KR101995238B1 - Composition for preventing, improving or treating diabetes mellitus comprising Rosa davurica extract as effective component - Google Patents

Abstract

The extract of Rhodiola extract of the present invention is remarkably superior in antioxidative activity, α-glucosidase inhibitory activity and glucose absorption ability than extracts of each of the fruits, leaves and roots of the ear buds known to have antidiabetic effect, Since the morphology of the damaged pancreatic islets is restored and the effect of increasing the number of beta cells, which are reduced insulin secretory cells, is enhanced when the genital extract is administered to the animal model of type 1 diabetes, And can be usefully used for health functional foods and medicines for improvement or treatment.

Description

[0001] The present invention relates to a composition for preventing, ameliorating or treating diabetes mellitus,

The present invention relates to a composition for preventing, ameliorating, or treating diabetes mellitus containing an extract of green ginger extract as an active ingredient.

Diabetes is a chronic wasting disease characterized by an abnormal glucose tolerance due to an absolute deficiency or resistance of insulin. It is a disease that causes various complications such as systemic weakness and resistance, vascular disorder, cerebral infarction, and myocardial infarction. Diabetes mellitus is classified into two types according to its cause and pathogenesis: insulin-dependent type 1 diabetes mellitus caused by the destruction of insulin-secreting cell, beta cell, by the immunological mechanism of lymphocyte, and function of insulin-secreting beta cells There is an insulin-independent adult type 2 diabetes mellitus induced by decreased and increased resistance to insulin in peripheral target organs.

Type 1 diabetes occurs mainly in children and adolescents, and the cause is unknown, but it is known to be caused by genetic, environmental, viral and chemical agents. Symptoms usually appear suddenly, and severe symptoms can include increased thirst, frequent urination, weight loss, extreme hunger, vomiting, abdominal pain, or fatigue. The destruction of pancreatic beta cells in type 1 diabetes results in insulin deficiency and eventually destroys glucose homeostasis in the body. Because insulin lowers blood sugar by transporting glucose in the blood into cells and by converting glucose into glycogen in the liver, the insulin deficiency resulting from the destruction of beta cells leads to glucose homeostasis in the blood, leading to hyperglycemia and metabolic disorders Lt; / RTI >

Rosa davurica is a perennial plant belonging to Rosaceae. It is a deciduous broad-leaved shrub distributed in the central and northern part of Korea, Japan, Manchuria, and Siberia. It is 1 ~ 1.5m high, with straight stem, reddish brown and spiny. Leaves are alternate phyllotaxis, elliptical or elliptical, pointed at both ends, 1 ~ 3cm long, with serrate on the edge and prefixed on the back. Flowers are rose-colored and fragrant. Fruits are spherical and ripen in yellowish red in September. In the private sector, fruits are used for food, and roots and fruits have been used for the treatment of indigestion, stomach pain, and menstrual relief. It has also been used in the private sector to increase vitamin D deficiency, resistance to various diseases,

Korean Patent Laid-Open No. 2010-0111088 discloses a composition for preventing and treating diabetes mellitus or a complication caused by diabetes mellitus containing the herbal extract as an active ingredient, Korean Patent No. 1043813 discloses a composition containing a bioartificial extract Discloses a cosmetic composition. However, a composition for preventing, ameliorating or treating diabetes mellitus containing the raw juice extract of the present invention as an active ingredient has not yet been disclosed.

The present invention has been made in view of the above-mentioned needs, and it is an object of the present invention to provide a method for preparing an extract for each of the raw nematodes (stem, fruit, leaf and root) -glucosidase inhibitory activity and glucose uptake activity of the extracts of Staphylococcus aureus and Streptozotocin (STZ) were significantly higher than those of extracts of fruit, Induced type 1 diabetic animal model resulted in the recovery of the form of pancreatic islets due to type 1 diabetes and the increase in the number of reduced insulin secreting beta cells , The present invention has been completed.

In order to solve the above-mentioned problems, the present invention provides a pharmaceutical composition for preventing or treating diabetes comprising Rosa davurica extract as an active ingredient.

In addition, the present invention provides a health functional food composition for preventing or ameliorating diabetes containing Rosa davurica extract as an active ingredient.

The extract of Rhodiola extract of the present invention is remarkably superior in antioxidative activity, α-glucosidase inhibitory activity and glucose absorption ability than extracts of each of the fruits, leaves and roots of the ear buds known to have antidiabetic effect, Since the morphology of the damaged pancreatic islets is restored and the effect of increasing the number of beta cells, which are reduced insulin secretory cells, is enhanced when the genital extract is administered to the animal model of type 1 diabetes, And can be usefully used for health functional foods and medicines for improvement or treatment.

