KR20180061062A - Composition for preventing or improving diabetes mellitus comprising momrdica charantia (l.) extract, chrysanthemum zawadskii var. latilobum and paeonia lactiflora extract as an effective ingredient - Google Patents

Abstract

Disclosed in the present specification are a composition for preventing or alleviating diabetes comprising extracts of Momordica charantia, Chrysanthemum zawadskii, Paeonia lactiflora as effective ingredients and a composition for regulating blood sugar. The composition according to the present invention can diversely control the activity of diverse factors related to diabetes and has excellent effects of preventing or alleviating the diabetes. Specifically, the compositions of the present invention inhibit the activity of alpha-amylase and glucose uptake in small intestine cells, and promote the absorption of glucose in muscle cells, thereby being effective in preventing or alleviating the diabetes.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for preventing or improving diabetes, which comprises as an active ingredient an extract of Lilium japonica, a Lilium extract, LATILOBUM AND PAEONIA LACTIFLORA EXTRACT AS AN EFFECTIVE INGREDIENT}

The present invention relates to a composition for preventing or alleviating diabetes, comprising as an active ingredient an extract of Lilac, Rhododendron japonica extract and Peony root extract.

Diabetes is a disease in which glucose present in the blood is excreted through the urine and is one of the chronic cures that are not fundamentally healed. The life expectancy is prolonged due to changes in dietary life due to rapid economic development, and chronic diseases such as diabetes are increasing. The prevalence rate of diabetes in Korea is estimated to reach 5 ~ 10%. In 2007, the number of diabetic patients exceeded 4 million, 8.3 out of 100 population is diabetic, but it is predicted that diabetic patients will increase to about 6.8 million by 2025 have. Diabetes mellitus is the fifth most common cause of death in Korea, including complications, due to various complications, shortening the patient's life span by 5 to 10 years.

Accordingly, various attempts have been made to treat diabetes, and drugs such as Acarbose and voglibose are generally used. These drugs cause side effects such as abdominal bloating, vomiting and diarrhea . Therefore, in order to minimize the side effects of drugs, the development of a diabetic therapeutic agent using natural products is still under development, and most of the studies are conducted only on natural substances, because it is not easy to find a combination of natural substances exhibiting synergistic effects. Natural substances have fewer side effects than artificial compounds, but their therapeutic effect is weak. Therefore, it is very important to find combinations that show synergistic effects.

 Results of the Cause of Death in 2015, National Statistical Office, 2016.9.

In one aspect, an object of the present invention is to control blood sugar and prevent or improve diabetes.

In one aspect, an object of the present invention is to provide a composition capable of modulating various factors associated with diabetes.

In one aspect, the object of the present invention is to inhibit the activity of alpha-amylase, inhibit glucose uptake in small intestine cells, and promote uptake of glucose in muscle cells.

In one aspect, an object of the present invention is to provide a content ratio that can provide an optimum effect of a combination of the extract of Kjellmani, Rhizoma extract and Peony root extract.

In order to achieve the above object, the present invention provides, in one aspect, a composition for preventing or improving diabetes or a composition for controlling blood glucose, which comprises an extract of Lachrymum, Rhododendron japonica extract and Peony root extract as an active ingredient.

The composition comprising the extract of Liliaceae, Rhododendron japonica extract and Peony root extract according to one aspect of the present invention as an active ingredient has various diabetes-related effects on various diabetes-related factors to prevent or improve diabetes. Specifically, it inhibits the activity of alpha-amylase, inhibits absorption of glucose in small intestine cells, and promotes absorption of glucose in muscle cells, thus being effective in preventing or improving diabetes.

FIG. 1 shows the result of confirming the activity of alpha-glucosidase inhibition after treatment of Yeoju, Jangsucho and Peony root extract.
FIG. 2 shows the result of confirming the inhibitory effect of alpha-amylase activity after treatment of Yeoju, Jangsucho and Peony root extract.
FIG. 3 is a graph showing the results of confirming the activity of inhibiting glucose absorption in small intestine cells after treatment with Yeoju, Quercus acutissima, and Peony root extract.
FIG. 4 is a graph showing the glucose uptake activity of muscles after treatment with Yeojoo, Kwangjoocho and peony extracts.
FIGS. 5 to 7 show animal test results. FIG. 5 is a graph showing the results of animal experiments in which the blood sugar-regulating effect of Yeoju, Korean persimmon and peony extracts was examined. FIG. 6 shows the oral glucose tolerance of Yeoju, FIG. 7 is a graph showing blood glucose levels (FIG. 7A) and HbA 1c concentrations (FIG. 7B).
FIGS. 1B, 2B, 3B and 4B show the results of treatment of a mixture of Yeoju, Rhus japonica and Peony Extract, which contains an excess of Yeast Extract, Figures 1c, 2c, 3c and 4c illustrate the case where the peony root extract is contained in an excessive amount, and Figures 1d, 2d, 3d and 4d show the case where the peony root extract is contained in an excessive amount.

Hereinafter, the present invention will be described in detail.

In one aspect, the present invention is a composition for preventing or improving diabetes, comprising Momordica charantia L. extract, Chrysanthemum zawadskii var. Latilobum extract and Paeonia lactiflora extract as an active ingredient.

In one aspect, the present invention is a composition for controlling blood glucose level, comprising Momordica charantia L. extract, Chrysanthemum zawadskii var. Latilobum extract and Paeonia lactiflora extract as active ingredients.

