KR101949153B1 - A pharmaceutical composition comprising viscozyme treated extract of Morus alba leaves for preventing or treating diabetes mellitus - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating diabetes containing an extract of mulberry leaves which is bioconverted by treating viscozyme as an active ingredient. The extract has an excellent effect of increasing glucose absorption capacity and insulin secretion ability if treating on muscle cells, fat cells or pancreas cells. In addition, an administration of the extract to diabetic-induced animal models significantly reduces blood sugar, glycated hemoglobin production and insulin resistance. Therefore, the extract can be usefully used as the pharmaceutical composition for preventing or treating diabetes.

Description

TECHNICAL FIELD [0001] The present invention relates to a pharmaceutical composition for preventing or treating diabetes mellitus comprising an extract of mulberry leaf,

The present invention relates to a pharmaceutical composition for the prevention or treatment of diabetes mellitus comprising an extract of mulberry leaf which is bioconverted by treatment with viscozyme as an active ingredient.

Diabetes is caused by insulin secretion problems such as decreased insulin secretion and resistance due to genetic and environmental reasons, or abnormal function of insulin, glucose in the blood can not be transferred to / stored in the cells and excessively increased in the blood, It is a metabolic disease with hyperglycemia which is much higher than normal people. Diabetes mellitus, hypertension, stroke, chronic renal failure, diabetic ulcer, diabetes mellitus, diabetes mellitus, hypertension, diabetes mellitus, diabetes mellitus, Retinopathy (WHO, Diabetes Fact sheet No.312, 2013), and it is the fourth most common cause of death in Korea.

Type 1 Diabetes (Type 1 Diabetes, Type 1 Diabetes, Type 1 Diabetes, which is a type 1 diabetes that produces little or no insulin in the body due to inherited genetic factors, and is caused by environmental factors such as obesity, lack of exercise, Type 2 diabets, gestational diabetes, and other forms of diabetes, which do not produce enough insulin in the body or cause insulin resistance to cells that do not respond to insulin.

Type 2 diabetes, which accounts for the majority of diabetes, is known to cause chronic hyperglycemia due to a combination of two causes: insulin secretory disorder and insulin resistance. Among them, insulin secretory disorder refers to a situation in which insufficient amount of insulin is secreted from the beta cells of the pancreas depending on the concentration of glucose, and includes both a quantitative decrease in insulin-releasing beta cells and a functional distribution disorder. On the other hand, insulin resistance means that the secreted insulin reaches the target organ in the bloodstream, but the insulin action and sensitivity of the target cell are lowered. Such causes of insulin resistance include genetic causes, obesity, decreased physical activity, and hyperglycemia or dyslipidemia. Type 2 diabetes is caused by a combination of the two etiologic causes of the disease, like cogs. That is, insulin resistance requires that a greater amount of insulin is secreted to overcome resistance, and conversely, hyperglycemia, which is caused by insufficient insulin secretion, may again exacerbate insulin resistance (International Patent Publication No. 2015-016682 ).

In general, as the duration of diabetes increases, blindness, end-stage renal failure, neurological disease, amputation and infectious diseases due to chronic complications will increase rapidly. In particular, cerebrovascular disease and cardiovascular disease are the most common complications of diabetes. About 3/4 of the deaths are deaths due to diabetic complications, and the risk of death due to cardiovascular complications is also increased by 10 years It was reported to increase by 24% every time. The prevalence of coronary artery disease is two times higher in diabetic patients than in normal controls, and the prevalence of peripheral vascular disease is about three times higher than that of normal subjects (Koboram et al., 21 (5), 382-388, 2006; Schneider , SH et al., Metabolism, 37 (10), 924-929, 1988). The cause of such atherosclerosis in diabetes is hyperglycemia, lipid metabolism abnormality, hyperinsulinemia, hypertension, changes in blood coagulation mechanism (Aronoff, S. et al., Diabetes Care, 23 (11), 1605-1611, 2000). These complications are associated with a decline in the quality of life of individual individuals, as well as significant social and economic losses.

