KR101204427B1 - Composition comprising Ganoderma lucidum extracts for DPP-IV inhibition - Google Patents

Abstract

The present invention relates to a composition for inhibiting DPP-IV comprising Ganoderma lucidum extract as an active ingredient and its use.

Ganoderma lucidum, diabetes, blood sugar, glycated hemoglobin, dipeptidyl peptidase-IV (DPP-IV).

Description

Composition comprising Ganoderma lucidum extracts for DPP-IV inhibition}

The present invention relates to a composition for inhibiting DPP-IV comprising Ganoderma lucidum extract as an active ingredient and its use.

The present invention is derived from a study conducted as part of the Gyeonggi-do Strategic Technology Development Project.

[Project number: A08081910, Title: Development of anti-diabetic functional material using mushrooms, mushroom mycelium and spores]

As the standard of living increases, metabolic diseases such as diabetes, osteoporosis, hypertension, and hyperlipidemia are rapidly increasing due to the increase in elderly population, decrease in physical activity, and increased fat intake. Among these metabolic diseases, diabetes is the third most serious disease in the world. Currently, 250 million people of the world population are estimated to be diabetics, and between 3 million and 5 million people in Korea are estimated to be diabetics.

Diabetes mellitus is generally defined as a chronic hyperglycemic state, but is actually one of the metabolic syndromes that affects the metabolism of carbohydrates, lipids and proteins, which are the main energy sources. Hyperglycemia is measured after 12 hours of fasting with a glucose concentration of at least 140 mg / dL of venous blood, or at a plasma glucose concentration of at least 200 mg / dL at any time of the day, or plasma glucose concentration at 2 hours after 75 g glucose administration. Is defined as the case of more than 200 mg / dL. The normal value of fasting plasma glucose concentration is 70-115 mg / dL, and the upper limit of normality is 140 mg / dL 2 hours after 75 g glucose administration.

The main cause of diabetes is a lack of insulin, including a lack of absolute amount of insulin or a relatively low amount of insulin due to the secretion of a number of hormones other than insulin. Diabetes mellitus metabolic disorders, as well as chronic hyperglycemia due to diabetes induces secondary complications of diabetic retinopathy, neuropathy, nephropathy and atherosclerosis, so the importance of glycemic control in the treatment of diabetic patients is very important.

Diabetes is the main cause of autoimmune processes that destroy pancrease β-cells, resulting in insulin-dependent diabetes (type 1 diabetes) caused by insufficient insulin secretion, insulin resistance, genetic causes, It is divided into insulin-independent diabetes mellitus (type 2 diabetes) due to decreased function.

In the case of insulin-dependent diabetes, insulin is a common treatment. In the case of insulin-independent diabetes, blood sugar is controlled primarily by diet and exercise.However, when blood glucose is not controlled by this method, drugs for lowering blood glucose or It is a common practice to use insulin and drugs together. However, insulin-independent type 2 diabetes mellitus is a progressive disease caused by deficiency of insulin secretion and insulin resistance, and thus increases blood sugar level despite administration of hypoglycemic agents due to deterioration of beta cell function. Recently, the increase in blood glucose caused by glucagon in type 2 diabetes has been recognized as a major contributor to hyperglycemia. Therefore, dipeptidyl peptidase IV (DPP-IV), which acts on incretin as a therapeutic target for type 2 diabetes, has attracted attention. Incretin is a hormone that promotes insulin secretion in response to digestion of nutrients in the body and includes GIP (Glucose-dependent insulinotropic polypeptide) and GLP-1 (Glucagon like peptide-1). GLP-1 is secreted in response to digestion and is involved in metabolic homeostasis such as insulin secretion associated with glucose metabolism, synthesis of insulin, and inhibition of glucagon secretion. Both GIP and GLP-1 are essential hormones to maintain normal glucose metabolism, but in patients with type 2 diabetes, blood levels of GIP remain nearly normal, but GLP-1 secretion is known to decrease. In particular, GLP-1 synthesized by endocrine cells becomes GLP-1 (9-36) amide and loses activity by cleaving two N-terminal amino acids by DPP-IV in the body. Inhibition of DPP-IV activity against GLP-1 may prolong GLP-1's action to improve glycemic control. Thus, DPP-IV inhibitors are being developed as promising therapeutic agents in patients with type 2 diabetes.