Yeoju ( Momordica charantia L.) is a perennial vine plant that is widely grown in Asia. Yeoju contains vitamin C, which has antioxidant effect, and contains carantin, carotene, calcium, iron, folic acid, pantothenic acid, plant fiber and mamordecin. It has anticancer effect, malaria treatment, weight loss and digestion And is widely used as a material for one-shot.

Chrysanthemum zawadskii var latilobum is a perennial dicotyledonous plant belonging to the family Asteraceae, which grows wild in mountainous and highland areas around the country and grows in China, Russia, Mongolia and Japan. It is used as a folk remedy for amenorrhea, pneumonia, bronchitis, cold, and pharyngitis.

Paeonia lactiflora is the root of the perennial herbaceous plant of the buttercups and is mainly used as a medicinal plant. Peonies include, but are not limited to, Paeoniflorin, Paeonel, Paeonin, Tannin, β-sitosterol, triterpenoid, essential oils, It is used for prevention and treatment of diseases due to antioxidant, inflammation, analgesic effect, blood reinforcement and immunity strengthening.

In the present specification, the term " diabetes " is a type of metabolic disorder such as insufficient secretion of insulin or a failure to perform a normal function. It is characterized by hyperglycemia in which the concentration of glucose in the blood is increased. Various symptoms and signs are caused by hyperglycemia, Which may be a disease that causes glucose to be excreted in the body.

Type 1 diabetes is referred to as 'childhood diabetes' and can refer to diabetes that is genetically unable to produce any insulin at all. Type 2 diabetes can be diabetes due to acquired causes, which is characterized by insulin resistance (failure of insulin to lower blood sugar and cells fail to burn glucose effectively). In this aspect, the diabetes may include Type 1 diabetes or Type 2 diabetes.

In the present specification, " blood glucose control " means lowering the concentration of blood glucose to the level of a person without diabetes.

In one aspect, the rumen extract, Rhizoma extract, and Peony root extract may be contained at a weight ratio of 1 to 100: 1 to 100: 1 to 100, respectively.

The weight ratio of the yeast extract, the herb extract, and the peony extract is 5 to 15: 0.5 to 2: 0.5 to 2, 0.5 to 2: 5 to 15: 0.5 to 2, or 0.5 to 2: Preferably from 8 to 12: 0.8 to 1.2: 0.8 to 1.2, 0.8 to 1.2: 8 to 12: 0.8 to 1.2, or 0.8 to 1.2: 0.8 to 1.2 : 8 to 12 can be included. When the weight ratio of Yeoju extract, Rhizoma extract, and Peony extract is included in the weight ratio, a remarkably superior diabetic improvement effect can be obtained than that of Yeoju extract, Rhizoma extract and Peony root extract due to the synergistic effect according to the combination of each extract. In the above ratio, if the content ratio of any one of the extracts of Y. japonica, Rhododendron japonica extract and Peony root extract is set to be high, even when the content of the other two kinds of extracts is set low, A superior diabetic improvement effect can be obtained.

In one embodiment, the composition comprises alpha-glucosidase activity inhibition; Inhibiting glucose uptake of small intestinal cells; Decrease in blood glucose concentration; Enhancing glucose metabolism activity; And enhancing glucose uptake of muscle cells.

In addition, in the above aspect, the composition may contain 0.1 to 99.9% (w / w), preferably 0.5 to 55% (w / w) based on the total weight of the composition, % (w / w), more preferably 3 to 55% (w / w).

In one aspect, the extraction solvent of the extracts may be water, an organic solvent, or a mixture thereof. The organic solvent may include, but is not limited to, alcohol, acetic acid, dimethyl sulfoxide (DMSO), acetone, acetonitrile, ethyl acetate, pentane, hexane, chloroform, diethyl ether and carbon tetrachloride. The alcohol may be a C _1-4 alcohol, and may be, for example, methanol, ethanol, propanol, n-butanol or iso-butanol. The organic solvent may preferably be ethanol. When the plant extracts are extracted with ethanol, the extraction efficiency can be maximized and the effect of the extracts can be maximized.

As used herein, an " extract " may include not only a crude extract, but also fractions obtained by fractionating the crude extract. That is, the extract includes not only those obtained using the above-described extraction solvent but also those obtained by further applying a purification process. The extract of the present invention thus prepared can be implemented in various forms such as solid and liquid.

The above-mentioned Yeast extract, Rhizoma extract and Peony root extract may be stem, flower, root, leaf, fruit, or pupa extract of a plant, respectively. Preferably, the extracts of Y. japonica, Rhododendron sp.

In one embodiment, the composition may be a pharmaceutical composition or a food composition.

The pharmaceutical composition of the present invention may be various oral or parenteral formulations. 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 one or more excipients such as starch, calcium carbonate, sucrose or lactose lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like are also used.

Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have.

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The preferred dosage of the pharmaceutical composition according to the present invention varies depending on the condition and the weight of the patient, the degree of the disease, the drug form, the administration route and the period, but can be appropriately selected by those skilled in the art. However, for the desired effect, the composition of the present invention can be administered at 0.001 to 100 mg / kg / day.

