Composition for treating fatty liver comprising lactococcus lactis strain, amino acids, and vitamin

Abstract

The present invention relates to a composition for treating, ameliorating and preventing liver diseases, comprising a Lactococcus lactis strain, amino acids, and a vitamin. Specifically, the present invention relates to a composition for treating, ameliorating and preventing fatty liver, comprising Lactococcus lactis KF140, tryptophan, histidine, and vitamin E.

Description

Composition for treating fatty liver comprising Lactococcus lactis strain, amino acids and vitamins

[0001] The present invention relates to a composition for treating, improving, and preventing liver disease, comprising a Lactococcus lactis strain, amino acids, and vitamins. Specifically, the present invention relates to a composition for treating, improving, and preventing fatty liver disease, comprising Lactococcus lactis KF140, tryptophan, histidine, and vitamin E.

[0002]

[0003] Fatty liver disease refers to the abnormal accumulation of triglycerides within liver cells, and can lead to progressive liver diseases such as fibrosis and cirrhosis. Fatty liver disease also includes alcoholic fatty liver disease and non-alcoholic fatty liver disease.

[0004] Non-alcoholic fatty liver disease (NAFLD) is a type of disease that is not related to liver damage caused by alcohol, but histologically shows findings similar to alcoholic liver disease. This disease is a type of metabolic syndrome that includes a wide range of categories, from non-alcoholic fatty liver (NAFL) to non-alcoholic steatohepatitis (NASH) with inflammation or fibrosis, cirrhosis, and hepatocellular carcinomas. Non-alcoholic fatty liver disease has been steadily increasing recently due to a westernized diet, lack of exercise, and changes in lifestyle habits, and is increasing along with the increase in the obese and diabetic population. Currently, the incidence rate is about 30%, similar to that in the West.

[0005] As new knowledge and information about the causes and mechanisms of the disease accumulate, opinions have begun to emerge that the term "non-alcoholic fatty liver disease (NAFLD)" does not reflect the heterogeneous nature and diverse course of the disease. Because it is difficult to completely exclude alcohol consumption, and various studies have examined the effects of alcohol consumption on patients with NAFLD, a new term, "metabolic dysfunction associated fatty liver disease" (MAFLD), has recently been proposed to address these limitations. MAFLD can be diagnosed when steatosis is accompanied by overweight / obesity, diabetes, and metabolic abnormalities, and can coexist with other liver diseases.

[0006] Among the substances contributing to the development of nonalcoholic fatty liver disease (NAFLD) are advanced glycation end products (AGEs). These AGEs are biologically active compounds formed through nonenzymatic reactions between reducing sugars and proteins, lipids, and nucleic acids. A concept first discovered by Camille Maillard, AGEs are formed through the nonenzymatic reaction between reducing sugars and proteins under conditions of hyperglycemia. Once formed, they are difficult to break down. Even when blood sugar levels return to normal, they remain intact and bind to blood proteins and various tissues, causing organ damage. Furthermore, AGEs, formed through glycation reactions, are known to be associated with diseases such as aging, diabetic complications, and arteriosclerosis, in addition to their association with NAFLD, as previously mentioned.

[0007] Methylglyoxal (MGO), one of the substances that cause advanced glycation end products (AGEs), is known to be a highly reactive toxic substance in the body and has been raised as a cause of diabetes. Accordingly, there has been a recent demand for searching for substances that scavenge the main cause of AGEs. The most abundant AGE contained in the food we consume is CML (Carboxymethyl Lysine). Since CML accumulates in the largest amount in the liver, it has a very high possibility of being correlated with MAFLD or NASH. It also increases the malignancy of liver cancer cells by inducing oxidative stress by binding to the AGE receptor, which is an AGE receptor.

[0008] Nonalcoholic steatohepatitis (NASH) is characterized by abnormal fat accumulation or deposition in the liver (hepatic steatosis), liver inflammation, and liver damage or tissue damage (fibrosis). The prevalence of NASH is estimated at 2-4% worldwide (3-12% of US adults). While simple steatosis exhibits a slow histological progression, NASH exhibits a more rapid histological progression and can progress to cirrhosis. Approximately 5-10% of individuals diagnosed with fatty liver disease are also diagnosed with steatohepatitis.

