Composition comprising placenta extract for preventing or treating liver disease and improving liver function

Abstract

The present invention relates to a composition comprising a placenta extract for preventing or treating liver diseases and improving liver function. More specifically, by comprising a placenta extract, the present invention can reduce fat accumulation associated with fatty liver and reduce serum AST activity and ALT activity, and thus can exhibit a prophylactic or therapeutic effect on fatty liver diseases such as non-alcoholic fatty liver or alcoholic fatty liver, and can exhibit an effect of improving liver health.

Description

[0001] This application is a divisional application of a Chinese patent application with an application date of March 12, 2021, an application number of 202180020552.7, and an invention title of "Composition Comprising Placental Extract for Preventing or Treating Liver Diseases and Improving Liver Function". Technical Field

[0002] The present invention relates to a composition for preventing or treating liver diseases and for improving liver function. Background Art

[0003] Non-alcoholic fatty liver disease (NAFLD) is defined as the accumulation of triglycerides, which are neutral fats, in hepatocytes without excessive alcohol or drug-induced fatty liver. Non-alcoholic fatty liver disease continues to increase due to the nutritional excess associated with the high-fat and high-carbohydrate intake of modern people. It has been reported that 80% of adults with non-alcoholic fatty liver disease develop metabolic diseases such as insulin-resistant diabetes and heart diseases.

[0004] Non-alcoholic fatty liver disease refers to a wide range of liver diseases, including non-alcoholic simple steatosis, non-alcoholic steatohepatitis (NASH), and other liver diseases that progress to cirrhosis associated with non-alcoholic fatty liver. Pathologically, if not paid attention to for a long time, non-alcoholic fatty liver diseases such as non-alcoholic simple steatosis and non-alcoholic steatohepatitis with inflammation may progress to severe liver diseases such as hepatitis, liver fibrosis, or cirrhosis. Non-alcoholic fatty liver disease is characterized by fat accumulation (fat infiltration) in hepatocytes. Non-alcoholic simple steatosis may progress to non-alcoholic steatohepatitis. Fat accumulation in non-alcoholic steatohepatitis is associated with varying degrees of liver inflammation and scar formation, and is often associated with insulin resistance, dyslipidemia, and hypertension.

[0005] The pathogenesis of non-alcoholic steatohepatitis has been explained by the two-hit hypothesis. First, fat accumulation occurs in liver tissue, and then, when the fat accumulation in liver tissue becomes severe, an inflammatory reaction occurs, which leads to an exacerbation of lipid peroxidation and inflammation. Although the number of patients with non-alcoholic steatohepatitis is increasing rapidly, there is no excellent therapeutic agent for non-alcoholic steatohepatitis. Since the number of non-alcoholic steatohepatitis patients increases with the increase in the obese population, the market for non-alcoholic steatohepatitis therapeutics has grown to a huge market size. In addition, since the cause and mechanism of non-alcoholic steatohepatitis have been disclosed, the development of therapeutics for non-alcoholic steatohepatitis is currently attracting attention. However, the development of safe and long-term administrable therapeutics for non-alcoholic steatohepatitis is still at a negligible level. Generally, for the treatment of non-alcoholic simple steatosis or non-alcoholic steatohepatitis, anti-obesity drugs, insulin resistance drugs, hyperlipidemia drugs, hepatoprotective agents, and antioxidants are used. However, these drugs are used as symptomatic improvers rather than as essential therapeutics for non-alcoholic simple steatosis or non-alcoholic steatohepatitis, and they involve side effects when taken for a long time. Therefore, there is an increasing demand for the development of new therapeutic compositions that are safer and can be taken for a long time and are ultimately suitable for treating non-alcoholic simple steatosis or non-alcoholic steatohepatitis as chronic diseases.

[0006] In particular, although the prevalence of non-alcoholic simple steatosis and non-alcoholic steatohepatitis is high in Korea, the development of drugs for preventing, improving, alleviating, or treating non-alcoholic simple steatosis or non-alcoholic steatohepatitis is substantially at a low level. Considering these problems, research on non-alcoholic simple steatosis or non-alcoholic steatohepatitis using natural products is continuing. In the case of therapeutics for non-alcoholic simple fatty liver and non-alcoholic steatohepatitis made from these natural products, since the content of active ingredients in natural extracts is low, a large amount needs to be used to obtain a therapeutic effect on non-alcoholic simple fatty liver or non-alcoholic steatohepatitis. In fact, most of the above-mentioned therapeutics are only marketed for their use of natural ingredients. More scientific research is needed on the actual efficacy of the treatment of non-alcoholic simple fatty liver or non-alcoholic steatohepatitis.