Dosage forms of the pharmaceutical compositions according to the present invention may be used in the form of their pharmaceutically acceptable salts and may also be used alone or in combination with other pharmaceutically active compounds as well as in suitable aggregates.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, and humans in various routes. All modes of administration may be expected, for example, by oral, intraperitoneal, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebral injection.

The food composition according to the embodiment of the present invention can be used alone or in combination with another food or other food ingredient, and can be suitably used according to a conventional method. The food composition according to the present embodiment includes components that are ordinarily added during the manufacture of food, and includes, for example, proteins, carbohydrates, fats, nutrients, and seasonings. In addition, there is no particular limitation on the type of food. However, there is no particular limitation on the type of food, but dairy products such as meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen and other noodles, gums, ice cream, Alcoholic beverages, vitamin complexes, and the like, all of which include health foods in a conventional sense.

The food composition according to the present embodiment may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The above-mentioned natural carbohydrates are sugar saccharides such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and xylitol, sorbitol and erythritol. As the sweetening agent, natural sweetening agents such as tau Martin and stevia extract, synthetic sweetening agents such as saccharine and aspartame, and the like can be used.

Further, the food composition of the present embodiment can be used as a food composition containing various nutrients, vitamins, electrolytes, flavors, colorants, pectic acids and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, A carbonating agent used in a carbonated beverage, and the like. In addition, the food composition of this example may contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination.

Hereinafter, the present invention will be described in detail with reference to examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

[Example 1] Preparation of Yeoju, Quercus mongolica and Peony root extracts

[Example 1-1] Preparation of extracts of each of Yeoju, Jungbucho and Peony

The extracts were obtained by boiling and chopping the herb medicines of each of Yeoju, Seonjangcho, and Peonies, boiling them for 4 hours after adding 10 times the solvent of the same weight of each herbal medicine. The extract was filtered, concentrated with a vacuum concentrator, and lyophilized to prepare an extract. For the experiment, the extract was dissolved in 10% DMSO at a concentration of 10-100 mg / ml, filtered through a sterile filter paper (Millipore membrane, 0.45 μm) and kept sterile.

[Example 1-2] Preparation of mixture of Yeoju, Quercus mongolica and Peony root extract

The extracts were obtained by boiling the herb medicines of each of the mulberry, persimmon, and peony on shade, finely chopped and mixed at different weight ratios, adding 10 times the weight of the mixed medicinal herbs and boiling for 4 hours. The extract was filtered, concentrated with a vacuum concentrator, and lyophilized to prepare an extract. For the experiment, the extract was dissolved in 10% DMSO at a concentration of 10-100 mg / ml, filtered with a sterile filter paper (Millipore membrane, 0.45 μm) and kept sterile.

[Experimental Example 1] Alpha-glucosidase inhibitory activity

The experimental method for measuring the inhibitory activity of alpha-glucosidase was carried out using PNP-glucoside (substrate) and 1 unit of alpha-glucosidase (Sacchromyces cerevisiae). For each extract, a reaction mixture consisting of 50 μl of 0.1 M KPB (pH 6.9), 10 μl of Alpha-glucosidase, 20 μl of Yeoju, Cheongwolcho and Peony root extract was reacted at 37 ° C for 20 minutes. Yeoju, Korean persimmon and peony extracts were treated at concentrations of 0.05, 0.1, 0.25 and 0.5 mg / ml. 20 μl of PNP-glucoside was added to the reaction solution, and the absorbance after 20 minutes was measured by a spectrophotometer (405 nm). The results are shown in Table 1. In Example 1, the alpha-glucosidase inhibitory activity was determined by the following formula 1:

[Equation 1] Alpha-glucosidase inhibition

Inhibition effect (%) = [(C- (S-SB)) / (C-CB)] 100

C: Absorbance of the sample not treated with the sample

CB: Absorbance of the control not treated with the sample

S: Absorbance of the experimental group treated with the sample

SB: absorbance of the control treated with the sample

Inhibition activity (%) Sample Concentration (mg / ml) 0.02 0.04 0.08 0.16 0.5 Acarbose - - 93.86 ± 0.27 - Necklace 4.97 ± 1.71 8.28 ± 2.81 15.74 + 1.63 24.8 ± 0.34 37.25 + - 4.19 Yeoju 2.84 ± 0.77 5.47 ± 1.17 15.14 + - 0.92 25.46 +/- 1.47 28.49 + - 2.24 Peony 8.82 + - 0.54 26.43 + - 1.55 34.36 ± 3.73 52.2 ± 3.31 85.38 + - 0.42

In addition, alpha - glucosidase inhibitory activity was measured by controlling the content ratio of the Y. japonica extract and Rhizoma extract. As a result, it was confirmed that the inhibitory activity against the enzymes was increased by increasing the ratio of Yeoju to that of Yeoju Extract after the fixed ratio of. For example, an approximately 40% increase in activity was observed in the extract mixture at a 10: 1: 1 ratio compared to the treatment with the single 0.16 mg / ml yeast (Table 2).

