Pharmaceutical composition for preventing or treating obesity or insulin resistance syndrome, comprising strain lactobacillus plantarum HAC03 and rutin

Abstract

The present invention relates to a pharmaceutical composition and a functional health food composition for preventing or treating obesity or insulin resistance syndrome, comprising the strain Lactobacillus plantarum HAC03 and rutin as active ingredients. The combination of the strain Lactobacillus plantarum HAC03 and rutin of the present invention has effects of reducing weight gain and tissue weight, reducing tissue cell size, inhibiting fat synthesis, and increasing fat oxidation. In addition, the present invention has the effect of reducing blood glucose fluctuation and lowering fasting blood glucose and fasting insulin levels, and thus can be utilized as a therapeutic agent for obesity or insulin resistance syndrome, a functional health food, and the like.

Description

Pharmaceutical composition for preventing or treating obesity or insulin resistance syndrome comprising Lactobacillus plantarum HAC03 strain and rutin

[0001] The present invention relates to a pharmaceutical composition for preventing or treating obesity or insulin resistance syndrome and a health functional food composition comprising Lactobacillus plantarum HAC03 strain and rutin as active ingredients.

[0002] Obesity is a disease characterized by abnormal or excessive fat accumulation, which negatively impacts health. It is one of the ten major health risks identified by the WHO. Obesity not only causes weight gain, but also contributes to diseases such as type 2 diabetes, dyslipidemia, hypertension, fatty liver disease, coronary artery disease, and colon cancer, as well as psychological disorders such as psychological withdrawal and depression.

[0003] As of 2021, there are three obesity treatments approved by both the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA): liraglutide, orlistat, and a combination of liraglutide and bupropion. Liraglutide, known under the brand name Saxenda, received FDA approval in 2014, EMA approval in 2015, and approval from the Korean Ministry of Food and Drug Safety (MFDS) in 2017. Since then, liraglutide has maintained its leading position in the global obesity treatment market. Liraglutide is an analogue of glucagon-like peptide-1 (GLP-1), which binds to the GLP-1 receptor and transmits signals. GLP-1 is a hormone naturally secreted by the body in response to food intake. It stimulates insulin secretion from the pancreas to remove excess blood sugar and reduces glucagon secretion to regulate blood sugar levels. It plays a crucial role in maintaining normal blood sugar levels and reduces and suppresses appetite by binding to receptors in the brain and transmitting signals to the hypothalamus. However, some adverse effects have been reported, including diarrhea and vomiting, as well as adverse effects on other tissues, such as the liver, pancreas, and heart. Furthermore, increased incidence of certain cancers, such as breast and pancreatic cancer, has also been reported.

[0004] On the other hand, insulin resistance syndrome is a cluster syndrome in which several or more clinical features such as decreased glucose metabolism, resistance to glucose utilization, hyperinsulinemia, impaired glucose tolerance, hyperglycemia, hypertension, elevated triglycerides, and decreased HDL-cholesterol exist in a complex pattern. If insulin resistance persists, the insulin secretory capacity reaches its limit, insulin secretion decreases, and the postprandial increase in blood glucose cannot be suppressed, so fasting blood glucose rises above the normal range. If the amount of insulin secreted is not enough to overcome insulin resistance, it progresses to a state of impaired glucose tolerance, and if the insulin secretion deficiency is severe, it develops into diabetes mellitus with persistent hyperglycemia.

[0005] The most crucial aspect of discovering a substance that can treat or prevent a specific disease, such as obesity or insulin resistance syndrome, is ensuring a meaningful effect in reducing the severity of the disease. However, this must also be achieved without side effects. Based on this principle, various research teams worldwide are conducting research to discover treatments for obesity or insulin resistance syndrome that are both safe and proven to be effective.

[0006] Flavonoids are secondary metabolites produced by plants and are found in various fruits and vegetables. Flavonoids can be divided into two forms: glycosides, which contain sugars, and aglycones, which do not contain sugars. Most flavonoids found in nature are in the form of glycosides. When flavonoids are ingested orally, the glycosides are hydrolyzed to aglycones in the small or large intestine, and then absorbed. However, humans lack the enzymes to absorb or hydrolyze glycosides, so most glycosides are not absorbed in the small intestine. Instead, they are hydrolyzed to aglycones by intestinal microorganisms such as Bacteroides spp., Lactobacillus spp., and Enterococcus spp. in the large intestine, and then absorbed into intestinal cells. If the glycosides are not hydrolyzed and remain, most flavonoids are excreted from the body.

[0007] Flavonoids have been proven to have antioxidant, anti-inflammatory, and anticancer effects through various studies, but their low bioavailability limits their use as candidates for disease treatment. One way to address the low bioavailability of flavonoids is to utilize microorganisms that can hydrolyze flavonoids in the glycoside form into aglycones. The enzymes that play the most pivotal role in hydrolyzing rutin, a glycoside of quercetin, to form quercetin in the aglycone form are -L-rhamnosidase (EC 3.2.1.40) and aD-glucosidase (EC 3.2.1.21). In fact, there are research results showing that rutin was hydrolyzed into quercetin in vitro using various microorganisms, including certain lactic acid bacteria.

[0008]

[0009] The present inventors have completed the present invention by confirming that when Lactobacillus plantarum HAC03 strain and rutin, a type of flavonoid, are administered in combination, the anti-obesity effect and the insulin resistance improvement effect are remarkably excellent due to the synergistic effect of Lactobacillus plantarum HAC03 strain and rutin.

[0010] The problem to be solved by the present invention is to provide a pharmaceutical composition or health functional food composition having an effect of preventing or treating obesity or insulin resistance syndrome, which contains Lactobacillus plantarum HAC03 strain and rutin as active ingredients.

[0011] To solve the above problem, the present invention provides a pharmaceutical composition for preventing or treating obesity or insulin resistance syndrome, comprising Lactobacillus plantarum HAC03 strain (accession number: KCTC 13242BP) and Rutin as active ingredients.

[0012] The composition can reduce at least one selected from the group consisting of total cholesterol, triglyceride, low density lipoprotein cholesterol (LDL), alanine aminotransferase (ALT), and aspartate aminotransferase (AST).

[0013] The composition may exhibit an anti-obesity effect by regulating the expression level of one or more genes selected from the group consisting of FAS (fatty acid synthase), ACC (acetyl CoA carboxylase), PPARγ (peroxisome proliferator-activated receptor γ), SREBP1C (sterol regulatory element binding protein 1C), PPARα (peroxisome proliferator-activated receptor α), PGC1α (peroxisome proliferator-activated receptor γ coactivator 1 α), CPT1 (carnitine palmitoyltransferase 1), ACOX1 (acyl-CoA oxidase 1), UCP1 (uncoupling protein 1), and PRDM16 (PR / SET Domain 16).

[0014] The above anti-obesity effect may be at least one selected from the group consisting of inhibition of fat synthesis, increase in fat oxidation, and increase in body heat generation.

[0015] The composition may reduce blood sugar fluctuations and decrease fasting blood sugar and fasting insulin levels.

