KR102169089B1 - A composition for preventing or treating diabetes mellitus comprising Forsythia koreana extract or fermentated extract thereof - Google Patents

Abstract

The present invention relates to a composition for preventing or treating diabetes comprising a forsythia flower extract or a fermented product thereof, and specifically, a pharmaceutical composition for preventing or treating diabetes, comprising a forsythia flower extract or a fermented product thereof; It relates to a method for preventing or treating diabetes comprising administering a health functional food for preventing or improving diabetes and forsythia flower extract or a fermented product thereof to an individual other than a human. Since the forsythia flower extract or fermented product thereof according to the present invention has an excellent β-cell recovery effect, it can be easily used as a composition for preventing or treating diabetes.

Description

[A composition for preventing or treating diabetes mellitus comprising Forsythia koreana extract or fermentated extract thereof]

The present invention relates to a composition for preventing or treating diabetes comprising a forsythia flower extract or a fermented product thereof, and specifically, a pharmaceutical composition for preventing or treating diabetes, comprising a forsythia flower extract or a fermented product thereof; Health functional food for preventing or improving diabetes; It relates to a method for preventing or treating diabetes, comprising administering an extract or a fermented product thereof to a subject other than humans, and a method for separating a compound from a forsythia flower.

Currently, Korea's rapid economic growth has greatly changed the pattern of diseases due to Westernized dietary habits and an excess of nutrients. Unlike in the past, chronic degenerative diseases such as arteriosclerosis, high blood pressure, cancer, obesity and diabetes are the main causes of death. Has become.

In particular, the incidence rate of diabetes and its complications is increasing rapidly. The incidence rate of diabetes was less than 1% of the population in the 1970s. After the 1990s, 17.2 people per 100,000 people died from diabetes, and it is estimated that the incidence of diabetes will continue to increase in the future. Diabetes is a chronic metabolic disease caused by prolonged hyperglycemia and metabolic disorders. Diabetes is classified into type 1 and type 2, and type 1 　diabetes is caused by not producing insulin, and type 2 　 diabetes is insulin Is secreted, but occurs when cells have insulin resistance. Because there is no abnormality in the secretion of insulin in type 2 　 diabetes, it is also called non-insulin dependent diabetes mellitus. Type 2 　 diabetes often appears in combination with obesity due to intake of high calorie and high protein diets and lack of exercise, and can also be caused by genetic factors, infection, certain drugs, pancreatic surgery, etc. The exact cause and pathogenesis of type 2 diabetes have not been fully elucidated.

Recently 　With the development of diabetes treatment methods 　The life expectancy of diabetic patients is prolonged and the mortality rate from acute metabolic complications is rapidly decreasing 　Due to the increase in the incidence of diabetic chronic complications 　 Diabetes complications 　The diabetic complication is greater than the diabetes treatment itself It is being pointed out as a problem.

Diabetes treatment includes drug therapy, exercise therapy, and diet, and insulin drugs and various blood sugar drugs are used depending on the patient's symptoms. However, 　diabetes is a complex disease characterized by excessive sugar production in the liver, insulin resistance, and decreased sugar processing capacity in muscle and fat cells, so specific treatment alone cannot prevent the induction of various side effects. Among these, drug therapy uses insulin and chemicals, so it is a constant problem with side effects and patient tolerance depending on the drug intake, so recently 　in the treatment of diabetes, using natural products with fewer side effects 　There is a need for research for treatment.

Under this background, the present inventors have completed the present invention by confirming that, as a result of researching to develop a composition for treating diabetes, a forsythia flower extract or a fermented product thereof restores damaged β-cells of Langerhans Island.

One object of the present invention is to provide a pharmaceutical composition for preventing or treating diabetes, including forsythia flower extract or fermented product thereof.

Another object of the present invention is to provide a health functional food for preventing or improving diabetes, including forsythia flower extract or fermented product thereof.

Another object of the present invention is to provide a method for preventing or treating diabetes comprising administering an extract or a fermented product thereof to an individual other than a human.

In order to achieve the above object, one aspect of the present invention provides a pharmaceutical composition for preventing or treating diabetes, including forsythia flower extract or a fermented product thereof.

In the present invention, the term "forsythia flower" means a flower of Forsythia koreana . It has been known that the extract of the forsythia flower has an antioxidant effect, but no diabetes treatment effect is known, and was first identified by the present inventors.

