JP5421348B2 - Pharmaceutical composition for prevention and treatment of diabetes containing as an active ingredient an extract of chrysanthemum fermented with Lactobacillus microorganisms - Google Patents

Description

  The present invention relates to a composition for the prevention and treatment of diabetes containing an extract of fermented chrysanthemum using Lactobacillus microorganisms. More specifically, the present invention relates to a composition having a preventive, ameliorating and / or therapeutic effect for diabetes, which contains a fermented chrysanthemum mash fermented with Lactobacillus sp.

  Due to the recent rapid improvement in living standards, the state of illness is gradually becoming Western Europe due to the excessive intake of high-calorie food and drink, lack of exercise and the sophistication of industrial society. The incidence of adult diseases is increasing, and typically includes hypertension, diabetes, obesity, hyperlipidemia, and the like. In particular, diabetes is the cause of all chronic diseases.

  Diabetes mellitus is a disease in which insulin secreted from the beta cells of the pancreas is insufficient or cannot fully function, and glucose cannot be used as an energy source in the body, and it remains in the blood and is excreted by urine. . However, such a phenomenon is only an acute symptom that appears in the early stage of diabetes, and when it becomes chronic, it causes blood circulation disorder, chronic fatigue, hypertension, myocardial infarction, diabetic neuropathy, retinopathy (visual impairment, blindness). Retinal hemorrhage), cataracts and other complications, leading to decreased metabolic and sensory functions throughout the human body, resulting in fatal consequences. Currently used therapeutic agents for diabetes are roughly classified into hypoglycemic agents for oral administration and insulin. In general, dietary therapy and exercise therapy are also used for insulin-dependent diabetics who do not have insulin secretion in the body, gestational diabetics, and non-insulin-dependent diabetics whose blood sugar is not regulated by oral hypoglycemic agents. Regardless, proper blood glucose control could not be achieved. Therefore, it is common for non-insulin dependent diabetics to take hypoglycemic agents.

  Jerusalem artichoke is native to North America and is a perennial plant of Chrysanthemum family that grows without fertilizer because it is a wild plant. It grows well in disease conditions that are strong against diseases and pests. It is known as a bulbous crop that is beneficial to the human body as it has been used as an important medicine for Kampo since ancient times, it contains a large amount of inulin components and does not decompose in the body, so it does not increase blood sugar after ingestion, conversely It is known to promote insulin secretion by lowering and strengthening the pancreas. Natural plant materials like chrysanthemums contain various physiologically active substances and antioxidants, and show various effects such as anti-diabetes, anti-aging, anti-cancer, anti-flame, and improvement of immune function. Sometimes it is unstable and sometimes irritating, so it can be toxic to the human body. Therefore, recently, attempts have been made to stabilize natural extracts, reduce toxicity, or convert them to stable derivatives to enhance the effect. As part of this, biological conversion methods using microorganisms and enzymes have been developed, and a typical example is fermentation. Fermentation is a process in which a substance that can be usefully used in human life is produced by degrading organic matter using enzymes possessed by microorganisms. Typical fermenting microorganisms that produce substances useful for the human body include lactic acid bacteria, yeast, and the like, and a variety of foods are produced by the fermentation process. Attempts to maximize the effectiveness of natural plant materials by microbial fermentation such as lactic acid bacteria and yeast or to solve safety problems have important implications in the development of various foods, medicines and the like.

  The fact that chrysanthemums are effective for diabetes is already known, but the therapeutic effect of chrysanthemums fermented with lactic acid bacteria has not yet been reported.

  While the present inventors are researching new compositions for preventing, improving and / or treating diabetes, when the chrysanthemums are fermented using Lactobacillus microorganisms, the inhibitory activity of alpha glucosidase is further increased. The present invention was completed by confirming that a fermented product with excellent preventive and therapeutic effects on diabetes was produced.

An object of the present invention is to provide a composition for the prevention and treatment of diabetes comprising, as an active ingredient, a chrysanthemum fermented extract fermented with Lactobacillus microorganisms.
Another object of the present invention is to provide a health food composition for preventing and ameliorating diabetes comprising an extract of chrysanthemum fermented with Lactobacillus microorganisms as an active ingredient.

In order to achieve the above-mentioned object, the present invention provides a pharmaceutical composition for the prevention and treatment of diabetes comprising, as an active ingredient, a chrysanthemum fermented extract fermented with a Lactobacillus microorganism.
In order to achieve another object of the present invention, the present invention provides a functional food composition for prevention and improvement of diabetes comprising, as an active ingredient, a chrysanthemum fermented extract fermented with a Lactobacillus microorganism.