Diabetes management is the most basic control of blood sugar, exercise and diet therapy to regulate blood sugar. If blood sugar is not controlled in this way, drug therapy is started. Type 2 diabetes is managed by administering insulin (WHO, Diabetes Fact sheet No.312, 2013) and type 2 diabetes is controlled by oral hypoglycemic agents or insulin administration (Kim Dong-rim, KRC, 2012, Konkuk University Hospital) . However, in the case of existing oral hypoglycemic agents, various side effects such as hypoglycemia, diarrhea, abdominal bloating, weight gain, lactic acid blood, cardiac toxicity and liver toxicity are caused not only in the positive aspect of maintaining normalization of blood glucose continuously, Pancreatic beta cells that function as insulin secretion are irreversibly damaged and destroyed, resulting in insulin resistance. In addition, insulin, which is the most commonly used drug for diabetes, has a disadvantage of incontinence and rejection because it has to be subcutaneously injected 2 or 3 times a day, which also has a great possibility of inducing hypoglycemia. Therefore, it is necessary to develop a more effective and safe drug which has a pancreatic β-cell protection function without any hypoglycemic effect and can significantly reduce the blood glucose level and drastically solve the insulin resistance even in the long-term use.

Morus alba is a generic term for deciduous trees or shrubs belonging to the genus Chrysanthemum morifolium and the mulberry of the dicotyledonous plant Nettles, and its origin is temperate and subtropical. Leaves are used for silkworm (silkworm feeding), bark for medicinal or paper use, and fruit for food and medicinal use.

Mulberry leaves have various physiological activities such as anticancer, antihypertensive, anti-diabetic, antihyperlipidemic, antioxidant and heavy metal removal ability, and are effective for antihypertensive, cholesterol lowering and body fat accumulation inhibition, Various functions such as intestinal function, immunity and anti-inflammatory activity are being studied.

Mulberry leaves generally contain a large amount of minerals such as water, carbohydrates, proteins, steroids, amino acids and vitamins. Among them, the major physiologically active ingredient is 1- (1-deoxynojirimycin, 1-DNJ), flavonoids including the blood pressure lowering component GABA (γ-aminobutyric acid) and the antioxidant component rutin, and phenolic acid phenolic acids, stilbene, and 2-arylbenzofuran derivatives.

Meanwhile, as a prior art related to a composition for the prevention or treatment of diabetes mellitus comprising an extract of mulberry leaf treated with viscose, the above-mentioned prior art [Kim, Young Wook, Dept. of Master's dissertation of Pusan National University, 2017] Korean Patent No. 10-0653460 discloses an antidiabetic composition containing a mulberry leaf extract and a fenugreek extract, Korean Patent No. 10-1274664 discloses an antidiabetic composition comprising a mulberry leaf extract or a fermented product Korean Patent No. 10-1851195 discloses a fermented mulberry leaf, a fermented mulberry leaf extract and use thereof, and Korean Patent Laid-open No. 10-2017-0061753 discloses an antioxidant and anti-inflammatory activity A method for producing an increased fermentation mulberry extract and a fermented mulberry extract prepared as described above have been disclosed.

However, as in the present invention, the mulberry leaf extract treated with biscotti extract of mulberry leaf extract has excellent glucose absorption ability and insulin secretion ability, and in particular, it has an effect of lowering blood glucose level, reducing glycated hemoglobin production, There is no previous report disclosing that the effect of reducing insulin resistance is excellent.

Korean Registered Patent No. 10-0653460, an antidiabetic composition containing mulberry leaf extract and fenugreek extract, registered on Nov. 27, 2006. Korean Registered Patent No. 10-1274664, beverage composition containing mulberry leaf extract or fermented product, registered on June 07, 2013. Korean Registered Patent No. 10-1851195, fermented mulberry leaf, fermented mulberry leaf extract and use thereof, registered on Apr. 17, 2018. Korean Patent Laid-Open No. 10-2017-0061753, a method for preparing fermented mulberry extract with increased antioxidant and anti-inflammatory activity, and a fermented mulberry extract prepared as described above, published Jun. 07, 2017. International Patent Publication No. 2015-016682, pharmaceutical composition for preventing or treating diabetes, method for preventing or treating diabetes, screening method for treating diabetes mellitus, published on February 05, 2015.

Correlation between ankle-brachial index and diabetic peripheral vascular disease, such as Gobalam, Journal of Endocrinology, 21 (5), 382-388, 2006. Kim, Dong-Rim, Drug Therapy for Type 2 Diabetes, Konkuk University Hospital, KRC, 2012. Kim, Young Wook, Bioconversion of Mulberry Leaf Using Viscozyme L, Master's Thesis, Pusan National University, 2017. Aronoff, S. et al., Pioglitazone hydrochloride monotherapy improves glycemic control in patients with type 2 diabetes: a 6-month randomized placebo-controlled dose-response study. The Pioglitazone 001 Study Group, Diabetes Care, 23 (11), 1605-1611, 2000. Han, J. H. et al., The effects of propionate and valerate on insulin responsiveness for glucose uptake in 3T3-L1 adipocytes and C2C12 myotubes via G protein-coupled receptor 41, PLoS One, 9 (4), e95268, 2014. Kitabachi, A. E. et al., Hyperglycemic crises in adult patients with diabets, Diabetes care, 32 (7), 1335-1345, 2009. Nakamura, T. et al., Establishment and pathophysiological characterization of type 2 diabetic mouse model produced by streptozotocin and nicotinamide, Biol Pharm Bull., 29 (6), 1167-1174, 2006. Nan, M. H. et al., Construction of adiponectin-encoding plasmid DNA and gene therapy of non-obese type 2 diabetes mellitus, J Drug Target., 18 (1), 67-77, 2010. Schneider, S. H. et al., Impaired fibrinolytic response to exercise in type II diabetes mellitus: Effect of exercise and physical training, Metabolism, 37 (10), 924-929, 1988. WHO, Diabetes Fact sheet No.312, 2013.

