KR20160011570A - A composition comprising extract of Lonicerae Flos for enhancing the therapy of diabetes mellitus and obesity - Google Patents

Abstract

The present invention relates to a composition comprising a Lonicerae flos extract for enhancing antidiabetic effects and anti-obesity effects. The composition can be usefully used for effectively treating diabetes, by confirming enhanced antidiabetic effects through combined administration of the Lonicerae flos extract of the present invention and Metformin as a representative antidiabetic drug, the reduction in side effects generated by Metformin, and effects in treating obesity through the inhibition of fat accumulation therewith.

Description

TECHNICAL FIELD The present invention relates to a composition for enhancing antidiabetic and anti-obesity effects,

The present invention relates to a composition for promoting an anti-diabetic effect and an anti-obesity effect, More particularly, the present invention relates to a composition comprising Metfonin, an anti-diabetic agent, which enhances the therapeutic effect of diabetes and treats obesity, including Lonicerae Flos extract.

Diabetes is a disease characterized by hyperglycemia resulting from the absolute or relative deficiency of insulin and a decrease in insulin action in tissues and concomitant metabolic disorders. Type 2 Diabetes Mellitus is a type 1 diabetes mellitus (Type 1 Diabetes Mellitus), a type 1 diabetes mellitus, in which insulin secretion is absolutely insufficient, due to changes in dietary patterns and lifestyle changes due to the development of human civilization. ), Insulin resistance is a major pathophysiological feature. Insulin resistance is closely linked to genetic factors and dietary patterns that reduce insulin sensitivity in peripheral tissues, lifestyle such as obesity, lack of exercise, and stress. There are many reports that the decrease of insulin sensitivity is highly related to obesity, and the inflammatory response in obese state reduces insulin sensitivity.

Currently, there are sulfonlurea drugs that increase insulin secretion, and pioglitazone and rosiglitazone, a peroxisome proliferator activated receptor gamma (PPAR-γ) agonist that improves insulin action. In addition, there is a drug called acarbose, which prevents the increase of postprandial blood glucose by interfering with digestion and absorption of Metformin system drugs, carbohydrates, which reduce your biosynthesis in the liver.

Among these drugs, Metformin has the advantage of less side effects such as hypoglycemia and weight gain than other oral hypoglycemic agents and is currently being used as the first drug therapy for type 2 diabetes. Presently, Metformin is marketed as its hydrochloride salt in the form of a tablet of GLUCOPHAGE (registered trademark, Bristol-Myers Squibb Company). Commercially available glucophage tablets contain 500 mg, 850 mg, or 1000 mg of Metformin hydrochloride, and its administration is within a range that does not exceed the maximum required dose of 2,550 mg per day, taking into consideration both efficacy and tolerance. .

Metformin has been used in Europe since 1957 as a major component of French lilac and has been approved for use in the United States since 1994, but its mechanism of action has been relatively recent. It has been reported that the typical mechanism of action thus far is to induce activation of AMP-activated protein kinase (AMPK), which is involved in energy regulation of the cell, thereby suppressing the hepatogenic activity and promoting fatty acid oxidation in muscles and liver. Recent studies have shown that LKB1, an upper kinase for phosphorylation of AMPK, is required for the hypoglycemic action of Metformin, and it has been shown that LKB1 inhibits your life by phosphorylation of the transcriptional co-activator TORC2.

However, as a side effect caused by Metformin, anorexia, abdominal bloating, nausea and diarrhea have been reported in 20 to 30% of patients taking it. It is rarely reported to cause lactic acidosis, and caution should be exercised when accompanied by renal disease, liver disease, hypoxia, severe infection, alcoholism, and the like. Such side effects can be partially resolved by reducing the minimum and / or sustained dose, by reducing the number of dosing regimens, or by co-administering with other drugs.

Therefore, the increase of the therapeutic effect of Metformin through the combination of drugs or the mixing thereof and the reduction of side effects are the main problems, and related studies have been made (Korea Patent Publication No. 10-2011-0123908).

Disclosure of the Invention The present invention has been conceived to solve the problems as described above. The present inventors have confirmed that the combination of Metformin, an anti-diabetic agent, and Ganoderma lucidum extract improves the antidiabetic effect, reduces side effects and inhibits fat accumulation, Thereby completing the present invention.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for enhancing an antidiabetic effect comprising a ginger extract, which is used in combination with Metformin, an anti-diabetic agent.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to accomplish the object of the present invention as described above, the present invention provides a pharmaceutical composition for enhancing an antidiabetic effect comprising a gingko extract, which is used in combination with Metformin, an anti-diabetic agent.

In one embodiment of the present invention, the composition is administered simultaneously, separately or sequentially with Metformin, the anti-diabetic agent.

In another embodiment of the present invention, the composition is characterized by inhibiting adipocyte differentiation.

In another embodiment of the present invention, the gingival oyster extract is characterized by being extracted with at least one solvent selected from the group consisting of water, an alcohol having 1 to 4 carbon atoms, and a mixed solvent thereof.

In another embodiment of the present invention, the composition is characterized in that the expression of one or more genes selected from the group consisting of P-AMPK, SirT1, AMPK-alpha, PPAR-alpha and PPAR-y is increased.

In another embodiment of the present invention, the composition is characterized in that expression of one or more genes selected from the group consisting of XBP-1, TNF-a and IL-6 is reduced.

The present invention provides a method of treating hyperlipemia and an obesity treatment method comprising the step of administering the pharmaceutical composition to a subject.