Hereinafter, the present invention will be described in detail.
The present invention relates to a pharmaceutical composition for the prevention and treatment of diabetes comprising, as an active ingredient, a chrysanthemum fermented extract fermented with a Lactobacillus microorganism.

  Chrysanthemum salmon is evaluated as a natural insulin food, and chrysanthemum salmon contains 15-20% inulin component, but it does not contain starch and cannot be digested by human digestive enzymes and is not absorbed into the blood. It is discharged as it is. In addition, foods taken together act to delay carbohydrate absorption in the small intestine, thereby suppressing an increase in blood sugar.

  Fermentation is the process of degrading organic matter using enzymes contained in microorganisms. In the case of Chinese medicines such as carrots with medicinal ingredients, fermentation is known to increase the absorption efficiency in the body after fermentation. ing. In the present invention, the microorganism of the genus Lactobacillus is not limited thereto, but Lactobacillus rhamnosus, Lactobacillus acidophlus, Lactobacillus plantarum, Lactobacillus plantarum, Lactobacillus bulgaricus), Lactobacillus casei, Lactobacillus lactis, Lactococcus confusus, Lactobacillus reutri, Lactobacillus reutri, Lactobacillus brevis, Lactobacillus brevis It is also Lactobacillus delbruekii, Lactobacillus johnsonii, Lactobacillus rhamnosus, Lactobacillus sakei or Lactobacillus buchneri. Desirably, it is also Lactobacillus acidophlus or Lactobacillus plantarum.

  In one embodiment of the present invention, the change of the active ingredient is caused by fermentation, and in order to confirm whether or not the effect of the medicinal component is improved, the unfermented chrysanthemums and chrysanthemums As a result of measuring the inhibitory effect of alpha glucosidase, which is a fermented chrysanthemum fermented with Lactobacillus plantaroom, Lactobacillus acidophilus, and Bacillus subtilis, fermented chrysanthemum fermented with Lactobacillus microorganisms It has been confirmed that the effect is far superior to that of fermented chrysanthemum or non-chrysanthemum fermented with other microorganisms.

  In another embodiment of the present invention, the change of the active ingredient is caused by fermentation, and in order to confirm whether the effect of the medicinal component is improved, the fermented components of the fermented chrysanthemum and the fermented chrysanthemum are compared. As a result of conducting an experiment comparing thin film chromatography and fructan content, in the case of chrysanthemum lactobacillus plantaroom fermented product, the high fluidity component was increased. It was found that some components changed during fermentation.

  In order to confirm the therapeutic effect of diabetes in the animal model of the Lactobacillus plantaroom fermentation extract of chrysanthemums of the present invention, in one embodiment of the present invention, the rat induced chrysanthemum lactobacilli plantaroom fermentation of the present invention was used. A diet containing the extract was administered, and blood glucose, body weight change, postprandial blood glucose change, small intestinal glucosidase activity, blood analysis, and the like were performed. From the result of the experiment, it was confirmed that the fermented extract of chrysanthemum lactobacillus plantaroom has a therapeutic effect for diabetes.

The present invention provides a pharmaceutical composition for the prevention and treatment of diabetes comprising as an active ingredient a chrysanthemum fermented extract fermented with a Lactobacillus microorganism.
The fermented chrysanthemum fermented with the chrysanthemum Lactobacillus microorganism of the present invention can be used as such or in the form of a pharmaceutically acceptable salt. The term “pharmaceutically acceptable” means that it is physiologically acceptable and does not normally cause an allergic reaction or a similar reaction when administered to a human, and the salt is pharmaceutically acceptable. Acid addition salts formed with free acids are preferred. The free acid may be an organic acid or an inorganic acid. The organic acid is not limited thereto, but citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, metasulfonic acid, glycolic acid, Contains succinic acid, 4-toluenesulfonic acid, glutamic acid and aspartic acid. The inorganic acid includes, but is not limited to, hydrochloric acid, bromic acid, sulfuric acid and phosphoric acid.

  In the case of the pharmaceutical composition, the fermented chrysanthemum fermented with the microorganism of the genus Lactobacillus alone is included, or one or more pharmaceutically acceptable carriers, excipients or diluents are added. Can be included. The term “pharmaceutically acceptable” refers to a composition that does not normally cause an allergic reaction or a similar reaction when administered to a human being biologically acceptable.