Currently, therapeutic agents such as sulfonylureas, biguanides, and amaryl have been developed and used for the treatment of diabetes mellitus, but these therapeutic agents have limited effects and have risks of causing side effects such as hypoglycemia, hepatotoxicity, and lactic acidosis. Thus, there is still a need for anti-diabetic agents with low risk of side effects and high therapeutic efficacy and safety. The cure for diabetes has not been established yet, and the use of many therapeutic agents has many limitations due to side effects. Therefore, a natural drug having relatively few side effects is used to control blood sugar, thereby preventing and treating diabetes. And research to develop a functional food material has attracted a lot of attention.

Therefore, the present inventors conducted a study on a substance that works safely in the body while having an excellent effect of preventing and improving diabetes through inhibiting the activity of DPP-IV, which is attracting attention as a target of new diabetes treatment, ganoderma lucidum and / or ganoderma lucidum The mushroom spore extract was found to lower blood sugar through the inhibition of DPP-IV activity to complete the present invention.

It is an object of the present invention to provide an antidiabetic composition comprising Ganoderma lucidum extract.

Still another object of the present invention is to provide an antidiabetic composition comprising a compound isolated and purified from Ganoderma lucidum extract.

In order to achieve the above object, the present invention provides a composition for inhibiting DPP-IV comprising Ganoderma lucidum extract as an active ingredient.

Ganoderma lucidum is a genus of fungus fungi, which is a year-old mushroom that grows from the roots of hardwoods and rises to the ground. It has been called Bulchocho since ancient times, and it is used as a decorative or medicinal herb in Japan. Ganoderma lucidum contains polysaccharides, triterpenes, nucleosides, steroids, fatty acids, alkaloids, proteins, amino acids, and inorganic salts as main components.In particular, various kinds of polymers such as polysaccharides and low-molecular substances such as triterpenes It has received a lot of attention because of its promising pharmacological activity.

As used herein, the term "manor mushroom extract" is intended to mean an extract from Ganoderma lucidum or Ganoderma lucidum spores or a mixture of Ganoderma lucidum and Ganoderma lucidum spores.

Ganoderma lucidum extract has DPP-IV inhibitory activity, which has been noted as a new therapeutic agent for diabetes, especially type 2 diabetes, thereby reducing blood sugar and glycated hemoglobin levels and inhibiting the weight loss commonly experienced by diabetics. It was confirmed. Hemoglobin in red blood cells binds to oxygen and transports oxygen to various organs in the body. However, when the glucose content in the blood is high, as in diabetics, hemoglobin binds to glucose, such as glucose, to form glycated hemoglobin (HbA1c). The higher the blood sugar, the more hemoglobin binds to the sugar, which stays about 3 months in this bound state. Therefore, lowering the glycated hemoglobin level means that the blood glucose is continuously lowered rather than temporarily lowered, thus reducing the glycated hemoglobin level may be an effective indicator of the antidiabetic effect.

In one embodiment of the present invention, the extract may be extracted by water, a hydrophilic organic solvent or a mixed solvent thereof, preferably an ethanol extract extracted with ethanol. The hydrophilic organic solvent includes, but is not limited to, lower alcohols having 1 to 4 carbon atoms such as methanol, ethanol, propanol, butanol, acetone, ethyl acetate, chloroform, 1,3-butylene glycol, hexane and diethyl ether . The extract may be an extract obtained by extraction, a diluent or concentrate of the extract, a dried product obtained by drying the extract or a crude or purified product of the extract.

In one embodiment of the present invention, the extract may be an ethanol extract prepared by adding about 5 times the total weight of ethanol and extracted three times at 70 ℃ for 4 hours, and concentrated the recovered extract under reduced pressure.

Ganoderma lucidum extract in the composition for inhibiting DPP-IV according to the present invention may be 0.1 mg / kg to 100 mg / kg based on the total weight of the composition. If the content of Ganoderma lucidum extract in DPP-IV inhibitory composition is less than 0.1 mg / kg, it is difficult to expect a clear hypoglycemic effect, and if the content of more than 100 mg / kg does not increase the effect of increasing the content. When ganoderma lucidum extract is used for antidiabetic effect through DPP-IV inhibition, the antidiabetic composition may be prepared by varying its concentration according to the purpose of use.

The present invention also provides a composition for inhibiting DPP-IV comprising the compound of formula I as an active ingredient:

Formula I: 22 (E) -Ergostar-5,7,22-trien-3β-ol

In one embodiment of the invention, the compound of formula I may be a compound of formula II substituted with a methyl group at carbon 23:

Formula II: 22 (E) -23-Methylergosta-5,7,22-trien-3β-ol

In one embodiment of the invention, the compound of formula I may be derived from Ganoderma lucidum extract.

