KR102246783B1 - The composition for anti-obesity and anti-diabets, comprising the extract of aralia excelsa - Google Patents

Abstract

The present invention relates to an anti-obesity and anti-diabetic composition comprising an Aralia excelsa (SE) extract or a fraction thereof as an active ingredient. The extracts and fractions of Aralia excelsa inhibited differentiation into adipocytes and glucose uptake in vitro, and it was confirmed that they have anti-diabetic efficacy as a result of the evaluation of OGTT (Oral glucose tolerance test) in vivo. Therefore, the composition comprising the Aralia excelsa extract or a fraction thereof of the present invention as an active ingredient exhibits anti-obesity and anti-diabetic effects, and thus can be usefully used in the prevention or treatment of obesity or diabetes.

Description

A composition for preventing and treating anti-obesity and anti-diabetes containing ARALIA EXCELSA extract and its fractions {THE COMPOSITION FOR ANTI-OBESITY AND ANTI-DIABETS, COMPRISING THE EXTRACT OF ARALIA EXCELSA}

The present invention relates to a pharmaceutical composition or a health functional food composition having anti-obesity and anti-diabetic effects comprising an Aralia excelsa extract or fraction as an active ingredient.

There have also been many changes in eating habits with changes in lifestyle and economic growth. Particularly, busy modern people consume a lot of high-calorie foods such as fast food, but excessive weight and obesity are increasing due to a small amount of exercise. Excessive weight means that the body mass index (BMI, weight divided by the square of height) is 25 or more and less than 30, and obesity means when the body mass index is 30 or more.

Excessive weight increases blood pressure and cholesterol levels, increasing the incidence of various adult diseases such as heart disease. In particular, the effect of obesity on various diseases is very serious, and it has been reported that 80% of diabetic patients and 21% of heart diseases worldwide are the cause of obesity. In addition, various cancers such as uterine cancer, kidney cancer, and breast cancer are also directly or indirectly related to obesity, and in the case of obesity or overweight, the incidence of sleep apnea, osteoarthritis, and cholelithiasis is also higher than that of normal people (Stein CJ, The epidemic of obesity. J Clin Endocrinol Metab 2004; 89: 2522-2525.).

Obesity is not a disease that occurs as a single cause, but is difficult to treat by any one method because it is caused by a combination of genetic, environmental-social, and mental factors. Currently, methods for treating obesity can be divided into dietary, exercise, behavioral, and other lifestyle correction methods, drug treatment, and surgical treatment. In severe cases of obesity, drug treatment should be combined.

Drugs for the treatment of obesity are mainly related to mechanisms of suppressing appetite or suppressing fat absorption. Obesity treatments developed through a mechanism to suppress appetite include Sibutramine, Phentermine, Phendimetrazine, Phentermine/Topiramate combination, and Locaserin. , Since the above drugs can cause central nervous system side effects or cardiovascular side effects, most of the drugs are prohibited or are used extremely limitedly.

On the other hand, diabetes is one of the important adult diseases worldwide. In particular, the prevalence of diabetes is on the rise as the economy grows and changes in dietary habits and diseases such as obesity increase. If diabetes is left unattended, it cannot be avoided from developing into complications. Usually, 10 to 20 years after diabetes, almost all organs in the body are damaged, resulting in diabetic retinopathy, diabetic cataract, diabetic nephropathy, and diabetic neuropathy. neuropathy), heart disease, cancer, osteoporosis, etc. Therefore, various drugs are being developed to treat diabetes.

Stein CJ et al., The epidemic of obesity. J Clin Endocrinol Metab 2004; 89: 2522-2525.

It is an object of the present invention to provide a plant extract effective against diabetes and obesity, and specifically, by confirming that the Aralia excelsa extract or a fraction thereof has anti-diabetic and anti-obesity effects, the present invention was completed.

To achieve the above object,

In one aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating diabetes, comprising an Aralia excels extract or a fraction thereof as an active ingredient.

Another aspect of the present invention provides a pharmaceutical composition for preventing or treating obesity comprising the SE extract or a fraction thereof as an active ingredient.

Another aspect of the present invention provides a health functional food composition for preventing or improving diabetes comprising the SE extract or a fraction thereof as an active ingredient.