  The pharmaceutically acceptable carrier can additionally contain, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration can include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. In addition, carriers for parenteral administration can contain water, suitable oils, saline, aqueous glucose, glycols, and the like, and can contain additional stabilizers. Suitable stabilizers include sulfating agents such as sodium bisulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl or propylparaben and chlorobutanol. For other pharmaceutically acceptable carriers, the description in the following literature can be referred to (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

  The pharmaceutical composition for preventing or treating diabetes of the present invention can be administered to mammals including humans by any method. For example, it can be administered orally or parenterally. Parenteral administration methods include but are not limited to intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual or It can also be administered rectally. The pharmaceutical composition of the present invention can be administered transdermally. The aforementioned “transdermal administration” means that the active ingredient contained in the pharmaceutical composition for preventing or treating diabetes is transmitted into the skin by administering the pharmaceutical composition of the present invention to cells or skin. To do. For example, the pharmaceutical composition of the present invention can be prepared into an injection-type preparation and administered by a method of pricking the skin with a thin 30-gauge needle, or a method of directly applying to the skin.

The pharmaceutical composition can be formulated into a preparation for oral administration or parenteral administration depending on the administration route as described above.
In the case of preparations for oral administration, the composition of the present invention is widely known in the art as powders, granules, tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. The dosage form can be formed using the prepared method. For example, for oral preparations, tablets or dragees can be obtained by blending the active ingredient with a solid excipient and crushing it, then adding suitable adjuvants and processing into granule mixtures. Examples of suitable excipients include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, and starches including corn starch, wheat starch, rice starch and potato starch, Fillers such as cellulose, cellulose including methylcellulose, sodium carboxymethylcellulose and hydroxypropylmethylcellulose, gelatin, polyvinylpyrrolidone and the like can be included. In some cases, cross-linked polyvinyl pyrrolidone, agar, alginic acid or sodium alginate can be added as a disintegrant. The pharmaceutical composition of the present invention can contain anti-aggregating agents, lubricants, wetting agents, perfumes, emulsifiers and the like.

In the case of preparations for parenteral administration, the preparations are in the form of injections, creams, lotions, ointments for external use, oils, moisturizers, gels, aerosols, and nasal inhalants by methods well known in the art. Can be These formulations are generally known in the formulary to all pharmaceutical chemistry literature ^{(Remington's Pharmaceutical Science, 15 th} Edition, 1975 Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87:. Blaug, Seymour) are described in .

  The total effective amount of the fermented chrysanthemum Lactobacillus splatterum of the present invention can be administered to a patient at a single dose, and can be administered at multiple doses for a long period of time in a divided treatment method ( Can be administered by fractionated treatment protocol). The pharmaceutical composition of the present invention can have different contents of active ingredients depending on the degree of the disease. The total dose of the fermented chrysanthemum fermented with the Lactobacillus microorganism of the present invention is about 0.01 to 1,000 mg, preferably 0.1 to 100 mg per kg of patient body weight per day. The dose of fermented Kikuto Lactobacillus splatterum is not only the route of administration of the pharmaceutical composition and the number of treatments, but also various factors such as patient age, body weight, health status, sex, disease severity, diet and excretion rate. The effective dosage for a patient is determined in consideration of the above, so when considering such points, a person with ordinary knowledge in the field can use a fermented chrysanthemum fermented with the aforementioned Lactobacillus microorganism for diabetes. An appropriate effective dose associated with a particular use as a prophylactic or therapeutic agent can be determined. As long as the pharmaceutical composition according to the present invention exhibits the effects of the present invention, its dosage form, administration route and administration method are not limited.

  The pharmaceutical composition may additionally contain anti-aggregating agents, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like.

In the case of a preparation for parenteral administration, it can be formulated in the form of an injection by a method widely known in the art. These dosage forms literature generally known formulary to all pharmaceutical chemistry ^{(Remington's Pharmaceutical Science, 15 th} Edition, 1975 Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87:. Blaug, Seymour) is described in Yes.

  The present invention relates to a health food composition for the prevention and improvement of diabetes containing a chrysanthemum fermented extract fermented with a Lactobacillus microorganism as an active ingredient.

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

  The type of food composition can be manufactured in various forms by a general method widely known in the art. Although not limited thereto, for example, for health foods, fermented chrysanthemums fermented with Lactobacillus microorganisms can be liquefied, granulated, encapsulated and powdered so that they can be produced in the form of tea, juice and drink Can be taken. Further, it can be mixed with a fermented chrysanthemum fermented with Lactobacillus microorganisms and a known active ingredient known to be effective for diabetes to produce a composition. Functional foods include but are not limited to beverages (including alcoholic beverages), fruits and processed foods (eg, canned fruits, bottling, jams, malamates, etc.), fish, meat and processed foods (eg, processed foods) : Ham, sausage, corn beef, etc.), bread and noodles (eg, udon, soba, ramen, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, strawberries, dairy products (eg, barter, cheese, etc.), edible It can be produced by adding fermented chrysanthemum fermented with Lactobacillus microorganisms to vegetable fats and oils, margarine, vegetable protein, retort food, frozen food, various seasonings (eg, miso, soy sauce, sauce, etc.). Furthermore, in order to use the fermented chrysanthemum fermented with Lactobacillus microorganisms in the form of a food additive, it can be used in the form of powder or concentrated liquid.

