the DC group compared to the NC group and increased in the MT group compared to the DC group.
도9에서 알 수 있듯이 혈장 내 free fatty acid(FFA) 농도는 NC군과 비교했을 때 DC, MT 및 MRB군에서 감소하였고 세군 사이에서 유의적인 차이는 없었다.As can be seen in FIG. 9, free fatty acid (FFA) concentrations in plasma were decreased in the DC, MT and MRB groups compared to the NC group, and there was no significant difference between the three groups.
실시예4. 간 조직 TG 및 T-CHO 함량Example 4. Liver tissue TG and T-CHO content
상지 및 상백피 열수추출물 투여에 따른 간에서 TG 및 T-CHO 농도를 측정하기 위해 상기 실시예5의 방법으로 얻은 간 조직을 이용하여 TG, T-CHO 및 FFA 농도를 측정하였다. TG, T-CHO and FFA concentrations were measured using liver tissue obtained by the method of Example 5 to determine TG and T-CHO concentrations in the liver following administration of the upper limbs and epithelial hot water extract.
실험 결과, 도10에서 알 수 있듯이 간 TG 농도는 NC군이 9.5, DC군이 19.4, MT군이 8.5, MRB군이 22.5mg/g으로 측정되어 NC군에 비해 DC군이 204% 증가하였고, DC군에 비해 MT군이 44%로 정상수준으로 감소하였다. 간 T-CHO 농도는 NC군이 2.6, DC군이 2.9, MT군이 2.5, MRB군이 1.9mg/g으로 NC군에 비해 DC군이 증가하였고 DC군에 비해 MT군 및 MRB군이 감소하였으나 유의적인 차이는 없었다. FFA 함량은 NC군에 비해 DC군, MT군 및 MRB군 모두 감소하였고 감소한 세군 사이의 차이는 없었다. 결론적으로 MT군이 DC군에 비해 간의 FFA 농도에는 효과가 없었으나 간 조직 TG 농도를 정상수준으로 감소시켰으며 T-CHO 수준을 감소시켜 간 조직의 지질수준 개선 효과를 나타내었다.As a result, as shown in FIG. 10, the liver TG concentration was measured to be 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. Compared with DC group, MT group decreased to normal level by 44%. The liver T-CHO concentrations were 2.6 in the NC group, 2.9 in the DC group, 2.5 in the MT group, and 1.9 mg / g in the MRB group. The DC group increased compared to the NC group and the MT and MRB groups decreased compared to the DC group. There was no significant difference. The FFA content was decreased in the DC, MT and MRB groups compared to the NC group, and there was no difference between the three groups. In conclusion, MT group had no effect on liver FFA level compared to DC group, but decreased TG concentration to normal level and decreased T-CHO level to improve liver lipid level.
실험예5. 소장 점막의 이당류분해효소 활성Experimental Example 5. Disaccharide Enzyme Activity of Small Intestinal Mucosa
상지 및 상백피 열수추출물 투여에 따른 MT군의 혈당강하 효과가 당이 대사되어 흡수되는 과정에서 차이가 있었는지 확인하기 위해 상기 실시예5에서 얻은 소장을 이용하여 소장 점막증의 lactase, sucrase 및 maltase 활성을 Dahlqvist의 방법으로 측정하였다.Lactase, sucrase and maltase activity of small intestinal mucosa using the small intestine obtained in Example 5 to determine whether the hypoglycemic effect of MT group according to the administration of the upper limb and epithelium hot water extract was different Was measured by Dahlqvist's method.