The forsythia flowers may be purchased commercially, or may be harvested or grown in nature.

In the present invention, the term "extract" refers to an extract obtained by extracting the forsythia flowers, a diluent or concentrate of the extract, a dried product obtained by drying the extract, a preparation or purified product of the extract, or a mixture thereof, etc. It includes extracts of all formulations that can be formed using itself and extracts.

The method of extracting the forsythia flowers is not particularly limited, and may be extracted according to a method commonly used in the art. Non-limiting examples of the extraction method include a hot water extraction method, an ultrasonic extraction method, a filtration method, a reflux extraction method, and the like, and these may be performed alone or in combination of two or more methods.

In addition, the kind of the extraction solvent used to extract the forsythia flowers is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the extraction solvent include water, an alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof, and these may be used alone or in combination of one or more. Specifically, methanol or ethanol aqueous solution may be used, and the methanol or ethanol aqueous solution is 70 to 90% (v/v), more specifically 75 to 85% (v/v), most specifically 80% (v/v) ) May be methanol or ethanol, but is not limited thereto.

In the present specification, the forsythia flower extract may be mixed with MeOH extract from F. koreana flowers (FKFM).

In the present invention, the term "fermented product" may be obtained by fermenting the forsythia flower extract, and specifically, may be obtained by reacting the coenzyme of E. coli, more specifically, the BGP-1 enzyme.

The fermented product may have an increased content of actigenin or matairesinol compared to the extract. Specifically, in the fermentation process of the extract, actin or matairesinoside, which is a lignan glycoside, loses sugar through an enzymatic reaction and is converted to actigenin or matairesinol, which is a lignin aglycone. It may be that the content of actigenin or matairesinol is increased.

In one embodiment of the present invention, as a result of measuring the compound content of the forsythia flower extract and the fermented product, it was confirmed that the content of actigenin and matai resinol was increased in the fermented product of the forsythia flower extract compared to the forsythia flower extract (FIG. 2) .

In the present specification, the fermented forsythia flower extract may be mixed with FKFM-BGP1.

The forsythia flower extract or a fermented product thereof may include actigenin, actin, matairesinol, or matairesinoside.

In a specific embodiment of the present invention, the forsythia flower extract or a fermented product thereof was fractionated through column chromatography to separate four compounds of actigenin, actin, matairesinol, and matairesinoside (FIG. 1).

In the present invention, the term "arctigenin" may be represented by the following formula (1). Actigenin is a lignan non-saccharide and is known to be contained in the roots of burdock.

[Formula 1]

In the present invention, the term "actin (arctiin)" may be represented by the following formula (2). Actin is a 2,3-dibenzylbutyrolactone-based lignan glycoside, and is known to be contained in burdock seeds, and hydrolysis of it produces D-glucose and actigenin.

[Formula 2]

In the present invention, the term "matairesinol" may be represented by the following formula (3). Matairesinol is a lignan non-saccharide and is known to be contained in the pine family and safflower seeds.

[Formula 3]

In the present invention, the term "matairesinoside" may be represented by the following formula (4). Matairesinoside is a lignan glycoside, which is isolated from plants such as masak line and sugar lily.

[Formula 4]

The lignan is a polar solvent such as water or alcohol having 1 to 4 carbon atoms in the forsythia flower extract or fermented product thereof; Non-polar solvents such as hexane and ethyl acetate; Or it may be a fraction obtained by fractionation with a mixed solvent thereof.

In the present invention, the term "fraction" means a result obtained by performing fractionation in order to separate a specific component or a specific component group from a mixture containing several different constituents.

The fractionation method for obtaining the fraction is not particularly limited, and may be performed according to a method commonly used in the art. Non-limiting examples of the fractionation method include a solvent fractionation method performed by treating various solvents, an ultrafiltration fractionation method performed by passing an ultrafiltration membrane having a constant molecular weight cut-off value, and various chromatography (size, charge, hydrophobicity). Or a chromatographic fractionation method for performing separation according to affinity), and combinations thereof.

In addition, any solvent known in the art may be used as the kind of fractionation solvent used to obtain the fraction. Non-limiting examples of the fractionation solvent include polar solvents such as water and alcohols having 1 to 4 carbon atoms; Non-polar solvents such as hexane and ethyl acetate; Or a mixed solvent of these, etc. are mentioned. These may be used alone or in combination of one or more, but are not limited thereto. Specifically, water, methanol, butanol, hexane, ethyl acetate, or a mixed solvent thereof may be used.

