KR20200069249A - Composition for preventing, improving or treating diabetes comprising Paeonia lactiflora extract fermented by lactic acid bacteria as effective ingredients - Google Patents

Abstract

The present invention relates to a composition for preventing, ameliorating, or treating diabetes comprising a lactic acid bacterium fermented product of a Paeonia lactiflora extract as an effective ingredient and, more particularly, to a pharmaceutical composition for preventing or treating diabetes comprising a lactic acid bacterium fermented product of a Paeonia lactiflora extract as an effective ingredient, a food composition thereof, and a method for converting paeoniflorin. According to the present invention, the lactic acid bacterium fermented product of a Paeonia lactiflora extract can effectively increase a glucose absorption rate by moving glucose to muscle cells, and cause no side effect to the human body by using Paeonia lactiflora and kimchi-derived lactic acid bacteria, which are natural materials, and thus can be useful in food and drug medicines for preventing, ameliorating or treating diabetes.

Description

Composition for preventing, improving or treating diabetes comprising Paeonia lactiflora extract fermented by lactic acid bacteria as effective ingredients}

The present invention relates to a composition for preventing, improving, or treating diabetes comprising lactic acid bacteria fermentation product of peony extract as an active ingredient, and more specifically, preventing or treating diabetes containing lactic acid bacteria fermentation product of peony extract as an active ingredient. Pharmaceutical compositions, food compositions and methods for converting paniflorine.

Diabetes mellitus is a type of metabolic disease in which insulin secretion is insufficient or normal function is not achieved, and is characterized by high blood glucose concentration.

Diabetes is divided into type 1 and type 2, and type 1 diabetes was previously called'pediatric diabetes' and is a disease caused by the inability to produce insulin at all. Type 2 diabetes, which is relatively insufficiency of insulin, is characterized by insulin resistance (the inability of cells to effectively burn glucose due to poor insulin function to lower blood sugar). Although type 2 diabetes seems to have a significant effect on environmental factors such as high calorie, high fat, high protein diet, lack of exercise, and stress due to westernization of diet, diabetes may occur due to defects in certain genes, pancreatic surgery, It can also be caused by infection or medication. In weak hyperglycemia, it is difficult for most patients to perceive symptoms as diabetes because they do not feel symptoms or are ambiguous. If your blood sugar increases, you will be thirsty and drink a lot of water, urine volume will increase, you will often go to the bathroom, and you will lose weight. If the state of hyperglycemia is maintained for a long period of time, various complications such as retinopathy, renal dysfunction, and neuropathy (agitation, pain) occur in the body or the risk of cardiovascular disease increases.

In the case of type 1 diabetes, insulin treatment is necessary, and the most effective treatment of type 2 diabetes is to achieve the optimal blood sugar level for the number of meals. In particular, delayed absorption of monosaccharides through inhibition of α-glucosidase activity has been suggested as a beneficial strategy for the treatment of type 2 diabetes.

On the other hand, peony ( Paeonia lactiflora ) is a perennial plant of the buttercup family, which grows mainly in the foothills or valleys. Several stems stand up straight from one aeration, about 60cm high, without hair on leaves and stems. There are several roots, but they are thin and thick with a pointed cylindrical shape with long ends. Flowers bloom from May to June at the end of the stem, which is large and beautiful, and is cultivated about 10 cm in diameter. The flower color varies from red to white, and there are many horticultural varieties. The roots are used as medicines such as pain relief, abdominal pain, menstrual pain, amenorrhea, hemolysis, anemia, and bruises. In addition, when the peony root is used as a drug, it can be divided into red peony and count peony according to the pre-processing method. The red peony is dried by removing the roots of the beard, and the county is removed by removing the roots of the beard and boiled in boiling water, and then the boil is removed or boiled again.

On the other hand, Korean Patent Publication No. 2016-0006812 discloses'a composition for preventing and treating diabetes including peony extract', and paeoniflorin is known as an indicator of peony or peony extract, but their diabetes There was a limitation that was not sufficient to exhibit a physiologically effective effect of the related activity.

Accordingly, the present inventors were studying to find an effective method for the treatment of diabetes by metabolizing or improving peony extract, and when fermenting the peony extract with Leuconostoc sp.LN180020 (KCTC 13719BP) isolated from kimchi Oniflorin was converted to another substance, and the present invention was completed by confirming that the anti-diabetic activity of the lactic acid bacteria fermentation product of the peony extract containing it was significantly improved compared to that of other lactic acid bacteria.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of diabetes containing a lactic acid bacteria fermentation product of a peony (Paeonia lactiflora) extract as an active ingredient.

Another object of the present invention is to provide a food composition for preventing or improving diabetes containing lactic acid bacteria fermentation product of peony extract as an active ingredient.

Another object of the present invention (a) inoculating a composition containing peoniflorin leuconostoc (Leuconostoc sp.) lactic acid bacteria; And (b) to provide a method for converting the peony florin in peony or peony extract comprising the step of culturing and fermenting the product of step (a).

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of diabetes containing lactic acid bacteria fermentation product of peony extract as an active ingredient.

In order to achieve another object of the present invention, the present invention provides a food composition for preventing or improving diabetes containing lactic acid bacteria fermentation product of peony extract as an active ingredient.