[0016] The above composition can exhibit an effect of reducing insulin resistance by regulating the expression level of G6P (glucose-6-phosphate dehydrogenase) or PEPCK (phosphoenolpyruvate carboxykinase) genes.

[0017] In addition, the present invention provides a health functional food composition for preventing or improving obesity or insulin resistance syndrome, comprising Lactobacillus plantarum HAC03 strain (Accession No.: KCTC 13242BP) and Rutin as active ingredients.

[0018] The composition can reduce at least one selected from the group consisting of total cholesterol, triglyceride, low density lipoprotein cholesterol (LDL), alanine aminotransferase (ALT), and aspartate aminotransferase (AST).

[0019] The composition may exhibit an anti-obesity effect by regulating the expression level of one or more genes selected from the group consisting of FAS (fatty acid synthase), ACC (acetyl CoA carboxylase), PPARγ (peroxisome proliferator-activated receptor γ), SREBP1C (sterol regulatory element binding protein 1C), PPARα (peroxisome proliferator-activated receptor α), PGC1α (peroxisome proliferator-activated receptor γ coactivator 1 α), CPT1 (carnitine palmitoyltransferase 1), ACOX1 (acyl-CoA oxidase 1), UCP1 (uncoupling protein 1), and PRDM16 (PR / SET Domain 16).

[0020] The above anti-obesity effect may be at least one selected from the group consisting of inhibition of fat synthesis, increase in fat oxidation, and increase in body heat generation.

[0021] The composition may reduce blood sugar fluctuations and decrease fasting blood sugar and fasting insulin levels.

[0022] The above composition can exhibit an effect of reducing insulin resistance by regulating the expression level of G6P (glucose-6-phosphate dehydrogenase) or PEPCK (phosphoenolpyruvate carboxykinase) genes.

[0023] The combination of the Lactobacillus plantarum HAC03 strain of the present invention and rutin has the efficacy of reducing body weight gain and tissue weight, reducing tissue cell size, inhibiting lipogenesis, and increasing fat oxidation. Furthermore, it has the efficacy of reducing blood sugar fluctuations and decreasing fasting blood sugar and insulin levels, making it suitable for use as a treatment for obesity or insulin resistance syndrome, as well as in health functional foods.

[0024] Figure 1 schematically illustrates the animal experiment method.

[0025] Figure 2 shows the change in body weight gain when Lactobacillus plantarum HAC03 strain and rutin were administered in combination.

[0026] Figure 3 shows the change in tissue weight when Lactobacillus plantarum HAC03 strain and rutin were administered in combination. A represents the change in weight of liver, B represents subcutaneous adipose tissue (SAT), C represents epididymal adipose tissue (EAT), D represents visceral adipose tissue (MAT), and E represents brown adipose tissue (BAT).

[0027] Figure 4 shows changes in tissue cell size when combined administration of Lactobacillus plantarum HAC03 strain and rutin. A represents changes in cell size of liver, B represents subcutaneous adipose tissue (SAT), C represents epididymal adipose tissue (EAT), D represents mesenteric adipose tissue (MAT), and E represents brown adipose tissue (BAT).

[0028] Figure 5 shows changes in biochemical indicators related to obesity when combined administration of Lactobacillus plantarum HAC03 strain and rutin. A represents changes in total cholesterol, B represents changes in triglycerides, and C represents changes in low-density lipoprotein cholesterol (LDL).

[0029] Figure 6 shows the changes in cytokines involved in fat synthesis in the extracted adipose tissue when combined administration of Lactobacillus plantarum HAC03 strain and rutin. A represents the change in the expression levels of FAS (fatty acid synthase), B represents ACC (acetyl CoA carboxylase), C represents PPARγ (peroxisome proliferator-activated receptor γ), and D represents SREBP1C (sterol regulatory element binding protein 1C).

[0030] Figure 7 shows the changes in cytokines involved in fat oxidation (β-oxidation) in the extracted adipose tissue when combined administration of Lactobacillus plantarum HAC03 strain and rutin was performed. A represents the change in the expression level of PPARα (peroxisome proliferator-activated receptor α), B represents PGC1α (peroxisome proliferator-activated receptor γ coactivator 1 α), C represents CPT1 (carnitine palmitoyltransferase 1), and D represents ACOX1 (acyl-CoA oxidase 1).

[0031] Figure 8 shows changes in cytokines involved in thermogenesis in brown adipose tissue (BAT) when combined administration of Lactobacillus plantarum HAC03 strain and rutin. A shows changes in the expression levels of UCP1 (uncoupling protein 1), and B shows changes in the expression levels of PRDM16 (PR / SET Domain 16).

[0032] Figure 9 shows the blood sugar changes in an oral glucose tolerance test when Lactobacillus plantarum HAC03 strain and rutin were administered in combination. A represents the blood sugar level, and B represents the AUC (area under curve) value.

[0033] Figure 10 shows changes in fasting blood sugar and insulin levels when combined with Lactobacillus plantarum HAC03 strain and rutin. A represents fasting blood sugar, B represents changes in insulin levels, and C represents HOMA-IR levels.

[0034] Figure 11 shows changes in biochemical indicators related to liver health when combined administration of Lactobacillus plantarum HAC03 strain and rutin. A represents changes in alanine aminotransferase (ALT), and B represents changes in aspartate aminotransferase (AST).

[0035] Figure 12 shows changes in cytokines related to insulin resistance in excised adipose tissue when combined administration of Lactobacillus plantarum HAC03 strain and rutin was performed. A represents changes in the expression levels of G6P (glucose-6-phosphate dehydrogenase), and B represents changes in the expression levels of PEPCK (phosphoenolpyruvate carboxykinase).

[0036] Figures 13 and 14 show the results of measuring intestinal microbial diversity. Figure 13 shows the alpha diversity recovery results, and Figure 14 shows the beta diversity recovery results.

[0037] Figure 15 shows the results of observing the relative distribution of intestinal microorganisms at the Phylum (A) or Class (B) level.

[0038] Figure 16 shows an analysis of the relative distribution of intestinal microorganisms. A represents Oscillospiraceae, B represents Lachnospiraceae, and C represents Eubacterium Coprostanoligenes.

[0039]

[0040] The present inventors completed the present invention by confirming that when the Lactobacillus plantarum HAC03 strain and rutin are administered in combination, the anti-obesity effect and the insulin resistance improvement effect are remarkably excellent due to the synergistic effect of the Lactobacillus plantarum HAC03 strain and rutin. Specifically, it was experimentally confirmed that when the strain and rutin are administered in combination, the anti-obesity effect is remarkably superior to when the strain or rutin is administered alone due to the synergistic effect of the anti-obesity effect of the strain itself and the anti-obesity effect of rutin, and the insulin resistance improvement effect is also remarkably excellent.

[0041] Therefore, from one aspect, the present invention relates to a pharmaceutical composition for preventing or treating obesity or insulin resistance syndrome, comprising Lactobacillus plantarum HAC03 strain (Accession No.: KCTC 13242BP) and Rutin as active ingredients.