It is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of diabetes mellitus comprising the biologically converted mulberry leaf extract as an active ingredient by treating viscozyme.

The present invention relates to a pharmaceutical composition for preventing or treating diabetes mellitus comprising an extract of mulberry leaf treated with viscozyme as an active ingredient.

The viscose is added to the mulberry leaf extract in an amount of 1 to 3 wt%, treated at 40 to 60 ° C for 10 to 24 hours, preferably 1 to 2 wt% of biscotti to the mulberry leaf extract having a pH of 4 to 7 It is treated at 40 ~ 50 ℃ for 10 ~ 20 hours. When the biscottin is treated out of the above-mentioned conditions, the enzyme reaction does not sufficiently take place or it occurs excessively to lower the anti-diabetic activity, or the anti-diabetic activity increase rate is not so large, which is not economical.

The viscose can be used as a commercial enzyme such as arabinase derived from Aspergillus aculeatus , carbohydrase, cellulase, beta-glucanase, Hemicellulase, and xylanase, and is a complex enzyme having strong pectinytic activity (pectolytic activity).

The mulberry leaf extract may be selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol, and isobutanol. The mulberry leaf extract is an extract of mulberry leaves extracted with water, C1 to C4 alcohol or a mixed solvent thereof. have.

Water, C1 to C4 alcohol, or a mixed solution thereof used in the production of the mulberry leaf extract may be used in an amount of 5 to 100 times the volume (5 to 100 L based on 1 kg of mulberry leaves) based on the weight of mulberry leaves used, Can be used in the range of 5 to 50 times. In addition, the above procedure can be repeated 1 to 4 times.

Although the extraction time of the mulberry leaf extract is not particularly limited, it is preferable to extract the mulberry leaf extract within 10 minutes to 1 day. The extraction method may be any extraction method commonly used, for example, pressure extraction, immersion (cold and warm) Extraction, and reflux extraction may be used, but the pressure is preferably extracted at 40 to 121 ° C for 1 to 5 hours.

The biscuit-treated mulberry leaf extract obtained from the above process may be dried using a conventional drying method such as vacuum drying, spray drying, or freeze drying, and then pulverized to about 50 to 70 mesh.

The pharmaceutical composition according to the present invention can be formulated into a suitable form together with a commonly used pharmaceutically acceptable carrier. &Quot; Pharmaceutically acceptable " refers to a composition that is physiologically acceptable and, when administered to humans, does not normally cause an allergic reaction such as gastrointestinal disorders, dizziness, or the like.

The pharmaceutical compositions may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, external preparations, suppositories and sterilized injection solutions according to a conventional method . Examples of carriers, excipients and diluents that can be contained in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, But are not limited to, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl paraoxybenzoate, propylparaxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, it is prepared using diluents or excipients such as fillers, stabilizers, binders, disintegrants, surfactants and the like which are 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 and the like. 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 witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The pharmaceutical composition containing the extract disclosed in the present invention as an active ingredient can be administered to mammals such as rats, livestock, and humans in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection. The dosage will depend on the age, sex, body weight, the particular disease or condition being treated, the severity of the disease or condition, the time of administration, the route of administration, the absorption, distribution and excretion of the drug, It depends on judgment. Dosage determinations based on these factors are within the level of ordinary skill in the art and generally the dosage ranges from 0.001 mg / kg / day to 2000 mg / kg / day. A more preferred dosage is 0.01 mg / kg / day to 500 mg / kg / day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

The present invention also provides a health functional food for preventing or ameliorating diabetes, comprising a mucilast leaf extract treated with viscose and a food-acceptable food-aid additive. The health functional food refers to a food prepared or processed by using a raw material or a component having useful functionality and includes, for example, a health supplement food, a functional food, a nutrient, and an auxiliary agent.