Another aspect of the present invention provides a health functional food composition for preventing or improving obesity, comprising the SE extract or a fraction thereof as an active ingredient.

In order to confirm whether the SE extract or a fraction thereof has an antidiabetic effect, the effect of the SE extract or fraction on the differentiation of adipocytes in vitro was observed. In addition, the effect of the extract or fraction on glucose uptake in the blood in vivo was measured. As a result, it was confirmed that the group treated with the SE extract or fractions thereof had excellent anti-diabetic and anti-obesity effects.

Figure 1 shows that the adipocyte differentiation inhibitory activity of Aralia excelsa extract obtained from Costa Rica or Nicaragua is equivalent to the results of UPLC-MS analysis.
Figure 2 shows the in vitro adipocyte differentiation inhibitory activity of Aralia excelsa extract obtained from Costa Rica or Nicaragua.
Figure 3 shows the MPLC chromatogram of the extract of Aralia excelsa (Nicaragua).
Figure 4 shows the CAD chromatogram of the SE methanol extract and the column fraction.
5 shows a chromatogram of SE methanol extract and column fractions at a wavelength of 210 nm.
6 shows a chromatogram of SE methanol extract and column fractions at a wavelength of 254 nm.
7 shows a chromatogram of a SE methanol extract and a column fraction at a wavelength of 280 nm.
8 shows a chromatogram of SE methanol extract and column fractions at a wavelength of 360 nm.
9 and 10 are analysis of SE methanol extract and column fractions using UPLC-MS.
11 and 12 are analysis of SE methanol extract and column fractions through a diode array (PDA).
13 is a schematic diagram of a method of obtaining an SE solvent fraction.
14 shows the equivalence and index components of the column fraction and the solvent fraction.
15 shows the cytotoxicity of the SE methanol extract and the column fraction was confirmed in L6 cells.
Figure 16 shows the cytotoxicity of the SE methanol extract and the solvent fraction confirmed in L6 cells.
17 shows the degree of the SE methanol extract and the column fraction on glucose uptake of L6 muscle cells.
18 shows the degree of the SE methanol extract and the solvent fraction on glucose uptake by L6 muscle cells.
19 shows the postprandial hypoglycemic activity of the SE methanol fraction and the solvent fraction.
Figure 20 shows the cytotoxicity of the SE methanol extract and the column fraction was confirmed in 3T3-L1 cells.
21 and 22 are graphs and images showing the effect of the SE methanol extract and the column fraction on the differentiation and lipid accumulation of 3T3-L1 cells.
23 shows the cytotoxicity of the SE methanol extract and the solvent fraction confirmed in 3T3-L1 cells.
FIG. 24 shows the effect of SE methanol extract and solvent fraction on lipid accumulation in 3T3-L1 cells.

Hereinafter, the present invention will be described in detail.

One aspect of the present invention relates to a pharmaceutical composition for preventing or treating diabetes, comprising an Aralia excelsa (hereinafter referred to as SE) extract or a fraction thereof as an active ingredient.

As used herein, the term "extract" refers to obtaining components contained in Aralia excelsa using a solvent. When obtaining the extract of Aralia excelsa, Aralia excelsa may be used without limitation, such as cultivated or commercially available, and Aralia excelsa may have leaves, stems, or roots all available, and specifically leaves, It is not limited to this.

In the obtaining step, water, alcohol, or a mixture thereof may be used as the extraction solvent, and _{a lower alcohol of C 1} to C ₃ may be used as the alcohol. Methanol, ethanol or propanol can be used as the lower alcohol. The extraction method may be an extraction method such as hot water extraction, immersion extraction, reflux cooling extraction, or ultrasonic extraction, but is not limited thereto.

The extraction can be extracted by adding 1 to 20 times the solvent to the dry weight of the dried Aralia Excel company, and specifically, it can be extracted by adding 2 to 5 times the solvent. The extraction temperature may be 20°C to 100°C, specifically 20°C to 40°C, and more specifically room temperature, but is not limited thereto. In addition, the extraction time may be 10 to 48 hours, specifically 15 to 30 hours, more specifically 24 hours, but is not limited thereto. In addition, the number of extractions may be 1 to 5 times, may be repeatedly extracted 3 to 4 times, and specifically 3 times, but is not limited thereto.

