KR20180088537A - Composition of Extracts and Fractions of Prunus mume Leaves having blood glucose control activity and/or insulin resistance - Google Patents

Abstract

The present invention relates to a pharmaceutical composition or a health food composition comprising the extracts or fractions of prunus mume leaves as an active ingredient which has alpha-glucosidase inhibitory effect or peroxisome proliferator activated receptor-gamma (PPAR gamma) agonist efficacy and has blood glucose controlling function or insulin resistance. The extracts or fractions of prunus mume leaves is effective for treating, preventing or improving diabetes or hyperlipidemia because it exhibits a high action mechanism for reducing blood sugar level and neutral fat content in blood.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] The present invention relates to a composition comprising a mulberry leaf extract or fraction thereof having glucose control function or insulin resistance,

The present invention relates to a pharmaceutical composition comprising a sheet-form extract having a glucose-regulating function or insulin resistance or a fraction thereof as an active ingredient by having an alpha-glucosidase inhibitory effect or a peroxisome proliferator activated receptor-gamma (PPAR y) Compositions or health food compositions.

Due to the recent rapid improvement in living standards, the overconsumption of high calorie foods, lack of exercise, and the stress caused by the advancement of the industrial society are leading to the increasingly serious diseases. Representative adult diseases include hypertension, diabetes, obesity, and hyperlipemia. In particular, diabetes is the cause of all chronic vascular diseases.

Diabetes is the most common non-communicable chronic disease occurring in modern people. It is caused by insufficient secretion of insulin secreted from the pancreas or insulin receptor, resulting in failure to absorb insulin. In diabetic patients, the sugar consumed through the meal is not properly stored in the liver, muscle, or fat cells, and accumulates in the blood to maintain high blood sugar. Excessive amounts of blood sugar may damage the gut syringe, It accumulates in the blood as cholesterol or triglycerides. However, these symptoms are only acute symptoms that occur early in the onset of diabetes. Diabetes mellitus is a chronic disease that is delayed in treatment rather than the severity of the disease itself, such as complications such as blood circulation disorder, chronic fatigue, hypertension, myocardial infarction, diabetic neuropathy, retinopathy (visual disturbance, blindness, ), Cataract, nephrotic syndrome, etc., and the overall metabolic function and sensory function of the human body are deteriorated, which can cause fatal results in the past. Therefore, people with diabetes should be able to slowly absorb the carbohydrates of the food they eat so that sudden increase in blood sugar is prevented. Alpha-Glucosidase enzyme is an enzyme that hydrolyzes oligosaccharide, which is digested by meals into small intestine, into monosaccharide. This enzyme inhibitor slows the postprandial increase of blood sugar by delaying the absorption of carbohydrates. Examples of such alpha-glucosidase inhibitors include acarbos and miglitol, which were developed by Bayer, Germany in 1977. They were developed as diabetic and obesity treatment agents, .

Peroxisome Proliferator Activated Receptor γ (PPARγ) is a member of the nuclear hormone receptor family. It is known that Thiazolidinedione (TZD), which increases insulin sensitivity in energy metabolism, It is known as a receptor for the family. Peroxisome proliferator activated receptor-gamma plays an important role in the lipid metabolism of adipose tissue according to the nutritional status. In particular, the postprandial expression increases and the activity of the gene that transfers the absorption and capture of the fatty acid is increased, Cells should be stored. Insulin resistance patients are difficult to use glucose. In order to increase the energy efficiency in the body, free fatty acids are released from adipocytes. Thiazolidinediones absorb and capture these free fatty acids to lower the free fatty acid concentration in the body , And increases insulin sensitivity.

Type 2 diabetes mellitus is favored by a combination of diabetes treatment with other mechanisms rather than monotherapy, which is clinically treated with one drug, due to the specificity of the disease. Especially, PPARgamma agonists have excellent antidiabetic effect, but it is preferable to create a synergistic effect by the combined administration rather than the single administration therapy because the duration of the effect is long.