Inhibition activity (%) Yeoju: the phrase: peony
(Ratio) Concentration (mg / ml) 0.05 0.1 0.25 0.5 Acarbose 90.95 ± 0.97 1: 1: 1 15.22 + - 5.58 22.9 ± 2.91 38.9 ± 4.22 52.07 ± 5.49 2: 1: 1 18.06 + - 7.22 24.89 + - 6.49 47.81 + - 3.6 61.35 ± 3.27 5: 1: 1 17.91 ± 3.68 23.12 + - 2.57 49.77 ± 3.2 63.64 ± 3.96 10: 1: 1 18.31 ± 1.19 27.48 ± 4.96 52.47 ± 2.85 65.8 ± 5.06

In addition, it was confirmed that the enzyme inhibitory activity was increased as the ratio of the perianth gum was increased in the experiment in which the ratio of the fenugreek and the peony extract was fixed and then the mixture of the perennial root extract was mixed. For example, an approximately 34% increase in activity was observed in the extract mixture at a ratio of 1: 10: 1 compared to the 0.16 mg / ml single treatment (Table 3).

Inhibition activity (%) Yeoju: the phrase: peony
(Ratio) Concentration (mg / ml) 0.05 0.1 0.25 0.5 Acarbose 103.32 ± 0.2 1: 1: 1 15.22 + - 5.58 22.9 ± 2.91 38.9 ± 4.22 52.07 ± 5.49 1: 2: 1 16.28 ± 5.09 21.03 + - 2.24 32.71 + - 0.74 49.78 ± 2.77 1: 5: 1 7.16 ± 2.78 21.98 + 1.31 34.25 + - 3.27 55.21 ± 1.77 1: 10: 1 10.11 ± 0.97 22.75 ± 3.42 32.11 + - 3.4 58.29 ± 1.55

In addition, it was confirmed that the enzymatic inhibitory activity was increased with the increase of the ratio of peony in the experiment in which the ratio of quercetin and Quercus mongolica extract was fixed and the peony extract was mixed by the ratio. For example, a 1: 1: 10 ratio of extract mixture showed about a 29% increase in activity compared to the single 0.16 mg / ml peanut (Table 4).

Inhibition activity (%) Yeoju: the phrase: peony
(Ratio) Concentration (mg / ml) 0.05 0.1 0.25 0.5 Acarbose 103.32 ± 0.2 1: 1: 1 15.22 + - 5.58 22.9 ± 2.91 38.9 ± 4.22 52.07 ± 5.49 1: 1: 2 16.58 ± 2.63 20.56 ± 5.46 40.9 ± 2.62 63.11 + - 12.46 1: 1: 5 16.87 ± 1.66 24.07 ± 2.82 41.17 ± 3.77 77.57 + - 4.77 1: 1: 10 15.74 ± 2.03 24.01 + - 1.76 42.26 ± 3.16 81.27 ± 3.17

[Experimental Example 2] Inhibition of alpha-amylase activity

The test method for measuring the inhibitory activity of alpha-amylase was performed using 0.5% starch solution of substrate and α-amylase of 1 mg / 10 ml concentration. For each of the extracts, 100 μl of 20 mM sodium phosphate buffer (pH 6.9), 100 μl of α-amylase, 100 μl of a sample of Yeoju, Cheongwolcho and Peony root extract, and 200 μl of a 0.5% starch solution were added, And reacted at 37 ° C. Yeoju, Korean persimmon and peony extracts were treated by concentration (0.05, 0.1, 0.25, 0.5 mg / ml). To this reaction solution, 200 μl of a coloring solution of DNS (3,5-dinitrosalicylic acid) was added, followed by color development at 95 ° C. or higher for 15 minutes, and the absorbance was measured by a spectrophotometer (540 nm). The results are shown in Table 5.

Alpha-amylase activity inhibition was determined by the following equation:

&Quot; (2) "

Inhibition effect (%) = [(C- (S-SB)) / (C-CB)] 100

C: Absorbance of the sample not treated with the sample

CB: Absorbance of the control not treated with the sample

S: Absorbance of the experimental group treated with the sample

SB: absorbance of the control treated with the sample

It was confirmed that the enzyme activity was inhibited in a concentration - dependent manner when Yeoju, Korean persimmon and peony extracts were treated respectively. For example, at a concentration of 0.16 mg / ml, the inhibitory activity was about 28% in the foliation-treated group, about 32% in the foliation-treated group, and about 30% in the peony-treated group (Table 5).

Inhibition activity (%) Sample Concentration (mg / ml) 0.02 0.04 0.08 0.16 0.5 Acarbose 58.09 ± 1.26 Necklace 14.81 ± 2.4 19.62 ± 1.9 23.46 ± 3.19 32.86 ± 2.64 35.12 ± 3.40 Yeoju 4.6 ± 0.57 6.45 ± 0.49 21.59 ± 0.5 28.32 + 0.79 34.97 ± 0.89 Peony 8.91 ± 0.24 11.45 ± 0.23 16.52 ± 1.2 30.42 ± 0.96 39.32 + - 0.59

In addition, it was confirmed that the inhibitory activity against the enzymes was increased as the ratio of the female ryegrass and peony root extract was fixed and the ratio of the female ryegrass was increased in the experiment in which the ryegrass extract was mixed by the ratio. For example, an increase in activity of about 17% was observed in the extract mixture at a ratio of 10: 1: 1 compared to the control group treated with a single yeast 0.16 mg / ml (Table 6).