The extract may be added in an amount of preferably 0.001% by weight to 50% by weight, more preferably 0.001% by weight to 30% by weight, and most preferably 0.001% by weight to 10% by weight, based on the total weight of the whole food. The health functional food of the present invention includes forms such as tablets, capsules, pills, and liquids. Examples of the foods to which the extract of the present invention can be added include various foods, beverages, gums, tea, vitamins .

In yet another aspect,

(Step 1) adding an extraction solvent having a volume of 5 to 100 times the volume of mulberry leaves;

(Step 2) Extracting the mixture of Step 1 at 40 to 121 캜 for 1 to 5 hours to obtain a mulberry leaf extract; And

(Step 3) Adding 1 to 3% by weight of viscose to the mulberry leaf extract of the above two steps and treating at 40 to 60 ° C for 10 to 24 hours; And a method for producing the mulberry leaf extract.

The present invention relates to a pharmaceutical composition for the prevention or treatment of diabetes mellitus comprising an extract of mulberry leaf which is bioconverted by treatment with viscozyme as an active ingredient. When the extract is administered to an animal model in which diabetes has been induced, it has been found that treatment of the extract with muscle cells, adipocytes or pancreatic cells has an excellent effect of increasing glucose uptake ability and insulin secretion ability, And can be usefully used as a pharmaceutical composition for preventing or treating diabetes.

Brief Description of the Drawings Fig. 1 is a graph showing changes in glucose absorption ability in muscle cells (C2C12) according to the treatments of Examples and Comparative Examples of the present invention.
Fig. 2 is a graph showing changes in glucose uptake ability according to the treatment of Examples and Comparative Examples of the present invention in adipocytes (3T3-L1). Fig.
3 is a graph showing changes in insulin secretion ability in pancreatic cells (HIT-T15) according to the treatments of Examples and Comparative Examples of the present invention.
FIG. 4 is a graph showing cytotoxicity of muscle cells (C2C12), adipocytes (3T3-L1), and pancreatic cells (HIT-T15) according to the treatment of Examples and Comparative Examples of the present invention.
FIG. 5 is a graph showing blood glucose levels measured in accordance with oral glucose load in Examples and Comparative Examples of the present invention in an animal model in which diabetes has been induced.
FIG. 6 is a graph showing blood glucose levels according to insulin administration in the diabetes-induced animal model of the present invention and Comparative Example.
FIG. 7 is a graph showing blood glucose levels measured in an animal model in which diabetes has been induced according to the treatment of Examples and Comparative Examples of the present invention. FIG.
FIG. 8 is a graph showing an analysis result of plasma insulin levels according to the treatments of Examples and Comparative Examples of the present invention in an animal model in which diabetes was induced.
FIG. 9 is a graph showing the results of analysis of insulin resistance values according to the treatments of Examples and Comparative Examples of the present invention in an animal model in which diabetes was induced.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Rather, the intention is to provide an exhaustive, complete, and complete disclosure of the principles of the invention to those skilled in the art.

Example 1 Preparation of Miscanthus Leaf Extract Treated with Viscot Bean

To 100 kg of mulberry leaves, 1800 L of water was added, and 19 kg of mulberry leaf extract was obtained by pressure extraction at 121 ° C for 3 hours. 100 kg of the mulberry leaf extract obtained by repeating the above procedure was adjusted to pH 5.0 with dilute hydrochloric acid and treated with 1 kg of viscose (Celluclast 1.5 L, Novozymes A / S, Denmark). This was reacted at 45 캜 for 15 hours to obtain a viscose-treated mulberry leaf extract of Example 1 of the present invention.

≪ Comparative Example 1 > Preparation of comparative mulberry leaf extract (1)

The procedure of Example 1 was followed, except that 1.8 g of water was added to 100 g of mulberry leaves, followed by pressure extraction at 121 ° C for 3 hours to obtain a mulberry leaf extract of Comparative Example 1. [

≪ Comparative Example 2 > Preparation of comparative viscose-treated mulberry leaf extract &

100 g of mulberry leaves were suspended in 1 L of water and suspended, treated with 5% biscotti, and reacted at 45 DEG C for 48 hours at 150 rpm. Three times volume of methanol was added to the reaction mixture, Mulberry leaf extract was obtained.

EXPERIMENTAL EXAMPLE 1 Confirmation of change in glucose uptake capacity and insulin secretion &

(3T3-L1) and pancreatic cells (HIT-T15), the glucose uptake ability of muscle cells (C2C12), adipocytes (3T3-L1) and pancreatic cells (HIT-T15) were determined by reference to the previous article [Han, JH et al, PLoS One, 9 (4), e95268, 2014] Insulin secretion ability was confirmed.