In one embodiment of the present invention, a dried forsythia flower was obtained using 80% methanol to obtain a forsythia flower extract, and the extract was fractionated with water, methanol, butanol, hexane, ethyl acetate, or a mixed solvent thereof, and actigenin and actin , Matairesinol and matairesinoside were isolated (FIG. 1).

In the present invention, the term "lignan" is a part of polyphenol found in plants, and is a steroid compound synthesized from cholesterol with a chemical structure similar to estrogen. It is present in more than 55 plant groups and acts as a defense molecule against antibiotics and pathogens and bacteria. Lignan is a type of phytoestrogen, a phytoestrogen that plays a similar role to estrogen. Lignans are a single cell layer beneath the fibrous layer and are abundant in the skins of fruits that are tightly bound to the fibrin. Epidemiologic and physiological studies in the human body have shown that lignan may have a positive effect on preventing type II diabetes and cancer, and may reduce the incidence of estrogen-related cancer. It can also reduce or prevent osteoporosis in the human body. Types of lignan include enterlignan, enterodiol, and enterolactone.

In the present invention, the term "diabetes" refers to a chronic metabolic disease in which hyperglycemia and metabolic disorders resulting therefrom persist for a long time due to symptoms such as insufficient secretion of insulin or inability to function normally, and diabetes is type 1 and 2 It is divided into types, type 1 　 diabetes is caused by the inability to produce insulin, and type 2 　 diabetes occurs because insulin is secreted but cells have resistance to insulin (insulin resistance).

In particular, in the case of type 1 diabetes, it is a diabetes caused by the inability to produce insulin due to damage to β-cells that constitute the pancreatic Langerhanssem and synthesize insulin due to factors such as metabolic disorders.

The forsythia flower extract or fermented product thereof according to the present invention may be used to restore damaged β-cells to treat diabetes.

In one embodiment of the present invention, as a result of treating a zebrafish inducing diabetes with alloxane and analyzing the pancreas of a zebrafish, a forsythia flower extract or a fermented product thereof was obtained after inducing diabetes. In the treated experimental group, the size of the pancreas Langerhans islet increased compared to the control group in which diabetes was induced, and in particular, it was confirmed that the fermented forsythia flower extract exhibited superior recovery effect than the forsythia flower extract (FIG. 4).

In addition, the diabetes treatment effect of the forsythia flower extract or fermented product thereof may be an effect of actigenin or matairesinol.

In one embodiment of the present invention, as a result of confirming the effect of recovering Langerhans islands of four lignans isolated from the forsythia flower extract or fermented product thereof, it was confirmed that actigenin and matai resinol exhibited a significant effect of recovering Langerhans islands (FIG. 3 ). .

In another embodiment of the present invention, as a result of analyzing the content of four kinds of lignans of the forsythia flower extract and its fermented product, the content of actigenin and matai resinol in the forsythia flower extract fermentation was significantly increased compared to the forsythia flower extract. It was confirmed (Fig. 2).

In the present invention, the term "prevention" refers to any action of suppressing or delaying the symptoms of diabetes by administration of a composition comprising the forsythia flower extract or fermented product thereof according to the present invention.

In the present invention, the term "treatment" refers to any action in which symptoms of diabetes are improved or beneficially changed by administration of a composition comprising the forsythia flower extract or fermented product thereof according to the present invention.

The term "pharmaceutical composition" as used in the present invention means that it is prepared for the purpose of preventing or treating diseases, and may be formulated and used in various forms according to conventional methods. For example, it can be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, and the like, and can be formulated in the form of external preparations, suppositories, and sterile injectable solutions. Specifically, it may be formulated and used in a form suitable for eye drop administration, for example, eye drops, creams, ointments, gels or lotions.

The composition of the present invention may be prepared in the form of a pharmaceutical composition for preventing or treating wounds, which further comprises an appropriate carrier, excipient, or diluent commonly used in the preparation of pharmaceutical compositions, the carrier being an unnatural carrier ( non-naturally occuring carrier).

In the present invention, the carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, Calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils.