The present invention provides a composition for improving the therapeutic effect of diabetes mellitus and a therapeutic use of obesity.

The composition according to the present invention contains the ginger eels extract as an active ingredient and the combination of the ginger extract and Metformin, an anti-diabetic agent, has been shown to improve diabetic and pre-diabetic therapeutic effects and reduce side effects. And is expected to be useful as a pharmaceutical composition. In addition, as a result of confirming the fat accumulation inhibitory effect as well as the diabetic therapeutic effect, it can be expected to prevent or treat obesity together with the treatment of diabetes.

FIG. 1 shows the cell survival rate of 3T3-L1 cells by administration of Ganoderma lucidum extract (GEH; water extract, GEH30; 30% ethanol extract, GEH100; 100% ethanol extract).
FIG. 2 shows the cell survival rate of 3T3-L1 cells by the combination of Ganoderma lucidum extract (GEH; water extract, GEH30; 30% ethanol extract, GEH100; 100% ethanol extract) and Metformin.
FIG. 3 shows the results of confirming cell viability by administration of various concentrations of Ganoderma lucidum extract (20, 50, 100, 200 / / ml) in 3T3-L1 cells.
Figure 4 shows the changes in the activity of reactive oxygen species (ROS) in the HepG2 cells by the combination of gigantospermicum extract (GEH; water extract, GEH30; 30% ethanol extract, GEH100; 100% ethanol extract) .
FIG. 5 shows the results of confirming the inhibitory effect on the production of nitrogen monoxide by administration of Ganoderma lucidum extract (GEH; water extract, GEH30; 30% ethanol extract, GEH100; 100% ethanol extract) in RAW 264.7 cells.
Figure 6 shows the activity of intracellular reactive oxygen species (ROS) in RAW 264.7 cells by the combined administration of Ganoderma lucidum extract (GEH; water extract, GEH30; 30% ethanol extract, GEH100; 100% ethanol extract) This is the result of confirming the change.
FIG. 7 shows the results of confirming the inhibitory effect of 3 eq. 3T3-L1 cells on the adipocyte differentiation inhibition by the combination of Ganoderma lucidum extract (GEH; water extract, GEH30; 30% ethanol extract, GEH100; 100% ethanol extract) and Metformin.
FIG. 8 shows the effect of increasing the glucose uptake capacity of L6 rat myoblast cells in combination with 100% ethanol extract of Geum Eul-ju (GEH) and Metformin.
FIG. 9 shows the results of insulin secretion by RN-m5F insulinoma cells in combination with Ganoderma lucidum extract (GEH; water extract, GEH30; 30% ethanol extract, GEH100; 100% ethanol extract) and Metformin.
FIG. 10 shows the results of confirming whether insulin resistance was improved by the combination of Ganoderma lucidum extract and Metformin (GEH + met1) in undifferentiated L6 rat myoblast cells.
FIG. 11 shows the results of a change in the expression level of DPP-4 (dipeptidyl peptidase-4) protein in combination with 100% ethanol extract of Geum Eul-ju (GEH100) and Metformin in 3T3-L1 cells.
FIG. 12 shows the results of a change in the expression level of PPAR-γ protein in the 3T3-L1 cells by the combined use of 100% ethanol extract of Ganoderma lucidum (GEH100) and Metformin.
FIG. 13 shows the results of a change in the expression level of PPAR-γ protein in the 3T3-L1 cells by the combined administration of various concentrations of Ganoderma lucidum extract (50, 100, 200 μg / ml) and Metformin.
FIG. 14 shows the results of the changes in the amounts of SirT1 and p-AMPK protein expressed by Metformin (M), 30% ethanol extract of Ganoderma lucidum (GEH) or 30% ethanol extract of Ganoderma lucidum and Metformin (M + GEH) in RAW 264.7.
15 shows changes in the expression level of AMPK-alpha gene in RAW 264.7 cells by Metformin (M), 30% ethanol extract of Ganoderma lucidum and Metformin (M + GEH) The result is confirmed.
16 shows changes in the expression level of PPAR-α gene in RAW 264.7 cells by Metformin (M), 30% ethanol extract of Ganoderma lucidum and Metformin (M + GEH) The result is confirmed.
17 shows changes in the expression level of PPAR-γ by metformin (M), 30% ethanol extract of Ganoderma lucidum, and metformin (M + GEH) or gemcitabine extract and Metformin combination (M + GEHW) in RAW 264.7 cells The result is confirmed.
FIG. 18 shows changes in the expression level of XBP-1 gene in RAW 264.7 cells by Metformin (M), 30% ethanol extract of Ganoderma lucidum, and Metformin (M + GEH) or Metformin (M + GEHW) The result is confirmed.
FIG. 19 shows changes in the amount of TNF-α gene expression in RAW 264.7 cells by Metformin (M), 30% ethanol extract of Ganoderma lucidum and Metformin (M + GEH) The result is confirmed.
FIG. 20 shows changes in the expression level of IL-6 gene in RAW 264.7 cells by Metformin (M), 30% ethanol extract of Ganoderma lucidum and Metformin (M + GEH) The result is confirmed.
FIG. 21 shows the results of (a) changes in insulin resistance due to administration of gingko frutescale extract and Metformin (GEH + Met), and (b) blood glucose changes over time in OLETF / LETO rats at 4 weeks of age.
22 shows the change in the concentration of Metformin in the blood after (a) 1st day, 7th day, or (b) 28th day according to the passage of time (120, 240, 360, 380, 600, 720 min) .
FIG. 23 shows the results of confirming the uptake change of Metformin according to the administration of Gingko nuts extract and Metformin.