Experimental Example 1-1. Cell culture

(C2C12, Cell Applications Inc., San Diego, Calif., USA), adipocytes (3T3-L1, Cell Applications), and the like were used to examine the antidiabetic effect of the biotransformed mulberry leaf extract (HIT-T15, Cell Applications Inc., San Diego, CA, USA) and pancreatic cells (HIT-T15, San Diego, CA, USA)

C2C12 cells, which are muscle cells, were placed in a 4.5 g / L D-glucose DMEM medium supplemented with 10% FBS (Fetal Bovine Serum, GIBCO) and 1% penicillin / streptomycin, % CO ₂ conditions (CO ₂ cell incubator, Heracell 150i, Thermo) was subcultured in 2-3 day intervals, the cells were used for passage to the number of 8-10. Cells were isolated using 0.25% trypsin-EDTA (GIBCO) when C2C12 muscle cells were about 80-90% confluence in a cell culture dish. Thereafter, the cells were centrifuged to allow the cells to settle, diluted to 200,000 cells per ml using 10% FBS DMEM medium, and then added to a 24-well plate at a rate of 500 μl until the plate was filled. When the cells were filled, differentiation was induced by adding 1% FBS and 1% penicillin / streptomycin to 4.5 g / L D-glucose DMEM. The differentiation medium was changed 3 times at intervals of 36 hours. After 24 hours from the third replacement, the final differentiation medium was replaced and the experiment was started 12 hours later.

3T3-L1 cells expressing mouse lipid precursors were cultured in DMEM medium containing 10% BS (Bovine Serum, Gibco, USA) and 1% penicillin / streptomycin at 37 ° C and 5% CO ₂ . When the cultured cells were inoculated into a 24-well plate and the proliferation was 90% or more, IBMX (3-isobutyl-1-methylxanthine), a differentiation inducing substance, was added to DMEM medium containing 10% FBS and 1% penicillin / streptomycin, , Dexamethasone and insulin were induced to induce differentiation for 3 days.

HIT-T15 cells, pancreatic cells, were subcultured in RPMI-1640 medium containing 11.1 mM glucose and 10% FBS (Fetal Bovine Serum) at intervals of 2 to 3 days at 37 ° C and 5% CO ₂ , Cells with passage numbers 8-10 were used. When HIT-T15 pancreatic cells were about 80-90% confluent in cell culture dishes, cells were isolated using 0.25% trypsin-EDTA. Thereafter, the cells were centrifuged to allow the cells to settle, diluted to a concentration of 200,000 cells / ml using 10% FBS DMEM medium, and 200,000 cells / well were inoculated on a 24-well plate and cultured for 2 days. .

Experimental Example 1-2. Identification of changes in glucose uptake in muscle cells

After the C2C12 muscle cells of Experimental Example 1-1 were completely differentiated, the glucose uptake experiment was started. The differentiated cells were washed twice with 1X PBS (phosphate buffered saline), and then cultured in DMEM supplemented with 5% BSA DMEM, 1 g / L D-glucose, GIBCO) and stabilized for 2 h at 37 ° C and in a CO ₂ cell incubator. After 2 hours, the samples dissolved in 20% DMSO (Example 1, Comparative Example 1 and Comparative Example 2) were treated with concentration (5, 10 占 퐂 / ml) for 1 hour and then DMEM medium was removed and KRPH buffer 1.2 mM KH ₂ PO ₄ , 1.2 mM MgSO ₄ , 1.3 mM CaCl ₂ , 118 mM NaCl, 5 mM KCl, 30 mM HEPES, pH 7.5). Controls and samples (Example 1, Comparative Example 1 and Comparative Example 2) were diluted with KRPH buffer containing 5% BSA, treated once more with the washed cells, and reacted for 30 minutes.

Cells were treated with 1 mM insulin for 30 min and radioactive cocktail (KRPH buffer, 2-deoxyglucose, [3H] -glucose) was added and reacted for 10 min. Then cytochalasin B ), And then washed twice with cold PBS. Then, lysis buffer (PBS, 10N NaOH, 10% SDS solution) was added and the cells were lysed at 4 ° C for 2 hours. The cell lysate was placed in a special container together with a liquid scintillation cocktail (Ultima Gold, PerkinElmer), and the value of [3H] -glucose introduced into the cell was measured using a liquid scintillation counter (1600 TR, Packard) to determine the glucose uptake capacity, which is shown in Fig.

Referring to FIG. 1, the mulberry leaf extract treated with viscose of Example 1 of the present invention was treated with biscotti extract and mulberry leaf extract, The extracts showed increased sugar uptake in a concentration dependent manner.