In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient, such as starch, calcium carbonate, in the upper leaf extract and its fractions. , Sucrose (sucrose) or lactose (lactose), gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include water and liquid paraffin, which are simple diluents commonly used for suspensions, liquid solutions, emulsions, syrups, etc., and various excipients such as wetting agents, sweetening agents, fragrances, and preservatives. have. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. As the non-aqueous solvent and suspension, 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, glycerogelatin, and the like may be used.

The pharmaceutical composition of the present invention can be administered orally, parenteral, rectal, topical, transdermal, intravenous, intramuscular, intraperitoneal, subcutaneous, etc. according to a desired method, and the active ingredient of the pharmaceutical composition is the age of the subject to be administered. , Sex, weight, pathological condition and severity, route of administration, or the judgment of the prescriber. Determination of the application amount based on these factors is within the level of those skilled in the art, and its daily dose is, for example, 0.1 mg/g/day to 100 mg/g/day, more specifically 5 mg/g/day to 50 mg/day. It may be g/day, and the frequency of administration of the composition of the present invention is not particularly limited thereto, but may be administered once a day or divided into several doses. The above dosage does not in any way limit the scope of the present invention.

Another aspect of the present invention provides a health functional food for preventing or improving diabetes, including forsythia flower extract or a fermented product thereof.

In the present invention, the terms "forsythia flower", "extract", "fermentation" and "diabetes" are as described above.

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. , The food may be prepared in various forms such as tablets, capsules, powders, granules, liquids, pills, etc. to obtain a useful effect in inhibiting cancer metastasis.

The health functional food of the present invention may further include a food pharmaceutically acceptable carrier.

There is no particular limitation on the type of food to which the composition containing the forsythia flower extract or fermented product thereof of the present invention can be added, and for example, various beverages, gums, teas, vitamin complexes, health supplement foods, and the like. Other ingredients that do not interfere with the diabetes treatment effect may be added to the food composition, and the type is not particularly limited. For example, it may contain various herbal extracts, food additives, or natural carbohydrates as an additional component, as in ordinary foods.

The food supplementary additives are added to prepare health functional foods of each formulation, and can be appropriately selected and used by those skilled in the art. For example, various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavors and natural flavoring agents, coloring agents and fillers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters , Stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, etc. are included, but the types are not limited by the above examples.

At this time, the content of the extract contained in the food is not particularly limited thereto, but may be included in 0.01 to 100% by weight, more preferably 1 to 80% by weight based on the total weight of the food composition.

When the food is a beverage, it may be included in a ratio of 1 to 30 g, preferably 3 to 20 g, based on 100 ml. In addition, the composition may include additional ingredients that are commonly used in food compositions to improve smell, taste, vision, and the like. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid, and the like may be included. In addition, minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), and copper (Cu) may be included. In addition, amino acids such as lysine, tryptophan, cysteine, and valine may be included. In addition, preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), disinfectants (bleaching and highly bleaching, sodium hypochlorite, etc.), antioxidants (butylhydroxyanisole (BHA), butylhydroxytoleuene ( BHT), etc.), colorants (tar colors, etc.), coloring agents (sodium nitrite, sodium nitrite, etc.), bleach (sodium sulfite), seasoning (MSG sodium glutamate, etc.), sweeteners (dulsin, cyclamate, saccharin, sodium, etc.) , Flavorings (vanillin, lactones, etc.), expanding agents (alum, D-potassium hydrogen stannate, etc.), reinforcing agents, emulsifiers, thickeners (thickening agents), coating agents, gum base agents, foam inhibitors, solvents, and food additives such as improving agents. ) Can be added. The additive is selected according to the type of food and used in an appropriate amount.

The health functional food of the present invention can be prepared by a method commonly used in the art, and at the time of the production, it can be prepared by adding raw materials and ingredients commonly added in the art. In addition, unlike general drugs, it has the advantage of not having side effects that may occur when taking drugs for a long time by using food as a raw material, and it can be excellent in portability.

Another aspect of the present invention provides a method for preventing or treating diabetes comprising administering an extract or a fermented product thereof to an individual other than a human.

In the present invention, the terms "forsythia flower", "extract", "fermentation" and "diabetes" are as described above.

The term "individual" used in the present invention may mean all animals including humans who have or are likely to develop diabetes. The animals may be mammals such as cattle, horses, sheep, pigs, goats, camels, antelopes, dogs, cats, etc. in need of treatment for symptoms similar to humans, but are not limited thereto.