  The content of the fermented chrysanthemum fermented with Lactobacillus microorganisms in the food composition of the present invention is not limited to this, but it is preferably 0.1 to 90% by weight in the finally produced food. More desirably, the food composition containing the fermented chrysanthemum fermented with the Lactobacillus genus microorganism of the present invention as an active ingredient is mixed with an active ingredient known to be particularly effective for diabetes to produce a health food form. be able to.

  The fermented chrysanthemums fermented with the Lactobacillus microorganism of the present invention is produced by adding distilled water and Lactobacillus microorganisms to chrysanthemum powder and fermenting at 20 ° C. to 40 ° C. for 1 to 60 hours. Although not limited thereto, the fermentation extraction is a step of adding distilled water and a Lactobacillus genus microorganism, specifically a Lactobacillus plantaroom, to fermented potato powder and fermenting at 35 ° C. to 40 ° C. for 12 to 60 hours; Sterilizing the dried chrysanthemum at 100 to 150 ° C. for 10 to 20 minutes and freeze-drying; adding water to the sterilized fermented chrysanthemum, extracting with a sonicator and centrifuging; and supernatant after the centrifugation It is manufactured by a process including a step of taking and drying.

  In the present invention, among the fermented lactic acid bacteria of chrysanthemums, fermented products were produced using Lactobacillus microorganisms, particularly Lactobacillus plantarooms or Lactobacillus acidophilus, which are the strains that showed the highest alpha glucosidase activity. . Preferably, the Lactobacils planter room utilized KCTC3103, more preferably the Lactobacils planter room.

  Lactobacillus plantaroom is a gonococcus and is a kind of lactic acid bacteria that are Gram-positive bacteria. It is a strain that is widely used for fermenting foods such as milk, cheese, barter, cereals and bread, and plays an especially important role in the fermentation of kimchi.

  The chrysanthemum fermented extract fermented with the Lactobacillus microorganism is extracted using a sonicator. Kikumochi is fermented with Lactobacillus microorganisms, the fermented product is freeze-dried, distilled water is added to the dried fermented product, and the mixture is extracted with a sonicator. The extraction is not limited to this, but it is desirable to extract at 40 to 50 ° C. for 30 to 90 minutes. In one embodiment of the present invention, the extraction was performed at 45 ° C. for 1 hour.

  The extract is separated, and only the water-soluble component of the fermented chrysanthemum koji is taken and lyophilized. Including, but not limited to, a process of centrifuging to obtain only water-soluble components, the centrifugation is preferably performed at 2 ° C. to 5 ° C. for 10 to 20 minutes. In one embodiment of the present invention, separation was performed at 4 ° C. for 15 minutes. The freeze-drying process is performed by a general freeze-drying process, and can be performed by the same method as the freeze-drying of the Lactobacillus microorganism fermentation product of chrysanthemum.

  As described above, the present invention relates to a composition having an effect of preventing, improving and / or treating diabetes, comprising a fermented chrysanthemum fermentation fermented with Lactobacillus microorganisms. The composition of the present invention has an antidiabetic effect superior to that of the unfermented chrysanthemum extract, and can be usefully used for the purpose of providing a composition for preventing, improving and / or treating new diabetes.

FIG. 1 is a diagram showing the results of thin film chromatography of a chrysanthemum extract before fermentation and an extract of chrysanthemum extract fermented with lactobacillus plantaroom. Fig. 2 shows the results of measurement of fasting blood glucose levels in the normal group (Lean), diabetes induction group (C), acarbose administration group (AB), and chrysanthemum lactobacillus plantaroom fermentation extract administration group (LJA1, LJA2) Each treatment group is as follows. Lean: Untreated group, C: Diabetes group, AB: Diet containing diabetes + acarbose 0.5g / kg, LJA1: Diet containing diabetes + LJA 1.5g / kg, LJA2: Diet group containing diabetes + LJP 3g / kg. Fig. 3 shows changes in body weight for 7 weeks in the normal group (Lean), the diabetes-inducing group (C), the acarbose administration group (AB), and the chrysanthemum lactobacillus plantaroom fermentation extract administration group (LJA1, LJA2). As shown, each treatment group is as follows. Lean: Untreated group, C: Diabetes group, AB: Diet containing diabetes + acarbose 0.5g / kg, LJA1: Diet containing diabetes + LJA 1.5g / kg, LJA2: Diet group containing diabetes + LJP 3g / kg. Figure 4 shows the postprandial blood glucose levels of the normal group (Lean), the diabetes induction group (C), the acarbose administration group (AB), and the chrysanthemum lactobacillus plantaroom fermented extract administration group (LJA1, LJA2) 30 minutes after meal The results of measurement at 60 minutes, 90 minutes and 120 minutes are shown, and each treatment group is as follows. Lean: Untreated group, C: Diabetes group, AB: Diet containing diabetes + acarbose 0.5g / kg, LJA1: Diet containing diabetes + LJA 1.5g / kg, LJA2: Diet group containing diabetes + LJP 3g / kg. FIG. 5 shows a comparison of the area under the curve (AUC) for each treatment group in FIG. FIG. 6 shows the results of measuring the glycosidase (maltase, lactase, sucrase) activity of the Proximal, middle, and distal parts of the small intestine.