Anti-diabetic substance having a hypoglycemic effect by DPP-IV inhibition previously contained in the Ganoderma lucidum extract of the present invention has not been identified, Ganoderma lucidum extract itself is mixed with other substances in addition to the anti-diabetic active substance and mutual The effect of the action on the antidiabetic effect is unknown. In general, when the crude crude extract itself is used as an active ingredient of the anti-diabetic composition, since the crude extract contains other components besides the anti-diabetic active substance, some of the components may be treated by an undesirable reaction with the anti-diabetic active substance. It may negatively affect diabetic activity or stability or may increase the amount of active ingredient to be added for the desired antidiabetic activity, which may be a limiting factor in the preparation of the antidiabetic composition. Therefore, it is advantageous to include the compound of formula (I), which is isolated and purified from crude extracts of Ganoderma lucidum mushroom and has been found to have antidiabetic activity by inhibiting DPP-IV activity, as an active ingredient of the antidiabetic composition.

In the composition for inhibiting DPP-IV according to the present invention, the compound of formula (I) may be chemically synthesized or extracted and purified from natural medicines including Ganoderma lucidum.

The composition for inhibiting DPP-IV of the present invention can be used for the preparation of a pharmaceutical composition for preventing or treating diabetes or a pharmaceutical composition for preventing or treating an adult disease caused by diabetes.

The pharmaceutical composition for preventing or treating diabetes or adult disease, including the composition for inhibiting DPP-IV according to the present invention, may further include appropriate carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions. Pharmaceutically acceptable carriers, excipients and diluents that may be included in the pharmaceutical composition according to one embodiment of the invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, Alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil Including, but not limited to.

The composition for inhibiting DPP-IV according to the present invention can be formulated and used in various formulations such as tablets, capsules, granules, suspensions, syrups and the like according to conventional methods known in the art.

The present invention also provides an anti-diabetic functional food comprising a composition for inhibiting DPP-IV comprising Ganoderma lucidum extract as an active ingredient. Anti-diabetic functional food comprising the composition for inhibiting DPP-IV according to the present invention may be in the form of fish, meat, beverage, chocolate, snacks, confectionary, ramen, other noodles, gum, ice cream, vitamin complex, fermented food, etc. Can be.

As used herein, "functional food" means a food manufactured and processed using raw materials or ingredients having a useful function to the human body, and "functional" means nutrients or physiological effects on the structure and function of the human body. Ingestion means the purpose of obtaining a beneficial effect for the same health use.

In one embodiment of the present invention, the anti-diabetic functional food comprising the composition for inhibiting DPP-IV is a conventional food additive that is acceptable in food production, for example, flavoring, flavoring, coloring, filler, stabilizer And the like may be further included.

Hereinafter, the present invention will be described in more detail by way of examples. However, the following examples are intended to illustrate the invention and are not intended to limit the scope of the invention.

The composition for inhibiting DPP-IV using Ganoderma lucidum extract according to the present invention as an active ingredient has an excellent effect in preventing or treating diabetes by inhibiting the rise of blood sugar and glycated hemoglobin through inhibition of DPP-IV activity. The composition for inhibiting DPP-IV according to the present invention can be safely and effectively administered as a pharmaceutical composition or ingested as a functional food without concern for side effects.

Example 1 Preparation of Ganoderma lucidum Extract

To spores of ganoderma lucidum and / or ganoderma lucidum, ethanol of 5 times its weight was added and extracted three times at 70 ° C. for 4 hours to recover the extract. The recovered extract was concentrated under reduced pressure to prepare an ethanol extract of Ganoderma lucidum.

Example 2. Dipeptidyl-peptidase IV Inhibitory Activity of Ganoderma lucidum Extract

Dipeptidyl-peptidase IV (DPP-IV) inhibitory activity of Ganoderma lucidum extract prepared in Example 1 was investigated.

DPPIV-GloTM Protease Assay (Cat.No., # G8351, Promega, WI, USA) kit was used to determine whether the Ganoderma lucidum extract can contribute to hypoglycemia by inhibiting DPP-IV activity. The Ganoderma lucidum extract prepared in Example 1 was successively diluted 5-fold in 45 µl of DPP-IV reaction buffer in a 96-well plate for fluorescence measurement, and the final concentration, 20 µg / ml, 4 µg / ml, 0.8 µg / ml and 0.16 μg / ml were added and the human DPP-4 enzyme was added to a final concentration of 1 ng / mL and reacted at 25 ° C. for 30 minutes. The product of the DPP-4 reaction was measured using a fluorescence detector (VICTOR ™ Light 1420, Perkin Elmer). The activity in the control group containing no Ganoderma lucidum extract was set to 100%, and the relative inhibition (CPS (counts per second) X 10 ⁴ ) of DPP-4 activity by Ganoderma lucidum extract was calculated. 1 shows that Ganoderma lucidum extract has a dose-dependent inhibitory effect on DPP-4 activity.