[0009] Currently, there is no commercially available treatment for nonalcoholic steatohepatitis. Due to the absence of a treatment, other treatments for metabolic syndrome such as abdominal obesity, hyperlipidemia, and diabetes, such as insulin resistance-improving drugs, antioxidants (e.g., vitamins C and E), dyslipidemia treatments, and hepatoprotectors, are being used. However, it is difficult to view these as direct treatments for nonalcoholic steatohepatitis.

[0010] Nonalcoholic steatohepatitis (NASH) is a complex disease, and regulatory authorities in the US and Europe have set strict approval requirements for therapeutic agents. As a result, many treatments have recently failed in clinical development. Consequently, multi-agent-based treatments that aim to improve various indicators simultaneously are emerging. Currently, multi-agent NASH treatments in clinical trials include AstraZeneca's MEDI0382, a GLP-1 / glucagon (GCG) dual agonist, Eli Lilly's LY3298176, a GIP / GLP-1 dual agonist, and Hanmi Pharmaceutical's HM15211, a glucagon / GIP / GLP-1 triple agonist.

[0011] Probiotics are a collective term for live microorganisms that improve the intestinal microbiota and have a beneficial effect on the host's health. Lactic acid bacteria, when ingested, live in symbiosis with the digestive system, contributing to the breakdown of fiber and complex proteins, thereby contributing to their important nutritional value. Lactic acid bacteria have been reported to maintain and improve intestinal flora, exert anti-diabetic effects, suppress colitis, and enhance the immune system.

[0012] Accordingly, the inventors of the present invention completed the present invention by confirming that a composition containing Lactococcus lactis KF140, tryptophan, histidine, and vitamin E has an excellent effect in suppressing fatty liver formation while having no side effects.

[0013]

[0014] [Prior Art Literature]

[0015] [Patent Document]

[0016] (Patent Document 1) Korean Patent Publication No. 10-2020-0131739

[0017] (Patent Document 2) Korean Patent No. 10-1841021

[0018] (Patent Document 3) Korean Patent Publication No. 10-2021-0095580

[0019]

[0020] The present invention aims to provide a composition for treating, preventing or improving fatty liver, comprising a Lactococcus lactis strain, amino acids and vitamins.

[0021] Specifically, the present invention aims to provide a pharmaceutical composition for treating or preventing fatty liver comprising Lactococcus lactis KF140 (accession number KCCM11673P), tryptophan, histidine, and vitamin E.

[0022] The present invention aims to provide a composition for animal feed for treating or improving fatty liver, comprising Lactococcus lactis KF140 (accession number KCCM11673P), tryptophan, histidine, and vitamin E.

[0023] The present invention aims to provide a food composition for treating or improving fatty liver, comprising Lactococcus lactis KF140 (accession number KCCM11673P), tryptophan, histidine, and vitamin E.

[0024]

[0025] The present invention relates to a composition for treating, preventing or improving fatty liver, comprising a Lactococcus lactis strain, amino acids and vitamins.

[0026] The above Lactococcus lactis strain may be Lactococcus lactis KF140 (accession number KCCM11673P).

[0027] Additionally, the amino acid may be one or more selected from the group consisting of tryptophan and histidine, but is not limited thereto.

[0028] Additionally, the vitamin may preferably be, but is not limited to, vitamin E.

[0029] The composition of the present invention can reduce levels of AST, ALT, HbA1C, or γ-GTP. In addition, the composition of the present invention can reduce levels of fibrosis or fatty liver.

[0030] In one embodiment, the present invention relates to a pharmaceutical composition for treating or preventing fatty liver comprising Lactococcus lactis KF140 (accession number KCCM11673P), tryptophan, histidine, and vitamin E.

[0031] In one embodiment, the present invention relates to an animal feed composition for improving or preventing fatty liver, comprising Lactococcus lactis KF140 (accession number KCCM11673P), tryptophan, histidine, and vitamin E.

[0032] In one embodiment, the present invention relates to a food composition for improving or preventing fatty liver comprising Lactococcus lactis KF140 (accession number KCCM11673P), tryptophan, histidine, and vitamin E.