FIG. 1 shows the result of measuring the DPPH radical scavenging activity of the extract of the raw nerve portion. VitC is a vitamin C as a positive control.
FIG. 2 shows cell viability of C2C12 cells treated with the extract of the raw virion.
FIG. 3 shows the results of measuring the inhibitory activity of? -Glucosidase from the extracts of the raw vertebrate part. Acarbose is a positive control.
FIG. 4 shows the results of measuring the glucose uptake ability of the extract of the genitourinary part. Con is a negative control without any treatment, and insulin is a positive control.
FIG. 5 shows the blood glucose lowering effect of the animal model when the extract of the present invention was administered to streptozotocin (STZ) -injected animal model of type 1 diabetes. Dia + R. davruica stem was administered with streptozotocin (STZ), and Dia + R. davruica stem was administered with streptozotocin to induce diabetes mellitus extract of the present invention at a concentration of 62.5 , 125, 250 and 500 mg / kg), respectively.
FIG. 6 is a graph showing a change in body weight of an animal model when a plant extract of the present invention was administered to a model animal of type 1 diabetes induced by administration of streptozotocin (STZ).
FIG. 7 is a graph showing changes in the dietary (A) and beverage (B) intakes of the animal model when the extract of the present invention was administered to streptozotocin (STZ) -treated model animals of type 1 diabetes Results.
FIG. 8 shows the results of measurement of blood insulin concentration when the extract of the present invention was administered to streptozotocin (STZ) -injected animal model of type 1 diabetes.
FIG. 9 is a graph showing the results of the Islets form of the pancreatic tissue of the animal model and the shape of the insulin-secreting cell of the animal model when the extract of the present invention was administered to the animal model of type 1 diabetes induced by streptozotocin (STZ) Beta cell production.

In order to accomplish the object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating diabetes comprising Rosa davurica extract as an active ingredient.

In the pharmaceutical composition for the prevention or treatment of diabetes according to the present invention, the biovarial extract may be, but is not limited to, Biovarial extract.

In addition, the diabetes may be type 1 diabetes but is not limited thereto.

In addition, the biopsied buds extract may be extracted with water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof, preferably methanol, but is not limited thereto.

The pharmaceutical compositions of the present invention may further comprise suitable carriers, excipients or diluents conventionally used in the manufacture of pharmaceutical compositions.

The pharmaceutical dosage forms of the compositions according to the invention may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable set.

The pharmaceutical composition according to the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method . Examples of carriers, excipients, and diluents that can be included in the pharmaceutical composition including the bioerodile extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium A variety of compounds or mixtures including phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, . In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, . In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include withexol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the drug form, the administration route and the period, but can be appropriately selected by those skilled in the art.

In addition, the present invention provides a health functional food composition for preventing or ameliorating diabetes containing Rosa davurica extract as an active ingredient.

The composition may be any one selected from powders, granules, pills, tablets, capsules, candies, syrups and beverages, but is not limited thereto.

When the health functional food composition of the present invention is used as a food additive, the health functional food composition may be added as it is, or may be used together with other food or food ingredients, and suitably used according to a conventional method. The active ingredient may be suitably used depending on its intended use (prevention or improvement). Generally, the health functional food composition of the present invention is added in an amount of not more than 15 parts by weight, preferably not more than 10 parts by weight based on the raw material, when the food or beverage is produced. However, in the case of long-term intake intended for health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount of more than the above range.

There is no particular limitation on the kind of the health functional food. Examples of the foods to which the health functional food composition can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen and other noodles, gums, ice cream, soups, Drinks, alcoholic beverages, and vitamin complexes, all of which include health foods in a conventional sense.

In addition, the health functional food composition of the present invention can be produced as a food, particularly a functional food. The functional food of the present invention may contain ingredients that are conventionally added. For example, proteins, carbohydrates, fats, nutrients, and seasonings. For example, in the case of a drink, a natural carbohydrate or a flavoring agent may be included as an additional ingredient in addition to the active ingredient. The natural carbohydrate may be selected from the group consisting of monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose etc.), oligosaccharides, polysaccharides (e.g., dextrin, cyclodextrin, , Xylitol, sorbitol, erythritol, etc.). The flavoring agent may be a natural flavoring agent (e.g., tau Martin, stevia extract, etc.) and a synthetic flavoring agent (e.g., saccharin, aspartame, etc.).