The prevention or treatment method of the present invention may specifically include the step of administering the composition in a pharmaceutically effective amount to an individual who has developed diabetes or is at risk of developing diabetes.

As used herein, the term "administration" means introducing the pharmaceutical composition of the present invention to a patient by any suitable method, and the route of administration of the composition of the present invention is oral or parenteral as long as it can reach the target tissue. It can be administered through a variety of routes.

Another aspect of the present invention provides a pharmaceutical composition for preventing or treating diabetes comprising a lignan compound selected from the group consisting of actgenin, actin, matai resinol and matai resinoside.

In the present invention, Yong-A, "Actigenin", "Actin", "Matai Resinol", "Matai Resinoside", "Lignan" are as described above.

The lignan compound may be isolated from forsythia flowers.

In a specific embodiment of the present invention, actigenin, actin, matai resinol and matai resinoside obtained by fractionating the forsythia flower extract were administered to zebrafish larvae treated with alloxane, and the size of Langerhans island was measured. It was confirmed that the size of Langerhans islands was increased in the experimental group administered with genin and matairesinol compared to the control group treated with only alloxane (FIG. 3).

Since the forsythia flower extract or fermented product thereof according to the present invention has an excellent β-cell recovery effect, it can be easily used as a composition for preventing or treating diabetes.

1 is a diagram showing the chemical structure of four compounds isolated from forsythia flower extract; arctigenin: actigenin, arctiin: actin, matairesinol: matairesinoside: matairesinoside, β-Glc: β-D-glucopyranosyl group.
2 is a graph analyzing the content of four compounds in forsythia flower extract and its fermentation product using LC-ESI-MS chromatogram; (A) lignan content analysis result of FKFM, (B) lignan content analysis result of FKFM-BGP1, FKFM: forsythia flower extract, FKFM-BGP1: forsythia flower extract fermented product.
3 is a graph confirming the recovery effect of β-cells by treatment with four kinds of lignans in zebrafish treated with alloxane; (A) Size of Langerhans island of each experimental group, (B) 2-NBCG absorption level of each experimental group, (C) Langerhans island image of each experimental group, Normal: Normal group, Alloxan: Alloxan (negative control), Glimepiride: Glimepiride (positive control) ).
4 is a graph confirming the recovery effect of β-cells by treatment of forsythia flower extract or a fermented product thereof in zebrafish treated with alloxane; (A) Size of Langerhans island of each experimental group, (B) 2-NBCG absorption level of each experimental group, (C) Langerhans island image of each experimental group, NOR: normal group, AX: alloxane (negative control), FKFM: forsythia flower extract, FKFM-BGP1: Fermented forsythia flower extract.

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples.

Example 1. Isolation of compounds derived from forsythia flower extract and fraction

Dried forsythia flowers (962g) were extracted for 24 hours at room temperature using 80% methanol (MeOH, 33LХ2), which was filtered and concentrated. Thereafter, water (500 mL) was successively poured into the obtained methanol extract, followed by extraction using n-butanol (n-BuOH, 450 mLХ2) and ethyl acetate (EtOAc, 500 mLХ2). The extracts were concentrated to obtain a residue of an EtOAc fraction (FKE, 45g), an n-BuOH fraction (FKB, 110g) and a water fraction (FKW, 395g).

After obtaining the fractions, the compound was separated using column chromatography.

Specifically, the EtOAc fraction was n -hexane-EtOAc (10:1→2:1→1:1, 14 L each) using SiO ₂ cc (SiO ₂ column chromatography, φ 12Х17 cm) → CHCl ₃ -MeOH -It was eluted with water (30:3:1→20:3:1→10:3:1→65:35:10, 15 ℓ each), and 12 fractions (FKE-1 to FKE-12) were obtained.

Among the 12 fractions, the FKE-10 fraction [1.9 g, eluent/total volume (Ve/Vt) 0.072-0.088] was converted to SiO ₂ cc (φ 4.5Х15 cm) using CHCl3-EtOAc (10:1→3: 1, respectively 5.6 L), and 19 fractions (FKE-10-1 to FKE-10-19) were obtained.