Hereinafter, the present invention will be described in detail with reference to examples.
However, the following examples only illustrate the present invention, and the contents of the present invention are not limited to the following examples.

<Example 1>
Manufacture of fermented chrysanthemums (1) Manufacture of fermented products by type of lactic acid bacteria The experimental material is 10-20 g of purple chrysanthemum koji powder, or 90-180 mL of distilled water or 150 L is added to 15 kg, and Aspergillus oryzae, basil Slicenifomis (Bacillus licheniformis), Lactobacillus plantarum (Lactobacillus Plantarum, KCTC3130) and Bacillus subtilis (Bacillus subtillis, KCTC1666) were added at 2 to 3% and fermented for 12 to 60 hours. After fermentation, it was sterilized at 80 ° C. to 125 ° C. for 10 to 20 minutes and freeze-dried to obtain a powder.
It was obtained by adding 500 mL of distilled water to 5% of the fermented powder and non-fermented raw material and extracting using a sonicator for 1 hour at 45 ° C and then centrifuging at 4 ° C and 10,000rpm for 15 minutes. The supernatant was freeze-dried to obtain a powder sample as shown in Table 1 below. There was almost no difference in yield between the unfermented chrysanthemum extract and the fermented extract.

(2) Fractane content measurement in fermented product This experiment utilized Megazyme Fructan assay procedure kit. Sample Blank is 200μl of solvent mixed with 100μl of 0.1M sodium aceto buffer (pH4.5), mixed with 5ml of PAHBAH Working Reagent, reacted at 80 ℃ for 6 minutes, cooled to 18-20 ℃ for 5 minutes, and measured at 410nm. did. For the measurement of D-pructose standard, 200 μl of D-pructose standard (1.5 mg / ml) and 900 μl of 0.1 M sodium acetoate buffer (pH 4.5) were mixed. Mix 200 µl of a part of the sample with 100 µl of 0.1M sodium acetoate (pH 4.5), mix with 5 ml of PAHBAH Working Reagent, react at 80 ° C for 6 minutes, cool at 18-20 ° C for 5 minutes, and measure at 410 nm did. For the four sample treatments, 200 μl of sucrase was added to 200 μl and reacted at 40 ° C. for 30 minutes, and 200 μl of sodium borohydride buffer (10 mg / mL) was added and reacted at 40 ° C. for 30 minutes. The reducing sugar produced during this reaction is converted to sugar alcohol. After the reaction, add 500 μl of 0.2 M aceto acid, add 100 μl of fructanase to 200 μl of this solution, add 100 μl of 0.1 M sodium aceto acid buffer (pH 4.5), and react for 20 minutes at 40 ° C. The fructan contained in is hydrolyzed with D-pructose and D-glucose. After the reaction, 5 ml of PAHBAH Working Reagent was mixed with the substance, reacted at 80 ° C. for 6 minutes, cooled at 18 to 20 ° C. for 5 minutes, and the absorbance was measured at 410 nm.

Calculation method (fructan% w / w as is) = ΔA × F × V / VW × 2.48
ΔA = Sample Absorbance−Sample Blank Absorbance F = D-Fructose Absorbance Change Factor (μg)
= (54.5μg D-Fructose) / (absorbance for 54.5μg D-Fructose)
V = Volume of solvent used (mL) (50 or 100 mL)
W = weight of sample extract (mg)

  As a result, as shown in Table 1 below, the content of fructan is slightly reduced when fermented in Lactobacillus plantaroom, compared to chrysanthemum without fermenting lactic acid bacteria. Aspergillus oryza and Bacillus ricenifomis And in the case of Bacillus subtilis fermented product, it was confirmed that there was almost no change.