In addition to the above health functional food composition, it is also possible to use various nutrients, vitamins, electrolytes, flavors, colorants, pectic acids and salts thereof, alginic acid and its salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, A carbonating agent used in beverages, and the like. Although the ratio of the above-mentioned ingredients is not critical, it is generally selected in the range of 0.01 to 0.1 part by weight based on 100 parts by weight of the health functional food composition of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited thereto.

Manufacturing example  One. Raw novae  Preparation of extracts by site

The livestock used in the present invention was cultivated in Jeongseon-gun Jeongseon-gun Jungseon-eup Gyeonggi-do (Gangseon-gun Jeongseon-eup Jeongseon-eup Jeongseon-eup) and Jeongseon-gun Agricultural Technology Center in Gangwon-do and classified into three parts (stem, fruit, leaf and root) Respectively. After drying each of the seedlings, fruit, leaves and roots were pulverized, 200 ml of methanol was added to each powder of about 10 g of raw nervous spots, and the mixture was stirred at 250 rpm at room temperature (25 ± 1 ° C) And extracted to obtain an extract. The methanol extract of the raw nervous area obtained above was passed through a Buchner funnel having a filter paper (No. 2, Whatman, Maidstone, England) to remove the residues, followed by filtration under reduced pressure. The filtrate was filtered through a rotary vacuum evaporator, Tokyo Rikakikai Co., Ltd., Tokyo, Japan) and then lyophilized to prepare a powder, which was stored in a freezer at -20 ° C, and used in the following examples.

Example  One. Raw novae  Of extracts DPPH  Radical scavenging activity

The antioxidative activities of the methanol extracts of the raw nematode (stem, fruit, leaf and root) were evaluated by the minimal modified DPPH (1,1-diphenyl-2-picryl-hydrazyl) method (Borse, B. et al., 2007, Journ al of Agricultural Food Chemistry, 55, 1750-1754). DPPH solution (6 × 10 ^-5 M) was dissolved in dimethyl sulfoxide (DMSO) and mixed uniformly with a vortex mixer. The prepared DPPH solution (900 μl), 100 μl of distilled water (control) or 100 Mu] l of the extracts of the raw nematode (experimental group) were mixed and the absorbance at 517 nm was measured to confirm DPPH radical scavenging activity.

For the sample group correction, 100 μl of Bovine Nodule extract was mixed with 900 μl of DMSO alone (sample group) in place of the DPPH solution. BHT (dibutyl hydroxy toluene) and L-ascorbic acid Were used as positive control. The control, experimental and specimen groups were transferred to a 96-well microplate and reacted for 1 hour in the dark. Absorbance was measured at 517 nm using a microplate reader (BioTek Instruments, Inc., Winooski, USA) The radical scavenging activity was calculated using the following equation.

DPPH radical scavenging activity (%) = [1 (A1 - (A2 - A3)) / A1] 100

A1 is the absorbance of the control group; A2 is the absorbance of the experimental group; A3 is the absorbance of the sample group

As a result, as shown in FIG. 1, it was confirmed that DPPH radical scavenging activity was increased in a concentration-dependent manner by the extract of Bovine Nodule, and the result was similar to that of the positive control vitamin C.

Example  2. Raw novae  Cell Viability Analysis by Extract Treatment

C2C12 (mouse muscle cell cell line) cells were maintained in at 37 ℃ and conditions of 5% CO ₂ heat inactivated (heat inactivation) with 10% FBS (fetal bovine serum) and antibiotics, the DMEM (Eagle's minimal essential medium) supplemented with and Respectively. For root canal differentiation, C2C12 cells were seeded at 6 × 10 ⁴ cells / well in a 6-well plate and after 48 hours (over 80% fusion) the medium was DMEM medium containing 1% (v / v) FBS For 2, 4, and 6 days. The crude extract methanol extract and insulin, a positive control, were processed after completion of cell differentiation of root canal. In this Example 2, in order to measure the cell viability according to the extract treatment of the raw nerve portion, C2C12 cells were inoculated on a 24-well plate and cultured for 24 hours, then replaced with Dulbecco's modified Eagle's medium, and MTT (3- (4, 5-dimethylthiazol-2yl) -2,5-diphenyl-2H-tetrazolium bromide) reagent was added to each well in an amount of 50 μl each. After reacting for 3 hours, the medium was removed and 500 μl of DMSO And the absorbance was measured at 540 nm using a microplate reader. The absorbance is the average value obtained by repeating the experiment three times or more for each treatment. As a result, as shown in FIG. 2, all the extracts of the raw nerve portion did not show cytotoxicity, and it was confirmed that cell proliferation was rather occurred.