In order to achieve another object of the present invention, the present invention comprises the steps of (a) inoculating a composition containing peoniflorin leuconostoc sp. And (b) provides a method of converting the peony florin in peony or peony extract comprising the step of culturing and fermenting the obtained product of step (a).

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for the prevention or treatment of diabetes containing the lactic acid bacteria fermentation product of the peony extract as an active ingredient.

In the present specification,'peony' refers to the root of a plant having the scientific name Paeonia lactiflora , preferably the root of Paeonia lactiflora used as a medicinal material. Peony as a medicinal herb is also called Paeoniae Radix or Peony root, and more specifically, it represents the root of the peony Paeonia lactiflora Pallas or other related plants (peony and Paeoniaceae) used as medicinal sites.

Peony extract may be extracted by a solvent extraction method known in the art. The extraction solvent is not limited thereto, but water, a lower alcohol having 1 to 6 carbon atoms (C1) to 6 (C6), an organic solvent, or a mixed solvent thereof may be used. The lower alcohol having 1 to 6 carbon atoms (C1) (C6) is not limited thereto, but may be methanol, ethanol, alcohol, propanol, isopropanol, butanol, pentanol, hexanol, and the organic solvent is not limited thereto. Acetone, ethyl acetate, n-nucleic acid, diethyl ether, acetone, and benzene. Preferably it can be extracted with water or an alcohol having 1 to 6 carbon atoms or a mixed solvent thereof, more preferably water, ethanol or alcohol. The alcohol may be a form in which a small amount of water (for example, 95% ethanol, 99% ethanol) is added to reflect physicochemical properties such as hygroscopicity.

The extraction temperature and time are not particularly limited as long as the purpose of extraction of the active ingredient is achieved, but can be extracted for 10 minutes to 12 hours at 30°C to 121°C. It may also be extracted by pressing to facilitate extraction of the active ingredient during extraction. Meanwhile, the extraction may be performed by supercritical extraction or subcritical extraction using an appropriate solvent such as carbon dioxide.

As used herein,'treatment' or'improvement' refers to suppressing the occurrence or recurrence of a disease, alleviating symptoms, reducing the direct or indirect pathological consequences of the disease, reducing the rate of disease progression, improving, improving, alleviating, or improving the disease state. Means prognosis. The term'prevention' used in the present invention means all actions that suppress the onset of disease or delay the progression.

In the present invention, "diabetes", which is the target of treatment or prevention, is a metabolic disorder syndrome characterized by hyperglycemia caused by defects in insulin hormone deficiency or insulin resistance, which are produced in the beta cells of the pancreas, and both. Such diabetes can be divided into insulin-dependent diabetes (IDDM, Type 1) and insulin-independent diabetes (NIDDM, Type 2) caused by insulin resistance and insulin secretion damage. In both type 1 and type 2 diabetes, various complications such as heart disease, intestinal disease, ophthalmic disease, neurological disease, stroke, etc. occur, which cause chronic neurological disease and cardiovascular disease due to elevated blood sugar and insulin levels over a long period of time. And short-term hypoglycemia and hyperglycemia reactions that cause acute complications. Diabetes mellitus causes chronic and persistent hyperglycemia, as well as lipid and protein metabolism disorders. The condition is diverse and is directly attributed to hyperglycemia, including diabetic peripheral neuropathy, diabetic retinopathy, diabetic nephropathy, diabetic cataract, keratosis, diabetic arteriosclerosis in the retina, kidney, nerve, cardiovascular system, and the like.

Accordingly, the diabetes in the present invention includes diabetes or diabetes complications due to diabetes, and more specifically, may be type 1 diabetes, type 2 diabetes, or gestational diabetes. In the case of diabetic complications, it may be a disease caused by the destruction or deterioration of Langerhans islet cells, and a disease caused by an increase in blood glucose concentration, but is not limited thereto, but is not limited to, diabetic peripheral neuropathy, diabetic retinopathy, diabetic nephropathy, Diabetic cataract, keratosis, diabetic arteriosclerosis.

In the present invention, the'lactic acid bacteria' may be Leuconostoc citreum or Leuconostoc sp. LN180020 with a deposit number of KCTC 13719BP, preferably Leuconostoc with a deposit number of KCTC 13719BP. ( Leuconostoc sp.) LN180020. The'lactic acid bacteria' is characterized by having a bioconversion ability to convert the paniflorine.

In an embodiment of the present invention, vegetable lactic acid bacteria derived from kimchi were isolated to search for useful lactic acid bacteria. As a result, 193 of the Lactobacillus genus (Lactobacillus), Petit O Rhodococcus genus (Pediococcus), Wei Cellar in (Weissella), flow Pocono stock in (Leuconostoc) and 39 kinds (species) contained in the Enterococcus genus (Enterococcus) The strain was isolated.

In one embodiment of the present invention, the isolated lactic acid bacteria strain was inoculated into a peony hot water extract and fermented to prepare a lactic acid bacteria fermentation product of the peony extract, and a strain in which the paeoniflorin was bioconverted was explored. As a result, it was confirmed that the peak of about 15 minutes, which is expected to be peoniflorin specifically in one of the isolated strains, decreases, and that the peak of the new material increases at 14.2 minutes.