(3) Measurement of glucosidase inhibitory activity Alpha-glucosidase, which is a kind of carbohydrate digestive enzyme secreted from the small intestinal mucosa of animals, is used as a substrate with p-nitrophenyl-α-D-glucopyranoside (p-nitrophenyl-α-D). -Use glucopyranoside). The enzyme inhibitory activity of the sample was evaluated by observing the change in absorbance of the control group in which only the solvent was added using alpha glucosidase and the treatment group in which the sample extract of a constant concentration was added and reacted. In other words, PBS was dissolved at a concentration of 0.4 mg / ml BSA (bovine serum albumin), 0.04 mg / ml NaN3, 0.5 U / ml alpha glucosidase enzyme was dissolved to make an enzyme solution, and p-nitrophenyl-α -A substrate solution was prepared by dissolving D-glucopyranoside in PBS at a concentration of 2 mM. Add 50 μl of sample to 300 μl of enzyme solution, add 650 μl of water, measure the absorbance of the sample at 405 nm, add 500 μl of water to the same amount of enzyme solution and sample, add 150 μl of substrate, and react at room temperature for 3 minutes. And the absorbance was measured at 405 nm. The degree of inhibition of enzyme activity was calculated by the following formula.
Enzyme activity inhibition rate (%) = (1- (S / C)) x 100
S: Absorbance change after sample addition C: Absorbance change of control group without addition of sample

  As a result, as shown in Table 1 below, it was confirmed that the alpha glucosidase inhibitory activity increased in the case of the fermented chrysanthemum fermented with lactobacillus plantaroom as compared with other strains. In particular, it was found that the suppression effect increases as the fermentation time is shorter. However, in the case of other strains, alpha glucosidase inhibitory activity does not increase compared to the case of fermentation with Lactobacillus plantaroom during fermentation, and there is a difference in the activity, when chrysanthemum is fermented with lactic acid bacteria. It was found that all of them did not have high alpha glucosidase inhibitory activity.

(4) Thin Film Chromatography Confirmation Chromatography (Thin Layer Chromatography) analysis was performed on the samples of the fermentation products before fermentation and lactic acid bacteria in order to compare and analyze the components during the chrysanthemum fermentation. Samples of the lactic acid bacteria untreated group, the Lactobacillus plantaroom fermentation group, the Bacillus subtilis KCTC-1666 fermentation group, and the Bacillus subtilis KFCC11492P fermentation group were subjected to TLC at a concentration of 10 mg / mL. The developing solvent used was a mixture of Acetonitrile and H2O in a 9: 1 ratio. As the coloring reagent, a mixture of 20 ml of solution A (0.2 g each of aniline and diphenylamine per 20 ml of 96% ethanol) and 2 ml of solution B (85% phosphoric acid) was used. For sulfuric acid coloration, 10% sulfuric acid methanol was injected and carbonized at high temperature.

  As a result, as shown in FIG. 1, it was confirmed that high-mobility materials containing fructose were produced by decomposition of the constituent components during fermentation.

<Example 2>
Production of fermented chrysanthemum meal with fermentation time Using the Lactobacils plantaroom with excellent alpha glucosidase inhibitory activity in Example 1, the change in alpha glucosidase inhibitory activity with chrysanthemum meal was measured and the result was obtained. The results are shown in Table 2 below.

  As shown in Table 2, the fermented chrysanthemum fermented with Lactobacillus plantaroom has an alpha glucosidase inhibitory effect that varies depending on the fermentation time, but has an excellent alpha glucosidase inhibitory effect until 12 to 60 hours. The therapeutic effect also gave excellent results.

<Experimental example 1>
Diabetes treatment effect of chrysanthemums
<1-1> Experimental animals and feed control Diabetes experimental animals were 19 male C57BLKS / J-db / db mice (30-35 g) 6 weeks old from the central experimental animal, and normal control group C57BLKS / J- 5 db / m + mouse animals were sold and adapted for one week and used for the experiment.
The breeding environment for animal experiments was maintained at a temperature of 23 ° C, humidity of 60%, noise of 60phone or less, odor of 20ppm or less, lighting of 150-300lux, and lighting time of 12 hours. Feeding (puriner, Korea) and water were given freely. Animal experiment rearing management was conducted with the approval of the Experimental Animal Ethics Committee of Yulin University, based on the standards for the Care and Use of Laboratory Animals.