On the other hand, plum tree ( Prunus mume Sieb. Et Zucc) is a deciduous tree of Rosaceae and is distributed mainly in East Asia. The plum trees are called differently for each part. The fruit is called plum, the flower is plum, the leaf is the sheet, the root is the root, It is also known that the effect of plum trees on different parts of plants is different. Among them, the leaves of plum (plum tree leaf) are reported to treat chronic diarrhea, vases, stomach damage and acute gastroenteritis in Dong-bo-gyung. In addition, it is known that in the private sector, it sucks up the sheets and diarrhea in the lower part, and suffers from sickness and sickness, and it has been known that these symptoms are serious and the muscles are lacking in water supply. However, only plum and plum are mainly used in the plum tree area, and the research on the sheet is hardly proceeded, and most of the sheet is discarded.

Korean Patent Publication No. 10-1431610 (Patent Document 1) discloses a method for reducing weight and visceral fat-reducing efficacy, a type 2 diabetes-treating effect, a cholesterol-reducing effect, and a triglyceride reducing effect on oat extract, The results are prepared. The omu is dried by fumigation of the less ripe fruit of the plum tree, which means that the root of the denture belonging to the dentistry is dried. According to the results of the above-mentioned Patent Document 1, it was confirmed that insulin resistance was low in the order of Omega Water Extract, Omega Seed Extract, Omega Seed Extract, and Omega Seed Extract. In order to activate the insulin action and reduce the fat synthesis, Or the extract of Ogawa and Prussia in comparison to the efficacy of the mixed extract is excellent. That is, according to Patent Document 1, it has been experimentally proved that the omep extract has a weak effect on treating type 2 diabetes mellitus in comparison with the mulberry extract or the mixed extract of mulberry and mulberry.

None of the published publications to date disclose that alcohol extracts or fractions thereof are effective for the treatment or prevention of diabetes.

Korean Patent Publication No. 10-1431610

The present inventors have continuously studied to increase the availability of the sheet which has been mostly disposed of, and as a result, by confirming that the sheet-extract or its fraction has alpha-glucosidase inhibitory effect and / or PPAR gamma agonist efficacy, It was completed. Particularly, considering that the prescription of the type 2 diabetes therapeutic agent is preferred to the combination therapy rather than the monotherapy, the bovine leaf extract and its fractions simultaneously having the alpha-glucosidase inhibitory effect and the PPAR gamma agonist efficacy are used as therapeutic agents for type 2 diabetes Commercialization is very likely.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating diabetes or hyperlipidemia, which comprises a leaf extract or a fraction thereof.

Another object of the present invention is to provide a health food composition for reducing the blood glucose level or the content of neutral fat in blood including the leaf extract or the fraction thereof.

In order to solve the above-mentioned problems, the present invention provides a pharmaceutical composition for prevention and treatment of diabetes or hyperlipidemia comprising an alcohol extract of sheet.

In addition, the present invention provides a pharmaceutical composition for preventing or treating diabetes or hyperlipidemia, which comprises a fraction obtained by sequentially fractionating an alcohol extract of sheet from n-hexane, dichloromethane, ethyl acetate and n-butanol.

In addition, the present invention provides a health food composition which reduces the blood glucose lowering or the blood fat content including alcohol extract of sheet.

The present invention also provides a health food composition for reducing the blood glucose level or the blood triglyceride content, comprising a fraction obtained by sequentially fractionating an alcohol extract of sheet from n-hexane, dichloromethane, ethyl acetate and n-butanol .

The extracts or fractions thereof as an active ingredient in the composition of the present invention have alpha-glucosidase inhibitory activity or PPAR gamma agonist activity or alpha-glucosidase inhibitory activity and PPAR gamma agonist activity simultaneously. Thus, the extracts or fractions thereof of the present invention exhibit the effect of inhibiting the increase of blood glucose level, the effect of decreasing triglyceride in blood, the effect of decreasing weight, and the effect of increasing insulin sensitivity.

Therefore, the sheet extract or its fractions can treat, prevent or ameliorate a disease in which a decrease in blood glucose level or a neutral fat content in blood is required. In particular, it can be used as an effective ingredient in medicines or health foods for the treatment, prevention or amelioration of type 2 diabetes, since it has the alpha-glucosidase inhibitory effect and the PPAR gamma agonist effect simultaneously.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a process diagram showing the process of obtaining an alcoholic extract or a solvent fraction thereof from a sheet.
FIG. 2 is a graph showing the results of measurement of the PPAR gamma agonist efficacy of the alcohol extract or the solvent fraction thereof on the sheet using the adipocyte differentiation ability.