이를 평가하기 위해 상기 실시예5의 방법으로 얻은 소장의 유문괄약근에서 10cm 떨어진 지점부터 맹장 직전까지 자른 후 3등분 하여 proximal, middle distal 부분으로 구분하여 생리식염수로 세척하였고 소장은 길이대로 잘라 편 후 차가운 생리식염수로 씻어 냉각 판에서 점만을 microscopic glass로 긁어서 무게를 측정 후 측정한 무게의 4배의 증류수와 함께 homogenizer로 균질화 시켜 4℃, 7000xg에서 10분간 원심분리하였다. 원심분리 후 상층액을 취해 효소활성 측정시료로 사용하였다. 희석시킨 효소 시료(lactase:1배, sucrase: 8배, maltase: 10배) 0.1mL, 기질 용액 0.056M disaccahride solution 및 0.1M sodium maltase buffer(pH 6.0) 0.1mL를 시험관에 넣고 잘 혼합해서 37℃ water bath에서 1시간 반응시킨 후 증류수 0.8mL를 넣고 2분간 끊는 물에 담근 후 420nm에서 흡광도를 측정하였다.To evaluate this, cut to 10cm from the pyloric sphincter of the small intestine obtained by the method of Example 5 until just before the cecum, divided into 3 parts, washed with physiological saline, divided into proximal and middle distal parts, and cut into small sections. After washing with physiological saline, only the points on the cooling plate was scraped with microscopic glass, weighed, and homogenized with homogenizer with 4 times the weight of distilled water, and centrifuged at 4 ℃ and 7000xg for 10 minutes. After centrifugation, the supernatant was taken and used as a sample for measuring enzyme activity. 0.1 mL of diluted enzyme samples (lactase: 1x, sucrase: 8x, maltase: 10x), substrate solution 0.056M disaccahride solution and 0.1mL 0.1M sodium maltase buffer (pH 6.0) were added to the test tube and mixed well. After reacting in a water bath for 1 hour, 0.8 mL of distilled water was added, soaked in water for 2 minutes, and the absorbance was measured at 420 nm.
실험 결과, 도11에서 알 수 있듯이 proximal의 lactase 활성은 MT군이 DC군에 비해 70% 감소하였다. sucrase 활성은 NC군에 비해 DC군이 120% 증가하였으며 DC군에 비해 MT 및 MRB군이 각각 67%, 75% 감소하였다. maltase 활성은 NC군에 비해 DC군이 138% 증가하였으며 MT군은 77% 감소하였다. MRB군은 DC군에 비해 70% 감소하였다. middle 부분의 lactase 및 sucrase 활성은 각 군의 유의적인 차이는 없었다. maltase 활성은 NC군에 비해 DC, MT 및 MRB군 모두 감소하였다. Distal 부분은 lactase, sucrase 및 maltase 활성은 NC군에 비해 DC, MT 및 MRB군 모두 감소하였다. As shown in FIG. 11, the lactase activity of proximal was 70% decreased in the MT group compared to the DC group. Sucrase activity was increased by 120% in DC group and 67% and 75% in MT and MRB groups, respectively. Maltase activity was increased by 138% in DC group and 77% in MT group. The MRB group was 70% less than the DC group. There was no significant difference in lactase and sucrase activity in the middle group. Maltase activity was decreased in DC, MT and MRB groups compared to NC group. In the distal portion, lactase, sucrase and maltase activities were decreased in DC, MT and MRB groups compared to NC group.
실험예6. 상지 지표성분 실험동물의 체중변화량Experimental Example 6. Weight Changes of Upper Limb Index Components
상지 지표성분의 항당뇨 효과 실험에서 NC, DC, ORTG, ORT 및 Met 그룹의 실험동물의 실험 시작 전 초기체중과 실험 종료 후 마지막 체중을 측정하여 비교하였다.In the antidiabetic effect test of the upper limb index component, the initial body weight of the experimental animals of NC, DC, ORTG, ORT and Met group was compared with the final body weight after the end of the experiment.
실험 결과, 하기 표6에서 알 수 있듯이 각 실험군간의 유의적인 차이는 없었다.As a result, as shown in Table 6, there was no significant difference between each experimental group.