[0042] The Lactobacillus plantarum of the present invention can be used synonymously with Lactoplantibacillus plantarum.

[0043] The above Lactobacillus plantarum HAC03 (Accession No.: KCTC 13242BP) strain was isolated from white kimchi, and has been confirmed to exhibit excellent acid and bile resistance, no antibiotic resistance, and no biogenic amine production ability (Domestic Patent No. 10-2266314). In addition, the above Lactobacillus plantarum HAC03 strain is characterized by including a 16s rDNA sequence represented by the following SEQ ID NO: 1.

[0044] (SEQ ID NO: 1)AGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTT GCGAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGTCCGTCACAACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGTAACC

[0045] In addition, the Lactobacillus plantarum HAC03 strain has a gene that expresses an enzyme that plays a central role in hydrolyzing rutin. Rutin is a glycoside in which rutinose (a disaccharide composed of glucose and rhamnose) is attached to the 3-carbon atom of quercetin. It was discovered in plants of the genus Ruta in the Rutaceae family, and was later isolated from many other plants, including the flower buds of Sophora japonica in the legume family and buckwheat (Fagopyrum esculentum in the knotweed family).

[0046] Through one embodiment of the present invention, it was confirmed that when Lactobacillus plantarum HAC03 strain and rutin were administered in combination, there was an anti-obesity effect (Example 1) and an insulin resistance improvement effect (Example 2), and it was confirmed that this effect was superior to when the strain or rutin was administered alone. Through this, it can be seen that a composition comprising Lactobacillus plantarum HAC03 strain and rutin as active ingredients can prevent or treat obesity or insulin resistance syndrome.

[0047] Specifically, when combined administration of Lactobacillus plantarum HAC03 strain and rutin resulted in decreased body weight gain, decreased tissue weight, and decreased tissue cell size. In addition, biochemical indicators related to obesity, such as total cholesterol, triglyceride, and low density lipoprotein cholesterol (LDL), were reduced. In addition, the expression levels of FAS, ACC, PPARγ, and SREBP1C, genes related to fat synthesis, were decreased, while the expression levels of PPARα, PGC1α, CPT1, and ACOX1, genes related to fat oxidation, were increased, and the expression levels of UCP1 and PRDM16, genes involved in thermogenesis in brown adipose tissue, were increased (Example 1).

[0048] In addition, when Lactobacillus plantarum HAC03 strain and rutin were combined, blood sugar fluctuations were reduced, fasting blood sugar and insulin levels were reduced, and alanine aminotransferase (ALT) and aspartate aminotransferase (AST), which are biochemical indicators related to liver health, were reduced. In addition, the expression levels of G6P and PEPCK, which are genes related to gluconeogenesis, were increased (Example 2).

[0049] In addition, when Lactobacillus plantarum HAC03 strain and rutin were administered in combination, the diversity and uniformity of microbial species increased, and showed an opposite trend to that of Eubacterium Coprostanoligenes, which significantly increased in obese patients, and Oscillospiraceae and Lachnospiraceae, which significantly decreased (Example 3).

[0050] In the present invention, the term “prevention” means any act of suppressing a disease or delaying its onset by administering a pharmaceutical composition.

[0051] The term "treatment" in the present invention includes abrogating, substantially inhibiting, slowing or reversing the progression of a disease, substantially improving clinical or aesthetic symptoms of the disease, or substantially preventing the appearance of clinical or aesthetic symptoms of the disease.

[0052] The above pharmaceutical composition may have any one dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, oral solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilizers, and suppositories, and may be in various oral or parenteral dosage forms. When formulated, it is prepared using diluents or excipients such as commonly used fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

[0053] In the present invention, the term "administration" refers to introducing the pharmaceutical composition of the present invention to a subject by any appropriate method, and the route of administration may be various routes, such as oral or parenteral, as long as it can reach the target tissue.

[0054] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations can be prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives can be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspending agents may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include witepsol, macrogol, Tween 61, cocoa butter, laurin butter, and glycerogelatin.

[0055] The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level can be determined based on factors including the type and severity of the individual, age, sex, type of disease, activity of the drug, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents. And it can be administered singly or in multiple doses. It is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects by taking all of the above factors into consideration, and this can be easily determined by those skilled in the art.

[0056] The pharmaceutical composition of the present invention is not particularly limited and can be applied to any subject for the purpose of treating obesity or insulin resistance syndrome. For example, it can be used for any non-human animals such as monkeys, dogs, cats, rabbits, guinea pigs, rats, mice, cows, sheep, pigs, goats, etc., humans, birds, and fish, and the pharmaceutical composition can be administered parenterally, subcutaneously, intraperitoneally, intrapulmonary, and intranasally, and for local treatment, it can be administered by any suitable method including intralesional administration if necessary. The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and body weight of the subject, the degree of the disease, the drug form, the route and duration of administration, but can be appropriately selected by those skilled in the art. For example, it can be administered orally, rectally, or by intravenous, intramuscular, subcutaneous, intrauterine, intradural, or intracerebrovascular injection, but is not limited thereto.

[0057] The appropriate total daily dose can be determined by treatment within the scope of sound medical judgment, and is generally administered once or several times daily in an amount of 0.001 to 1000 mg / kg, preferably 0.05 to 200 mg / kg, more preferably 0.1 to 100 mg / kg.

[0058]

[0059] From another perspective, the present invention relates to a health functional food composition for preventing or improving obesity or insulin resistance syndrome, comprising Lactobacillus plantarum HAC03 strain (Accession No.: KCTC 13242BP) and Rutin as active ingredients.

[0060] The above food composition refers to a natural product or processed product containing one or more nutrients, preferably one that has gone through a certain degree of processing to become directly edible, and in its general sense, refers to all of food, food additives, health functional foods, and functional beverages.

[0061] The food composition of the present invention may be in the form of a pill, powder, granule, infusion, tablet, capsule or liquid, and there is no particular limitation on the type of food, and examples thereof include various beverages, gum, tea, vitamin complexes, health supplements, etc.

[0062] The above food composition may contain other ingredients, the types of which are not particularly limited. For example, as with conventional foods, it may contain various herbal extracts, food science-acceptable food additives, or natural carbohydrates as additional ingredients, but is not limited thereto.

[0063] In the present invention, the term "food supplement additive" means a component that can be added to food as an auxiliary, and can be appropriately selected and used by those skilled in the art as added in the manufacture of health functional foods of each formulation. Examples of food supplement additives include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and fillers, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc., but the types of food supplement additives of the present invention are not limited by the above examples.

[0064] Examples of the above natural carbohydrates include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. In addition to the above, natural flavoring agents (thaumatin, etc.), stevia extracts (rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used as flavoring agents.

[0065] The food composition of the present invention may include a health functional food. As used herein, the term "health functional food" refers to a food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. using raw materials or ingredients having functionality useful to the human body. Here, functionality means obtaining a beneficial effect for health purposes, such as regulating nutrients for the structure and function of the human body or physiological functions. The health functional food of the present invention can be manufactured by a method commonly used in the art, and during the manufacturing process, raw materials and ingredients commonly added in the art can be added. In addition, unlike general drugs, it has the advantage of not having side effects that may occur with long-term use of drugs because it uses food as a raw material, and can be highly portable.