Hereinafter, the present invention will be described in detail so that those skilled in the art can easily carry out the present invention.

The present invention relates to a pharmaceutical composition or a health food composition comprising a sheet extract or a fraction thereof.

The extracts or fractions thereof inhibit alpha-glucosidase and inhibit the absorption of glucose in the blood, and act as a PPAR gamma agonist to increase insulin sensitivity. In addition, the extracts or fractions thereof of the present invention have both alpha-glucosidase inhibitory activity and PPAR gamma agonist efficacy, so that they can be safely used alone for type 2 diabetes using a therapeutic agent by a combination therapy.

A series of methods for obtaining the mulberry extract or its fractions from the leaves of Prunus mume Sieb. Et Zucc are as follows:

I) extracting the leaf of the plum tree with at least one extraction solvent selected from the group consisting of C _1-4 alcohol and C _1-4 alcohol aqueous solution to obtain an alcoholic extract;

Ii) suspending the alcohol extract in water, and then sequentially fractionating the mixture with n-hexane, dichloromethane, ethyl acetate and n-butanol to obtain fractions.

A method of obtaining a sheet-form extract or a fraction thereof from the leaves of Prunus mume Sieb. Et Zucc will be described in more detail with reference to the flowchart of Fig.

Step i) is a step of obtaining a sheet extract.

In the present invention, leaves of plum trees are used as raw materials. As described above, since the effect of plum trees varies depending on leaves, stems, branches, and roots, the leaves of plum trees are used as raw materials in order to maximize blood glucose control function and / or insulin resistance. According to the experimental results of the present inventors, there was a remarkable difference in the alpha-glucosidase inhibitory effect and the PPAR gamma agonist efficacy of each part of the plum tree, and the leaf extract of the plum was superior to the plum extract in its efficacy up to 10 times Respectively. [Refer to Reference Example 4 in Table 4] Accordingly, in the present invention, among the various parts of the plum tree, a leaf part is used as a raw material.

The leaves of the plum trees were washed thoroughly with water, dried and shredded. And extracted by immersing in at least one extraction solvent selected from the group consisting of C _1-4 alcohol and C _1-4 alcohol aqueous solution. The extraction solvent may be selected from methanol, ethanol, isopropanol, propanol, butanol, and an aqueous solution thereof. Preferably, ethanol, ethanol aqueous solution or alcohol is used, more preferably an ethanol aqueous solution of 50 to 90% , And most preferably an ethanol aqueous solution having a concentration of 70% by weight is used. The amount of the extraction solvent is preferably 1: 1 to 30 times by weight, more preferably 1:10 to 20 times by weight, based on the dry weight of the leaf of the plum tree. In the present invention, There is no restriction. The extraction temperature may be in the range of from room temperature to the reflux temperature of the extraction solvent, and specifically, it may be extracted at a temperature of 20 to 80 ° C. The extraction time is preferably 1 to 10 days, but is not limited thereto. The number of times of extraction may be one or more times, but since the yield of the active ingredient is significantly reduced as the extraction continues, it may not be economical to repeat the extraction more than five times. Therefore, the extraction is preferably repeated two to five times Do.

The extraction method is a method commonly known in the art, for example, a method using an extraction device such as a supercritical extraction, a subcritical extraction, a high-temperature extraction, a high-pressure extraction or an ultrasonic extraction, or a method using an adsorption resin containing XAD and HP- And the like can be used.

The extract is concentrated under reduced pressure using a vacuum rotary evaporator or the like to obtain an extract. The resulting extract may also be subjected to vacuum drying, vacuum drying, boiling drying, spray drying, room temperature drying, freeze drying, and the like, if necessary. In particular, when the freeze-drying method is applied, there is an advantage that loss of volatile organic substances in the extract can be reduced.

Step ii) is a step of extracting the leaf extract with an organic solvent to obtain fractions.

Specifically, the ethanol extract of the sheet obtained in the step (i) is suspended in water, and then fractionated by sequential fractionation with n-hexane, dichloromethane, ethyl acetate and n-butanol. The above fraction can be obtained by repeating from 1 time to 5 times, preferably 3 times.