This strain was identified and named Leuconostoc sp. LN180020 strain, and deposited with the Korea Resource Center (KCTC) as of November 20, 2018 (Accession No.: KCTC 13719BP).

In an embodiment of the present invention, it was confirmed whether the fermentation product of LN180020 in the genus Leuconostok has anti-diabetic activity through cytotoxicity and the ability to absorb glucose from myocytes, and whether this anti-diabetic activity is also caused by fermentation of other lactic acid bacteria. . As a result, although the cytotoxicity did not appear in the fermented product of lactic acid bacteria, only the fermented product of LN180020 in Leuconostock showed anti-diabetic activity. Particularly, it was confirmed that the fermentation product of LN180020 in the Leuconostock of the present invention functions to increase glucose absorption into muscle cells, which has anti-diabetic activity.

The pharmaceutical composition according to the present invention may be variously formulated according to the route of administration in a method known in the art with a pharmaceutically acceptable carrier for the treatment of diabetes. The carrier includes all kinds of solvents, dispersion media, oil-in-water or water-in-oil emulsions, aqueous compositions, liposomes, microbeads, and microsomes.

The pharmaceutical composition according to the present invention may be administered to a patient in a pharmaceutically effective amount, that is, an amount sufficient to prevent or alleviate symptoms and treat diabetes. For example, a typical daily dosage may be administered in the range of about 0.01 to 1000 mg/kg, preferably, in the range of about 1 to 100 mg/kg. The pharmaceutical composition of the present invention may be administered once or several times within a preferred dosage range. In addition, the dosage of the pharmaceutical composition according to the present invention can be appropriately selected by a person skilled in the art according to the administration route, administration target, age, sex weight, individual difference, and disease state.

The route of administration may be administered orally or parenterally. The parenteral administration method is not limited to this, but is not limited to intravenous, intramuscular, arterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, rectal, or pancreatic. It may be intra-administration, but is not limited thereto, and most preferably, may be administered orally.

When the pharmaceutical composition of the present invention is administered orally, powders, granules, tablets, pills, dragees, capsules, liquids, gels, syrups, suspensions, wafers, etc., according to methods known in the art, together with a suitable oral administration carrier It can be formulated in the form of. Examples of suitable carriers include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, starches including cellulose starch, wheat starch, rice starch and potato starch, cellulose, and the like. Fillers such as cellulose, gelatin, polyvinylpyrrolidone, and the like may be included, including methyl cellulose, sodium carboxymethyl cellulose, and hydroxypropyl methyl cellulose. In addition, if necessary, cross-linked polyvinylpyrrolidone, agar, alginic acid or sodium alginate may be added as a disintegrant. Furthermore, the pharmaceutical composition may further include an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and a preservative.

In addition, when administered parenterally, the pharmaceutical composition of the present invention may be formulated according to methods known in the art in the form of injections, transdermal administrations, and nasal inhalants, together with suitable parenteral carriers.

In addition, the pharmaceutical composition can be administered by any device capable of transporting the active substance to target cells. Preferred modes of administration and preparations are intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, or drip injections. Injectables include aqueous solvents such as physiological saline solutions or ring gel solutions, vegetable oils, higher fatty acid esters (e.g., oleic acid, etc.), and non-aqueous solvents such as alcohols (e.g., ethanol, benzyl alcohol, propylene glycol or glycerin). Stabilizer to prevent deterioration (e.g., ascorbic acid, sodium hydrogen sulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), emulsifier, buffer for pH adjustment, to prevent microbial growth And a pharmaceutical carrier such as a preservative (eg, phenyl mercuric nitrate, chimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.).

The injections must be sterile and protected from contamination of microorganisms such as bacteria and fungi. For injections, examples of suitable carriers include, but are not limited to, solvents or dispersion media including water, ethanol, polyols (eg, glycerol, propylene glycol and liquid polyethylene glycol, etc.), mixtures thereof and/or vegetable oils. Can be. More preferably, suitable carriers include Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) with triethanol amine or sterile water for injection, isotonic solutions such as 10% ethanol, 40% propylene glycol and 5% dextrose. Etc. can be used. In order to protect the injection from microbial contamination, various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid, thimerosal, etc. may be further included. In addition, the injection may additionally include an isotonic agent such as sugar or sodium chloride in most cases.

As other pharmaceutically acceptable carriers, reference may be made to those described in the following documents (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

Pharmaceutical compositions according to the invention may include one or more buffers (e.g. saline or PBS), carbohydrates (e.g. glucose, mannose, sucrose or dextran), antioxidants, bacteriostatic agents, chelating agents (e.g. For example, EDTA or glutathione), adjuvants (eg, aluminum hydroxide), suspending agents, thickening agents and/or preservatives may be further included.

In addition, the pharmaceutical composition of the present invention can be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal.

In addition, the pharmaceutical composition of the present invention may be administered alone or in combination with a known compound having an effect of treating diabetes.

On the other hand, the present invention provides a food composition for preventing or improving diabetes containing lactic acid bacteria fermentation product of peony extract as an active ingredient.

The food composition of the present invention includes all forms of functional food, nutritional supplement, health food, and food additives.