표 6
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
Table 6
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
실험예7. 혈당 및 혈장 인슐린 함량 측정Experimental Example 7. Blood glucose and plasma insulin content determination
상지 지표성분의 항당뇨 효과를 확인하기 위해 실험동물에 실험 시료 투여 19일 후에 12시간 절식시키고 다음날 오전에 실험 시료 투여 전 공복혈당을 측정하였다. 혈당 측정은 미정맥으로부터 소량의 혈액을 채취하여 혈당계(blood glucose monitoring kit, ARKRAY)를 이용하여 측정하였다. In order to confirm the antidiabetic effect of the upper extremity indicator component, the animals fasted 12 hours after 19 days of administration of the test sample, and fasting blood glucose was measured before administration of the test sample the next morning. Glucose was measured using a blood glucose monitoring kit (ARRKRAY) with a small amount of blood taken from the vein.
실험 결과, 도12에서 알 수 있듯이 경구투여 19일 경과 후 공복혈당은 NC군에 비해 DC군이 232% 증가하였고, DC군에 비해 ORTG군, ORT군 및 Met군 모두 감소하였다.As shown in FIG. 12, fasting blood glucose was increased by 232% in the DC group compared to 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.
또 도13에서 알 수 있듯이 실험동물 희생 후 혈장에서 측정한 혈당은 NC군에 비해 DC군이 증가하였고 DC군에 비해 ORT군이 43% 감소하여 정상수준으로 나타났다. ORTG군 및 Met군은 유의적인 차이는 없었다. 혈장 인슐린 농도는 NC군에 비해 당뇨를 유발한 DC, ORT, ORTG 및 Met군 모두 유의적인 감소를 나타냈고 네군 사이의 차이는 없었다. 따라서 각 군의 혈당 농도의 변화는 인슐린 농도의 변화로 인한 것이 아닌 것으로 나타났다.As shown in FIG. 13, the blood glucose measured in plasma after sacrifice of experimental animals was increased in the DC group compared to the NC group and decreased by 43% in the ORT group compared to the DC group. There was no significant difference between ORTG group and Met group. Plasma insulin concentrations were significantly decreased in the DC, ORT, ORTG, and Met groups that induced diabetes compared to the NC group, and there was no difference between the four groups. Therefore, it was shown that the change in blood glucose level of each group was not due to the change in insulin concentration.
실험예8. 장기 무게 측정Experimental Example 8. Organ weight measurement
상지 지표성분의 항당뇨 실험에서 상기 실시예5의 방법으로 얻은 시료의 무게를 측정하였다.The weight of the sample obtained by the method of Example 5 in the antidiabetic experiment of the upper extremity indicator component was measured.
실험 결과, 하기 표7에서 알 수 있듯이 간과 췌장의 무게는 각 실험군간의 유의적인 차이는 나타나지 않았다.As a result, as shown in Table 7, the weight of liver and pancreas did not show a significant difference between each experimental group.
표 7
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
TABLE 7
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
실험예9. 혈중지질 농도 측정Experimental Example 9. Blood lipid concentration measurement
상지 지표성분 투여에 따른 혈중 지질농도를 측정하기 위해 상기 실시예5에서 얻은 혈장을 이용하여 혈중지질 농도를 측정하였고 혈장 포도당 함량 측정을 위해 포도당 측정용 시액(Asan kit)을 사용하였다. 혈장 인슐린은 mouse Insulin ELISA kit(U-type, Shibayagi Co.)를 사용하여 측정하였다.Plasma lipid concentration was measured by using the plasma obtained in Example 5 to measure blood lipid concentrations according to the administration of the upper limb index component, and a glucose measuring solution (Asan kit) was used to measure plasma glucose content. Plasma insulin was measured using a mouse Insulin ELISA kit (U-type, Shibayagi Co.).