The fraction is concentrated under reduced pressure using a vacuum rotary evaporator or the like to obtain an extract. The resulting extract may also be subjected to vacuum drying, vacuum drying, boiling drying, spray drying, room temperature drying, freeze drying, and the like, if necessary. In particular, when the freeze-drying method is applied, there is an advantage that the loss of volatile organic substances in the fraction can be reduced.

Solvent fractions obtained by the above method or extracts of the extract with an organic solvent have alpha-glucosidase inhibitory activity, PPAR gamma agonist activity or alpha-glucosidase inhibitory activity and PPAR gamma agonist activity simultaneously. As a result, the leaf extract or fraction thereof exhibits the effect of inhibiting the increase of blood glucose level, the effect of decreasing the triglyceride in blood, the effect of decreasing weight, and the effect of increasing insulin sensitivity. Therefore, the leaflet extract or the fraction thereof can be used as an effective ingredient in medicines or health foods used for the treatment, prevention, or improvement of diseases in which blood glucose lowering or neutral fat content reduction in blood is required.

The pharmaceutical composition according to the present invention comprises a leaf extract or a fraction thereof as an active ingredient, and the active ingredient may be contained in an amount of 0.1 to 50% by weight based on the total weight of the composition, and the present invention is not limited thereto.

When the pharmaceutical composition is used clinically, it may be formulated together with carriers customary in the pharmaceutical field to prepare pharmaceutical preparations customary in the pharmaceutical field, such as tablets, capsules, powders, granules, pills, liquids and suspensions Preparations for oral administration; Injectable preparations in the form of injectable solutions or suspensions, or ready-to-use injectable dry powders which can be used as distilled water for injection for injection; Or ointments, and the like. The pharmaceutical preparations comminuted with conventional carriers may be administered orally or parenterally, for example intravenously, subcutaneously, intraperitoneally or topically. Accordingly, the pharmaceutical composition of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of medicaments.

Examples of carriers, excipients and diluents that can be contained in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methylcellulose, hydroxymethylcellulose, microcrystalline cellulose, silicified microcrystalline cellulose, povidone, crospovidone, croscarmellose sodium, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, Colloidal silicon dioxide, lactose, talc, magnesium stearate, colloidal stearyl magnesium, mineral oil, and the like.

 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, troches, rosin, capsules and the like, which may contain at least one excipient such as lactose, saccharose, sorbitol , Mannitol, starch, amylopectin, cellulose, calcium carbonate, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, elixirs, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like are used in addition to water and liquid paraffin, 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. As a suppository base, witepsol, macrogol, tween 61, cacao paper, laurin, glycerol gelatin and the like can be used. Parenteral administration may be by subcutaneous injection, intravenous injection, intramuscular injection or intra-thoracic injection.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the age, body weight, degree of disease, drug form, route of administration, and duration of the patient, but can be appropriately selected by those skilled in the art. However, for the desired effect, the pharmaceutical composition of the present invention may be administered at a dose of 0.01 mg / kg to 10 g / kg, preferably 1 mg / kg to 1 g / kg per day. The administration can be administered several times a day, preferably once or six times, at a predetermined time interval according to the judgment of a doctor or a pharmacist.

In addition, the health food composition according to the present invention may contain a leaf extract or a fraction thereof as an active ingredient, and may be ingested for the purpose of lowering blood glucose levels or reducing the content of triglycerides in blood. The active ingredient may be ingested as food prepared from tablets, capsules, powders, granules, pills, liquid preparations, suspensions, etc., or may be added to general foods. Unlike conventional medicines, the health food uses food as a raw material, so there is no side effect that may occur when a medicine is taken for a long time. The content of the active ingredient can be suitably determined according to its use purpose (for prevention or improvement). Generally, the active ingredient in the health food composition may be contained in an amount of 0.1 to 90% by weight. However, in the case of long-term ingestion intended for health and hygiene purposes or for health control purposes, 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 exceeding the above range.

There is no particular limitation on the kind of the food. Examples of foods to which the active ingredient can be added include dairy products including dairy products, meat, sausage, bread, biscuits, rice cakes, chocolates, snacks, confectionery, pizza, ramen noodles, other noodles, gums, ice cream, , Beverages, alcoholic beverages and vitamin complexes, dairy products, and dairy products, all of which include health functional foods in a conventional sense. The shape of the food is not particularly limited, and it may be a solid, semi-solid, gel, liquid, or powder.