Food compositions of this type can be prepared in various forms according to conventional methods known in the art. Although not limited thereto, for example, as a healthy food, lactic acid bacteria fermentation products of peony extract may be prepared in the form of tea, juice, and drink, and liquefied, granulated, encapsulated, and powdered to be consumed. In addition, it can be prepared in the form of a composition by mixing with the lactic acid bacteria fermentation of the peony extract and a known active ingredient known to be effective in diabetes. In addition, functional food products include, but are not limited to, beverages (including alcoholic beverages), fruits and processed foods thereof (eg, canned fruits, canned fruits, jams, marmalade, etc.), fish, meat, and processed foods thereof (eg ham, sausages) Corn beef, etc.), breads and noodles (e.g. udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), juice, various drinks, cookies, syrup, dairy products (e.g. butter, cheese, etc.), edible vegetable oil, margarine, vegetable It can be prepared by adding lactic acid bacteria fermentation product of peony extract to protein, retort food, frozen food, various seasonings (eg, miso, soy sauce, sauce, etc.). In addition, in order to use the fermented lactic acid bacteria of the peony extract in the form of an additive, it can be prepared and used in the form of a powder or a concentrate.

The preferred content of the fermentation product of lactic acid bacteria in the peony extract in the food composition of the present invention is not limited to this, but is preferably 0.1 to 90% by weight in the final manufactured food. More preferably, the food composition containing the lactic acid bacteria fermentation product of the peony extract of the present invention as an active ingredient, in particular, can be prepared in the form of a health functional food or dietary supplement by mixing with an active ingredient known to be effective for diabetes. have.

The health functional food composition for preventing or improving diabetes of the present invention contains lactic acid bacteria fermentation product of a peony extract that promotes glucose absorption into muscle cells as an active ingredient, and is excellent in preventing or improving diabetes. do.

Meanwhile, in one embodiment of the present invention, LN180020 lactobacillus in Leuconostock was identified through 16S rRNA sequencing, and a new substance that showed an increase in anti-diabetic activity was attempted. As a result, LN180020 lactic acid bacteria in the genus Leuconostoc showed the highest homology to the sequence of Leuconostoc citreum ( L. citreum ), and the peony extract as a marker of the peony extract had a molecular weight of 480.3 and a molecular weight of 642.3. It was found that the bioconversion to a new useful ingredient material.

Accordingly, the present invention provides a method for converting paoniflorine in a composition comprising paoniflorine comprising the following steps:

(a) inoculating a composition comprising paoniflorin with Leuconostoc sp. And

(B) step of culturing the obtained product of step (a) to ferment.

In the present invention, the composition containing paoniflorine may be an extract of a raw material containing paoniflorine, and preferably may be an extract of a peony or peony plant (Paeonia). In order to facilitate cultivation, preferably, the composition comprising paoniflorine may be liquid.

Leukonostoc sp. lactic acid bacteria in the method of converting paoniflorin of the present invention preferably has Leukonostoc citreum or a deposit number of KCTC having a bioconversion ability to convert paniflorine. 13719BP Liu Kono stock in (Leuconostoc sp.) can be LN180020, and preferably the deposit number KCTC 13719BP Liu Kono stock in (Leuconostoc sp.) may be LN180020.

The amount of lactic acid bacteria to be inoculated can be added to the lactic acid bacteria strain of 1 x 10 ⁸ to 1 x 10 ¹¹ to the composition itself or to a medium containing the composition, or to a pre-cultured lactic acid bacteria culture medium at about 1 to 5% (v/v).

Culture of step (b) after inoculation of lactic acid bacteria in the genus Leuconostock according to the present invention may be performed by a method for culturing lactic acid bacteria known in the art. In addition, as the culture medium, a medium composed of a carbon source, a nitrogen source, vitamins and minerals, or whey medium using milk components, skim milk medium, or the like can be used. In the medium for producing the culture of the present invention, one or more selected from the group consisting of glucose, sucrose, maltose, fructose, lactose, xylose, galactose, arabinose, and combinations thereof, It is preferably one or more selected from the group consisting of sucrose, fructose, glucose, galactose, arabinose, and lactose, and more preferably glucose. What can be used as the nitrogen source in the medium for producing the culture of the present invention is one selected from the group consisting of yeast extract, soytone, peptone, beef extract, tryptone, casitone, and combinations thereof. It is the above, Preferably it is one or more selected from the group which consists of yeast extract, peptone, tryptone, and soytone, More preferably, it is a yeast extract or soytone.

The culture and fermentation of lactic acid bacteria may be performed according to milk-containing media and culture conditions known in the art. Those skilled in the art can be easily adjusted and used according to the selected strain. Various such methods have been disclosed in various literature (eg, James et al., Biochemical Engineering, Prentice-Hall International Editions). Depending on the cell growth method, the suspension culture and the adhesion culture can be used in batch, fed-batch, and continuous culture methods depending on the culture method.

In the present invention, the conversion of the paniflorine results in the conversion of the paniflorine to another active substance, resulting in a reduction of the paniflorine.

Therefore, the peony extract lactic acid bacteria fermentation product of the present invention can effectively increase the glucose absorption rate by moving glucose to muscle cells, and there is no side effect on the human body using peony and kimchi-derived lactic acid bacteria derived from natural materials, thereby preventing and improving diabetes Or, it is expected to be useful in therapeutic foods and medicines.