Among the 19 fractions, the FKE-10-10 fraction [127.8 mg, Ve/Vt 0.043-0.050] was prepared in acetone-water (1:3→1:1, each 730 ml) using ODS cc (φ 3Х6 cm). Eluted with, to obtain 6 fractions (FKE-10-10-1 to FKE-10-10-6). Then, in the FKE-10-10-2 fraction of the six fractions, arctigenin [119.2 mg, Ve/Vt 0.082-0.110, TLC (Kieselgel 60 F ₂₅₄ ) R _f 0.62, CHCl ₃ -EtOAc (1: 1), TLC (RP-18F _254S ) R _f 0.72, acetone-water (3:1)] was separated.

In addition, of the 19 fractions, FKE-10-14 fraction [220.0mg, Ve/Vt 0.736-0.780] was prepared by using ODS cc (φ 2.5Х5 cm) in acetone-water (2:1→1:1, respectively 2.2 ℓ), and to obtain 8 fractions (FKE-10-14-1 to FKE-10-14-8), FKE-10-14-2 fraction [69.0 mg, Ve/ Vt 0.736-0.780] was eluted with CHCl ₃ -EtOAc (5:1, 500 ml) using a SiO ₂ (φ 2Х10 cm) column, and three fractions (FKE-10-14-2-1 ~ FKE-10 -14-2-3) was obtained. Thereafter, in the FKE-10-14-2-3 fraction of the three fractions, matairesinol [12.9 mg, Ve/Vt 1.000, TLC (Kieselgel 60 F ₂₅₄ ) R _f 0.50, CHCl ₃ -EtOAc (3 :1), TLC(RP-18F _254S ) R _f 0.53, acetone-water (2:1)] was separated.

In addition, the n-BuOH fraction was chromatographed using a SiO ₂ resin (Φ 11 x 15 cm) and CHCl ₃ -MeOH-H ₂ O (30:3:1→20:3:1→10:3) :1, each 43 L) → EtOAc- n -BuOH-H ₂ O (4:5:1, 45 L) eluted to give 15 fractions (FKB-1 ~ FKB-15), actin (arctiin) (8.0 g, Ve/Vt 0.038-0.070, TLC (Kieselgel 60 F ₂₅₄ ) R _f 0.45, CHCl ₃ -MeOH-H ₂ O (15:3:1), TLC (RP-18 F _254S ) R _f 0.65, Acetone-water (1:1)] was separated.

In addition, of the 15 fractions, the FKB-3 fraction [20.8 g, Ve/Vt 0.071-0.122] was used as SiO ₂ cc (Φ 2 x 10 cm) using CHCl ₃ -MeOH-H ₂ O (25:3: 1→10:3:1, 3 L each) and 14 fractions (FKB-3-1 ~ FKB-3-14) were obtained, matairesinoside [8.8 g, Ve/Vt 0.855 -0.909, TLC (Kieselgel 60 F ₂₅₄ ) R _f 0.45, CHCl ₃ -MeOH-H ₂ O (10:3:1), TLC (RP-18 F _254S ) R _f 0.52, acetone-water (2:3) ] Was separated.

The structure of the four types of lignans separated in Example 1 is as shown in FIG. 1.

Example 2. Fermentation of methanol extract (FKFM) of forsythia flowers

2-1. Cultivation of strains

The E. coli seed culture was aseptically transferred to a 5 L fermentor using a flame inoculation method. Fermentation medium is peptone 20g·L ^-1 , yeast X 12g·L ^-1 , NaCl 10g·L ^-1 , KH2PO4 1.5g·L ^-1 , K2HPO4 2.3g·L ^-1 and MgSO ₄ ·7H ₂ O 0.25g· It consists of L ^-1 . E. coli cultures were engineered to optimize protein production. The pH was adjusted to 7.0±0.2 with 4 mol·L ^-1 and 1 mol·L ^-1 HCl. For dissolved oxygen (DO), the stirrer rpm was first increased, and then the air was set to 20% or more. The temperature was adjusted at 37°C for 9 hours and then changed to 30°C. Protein expression was induced by the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG). Dry cell weight (DCW) samples were taken periodically for analysis.

2-2. Preparation of coenzyme BGP1

Cells were harvested for 15 minutes at 4° C. and 6000 rpm using a centrifuge (Ihanil, Daejeon, Korea). Thereafter, the cell pellet was suspended in a 3ХM(g) volume of pre-cool 1X PBS buffer (pH 7.0), and the enzyme was extracted three times at 4°C with a high pressure homogenizer. After enzyme extraction, the sample was centrifuged at 4° C. and 10,000 rpm for 20 minutes, and the supernatant was collected as a coenzyme solution.