In particular, although not shown in FIG. 1, in the case of treating biscocolamine at 1% as in the case of the present invention, instead of treating biscocolamine at 5% in the same manner as in Comparative Example 2, And the enzyme reaction was inhibited by the components. Thus, it was confirmed that the antidiabetic activity was very low.

Thus, as in Example 1 of the present invention, the composition obtained by treating biscotti extract with mucilast leaf extract had a smaller amount of viscose and a sufficient amount of enzymes as compared with the composition extracted with a solvent after biscottin treatment in mulberry leaf powder as in Comparative Example 2 As a result, the enzyme activity is maximized while remarkably reducing the time and cost, and thus it was found that the composition is a diabetic therapeutic composition having excellent antidiabetic activity.

Experimental Example  1-3. Sugar in fat cells Absorption capacity  Confirm change

The medium was removed from the differentiated 3T3-L1 adipocytes of Experimental Example 1-1, washed with PBS, and replaced with DMEM supplemented with 5% BSA in DMEM medium for 1 hour at 37 ° C and in a CO ₂ cell incubator . After 1 hour, the samples (Example 1, Comparative Example 1 and Comparative Example 2) were treated with concentration (10, 20 占 퐂 / ml) for 1 hour and then the medium was removed and washed twice with KRPH buffer. After washing, 100 nM insulin was treated for 30 minutes to induce glucose uptake, radioactively cocktail (KRPH buffer, 2-deoxyglucose, [3H] -glucose) was added and the reaction was allowed to proceed for 10 minutes. B containing PBS was added to stop the glucose uptake reaction. The reaction stop solution was removed and the lysis buffer was added to dissolve the cells at 4 ° C for 2 hours. The cell lysate was placed in a special container together with the cocktail solution, and the value of [3H] -glucose introduced into the cell was measured with a liquid scintillation counting device to calculate glucose absorption capacity, which is shown in FIG.

2, the mulberry leaf extract treated with viscose of Example 1 of the present invention was treated with biscotti mulberry leaf extract and mulberry leaf powder which had not undergone bioconversion and did not undergo bioconversion, It was confirmed that the sugar uptake ability in adipocytes was increased in a concentration dependent manner compared to the extracted extracts.

Therefore, when the biscozymes of Example 1 of the present invention are treated to treat muscle cells and adipocytes in a biotransformed mulberry leaf extract according to the results of FIGS. 1 and 2, It can be seen that

Experimental Example  1-4. In pancreatic cells  insulin Secretory function  Confirm change

Experimental Example 1-1 HIT-T15 cells in 24-well plates to about 80%, ie as equally proliferation, KRBH buffer that does not contain glucose (NaCl 119mM, KCl 4.74mM _{of, CaCl 2 · 2H 2 O 2.54mM} , MgCl 2 _{· 6H 2 O 1.19mM, KH 2} PO 4 1.19mM, NaHCO ₃ 25 mM, HEPES 7 mM, pH 7.4), and then KRBH buffer supplemented with 0.3% BSA was added thereto and cultured for 30 minutes. Then, KRBH buffer containing 11.1 mM glucose was replaced, and the samples (Example 1, Comparative Example 1 and Comparative Example 2) were added for each concentration (2.5, 5 / / ml) and treated for 1 hour. The medium containing the cells is shown in FIG. 3 by measuring insulin secreted from cells with a mouse insulin kit.

Referring to FIG. 3, the mulberry leaf extract treated with viscose treatment of Example 1 of the present invention was subjected to viscozyme treatment and then extracted with a solvent after treatment with biscotti extract and mulberry leaf extract, The insulin secretion in pancreatic cells was increased in a dose - dependent manner.

Therefore, it was confirmed that the mulberry leaf extract with bioconversion treated with viscose of Example 1 of the present invention is an effective composition for treating diabetes because of its excellent effect of promoting insulin secretion.

Experimental Examples 1-5. Toxicity assessment

(50 μg / ml) were added to the cells cultured under the same conditions as those for the glucose uptake and insulin secretion evaluations. Then, C2C12 and 3T3-L1 cells were reacted for 2 hours, and HIT-T15 cells The cells were reacted for 1 hour and then cytotoxicity was assessed by MTT [3- (4,5-dimethylthiazole-2-yl) -2,5-diphenyltetrazolium bromide) assay.

The MTT reagent was added to each well at a rate of 500 g / ml and reacted for 4 hours. Then, the MTT reagent was removed and the resulting formazan crystal was dissolved in DMSO. The absorbance at 450 nm was measured with a microplate reader and is shown in Fig.