1 is an HPLC chromatogram showing the content of indicators and new useful substances in non-fermented peony extract (non-fermented, (a)) and lactic acid bacteria fermented (b) of peony extract.
Figure 2 is a result of confirming the cell viability and relative glucose uptake of lactic acid bacteria fermentation products and non-fermented peony extracts of peony extracts into muscle cells (A) and (B) in Leuconostock ( Leuconostoc sp.) LN180020 (KCTC 13719BP) is a result of treating the lactic acid bacteria fermentation product of the peony extract, (C) and (D) Lactobacillus plantarum subspecies plantarum ( Lactobacillus plantarum subsp. plantarum ; Df2) ) LN180155 or Lactobacillus paraplantarum KACC 12373; Df3). INS; Insulin (porcine insulin).
FIG. 3 is a schematic diagram of the analysis of the system of Leuconostoc sp. LN180020 (KCTC 13719BP).
Figure 4 is a non-fermented peony extract (non-fermented) and peony extract fermentation (fermented) of the peony extract, respectively, before and after bioconversion peony indicator material (paoniflorin, paeoniflorin) and bioconversion after separation and purification of LC-MS This is the result.

Hereinafter, the present invention will be described in detail by examples.

However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

<Example 1>

Separation of vegetable lactic acid bacteria from kimchi

In order to separate vegetable lactic acid bacteria derived from kimchi, various samples of kimchi are diluted stepwise with sterile water, plated on MRS (Man Rogosa and Sharpe) medium, incubated at 28°C for 2 days, and then lactic acid bacteria are separated through morphological features. The isolated strains were identified through 16s rRNA sequence analysis and stored at -70°C using 20% glycerol.

Strain Identification result, 193 of the Lactobacillus genus (Lactobacillus), Petit O Rhodococcus genus (Pediococcus), Wei Cellar in (Weissella), flow Pocono stock in (Leuconostoc) and 39 kinds (species) contained in the Enterococcus genus (Enterococcus) Dog strains were isolated.

<Example 2>

Preparation of lactic acid bacteria fermentation product of peony extract

After crushing the dried peony root and pulverizing it, 10 times of water was mixed with powder to extract hot water at 121°C for 15 minutes, and the hot water extract was treated with sodium carbonate (Na ₂ CO ₃ ) to pH 3.6-6.8. I matched it. Lactic acid bacteria were incubated at 28° C. for 48 hours, and then inoculated into peony hot water extract at a concentration of 6×10 ⁹ CFU/ml, followed by static fermentation at 28° C. for 48 hours to obtain a supernatant (lactic acid fermentation product of peony extract). Thereafter, the obtained peony extract and the lactic acid bacteria fermentation product of the peony extract were concentrated under reduced pressure and powdered.

<Example 3>

Identification of Lactic Acid Bacteria Strains with Paoniflorin Metabolism

The inventors of the peony extract or peoniflorin (paeoniflorin), which is one of the indicator components of the peony, are known to have diabetes-related activity, but it was determined that the therapeutic activity for diabetes is not sufficient. I wanted to search.

To search for these strains, 10 times (w/v) water was added to 1 g of peony dry weight to extract peony extracts at 121 degrees for 15 minutes to prepare 2 x 10 ⁹ CFU of each strain isolated in Example 1. Inoculated at /ml concentration and allowed to stand for 48 hours in a 28-degree culture room. Then, the culture (supernatant) was extracted with 50% ethanol and analyzed for chromatogram changes under UV 235nm, 15-100% methanol gradient conditions through HPLC using a TSK-GEL ODS-100V 5㎛ column (4.6mm×15cm). (See Figure 1).

As a result, among the isolated strains, it was confirmed that the peak of LN180020 in Leukonostock specifically decreases the peak around 15 minutes, which is expected to be a phonyflorin, and the peak of a new material increases at 14.2 minutes (see FIG. 1).

<Example 4>

Peony extract in the genus Leuconostock ( Leuconostoc sp.) LN180020 Fermentation cytotoxicity and anti-diabetic effect

<4-1> Confirmation of cytotoxicity

To confirm the cytotoxicity of the lactic acid bacteria fermentation product of the peony extract, the lactic acid bacteria fermentation product of the peony extract in mouse L6 myotube was treated by concentration (3 and 30 µg/ml) and MTT assay was performed after 24 hours. .

As a result, it was confirmed that there was no significant effect on cell proliferation in both the untreated control group, the non-fermented peony extract treated group and the lactic acid bacteria fermented product treated group of peony extract in mouse L6 root canal cells, through which the peony extract was introduced into the leuconostock ( Leuconostoc sp.) LN180020 (KCTC 13719BP) was found to be non-cytotoxic (Fig. 2A).

<4-2> Confirm anti-diabetic effect

To confirm the anti-diabetic effect of lactic acid bacteria fermentation of peony extract, dispensing mouse L6 myoblasts into 5×10 ⁵ cells/mL in a 24-well culture container, followed by differentiation once every 2 days (low glucose (5mM) DMEM, 2% Fetal Bovine Serum, 1% penicillin/streptomyc) and cultured. On the 7th day after cultivation, the lactic acid bacteria fermentation product of the peony extract was pretreated by concentration (3 and 30 µg/ml) and reacted for 30 minutes, followed by [ ¹⁴ C]2-Deoxy-D-glucose treatment for 5 minutes. After the reaction was over, glucose uptake was measured by measuring [ ¹⁴ C]2-deoxy-D-glucose-6-phosphate, which was absorbed into mouse L6 progenitor cells and converted into a form. As a positive control, 100 nM concentration of insulin (porcine insulin) was used.