As an example of the extraction method, polymerization liquid chromatography (MPLC) may be used. As an example, after extracting Aralia excelsa using purified water, an MPLC fraction may be obtained using methanol.

In the obtaining step, a process of concentrating the extract under reduced pressure may be additionally included, and the concentration under reduced pressure may be performed using a vacuum vacuum concentrator or a vacuum rotary evaporator, but is not limited thereto. In addition, the manufacturing step may further include drying the extract that has undergone the vacuum concentration process. The drying may be vacuum drying, vacuum drying, boiling drying, spray drying, or freeze drying, but is not limited thereto.

In addition, the extract includes an extract, a dilution or concentrate of the extract, a dried product obtained by drying the extract, or all of these preparations or purified products.

As used herein, the term "fraction" refers to a product obtained by a fractionation method for separating a specific component or a specific group from a mixture containing various constituents. Specifically, it may be hexane, chloroform, ethyl acetate, butanol, or a mixed solvent fraction thereof. The fractionation method in the present specification is not limited to the method as long as it is a method for obtaining a substance used in the prevention or treatment of diabetes.

_{Aralia excelsa fraction is suspended in a polar solvent of a lower alcohol of C 1} to C ₃ such as water, methanol, ethanol and butanol by suspending the above-described Aralia excelsa extract in an organic solvent such as hexane, dichloromethane or butanol. The fractionation process can be obtained by repeating distribution several times. As an example, the methanol extract of Aralia excel was fractionated three times with hexane to obtain a hexane fraction, and the remaining water layer fraction was repeatedly fractionated three times with a chloroform solvent to obtain a chloroform fraction. The water layer fraction remaining above may be repeatedly distributed with butanol three times to obtain a butanol fraction.

The term "diabetes" as used herein refers to a disease that occurs when the secretion of insulin is insufficient or the action and function of insulin are not sufficiently performed. In the case of this disease, excessive decomposition of glycogen, protein and fat causes abnormal increase in glucose concentration in the liver or blood, causing diabetes and ketonuria, and circulation disorder with blood concentration due to electrolyte loss due to abnormal water and electrolyte metabolism. , Kidney disorders, and other pathological conditions. Diabetes is divided into insulin-dependent diabetes (type I) and insulin-independent diabetes (type II). Diabetes can be diagnosed generally by measuring blood glucose levels, but there are differences according to the criteria. In humans, diabetes is generally diagnosed when glucose in the blood is usually 200 mg/dl or more and 140 mg/dl or more when fasting.

Another aspect of the present invention relates to a pharmaceutical composition for preventing or treating obesity comprising an Aralia excelsa (SE) extract or a fraction thereof as an active ingredient.

In this case, the extract _{may be extracted with water, a lower alcohol of C 1} to C ₃ or a mixture thereof, and specific details are as described above. In addition, the fraction may be obtained through polymerization liquid chromatography of the methanol extract. In addition, the fraction may be a hexane fraction, a chloroform fraction, and a butanol fraction obtained through the above-described solvent fractionation method. Details are as described above.

As used herein, the term "obesity" refers to an excessive accumulation of body fat. Criteria for obesity may be described as obesity if the body fat is 25% or more of body weight, women more than 30-35%, body mass general measurement methods as is shown by the weight (kg) / height (m) ² index (BMI; Body Mass Index) is widely used. When Western BMI 30kg / m ² is exceeded obesity, 25 ~ 30kg / m ² If the In and defined as overweight, in the case of the Orientals, the than about two lower 28 kg / when m ² is exceeded obesity, 23 ~ 28 kg / m ² can be said to be overweight. In addition, obesity can be defined based on the waist to hip ratio (WHR) or the amount of abdominal fat, and in the present invention, all cases of obesity defined by other general criteria including the above criteria are included. do.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable additive, wherein the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, Lactose, mannitol, syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, opadry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, stearic acid Calcium, sucrose, dextrose, sorbitol, talc, and the like can be used. The pharmaceutically acceptable additive according to the present invention may be included in an amount of 0.1 to 90 parts by weight with respect to the composition, but is not limited thereto.