In the present invention, P-AMP-activated protein kinase (P-AMPK), Sirt1 (sirtuin 1), AMPK-alpha (AMP- (PPAR-alpha) and PPAR-gamma (Peroxisome proliferator-activated receptor-gamma) In addition, the decrease of XBP-1 (X-box binding protein 1), TNF-alpha (tumor necrosis factor-alpha) and IL-6 gene expression associated with adverse effects of Metformin was confirmed.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for enhancing an antidiabetic effect comprising a ginger extract, which is used in combination with Metformin, an anti-diabetic agent.

In the present invention, the Ganoderma lucidum extract can be extracted using a conventional solvent known in the art for extracting an extract from a natural product, that is, under ordinary temperature and pressure conditions. For example, the extract of Ganoderma lucidum in the present invention may be extracted using at least one solvent selected from the group consisting of water, an alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, preferably ethanol. In addition, the method for extracting the extract from gold or silver can be extracted through various methods such as hot water extraction, cold extraction, reflux extraction, ultrasonic extraction, etc., but is not limited thereto.

The extract thus prepared may be filtered, concentrated or dried to remove the solvent, and may be subjected to both filtration, concentration and drying. For example, the filtration can be performed using a filter paper or a vacuum filter, the concentration can be carried out using a vacuum concentrator, and the lyophilization can be carried out, but the present invention is not limited thereto.

Further, the extract extracted with the solvent may be further fractionated with a solvent selected from the group consisting of hexane, methylene chloride, acetone, ethyl acetate, ethyl ether, chloroform, water, and mixtures thereof. The fractionation temperature may be 4 캜 to 120 캜, but is not limited thereto.

As used herein, the term "treatment" refers to any action that improves or alters the symptoms of diabetes by administration of the pharmaceutical composition according to the present invention.

"Diabetes Mellitus", a preventive and therapeutic disease to be treated and prevented by the composition of the present invention, is a chronic metabolic disease, and it causes a vascular disorder and dysfunction such as nerve, kidney and retina, . Diabetes mellitus is divided into insulin-dependent diabetes mellitus (type 1 diabetes) and non-insulin dependent diabetes mellitus (type 2 diabetes mellitus) according to a mechanism that occurs largely, and the present invention preferably means non-insulin dependent diabetes mellitus. The non-insulin-dependent diabetes mellitus is generally resistant to insulin, and the hyperglycemic state is usually sustained due to insulin action. Chronic hyperglycemia causes damage to pancreatic beta cells, leading to apoptosis, so effective glycemic control is required for the treatment of type 2 diabetes.

The antidiabetic agent used in combination with the composition of the present invention may be gliclazide, glibenclamide, repaglinide, nateglinide, mitiglinide, rosiglitazone, pioglitazone, acarbose, voglibose and the like, preferably Metformin, but is not limited thereto.

For example, the anti-diabetic agent "Metformin" used in the present invention is a biguanide-based drug, which is used as a first-line treatment for patients with type 2 diabetes, but it has been reported that anorexia, abdominal bloating, Diarrhea, skin rash, etc., it is necessary to pay special attention to this.

Accordingly, the composition according to the present invention is characterized in that it is administered simultaneously, separately or sequentially with an anti-diabetic agent in order to enhance the anti-diabetic effect and reduce the side effects by the administration of the anti-diabetic agent .

In addition, the composition according to the present invention is characterized by preventing or treating obesity while enhancing the antidiabetic effect.

The term "obesity" which is a preventive and therapeutic disease caused by the composition of the present invention means a state in which the fat cells multiply and differentiate in the body due to metabolic disorders and the fat is accumulated excessively, In the case of relative increase, the number and volume of adipocytes are increased, resulting in an increase in the mass of adipose tissue. Obesity at the cellular level means an increase in the number and volume of adipocytes due to promotion of proliferation and differentiation of adipocytes.

Obesity is closely associated with increased insulin resistance, a major pathophysiological feature of type 2 diabetes. Insulin resistance means a decrease in insulin sensitivity with no blood glucose lowering even with a large amount of insulin injections. This decrease in insulin sensitivity is due to the irregular secretion of adipokines and free fatty acids, which causes fatty acids to accumulate in insulin-sensitive tissues such as the beta cells, kidney, liver, and heart, resulting in lipotoxicity It is known to occur because of.

Also, the composition according to the present invention is characterized in that the expression of one or more genes selected from the group consisting of P-AMPK, SirT1, AMPK-alpha, PPAR-alpha and PPAR-y is increased.

AMP-activated protein kinase (AMPK) is a gene that is associated with the inhibitory effect of anti-diabetic and fat accumulation, such as P-AMPK, SirT1, AMPK-alpha, PPAR-alpha and PPAR- (A situation where AMP is increased compared with that of normal cells), and the process of consuming ATP (restricting the synthesis of fatty acids, cholesterol, etc. and reversing production of ATP) in order to restore normal energy balance, , SirT1 has deacetylation functions of various transcription factors related to histone protein and cell growth, stress response, and endocrine regulation. In addition, PPAR-α regulates glycolipid metabolism involved in the degradation of triglycerides and lowers the level of triglyceride (TG) through lipoprotein lipase (LPL). PPAR-γ is one of the transcriptional regulatory molecules of adipocytes It plays an important role not only in regulating the expression of enzymes involved in differentiation and lipid synthesis and storage of adipocytes but also in enhancing insulin sensitivity.