[0033] In the present invention, fatty liver may be, but is not limited to, alcoholic fatty liver disease, non-alcoholic fatty liver disease (NAFLD), steatohepatitis, liver fibrosis, cirrhosis or liver cirrhosis.

[0034]

[0035] The composition of the present invention exhibits excellent effects in suppressing the activity of AST, ALT, HbA1C, or γ-GTP, and reducing fatty liver and fibrosis levels. Therefore, the complex composition of the present invention can be effectively used for the treatment, improvement, and prevention of fatty liver disease.

[0036]

[0037] Figure 1 presents an overview of the design of a randomized, double-blind, placebo-controlled parallel trial.

[0038] Figure 2 shows the change in AST (Aspartate Aminotransferase) levels in the control group (placebo), test group 1 (administration of KF140 alone), and test group 2 (administration of KF140, tryptophan, histidine, and vitamin E combination) in a clinical trial targeting patients with fatty liver disease.

[0039] Figure 3 shows the change in ALT (Alanine Aminotransferase) levels in the control group (placebo), test group 1 (administration of KF140 alone), and test group 2 (administration of KF140, tryptophan, histidine, and vitamin E combination) in a clinical trial targeting patients with fatty liver disease.

[0040] Figure 4 shows the change in HbA1C levels in the control group (placebo), test group 1 (administration of KF140 alone), and test group 2 (administration of KF140, tryptophan, histidine, and vitamin E combination) in a clinical trial targeting patients with fatty liver disease.

[0041] Figure 5 shows the change in γ-GTP levels in the control group (placebo), test group 1 (administration of KF140 alone), and test group 2 (administration of KF140, tryptophan, histidine, and vitamin E combination) in a clinical trial targeting patients with fatty liver disease.

[0042] Figure 6 shows the change in fibrosis levels in the control group (placebo), test group 1 (administration of KF140 alone), and test group 2 (administration of KF140, tryptophan, histidine, and vitamin E combination) in a clinical trial targeting patients with fatty liver disease.

[0043] Figure 7 shows the change in the hepatic steatosis index in the control group (placebo), test group 1 (administration of KF140 alone), and test group 2 (administration of KF140, tryptophan, histidine, and vitamin E combination) in a clinical trial targeting patients with fatty liver disease.

[0044] Figure 8 shows the results of evaluating the efficacy of inhibiting fat accumulation in liver cells.

[0045] Figure 9 shows the results of evaluating ALT inhibition efficacy in hepatocytes.

[0046]

[0047] Hereinafter, with reference to the attached drawings, embodiments and examples of the present invention will be described in detail so that those skilled in the art can easily implement the present invention. However, the present invention may be implemented in various forms and is not limited to the embodiments and examples described herein.

[0048] Throughout this specification, whenever a part is said to "include" a component, this means that it may include other components, but not to the exclusion of other components, unless otherwise stated.

[0049] The "Lactococcus lactis KF140" of the present invention was deposited with the Korea Culture Collection for Microorganisms (KCCM), an international microorganism depository, and was assigned the deposit number KCCM 11673P.

[0050] The term "prevention" in the present invention means any act of inhibiting or delaying the onset of a disease by administering a composition, "treatment" means any act of improving or beneficially changing the symptoms of a subject suspected of or suffering from a disease by administering a composition, and "improvement" means any act of at least reducing a parameter related to a condition, for example, the degree of a symptom, by administering a composition.

[0051] The animal feed composition can be fed to all non-human animals, such as non-human primates, sheep, dogs, cows, horses, etc.

[0052] The present invention will be described in more detail through the following examples; however, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0053]

[0054] [Example 1]

[0055] Preparing food for testing

[0056]

[0057] A test food was prepared using the ingredient combination shown in Table 1 below. The raw material for the food used as a control group (placebo) was a mixture of dextrin and soybean oil, excluding Lactococcus lactis KF140 and amino acids from the test food raw material, and excluding vitamin E.

[0058]

[0059]

[0060]

[0061] The above test food was already in the form of hard and soft capsules without any off-flavor, and the capsules (live bacteria 3.0Х10^10 CFU / capsule / once a day, amino acid 200mg / capsule / twice a day, vitamin E 400IU / capsule / once a day) were stored at 2-8℃.