[0066] In general, the amount of Lactobacillus plantarum HAC03 strain or rutin included in a food composition may be added in an amount of 0.1 to 90 wt% of the total food weight. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be below the above range. In addition, the food composition according to the present invention may contain other ingredients that can preferably provide a synergistic effect to the main effect, as long as the main effect aimed at by the present invention is not impaired, in addition to the Lactobacillus plantarum HAC03 strain or rutin.

[0067]

[0068] Hereinafter, the composition and effects of the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and the scope of the present invention is not limited by these examples.

[0069]

[0070] <Experimental Method>

[0071] 1. Isolation and identification of strains

[0072] After purchasing white kimchi that was fermenting for approximately 10 to 15 days, the pH was measured to confirm a decrease in pH due to lactic acid production, and then lactic acid bacteria were isolated. Ninety milliliters of saline solution (0.85% NaCl / L) was placed in an autoclaved plastic bag, 10 g of white kimchi sample was added, and mixed uniformly at 200 rpm for 5 minutes (Stomacher® 400 Circulator, Seward, UK). 1 ml was dispensed, serially diluted 10-fold in 9 ml of saline solution (0.85% NaCl / L), and plated on MRS agar (5.5% Lactobacillus MRS broth, BD Difco, USA and 1.5% Bacteriological Agar, Affymetrix, USA). The cultures were then grown in an incubator at 37°C for 24 to 48 hours, and isolated and identified.

[0073] The grown strains were then cultured in 3% hydrogen peroxide at 37°C for 24–48 h, and catalase activity and Gram staining were analyzed. The taxonomic species of catalase-negative strains were identified by analyzing the bidirectional 16S rDNA gene base sequence. The base sequence identification of the strains was performed by SolGent Co., Ltd. (Korea).

[0074]

[0075] As a result, the strain of the present invention isolated from white kimchi was identified as Lactobacillus plantarum, a catalase-negative, gram-positive, rod-shaped strain, and was named Lactobacillus plantarum HAC03. The strain was deposited with the Korea Research Institute of Bioscience and Biotechnology on December 28, 2020 (Accession Number: KCTC 13242BP).

[0076]

[0077] 2. Animal testing

[0078] Animal experiments were conducted to determine the preventive and ameliorating effects of Lactobacillus plantarum HAC03 strain and rutin on obesity and insulin resistance syndrome. Figure 1 schematically illustrates the animal experiment method.

[0079]

[0080] Five-week-old male C57BL6 mice were purchased and acclimated for one week. Thereafter, they were divided into groups (n = 10) and fed either a low-fat or high-fat diet. After one week of low-fat or high-fat diet, each group was administered Lactobacillus plantarum HAC03, quercetin, or rutin orally once daily for 14 weeks. The test groups were established as shown in Table 2.

[0081]

[0082] Group Individual Diet and Oral Administration Substances LFD10 Low-fat diet (10% fat) HFD10 High-fat diet (60% fat) Q10 High-fat diet + Quercetin (100 mg / kg) (Positive control) R10 High-fat diet + Rutin (100 mg / kg) H10 High-fat diet + Lactobacillus plantarum HAC03 strain (3x10 8 CFU / mouse)HR10 high-fat diet + Lactobacillus plantarum HAC03 strain (3x10 8 CFU / mouse) + Rutin (100 mg / kg)HQ10 High-fat diet + Lactobacillus plantarum HAC03 strain (3x10 8 CFU / mouse) + quercetin (Quercetin, 100 mg / kg) Total 70

[0083] Body weight and food intake were measured weekly for each group. After oral administration, an oral glucose tolerance test was performed at week 12 by oral administration of glucose. After oral administration, the mice were euthanized with carbon dioxide gas at week 14 and body weight and tissue weight were measured. In addition, blood was extracted from the heart, serum was separated, and biochemical indices related to obesity and liver health were measured in the serum. Total cholesterol, triglycerides, and low-density lipoprotein cholesterol (LDL) were used as biochemical indices related to obesity, and alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were used as biochemical indices related to liver health. In addition, adipose tissue was extracted, H&E staining was performed, and the size and number of adipocytes were measured.

[0084]

[0085] 3. Cytokine measurement

[0086] To identify the molecular mechanisms involved in the prevention and improvement effects of Lactobacillus plantarum HAC03 strain and rutin on obesity or insulin resistance syndrome, RNA was isolated and cDNA was synthesized from adipose tissue, and then the expression levels of genes related to lipogenesis were measured at the mRNA level using qPCR.

[0087] Specifically, the mouse colon of each experimental group was homogenized and dissolved in Trizol® (RNA extraction buffer, Thermo Fisher), and then RNA was extracted according to the protocol provided by the manufacturer. The extracted RNA was quantified, and the same amount of RNA was reverse transcribed using the SuperiorScript III cDNA synthesis Kit (Enzynomics, Daejeon, South Korea) to synthesize cDNA. Afterwards, real-time PCR was performed using the gene-specific primers in Table 3, TOPreal qPCR 2X premix (SYBR Green with high ROX) (Enzynomics, Daejeon, South Korea), and cDNA. Real-time PCR was performed using the ABI 7500 Fast Real time PCR system (Applied Biosystems, Waltham, MA, USA). The relative gene expression level was calculated using the △△Ct method.

[0088]

[0089] Target Forward (5' → 3') Reverse (5' → 3') FASCTGGACTCGCTCATGGGTG (SEQ ID NO: 2) CATTTCCTGAAGTTTCCGCAG (SEQ ID NO: 3) ACCTGACAGACTGATCGCAGAGAAAG (SEQ ID NO: 4) TGGAGAGCCCCACACA (SEQ ID NO: 5) PPARγ AGTGGAGACCGCCCAGG (SEQ ID NO: 6) GCAGCAGGTTGTCTTGGATGT (SEQ ID NO: 7) SREBP1 CAGCAGCCCCTAGAACAAACAC (SEQ ID NO: 8) CAGCAGTGAGTCTGCCTTGAT (SEQ ID NO: 9) PPARGTACGGTGTGTATGAAGCCATCTT (SEQ ID NO: 10) GCCGTACGCGATCAGCAT (SEQ ID NO: 11) PGC1 CCTGAAGCCGGGAGAGAATG (SEQ ID NO: 12)TAGCCAGCAGAGACTGTGGA (SEQ ID NO: 13)CPT1TGAGTGGCGTCCTCTTTGG (SEQ ID NO: 14)TCAGCGAGTAGCGCATAGTCA (SEQ ID NO: 15)ACOX1GTGCAGCTCAGAGTCTGTCCAA (SEQ ID NO: 16)TACTGCTGCGTCTGAAAATCCA (SEQ ID NO: 17)UCP1CTTTGCCTCACTCAGGATTGG (SEQ ID NO: 18)ACTGCCACACCTCCAGTCATT (SEQ ID NO: 19)PRDM16GAAGTCACAGGAGGACACGG (SEQ ID NO: 20)CTCGCTCCTCAACACACCTC (SEQ ID NO: 21)G6PCGACTCGCTATCTCCAAGTGA (SEQ ID NO: 22)GTTGAACCAGTCTCCGACCA (SEQ ID NO: 23)PEPCKCTGCATAACGGTCTGGACTTC (SEQ ID NO: 24) CAGCAACTGCCCGTACTCC (SEQ ID NO: 25) Beta-actin GGCACCACACYTTCTACAATG (SEQ ID NO: 26) GGGGTGTTGAAGGTCTCAAAC (SEQ ID NO: 27)

[0090] 4. Insulin measurement

[0091] Serum insulin was measured using the Ultra Sensitive Mouse Insulin ELISA kit (MORINAGA Institute of Biological Science, Inc (MIoBS), Yokohama-shi, Japan).