The pharmaceutical composition of the present invention can be administered in various oral and parenteral dosage forms at the time of actual clinical administration.When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants are used. Can be prepared using.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations include at least one excipient, such as starch, calcium carbonate, and water in the mixed extract of the present invention. It can be prepared by mixing sucrose, lactose, or gelatin. In addition, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions and syrups, but may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to water and liquid paraffin, which are commonly used simple diluents. .

Formulations for parenteral administration may include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

The composition of the present invention can be administered orally or parenterally according to a desired method, and when administered parenterally, external use of the skin or intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection injection method You can choose. The dosage range may vary depending on the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate, and severity of disease.

The dosage of the composition of the present invention varies according to the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate, and severity of disease, and the daily dosage is the extract of the present invention. Based on the amount, it may be 0.0001 mg/kg to 100 mg/kg, specifically 0.001 mg/kg to 10 mg/kg, and may be administered once to 6 times a day, but is not limited thereto.

Another aspect of the present invention relates to a health functional food composition for preventing or improving diabetes comprising an Aralia excelsa (SE) extract or a fraction thereof as an active ingredient.

Another aspect of the present invention relates to a health functional food composition for preventing or improving obesity, comprising an Aralia excelsa (SE) extract or a fraction thereof as an active ingredient.

As used herein, the term "prevention" refers to any action that inhibits or delays the occurrence, spread, and recurrence of a target disease by administration of the pharmaceutical composition, and "improvement" refers to a target disease or target by administration of the composition. It refers to any action in which symptoms improve or are beneficially changed.

In the health functional food composition containing the composition of the present invention as an active ingredient, each component contained in the extract of the present invention is a material that is safe for the human body, and even when ingested, it is effective in preventing or improving diabetes, It can be used as food because it is effective in preventing or improving obesity.

As food, it can be used, for example, in various foods, beverages, gum, tea, vitamin complexes, health functional foods, and the like. The food can be prepared in formulations such as tablets, granules, powders, capsules, liquid solutions, and pills according to a known manufacturing method, and the content of the extract according to the present invention is 0.0001 based on the total weight of the food composition depending on the formulation. It can be adjusted to 100% by weight. Except for containing the extract according to the present invention as an active ingredient, there is no particular limitation on other ingredients, and various conventional flavoring agents or natural carbohydrates may be contained as additional ingredients.

Examples of the natural carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, and the like; And polysaccharides, for example, common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion 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 to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavors and natural flavoring agents, coloring agents and heavy weight agents (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid, and It may contain salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, and the like.

In addition, the food composition according to the present invention may contain flesh for the manufacture of natural fruit juice and fruit juice beverages and vegetable beverages. These components may be used independently or in combination. The proportion of these additives is not so critical, but may be selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

The term "functional food" is the same term as food for special health use (FOSHU), and refers to foods with high medical and medical effects that have been processed to efficiently display bioregulatory functions in addition to nutritional supply. do. Here, the term "function (sex)" means obtaining useful effects for health purposes such as controlling nutrients or physiological effects on the structure and function of the human body. The food product of the present invention can be prepared by a method commonly used in the art, and during the production, raw materials and ingredients commonly added in the art may be added to prepare it. In addition, the formulation of the food may also be prepared without limitation as long as it is a formulation recognized as a food. The food composition of the present invention may be prepared in various forms of formulation, and unlike general drugs, it has the advantage of not having side effects that may occur when taking a drug for a long time using food as a raw material, and is excellent in portability.

Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are for illustrative purposes only, and the scope of the present invention is not limited thereto.