[0062]

[0063] [Example 2]

[0064] Test design

[0065]

[0066] This trial was designed as a randomized, double-blind, placebo-controlled parallel trial, and an outline of the trial is briefly presented in Figure 1.

[0067] When a subject of a human application study who voluntarily signed the consent form for a human application study participated in this human application study, a demographic survey, a lifestyle survey, a medical history and medication history survey, vital signs (blood pressure, pulse) measurement, anthropometric measurements (height, weight), a physical examination, a clinical pathology test, a pregnancy test (only for women of childbearing age), a drinking habit questionnaire, a blood test (efficacy evaluation), and Fibroscan were conducted, and if they met the inclusion / exclusion criteria, they were enrolled as a subject of the human application study through random assignment. The subject of the human application study assigned to Test Group I, Test Group II, or the control group consumed the test food or the control food for a total of 26 weeks. The allocation ratio of each group was Test Group 1: Test Group 2: Control Group = 1:1:1.

[0068]

[0069] Control group: placebo

[0070] Test group 1: KF140 administered alone

[0071] Test group 2: KF140, tryptophan, histidine, and vitamin E combination administration

[0072]

[0073] [Example 3]

[0074] Selection of subjects for human application testing

[0075]

[0076] <Selection Criteria>

[0077] 1) Adult men and women aged 19 or older and under 75 years old

[0078] 2) Those who meet one of the following criteria according to the diagnostic criteria for fatty liver disease related to metabolic disorders: A. Those with 50 U / L ≤ ALT ≤ 250 U / L as of Visit 1 B. Those with Fibrosis stage F1 or F2 as of Visit 1 C. Those with BMI ≥ 23 kg / m2

[0079] 3) Those with glycated hemoglobin (HbA1c) ≥ 6.5% as of Visit 1

[0080] 4) A person who agrees to participate in the human application test and voluntarily completes the written informed consent form before the human application test begins.

[0081]

[0082] <Exclusion Criteria>

[0083] 1) Those currently receiving treatment for severe cardiovascular, immune, respiratory, gastrointestinal / liver and biliary, renal and urinary, nervous, musculoskeletal, psychiatric, infectious, and malignant tumor diseases.

[0084] 2) Patients with chronic alcoholism {within 4 weeks of visit 1, drinking alcohol equivalent to an average of 30 g or more per day (210 g / week) for men and an average of 20 g or more per day (140 g / week) for women}

[0085] 3) Those with a history of other chronic liver diseases (viral hepatitis B or C, autoimmune hepatitis, cholestasis, and hereditary metabolic liver disease)

[0086] 4) Patients with cirrhosis based on clinical criteria or liver histology or imaging techniques.

[0087] 5) Patients with HIV positive, hemochromatosis, active tuberculosis, active malaria, or inflammatory bowel disease

[0088] 6) Those with active bleeding

[0089] 7) Patients with uncontrolled thyroid disease (TSH is 0.1 μIU / mL or less or 10 μIU / mL or more)

[0090] 8) Patients with uncontrolled hypertension (systolic blood pressure of 160 mmHg or higher or diastolic blood pressure of 100 mmHg or higher, based on measurements taken after 10 minutes of rest in human subjects)

[0091] 9) Patients with uncontrolled diabetes (fasting blood sugar level 180 mg / dL or higher) Metacentre Therapeutics Co., Ltd. Clinical Study Report (Version 1.0) Protocol No.: MCT101-FL1 (Version 1.4) 48 / 229 NeoNutra co., Ltd. (www.NeoNutra.com) Confidential

[0092] 10) Those whose blood creatinine level is more than twice the upper limit of normal of the institution conducting the test.

[0093] 11) Those who have administered or consumed probiotics or lactic acid bacteria products continuously (more than 4 times a week) within 2 weeks of Visit 1

[0094] 12) Those who have been administered antibiotics or immunosuppressants within 2 weeks of Visit 1

[0095] 13) Those using thyroid hormones, estrogen, amiodarone, steroids, tamoxifen, and other drugs known to affect liver fat accumulation (However, those who have been taking thyroid hormones stably for more than 2 months as of Visit 1 may participate in the human application trial)

[0096] 14) Those who have taken obesity treatment medication within 1 month (30 days) prior to visit 1

[0097] 15) Those who are sensitive or allergic to the food ingredients used in this human application test.