The beverage can be prepared using the health food composition of the present invention. There are no particular restrictions on the selection of other ingredients other than the active ingredient as a component to be contained in the beverage, and it may contain various flavors or natural carbohydrates as additional ingredients such as ordinary beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. As other flavoring agents, natural flavoring agents (tau martin), stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) have. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition, the health food composition of the present invention may contain various additives such as various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and aging agents (cheese, chocolate, etc.) Salts, alginic acid and salts thereof, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks and the like. It can also contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not so important, but is generally selected in the range of 0.1 to 20% by weight in the health food composition of the present invention.

The present invention as described above is explained in more detail based on the following examples, but the present invention is not limited thereto.

[Example]

Example 1: Preparation of sheet extracts

Leaf of Prunus mume Sieb. Et Zucc collected from Sancheong, Gyeongsangnam - do was used as a raw material. The sheets were washed, dried and shredded. 230 g of the crushed sheet was immersed in 20 L of 70% ethanol and extracted repeatedly 2 to 5 times at room temperature. The extract was subjected to vacuum distillation to obtain 63 g (yield: 27.4%) of an ethanol extract of the sheet.

Example 2: Preparation of sheet fraction of solvent

Based on the flow chart shown in Fig. 1, solvent fractions were obtained from the sheet extracts. Namely, 63 g of the leaf extract obtained in Example 1 was suspended in 2 L of distilled water, and then 20 L of n-hexane, dichloromethane, ethyl acetate and n-butanol were sequentially fractionated and distilled under reduced pressure to obtain respective solvent fractions .

The yield of each solvent fraction is as follows: 6.0 g of n-hexane fraction, 3.4 g of dichloromethane fraction, 2.4 g of ethyl acetate fraction, 7.4 g of n-butanol fraction and 75.5 g of remaining water fraction.

[Experimental Example] Effectiveness of Extracts and Solvent Fractions

Experimental Example 1. Alpha-glucosidase inhibitory activity

Alpha-glucosidase inhibitory activity was measured on the sheet extracts and their solvent fractions obtained in Examples 1 and 2 above.

The enzyme alpha-glucosidase and the substrate para-nitrophenyl-glucopyranoside were purchased from Sigma. 0.1 M phosphate buffer solution (pH 6.8) was used directly.

10 μl each of the leaf extract or solvent fraction was added to 1 μl of 5 U alpha-glucosidase, and 84 μl of 0.1 M phosphate buffer and 5 μl of 50 mM para-nitrophenyl-glucopyranoside as a substrate were added thereto, And allowed to react at 37 ° C for 20 minutes. Then, 200 μl of 2M NaOH was added to stop the reaction, and the absorbance was measured at 405 nm. As a control, dimethylsulfoxide (DMSO) was used instead of the extract. The absorbance of each test solution on the basis of the control was measured to calculate the concentration (IC ₅₀ ) which inhibited 50% of alpha-glucosidase.

Table 1 summarizes the concentration (IC ₅₀ ) that inhibits 50% of alpha-glucosidase for each of the leaf extract and the solvent fraction.

division IC ₅₀ ([mu] g / mL) Sheet extract ethanol extract 70.6 ± 5.16
Mattress
Fraction n-hexane fraction - Dichloromethane fraction - Ethyl acetate fraction 68.2 ± 5.37 n-butanol fraction 75.8 ± 18.76 Water fraction -

According to the above Table 1, the leaf extract inhibited the alpha-glucosidase by 50% at a concentration of 70.6 g / mL, and the ethyl acetate fraction and the n-butanol fraction of the fractions showed alpha-glucosidase activity at the concentrations of 68.2 and 75.8 g / Sodium was inhibited by 50%.

Experimental Example 2: Efficacy as a PPARγ agonist

The efficacy of the extracts and fractions obtained in Examples 1 and 2 as PPARγ agonists was measured by the ability to differentiate into adipocytes.

The mouse whole cell line 3T3-L1 was purchased from the American Type Culture Collection (ATCC). DMEM medium, trypsin, FBS (Fetal bovine serum) and BCS (Bovine calf serum) were purchased from Gibco ( ^TM ) (Invitrogen corporation). AdipoRed ^TM Assay Reagent for measurement was purchased from Cambrex.