[0092]

[0093] 5. Statistics

[0094] All data are expressed as the mean ± standard error of the median (SEM). Statistical significance was established as follows:

[0095]

[0096] + p < 0.05, ++ p < 0.01 between R and HR.

[0097]

[0098] *# p < 0.05, ## p < 0.01, ### p < 0.001 between H and HR.

[0099] * p < 0.05, ** p < 0.01, *** p < 0.001 compared with HFD.

[0100]

[0101] [Example 1] Anti-obesity effect

[0102] 1. Changes in weight gain

[0103] Figure 2 shows the change in body weight gain when Lactobacillus plantarum HAC03 strain and rutin were administered in combination.

[0104]

[0105] Specifically, compared to the group that consumed only a high-fat diet (HFD), it was confirmed that the change in body weight gain was reduced in all groups that were administered a high-fat diet and Lactobacillus plantarum HAC03 strain or rutin, either singly or in combination. In addition, the group administered a combination of Lactobacillus plantarum HAC03 strain and rutin (HR) showed a significantly reduced change in body weight gain compared to the group administered Lactobacillus plantarum HAC03 strain (H) or rutin (R) alone (Fig. 2).

[0106]

[0107] 2. Changes in tissue weight

[0108] Figure 3 shows the change in tissue weight when Lactobacillus plantarum HAC03 strain and rutin were administered in combination. A represents the change in weight of liver, B represents subcutaneous adipose tissue (SAT), C represents epididymal adipose tissue (EAT), D represents visceral adipose tissue (MAT), and E represents brown adipose tissue (BAT).

[0109]

[0110] Specifically, compared to the group fed only a high-fat diet (HFD), liver weight was confirmed to decrease in all groups administered a high-fat diet and Lactobacillus plantarum HAC03 strain or rutin, either singly or in combination. In the group administered a combination of Lactobacillus plantarum HAC03 strain and rutin (HR), liver weight was significantly reduced compared to the group administered Lactobacillus plantarum HAC03 strain (H) or rutin (R) alone (Fig. 3A).

[0111]

[0112] In addition, compared to the group fed only a high-fat diet (HFD), we confirmed that the weight of subcutaneous fat tissue decreased in all groups administered a high-fat diet and Lactobacillus plantarum HAC03 strain or rutin, either singly or in combination. In the group administered a combination of Lactobacillus plantarum HAC03 strain and rutin (HR), the weight of subcutaneous fat tissue decreased more than in the group administered Lactobacillus plantarum HAC03 strain (H) or rutin (R) alone, but this was not statistically significant (Fig. 3B).

[0113]

[0114] In addition, compared to the group fed only a high-fat diet (HFD), all groups administered a high-fat diet and Lactobacillus plantarum HAC03 strain or rutin, either singly or in combination, showed a decrease in testicular adipose tissue weight. In the group administered a combination of Lactobacillus plantarum HAC03 strain and rutin (HR), testicular adipose tissue weight was significantly reduced compared to the group administered Lactobacillus plantarum HAC03 strain (H) alone (Fig. 3C).

[0115]

[0116] In addition, compared to the group fed only a high-fat diet (HFD), all groups administered a high-fat diet and Lactobacillus plantarum HAC03 strain or rutin, either singly or in combination, showed a decrease in visceral fat tissue weight. The group administered a combination of Lactobacillus plantarum HAC03 strain and rutin (HR) showed a significantly greater decrease in visceral fat tissue weight than the group administered Lactobacillus plantarum HAC03 strain (H) alone (Fig. 3D).

[0117]

[0118] In addition, compared to the group fed only a high-fat diet (HFD), we confirmed that the weight of brown adipose tissue decreased in all groups administered a high-fat diet and Lactobacillus plantarum HAC03 strain or rutin, either singly or in combination. In the group administered a combination of Lactobacillus plantarum HAC03 strain and rutin (HR), the weight of brown adipose tissue decreased more than in the group administered Lactobacillus plantarum HAC03 strain (H) or rutin (R) alone, but this was not statistically significant (Fig. 3E).

[0119]

[0120] 3. Changes in tissue cell size

[0121] Figure 4 shows changes in tissue cell size when combined administration of Lactobacillus plantarum HAC03 strain and rutin. A represents changes in cell size of liver, B represents subcutaneous adipose tissue (SAT), C represents epididymal adipose tissue (EAT), D represents mesenteric adipose tissue (MAT), and E represents brown adipose tissue (BAT).

[0122]

[0123] Specifically, compared to the group fed only a high-fat diet (HFD), all groups administered a high-fat diet and Lactobacillus plantarum HAC03 strain or rutin, either singly or in combination, showed a decrease in liver tissue cell size. In the group administered a combination of Lactobacillus plantarum HAC03 strain and rutin (HR), the liver tissue cell size was significantly reduced compared to the group administered Lactobacillus plantarum HAC03 strain (H) alone (Fig. 4A).

[0124]

[0125] In addition, compared to the group fed only a high-fat diet (HFD), all groups administered a high-fat diet and Lactobacillus plantarum HAC03 strain or rutin, either singly or in combination, showed a decrease in subcutaneous adipose tissue size. In the group administered a combination of Lactobacillus plantarum HAC03 strain and rutin (HR), the subcutaneous adipose tissue cell size was significantly reduced compared to the group administered Lactobacillus plantarum HAC03 strain (H) alone (Fig. 4B).

[0126]

[0127] In addition, compared to the group fed only a high-fat diet (HFD), we confirmed that the size of testicular adipose tissue decreased in all groups administered a high-fat diet and Lactobacillus plantarum HAC03 strain or rutin, either singly or in combination. In the group administered a combination of Lactobacillus plantarum HAC03 strain and rutin (HR), the size of testicular adipose tissue decreased more than in the group administered Lactobacillus plantarum HAC03 strain (H) or rutin (R) alone, but this was not statistically significant (Fig. 4C).

[0128]

[0129] In addition, compared to the group fed only a high-fat diet (HFD), all groups administered a high-fat diet and Lactobacillus plantarum HAC03 strain or rutin, either singly or in combination, showed a decrease in visceral fat tissue size. The group administered a combination of Lactobacillus plantarum HAC03 strain and rutin (HR) showed a significantly greater decrease in visceral fat tissue size than the group administered Lactobacillus plantarum HAC03 strain (H) alone (Fig. 4D).