[0098] 16) Pregnant or lactating women or those planning to become pregnant during the human application test period

[0099] 17) Those who have participated in another interventional clinical trial (including human application trials) within 3 months of Visit 1 or plan to participate in another interventional clinical trial (including human application trials) after the start of this human application trial.

[0100] 18) If the tester determines that the subject is unsuitable for participation in the human application test

[0101] Subjects for the human application test of the present invention were selected according to the above selection criteria, and those who had used hormonal preparations such as female hormones or similar hormonal preparations (plant extracts, etc.) within the past 3 months, those who were taking medication for the purpose of improving menopausal symptoms, or those who were being treated for a disease or taking medication were excluded. A total of 85 menopausal female subjects were selected, and the experimental period was 12 weeks.

[0102]

[0103] [Example 1]

[0104] Analysis of liver function improvement effects

[0105]

[0106] The blood samples collected from the subjects of the above test before and after consuming the food for 26 weeks were used to measure changes in blood AST (Aspartate Aminotransferase), ALT (Alanine Aminotransferase), γ-GTP, HbA1C, fibrosis index, and hepatic steatosis index.

[0107] While AST is also present in heart, muscle, and kidney cells, ALT is a hepatocyte-specific enzyme found only in the liver. Therefore, elevated ALT levels directly reflect hepatotoxicity. It can be easily measured in the blood and is widely used because it indicates the potential for liver damage.

[0108] HbA1c (glycated hemoglobin) reflects the blood sugar status for 2-3 months prior to the test, so it is used as a guideline for diabetes treatment and a good predictor of complications. It has little intra-individual variation, is not affected by diet, and is more convenient to test than insulin resistance tests or glucose tolerance tests, so it is widely used as a glucose homeostasis test during health checkups.

[0109] Most serum γ-GTP originates from liver and biliary tract epithelial cells. Recently, γ-GTP has been shown to be a marker of internal organ fat in patients with fatty liver disease. High levels of γ-GTP may be a risk factor for type 2 diabetes, and thus can be used as an indicator of internal organ fat, fatty liver disease, and insulin resistance. Combination Marker testing utilizes a variety of noninvasive methods to diagnose simple fatty liver disease and liver fibrosis.

[0110] The Hepatic Steatosis Index (HSI) is a tool for predicting hepatic steatosis, and calculates a score based on ALT and AST levels, BMI, gender, and the presence or absence of diabetes.

[0111] Measuring fibrosis using Fibroscan utilizes the fact that liver stiffness and liver fibrosis are related, and that liver elasticity is also related to liver stiffness. Consequently, liver stiffness and liver fibrosis have a deep correlation, and liver fibrosis can be predicted by measuring liver elasticity.

[0112] The results of the above experiment are shown in Figures 2 to 7. As shown in Figures 2 to 7, compared to the control group and test group 1, it was confirmed that test group 2 significantly reduced AST (Aspartate Aminotransferase), ALT (Alanine Aminotransferase), HbA1C, γ-GTP, fibrosis level, and hepatic steatosis index for 26 weeks.

[0113]

[0114] [Example 2]

[0115] Evaluation of the inhibitory effect on lipogenesis in hepatocytes

[0116]

[0117] For the proliferation of HepG2 cells, a human hepatoma cell line, cells were grown in DMEM (gilbco, Grand Island, NY) supplemented with 10% (v / v) Fetal Bovine Serum (FBS) and 1% (v / v) penicillin / streptomycin (P / S) in a 5% CO2 incubator at 37℃. When the confluency of HepG2 cells reached 70–80%, they were subcultured in 6-well plates. On the second day of subculture, samples (100 μg / mL each of amino acids and vitamin E or KF140 culture medium) were treated together with free fatty acids (0.25 mM). Oil red O staining was performed 24 hours after treatment with free fatty acids and samples.