Example One. Aralia excelsa (SE) Preparation of extract

Example 1.1. Medium pressure liquid chromatography (MPLC) SE methanol Fraction Produce

In order to obtain the methanol fraction of Aralia excelsa, medium pressure liquid chromatography (MPLC) was performed using Armen spot prepII. The specific method is as follows (Fig. 3):

Column 2 x 25 cm Resin YMC ODS AQ HG 100g, 10 uM Solvent DW(A), Methanol (B) Flow 18 ml/min Sample SE total extract Method 0 to 5 minutes 0% MeOH 5 to 60 minutes 100% MeOH 60 to 80 minutes 100% MeOH

Fraction weight SE Fr.1 325.4 SE Fr.2 24.7 SE Fr.3 19.1 SE Fr.4 22.9 SE Fr.5 21.2 SE Fr.6 410.5 SE Fr.7 22.3

Example 1.2. UPLC through MPLC Fraction Identification of indicator components

In order to analyze the fraction, using an ACQUITY ^TM Ultra Performance LC and a detector with a wavelength of UV 200 to 400 nm, charged aerosol detection (CAD), mass spectrometry (MS), and analysis of components of the fraction Was performed. In this case, 0.1% FA DW was used as the solvent A, and 0.1 FA ACN (acetonitrile) solvent was used as the solvent B. Gradient was analyzed as shown in Table 3 below.

Time(min) Flow(mL/min) %A %B 0.00 0.400 90 10 1.00 0.400 90 10 12.00 0.400 0 100 13.40 0.400 0 100 13.50 0.400 90 10 15.00 0.400 90 10

At this time, ACQUITY UPLC® BEH C18 1.7 μm, 2.1 X 100 mm was used as the column, and analysis was performed by irradiating UV with wavelengths of 280 nm, 254 nm, 230 nm, and 360 nm to measure absorbance. The dosage volume was 1 mg/ml, and 5 μl was administered (FIGS. 4 to 12 ).

Example 2. Preparation of SE fraction through solvent fractionation method

Example 2.1. Preparation of SE fraction through solvent fractionation method

A solvent fraction was obtained by the method of FIG. 13. Specifically, 69.8 g of SE methanol extract was mixed with 1 L of hexane, and the process of separating the hexane fraction was performed three times, and then dried under reduced pressure to obtain 1.41 g of a SE hexane fraction. In addition, the water layer from which the SE hexane fraction was removed was mixed with 1 L of chloroform to separate the chloroform fraction three times, and then dried under reduced pressure to obtain 15.71 g of a SE chloroform fraction. In addition, the water layer from which the SE chloroform fraction was removed was mixed with 1 L of butanol to separate the butanol fraction three times, and then dried under reduced pressure to obtain 11.09 g of the SE butanol fraction (FIG. 13).

Example 2.2. Checking the index components of the column fraction and solvent fraction through UPLC-MS

In order to analyze the column and solvent fractions, the equivalence comparison and index component analysis of the fractions whose activity was confirmed in the same manner as in the above-described Example 1.2. was performed (FIG. 14 and Table 4).

Peak [MH] ^- Rt (min) MS/MS Identity Column or solvent fraction One 911 6.30 3β-O-{α-L-rhamnopyranosyl-(1->2)[β-D-glucopyranosyl-(1->4)-α-L-arabinopyranosyl]} hederagenin (isomer) 4,5, DW 2 911 6.55 749,603,
471 3β-O-[β-D-glucopyranosyl-(1->3)-α-L-rhamnopyranosyl-(1->2)-α-L-arabinopyranosyl] hederagenin (isomer) 4,5, DW 3 749 6.66 3β-O-[α-L-rhamnopyranosyl-(1->2)-α-L-arabinopyranosyl] hederagenin (isomer) 4,5, DW 4 749 6.87 603,471 3β-O-[α-L-rhamnopyranosyl-(1->2)-α-L-arabinopyranosyl] hederagenin (isomer) 4,5, DW 5 749 6.99 603,471 3β-O-[α-L-rhamnopyranosyl-(1->2)-α-L-arabinopyranosyl] hederagenin (isomer) 4,5, DW 6 895 7.38 733,587,
455 3β-O-{α-L-rhamnopyranosyl-(1->2)[β-D-glucopyranosyl-(1->4)-α-L-arabinopyranosyl]} oleanolic acid (isomer) 4,5, DW 7 733 7.98 587,455 3β-O-[α-L-rhamnopyranosyl-(1->2)-α-L-arabinopyranosyl] oleanolic acid (isomer) 4,5, DW

Experimental Example 1. Confirmation of antidiabetic effect

Experimental Example 1.1. Confirmation of cytotoxicity: in vitro experiment

After completion of the L6-GLUT4myc cell differentiation, it was exchanged with a serum-free medium and cultured for 6 hours. SE methanol extract (SE-To) or fractions 1 to 7 (SE_Fr. 1 to 7) were treated at a concentration of 20 μM or 40 μM for 24 hours, and then 3-[4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide (MTT) reagent was treated with a final concentration of 50 μM and incubated for an additional hour. Thereafter, all the medium was discarded, and the non-aqueous formazan produced in the cells was dissolved in DMSO, and the absorbance was measured at 540 nm.