4, it was confirmed that the extracts of Example 1 and Comparative Example 1 did not cause apoptosis in both muscle cells, adipocytes and pancreatic cells.

Therefore, Example 1 of the present invention was found to be useful as a composition for treating diabetes without cytotoxicity and excellent glucose absorption ability and insulin secretion ability.

≪ Experimental Example 2: Antidiabetic effect confirmation in animal model >

First, 5-week-old male C57BL / 6 mice were purchased and purified for one week. Twenty-two-hour cycles of light and humidity were maintained at a temperature of 22 ± 2 ° C and a humidity of 50 ± 5%. High fat diet (60% Kcal) was fed from the 6th week of age and the general diet was fed to the control group. After an 8-week high-fat diets or diets were fed, a previous study [Nakamura, T. et al., Biol Pharm Bull., 29 (6), 1167-1174, 2006; According to Nan, M. H. et al., J Drug Target., 18 (1), 67-77, 2010], streptomycin and nicotinamide were administered intraperitoneally to induce type 2 diabetes. Fasting blood glucose was measured one week after the induction, and the fasting blood glucose level was measured for 7 weeks. The results are shown in Table 1, Example 1 (high-fat diet and mulberry leaf extract administered with biocyclovir), Comparative Example 1 (high fat diet and mulberry leaf extract- (High-fat diet) and Comparative Example 2 (high fat diet and mulberry leaf powder) were orally administered to each group. The blood samples collected after the experiment were analyzed to determine the levels of glycated hemoglobin and insulin levels And blood glucose were measured.

Experimental Example 2-1. Oral glucose tolerance test (OGTT)

For the test of oral glucose tolerance in animal models, each animal group divided into the control group (the type 2 diabetes-induced mouse was high-fat diet), Example 1, and Comparative Example 1, Blood was collected from the rats at 0, 15, 30, 45, 60, and 120 minutes after oral administration of glucose solution (2 g / kg). The blood was measured and plotted with a blood glucose meter (Acuu-Check, Roche Diagnostics Co., Ltd., Seoul, Korea) and shown in FIG.

5, in Example 1 and Comparative Example 1, blood glucose level was controlled within 2 hours according to glucose load. In particular, Example 1 showed excellent blood glucose control ability as compared with Comparative Example 1. Thus, it can be seen that Example 1 of the present invention is an effective composition for managing blood glucose in diabetic patients.

Experimental Example 2-2. Insulin tolerance test (ITT)

Three weeks before the end of the experiment (6th week, 4th day), each animal group was fasted for 16 hours before insulin resistance test. Then glucose solution (2g / kg) was orally administered and blood was collected from the tail Blood glucose was measured. After 30 minutes, insulin (1 U / kg) was injected subcutaneously, and blood was collected from the tail of mice at 0, 15, 30, 60, and 120 minutes, and blood glucose was measured and shown in FIG.

6, in Example 1 and Comparative Example 1 of the present invention, insulin administration regulated blood glucose control. In particular, in the group administered with 600 mg / kg of Example 1, blood glucose similar to that of metformin 300 mg / Respectively.

Thus, it was confirmed that the administration of Example 1 of the present invention resulted in normal blood glucose control by administration of insulin in the type 2 diabetic model animal.

Experimental Example 2-3. Blood glycated hemoglobin level measurement

During the 7-week experiment, the animals were fasted 16 hours before the end of the experiment, and the blood was orbitalized. Fasting glycated hemoglobin (HbA _1C ) levels were measured by absorbance at 700 nm of a microplate reader using a mouse hemoglobin A _1C kit (Crystal Hemoglobin A _1C kit, Crystal Chem, Downer Grove, USA) .

Referring to FIG. 7, it was confirmed that Example 1 of the present invention significantly inhibited the blood glycosylated hemoglobin level as compared with Comparative Example 1 and Comparative Example 2. From this, it can be seen that when Example 1 is administered, blood glycosylated hemoglobin production is reduced by reducing blood glucose in an animal model of type 2 diabetes.

Experimental Example 2-4. Plasma Insulin Level Measurement

In the 7-week experiment, the animals were fasted 16 hours before the end of the experiment, and the blood was orbitalized and centrifuged at 4000 rpm at 4 ° C for 20 minutes to separate the plasma. Fasting insulin levels in the separated plasma were measured using a mouse insulin ELSIA kit (mouse insulin ELSIA kit, Shibayagi, Shibukawa, Japan) at 450 nm of a microplate reader and shown in Fig.

FIG. 8 shows that Example 1 of the present invention was superior to Comparative Example 1 in increasing the plasma insulin level, and it was found that diabetic symptoms could be alleviated by increasing insulin secretion in the type 2 diabetic model animal.