실험 결과, 도14에서 알 수 있듯이 혈장 TG농도와 GDL-CHO 농도는 실험군간의 차이가 없었다. T-CHO 농도는 DC군에 비해 ORTG군, ORT군 및 Met군에서 감소하였고 NC군과 DC군간의 차이는 없었다. HDL-CHO/T-CHO 비율은 다른 실험군에 비해 Met군에서 크게 증가하였다.As shown in FIG. 14, the plasma TG concentration and the GDL-CHO concentration did not differ between the experimental groups. T-CHO concentration was decreased in ORTG, ORT and Met groups compared to DC group, and there was no difference between NC and DC groups. The HDL-CHO / T-CHO ratio was significantly increased in the Met group compared to the other experimental groups.
또 도15에서 알 수 있듯이 혈장 내 free fatty acid(FFA) 농도는 NC군과 비교했을 때 DC, ORTG, ORT 및 Met군에서 감소하였고 네군 사이에서 유의적인 차이는 없었다.As can be seen in FIG. 15, free fatty acid (FFA) concentration in plasma was decreased in the DC, ORTG, ORT and Met groups compared to the NC group, and there was no significant difference between the four groups.
실험예10. 소장 점막의 이당류분해효소 활성Experimental Example 10. Disaccharide Enzyme Activity of Small Intestinal Mucosa
상지 지표성분 투여에 따른 혈당강하 효과가 당이 대사되어 흡수되는 과정에서 차이가 있었는지 확인하기 위해 상기 실시예5에서 얻은 소장을 이용하여 소장 점막증의 lactase, sucrase 및 maltase 활성을 Dahlqvist의 방법으로 측정하였다.In order to determine whether the hypoglycemic effect according to the administration of the upper limb index component was different in the process of metabolizing and absorbing glucose, the lactase, sucrase and maltase activity of small intestinal mucosa were examined using Dahlqvist's method. Measured.
이를 평가하기 위해 상기 실시예5의 방법으로 얻은 소장의 유문괄약근에서 10cm 떨어진 지점부터 맹장 직전까지 자른 후 3등분 하여 proximal, middle distal 부분으로 구분하였다. 생리식염수로 세척하였고 소장은 길이대로 잘라 편 후 차가운 생리식염수로 씻어 냉각 판에서 점만을 microscopic glass로 긁어서 무게를 측정 후 측정한 무게의 4배의 증류수와 함께 homogenizer로 균질화 시켜 4℃, 7000xg에서 10분간 원심분리하였다. 원심분리 후 상층액을 취해 효소활성 측정시료로 사용하였다. 희석시킨 효소 시료(lactase:1배, sucrase: 8배, maltase: 10배) 0.1mL, 기질 용액 0.056M disaccahride solution 및 0.1M sodium maltase buffer(pH 6.0) 0.1mL를 시험관에 넣고 잘 혼합해서 37℃ water bath에서 1시간 반응시킨 후 증류수 0.8mL를 넣고 2분간 끊는 물에 담근 후 420nm에서 흡광도를 측정하였다. 상기 실험예5의 결과에서 proximal 부분에서만 이당류분해효소 활성 효과가 나타났기 때문에 proximal 부분만 이당류분해효소 활성을 측정하였다.In order to evaluate this, cut from the point of 10 cm from the pyloric sphincter of the small intestine obtained by the method of Example 5 until just before the cecum, divided into three parts and divided into proximal and middle distal parts. After washing with physiological saline, cut the small intestine to the length, wash with cold physiological saline, scrape only the points on the cooling plate with microscopic glass, measure the weight, and homogenize with homogenizer with 4 times distilled water of the measured weight. Centrifuged for a minute. After centrifugation, the supernatant was taken and used as a sample for measuring enzyme activity. 0.1 mL of diluted enzyme samples (lactase: 1x, sucrase: 8x, maltase: 10x), substrate solution 0.056M disaccahride solution and 0.1mL 0.1M sodium maltase buffer (pH 6.0) were added to the test tube and mixed well. After reacting in a water bath for 1 hour, 0.8 mL of distilled water was added, soaked in water for 2 minutes, and the absorbance was measured at 420 nm. Since the disaccharide dehydrogenase activity effect was shown only in the proximal part in the results of Experimental Example 5, only the proximal part was measured for disaccharide dehydrogenase activity.