Example 3 In Vivo Antidiabetic Effect of Ethanol Extract of Ganoderma lucidum Mushroom

In this example, the in vivo antidiabetic effect of Ganoderma lucidum extract prepared in Example 1 using type 2 diabetic mice (C57BL / KsJ db / db) was investigated.

(1) Breeding of the target animal

18 male C57BLKs db / db mice of 5 weeks old were purchased from the central experimental animals and used for the experiment after one week of adaptation. Mice were divided into 6 groups in each group, control group and experimental group . For each group, weight change, dietary intake, blood glucose and glycated hemoglobin (HbA1c) were measured for 11 weeks. The room temperature was maintained at 22 ± 1 ℃, the humidity at 50 ± 5%, and the contrast was adjusted at 12 hour intervals.

As a feed, Purina feed (# 38057) for mice purchased from a central laboratory animal was used, and the experimental group was orally administered with Ganoderma lucidum extract prepared in Example 1 twice a day at 20 mg / kg. Diet and water were freely supplied without restriction.

(2) blood analysis

Normal and control groups were bred as in (1), and the glucose content in blood and the amount of glycated hemoglobin were measured.

To measure blood glucose and glycated hemoglobin, blood was collected from the tail vein after fasting for 12 hours. Hemoglobin in red blood cells binds to oxygen and transports oxygen to various organs in the body. However, when the glucose content in the blood is high, as in diabetics, hemoglobin binds to glucose, such as glucose, to form glycated hemoglobin (HbA1c). The higher the blood sugar, the more hemoglobin binds to the sugar, which stays about 3 months. Therefore, lowering the glycated hemoglobin level means that the blood sugar is continuously lowered rather than temporarily lowered, thus reducing the glycated hemoglobin level may be an indicator of the antidiabetic effect.

Blood glucose levels were measured weekly during the breeding period by applying whole blood to glucose strips (Accuchek Sensor, Roche, Basel, Switzerland) based on the glucose-oxidase method. The blood glucose reduction of the experimental group receiving the Ganoderma lucidum extract compared to the control is shown in FIG. 2.

Glycated hemoglobin was measured using the HbA1C kit (# 11045, Biosystems S.A., Barcelona, Spain) based on affinity chromatography. Glycosylated hemoglobin was formed by the binding of sugar in the blood and hemoglobin, a blood pigment, and the binding state was maintained for about 3 months, so it was measured at weeks 1, 9 and 11 during the breeding period. The change in the amount of glycated hemoglobin according to the administration of the extract of Example 1 during the breeding period is shown in FIG. 3.

The decrease in blood sugar and the amount of glycated hemoglobin in administration of the Ganoderma lucidum extract confirmed as described above is believed to be due to the inhibition of DPP-4 activity of Ganoderma lucidum extract confirmed in Example 2.

(3) weight analysis

In general, diabetes mellitus causes changes in glucose metabolism, such as the lack of insulin and decreased functionality, resulting in uncontrolled blood sugar and the inability of sugar to be supplied into the tissues, resulting in the active generation of glucose in muscle and liver tissue to create energy sources. Weight loss is induced (Pain VM, et al. 1974. J Biol Chem 249: 4510-4514; Francesca P, et. Al. 2007. International Congress Series 1303: 138-145). In order to investigate the effect of administration of Ganoderma lucidum extract having a hypoglycemic effect on weight loss, body weight was measured with blood sugar at a weekly interval while breeding as described in (1) above. The results are shown in FIG. Following administration of the Ganoderma lucidum mushroom extract prepared in Example 1, body weight, which was decreased after 9 weeks, was increased by 16.3% again. These results show that Ganoderma lucidum mushroom extract can induce smooth glucose metabolism in the body to suppress rapid weight loss due to diabetes and to control blood glucose.

Example 4 Isolation and Identification of Inhibitors of DPP-IV Activity

In the Ganoderma lucidum extract prepared in Example 1, a DPP-IV activity inhibitor was isolated and identified.

(1) Isolation and Identification of Compound I Inhibiting DPP-IV Activity

137.6 g of the ethanol extract of Ganoderma lucidum mushroom prepared in Example 1 was suspended in 1000 ml of distilled water, and hexane, ethyl acetate, and butanol were used for systematic extraction in a sequential direction. First, the ethanol extract was fractionated with hexane to obtain 15.0 g of hexane fraction, the remaining aqueous layer was fractionated with ethyl acetate to give 59.7 g, and the remaining aqueous layer was fractionated with the most polar butanol to obtain 32.6 g of fraction. .