As a result, in the group treated with the fermented lactic acid bacteria of the peony extract, the content of [ ¹⁴ C]2-deoxy-D-glucose-6-phosphate in L6 myoblasts was increased by about 55% to 80% compared to the untreated control. It was confirmed that the fermentation peony extract treatment group increased by about 25-35%. This indicates that when the peony extract is fermented using lactic acid bacteria, the anti-diabetic activity can be increased by promoting glucose absorption into muscle cells compared to the non-fermented peony extract. Moreover, 100 nM of insulin (porcine insulin) used as a positive control group has an anti-diabetic effect almost equal to 10 μM (3.57 μg/ml) of rosiglitazone, which is actually used as a therapeutic agent for diabetes. The content of [ ¹⁴ C]2-deoxy-D-glucose-6-phosphate in the group treated with the lactic acid bacteria fermentation product of the peony extract of ㎖) was similar to that of the insulin 100 nM treatment group, and the peony extract of high concentration (30 µg/ml) The content of [ ¹⁴ C]2-deoxy-D-glucose-6-phosphate in the group treated with the lactic acid bacteria fermentation product was significantly increased compared to the insulin 100 nM treatment group (FIG. 2B).

<Comparative Example 1>

Confirmation of cytotoxicity and anti-diabetic effect of other lactic acid bacteria fermentation of peony extract

<1-1> Cytotoxicity confirmation

Cytotoxicity of Lactobacillus plantarum subspecies plantarum ( Lactobacillus plantarum subsp. plantarum ) LN180155 (hereinafter Df2) or Lactobacillus paraplantarum KACC 12373 (hereinafter Df3) fermented lactic acid bacteria fermentation of peony extract To confirm, the cytotoxicity was confirmed in the same manner as in Example 4-1, except that Df2 and Df3 were treated with 3, 10, 30, and 100 μg/ml, respectively.

As a result, it was confirmed that Lactobacillus plantarum subsp. did not have a significant effect on cell proliferation in both the untreated control group and the lactic acid bacteria fermentation product group of peony extract in mouse L6 root canal cells. It was found that the lactic acid bacteria fermentation product of the peony extract fermented with plantarum (Df2) or Lactobacillus paraplantarum (Df3) had no cytotoxicity (FIG. 2C ).

<1-2> Anti-diabetic effect confirmation

In order to confirm the anti-diabetic effect of the fermented lactic acid bacteria of the peony extract, the glucose absorption rate was the same as in Example 4-2, except that the Df2 and Df3 were treated with 3, 10, 30, and 100 μg/ml, respectively. It was measured.

As a result, the group treated with 100 nM of insulin (porcine insulin) used as a positive control group significantly increased compared to the untreated control group, but the Lactobacillus plantarum subsp. [ ¹⁴ C]2-deoxy-D-glucose-6-phosphate in L6 progenitor cells in the group treated with lactic acid bacteria fermentation of peony extract fermented with Plantarum LN180155 (Df2) or Lactobacillus paraplantarum (Df3). It was confirmed that the content was almost the same as the untreated control. However, [ ¹⁴ C]2-deoxy-D-glucose-6-phosphate in L6 progenitor cells in the group treated with lactic acid bacteria fermentation of peony extract fermented with Lactobacillus paraplantarum (Df3) at a concentration of 100 μg/ml. The content was slightly increased compared to the untreated control group, but there was no significant difference (FIG. 2D).

As a result, when the peony extract is fermented using the Leuconostoc sp. LN180020 (KCTC 13719BP) lactic acid bacteria of the present invention, as a muscle cell compared to the lactic acid bacteria fermentation product of the non-fermented peony extract and peony extract fermented with other lactic acid bacteria It has been shown that it can increase the anti-diabetic activity by promoting the absorption of glucose. This effect is lost sludge Florin this Chapter Pocono stock in (Leuconostoc sp.) LN180020 (KCTC 13719BP) is switched to the new materials by the lactic acid appears to be the the anti-diabetic activity it increased and therefore, of the present invention peony extract, lactic acid bacteria fermented product is diabetes It was thought to be effective in the prevention or improvement of.

<Example 5>

In Ryukono Stock ( Leuconostoc sp.) LN180020 Systematic analysis of lactic acid bacteria

Accordingly, the Leuconostoc sp. LN180020 (KCTC 13719BP) of the genus Leuconostoc of the present invention was additionally identified, and a new substance was shown to increase anti-diabetic activity.

For the systematic analysis of Leuconostoc sp. LN180020 (KCTC 13719BP), genomic DNA of LN180020 in Leuconostoc was extracted. The 16S rRNA gene was amplified using a primer set (Table 1), and after confirming the base sequence (SEQ ID NO: 1) of the 16S rRNA, it was compared with a previously known sequence of Leukonostock.