[0130]

[0131] In addition, compared to the group fed only a high-fat diet (HFD), all groups administered a high-fat diet and Lactobacillus plantarum HAC03 strain or rutin, either singly or in combination, showed a decrease in brown adipose tissue size. In the group administered a combination of Lactobacillus plantarum HAC03 strain and rutin (HR), the brown adipose tissue size was significantly reduced compared to the group administered Lactobacillus plantarum HAC03 strain (H) or rutin (R) alone (Fig. 4E).

[0132]

[0133] 4. Changes in biochemical indicators

[0134] Figure 5 shows changes in biochemical indicators related to obesity when combined administration of Lactobacillus plantarum HAC03 strain and rutin. A represents changes in total cholesterol, B represents changes in triglycerides, and C represents changes in low-density lipoprotein cholesterol (LDL).

[0135]

[0136] Specifically, compared to the group fed only a high-fat diet (HFD), total cholesterol levels were reduced in all groups administered a high-fat diet and Lactobacillus plantarum HAC03 strain or rutin, either singly or in combination. The group administered a combination of Lactobacillus plantarum HAC03 strain and rutin (HR) showed a significantly greater reduction in total cholesterol levels than the group administered Lactobacillus plantarum HAC03 strain (H) or rutin (R) alone.

[0137]

[0138] In addition, compared to the group that consumed only a high-fat diet (HFD), neutral fat levels decreased in all groups that were administered a high-fat diet and Lactobacillus plantarum HAC03 strain or rutin, either singly or in combination. The group administered a combination of Lactobacillus plantarum HAC03 strain and rutin (HR) showed a significantly greater decrease in neutral fat levels than the group administered Lactobacillus plantarum HAC03 strain (H) or rutin (R) alone.

[0139]

[0140] In addition, compared to the group fed only a high-fat diet (HFD), low-density lipoprotein cholesterol (LDL) levels were reduced in all groups administered a high-fat diet and Lactobacillus plantarum HAC03 strain or rutin, either singly or in combination. The group administered a combination of Lactobacillus plantarum HAC03 strain and rutin (HR) showed a significantly greater reduction in LDL levels than the group administered Lactobacillus plantarum HAC03 strain (H) or rutin (R) alone.

[0141]

[0142] 5. Changes in gene expression

[0143] (1) Changes in the expression level of fat synthesis genes

[0144] Figure 6 shows the changes in cytokines involved in lipogenesis in the excised adipose tissue when combined administration of Lactobacillus plantarum HAC03 strain and rutin. A represents the change in the expression levels of FAS (fatty acid synthase), B represents ACC (acetyl CoA carboxylase), C represents PPARγ (peroxisome proliferator-activated receptor γ), and D represents SREBP1C (sterol regulatory element binding protein 1c). FAS, ACC, PPARγ, and SREBP1C are all genes involved in lipogenesis.

[0145]

[0146] Specifically, compared to the group fed only a high-fat diet (HFD), we confirmed that FAS expression levels decreased in all groups administered a high-fat diet and Lactobacillus plantarum HAC03 strain or rutin, either singly or in combination. In the group administered a combination of Lactobacillus plantarum HAC03 strain and rutin (HR), FAS expression levels decreased significantly more than in the group administered Lactobacillus plantarum HAC03 strain (H) or rutin (R) alone (Fig. 6A).

[0147]

[0148] In addition, compared to the group that consumed only a high-fat diet (HFD), it was confirmed that the expression level of ACC decreased in all groups that were administered a high-fat diet and Lactobacillus plantarum HAC03 strain or rutin, either singly or in combination. In the group administered a combination of Lactobacillus plantarum HAC03 strain and rutin (HR), the expression level of ACC decreased significantly more than in the group administered Lactobacillus plantarum HAC03 strain (H) alone (Fig. 6B).

[0149]

[0150] In addition, compared to the group fed only a high-fat diet (HFD), it was confirmed that the PPARγ expression level was significantly reduced in all groups administered a high-fat diet and Lactobacillus plantarum HAC03 strain or rutin, either singly or in combination, except for the group administered quercetin alone (Q). In the group administered a combination of Lactobacillus plantarum HAC03 strain and rutin (HR), the PPARγ expression level was significantly reduced compared to the group administered Lactobacillus plantarum HAC03 strain (H) alone (Fig. 6C).

[0151]

[0152] In addition, compared to the group that consumed only a high-fat diet (HFD), a significant decrease in SREBP1C expression was confirmed only in the group that consumed a low-fat diet (LFD) and the group that received a combination of Lactobacillus plantarum HAC03 strain and rutin (HR). In the group that received a combination of Lactobacillus plantarum HAC03 strain and rutin (HR), the SREBP1C expression level was significantly reduced compared to the group that received only Lactobacillus plantarum HAC03 strain (H) (Fig. 6D).

[0153]

[0154] (2) Changes in the expression of fat oxidation genes

[0155] Figure 7 shows the changes in cytokines involved in fat oxidation (β-oxidation) in the isolated adipose tissue when combined administration of Lactobacillus plantarum HAC03 strain and rutin was performed. A represents the change in the expression level of PPARα (peroxisome proliferator-activated receptor α), B represents PGC1α (peroxisome proliferator-activated receptor γ coactivator 1 α), C represents CPT1 (carnitine palmitoyltransferase 1), and D represents ACOX1 (acyl-CoA oxidase 1). PPARα, PGC1α, CPT1, and ACOX1 are all genes involved in fat oxidation.

[0156]

[0157] Specifically, compared to the group that consumed only a high-fat diet (HFD), the group administered a combination of Lactobacillus plantarum HAC03 strain and quercetin or rutin (HQ, HR) showed a significant increase in PPARα expression. The group administered a combination of Lactobacillus plantarum HAC03 strain and rutin (HR) showed a significantly greater increase in PPARα expression than the group administered Lactobacillus plantarum HAC03 strain or rutin alone (H, R) (Fig. 7A).

[0158]

[0159] In addition, a significant increase in PGC1 expression was confirmed in all groups except the group that consumed only quercetin (Q) compared to the group that consumed only a high-fat diet (HFD). In the group administered a combination of Lactobacillus plantarum HAC03 strain and rutin (HR), PGC1 expression increased significantly more than in the groups administered Lactobacillus plantarum HAC03 strain or rutin alone (H, R) (Fig. 7B).

[0160]

[0161] In addition, compared to the group that consumed only a high-fat diet (HFD), a significant increase in CPT1 expression was confirmed in the group that was administered only rutin (R) and the group that was administered a combination of Lactobacillus plantarum HAC03 and rutin (HR). In the group that was administered a combination of Lactobacillus plantarum HAC03 strain and rutin (HR), the CPT1 expression level increased significantly more than in the groups that were administered Lactobacillus plantarum HAC03 strain or rutin alone (H, R) (Fig. 7C).