Inhibition activity (%) Yeoju: the phrase: peony
(Ratio) Concentration (mg / ml) 0.05 0.1 0.25 0.5 Acarbose 57.89 + 1.84 1: 1: 1 26.72 + - 0.87 29.71 + - 4.02 32.59 ± 0.22 37.19 + - 3.32 2: 1: 1 28.53 + - 0.49 30.59 + - 0.85 32.84 ± 0.18 39.9 + 1.36 5: 1: 1 29.13 + - 2.41 30.2 ± 1.75 34.93 ± 0.18 41.33 ± 1.07 10: 1: 1 38.47 ± 2.91 40.82 ± 1.29 42.27 ± 2.07 45.47 ± 0.96

In addition, it was confirmed that the enzyme inhibitory activity was increased as the ratio of the perianth gum was increased in the experiment in which the ratio of the fenugreek and the peony extract was fixed and then the mixture of the perennial root extract was mixed. For example, an increase in activity of about 17% was observed in the extract mixture at a 1: 10: 1 ratio compared to the 0.16 mg / ml single treatment (Table 7).

Inhibition activity (%) Yeoju: the phrase: peony
(Ratio) Concentration (mg / ml) 0.05 0.1 0.25 0.5 Acarbose 59.67 ± 5.46 1: 1: 1 26.72 + - 0.87 29.71 + - 4.02 32.59 ± 0.22 37.19 + - 3.32 1: 2: 1 26.39 ± 1.56 32.4 ± 0.56 37.85 ± 2.08 40.36 ± 1.22 1: 5: 1 30.68 + - 2.53 33.62 + - 2.5 37.39 + 1.7 43.26 + - 0.43 1: 10: 1 35.05 ± 1.23 40.02 ± 3.32 43 ± 2.37 49.43 ± 0.92

In addition, it was confirmed that the enzymatic inhibitory activity was increased with the increase of the ratio of peony in the experiment in which the ratio of quercetin and Quercus mongolica extract was fixed and the peony extract was mixed by the ratio. For example, a 1: 1: 10 ratio of extract mixture showed an increase in activity of about 29% compared to the peanut single 0.16 mg / ml treated group (Table 8).

Inhibition activity (%) Yeoju: the phrase: peony
(Ratio) Concentration (mg / ml) 0.05 0.1 0.25 0.5 Acarbose 57.89 + 1.84 1: 1: 1 26.72 + - 0.87 29.71 + - 4.02 32.59 ± 0.22 37.19 + - 3.32 1: 1: 2 32.36 ± 1.4 33.67 ± 1.71 35.85 ± 1.59 44.55 + - 0.54 1: 1: 5 33.56 + - 0.37 37.38 + - 2.33 45.92 1.52 53.03 + - 1.56 1: 1: 10 39.99 + - 0.78 46.25 + 0.29 49.81 + - 0.75 59.6 ± 0.08

[Experimental Example 3] Confirmation of inhibition of glucose absorption in small intestine cells

The experimental method for measuring the inhibitory activity of glucose uptake into the blood in the small intestine was performed using the Caco-2 cell line and 2-NBDG (2- (N- (7-nitrobenz-2-oxa-1,3-diazol- Caco-2 was inoculated at a concentration of 1 × 10 ⁴ cells / well in a 96-well plate at 37 ° C for 14 days in a CO ₂ incubator, After incubation, the cells were washed twice with PBS. Then, 400 μl of glucose-free medium, 500 μl of 100 μM 2-NBDG, and 100 μl of samples (0.05, 0.1, 0.25, 0.5 mg / ml) were added and reacted at 37 ° C for 2 hours. After removing the reaction solution and washing the cells three times with ice-cold PBS, the fluorescence intensity of 2-NBDG was measured with a spectrophotofluorometer (excitation: 485 nm, emission: 535 nm). In order to compensate for the effect of cell concentration, MTT assay was performed as follows: 5 mg / ml MTT solution was added to separate the 50 ㎕ was measured at 540 nm after 2 hours of reaction at 37 * was used as a positive standard phlorizin.

The glucose uptake inhibitory activity of the samples was calculated according to the following equation and shown in Table 9 in the control group.

The glucose uptake inhibitory activity was determined by the following equation (3)

&Quot; (3) "

Inhibition effect (%) = [(Fc - Fs) / Fc] x 100

Fs: Fluorescence intensity of the specimen treated

Fc: Fluorescence intensity of the sample without treatment

It was confirmed that the treatment of Yeoju, Kwangseocho, and peony extract increased the glucose uptake inhibitory activity in small intestine cells in a concentration - dependent manner. For example, at a concentration of 0.16 mg / ml, about 13% inhibition activity was observed in about 13% of the treated group and about 30% in the peony treated group.

Uptake activity Sample Concentration (mg / ml) 0.02 0.04 0.08 0.16 Phlorizine (0.1 mg / ml) 0.76 + 0.07 Necklace 0.96 + 0.12 0.92 + 0.13 0.88 + 0.04 0.87 ± 0.07 Yeoju 1.01 0.08 0.94 + 0.14 0.89 ± 0.01 0.87 ± 0.05 Peony 0.96 + 0.03 0.89 + 0.03 0.78 ± 0.01 0.7 ± 0.05 * Glucose uptake ratio compared with Control (1.0)

In addition, it was confirmed that the activity of inhibiting glucose uptake in small intestine cells was increased as the ratio of yeast was increased in the experiment in which the ratio of citrus extract and peony root extract was fixed, For example, an inhibitory activity increase of about 26% was observed in the extract mixture at a ratio of 10: 1: 1 compared with the control group of 0.14 mg / ml of the yeast extract.