Primer sequence information Primer name Base sequence (5'→3') (SEQ ID NO) 27F-1492R_F AGAGTTTGATCCTGGCTCAG (2) 27F-1492R_R CGGTTACCTTGTTACGACTT (3)

The nucleotide sequence of the 16S rRNA of LN180020 in Leuconostock was confirmed through BigDye (R) Terminator v3.1 Cycle Sequencing Kits (Applied Biosystems), and the previously known Leuconostock sequence is NCBI database (https://www. Refer to ncbi.nlm.nih.gov/), EzTaxon-e server (https://www.eztaxon-e.ezcloud.net/) and LPSN (https://www.bacterio.net/nocardioides.html) databases The sequence comparison analysis was performed using BioEdit v7.2.6.1, MEGA7 program.

As a result, the genus LN180020 showed the highest 16S rRNA sequence homology to 99.86% with L. citreum ATCC 49370T, followed by L. holzapfelii BFE 7000 ^T and 99.78%; L. lactis JCM 6123 ^T and L. palmae TMW 2.694 ^T and 98.92%; L. kimchii IMSNU 11154 ^T and 98.49%; Leukono Stock Gelidum subsp. L. gelidum subsp. gasicomitatum LMG 18811 ^T and 98.14%; Leukonostock Mesenteroides subsp. L. mesenteroides subsp. mesenteroides ATCC 8293 ^T and 97.85%; L. pseudomesenteroides NRIC 1777 ^T and 97.71%; And L. rapi LMG 27676 ^T and 97.63% of 16s rRNA sequence homology. Through this, Kimchi-derived Leukonostock genus LN180020 is the closest phylogenetically to Leukonostock Citreum ATCC 49370T. Was found (Fig. 3).

<Example 6>

Analysis of bioconversion components of peony extract lactic acid bacteria fermentation

After extracting by adding the same amount of ethanol to the obtained supernatant, TSK-GEL ODS-100V 5 μm column (4.6 mm×15 cm), 25° C., UV 230 nm, 15~100% methanol gradient conditions under HPLC (High-performance) liquid chromatography) to analyze the presence or absence of bioconversion. The LC-MS (Liquid chromatography mass spectrometry) analysis for the identification of the bioconverted material dissolved the supernatant in methanol and the HPLC system connected with the Turbolon Spray source (AB SCIEX, Singapore) and QTrap 3200 (Luna C18 (2), 100 X 2.0 mm, 3 μm; guard cartridge system, security guard #KJ 0-4282; Phenomenex, USA). The column was maintained at 25°C with a flow rate of 1.0 ml/min of HPLC water (B) containing 100% methanol (A) and 0.04% trifluoroacetic acid, and the sample was 15% for the first 5 minutes. After flowing methanol, the concentration of methanol was sequentially increased from 15% to 100% from 5 to 20 minutes, maintained at 100% methanol for 20 to 25 minutes, and from 100% to 15% from 25 to 35 minutes. The methanol concentration was reduced until then, and it was measured while maintaining 15% methanol for 5 minutes (Table 2).

HPLC conditions HPLC retention time (min) A; MeOH(%) B; Water(%) 0-5 15 85 5-20 15-100 85-0 20-25 100 0 25-35 100-15 0-85 35-40 15 85

As a result of performing HPLC to analyze the components converted by LN180020 in Leuconostoc, the indicator material was reduced near the retention time of the peony extract fermentation for about 15 minutes (FIG. 1), corresponding to the new useful component at about 14.2 minutes. It was confirmed that the peak to be increased was increased. In addition, in order to confirm the reduced indicators and new useful components in the above, each of the corresponding peaks was developed using a thin layer chromatography method using silica gel 60G F254 glass plate in methanol:chloroform (1.5:5). As a result of separation and purification to perform LC-MS analysis, it was found that the indicator material in the vicinity of about 15 minutes was a paeoniflorin with a molecular weight of 480.3, and the new useful component material had a molecular weight of 642.3 after conversion at about 14.2 minutes. It was confirmed (Fig. 4).

<Formulation Example 1>

Preparation of pharmaceutical preparations

1. Preparation of powder

Ferment 2 g of peony extract according to the present invention

1 g of lactose

A powder was prepared by mixing the above components and filling the gas-tight fabric.

2. Preparation of tablets

Fermentation product 100 mg of peony extract according to the present invention

Corn starch 100 mg

Lactose 100mg

Magnesium stearate 2mg

After mixing the above ingredients, tablets were prepared by tableting according to a conventional tablet manufacturing method.

3. Preparation of capsules

Fermentation product 100 mg of peony extract according to the present invention

Corn starch 100 mg

Lactose 100mg

Magnesium stearate 2mg

After mixing the above components, the capsules were prepared by filling the gelatin capsules according to a conventional capsule preparation method.

4. Preparation of pills

Fermentation product of peony extract according to the invention 1 g

1.5 g lactose

Glycerin 1 g

Xylitol 0.5 g

After mixing the above components, it was prepared to be 4 g per ring according to a conventional method.

5. Preparation of granules

Fermentation product 150 mg of peony extract according to the present invention

Soybean extract 50 mg

Glucose 200 mg

Starch 600 mg

After mixing the above components, 100 mg of 30% ethanol was added, dried at 60° C. to form granules, and then filled into the fabric.