Silica gel column chromatography was performed on 15.0 g of hexane fraction. 15.0 g of the hexane fraction was coated on the surface of silica gel and loaded into a column filled with 100% of hexane, and six small fractions were obtained by sequentially introducing a hexane: chloroform composition and a chloroform: ethanol composition. White needle-shaped crystals were isolated from the sixth of six subfractions. After dissolving the sixth small fraction in a small amount of chloroform, crystals were formed while filling with ethanol and drying slowly. The mother liquor was separated before the filled ethanol was completely dried and the crystals washed with ethanol to give pure crystals. The DPP-IV inhibitory effect of the separated crystals was confirmed in the same manner as in Example 2, and the structure was confirmed by performing ¹ H-NMR and mass spectrometry on the separated crystals. 5 and 6 show ¹ H-NMR and mass spectrometry, respectively. As shown in the ¹ H-NMR spectrum of FIG. 5, a complex peak located between 0-2 ppm corresponds to a terpenoid structure, and a multiplet peak located at 3.6 ppm is a low-long field. It indicates the presence of a hydroxy group (-OH) that affects migration to. In addition, polyline peaks located at 5.1 ppm reflect the presence of double bonds in the chain structure, and double doublet peaks located at 5.3 and 5.5 ppm show the presence of double bonds, and the separation of peaks It shows that there is an interaction between hydrogens. 6 shows that the molecular weight of the separated crystal is m / z 396, it can be seen that the structure has a stable structure because the value of the molecular weight shows a stability of 100%. m / z 253 represents the molecular weight of the structure in which the H ₂ O and the side chain are removed from the crystal structure. And the same ¹ H-NMR spectrum and mass spectrum data (22 E) in the analysis and reference structures, such as to a separate decision based on a comparison of literature - Costa El -5,7,22- triene -3β- It was identified as ol ((22 E ) -Ergosta-5,7,22-trien-3β-ol).

(2) Isolation and Identification of DPP-IV Inhibitory Compound II

As in Example 1, 4.0 kg of ganoderma lucidum was extracted with ethanol (8 L x 4 times) to obtain 164.2 g of ethanol extract. 26.6 g of a large amount of white powdery crystals were isolated from the ethanol extract. It was confirmed that a large amount of white crystals were produced in the process of concentrating the extract obtained in the extraction process. After the concentration was completed, the concentrate was dissolved in ethanol again, and the undissolved crystals were filtered to obtain pure crystals. The DPP-IV inhibitory activity of the obtained crystals was confirmed in the same manner as in Example 2, and the structure thereof was confirmed through ¹ H-NMR analysis and comparison with references to the crystals separated above.

According to the ¹ H-NMR spectrum of FIG. 7, the complex peak located between 0-2 ppm corresponds to the terpenoid structure, and the multiline peak located at 3.6 ppm affects the migration to the low field. Indicates the presence of group (-OH). In addition, polyline peaks located at 5.1 ppm reflect the presence of double bonds in the chain structure, double double line peaks located at 5.3 and 5.5 ppm show the presence of double bonds, and separation of peaks interacts between hydrogens. Shows that there is. This spectrum is almost identical to the ¹ H-NMR spectrum of FIG. 5, but the presence of a single line near 1.3 ppm is judged to reflect the presence of a methyl group. It is inferred that the molecular weight of this crystal is m / z 410 based on the presence of methyl groups in the structure. ^{Based on 1} H-NMR analysis and bibliographic comparison, isolated crystals were prepared using (22 E ) -23-methylercosta-5,7,22-trien-3β-ol ((22 E )- 23-Methylergosta-5,7,22-trien-3β-ol).

Figure 1 shows the effect of inhibiting the dose-dependent DPP-IV activity of the composition for inhibiting DPP-IV according to an embodiment of the present invention.

Figure 2 shows the change in blood glucose according to the administration of the composition for inhibiting DPP-IV according to an embodiment of the present invention.

Figure 3 shows the change in glycated hemoglobin according to the administration of the composition for inhibiting DPP-IV according to an embodiment of the present invention.

Figure 4 shows the weight change according to the administration of the composition for inhibiting DPP-IV according to an embodiment of the present invention.

5 shows the ¹ H-NMR spectrum of a compound of Formula (I).

6 shows the MS spectrum of the compound of formula (I).

7 shows the ¹ H-NMR spectrum of a compound of Formula II.

Claims (7)

A composition for the prevention or treatment of diabetes comprising the compound of formula I as an active ingredient:

Expression I. delete delete delete delete delete delete

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