In addition, the composition according to the present invention is characterized in that expression of one or more genes selected from the group consisting of XBP-1, TNF-a and IL-6 is reduced.

The XBP-1 gene, the TNF-α and the IL-6 gene are related to the adverse effect of Metformin. The XBP-1 gene is involved in the stress of the endoplasmic reticulum. As a cytokine, it plays a role in increasing the inflammatory response.

It has been confirmed that cytotoxicity is not exhibited in the case of the present invention alone or in combination with metformin when administered alone or in combination with Metformin. In addition, it has been confirmed that the coexistence of intracellular reactive oxygen species, free radical elimination, (Example 1 to 5). In addition, it was confirmed that glucose uptake enhancement, insulin secretion increase, and insulin resistance improvement effect were obtained by the combined use of Ganoderma lucidum extract, and DPP-4 protein expression inhibition, PPARγ, P-AMPK and SirT1 protein expression were increased 6 to 9). In addition, the antidiabetic effect and lipid accumulation inhibitory effect were confirmed by the increase of AMPK-α, PPAR-α and PPAR-γ gene expression, and decreased expression of XBP-1, TNF-α and IL- . In vivo animal experiments, it was concluded that there was no change in the pharmacokinetic properties of Metformin by reducing insulin resistance and concomitant administration of Ganoderma lucidum extract and Metformin (Examples 10 to 12 ).

The pharmaceutical composition according to the present invention may contain, in addition to the active ingredient, a pharmaceutically acceptable carrier. Herein, pharmaceutically acceptable carriers are those conventionally used at the time of formulation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose But are not limited to, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Further, in addition to the above components, a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like may be further included.

The pharmaceutical composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) depending on the intended method, and the dose may vary depending on the condition and weight of the patient, The mode of administration, the route of administration, and the time, but may be appropriately selected by those skilled in the art.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will depend on the type of disease, severity, The sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, sequentially or concurrently with conventional therapeutic agents, and may be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the age, sex, condition, body weight, absorbency, inactivation rate and excretion rate of the active ingredient in the body, type of disease, 0.001 to 150 mg, preferably 0.01 to 100 mg, per 1 kg of body weight may be administered daily or every other day, or one to three divided doses per day. However, the dosage may be varied depending on the route of administration, the severity of obesity, sex, weight, age, etc. Therefore, the dosage is not limited to the scope of the present invention by any means.

In another aspect of the present invention, the present invention provides a method for treating diabetes comprising administering the above pharmaceutical composition to a subject. The term " individual "as used herein refers to a subject in need of treatment for a disease, and more specifically refers to a mammal such as a human or non-human primate, mouse, dog, cat, horse and cattle .

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

Example 1. Cytotoxicity experiment

3T3-L1 cells were plated at 3 × 10 ³ / well in 96-well plates and cultured for 24 hours in a CO ₂ incubator. Samples of various concentrations were added to each well and cultured for 24 hours, and 10 E of EZ-Cytox was added to each well. After incubation for 2 h in an incubator, the plate was shaken for 1 min before absorbance measurement and absorbance was measured at 450 nm using a 96-well plate reader. Extraction method (water (GEH), 30% ethanol (GEH 30), 100% ethanol extraction (GEH 100)) and the combination of Metformin and the concentration of Ganoderma lucidum extract (20, 50, 100, 200 ㎍ / ㎖ ) Were measured for cytotoxicity.

As a result, as shown in Fig. 1 to Fig. 3, no cytotoxicity was shown in all the groups regardless of whether they were used alone or in combination with Metformin and in the extraction method. In addition, cytotoxicity was not observed in spite of the increase of the concentration of the extract of Ganoderma lucidum extract.

Example 2. Measurement of intracellular reactive oxygen species activity

After culturing HepG2 cells in 6-well plate in 3 × 10 ⁵ CO ₂ incubator for 8 hours to Pipette 2mL to / well cells, Metformin was administered in combination with single dose or honeysuckle extract and Metformin, re-cultured for 6 hours After that, the cells were recovered. 1200g After centrifugation for 5 minutes, the supernatant was discarded, treated with 5 ug / mL DHR123 and incubated at 37 캜 for 30 minutes. After 5 minutes of centrifugation, the cells were washed 2 times with PBS, filtered, and the activity of intracellular reactive oxygen species was measured by FACS fluorescence.

As a result, as shown in FIG. 4, the activity of intracellular reactive oxygen species (ROS) tended to decrease in the Metformin administration group as compared with the normal group. In addition, the group treated with Ganoderma lucidum extract and Metformin decreased the activity of intracellular reactive oxygen species and showed the most excellent effect in 100% ethanol extract of Ganoderma lucidum (GEH 100% + Met).

Example 3. Measurement of DPPH free radical scavenging activity

40ul of sample was mixed with 760ul of 2,2-Diphenyl-1-picrylhydrazyl (DPPH) in 300uM, reacted at 37 ℃ for 30 minutes, and then divided into triplicate in 96-well. Absorbance was measured at 515nm wavelength in a microplate reader Respectively. Was used as the BHT as a positive control, in the present embodiment, three kinds of extraction methods DPPH in the honeysuckle extract according to (water, 30% ethanol, 100% ethanol extract), a free radical (DPPH free radical) removal was measured the ability calculate the IC ₅₀ Respectively.

As a result, it was measured as 113.85 占 퐂 / ml in the case of BHT as the control group. As shown in the following Table 1, IC _{50 values} of 143.36 μg / ml, 154.35 μg / ml and 146.93 μg / ml, respectively, were shown in the case of administration of the gum disulphide, 30% ethanol or 100% And water extracts showed the best effect.