Example  3. Raw novae  The extract of each part of α-glucosidase (α- glucosidase ) Inhibitory activity

The α-glucosidase inhibitory activity of the extract of the raw nematode was measured by the method of Aparna et al., 2009, Phytother Res., 23 (4), 591-596. The enzyme was dissolved in 100 mM phosphate buffer (pH 7.0) containing 0.2% BSA and 0.02% NaN ₃ to a concentration of 0.7 U / mL, and the enzyme solution was used as the enzyme solution. The? -Glucosidase (Sigma, USA) Nitrophenyl α-D-glucopyranoside (Sigma, USA) was dissolved in 100 mM phosphate buffer (pH 7.0) to a concentration of 5 mM and used as a substrate solution. More specifically, 10 .mu.l of each extract (hereinafter referred to as "sample") was added to 96-well plates at concentrations of 10, 30, 100, and 300 .mu.g / ml, (Acarbose, Sigma, USA), which is an inhibitor of α-glucosidase, was treated at the same concentration as that of the sample as a positive control. 50 μl of the enzyme solution was added to the plate containing the sample and 100 mM phosphate buffer (pH 7.0) containing 0.2% BSA and 0.02% NaN ₃ was added to the blank plate. ) Was preincubated at room temperature for 10 minutes, and 50 μl of the substrate solution was added thereto. The mixture was reacted at room temperature for 5 minutes, and the plate was immersed in a microplate reader (Molecular Devices, Sunnyvale, CA, USA) was used to measure the absorbance at 405 nm. The enzyme inhibition activity was calculated from the difference of the absorbance after the addition of the substrate.

Enzyme activity inhibition (%) = (1- (ODS-ODB) / C) 100

ODS: absorbance of sample, ODB: absorbance of blank without enzyme

C: Absorbance of the control without addition of the sample

As a result, as shown in Fig. 3, the extract of the raw nerve sparing portion significantly inhibited the activity of? -Glucosidase from the positive control group of acabose, and in particular, the extract of 100 and 300 μg / ml In the case of treatment with nematode stem extract, α-glucosidase inhibitory activity was higher than that of leaf extract, root extract and fruit extract, which are known to have antidiabetic effects.

Example  4. Raw novae  Glucose uptake activity of extracts by site

In Example 4, muscle cells were starved in serum-free DMEM medium for 4 hours and then washed with PBS. Subsequently, fresh serum-free DMEM medium was replaced with fresh serum-free DMEM medium. The extracts were then treated for 30 minutes and treated with insulin as a positive control for 10 minutes. Glucose uptake was measured by measuring the amount of glucose contained in the medium and calculating the amount of glucose transferred into the cells. As a result, it was confirmed that the glucose uptake into the muscle cells was increased when the extract of the raw nevre part was treated as shown in FIG. 4. In particular, when treated with 1000 μg / ml of the nevus extract, Glucose absorption effect similar to that of the control group, insulin. From these results, it was confirmed that the extract of Staphylococcus aureus has an effect of accelerating glucose uptake into muscle cells.

Example  5. In Type 1 diabetes-inducing animal models Raw novae  Antidiabetic effect of stem extract treatment

Male rats (mal, Nara Biotech, Korea) of 280-300 g were purchased and maintained at a temperature of 40-70% in a room temperature cage and tested in an environment where day and night alternated for 12 hours. Streptozotocin (STZ, Sigma, USA), which was dissolved in citrate buffer (2.5 ml / kg) at pH 4.5, was injected into the rat via the tail vein through the tail vein. Mg / kg. ≪ / RTI > In the normal group, only citrate buffer (2.5 ml / kg) was injected into the tail vein of the rats. Three days after the intravenous injection of STZ, blood was collected from the tail vein of the rats and blood glucose was measured using a blood glucose meter (SureStep, LIFESCAN INC., USA). The blood glucose level was measured to be 300 mg / dl or more Only rats were used in this experiment. Normal group was normal control group, and Dia group was diabetes control group in which diabetes was induced by streptozotocin administration. In the Dia + rhodobacterium extract-treated group, RDA of the present invention was taken at 62.5, 125, 250 and 500 mg / kg, respectively, after diabetes was induced by streptozotocin administration. The administration of the citrate buffer or nevus extract to the experimental animals was orally administered at a daily dose of 100 mg / kg for 4 weeks.