2-3. Enzymatic treatment of forsythia flower extract using BGP1

The activity of the BGP1 enzyme was measured using pNP-β-D-glucopyranoside (pNPG) contained in 1ХPBS. After reacting pNPG to the BGP1 enzyme, 50mM Na ₂ CO ₃ was added to stop the reaction. The release of pNP was determined immediately by measuring the absorbance of the reaction at 405 nm with a microplate reader. One unit of activity was defined as the amount of protein required to produce 1 μmol of pNP per minute. Thereafter, the BGP1 coenzyme was reacted with FKFM at pH 7.0 and 37°C. After reaction for 24 hours, bioconverted FKFM (FKFM-BGP1) was extracted with 200 μl of n-butanol saturated with water. The butanol layer was confirmed by TLC experiment.

2-4. Quantitative analysis of lignan in FKFM and FKFM-BGP1 through LC/MS experiments

Each of the four compounds obtained in Example 1 was precisely weighed and dissolved in MeOH to obtain a stock solution having a concentration of 1.0 mg/mL. Calibration curves were created for each standard at four concentrations (125, 50, 25, 12.5 μg/mL). The calibration equation was established by plotting the average area (n=3) generated from the standard solution against the concentration. HPLC experiments were performed as follows for quantitative analysis of each compound in both the n-BuOH layers of FKFM and FKFM-BGP1. The extract was filtered through a 0.22 μm membrane filter and evaporated in vacuo. A 10 μL aliquot of the fractionation solution (1.0 mg/mL) was injected into the HPLC system. Analysis was performed using an Agilent technology 1200 series (Tokyo, Japan) with an Agilent G1314B UV detector (280 nm). The column was YMC-triart C18 (100 mm Х 2.0 mm, particle size 3 μm). The mobile phase consisted of 0.1% FA in H ₂ O (solvent A) and 0.1% FA in acetonitrile (solvent B) and eluted with the following gradient elution and concentration of solvent B at a flow rate of 0.4 mL/min. 5% → 13% (15 minutes) → 17% (18 minutes) → 17% (20 minutes) → 25% (25 minutes) → 100% (37 minutes) → 100% (40 minutes). Quantitative analysis was repeated 3 times.

As a result, as shown in Figure 2, in the case of forsythia flower extract, it can be confirmed that the content of actin and matai resinoside is high and the content of actigenin and matai resinol is low, whereas in the case of forsythia flower extract fermented product, forsythia flower Compared to the extract, the contents of actin and matairesinoside were decreased, and the contents of actigenin and matairesinol were significantly increased.

Example 3. Analysis of recovery effect of forsythia-derived lignan in Langerhans Island induced by alloxane

In order to confirm the diabetes treatment effect of forsythia-derived lignan isolated in Example 1, treatment with alloxane, which is known as a diabetic chemical substance in the larvae of zebrafish, and reported to reduce the β-cell mass of Langerhans islets Thus, damage to Langerhans Island was induced, and the recovery effect of Langerhans Island was analyzed by treating each of four types of forsythia-derived lignan.

Specifically, zebrafish larvae were classified into normal group, alloxan induction group, and compound treatment group after alloxane induction, and wild zebrafish larvae 5 days after fertilization were placed in a 24-well plate, and the experimental group excluding the normal group was 600 μM. It was exposed to alloxane of 3 hours to induce damage to Langerhans Island. Then, 10 μM of each lignan was treated with zebrafish larvae for 12 hours and stained with 40 μM 2-NBDG for 30 minutes, followed by washing in 0.03% seawater solution for 20 minutes. The stained islets of Langerhans were observed with a fluorescence microscope and analyzed using Focus Lite and Image J software.

As a result, as shown in FIG. 3, the alloxane treatment significantly reduced the size of Langerhans Island by 41.4% (p <0.0001) compared to the normal group (FIGS. 3a, c). The size of Langerhans islets treated with glimepiride (positive control) increased by 78.8% (p <0.0001) compared to the alloxane-inducing group. The lignin glycosides actin and matairesinoside slightly increased the size of Langerhans islands compared to the alloxane-inducing group, but there was no statistical significance. However, the lignin aglycone, actigenin and matairesinol, increased the size of Langerhans islands to 65.8% (p = 0.0014) and 77.2% (p = 0.0001), respectively (Fig. 3a, c). This recovery effect was almost the same as the positive control, glimepiride.