실험 결과, 도16에서 알 수 있듯이 proximal 부분의 lactase, sucrase 및 maltase의 활성은 NC군에 비해 DC군, ORTG군, ORT군 및 Met군에서 모두 효소 활성이 증가하였으며 네군간의 유의적인 차이는 없었다. As can be seen from Figure 16, the activity of lactase, sucrase and maltase in the proximal part 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. .
실험예11. 간 조직 GLUT2 mRNA 발현량 확인Experimental Example 11. Confirmation of liver tissue GLUT2 mRNA expression level
상지 지표성분 투여에 따른 간으로 포도당 유입이 증가하였는지를 확인하기 위해 상기 실시예5의 방법으로 얻은 간 조직을 사용하여 포도당수송체인 glucose transporter 2(GLUT2)의 mRNA 발현량을 측정하였다.In order to determine whether glucose inflow increased to the liver following the administration of the upper limb index component, mRNA expression level of glucose transporter glucose transporter 2 (GLUT2) was measured using liver tissue obtained by the method of Example 5.
이를 평가하기 위해 간 조직을 액체질소가 담긴 막자사발에서 균질화하였고 대장 조직 0.1g당 RNAiso Plus(TaKaRa) 1mL을 첨가하였다. 상층액을 취해 chloroform 200uL를 첨가하여 4℃, 10,000rpm에서 5분간 원심분리하였다. 원심분리하여 얻은 상층액에 isopropanol 500uL를 첨가한 후 4℃, 14,000rpm에서 20분 동안 원심분리하여 RNA pellet을 얻었다. 상기 단계에서 얻은 RNA pellet을 75% ethanol로 washing하고 4℃, 9,500rpm에서 5분 동안 원심 분리한 후 air-dry 후 diethyl pyrocarbonate(DEPC)-treated water에 녹여 분석하였다.To evaluate this, liver tissue was homogenized in a mortar containing liquid nitrogen and 1 mL of RNAiso Plus (TaKaRa) was added per 0.1 g of colon tissue. The supernatant was taken and chloroform 200uL was added and centrifuged at 4 ° C. and 10,000 rpm for 5 minutes. After isopropanol 500uL was added to the supernatant obtained by centrifugation, RNA pellet was obtained by centrifugation at 4 ° C and 14,000 rpm for 20 minutes. The RNA pellet obtained in the above step was washed with 75% ethanol, centrifuged at 4 ° C. and 9,500 rpm for 5 minutes and then air-dryed and dissolved in diethyl pyrocarbonate (DEPC) -treated water for analysis.
실험 결과, 도17에서 알 수 있듯이 간의 mRNA 발현량은 NC군에 비해 DC군에서 감소하였고, DC군에 비해 ORTG군이 증가하였으나 유의적인 차이는 없었다. 그러나 DC군에 비해 ORT군 및 Met군은 유의적으로 증가하였다.As can be seen from Figure 17, mRNA expression of liver was decreased in the DC group compared to the NC group, ORTG group increased compared to the DC group, but there was no significant difference. However, ORT and Met groups were significantly increased compared to DC group.
실험예12. 간 조직 글리코켄 함량 확인Experimental Example 12. Determination of liver tissue glycogen content
상기 실험예11에서 GLUT2 mRNA 발현량의 차이를 확인하였기 때문에 상피 지표성분 투여에 따른 간 조직 내로 유입되는 포도당의 양에도 차이가 있을 것으로 생각하고 간 조직의 포도당 저장 형태인 글리코겐 양의 변화를 분석하였다.Since the difference in the expression level of GLUT2 mRNA was confirmed in Experiment 11, the amount of glucose introduced into the liver tissue according to the administration of the epidermal indicator component was considered to be different, and the change in the glycogen amount in the form of glucose storage of the liver tissue was analyzed. .