3T3-L1 preadipocytes were cultured in a DMEM medium containing 10% serum and antibiotics, in an incubator maintained at 37 ° C with 5 vol.% Carbon dioxide feed. 3T3-L1 cells were seeded in a 96-well plate at a concentration of 1 × 10 ⁴ cells / well and cultured until cells were fused to 100%. MD (0.5 mM 3-isobutyl-1-methylsynthine, 1 μM dexamethasone) cocktail was treated to measure adipocyte differentiation ability. After 2 days, the medium was replaced with a culture medium containing only 10% by weight of serum in DMEM medium, and further cultured for 6 days. For MDA treatment, extracts or solvent fractions were treated at 100, 50, 25, 12.5, 6.25, and 0 ㎍ / mL for MDA.

For 8 days, the differentiated and cultured adipocytes were fixed with 4% formaldehyde solution for 5 hours, washed twice with phosphate buffered saline (PBS) and treated with adipoRed for fat measurement. After 10 minutes, the fluorescence intensity of each extract or solvent fraction was measured with a fluorescence meter (excitation 485 nm; emission 535 nm). As a control, dimethylsulfoxide (DMSO) was used instead of the extract. The relative fat cell differentiation rate (%) of each extract or solvent fraction relative to the control group was calculated, and the result is shown in the graph of FIG.

As shown in FIG. 2, the extracts of Bacillus thuringiensis extract increased the adipocyte differentiation rate by 48.3% at a concentration of 50 ㎍ / mL and 89.6% and 62.4% at the concentration of 50 ㎍ / mL of the dichloromethane and ethyl acetate fractions, respectively , And 100 ㎍ / mL of ethyl acetate increased the fat cell differentiation rate by 70.1% compared to the control group.

When preparing the adipocyte differentiation rate of rogiglitazone, which is a positive control, the extracts or the solvent fractions of the present invention showed almost similar adipocyte differentiation efficacy. It has been found that Avandia ™ (active ingredient: rosiglitazone), which is marketed as a diabetic treating drug, has a high ability to differentiate into adipocytes and thus has an effect as a PPARγ agonist. Therefore, the extracts or the solvent fractions of the present invention have a glucose regulating action as a PPARγ agonist .

According to the results of Experimental Examples 1 and 2, the leaf extract of the present invention has an inhibitory effect on alpha-glucosidase and an activity to increase PPAR gamma simultaneously, and thus it has strong glucose regulating function.

Experimental Example 3. Analysis of total polyphenol content and flavonoid content

Total polyphenol content and flavonoid content were measured to analyze the antidiabetic effect and the correlation between the antioxidant efficacy of the extracts and fractions obtained in Examples 1 and 2 above.

(1) Analysis of total polyphenol content

Total polyphenol content was quantified by Folin-Ciocalteu method (Kaur & Kapoor, 2002). Each of the leaf extracts and the solvent fractions were dissolved in dimethylsulfoxide (DMSO) to a final concentration of 2 mg / mL, and 0.2 mL was added to the test tube. The primary extracts and the distilled water were added to make 3 mL, and 0.5 mL of Folin-Ciocalteu's phenol reagent was added And left at room temperature for 3 minutes. It was then mixed with 2 mL of a 20% aqueous solution of sodium carbonate and allowed to stand at room temperature for 1 hour. Absorbance was measured at 700 nm with a spectrophotometer and converted into total polyphenol content. The total polyphenol content was determined using a standard curve prepared from tannic acid as a standard product at a concentration of 0 to 600 μg / mL. TAE (Tannic Acid Equivalent) / g was used as the measurement unit.

(2) Analysis of flavonoid content

The flavonoid content was measured by the aluminum chloride method. Each of the leaf extracts and the solvent fractions were dissolved in dimethylsulfoxide (DMSO) to a concentration of 2 mg / mL, and 0.05 mL was added to the test tube. 1 mL of methanol and 0.3 mL of 5% sodium nitrite solution were added and stirred. Respectively. After adding 0.3 mL of 10% aluminum chloride solution, the mixture was allowed to stand at room temperature for 6 minutes, 2 mL of 1M sodium hydroxide solution was added, water was added to a total volume of 10 mL, and the mixture was allowed to stand at room temperature for 15 minutes. The supernatant solution was measured for absorbance at 510 nm using a spectrophotometer. At this time, the standard used for the calibration curve was a rutin, and the measurement unit was RE (Rutin Equivalents) / g.