Uptake activity Yeoju: the phrase: peony
(Ratio) Concentration (mg / ml) 0.05 0.1 0.25 0.5 Phlorizin 0.76 + 0.07 1: 1: 1 0.97 + 0.06 0.87 + 0.03 0.73 + 0.11 0.65 ± 0.05 2: 1: 1 0.95 + 0.06 0.87 ± 0.05 0.74 + 0.04 0.64 ± 0 5: 1: 1 0.87 + 0.14 0.81 + 0.06 0.71 + 0.07 0.62 ± 0.01 10: 1: 1 0.85 ± 0.08 0.8 ± 0.05 0.71 + 0.07 0.61 + 0.07 * Glucose uptake ratio compared with Control (1.0)

In addition, it was confirmed that the activity of inhibiting glucose uptake in small intestinal cells was increased as the ratio of pertussis increased in the experiment in which the ratio of goethite and peony extract was fixed and then the peony extract was mixed. For example, an inhibitory activity of about 27% was increased in the extract mixture at a ratio of 1: 10: 1 compared with the control group (0.16 mg / ml).

Uptake activity Yeoju: the phrase: peony
(Ratio) Concentration (mg / ml) 0.05 0.1 0.25 0.5 Phlorizin 0.76 + 0.07 1: 1: 1 0.97 + 0.06 0.87 + 0.03 0.73 + 0.11 0.65 ± 0.05 1: 2: 1 0.92 + 0.08 0.88 ± 0.08 0.73 ± 0.06 0.63 + 0.03 1: 5: 1 0.92 ± 0.05 0.83 0.06 0.72 + 0.1 0.62 + 0.03 1: 10: 1 0.91 + 0.03 0.81 + 0.04 0.74 ± 0.1 0.6 ± 0.01 * Glucose uptake ratio compared with Control (1.0)

In addition, it was confirmed that the activity of inhibiting glucose absorption in small intestine cells was increased as the ratio of peanut was increased in the experiment in which the ratio of quercetin and quercetin extract was fixed and then the peony extract was mixed in proportions. For example, an inhibitory activity of about 14% was increased in the extract mixture at a ratio of 1: 1: 10 compared to the control group (0.16 mg / ml).

Uptake activity Yeoju: the phrase: peony
(Ratio) Concentration (mg / ml) 0.05 0.1 0.25 0.5 Phlorizin 0.76 + 0.07 1: 1: 1 0.97 + 0.06 0.87 + 0.03 0.73 + 0.11 0.65 ± 0.05 1: 1: 2 0.82 ± 0.06 0.76 ± 0.01 0.7 ± 0.06 0.63 + - 0.1 1: 1: 5 0.81 + 0.07 0.7 ± 0.07 0.62 ± 0.01 0.57 + 0.03 1: 1: 10 0.76 + 0.09 0.68 + 0.04 0.62 + 0.04 0.56 ± 0.1 * Glucose uptake ratio compared with Control (1.0)

[Experimental Example 4] Confirmation of glucose absorption enhancement effect in muscle cells

Experimental methods for measuring glucose uptake activity from blood to muscle cells were L6 cell line and 2-NBDG (2- (N- (7-nitrobenz-2-oxa-1,3-diazol- To investigate the glucose uptake of each extract, L6 was cultured in a 96-well plate at 1 × 10 ⁴ cells / well at 37 ° C for 4 days in a CO ₂ incubator After incubation for 2 days, the cells were washed twice with PBS, then 400 μl of glucose-free medium, 500 μl of 100 μM 2-NBDG, and 0.05 μl of sample (0.05, 0.1, 0.25, 0.5 mg / ml) The reaction solution was removed and the cells were washed three times with ice-cold PBS. The fluorescence intensity of 2-NBDG was measured by a spectrophotofluorometer (excitation: 485 nm, emission: The MTT assay was performed in order to correct the effect of cell concentration on the measurement of glucose uptake inhibitory activity. 50 μl of MTT solution (5 mg / ml) was added to the well plate for fluorescence intensity measurement and reacted at 37 ° C for 2 hours, and the absorbance was measured at 540 nm. The activity of glucose uptake by the sample is calculated according to Equation (4) below and is shown in Table 13.

[Equation 4] Calculation of glucose uptake activity in muscle

Activation effect (%) = [(Fc - Fs) / Fc] × 100

Fs: Fluorescence intensity of the specimen treated

Fc: Fluorescence intensity of the sample without treatment

Uptake activity Sample Concentration (mg / ml) 0.02 0.04 0.08 0.16 Insulin (500 nM) 1.4 ± 0.02 Necklace 0.91 + 0.06 0.95 + 0.23 1.09 ± 0.12 1.11 + 0.08 Yeoju 0.94 + - 0.1 1.02 + 0.13 1.07 + 0.07 1.1 ± 0.14 Peony 0.98 ± 0.09 1.07 ± 0.11 1.15 ± 0.15 1.2 ± 0.07 * Glucose uptake ratio compared with Control (1.0)

As shown in Table 13, when the test for absorption-enhancing activity per muscle cell was performed, it was confirmed that the activity was increased in a concentration-dependent manner when Yeoju, Quercus acutissima and Peony root extract were treated. For example, at a concentration of 0.16 mg / ml, about 10% of the treatment group showed about 11% increase in the peritoneal treatment group and about 20%

In addition, it was confirmed that the glucose uptake activity of the muscle cells was increased as the ratio of Yeoju was increased in the experiment in which the ratio of the herb extract was fixed and the extract of Yeoju was mixed by the ratio. For example, an increase in activity of about 31% was observed in an extract mixture at a 10: 1: 1 ratio compared to the treatment with a single 0.16 mg / ml yeast (Table 14).