[0007] On the other hand, human placental extract (HPE) contains various growth factors, cytokines, and other physiologically active substances, and is widely used for relieving fatigue, antioxidant effects, etc. (Lee KK et al., Evid Based Complement Alternat. Med., 2012, Vol. (2012), p. 130875). However, despite the great interest in human placental extract, its functions have not been fully studied. SUMMARY OF THE INVENTION

[0008] Problems to be Solved by the Invention

[0009] An object of the present invention is to provide a pharmaceutical composition for preventing or treating fatty liver disease.

[0010] Another object of the present invention is to provide a food composition for improving liver health.

[0011] Solutions for Solving the Problems

[0012] 1. A pharmaceutical composition for preventing or treating fatty liver disease, which comprises a placental extract.

[0013] 2. The pharmaceutical composition according to 1 above, wherein the placental extract is a human placental extract.

[0014] 3. The pharmaceutical composition according to 1 above, wherein the placental extract is obtained by treating the placenta with pepsin and hydrochloric acid.

[0015] 4. The pharmaceutical composition according to 3 above, wherein the placenta is defatted by acetone treatment.

[0016] 5. The pharmaceutical composition according to 3 above, wherein, after the treatment with pepsin and hydrochloric acid, the placental extract is obtained by purifying more than once by anion exchange chromatography.

[0017] 6. The pharmaceutical composition according to 5 above, wherein the functional group of the anion exchange chromatography resin is selected from the group consisting of Q, QAE, TEAE, and DEAE.

[0018] 7. The pharmaceutical composition according to 6 above, wherein the anion exchange chromatography is carried out by using a column selected from the group consisting of a cross-linked agarose column, a polymethacrylate resin column, a hydrogel column, and a cross-linked polymethacrylate resin column.

[0019] 8. The pharmaceutical composition according to 1 above, wherein the pharmaceutical composition is administered by any one or more of the administration methods of intravenous (IV) injection, subcutaneous (SC) injection, and intramuscular (IM) injection.

[0020] 9. The pharmaceutical composition according to 1 above, wherein the fatty liver disease includes non-alcoholic fatty liver disease or alcoholic fatty liver disease.

[0021] Effects of the Invention

[0022] The pharmaceutical composition of the present invention reduces fat accumulation related to fatty liver and decreases the AST level and ALT level in serum. Therefore, it can exhibit the effects of preventing or treating fatty liver diseases such as non-alcoholic fatty liver and alcoholic fatty liver, and can show the effect of improving liver health. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a flowchart showing the process of preparing a placental extract according to an embodiment.

[0024] Figure 2 It shows a heatmap result confirming a decrease in the degree of fat accumulation in liver tissue when a tamoxifen-induced non-alcoholic fatty liver disease model is treated with placental extracts at different concentrations by LipidGreen2 staining.

[0025] Figure 3 It shows the quantitative analysis results confirming a decrease in the degree of fat accumulation in liver tissue when a tamoxifen-induced non-alcoholic fatty liver disease model is treated with placental extracts at different concentrations.

[0026] Figure 4 It shows the experimental results confirming the change in AST when a high-fat diet mouse model is treated with placental extract.

[0027] Figure 5 It shows the experimental results confirming the change in ALT when a high-fat diet mouse model is treated with placental extract. DETAILED DESCRIPTION

[0028] The present invention provides a pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease, which comprises a placental extract.

[0029] The term "placental extract" refers to an extract obtained from the placenta of a human or an animal. The extract can be obtained by treating the placenta provided by a human or an animal with an acid and / or an enzyme. At this time, the animal can include mammals, and specifically humans, cows, horses, sheep, or pigs, etc.

[0030] The term "placenta" refers to an organ produced during pregnancy, which supplies nutrients and enzymes from the mother to the fetus, discharges waste and carbon dioxide from the fetus to the mother, prevents pathogens or drugs as foreign substances in the living body from transferring to the fetus, and has a fetal endocrine regulation function.