실험 결과, 도18에서 알 수 있듯이 간의 글리코겐 함량은 NC군에 비해 DC군에서 감소하였고, DC군에 비해 ORTG군과 ORT군은 증가하였으나 유의적인 차이는 없었고, Met군은 유의적으로 증가하였다.As can be seen in Figure 18, the glycogen content in liver was decreased in the DC group compared to the NC group, ORTG and ORT group increased compared to the DC group, but there was no significant difference, Met group significantly increased.
결론적으로 상지 열수추출물이 소장 이당류분해효소 활성을 억제하여 혈당을 낮춰 혈장 T-CHO 농도를 감소시키고 간 조직 TG 농도를 감소시켜 지질 개선 효과가 나타났고 상백피 열수추출물과 비교했을 때 효능이 더 뛰어나게 나타났다. 상지 지표성분의 항당뇨 효과 실험에서는 상지의 주된 물질인 ORT의 혈당강하 효과를 확인하였고 그 기전으로 ORT가 간 조직의 GLUT2 mRNA 발현량을 증가시켜 간 조직 내로의 포도당 유입을 증가시키고 글리코겐의 합성을 증가시켜 혈당을 낮추는 것으로 나타났다. 또한 혈장 T-CHO 농도를 감소시켜 지질 개선에도 효과가 있다(도19).In conclusion, upper limb hydrothermal extracts inhibited the activity of small intestinal disaccharides, lowering blood glucose, reducing plasma T-CHO levels, and reducing liver tissue TG levels, resulting in lipid-improving effects and superior efficacy compared to epithelial hot water extracts. . The antidiabetic effect of the upper limb markers confirmed the hypoglycemic effect of ORT, the major substance in the upper limb.The mechanism of ORT increased the expression of GLUT2 mRNA in liver tissue, which increased glucose inflow into liver tissue and the synthesis of glycogen. It has been shown to increase blood sugar levels. It is also effective in improving lipids by reducing plasma T-CHO concentration (Fig. 19).
본 발명은 상지 열수추출물 및 상지 지표성분인 ORT를 함유하는 당뇨병 예방 및 치료용 조성물을 제공하는 뛰어난 효과가 있으므로 건강식품 및 의약품 산업상 매우 유용한 발명인 것이다.The present invention is a very useful invention for the health food and pharmaceutical industry because it has an excellent effect of providing a composition for the prevention and treatment of diabetes containing the upper limbs hot water extract and the upper limb index ORT.

Claims (5)

  1. 건조한 상지에 이온수를 가하여 초음파추출기에서 초음파 추출하는 단계와; 상기 단계에서 얻은 상지 추출물을 감압 농축하여 열수 추출하는 단계로 이루어지거나 또는 상기 단계에서 얻은 상지 열수추출물을 초음파분쇄기를 이용하여 에탄올 추출 단계와; 상기 단계에서 얻은 에탄올 추출물을 여과 및 감압농축하는 단계와; 상기 단계에서 얻은 농축물을 다시 에탄올에 용해한 후 헥산으로 분획하여 얻은 탈지 에탄올 추출물을 감압농축하는 단계와; 상기 단계에서 감압농축한 탈지 에탄올 추출물에 가수하여 CH2Cl2, EtOAC 및 n-BuOH로 순차적으로 분획하여 EtOAC 분획물로부터 oxyresveratrol aglycon을 분리 및 정제하는 단계로 이루어진 상지 지표성분 oxyresveratrol aglycon의 중 어느 하나를 함유하는 당뇨 예방 및 건강 기능성 식품 조성물.Ultrasonic extraction from the ultrasonic extractor by adding ionized water to the dry upper limbs; Concentrating the upper limb extract obtained in the above step under reduced pressure to extract the hot water or ethanol extraction step using the ultrasonic grinding machine for the upper limb hot water extract obtained in the step; Filtering and concentrating the ethanol extract obtained in the step; Concentrating the degreasing ethanol extract obtained by dissolving the concentrate obtained in the above step again in ethanol and fractionated with hexane; The hydrolyzed degreasing ethanol extract concentrated under reduced pressure in the above step is sequentially fractionated into CH 2 Cl 2 , EtOAC and n-BuOH to separate and purify the oxyresveratrol aglycon from the EtOAC fraction. Diabetic prevention and health functional food composition containing.