In FIG. 15, the cell growth was reduced to 20% compared to the control group in the 24-hour treatment group with a final concentration of 20 μM and 40 μM of the SE methanol extract, fraction 5 (SE_Fr.5) and fraction 6 (SE_Fr.6). Cytotoxicity could be confirmed. No cytotoxicity was observed in the remaining fractions.

16 shows the result of confirming the cell growth rate by fractionating the SE methanol extract (SE_Total) by solvent. The concentration-dependent growth rate of L6-GLUT4myc cells decreased in the SE methanol extract, nucleic acid layer (SE_Hex), chloroform layer (SE_CHCL3), and butanol layer (SE_BuOH) treatment group. It can be seen that cytotoxicity was not observed in the water layer (SE_DW) treatment group.

Experimental example 1.2. Antidiabetic Checking the effect: in vitro experiment

L6-GLUT4myc myoblasts were planted in a 24-well plate at ^{a concentration of 2 X 10 5} cells/ml, and differentiation was induced for 8 days by changing the medium every two days. After completion of differentiation, it was replaced with a serum-free medium and cultured for an additional 6 hours. Thereafter, the SE methanol extract or fractions 1 to 7 were treated at a concentration of 20 μM or 40 μM for 30 minutes, and at the same time, 100 nM pig-derived insulin (positive control) and 20 μM cytochalasin B (actin inhibitor) were each treated for 30 minutes. . After the reaction, 0.2 μCi/ml [ ¹⁴ C]-2-deoxy-D-glucose was treated for 5 minutes, and all substrates that were not absorbed in the cells were washed 3 times with KRB buffer. Dissolve the cells with 500 μl of 1 M sodium hydroxide, mix 200 μl lysate with 1 ml of LSC Cocktail and put it in a vial for measurement, and measure the DPM value with a scintillation counter (Liquid Scintillation Analyzer, PerkinElmer, Tri-Carb 2910 TR). Was calculated. In addition, GLUT4-independent glucose uptake was excluded by excluding the values of the cytochalasin B-treated group from all values.

In FIG. 17, treatment with 100 nM pig-derived insulin (positive control group) appeared to increase glucose uptake in L6-GLUT4myc myoblasts by about twice as much as that of the control group. Accordingly, in the group treated with SE methanol extract (SE_To), fraction 6 (SE_Fr.6), and fraction 7 (SE_Fr.7), concentration-dependent increase in intracellular glucose uptake was observed, but among them, SE, whose cytotoxicity was confirmed. Excluding the methanol extract and fraction 6, it is judged that 7 has an effective effect on increasing the glucose absorption capacity in muscle cells.

18 shows the result of confirming the glucose absorption ability by fractionating the SE methanol extract (SE_Total) by solvent. It was confirmed that glucose uptake in L6-GLUT4myc muscle cells was increased only in the water layer of SE.

Experimental example 1.3. Antidiabetic effect Check:In vivo Experiment

ICR female mice (5 weeks old) without a specific pathogen were acclimatized for 1 week and the experiment was carried out. The sample was dissolved in 10% DMSO/10% Tween-20, and the positive control, Metformin, was dissolved in PBS. The amount of liquid to be administered was 10 ml/kg. The acclimated mice were fasted for 16 hours before the test. On the day of the experiment, a sample was orally administered to mice and 90 minutes later, D-(+)-glucose was orally administered at a dose of 2 g/kg at 0.2 ml per head. The time point of D-(+)-glucose treatment was set to 0 minutes, and blood glucose was measured through tail blood sampling at 15, 30, 60, and 120 minutes thereafter.