Example 4. Measurement of inhibitory effect on nitrogen monoxide formation

In order to compare the anti-inflammatory function, LPS-induced NO production was measured using an in vitro model. NO measurements were performed on the cell supernatants based on the Griess reaction (Green et al., 1982). RAW 264.7 cells were inoculated at 1.5 × 10 ⁵ cells / mL, pretreated with diluted samples and treated with 1 μg / mL of lipopolysaccharide (LPS; Sigma, St Louis, MO, USA) And cultured for 24 hours. 50 μL of cell culture supernatant and 50 μL of 1% (w / v) sulfanilamide reagent were added to 96-well plate and incubated for 10 min at room temperature. (w / v) N-1-naphthylethylenediamine were mixed and shaded for 10 minutes. Absorbance was measured at 540 nm using a microplate reader (Molecular Device, CA, USA) within 30 min. The amount of NO production was calculated using a nitric oxide standard solution.

As a result, as shown in FIG. 5, the amount of nitrogen monoxide was decreased in Metformin alone (Met 0.5, Met 1, Met 2) as compared with LPS administration group (LPS) The production of nitrogen monoxide was decreased in the LPS - treated group (LPS) regardless of the extraction method. In addition, as shown in FIG. 6, it was confirmed that the amount of nitrogen monoxide production was further reduced in the group administered with Metformin and Ganoderma extract, as compared with the group administered with Metformin alone.

Example 5 Confirmation of Inhibitory Effect on Adipocyte Differentiation

3T3-L1 cells were seeded at a density of 5 × 10 ⁵ cells / well in a 6-well plate and cultured until the cells became full confluence. The cells were cultured for 48 hours in DMEM supplemented with 1 μM Dexamethasone, 0.5 mM IBMX and 10 μg / ml insulin, and then treated with DMEM (maturation media) containing 10 μg / ml insulin. The differentiation-induced adipocytes were treated with various concentrations of the sample or positive control, and the inhibitory effect of adipocyte differentiation was analyzed through Oil red O staining staining, TG, and TC assay.

As a result, as shown in Fig. 7, the formation of 3T3-L1, a pre-adipocyte, was suppressed in the Metformin alone administration group (Met) as compared with the control group. In addition, the inhibitory effect was better than that of Metformin and Ganoderma lucidum extract, and the best effect was obtained from 30% ethanol extract of Ganoderma lucidum (GEH 30% + Met).

Example 6. Measurement of Glucose uptake assay (Glucose uptake assay)

Undifferentiated L6 rat myoblast cells were induced to differentiate into myotube cells using 2% horse serum or incubated in a 96-well clear-bottom blackwell plate using HepG2 cells, and then the medium was changed to glucose free medium for 12 hours and incubated under glucose starvation conditions. After incubating for 6 ~ 12 hours, the cells were washed twice with DPBS, and then analyzed with a fluorescence microplate reader. Ex. Ex. 2-NBDG / Em = fluorescence was measured at 485/535 nm wavelength and Apigenin, which inhibits glucose uptake, was used as a control.

As a result, as shown in FIG. 8, glucose uptake capacity was increased in Metformin alone group (Met) compared with the control group. Compared with the Metformin group alone, glucose uptake ability in the group treated with 100% .

Example 7 Insulin uptake assay (Insulin uptake assay)

RIN-m5F the insulinoma cells, 10% FBS, 0.6% PS ( Penicillin Streptomycin), 300 mg / L of L-glutamine containing the RPMI 1640 Medium (WELGENE Inc, Korea ) and then treated with medium, 37 ℃, 5% CO ₂ Lt; / RTI > RIN-m5F cells were cultured for 3 days at a density of 3 × 10 ⁵ cells / well in a 12-well plate. Then, 0.75 mM of Metformin and GEA (GEH) were used in combination. After culturing for 2 days, the medium was removed and the modified Krebs ringer bicarbonate buffer (KRBB-HEPES, 134 mmol / L NaCl, 4.8 mmol / L KCl, 1 mmol / L CaCl ₂ , 1.2 mmol / L MgSO ₄ , 1.2 mmol / L KH _{2 PO 4, 5mmol / L NaHCO} 3, 10mmol / L HEPES, washed twice with 1mg / mL BSA, pH7.4), and translates to a KRBB-HEPES buffer solution containing 20mM glucose and incubated for 1 hour. The supernatant was centrifuged at 4 ° C for 10 minutes, and the supernatant was collected and stored at -20 ° C. The amount of insulin secreted was measured by a rat insulin ELISA kit (Mercodia, Sweden), and the amount of insulin secreted per gram of protein was calculated by measuring the cell protein concentration in each well.

As a result, as shown in FIG. 9, the insulin secretion amount was not significantly increased in the case of Metformin alone (MET1, MET2), but the insulin secretion amount was significantly increased in the combination of Metformin and Ganoderma extract. , And the best effect was confirmed.

Example 8. Measurement of insulin resistance (Insulin resistance assay)

Undifferentiated L6 rat myoblast cells were plated on a 96-well clear-bottom blackwell plate and differentiated into myotubes using 2% horse serum. Fluorescence results were then measured. The differentiated L6 cell medium was changed to glucose free medium for 2 hours to treat various concentrations of the sample in glucose starvation state. Subsequently, the medium was replaced with glucose-free media containing 5 mM of glucosamine and incubated for 6 to 12 hours to induce insulin resistance. After removing all supernatants, glucose-free media containing 2-NBDG at a concentration of 100 ug / ml was cultured for 6 hours, and then washed twice with DPBS. The fluorescence plate reader was irradiated with Ex / Em = 485/535 nm Absorbance was measured.