Uptake activity Yeoju: the phrase: peony
(Ratio) Concentration (mg / ml) 0.05 0.1 0.25 0.5 Insulin (500 nM) 1.4 ± 0.02 1: 1: 1 1.05 0.06 1.07 ± 0.06 1.16 ± 0.1 1.29 + 0.08 2: 1: 1 0.99 ± 0.06 1.15 ± 0.05 1.2 ± 0.16 1.3 ± 0.21 5: 1: 1 1.06 + - 0.14 1.12 + 0.06 1.15 ± 0.03 1.34 ± 0.1 10: 1: 1 0.99 ± 0.09 1.09 ± 0.11 1.28 ± 0.15 1.41 0.21 * Glucose uptake ratio compared with Control (1.0)

In addition, it was confirmed that the sugar absorption activity of muscle cells was increased as the ratio of pertussis increased in the experiments in which the ratio of the fennel extract and the fennel extract was fixed and then the mixture of the perennial extract was mixed. For example, a 1: 10: 1 ratio of extract mixture showed a 32% increase in activity compared to the 0.16 mg /

Uptake activity Yeoju: the phrase: peony
(Ratio) Concentration (mg / ml) 0.05 0.1 0.25 0.5 Insulin (500 nM) 1.4 ± 0.02 1: 1: 1 1.05 0.06 1.07 ± 0.06 1.16 ± 0.1 1.29 + 0.08 1: 2: 1 1 ± 0.08 1.02 + 0.18 1.17 ± 0.1 1.34 ± 0.1 1: 5: 1 0.99 ± 0.07 1.14 ± 0.33 1.29 + - 0.01 1.42 ± 0.24 1: 10: 1 1.02 + 0.02 1.15 ± 0.22 1.33 ± 0.01 1.43 ± 0.3 * Glucose uptake ratio compared with Control (1.0)

In addition, it was confirmed that the sugar absorption activity of muscle cells was increased as the ratio of peanut was increased in the experiment in which the ratio of quercetin and quercetin extract was fixed and then the peony extract was mixed with each other in proportions. For example, an increase in activity of about 31% was observed in the extract mixture at a ratio of 1: 1: 10 compared to the control group with the single pea 0.16 mg / ml.

Uptake activity Yeoju: the phrase: peony
(Ratio) Concentration (mg / ml) 0.05 0.1 0.25 0.5 Insulin (500 nM) 1.4 ± 0.02 1: 1: 1 1.05 0.06 1.07 ± 0.06 1.16 ± 0.1 1.29 + 0.08 1: 1: 2 1.08 ± 0.15 1.14 ± 0.13 1.21 ± 0.07 1.37 + 0.17 1: 1: 5 1.01 ± 0.18 1.11 ± 0.11 1.28 ± 0.26 1.45 ± 0.12 1: 1: 10 1.14 ± 0.05 1.22 ± 0.1 1.39 0.47 1.51 + - 0.23 * Glucose uptake ratio compared with Control (1.0)

[Experimental Example 5] Confirmation of blood glucose control effect

In this experiment, a type 2 diabetic mouse model induced by high fat diet and streptozotocin (STZ) was used. After a week of purifying period, high - fat diets were fed for 2 weeks, and STZ was administered once per day for 3 days.

This experiment was carried out in a mixture of normal (NC), diabetic (HFD + STZ), positive control (Metformin) and low dose groups (Rhizoma, 100 mg / kg), and high dose group (300 or 300 mg / kg of complex except Yeoju).

Metformin and test substances in the positive control group were orally administered for two weeks after the administration of STZ for 6 weeks. In the normal group and the diabetic group, the purified water was orally administered in the same manner as the test substance administration schedule.

Fasting blood glucose was measured once a week (1, 2, 3, 4, 5, 6 weeks) after the administration of the test substance, using whole blood flowing naturally from the vein of the experimental animals.

group 0 day 7 day 14 day 21 day 28 day 35 day 42 day NC 146.00 ± 13.77 143.75 ± 10.65 142.75 +/- 10.40 125.75 + - 6.88 120.88 + - 8.34 141.50 ± 8.30 142.38 + - 7.80 HFD + STZ 352.14 + - 89.90 373.71 ± 58.75 379.29 + - 77.54 383.29 ± 89.11 373.29 ± 58.59 378.43 ± 45.89 388.71 + - 90.08 HFD + STZ +
Metformin (200) 348.57 +/- 85.26 366.57 ± 90.83 352.71 + - 132.57 316.57 ± 97.49 261.71 + - 97.01 234.14 ± 58.10 221.43 ± 49.73 HFD + STZ +
Yeoju Outside Complex (100) 346.43 + - 79.06 370.29 + - 80.66 368.71 + - 74.51 358.29 + - 61.55 319.71 ± 92.67 301.14 ± 102.46 287.14 ± 80.23 HFD + STZ +
Complexes outside Yeoju (300) 346.71 + - 78.43 371.00 ± 101.35 360.86 ± 91.90 349.43 ± 97.93 303.43 ± 87.49 275.00 + - 71.37 270.71 + - 75.70

As a result of fasting blood glucose measurement, it was confirmed that blood glucose level was lower in the test substance group than in the diabetic group. It was also found that blood glucose was decreased by the amount of the test substance.