<1-2> Experimental design The experimental animals measured blood glucose from the tail vein with a blood glucose meter (Accucheck, Germany) after fasting for 16 hours. Animals showing fasting blood glucose of 250 mg / dL or more were used in the experiment assuming that diabetes was induced. In order to examine the antidiabetic activity of the sample, the sample was added to the feed and fed. Diabetes-induced animals were randomly divided into 4 test groups, 3 C (diabetic control), 6 AB Acarbose (200 mg / kg), LJA1 (Lactobacil plantaroom (KCTC3103)) fermentation A diet containing 1.5 g / kg of chrysanthemum candy), 7 LJA2 (food containing 3 g / kg of Lactobacils plantaroom (KCTC3103)) fermented chrysanthemum candy, etc., and C57BLKS / J-db / m + mouse5 Lungs were used to measure fasting blood glucose and body weight changes for 10 weeks. As the Lactobacillus plantaroom fermented chrysanthemum chrysanthemum, the fermented chrysanthemum fermented extract fermented in the Lactobacils plantaroom in Example 1 for 36 hours was used.

  As a result, as shown in FIG. 2, the control group fed only the general feed in the first week of feeding had a rapid increase in blood glucose, but it was slow in the group fed chrysanthemum fermented extract and Acarbose. Showed an increase. The group fed with chrysanthemum fermented extract during the whole feeding period showed a tendency of lower blood sugar than the control group, and the blood glucose of the LJA1 group became even lower in the last 7 weeks, which was not seen in other groups.

Regarding the change in body weight, as shown in FIG. 3, the change in the body weight during the experimental period of 7 weeks only had a difference in body weight between the normal mouse and the obese diabetic mouse, but there was no significant difference in the body weight of each treatment section.
Table 3 below shows the results of calculating the daily feed intake and feed efficiency between the groups. Similar to the results shown in FIG. 3, as shown in Table 4, there was only a difference between the normal group and obese diabetic mice, and there was no difference between the groups.

AB: Diet containing diabetes + Acarbose 0.5g / kg LJA1: Diet containing diabetes + LJA 1.5g / kg LJA2: Diet containing diabetes + LJP 3g / kg

  a, b means that different characters included in the same line are clearly different. (P <0.05); each value ± S.D, FER, food efficiency ratio = body weight gain (g) / food intake (g)

<1-3> Measurement of postprandial blood glucose change In order to measure the postprandial blood glucose lowering effect of the sample, the soluble starch (2 g / kg, Sigma Co., USA) was added to the C group and Lean group after 6 hours fasting in the 8th week of the experiment. In the Akabos administration group, soluble starch and Akabos (20 mg / kg were orally administered), and the fermented chrysanthemums were orally administered at low concentrations (LJA1,200 mg / kg) and high concentrations (LJA2,400 mg / kg). Blood glucose was measured from the tail vein at 30, 60 and 120 minutes after administration.

  As a result, as shown in FIG. 4, in the diabetic group, it was found that the blood glucose level increased 30 minutes after meal compared to the normal group, and in the chrysanthemum fermented extract administered group compared to the diabetic group to which nothing was administered, It was confirmed that the increase rate of blood glucose decreased in a concentration-dependent manner. Specifically, FIG. 5 compares and shows the area under the curve (AUC) of the graph of FIG. As a result, in the diabetic group C, the area was greatly increased to 15,000 or more, but the LJA1 and LJA2 which were the chrysanthemum administration group did not exceed 15000 and were shown to have values close to 10000. From the above results, it was found that the postprandial blood glucose increase rate was relatively small in the LJA administration group.

<1-4> Search for alpha glucosidase activity in small intestine The small intestine of the mouse to be experimented was removed, washed with physiological saline on ice, and the duodenum was divided into three equal parts, and each was proximal, middle, Obtained by centrifuging at 20000 g for 30 minutes after adding homogenization buffer (0.5M NaCl, 0.5M KCl, 5mM EDTA, pH7.0) Saline was added to the precipitate and centrifuged at 3000 rpm for 40 minutes, and the supernatant was used as an enzyme solution.

  0.1 ml of substrate (10 mm sucrose, 2 mM maltose, 1% starch in 0.1 M phosphate buffer, pH 7) mixed with 0,1 ml of enzyme solution separated in each part and 0.2 ml of 0.1 M phosphate buffer (pH 7) at 37 ° C. For 180 minutes, maltose for 40 minutes, starch for 120 minutes, and treated at 100 ° C. for 5 minutes to stop the reaction. 0.1 ml of the supernatant obtained by centrifuging each sample for 5 minutes at 3000 rpm was reacted with 0.75 ml of a glucose reagent at 37 ° C. for 30 minutes. After stopping the reaction with 0.5 ml of 1N HCl, the absorbance value was measured at 492 nm.