[0162]

[0163] In addition, compared to the group that consumed only a high-fat diet (HFD), a significant increase in ACOX1 expression was confirmed in the group administered only the Lactobacillus plantarum HAC03 strain (H) and the groups administered a combination of the Lactobacillus plantarum HAC03 strain and rutin (HQ, HR). In the group administered a combination of the Lactobacillus plantarum HAC03 strain and rutin (HR), the ACOX1 expression level increased significantly more than in the group administered rutin alone (R) (Fig. 7D).

[0164]

[0165] (3) Changes in the expression level of heat-generating genes

[0166] Figure 8 shows changes in cytokines involved in thermogenesis in brown adipose tissue (BAT) following combined administration of Lactobacillus plantarum HAC03 strain and rutin. A represents changes in the expression levels of UCP1 (uncoupling protein 1), and B represents changes in the expression levels of PRDM16 (PR / SET Domain 16). UCP1 and PRDM16 are involved in generating body heat and increasing energy use.

[0167]

[0168] Specifically, we confirmed that in brown adipose tissue (BAT), UCP1 expression levels increased in all groups administered a high-fat diet and Lactobacillus plantarum HAC03 strain or rutin, either singly or in combination, compared to the group fed only a high-fat diet (HFD). In the group administered a combination of Lactobacillus plantarum HAC03 strain and rutin (HR), UCP1 expression levels increased significantly more than in the group administered Lactobacillus plantarum HAC03 strain (H) or rutin (R) alone (Fig. 8A).

[0169]

[0170] In addition, we confirmed that the expression level of PRDM16 increased in the group that consumed only a high-fat diet (HFD) in brown adipose tissue (BAT), the group that consumed quercetin (Q), the group that received a combination of Lactobacillus plantarum HAC03 strain and quercetin (HQ), and the group that received a combination of Lactobacillus plantarum HAC03 strain and rutin (HR). In the group that received a combination of Lactobacillus plantarum HAC03 strain and rutin (HR), the expression level of PRDM16 increased more than in the group that received only Lactobacillus plantarum HAC03 strain (H) or rutin (R), but this was not statistically significant (Fig. 8B).

[0171]

[0172] [Example 2] Effect of improving insulin resistance

[0173] 1. Changes in blood sugar and insulin levels

[0174] (1) Confirmation of reduction in blood sugar changes

[0175] Figure 9 shows the blood sugar changes in an oral glucose tolerance test when Lactobacillus plantarum HAC03 strain and rutin were administered in combination. A represents the blood sugar level, and B represents the AUC (area under curve) value.

[0176]

[0177] Specifically, compared to the group fed only a high-fat diet (HFD), all groups administered a high-fat diet and Lactobacillus plantarum HAC03 strain or rutin, either singly or in combination, showed statistically significant decreases in the maximum blood glucose level (Fig. 9A) and AUC value (Fig. 9B) after glucose administration. In the group administered a combination of Lactobacillus plantarum HAC03 strain and rutin (HR), the AUC value was statistically significantly decreased compared to the group administered Lactobacillus plantarum HAC03 strain (H) alone.

[0178]

[0179] (2) Reduction of fasting blood sugar and insulin levels

[0180] Figure 10 shows changes in fasting blood sugar and insulin levels when combined with Lactobacillus plantarum HAC03 strain and rutin. A represents fasting blood sugar, B represents changes in insulin levels, and C represents HOMA-IR levels.

[0181]

[0182] Compared to the group fed only a high-fat diet (HFD), all groups administered a high-fat diet and Lactobacillus plantarum HAC03 strain or rutin, either singly or in combination, showed a decrease in insulin levels and HOMA-IR values ​​indicating insulin resistance. The group administered a combination of Lactobacillus plantarum HAC03 strain and rutin (HR) showed a statistically significant decrease in insulin levels and HOMA-IR values ​​compared to the group administered Lactobacillus plantarum HAC03 strain alone (H).

[0183]

[0184] 2. Changes in biochemical indicators

[0185] Figure 11 shows changes in biochemical markers related to liver health following combined administration of Lactobacillus plantarum HAC03 strain and rutin. A represents changes in alanine aminotransferase (ALT), and B represents changes in aspartate aminotransferase (AST). Liver enzymes ALT and AST leak into the bloodstream when liver cells die, making them important indicators of liver health.

[0186]

[0187] Specifically, compared to the group fed only a high-fat diet (HFD), ALT and AST levels decreased in all groups administered a high-fat diet and Lactobacillus plantarum HAC03 strain or rutin, either singly or in combination. In the group administered a combination of Lactobacillus plantarum HAC03 strain and rutin (HR), a significant decrease in ALT and AST levels was confirmed compared to the group administered Lactobacillus plantarum HAC03 strain (H) or rutin (R) alone.

[0188]

[0189] 3. Changes in gene expression

[0190] Figure 12 shows the changes in cytokines related to insulin resistance in the excised adipose tissue when combined administration of Lactobacillus plantarum HAC03 strain and rutin was performed. A represents the change in the expression level of G6P (glucose-6-phosphate dehydrogenase), and B represents the change in the expression level of PEPCK (phosphoenolpyruvate carboxykinase). G6P is a gene that expresses glucose-6-phosphate dehydrogenase, an enzyme that activates gluconeogenesis. When G6P increases, blood sugar increases. PEPCK is a gene that expresses phosphoenolpyruvate carboxykinase, an enzyme that activates gluconeogenesis. When PEPCK increases, blood sugar increases.

[0191]

[0192] Specifically, compared to the group that consumed only a high-fat diet (HFD), it was confirmed that the GP6 expression level decreased in all groups. In the group administered a combination of Lactobacillus plantarum HAC03 strain and rutin (HR), the G6P expression level decreased significantly more than in the group administered Lactobacillus plantarum HAC03 strain (H) or rutin (R) alone (Fig. 12A).

[0193]

[0194] In addition, compared to the group that consumed only a high-fat diet (HFD), it was confirmed that the expression level of PEPCK decreased in all groups. In the group administered a combination of Lactobacillus plantarum HAC03 strain and rutin (HR), the expression level of PEPCK was confirmed to be significantly reduced compared to the group administered Lactobacillus plantarum HAC03 strain (H) or rutin (R) alone (Fig. 12B).

[0195]

[0196] [Example 3] Results of improved intestinal microflora (changes in diversity recovery)

[0197] 1. Diversity measurement results

[0198] (1) Alpha diversity recovery results

[0199] Figure 13 shows the alpha diversity recovery results, where A is the Observed OTUs result, B is the Simpson index result, and C is the Shannon index result.

[0200]

[0201] Observed OTUs is a measure of the total number of operational taxonomic units (OTUs) or amplicon sequence variants (ASVs) present in a sample. The presence or absence of each OTU is not considered. By observing Observed OTUs, it was confirmed that the gut microbiota species of the combination group (HR) of Lactobacillus plantarum HAC03 strain and rutin increased compared to the group fed only a high-fat diet (HFD) (Fig. 13A).

[0202]

[0203] The Simpson index is a measurement method that considers the richness and evenness of species, and is a measurement method that places more weight on evenness.

[0204] Simpson index = 0: means infinite variety.