EXPERIMENTAL EXAMPLE 2-5. Homeostasis model assessment-insulin resistance (HOMA-IR)

When type 2 diabetes is induced, the increase in fasting blood glucose and resulting insulin secretion in the pancreatic? -Cells is excessive, but insulin action does not occur smoothly in end organs (adipose tissue, skeletal muscle, etc.) In the case of insulin resistance can be seen.

In the present invention, the HOMA-IR value, which is an index for evaluating the degree of insulin resistance and the function of the? -Cells (insulin secretory function), was used. After the end of the experiment of Experimental Example 2-5, the fasting glucose and plasma insulin 1 to calculate the insulin resistance index, which is shown in FIG.

[Equation 1]

9, the HOMA-IR level of Example 1 of the present invention was lower than that of Comparative Example 1. In particular, when Example 1 was administered at 600 mg / kg, metformin 300 mg / kg was administered as a positive control It can be confirmed that the composition is insulin resistance-reduced to a degree similar to the case, and thus it can be usefully used as a composition for the treatment of diabetes.

≪ Preparation Example 1: Preparation of tablets >

20 g of the Miscanthus orientalis extract obtained in Example 1 of the present invention was mixed with 175.9 g of lactose, 180 g of potato starch and 32 g of colloidal silicic acid. To this mixture was added a 10% gelatin solution, which was pulverized and passed through a 14-mesh sieve. This was dried, and a mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into tablets.

≪ Formulation Example 2: Preparation of capsules >

100 mg of corn starch, 100 mg of lactose, and 2 mg of magnesium stearate were mixed with 100 mg of mucilast leaf extract treated with viscose according to Example 1 of the present invention, and the above components were mixed according to a conventional capsule preparation method. To prepare a capsules.

≪ Formulation Example 3: Preparation of injection &

1 g of the mossberry leaf extract treated with viscose treatment of Example 1 of the present invention, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. This solution was placed in a bottle and sterilized by heating at 20 DEG C for 30 minutes.

Formulation Example 4. Preparation of Health Functional Foods [

20 g of mucilast leaf extract of Example 1 of the present invention treated with a vitamin mixture, 70 g of vitamin A acetate, 1.0 mg of vitamin E, 0.13 mg of vitamin B1, 0.15 mg of vitamin B2, 0.2 mg of vitamin B6, 0.2 g of vitamin B12, 10 mg of vitamin C, 10 μg of biotin, 1.7 mg of nicotinic acid amide, 50 μg of folic acid, 0.5 mg of calcium pantothenate, 1.75 mg of ferrous sulfate, 0.82 mg of zinc oxide, 25.3 mg of magnesium carbonate, , 55 mg of calcium secondary phosphate, 90 mg of potassium citrate, 100 mg of calcium carbonate, and 24.8 mg of magnesium chloride were mixed to prepare granules. In addition, the composition ratio of the above-mentioned vitamin and mineral mixture may be arbitrarily modified, and the above components may be mixed according to a conventional health functional food manufacturing method.

< Formulation example  5. Preparation of Health Functional Drink>

1 g of mucilus leaf extract of Example 1 of the present invention, 0.1 g of citric acid, 100 g of fructooligosaccharide, and 900 g of purified water were mixed and stirred, heated, filtered, sterilized and refrigerated according to a conventional beverage preparation method to prepare a drink.

Claims (7)

The present invention relates to a method for preventing or preventing diabetes mellitus comprising, as an active ingredient, a viscose-treated mulberry leaf water extract obtained by adding 1 to 3% by weight viscozyme to a mulberry leaf water extract and treating the mulberry leaf water extract at 40 to 60 ° C for 10 to 24 hours. A pharmaceutical composition for therapeutic use. delete delete The present invention relates to a method for preventing or preventing diabetes mellitus comprising, as an active ingredient, a viscose-treated mulberry leaf water extract obtained by adding 1 to 3% by weight viscozyme to a mulberry leaf water extract and treating the mugwort water extract at 40 to 60 ° C for 10 to 24 hours. Health functional foods for improvement. delete delete (Step 1) adding water as an extraction solvent having a volume of 5 to 100 times the volume of mulberry leaves;
(Step 2) Extracting the mixture of Step 1 at 40 to 121 ° C for 1 to 5 hours to obtain a mulberry leaf water extract; And
(Step 3) Adding 1 to 3% by weight of viscose to the mulberry leaf water extract of the above two steps and then treating at 40 to 60 ° C for 10 to 24 hours;
To obtain a water-extract of mulberry leaves.

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