[0031] The placenta contains amino acids, proteins, sugars, nucleic acids, lipids, minerals, enzymes, hormones, etc. At this point, the amino acids can include, for example, aspartic acid, glutamic acid, leucine, lysine, glycine, alanine, serine, threonine, phenylalanine, tyrosine, methionine, histidine, etc. The proteins and enzymes can include, for example, albumin, globulin, acidic and alkaline phosphatase, hyaluronidase, etc. The sugars can include, for example, glucose, galactose, ribose, etc. The nucleic acids can include, for example, uracil, xanthine, hypoxanthine, etc. In addition, the lipids can include, for example, lauric acid, palmitic acid, linoleic acid, etc. The minerals can include Na, K, Ca, P, Fe, Cl, etc., and the hormones can include gonadotropin, prolactin, steroid hormones, etc.

[0032] The placental extract can be obtained by treating the placenta with enzymes and / or acids.

[0033] According to an embodiment, the placental extract can be obtained by treating the placenta with pepsin and hydrochloric acid.

[0034] The placental extract can be obtained by treating the placenta with pepsin and hydrochloric acid and then purifying it by chromatography more than once.

[0035] The placental extract prepared by chromatographic purification can be effectively obtained, which has reduced impurities compared with the placental extract obtained without chromatographic purification.

[0036] The chromatography used herein can be anion-exchange chromatography or cation-exchange chromatography, but is not limited thereto.

[0037] According to an embodiment, the chromatography can be anion-exchange chromatography.

[0038] The functional groups of the anion-exchange chromatography resin can be selected from the group consisting of Q, QAE, TEAE, and DEAE.

[0039] The anion-exchange chromatography can be carried out by using a column selected from the group consisting of a cross-linked agarose column, a polymethacrylate resin column, a hydrogel column, and a cross-linked polymethacrylate resin column.

[0040] The placenta can be defatted. According to an embodiment, the placenta treated with enzymes and / or acids can be defatted by acetone treatment.

[0041] The placenta can be prepared in a ground form. The ground placenta can be obtained by finely chopping or grinding the placenta provided from a human or an animal according to any method applicable to those skilled in the art.

[0042] Fatty liver disease refers to a disease in which fat accumulates in the liver at a content of more than about 5% of the liver weight, and may include non-alcoholic fatty liver disease caused by non-alcoholic reasons or alcoholic fatty liver disease caused by alcoholic reasons.

[0043] Non-alcoholic fatty liver disease (NAFLD) is a disease involving the following medical viewpoints: in the absence of significant alcohol intake, administration of drugs causing fatty liver, liver diseases caused by other accompanying reasons, etc., according to radiological imaging tests or biopsies, fat accumulates in the liver.

[0044] Non-alcoholic fatty liver disease may include, for example, non-alcoholic steatosis, non-alcoholic simple steatosis disease, non-alcoholic nutritional fatty liver disease, non-alcoholic starvation fatty liver disease, non-alcoholic obesity fatty liver disease, non-alcoholic diabetic fatty liver disease, non-alcoholic steatohepatitis (NASH), non-alcoholic hepatocirrhosis, non-alcoholic liver fibrosis, or non-alcoholic liver cirrhosis, etc., but is not limited thereto.

[0045] The term "treatment" refers to any action that improves or beneficially changes the symptoms of an individual suspected of having fatty liver and / or affected by fatty liver.

[0046] The term "prevention" refers to any action that inhibits or delays fatty liver.

[0047] According to an embodiment of the present invention, the preventive and therapeutic effects of liver diseases using placental extract are observed on a liver disease model. According to an embodiment of the present invention, the liver function improvement activity is confirmed by treating a fatty liver animal model with placental extract.

[0048] The pharmaceutical composition of the present invention may contain placental extract in an amount of 0.01% to 1% by volume ("vol. %") (v / v) based on the total volume of the composition.

[0049] The pharmaceutical composition of the present invention may contain placental extract in an amount of 0.01% to 1% by volume, 0.01% to 0.9% by volume, 0.01% to 0.8% by volume, 0.01% to 0.7% by volume, 0.01% to 0.6% by volume, 0.01% to 0.5% by volume, 0.01% to 0.4% by volume, 0.01% to 0.3% by volume, 0.01% to 0.2% by volume, or 0.01% to 0.1% by volume (v / v) based on the total volume of the composition.

[0050] When the pharmaceutical composition contains an excessive amount of placental extract, cytotoxicity appears and may cause difficulties in using the pharmaceutical composition. On the other hand, when the pharmaceutical composition contains a trace amount of placental extract, the desired medical effect may not be obtained.

[0051] The preparation of the pharmaceutical composition according to the present invention may be in the form of an injection or a skin application agent, for example, any form selected from the group consisting of injections, micro-needles, rollers, oral tablets, oral capsules, and oral granules and combinations thereof.