As a result, as shown in FIG. 10, excellent glucose uptake was confirmed in the group administered with insulin and glucosamine and in the group administered with gingival extract and metfomrin in comparison with the group administered with Metformin alone, that is, synergic improvement of insulin resistance Effect.

Example 9. Confirmation of Expression Amount of Related Protein

9-1. DPP-4, PPARgamma protein expression

3T3-L1 preadipocytes were treated with DMEM (WELGENE Inc, Korea) containing 10% FBS and 1% PS (Penicillin Streptomycin) and cultured in a 5% CO ₂ incubator at 37 ° C. The cells were seeded in a 6-well plate for cell culture at 8 × 10 ⁴ / well. To induce cell differentiation, cells were incubated with 0.5 mM IBMX, 1 μM Dexamethasone, 10 μg / ml insulin / DMEM differentiation induction medium containing 10% FBS was added and cultured for 3 days. After 3 days, the cells were cultured in DMEM supplemented with 10 μg / mL insulin / 10% FBS, and the medium was changed every 2 days. After 5 days of differentiation, the cells were cultured for 24 hours. Cells in 6-well plates were washed twice with PBS and resuspended in RIPA buffer (50 mM Tris-HCl pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM NaF, 1 mM sodium , 1 μg / ml aprotinin, leupeptin, pepstatin) and centrifuged at 12,000 rpm for 20 minutes to obtain supernatant containing protein. After quantification according to BCA (Thermo Scientific, USA), 10% polyacrylamide gel electrophoresis was performed. After electrophoresis, gel proteins were transferred to a PVDF membrane at 200 mA for 1.5 h, blocked with 5% skim milk or 5% BSA for nonspecific protein, the primary antibody was treated overnight at 4 ° C, and TBS-T Lt; / RTI > for 10 minutes. Subsequently, the secondary antibody was treated at room temperature for 1 hour, washed three times for 10 minutes with TBS-T, treated with ECL (NEURONEX, Korea) solution and observed for protein expression with LAS-3000 (FUJIFILM, Japan).

As a result, as shown in Figs. 11 to 13, the expression level of DPP-4 (dipeptidyl peptidase-4) was inhibited in the group treated with Metformin and Ganoderma extract, compared with that of Metformin alone (MET1 and MET2) Was significantly increased. In this example, DPP-4 is a protein associated with the DPP-4 enzyme responsible for the decomposition of incretins. When the expression of DPP-4 is inhibited, insulin synthesis / secretion is promoted and glucagon inhibition and glucose It is known that PPARγ plays a role in increasing insulin sensitivity as the expression level increases. Therefore, this result means that the antidiabetic effect can be further enhanced by the combined use of Metformin and Gingko Euphorbia extract.

9-2. P- AMPK , Sirt1  Protein expression

RAW 264.7 cell line, a macrophage cell line, was used in a Korean cell line bank (KCLB, Seoul, Korea) and cell culture was performed in DMEM supplemented with 10% FBS, 2 mML-glutamine, 100 U / ml penicillin, and 100 lg / ml streptomycin Was used. CO ₂ incubator The cells were cultured under the conditions of 37 ° C, 5% CO _{2 and} 95% O ₂ . The Euglena extract (100% water, 30% ethanol) used in the experiment was supplied by the College of Pharmacy, Dongguk University. Each of the cells was divided into four groups as normal (N), Metformin (M), Ganoderma lucidum (30%), and Metformin + .

RAW 264.7 cells were cultured in DMEM with culture medium containing 10% FBS, 2 mML-glutamine, 100 U / ml penicillin, and 100 lg / ml streptomycin at 37 ° C, 5% CO ₂ , and 90% humidity . The cultured cells were cultured with the culture medium changed every 2-3 days. When the cell differentiation reached its maximum, the cells were washed with phosphate buffered saline (PBS), and the cells adhered with trypsin-EDTA solution were separated Cells were collected by centrifugation, and cells and medium were mixed well and subcultured.

Cells treated for cell lysate preparation were washed with 10 mM phosphate buffer (pH 7.4) containing 150 mM NaCl (PBS), and dissolved in PBS solution containing 0.1% SDS and 10 mM β-mercaptoethanol. Cells were collected and placed in 8% SDS-polyacrylamide gels. Protein bands were incubated on nitrocellulose membranes (Schleicher and Schull, Dassel, Germany) using a semi-dry blotter (MilliBlot-SDE system, Millipore, Bedford, I dropped it. The membranes were washed once with 10 mM Tris-buffered saline (TBS, pH 7.2) containing 0.1% tween-20 (TBS-T) and resuspended in Tris-buffered saline 7.2) and blocked for 1 hour at room temperature. anti-Sirt1 antibody, anti-p-AMPK antibody, anti-AMPK antibody (Cell Signaling Technology, DV, USA) or anti-beta actin antibody. After incubation for 2 h, horseradish peroxidase-conjugated goat anti-rabbit IgG (Santa Cruz, CA, USA) (diluted 1: 1000) was used as a secondary antibody. Then, it was treated with an enhanced chemiluminescence solution (ECL) (Amersham Corp., Newark, NJ, USA) and analyzed with an image reader (LAS-3000, Fuji Photo Film, Tokyo, Japan). The intensity of the resultant protein band was measured by densitometry, and the amount of protein was analyzed with reference to beta-actin.