[0118]

[0119] Control: Palmitic Acid (0.25 mM)

[0120] Test group 1: Palmitic acid + tryptophan

[0121] Test group 2: Palmitic acid + histidine

[0122] Test group 3: Palmitic acid + vitamin E

[0123] Test group 4: Palmitic acid + tryptophan, histidine, and vitamin E

[0124] Test group 5: Palmitic Acid + KF140

[0125] Test group 6: Palmitic acid + tryptophan, histidine, vitamin E, KF140

[0126]

[0127] The results of the above experiment are shown in Table 2 and Fig. 8.

[0128]

[0129]

[0130]

[0131]

[0132] As shown in Table 2 and Figure 8, it was confirmed that the fat accumulation inhibition effect was significantly superior when KF140, tryptophan, histidine, and vitamin E were administered separately, compared to when tryptophan, histidine, and vitamin E were administered in combination.

[0133]

[0134] [Example 3]

[0135] Evaluation of the efficacy of inhibiting ALT activity in hepatocytes

[0136]

[0137] For the proliferation of HepG2 cells, a human hepatoma cell line, cells were grown in DMEM (gilbco, Grand Island, NY) supplemented with 10% (v / v) Fetal Bovine Serum (FBS) and 1% (v / v) penicillin / streptomycin (P / S) in a 5% CO2 incubator at 37℃. When the confluency of HepG2 cells reached 70–80%, they were subcultured in 6-well plates. On the second day of subculture, samples (100 μg / mL each of amino acids and vitamin E or KF140 culture medium) were treated together with free fatty acids (0.25 mM). The inhibitory efficacy of ALT activity was evaluated 24 hours after treatment with free fatty acids and samples.

[0138]

[0139] Control: Palmitic Acid (0.25 mM)

[0140] Test group 1: Palmitic acid + tryptophan

[0141] Test group 2: Palmitic acid + histidine

[0142] Test group 3: Palmitic acid + vitamin E

[0143] Test group 4: Palmitic acid + tryptophan, histidine, and vitamin E

[0144] Test group 5: Palmitic Acid + KF140

[0145] Test group 6: Palmitic acid + tryptophan, histidine, vitamin E, KF140

[0146]

[0147] The results of the above experiment are shown in Table 3 and Fig. 9.

[0148]

[0149]

[0150]

[0151] As shown in Table 3 and Figure 9, it was confirmed that the ALT inhibition efficacy was significantly superior when KF140, tryptophan, histidine, and vitamin E were administered separately, compared to when tryptophan, histidine, and vitamin E were administered in combination.

Claims

1. A composition for treating, preventing or improving fatty liver comprising a Lactococcus lactis strain, amino acids and vitamins.

2. A composition for treating, preventing or improving fatty liver, wherein the Lactococcus lactis strain in paragraph 1 is Lactococcus lactis KF140 (accession number KCCM11673P).

3. A composition for treating, preventing or improving fatty liver, wherein the amino acid in paragraph 1 is at least one selected from the group consisting of tryptophan and histidine.

4. A composition for treating, preventing or improving fatty liver, wherein the vitamin in paragraph 1 is vitamin E.

5. A composition for treating, preventing or improving fatty liver, which reduces one or more values ​​selected from the group consisting of AST, ALT, HbA1C, and γ-GTP in the first paragraph.

6. A composition for treating, preventing or improving fatty liver, which reduces fibrosis or fatty liver index in accordance with paragraph 1.

7. A pharmaceutical composition for treating or preventing fatty liver, comprising Lactococcus lactis KF140 (accession number KCCM11673P), tryptophan, histidine, and vitamin E in the first paragraph.

8. A composition for animal feed for improving or preventing fatty liver, comprising Lactococcus lactis KF140 (accession number KCCM11673P), tryptophan, histidine, and vitamin E in the first paragraph.

9. A food composition for improving or preventing fatty liver, comprising Lactococcus lactis KF140 (accession number KCCM11673P), tryptophan, histidine, and vitamin E in the first paragraph.

10. A composition according to claim 1, wherein the fatty liver is at least one selected from the group consisting of alcoholic fatty liver disease, non-alcoholic fatty liver disease (NAFLD), steatohepatitis, liver fibrosis, cirrhosis, and liver cirrhosis.

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