[0052] The administration routes of the pharmaceutical composition according to the present invention may include oral, intravenous, intramuscular, intra-arterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal routes, but are not limited thereto.

[0053] The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, skin application or intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection can be selected for administration.

[0054] The pharmaceutical composition of the present invention can be administered at a dose of 1 to 10 mL per day. For example, the composition can be administered in vivo at a dose of 1 to 10 mL, 1 to 8 mL, 1 to 6 mL, 1 to 4 mL, or 1 to 2 mL per day.

[0055] The pharmaceutical composition of the present invention can be administered once a day, twice a day, three times a day, four times a day, or more frequently.

[0056] In addition, the present invention provides a food composition for improving liver health, which contains placental extract.

[0057] The content of the placental extract has been described above, and thus will not be described in detail.

[0058] The food composition according to the present invention can have any preparation selected from the group consisting of functional foods, nutritional supplements, health foods, food additives, livestock feeds, and combinations thereof.

[0059] The food composition of the present invention can contain placental extract in an amount of 0.01% to 1% (v / v) based on the total volume of the composition.

[0060] The food composition of the present invention may contain placental extract in an amount of 0.01% to 1% by volume, 0.01% to 0.9% by volume, 0.01% to 0.8% by volume, 0.01% to 0.7% by volume, 0.01% to 0.6% by volume, 0.01% to 0.5% by volume, 0.01% to 0.4% by volume, 0.01% to 0.3% by volume, 0.01% to 0.2% by volume, or 0.01% to 0.1% by volume (v / v) based on the total volume of the composition.

[0061] When the food composition contains an excessive amount of placental extract, cytotoxicity occurs and may cause difficulties in using the food composition. On the other hand, when the food composition contains a trace amount of placental extract, the effect of improving liver health may not be obtained.

[0062] Hereinafter, the present invention will be described in more detail by way of the following examples. However, the following examples are provided for illustrative purposes only, and the scope of the present invention is not particularly limited thereto.

[0063] Example 1. Preparation of Placenta Extract

[0064] After defatting the placenta with acetone, the placenta was sufficiently hydrolyzed by pepsin and hydrochloric acid treatment to prevent the production of incompletely hydrolyzed products, thereby preparing a placental extract (see Figure 1 ). In addition, the placental extract was purified by anion exchange chromatography. This process is an example of a method for treating the placenta to prepare a placental hydrolyzate, and thus, the method for preparing a placental hydrolyzate is not particularly limited thereto.

[0065] Experimental Example 1. Confirmation of Reduction in Fatty Liver Size of Placenta Extract in Non-alcoholic Fatty Liver Zebrafish Model Effect

[0066] 1. Experimental procedure

[0067] To study the effect of reducing fat accumulation in liver tissue through placental extract, a zebrafish model with tamoxifen-induced non-alcoholic steatosis was used to confirm the medical effect. Zebrafish embryos at 5 days post-fertilization were distributed in 24-well plates, 5 to 7 per well, and then 5 μM tamoxifen was combined with placental extract diluted to an appropriate concentration in a 0.06% sea salt solution. When zebrafish were treated with the placental extract of the present invention at a concentration of 1% or more, it was found that the zebrafish individuals did not survive but died. Therefore, this experiment was carried out using the product of the present invention at concentration ranges of 0.05%, 0.1%, and 0.5% respectively. After 24 hours of treatment (6 days post-fertilization), the product was replaced with 5 μM LipidGreen 2 solution, and then the reaction was carried out for 30 minutes under light-shielded conditions. After anesthetizing the zebrafish embryos with tricaine, the embryos were fixed in 3% methylcellulose, and the degree of staining of the liver part was imaged using a fluorescence microscope. In addition, the area and fluorescence intensity of the fluorescently stained liver were quantitatively analyzed through the Gen5 (BioTek) program. In addition, the Edit LUT function in the Image J (NIH) program was used to perform heat map image conversion.

[0068] 2. Experimental results

[0069] Figure 2 Shows the liver region of zebrafish at 6 days post-fertilization after completion of fat staining.

[0070] Figure 2 The upper row of images (black / white images) in [reference] is the images obtained through the GFP filter (469 nm and 525 nm), which means that fat accumulation increases towards the white side. The dotted part in each image indicates the liver part of the zebrafish. According to Figure 2 In the upper row of [reference], it can be confirmed that steatosis accumulates through tamoxifen ( Figure 2 In [reference], tamoxifen + 0% placental extract). In addition, Figure 2 The lower row of images (images represented from blue to red) in [reference] is the images converted into heat map form through the ImageJ program, so as to more easily show the fluorescence degree of the upper row of images. The part close to red in the liver part indicates fatty liver.