As a result, as shown in FIG. 14, compared with the combination of the gingham extract (ethanol 30%) alone (GEH) and Metformin alone, the combination of Metformin and Ganoderma lucidum extract (ethanol 30%) Significantly, it was confirmed that Sirt1 and p-AMPK proteins were increased.

Example 10. Confirmation of Change in Expression Quantity of Related Gene

10-1. Antidiabetic effect related gene expression

To determine the effect of the combination of Metformin and Ganoderma lucidum extract on the antidiabetic effect and expression of the related genes, Metformin + gumulhwa (30% or 100% water) was administered with Metformin (M) and RAW 264.7 cells alone AMPK-α, PPAR-α, and PPAR-γ gene expression in RAW 264.7 cells were compared by real-time PCR. RAW 264.7 cells were obtained in the same manner as in Example 9-2, except that each cell was treated with the normal (N), Metformin (M), Metformin + (Water extract) treated group (M + GEHW).

Total RNA was isolated and purified according to the protocol using Trisure (Bioline, USA). 1 μg of total RNA was reverse transcribed according to the protocol using cDNA synthesis kit (Sprint TMRT Complete Oligo- (dT) 18, Clontech, MountainView, CA, USA) to obtain first strand cDNA. RT-PCR was performed on a Light Cycler instrument (Roche Applied Science) with a final reaction volume of 20 μL using Light Cycler-Fast Start DNA Master SYBR Green (Roche Applied Science, Indianapolis, ID, USA).

The DNA sequence of the primer used in the above example is as follows.

PCR amplification was carried out at 95 ° C for 10 min in C / EBPα for 10 min, followed by 45 cycles of amplification (denaturation at 95 ° C for 10 sec, annealing at 52 ° C for 10 sec, extension at 72 ° C for 15 sec) After 10 minutes of prior incubation, 35 cycles of total RNA were isolated and purified according to the protocol using Trisure (Bioline, USA). 1 μg of total RNA was reverse transcribed according to the protocol using cDNA synthesis kit (Sprint TMRT Complete Oligo- (dT) 18, Clontech, MountainView, CA, USA) to obtain first strand cDNA. RT-PCR was performed on a Light Cycler instrument (Roche Applied Science) with a final reaction volume of 20 μL using Light Cycler-Fast Start DNA Master SYBR Green (Roche Applied Science, Indianapolis, ID, USA).

As a result, as shown in Fig. 15, the AMPK-alpha gene expression was measured to be 0.80 in the normal group (N) and 0.76 in the Metformin administration group (M). The expression of AMPK-α gene was increased in M + GEH, Metformin and water (100% water) (M + GEHW) The GEH group was 2.70, indicating a significant increase in expression.

As shown in FIG. 16, the expression of PPAR-α gene was 1.01 in the normal group (N), 0.68 in the metformin administration group (M), and the expression of Metformin and Ganoderma lucidum (30% The expression of the PPAR-α gene was confirmed in the combined treatment group (M + GEH), Metformin, and ginger extract (water 100%) (M + GEHW).

In addition, as shown in Fig. 17, PPAR-gamma gene expression was measured to be 1.03 in the normal group (N) and 0.83 in the metformin administered group (M). (M + GEHW) was measured to be 0.90 and the increase of expression of PPAR-γ gene was confirmed in the combination of Metformin and Ganoderma lucidum extract (water 100%).

10-2. Metformin Side Effects Related Gene Expression

To investigate the effect of Metformin and EE on Metformin-induced side effects and expression of related genes, Metformin (M) and Metformin + germanium (30% ethanol or water 100% The expression of XBP-1, TNF-α and IL-6 genes in RAW 264.7 cells was compared by real-time PCR. RAW 264.7 cells were obtained in the same manner as in Example 9-2, except that each cell was treated with the normal (N), Metformin (M), Metformin + (Water extract) treated group (M + GEHW).

Real-time PCR was carried out in the same manner as described in Example 10-1 except for the primer in order to confirm the expression of XBP-1, TNF-alpha and IL-6 gene.

The DNA sequence of the primer used in the above example is as follows.

As a result, as shown in Fig. 18, XBP-1 gene expression was measured to be 1.00 in normal group (N) and 1.01 in metformin administration group (M). (M + GEH) and Metformin and Gingko nucifera extract (ethanol + 30%) were 0.41 and 0.53, respectively. The decrease of XBP-1 gene expression was confirmed Respectively.

In addition, as shown in Fig. 19, TNF-alpha gene expression was measured to be 1.01 for the normal group (N) and 1.34 for the metformin administered group (M). (M + GEH) and metformin and gingerol extract (ethanol + 30%) were 0.66 and 0.97, respectively. The decrease of TNF-α gene expression was observed Respectively.

As shown in Fig. 20, IL-6 gene expression was measured to be 1.11 in the normal group (N) and 1.91 in the metformin administered group (M). (M + GEHW) and Metformin and Ganoderma lucidum extracts (M + GEHW) were measured to be 0.59 and 0.35, respectively, and the reduction of IL-6 gene expression was confirmed Respectively.