[Experimental Example 6] Oral glucose tolerance test

In this experiment, oral glucose tolerance was measured to confirm smooth glucose metabolism.

Oral glucose tolerance test (OGTT) was performed at the end of the last week for 12 hours and glucose was orally administered at a dose of 2 g / kg. Glucose was measured immediately before and 15, 30, 45, 60, 90 and 120 minutes after administration.

ru0 min 15 min 30 min 45 min 60 min 90 min 120 min 153.71 ± 9.69 420.43 + - 33.78 295.00 ± 42.86 233.43 ± 17.73 187.4 ± 17.38 172.00 ± 9.63 159.29 + - 5.79 F + T384.14 + - 53.22 668.14 ± 50.76 619.29 + - 41.95 556.57 ± 27.28 522.29 ± 25.97 459.71 ± 2239 427.86 +/- 22.95 ++
fi0265.00 ± 48.49 575.86 + - 38.41 499.86 + - 56.03 452.86 + - 40.01 429.86 + - 65.16 370.49 + - 47.67 313.14 + - 54.68 +
Except 0341.43 ± 87.04 678.29 ± 70.19 605.00 + - 24.93 526.57 ± 56.81 506.43 ± 30.95 445.43 ± 19.00 397.86 + 54.27 +
0336.43 ± 92.21 645.71 + - 42.18 567.43 + - 44.50 508.57 ± 38.50 484.29 + - 42.10 416.43 ± 28.78 370.29 ± 30.30

 In the diabetic group, oral glucose tolerance test was 384.14 ± 53.22 mg / dL for fasting and 668.14 ± 50.76 mg / dL for 15 minutes after fasting and 427.86 ± 22.95 mg / dL after 120 minutes. In the control group and the group administered orally with the test substance, it was confirmed that the blood glucose level was lower than that of the diabetic group after 30 minutes.

[Experimental Example 7] Experimental Example 7: Glucose, HbA1c,

In this experiment, the concentration of glucose and HbA1c, which is a part of hemoglobin erythrocyte, were measured.

Experimental animals were fasted for 6 weeks and blood samples were collected. The contents of glucose and HbA1c were analyzed using a blood biochemical analyzer.

Group GLU HbA1c NC 171.49 ± 6.10 3.20 ± 0.00 HFD + STZ 405.44 + - 130.54 5.59 ± 1.29 HFD + STZ +
Metformin (200) 278.71 + - 69.12 4.23 + - 0.68 HFD + STZ + Yeoju Complex (100) 394.07 ± 145.37 5.03 ± 1.17 HFD + STZ + Yeoju Complex (300) 359.31 + - 123.43 4.89 ± 0.93

As a result, blood glucose levels were 171.49 ± 6.10 mg / dl, 405.44 ± 130.54 mg / dl, 278.71 ± 69.12 mg / dl, and 394.07 ± 145.37 mg / dl, respectively, in the normal group, the diabetic group, the positive control group, And 123.43 mg / dl, respectively, compared to the diabetic group.

Serum HbA1c levels were 3.20 ± 0.00%, 5.59 ± 1.29%, 4.23 ± 0.68%, 5.03 ± 1.17%, and 4.89 ± 0.93% in the normal, diabetic, and test substance groups, respectively. The diabetic group showed lower HbA1c levels than the diabetic group.

Claims (9)

A composition for preventing or improving diabetes comprising Momordica charantia L. extract, Chrysanthemum zawadskii var. Latilobum extract and Paeonia lactiflora extract as an active ingredient. A composition for controlling blood glucose level comprising Momordica charantia L. extract, Chrysanthemum zawadskii var. Latilobum extract and Paeonia lactiflora extract as an active ingredient. 3. The method according to claim 1 or 2,
The above-mentioned extracts,
1 to 100: 1 to 100: 1 to 100, respectively. 3. The method according to claim 1 or 2,
The above-mentioned extracts,
Wherein the composition is contained in a weight ratio of 5 to 15: 0.5 to 2: 0.5 to 2, a weight ratio of 0.5 to 2: 5 to 15: 0.5 to 2 or a weight ratio of 0.5 to 2: 0.5 to 2: 5 to 15, respectively. 3. The method according to claim 1 or 2,
Wherein the composition comprises at least one feature selected from the group consisting of:
Alpha-glucosidase activity inhibition; Inhibiting glucose uptake of small intestinal cells; Decrease in blood glucose concentration; enhance glucose metabolism activity; And enhancement of glucose uptake in muscle cells. 3. The method according to claim 1 or 2,
The composition is prepared by mixing the extract of Lady Lily,
Wherein the composition comprises from 0.1 to 99.9% (w / w) based on the total weight of the composition, 3. The method according to claim 1 or 2,
Wherein the extraction solvent of the extract is water, an organic solvent or a mixture thereof. The method according to claim 1,
Wherein said diabetes comprises type 1 diabetes or type 2 diabetes. 3. The method according to claim 1 or 2,
Wherein the composition is a pharmaceutical composition or a food composition.

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