[0071] Figure 3 Is a figure showing the results of Figure 2 , where statistical significance is defined as *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001.

[0072] Although there was no difference in liver size between normal individuals (DMSO-treated group) and steatotic individuals (tamoxifen-treated group), the fluorescence intensity representing fat accumulation was quantified, and this fluorescence intensity was approximately 2-fold higher in steatotic individuals (tamoxifen-treated group) than in normal individuals (DMSO-treated group) (see Figure 3 left side).

[0073] As a result of studying the fat inhibitory effect of the placental extract of the present invention in a zebrafish steatosis model, there was little difference in liver size. However, regarding the fluorescence intensity representing fat accumulation, it was confirmed that the model treated with the placental extract of the present invention at a concentration of 0.05% to 0.1% (tamoxifen + 0.05% or 0.1% placental extract treatment) showed a fluorescence intensity reduced to approximately 60% of that of the steatosis model (tamoxifen-treated group). Therefore, it was confirmed that the placental extract at a concentration within the above range can exhibit a medical effect of inhibiting fat accumulation in the steatosis model.

[0074] Experimental Example 2. Confirmation of Reduction Effects of ALT and AST in High-fat Diet Mouse Model

[0075] 1. Experimental procedure

[0076] In this experiment, 6-week-old male C57BL / 6J mice were fed a normal fat diet and a high-fat diet. The mice used in the experiment were randomly divided into groups of 6 mice each. Then, the high-fat diet group was further divided into a saline administration group, a placental extract (Laennec, 1.8 mL / kg) administration group, and a metformin (300 mg / kg) administration group. The saline administration group and the placental extract administration group were subjected to intravenous (IV) injection, while the metformin administration group was subjected to oral administration (PO) for 21 days. At this time, metformin used in this article is widely known as an antidiabetic agent and is used as a positive control in this article to inhibit fat accumulation in liver tissue. The mice at the end of the experiment were fasted for 16 hours, then anesthetized with CO2 gas, and then blood was collected from the abdominal aorta. The blood was allowed to stand at room temperature for 1 hour, and then centrifuged at 3000 rpm and 4 °C for 10 minutes to obtain serum. The collected serum was stored at -70 °C until analysis, and an automatic biochemical analyzer was used to analyze aspartate aminotransferase (AST) and alanine aminotransferase (ALT). The aspartate aminotransferase (AST) and alanine aminotransferase (ALT) measured at this time are biomarkers useful for liver injury and liver toxicity. Therefore, in order to confirm the liver injury improvement effect of the placental extract according to the present invention, the AST level and the ALT level were measured.

[0077] 2. Experimental results

[0078] In mice fed a high-fat diet, the levels of AST and ALT enzymes were significantly increased. When the placental extract of the present invention was administered to mice fed a high-fat diet, it was confirmed that the levels of AST and ALT enzymes were significantly inhibited. On the other hand, for the administration of metformin as a positive control, the AST level in the blood was not substantially different from that in the high-fat diet group (saline administration), but it was confirmed that only the ALT level was significantly inhibited (see Figure 4 and Figure 5 ). Therefore, as a result of observing the efficacy of the placental extract in a steatosis model by using blood biochemical evaluation, it was found to be effective in suppressing liver damage caused by steatosis, and thus a higher level of therapeutic effect was confirmed compared to the metformin administration group as a positive control.

Claims

1. A pharmaceutical composition for preventing or treating fatty liver disease, which comprises a placenta extract, defatting the placenta by acetone treatment, the placenta extract is obtained by purifying more than once by anion exchange chromatography after treating the placenta with pepsin and hydrochloric acid, wherein the functional group of the anion exchange chromatography resin is selected from the group consisting of Q, QAE, TEAE, and DEAE, and, wherein the anion exchange chromatography is carried out by using a column selected from the group consisting of a cross-linked agarose column, a polymethacrylate resin column, a hydrogel column, and a cross-linked polymethacrylate resin column.

2. The pharmaceutical composition according to claim 1, wherein the placenta extract is obtained from a human placenta.

3. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is administered by any one or more of the following administration methods: intravenous (IV) injection, subcutaneous (SC) injection, and intramuscular (IM) injection.

4. The pharmaceutical composition according to claim 1, wherein the fatty liver disease includes non-alcoholic fatty liver disease or alcoholic fatty liver disease.

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