Example 11. Intraperitoneal insulin tolerance test (IPITT)

In order to confirm the effect of metformin and gingkofloccus extract on diabetes mellitus, four-week-old OLETF rats, LETO rats (Otsuka Pharmaceutical, Japan) were distributed and received either Metformin alone at a dose of 100 mg / kg or 200 mg / The extract was combined with 100 mg / kg of Metformin. Weekly dietary intake, weight, and status were checked. At week 24, IPITT was performed by collecting blood from the tail vein. After 12 weeks, the animals were sacrificed by anesthetizing with zoletil / rumpun IP, and fat and each organ sample and serum sample were separated. One week prior to the end of the experiment, OLETF / LETO was fasted for 15 hours and insulin IP was injected at a concentration of 1 U / kg. Thereafter, a small amount of bleeding was performed in the tail vein of each individual at 0 min, 30 min, 60 min, 90 min and 120 min Blood glucose was measured using an Accucheck blood glucose meter (Roche, USA) in the blood. The results were analyzed using AUC (area under curve).

As a result, as shown in Fig. 21, insulin resistance was observed to be higher in the OLEFT group than in the LETO group, and the resistance tended to decrease through Metformin alone treatment. In addition, it was confirmed that insulin resistance was significantly reduced in the group treated with Metformin and Ganoderma lucidum extracts compared to the group administered with Metformin alone.

Example 12. Measurement of pharmacokinetic change of Metformin by concomitant administration

12-1. Pharmacokinetic Changes of Metformin over Combined Periods

After rats were anesthetized, a cannula was inserted into the artery, and anesthesia was initiated. The patient was divided into two groups: Metformin 100 mg / mg alone or Metformin 100 mg / kg and 200 mg / kg combination. For 1, 7, or 4 weeks. After administration Blood samples were taken at regular time intervals and urine was collected for 24 hours. Gastrointestinal samples were taken at 24 hours after lapse of time to measure the amount of metformin remaining in the gastrointestinal tract. In addition, the amount of Metformin remaining in the blood concentration profile, urine, and gastrointestinal tract was calculated by quantification using LC / MSMS.

As a result, as shown in the following Table 4 and FIG. 22, significant pharmacokinetic parameters such as the accumulation effect of Metformin were observed in the combination group of Metformin and Ganoderma extract (single, 7 days, or 4 weeks) Were not observed.

12-2. Metformin uptake changes by inhibition of OCT 1 and OCT 2

Metformin uptake changes were observed in the OCT transporter cell products due to the coexistence of Metformin and Ganoderma lucidum extract. 30uM and 300uM verapamil were used as inhibitors of OTC1 and OTC2, and 10uM Metformin was used as the substrate of OCT1,2.

As a result, as shown in Fig. 23, the uptake of Metformin was significantly decreased in the 30 uM verapamil or 300 uM verapmil-treated group, which is an inhibitor of OCT1 and OCT2, while the uptake of Metformin was decreased in the combination administration of Metformin and Ganoderma lucidum extract Respectively.

Taken together, these results indicate that the combined use of Ganoderma lucidum extract and Metformin, an anti-diabetic agent, has no effect on the absorption and action of Metformin drug itself.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

<110> Dongguk University Gyeongju Campus Industry-Academy Cooperation Foundation <120> A composition comprising extract of Lonicerae Flos for enhancing          the therapy of diabetes Mellitus and obesity <130> MP15-042 <150> KR 10-2014-0092194 <151> 2014-07-21 <160> 14 <170> KoPatentin 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> beta actin primer_forward <400> 1 gcaagtgctt ctaggcggac 20 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> beta actin primer_reverse <400> 2 aagaaagggt gtaaaacgca gc 22 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> AMPK alpha1 primer_forward <400> 3 aagccgaccc aatgacatca 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> AMPK alpha1 primer_reverse <400> 4 cttccttcgt acacgcaaat 20 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> PPAR-alpha primer_forward <400> 5 gcctgtctgt cgggatgt 18 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PPAR-alpha primer_reverse <400> 6 ggcttcgtgg attctcttg 19 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PPAR-gamma primer_forward <400> 7 gccctttggt gactttatgg a 21 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PPAR-gamma primer_reverse <400> 8 gcagcaggtt gtcttggatg 20 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> XBP-1 primer_forward <400> 9 tggccgggtc tgctgagtcc g 21 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> XBP-1 primer_reverse <400> 10 gtccatggga agatgttctg g 21 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TNF-alpha primer_forward <400> 11 gaactggcag aagaggcact 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TNF-alpha primer_reverse <400> 12 agggtctggg ccatagaact 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL-6 primer_forward <400> 13 agttgccttc ttgggactga 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> IL-6 primer_reverse <400> 14 cagaattgcc attgcacaac 20

Claims (6)

A pharmaceutical composition for enhancing an anti-diabetic effect in combination with an anti-diabetic agent,
Wherein the composition comprises Lonicerae Flos extract and the anti-diabetic agent is Metformin.
The method according to claim 1,
Wherein said composition is administered simultaneously, separately or sequentially with said anti-diabetic agent.
The method according to claim 1,
Wherein said composition inhibits the differentiation of adipocytes.
The method according to claim 1,
The above- Wherein the composition is extracted with at least one solvent selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, and mixed solvents thereof.
The method according to claim 1,
The composition may further comprise at least one selected from the group consisting of P-AMP-activated protein kinase, SirTl (sirtuin 1), AMP-activated protein kinase-alpha (AMPK-a), Peroxisome proliferator- gamma (Peroxisome proliferator-activated receptor-gamma). &lt; / RTI &gt;
The method according to claim 1,
Wherein the composition reduces expression of any one or more of the genes selected from the group consisting of X-box binding protein 1 (XBP-1), tumor necrosis factor alpha (TNF-alpha) and IL-6.

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