  As a result, as shown in FIG. 6, when the chrysanthemum fermented extract was fed, the activity of maltase was slightly lower in proximal than in control, and there was no difference in other parts. On the other hand, the inhibitory activity of lactase was expressed in all parts of the small intestine. In particular, it appeared specifically in middle and distal, and the inhibitory activity increased with increasing concentration of fermented chrysanthemums extract. The fermented product of the present invention did not affect the activity of sucrase, but showed significantly lower activity than control in all LJA1 and LJA2 treated groups at the distal site.

<1-5> Blood analysis After the experiment, blood was collected from the orbit of the subject mouse. HbAlc was measured using glycated hemoglobin (HLCr-723GHbC7, Tosoh, Denmark) to confirm the long-term maintenance blood glucose level from blood. Plasma obtained by centrifuging blood glucose concentration from blood at 3,000 rpm for 15 minutes at 4 ° C. was measured with a blood biochemistry measuring instrument (Thermo, USA). Blood insulin and adiponectin contents were measured using mouse insulin ELISA Kit (Shibay, Japan) and mouse adiponectin / Acrp30 kit (R & D system, UK), respectively.

  As a result, as shown in Table 4, the blood glucose level was not different between each treatment group and control, but in the case of HbAlc, all of the LJA1 and LJA2 groups fed with the chrysanthemum fermented extract were used. It was confirmed that a significantly low level was maintained. Furthermore, regarding the insulin content in plasma, the group fed with chrysanthemum fermented products (LJA1, LJA2) appeared to be characteristically higher than the control, but it was found that insulin secretion promotion or damage to the insulin secreting organ was suppressed. It was.

AB: Diet containing diabetes + acarbose 0.5g / kg LJA1: Diet containing diabetes + LJA 1.5g / kg LJA2: Diet containing diabetes + LJP 3g / kg

“a”, “b”, “c”, and “d” mean that different characters included in one line are clearly different.
(P <0.05); each value

  Furthermore, the amount of Triglyceride (TG) and T-CHOL in plasma was measured with a blood biochemical measuring device (Thermo, USA) from plasma obtained by centrifugation at 3,000 rpm for 15 minutes at 4 ° C. HDL-CHO and serum free fatty acid (Nonesterified fatty acid, NEFA) were measured with HDL-Cholesterel Kit and NEFA-HR2 kit (Asan, Korea), respectively. HTR is expressed by the ratio of HDL-CHO-CHO to T-CHOL, HDL-CHO / T-CHOL, and displayed so that the ratio of HDL cholesterol in the total cholesterol can be grasped.

  As a result, as shown in FIG. 5, it was found that the amount of triglyceride in blood was significantly reduced in the group administered with LJA1 and LJA2, compared to control, and there was no significant difference between LJA1 and LJA2. I was able to confirm it. It was found that the serum level of NEFA was also decreased by 14% and 16%, respectively, in the LJA1 and LJA2 administration groups compared to control. Furthermore, the total blood cholesterol level was almost the same in each group, but the HDL total cholesterol ratio (HTR) was higher in the LJA administration group than in the control group. In particular, the highest HTR was confirmed in the LJA2 group administered with 3 g of chrysanthemum fermented product per kg, which was measured at 138.9% of the control HDL cholesterol level.

Lean, C: Diabetes group AB: Diet containing diabetes + Acarbose 0.5g / kg LJA1: Diet containing diabetes + LJA 1.5g / kg LJA2: Diet containing diabetes + LJP 3g / kg

“a”, “b”, “c”, and “d” mean that different characters included in one line are clearly different.
(P <0.05); each value; HTR (%) = (HDL-Choresterol / totalcholestew) 100

  The present invention relates to a composition having a preventive, ameliorating and / or therapeutic effect for diabetes, which contains an extract of fermented chrysanthemum using Lactobacillus microorganisms. More specifically, the present invention relates to a composition having a preventive, ameliorating and / or therapeutic effect on diabetes, which contains an extract of fermented chrysanthemum fermented with Lactobacillus microorganisms. The composition of the present invention has a significantly superior antidiabetic effect than the unfermented chrysanthemum extract, and can be usefully used for the purpose of providing a composition for preventing, improving and / or treating new diabetes. it can.

Claims (2)

A pharmaceutical composition for the prevention and treatment of diabetes comprising, as an active ingredient, a chrysanthemum fermented extract fermented with a Lactobacillus acidophilus Lactobacillus acidophilus Lactobacillus acidophilus microorganism. The fermented chrysanthemum extract is prepared by adding distilled water and lactobacillus microorganisms such as Lactobacillus plantarum or Lactobacillus acidophilus to chrysanthemums powder at 20 to 40 ° C. for 1 to 60 ° C. 2. The composition according to item 1 , which is produced by time fermentation.