Table 2 summarizes the analysis results of total polyphenol content and flavonoid content for each of the leaf extracts and solvent fractions.

division yield
(%, w / w) Total polyphenol content
(mg TAE / g) Flavonoid content (mg RE / g) Ethanol extract 27.4 428.0 ± 10.8 389.6 ± 7.2 n-hexane fraction 9.5 112.1 ± 8.2 87.0 ± 4.1 Dichloromethane fraction 5.4 168.6 ± 4.6 134.0 ± 5.9 Ethyl acetate fraction 3.8 883.5 ± 14.9 820.1 + - 7.7 n-butanol fraction 11.7 557.2 ± 7.1 503.3 8.3 Water fraction 66.9 130.8 ± 6.6 115.5 ± 2.9

As shown in Table 2, the contents of total polyphenols and flavonoids were very high in the leaf extract, ethyl acetate fraction and n-butanol fraction.

Experimental Example 4. Antidiabetic evaluation in an animal model of type II diabetes

Long-term high-fat diets induce insulin resistance, but do not accompany diabetes (Chalkley et al., 2002) . Thus, the type 2 diabetes study suggests that diets high in fat diet and low in streptozotocin (STZ) One diabetic animal is widely used (Mu et al., 2006; Srinivasan et al., 2005).

Five-week-old ICR mice (males) were obtained from Raon Bio and purified for one week with free, regular solid feed and water. The environmental conditions of the animal room were set at a temperature of 23 ± 3 ° C, a relative humidity of 50 ± 10%, a lighting time of 12 hours (8:00 am to 8:00 pm), a ventilation frequency of 10 to 20 times / . During the experiment, the temperature and humidity of the animal room were automatically controlled by constant temperature humidification.

2) STZ treatment group, 3) high fat diet group, 4) STZ treatment group and high fat diets induction group, 5) diabetic group, 5 mg / kg, 6) 25 mg / kg of the extracts from the leaves after induction of diabetes, and 7) 50 mg / kg of the extracts from the leaves of the leaves after diabetic induction.

To induce diabetes in experimental animals, obese type 2 diabetes mellitus was induced by feeding high fat diet for 3 weeks, which is recovery period after induction of diabetes by low dose STZ. Blood glucose and body weight were measured at intervals of one week while oral extracts were administered to diabetic experimental animals for 4 weeks.

After the completion of the experiment, blood was collected and the serum was separated. Then, serum glucose, total cholesterol, triglyceride, body weight, and feed intake were measured. The results are summarized in Table 3 below.

division Blood sugar
(mg / dL) Total cholesterol
(mg / dL) Triglyceride
(mg / dL) weight
(g) Feed intake
(g / day) General dietary intake group 130.4 ± 9.24 538.9 ± 5.62 143.0 ± 15.55 33.5 ± 1.49 33.5 ± 1.49 High fat diet group 207.8 ± 12.61 946.4 ± 7.85 246.8 ± 10.49 40.3 ± 2.06 40.3 ± 2.06 Streptozotocin-treated group 284.6 ± 10.98 675.4 ± 6.88 232.6 ± 8.98 32.6 ± 1.14 32.6 ± 1.14
Party
Urine
U
foot
group Control group 447.6 ± 15.87 1045.6 ± 12.63 494.9 ± 1480 37.8 ± 1.63 37.8 ± 1.63 Sheet extract ethanol extract
25 mg / kg 323.3 ± 29.72 * 949.1 ± 22.33 336.0 + - 23.58 * 32.9 ± 1.90 * 32.9 ± 1.90 * Sheet extract ethanol extract
50 mg / kg 264.2 ± 26.73 ** 1035.2 + - 18.80 320.7 ± 20.73 * 29.9 ± 0.56 ** 29.9 ± 0.56 ** * p < 0.05, ** p < 0.01

As shown in Table 3, in comparison with the control group, the group administered with the mulberry extract has been remarkably improved in the serum glucose, total cholesterol, triglyceride and weight loss. In particular, the 50 mg / kg group of extracts from the leaf extracts decreased the blood sugar by about 59% and decreased the triglyceride in the blood by 64.8%.