  2. 건조한 상지에 이온수를 가하여 초음파추출기에서 초음파 추출하는 단계와; 상기 단계에서 얻은 상지 추출물을 감압 농축하여 열수 추출하는 단계로 이루어지거나 또는 상기 단계에서 얻은 상지 열수추출물을 초음파분쇄기를 이용하여 에탄올 추출 단계와; 상기 단계에서 얻은 에탄올 추출물을 여과 및 감압농축하는 단계와; 상기 단계에서 얻은 농축물을 다시 에탄올에 용해한 후 헥산으로 분획하여 얻은 탈지 에탄올 추출물을 감압농축하는 단계와; 상기 단계에서 감압농축한 탈지 에탄올 추출물에 가수하여 CH2Cl2, EtOAC 및 n-BuOH로 순차적으로 분획하여 EtOAC 분획물로부터 oxyresveratrol aglycon을 분리 및 정제하는 단계로 이루어진 상지 지표성분 oxyresveratrol aglycon의 중 어느 하나를 함유하는 당뇨 예방 및 치료용 약학적 조성물.Ultrasonic extraction from the ultrasonic extractor by adding ionized water to the dry upper limbs; Concentrating the upper limb extract obtained in the above step under reduced pressure to extract the hot water or ethanol extraction step using the ultrasonic grinding machine for the upper limb hot water extract obtained in the step; Filtering and concentrating the ethanol extract obtained in the step; Concentrating the degreasing ethanol extract obtained by dissolving the concentrate obtained in the above step again in ethanol and fractionated with hexane; The hydrolyzed degreasing ethanol extract concentrated under reduced pressure in the above step is sequentially fractionated into CH 2 Cl 2 , EtOAC and n-BuOH to separate and purify the oxyresveratrol aglycon from the EtOAC fraction. Pharmaceutical composition for preventing and treating diabetes.
  3. 제 1항에 있어서, 상기 식품 조성물은 음료, 껌, 차 및 선식 중 어느 하나의 제형인 것이 특징인 식품조성물.According to claim 1, wherein the food composition is a food composition, characterized in that the formulation of any one of beverages, gum, tea and wire.
  4. 제 2항에 있어서, 상기 약학적 조성물은 정제, 환제, 산제, 과립제, 캅셀제, 현탁제, 유제, 시럽제, 비수성용제, 현탁제 및 동결건조제제로 이루어진 군으로부터 선택되는 어느 하나의 제형을 가지는 것을 특징으로 하는 약학적 조성물.The method of claim 2, wherein the pharmaceutical composition is a tablet, pills, powder, granules, capsules, suspensions, emulsions, syrups, non-aqueous solvents, suspensions and lyophilized formulations, characterized in that any one selected from the group consisting of Pharmaceutical compositions.
  5. 제 2항에 있어서, 상기 조성물은 약학적으로 허용되는 담체, 부형제 또는 희석제를 추가로 더 포함하는 것이 특징인 약학적 조성물.The pharmaceutical composition of claim 2, wherein the composition further comprises a pharmaceutically acceptable carrier, excipient or diluent.
PCT/KR2016/003209 2016-03-17 2016-03-29 Antidiabetic functional composition containing hot water extract of ramulus mori and oxyresveratrol as active ingredients and method for producing thereof WO2017159912A1 (en)

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