As described above, when fermenting the peony extract using LN180020 (KCTC 13719BP) lactic acid bacteria in the genus Leuconostock of the present invention, glucose absorption into muscle cells is compared to the lactic acid bacteria fermentation products of the non-fermented peony extract and peony extract fermented with other lactic acid bacteria. It was found that it can promote anti-diabetic activity. Since the lactic acid bacteria fermentation product of the peony extract of the present invention has no side effects on the human body using peony and kimchi derived from natural materials, it can be usefully used in health functional foods and medicines for preventing, improving or treating diabetes.

Korea Research Institute of Bioscience and Biotechnology KCTC13719BP 20181120

<110> Korea Research Institute of Bioscience and Biotechnology <120> Composition for preventing, improving or treating diabetes comprising Paeonia lactiflora extract fermented by lactic acid bacteria as effective ingredients <130> NP19-0121P <150> KR 1020180155698 <151> 2018-12-06 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 1474 <212> RNA <213> Artificial Sequence <220> <223> Leuconostoc sp. LN180020 <400> 1 ucaggaugaa cgcuggcggc gugccuaaua caugcaaguc gaacgcgcag cgagaggugc 60 uugcaccuuu caagcgagug gcgaacgggu gaguaacacg uggauaaccu gccucaaggc 120 uggggauaac auuuggaaac agaugcuaau accgaauaaa acuuaguauc gcaugauauc 180 aaguuaaaag gcgcuacggc gucaccuaga gauggauccg cggugcauua guuaguuggu 240 gggguaaagg cuuaccaaga cgaugaugca uagccgaguu gagagacuga ucggccacau 300 ugggacugag acacggccca aacuccuacg ggaggcugca guagggaauc uuccacaaug 360 ggcgcaagcc ugauggagca acgccgcgug ugugaugaag gcuuucgggu cguaaagcac 420 uguuguaugg gaagaaaugc uaaaauaggg aaugauuuua guuugacggu accauaccag 480 aaagggacgg cuaaauacgu gccagcagcc gcgguaauac guaugucccg agcguuaucc 540 ggauuuauug ggcguaaagc gagcgcagac gguugauuaa gucugaugug aaagcccgga 600 gcucaacucc ggaauggcau uggaaacugg uuaacuugag uguuguagag guaaguggaa 660 cuccaugugu agcgguggaa ugcguagaua uauggaagaa caccaguggc gaaggcggcu 720 uacuggacaa caacugacgu ugaggcucga aagugugggu agcaaacagg auuagauacc 780 cugguagucc acaccguaaa cgaugaauac uagguguuag gagguuuccg ccucuuagug 840 ccgaagcuaa cgcauuaagu auuccgccug gggaguacga ccgcaagguu gaaacucaaa 900 ggaauugacg gggacccgca caagcggugg agcauguggu uuaauucgaa gcaacgcgaa 960 gaaccuuacc aggucuugac auccuuugaa gcuuuuagag auagaagugu ucucuucgga 1020 gacaaaguga cagguggugc auggucgucg ucagcucgug ucgugagaug uuggguuaag 1080 ucccgcaacg agcgcaaccc uuauuguuag uugccagcau ucaguugggc acucuagcga 1140 gacugccggu gacaaaccgg aggaaggcgg ggacgacguc agaucaucau gccccuuaug 1200 accugggcua cacacgugcu acaauggcgu auacaacgag uugccaaccu gcgaagguga 1260 gcuaaucucu uaaaguacgu cucaguucgg acugcagucu gcaacucgac ugcacgaagu 1320 cggaaucgcu aguaaucgcg gaucagcacg ccgcggugaa uacguucccg ggucuuguac 1380 acaccgcccg ucacaccaug ggaguuugua augcccaaag ccgguggccu aaccuucggg 1440 agggagccgu cuaagcagga cagaugacug gggu 1474 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 27F-1492R_F <400> 2 agagtttgat cctggctcag 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 27F-1492R_R <400> 3 cggttacctt gttacgactt 20

Claims (9)

A pharmaceutical composition for the prevention or treatment of diabetes, containing a fermented product of lactic acid bacteria of Paeonia lactiflora extract as an active ingredient.
The pharmaceutical composition for preventing or treating diabetes according to claim 1, wherein the lactic acid bacteria are Leuconostoc citreum .
The pharmaceutical composition for preventing or treating diabetes according to claim 1, wherein the lactic acid bacteria are Leuconostoc sp. LN180020 having accession number KCTC 13719BP.
The pharmaceutical composition for preventing or treating diabetes according to claim 1, wherein the diabetes is selected from the group consisting of type 1 diabetes, type 2 diabetes, and gestational diabetes.
The method of claim 1, wherein the lactic acid bacteria fermentation product is a pharmaceutical composition for the prevention or treatment of diabetes, characterized in that to increase glucose absorption into muscle cells.
The pharmaceutical composition for preventing or treating diabetes according to claim 1, wherein the peony extract is extracted using water, a lower alcohol of C1 to C6, an organic solvent, or a mixed solvent thereof.
Food composition for preventing or improving diabetes, which contains the fermentation product of lactic acid bacteria of peony extract as an active ingredient.
(a) inoculating a composition comprising paoniflorin with Leuconostoc sp.
(B) a method for converting paoniflorin comprising the step of culturing and fermenting the product of step (a).
10. The method of claim 8, wherein the conversion of the paoniflorin is a reduction of the paoniflorine.

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