In FIG. 19, when the SE methanol extract and the solvent fraction were pretreated at a concentration of 500 mg/kg, the degree of change in blood glucose during 30 minutes after administration of D-(+)-glucose was examined. The blood glucose increase rate was shown in a graph by subtracting the blood glucose level of 0 minutes from the blood glucose level at 30 minutes, and the results showed that the effect of lowering blood glucose was excellent in the order of methanol extract, hexane layer, butanol layer, chloroform layer, and water layer. At this time, Metformin, which was used as a positive control, reduced the blood glucose rise rate by about 50%.

Experimental example 2. Anti-obesity Checking the effect: in vitro experiment

Differentiation medium (DM, differentiation medium) was used to induce differentiation for 6 days using 3T3-L1 cells, which are rat adipocytes. Adipose differentiation induction process was performed using differentiation media (DM) containing 500 μM IBMX, 5.2 μM DEX, and 167 nM insulin in DMEM supplemented with 10% (v/v) FBS and 1% (v/v) penicillin-streptomycin. Treatment was performed for 48h from day 0 to day 2. After induction of differentiation, post-differentiation media (Post-DM) was treated from day 2 to day 4 by adding only 167 nM insulin to DMEM supplemented with 10%(v/v) FBS and 1%(v/v) penicillin-streptomycin. I did. From day 4 to day 6, only DMEM supplemented with 10% (v/v) FBS and 1% (v/v) penicillin-streptomycin was treated for 48 h, and adipocyte differentiation was terminated on day 6.

In order to check the effect of the drug during the fat differentiation process, the extract and the column fraction (10-80 μg/mL) were treated together whenever the medium was changed. Before evaluating the anti-obesity activity against the SE extract and the column fraction, the cytotoxicity of the SE methanol extract and the column fraction on 3T3-L1 cells, which is a rat adipocyte, was measured using the CCK-8 (Dojindo) kit. As a result, it was confirmed that there was no effect on cell viability at a concentration of 40 μg/mL or less except for the extract and column fraction 6 (FIG. 20).

Based on these results, SE methanol extract and column fraction were quantitatively determined by staining with Oil Red O, a triglyceride dyeing reagent, and then destaining to determine whether the content of triglycerides in mast cells decreases in a concentration-dependent manner up to 10-40 μg/mL. As a result of measurement, it was confirmed that lipid accumulation in mast cells was inhibited in column fractions 4 and 5 (FIGS. 21 and 22). In addition, as a result of the experiment using the methanol extract and the solvent fraction, it was confirmed that lipid accumulation was suppressed without affecting the cell viability in the methanol extract and the solvent fraction (FIGS. 23 and 24).

Claims (11)

A pharmaceutical composition for preventing or treating diabetes, comprising an Aralia excelsa extract or a fraction thereof as an active ingredient. The method of claim 1,
The extract will _{be extracted with water, C 1} to C ₃ lower alcohol or a mixture thereof, a pharmaceutical composition for preventing or treating diabetes. The method of claim 2,
The lower alcohol is methanol, ethanol, or propanol, which is a pharmaceutical composition for preventing or treating diabetes. The method of claim 1,
The fraction is obtained through a methanol extract polymerized liquid chromatography, a pharmaceutical composition for preventing or treating diabetes. The method of claim 1,
The fraction is a hexane fraction, a chloroform fraction, or a butanol fraction, a pharmaceutical composition for preventing or treating diabetes. A pharmaceutical composition for preventing or treating obesity comprising an Aralia excelsa extract or a fraction thereof as an active ingredient. The method of claim 6,
The extract is _{extracted with water, C 1} to C ₃ of lower alcohol or a mixture thereof, a pharmaceutical composition for preventing or treating obesity. The method of claim 6,
A pharmaceutical composition for preventing or treating obesity, wherein the fraction is obtained through polymerization liquid chromatography of a methanol extract. The method of claim 6,
The fraction is a hexane fraction, a chloroform fraction, or a butanol fraction, wherein the pharmaceutical composition for preventing or treating obesity. A health functional food composition for preventing or improving diabetes comprising an Aralia excelsa extract or a fraction thereof as an active ingredient. A health functional food composition for preventing or improving obesity, comprising an Aralia excelsa extract or a fraction thereof as an active ingredient.

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