[Reference Example]

Effectiveness of plum extract and mulberry extract

In this experiment, the extracts and solvent fractions of the plum (plum) and leaves (sheet) of plum tree were compared for the alpha-glucosidase inhibitory effect and the PPAR gamma agonist activity.

The extracts and solvent fractions of plum or sheet were prepared as described in Examples 1 and 2, respectively. The alpha-glucosidase inhibitory potency assay according to Experimental Example 1 and the PPAR gamma agonist according to Experimental Example 2 were each analyzed.

In Table 4, the alpha-glucosidase inhibitory effect and the PPAR gamma increase effect on the extract or solvent fraction of the plum and the extract or solvent fraction of the sheet were compared and shown.

division Alpha-glucosidase inhibition,
IC ₅₀ ([mu] g / mL) PPAR [gamma] increase,
EC ₅₀ ([mu] g / mL) plum Mattress plum Mattress Ethanol extract 352.2 + - 11.23 70.6 ± 5.16 - 62.1 ± 3.22 n-hexane fraction - - - - Dichloromethane fraction - - - 30.2 ± 1.21 Ethyl acetate fraction 203.6 ± 6.87 68.2 ± 5.37 412.6 + - 21.63 39.6 ± 1.53 n-butanol fraction 232.1 ± 13.57 75.8 ± 18.76 - - Water fraction - - - -

According to the above Table 4, it can be seen that the leaf extract (or fraction) has an excellent inhibitory effect on alpha-glucosidase and an excellent effect of increasing PPAR.gamma. In comparison with the plum extract (or fraction). As shown in Table 4, the plum extract (or fraction) showed an inhibitory effect on alpha-glucosidase and an increase effect on PPAR gamma Remarkably low. Considering this fact, the extracts (or fractions) of the mulberry extracts are effective as a therapeutic agent for diabetes or hyperlipidemia by overcoming the limitations of plum extract (or fraction) and exhibiting a high action mechanism for reducing blood glucose level and neutral fat content in blood .

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, Of the right.

Claims (11)

A pharmaceutical composition for prevention and treatment of diabetes or hyperlipidemia comprising an alcohol extract of sheet.
A pharmaceutical composition for the prevention and treatment of diabetes or hyperlipidemia comprising fractions obtained by sequentially fractionating an alcohol extract of a sheet with n-hexane, dichloromethane, ethyl acetate and n-butanol.
3. The method according to claim 1 or 2,
Wherein the alcohol used for the extraction is a C _1-4 alcohol or an aqueous solution of C _1-4 alcohol in a concentration of 50-90% by weight.
3. The method according to claim 1 or 2,
Wherein the extract or fraction is a liquid or lyophilized powder.
3. The method according to claim 1 or 2,
A pharmaceutical composition for the prevention and treatment of diabetes or hyperlipidemia characterized in that the diabetes is type 2 diabetes.
3. The method according to claim 1 or 2,
A pharmaceutical composition for preventing or treating diabetes mellitus or hyperlipidemia, which is prepared in any one form selected from the group consisting of tablets, capsules, powders, granules, pills, liquids and suspensions.
The present invention relates to a health food composition containing a sheet of alcoholic extract and reducing blood glucose level or neutral fat content in blood.
A fraction obtained by sequentially fractionating a sheet of alcoholic extract with n-hexane, dichloromethane, ethyl acetate and n-butanol, and reducing the blood glucose level or the content of triglyceride in blood.
9. The method according to claim 7 or 8,
The alcohol is C _{1 ~ 4} alcohol or 50 to 90% by weight of C _{1 ~ 4} health food composition for reducing the blood glucose lowering or blood content in the triglycerides, characterized in that the alcohol aqueous solution used in the extraction.
9. The method according to claim 7 or 8,
Wherein the extract or fraction is in a liquid or lyophilized powder form to reduce the blood glucose lowering or the blood triglyceride content.
9. The method according to claim 7 or 8,
Wherein the food is prepared in any one form selected from the group consisting of tablets, capsules, powders, granules, pills, liquid preparations and suspensions, wherein the composition reduces the blood glucose level or the blood triglyceride content.

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