1) Blood sugar lowering effect

In the case of Streptozotocin (STZ) -induced type 1 diabetic animal model, in which the antidiabetic effect was most excellent in Examples 3 to 4, Diabetic effect was confirmed. As a result, as shown in FIG. 5, the blood glucose level was significantly increased in the diabetic control group (Dia) compared to the normal group, whereas the test group treated with the extract of the present invention after diabetes induction by streptozotocin administration (Dia + R.davruica stem) showed a significant decrease in blood glucose level compared to the diabetic control group in a concentration-dependent manner.

2) Weight loss inhibition effect

After the diabetes was induced by streptozotocin administration, the body weight changes of the experimental group and the control group (Normal and Dia) of the present invention were compared. As a result, as shown in FIG. 6, in the diabetic control group (Dia), the body weight decreased from 3 days after the administration and after 12 days, the body weight decreased by about 20% compared with the normal group. On the other hand, when the extract of Dia + R. davruica stem according to the present invention was administered after the induction of diabetes by the administration of streptozotocin, the effect of suppressing the weight loss was suppressed compared to the diabetic control group (Dia). Therefore, in the animal model of type 1 diabetes induced by STZ administration, glucose utilization of the cells is decreased and metabolic characteristics of starvation state are exhibited. Therefore, the effect of suppressing the weight loss phenomenon in the experimental group administered with the extract of Rhizoma of the present invention is limited .

3) In drinking water and feed intake Raw novae  Influence of stem extracts

In type 1 diabetes induced by streptozotocin (STZ) administration, hyperglycemia, diabetic and polyuric characteristic diabetic symptoms are seen. The influence of the extract of the present invention on the beverage and the dietary intake of the animal model of type 1 diabetes was measured for more than 4 weeks and calculated based on the average daily intake, and is shown in Fig. The normal group showed no significant difference in drink and dietary intake over the entire period of the experiment, but the diabetes control group (Dia) induced by streptozotocin administration was 4 times more drinkable, 1.5 times more than the dietary intake Respectively. On the other hand, in the experimental group (Dia + R.davruica stem) administered with the extract of the present invention after diabetes induction by streptozotocin administration, the drink and dietary intake were significantly decreased in a concentration-dependent manner compared to the diabetic control group.

4) Insulin concentration in blood Raw novae  Effect of stem extract

The insulin concentration in blood was measured using the mouse insulin ELISA kit according to the present invention. Specifically, insulin levels were measured after administration of streptozotocin (STZ) for 3 days from the induction of diabetes mellitus of the present invention over 4 weeks. As a result, as shown in FIG. 8, Dia), the insulin concentration was significantly reduced in the group treated with 500 mg / kg of niacin extract (Dia + R. davruica stem), about 10 times that of the diabetic control group, Normal).

5) Raw novae  Effect of stem extract on pancreatic insulin-producing cells

Insulin is produced by beta cells present in pancreatic islets, which are the most important hormones in the body's blood sugar control. When streptozotocin (STZ) is administered to experimental animals, beta cells of the pancreas are destroyed and abnormal pancreas It is known to cause small form. Therefore, the effect of the extract of Staphylococcus aureus on the insulin secretion of Islets and beta cells of pancreatic tissues was confirmed based on the result that the extract of Staphylococcus aureus induces an increase in blood insulin concentration. Rhodiola extract was administered to streptozotocin-induced type 1 diabetes model for more than 4 weeks and hematoxylin / Eosin staining was performed to examine the islets by taking the pancreas As a result, pancreatic islets and insulin-producing beta cells were markedly damaged in the diabetic control group (Dia) as shown in FIG. 9, whereas the streptozotocin-treated diabetic rats extract (Dia + R If the administration of davruica stem), addition to, increasing the area of the islet (islets) was confirmed that the increase in the number of beta cells, especially with increasing dosage levels of the green yeolgwi stem extract that is positive in the beta-cells cell growth Could know.

Claims (8)

A pharmaceutical composition for preventing or treating type 1 diabetes containing Rosa davurica stem extract as an active ingredient. delete delete The pharmaceutical composition for preventing or treating type 1 diabetes according to claim 1, wherein the extract is extracted using water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof. The pharmaceutical composition for preventing or treating type 1 diabetes according to claim 1, further comprising a pharmaceutically acceptable carrier, excipient or diluent in addition to the active ingredient. The pharmaceutical composition for the prevention or treatment of type 1 diabetes according to claim 1, wherein the composition is prepared by any one of formulations selected from the group consisting of capsules, powders, granules, tablets, suspensions, emulsions, syrups and aerosols. A health functional food composition for preventing or ameliorating type 1 diabetes containing Rosa davurica stem extract as an active ingredient. The composition according to claim 7, wherein the composition is prepared from any one of powder, granule, ring, tablet, capsule, candy, syrup and beverage. .

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