In addition, glucose uptake in the zebrafish model using 2-[N-(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG) Was evaluated. 2-NBDG is a fluorescent dye derived from glucose with a modified amino group at the C-2 position (Oshioka et al., 1996), and is widely used in diabetes studies to measure the ability of cells to absorb glucose. To measure the 2-NBDG absorption level of Langerhans Island, a histogram of Image J software representing the pixel intensity (green) level was used in the range of 0 to 255.

The alloxane-treated group significantly decreased 2-NBDG absorption compared to the normal group ( p =0.0002), whereas the other experimental groups were 47.19% in the glimepiride-treated group compared to the alloxane-treated group ( p =0.0015), and actinoids is 51.39% genin-treated group (p = 0.0002), Mathai Lecinol treatment group is 61.28% (p = 0.0003), Mata seven Sino is 35.56% side-treated group (p = 0.0265), the actin-treated group is 32.28% (p It was confirmed that the 2-NBDG absorption increased by as much as =0.0203) (Fig. 3b, c). Through this experiment, the efficacy of four lignans as therapeutic substances against alloxane-induced Langerhans island damage in zebrafish was confirmed.

Example 4. Analysis of the recovery effect of FKFM or FKFM-BGP1 in Langerhans Island induced by alloxane

In order to confirm the diabetes treatment effect of FKFM or FKFM-BGP1, the same method as in Example 3 was used, and FKFM or FKFM-BGP1 was treated with 1 μg/mL to confirm the effect of recovering Langerhans Island.

As a result, as shown in FIG. 4, the size of Langerhans islet was significantly increased by 28.3%, p=0.0495 and 58.5%, p=0.0010 in the FKFM or FKFM-BGP1 treatment group compared to the alloxane-inducing group. In particular, FKFM-BGP1 increased the size of Langerhans islets by 23.6% more than FKFM (Figure 4a, c), and the level of 2-NBDG uptake was also 49.10% in the FKFM and FKFM-BGP1 treated groups compared to the alloxane treatment group ( p = p = 0.0047) and 64.86% ( p = 0.0004) showed a significant increase (Fig. 4b, c), FKFM or FKFM-BGP1 also confirmed the therapeutic effect on the Langerhans Island injury induced by alloxane.

Taken together, FKFM or FKFM-BGP1 has a therapeutic effect on alloxane-induced Langerhans islet damage, which is caused by the lignans of actigenin, actin, matairesinol and matairesinoside, which are included in FKFM or FKFM-BGP1. It can be inferred. In addition, in the case of FKFM-BGP1, which is a fermented product of FKFM, it can be confirmed that the effect is higher than that of FKFM, and it can be inferred that the effect is due to an increase in the content of actigenin and matairesinol in the fermentation process.

Therefore, the composition comprising FKFM, FKFM-BGP1 or lignan derived therefrom according to the present invention has a therapeutic effect on Langerhans Island damage, and can be used as a composition for treating diabetes.

In the present specification, details that can be sufficiently recognized and inferred by those of ordinary skill in the technical field of the present invention have been omitted, and the technical spirit or essential configuration of the present invention other than the specific examples described in the present specification is changed More various modifications are possible within the range that does not. Accordingly, the present invention may be implemented in a manner different from that specifically described and illustrated in the present specification, and this is a matter that can be understood by those of ordinary skill in the technical field of the present invention.

Claims (9)

A pharmaceutical composition for preventing or treating diabetes, comprising a fermented product obtained by reacting BGP-1 (β-glucosidase) enzyme with forsythia flower extract.
The method of claim 1, wherein the fermented product obtained by reacting the BGP-1 enzyme with the forsythia flower extract comprises actigenin, actin, matairesinol, or matairesinoside. That is, the pharmaceutical composition.
The pharmaceutical composition of claim 1, wherein the extract is an extract extracted with a solvent selected from the group consisting of water, a lower alcohol having 1 to 4 carbon atoms, and a mixed solvent thereof.
delete The pharmaceutical composition of claim 1, wherein the fermented product has an increased content of actigenin or matairesinol compared to the extract.
Health functional food for preventing or improving diabetes, comprising a fermented product obtained by reacting BGP-1 enzyme with forsythia flower extract.
A method for preventing or treating diabetes, comprising administering a fermented product obtained by reacting a BGP-1 enzyme with a forsythia flower extract to an individual other than a human.
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