[0205] Simpson index = 1: means no diversity.

[0206] The Inverse Simpson index is simply the inverse of the Simpson index, and is set for easy comparison with other indices. By observing the Inverse Simpson index, we can confirm that the distribution of HR's gut microbiota increased not only in species but also in a more even distribution (Figure 13B).

[0207]

[0208] The Shannon index is a measure of species richness and evenness, placing greater emphasis on diversity. A higher value indicates greater species richness.

[0209] Shannon index = 0: means no diversity.

[0210] Shannon index = 1: means infinite diversity.

[0211] By observing the Shannon index, it can be confirmed that the distribution of intestinal microorganisms in HR increased not only in number of types but also in distribution evenly (Fig. 13C).

[0212]

[0213] Through this, it was confirmed that the diversity and uniformity of species increased in the group (HR) administered with the combination of Lactobacillus plantarum HAC03 strain and rutin.

[0214]

[0215] (2) Beta diversity recovery results

[0216] Figure 14 shows the results of beta diversity recovery. Beta diversity is an indicator of the similarity between microbial groups. When looking at the PCoA1 axis, the difference between the group that consumed a low-fat diet (LFD) and the group that received a combination of Lactobacillus plantarum HAC03 strain and rutin (HR) was the smallest. When looking at the Figure composed of PCoA1 and PCoA3, the difference between the group that received a combination of Lactobacillus plantarum HAC03 strain and rutin (HR) and the group that consumed a low-fat diet (LFD) was also small.

[0217]

[0218] Additionally, Figure 15 shows the results of observing the relative distribution of intestinal microorganisms at the Phylum (A) or Class (B) level.

[0219]

[0220] (3) Results of relative distribution of intestinal microorganisms

[0221] Figure 16 shows an analysis of the relative distribution of intestinal microorganisms. A represents Oscillospiraceae, B represents Lachnospiraceae, and C represents Eubacterium Coprostanoligenes.

[0222]

[0223] Oscillospiraceae is a genus whose abundance is significantly reduced in obese patients (Yang, Jingpeng, et al. "Oscillospira—a candidate for the next-generation probiotics." Gut Microbes 13.1 (2021): 1987783). Compared to the group that consumed only a high-fat diet (HFD), the group administered a combination of Lactobacillus plantarum HAC03 strain and rutin (HR) showed a significant increase in the relative distribution of bacteria (Fig. 16A).

[0224]

[0225] Lachnospiraceae is also a bacterium that significantly decreases in obese patients (Lippert, Katrin, et al. "Gut microbiota dysbiosis associated with glucose metabolism disorders and the metabolic syndrome in older adults." Beneficial microbes 8.4 (2017): 545-556.). Compared to the group that consumed only a high-fat diet (HFD), the group administered a combination of Lactobacillus plantarum HAC03 strain and rutin (HR) showed a significant increase in the relative distribution of bacteria (Fig. 16B).

[0226]

[0227] On the other hand, Eubacterium Coprostanoligenes is a bacterium abundant in patients with type 2 diabetes induced by obesity (Ahmad, Aftab, et al. "Analysis of gut microbiota of obese individuals with type 2 diabetes and healthy individuals." PloS one 14.12 (2019): e0226372.). Compared to the group that consumed only a high-fat diet (HFD), the relative distribution of bacteria was significantly reduced in the group administered a combination of Lactobacillus plantarum HAC03 strain and rutin (HR) (Fig. 16C).

[0228]

[0229] That is, when Lactobacillus plantarum HAC03 strain and rutin were administered in combination, it was confirmed that the microorganisms that significantly increased (Eubacterium Coprostanoligenes) or decreased (Oscillospiraceae, Lachnospiraceae) in obese patients showed an opposite tendency.

[0230]

[0231] Through this, it can be seen that combined administration of Lactobacillus plantarum HAC03 strain and rutin can effectively treat or prevent obesity or insulin resistance syndrome.

[0232]

[0233]

Claims

1. A pharmaceutical composition for preventing or treating obesity or insulin resistance syndrome, comprising Lactobacillus plantarum HAC03 strain (Accession No.: KCTC 13242BP) and Rutin as active ingredients. The pharmaceutical composition reduces at least one selected from the group consisting of total cholesterol, triglyceride, low density lipoprotein cholesterol (LDL), alanine aminotransferase (ALT), and aspartate aminotransferase (AST). A pharmaceutical composition for reducing blood sugar fluctuations, fasting blood sugar levels and fasting insulin levels.

2. In the first paragraph, the composition exhibits an anti-obesity effect by regulating the expression level of one or more genes selected from the group consisting of FAS (fatty acid synthase), ACC (acetyl CoA carboxylase), PPARγ (peroxisome proliferator-activated receptor γ), SREBP1C (sterol regulatory element binding protein 1C), PPARα (peroxisome proliferator-activated receptor α), PGC1α (peroxisome proliferator-activated receptor γ coactivator 1 α), CPT1 (carnitine palmitoyltransferase 1), ACOX1 (acyl-CoA oxidase 1), UCP1 (uncoupling protein 1), and PRDM16 (PR / SET Domain 16). A pharmaceutical composition characterized in that the anti-obesity effect is at least one effect selected from the group consisting of inhibition of fat synthesis, increase in fat oxidation, and increase in body heat generation.

3. A pharmaceutical composition according to claim 1, characterized in that the composition reduces insulin resistance by regulating the expression level of G6P (glucose-6-phosphate dehydrogenase) or PEPCK (phosphoenolpyruvate carboxykinase) genes.

4. A health functional food composition for preventing or improving obesity or insulin resistance syndrome, comprising Lactobacillus plantarum HAC03 strain (Accession No.: KCTC 13242BP) and Rutin as active ingredients. The above health functional food composition reduces at least one selected from the group consisting of total cholesterol, triglyceride, low density lipoprotein cholesterol (LDL), alanine aminotransferase (ALT), and aspartate aminotransferase (AST). A health functional food composition that reduces blood sugar fluctuations, fasting blood sugar levels, and fasting insulin levels.

5. In the fourth paragraph, the composition exhibits an anti-obesity effect by regulating the expression level of one or more genes selected from the group consisting of FAS (fatty acid synthase), ACC (acetyl CoA carboxylase), PPARγ (peroxisome proliferator-activated receptor γ), SREBP1C (sterol regulatory element binding protein 1C), PPARα (peroxisome proliferator-activated receptor α), PGC1α (peroxisome proliferator-activated receptor γ coactivator 1 α), CPT1 (carnitine palmitoyltransferase 1), ACOX1 (acyl-CoA oxidase 1), UCP1 (uncoupling protein 1), and PRDM16 (PR / SET Domain 16). A health functional food composition characterized in that the above anti-obesity effect is at least one effect selected from the group consisting of inhibition of fat synthesis, increase in fat oxidation, and increase in body heat generation.

6. A health functional food composition according to claim 4, characterized in that the composition reduces insulin resistance by regulating the expression level of G6P (glucose-6-phosphate dehydrogenase) or PEPCK (phosphoenolpyruvate carboxykinase) genes.

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