KR20230167235A - Composition for Improving Fatty Liver Disease Using an Extract of Phanera penicilliloba - Google Patents

Abstract

The present invention is an extract of Panera penicillilloba, which has the activity of suppressing neutral fat accumulation, promoting neutral fat decomposition, and preventing damage to liver cells due to oxidative stress in a cell test using HepG2 cells, which are human liver cancer cells. Disclosed is a composition for improving fatty liver disease using .

Description

Composition for Improving Fatty Liver Disease Using an Extract of Phanera penicilliloba}

The present invention relates to a composition for improving fatty liver disease using Phanera penicilliloba extract.

Fatty liver refers to a condition in which fat is excessively accumulated within liver cells, and the main causes of its development include drinking, obesity, diabetes, and hyperlipidemia.

Depending on the cause, fatty liver can be divided into alcoholic fatty liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD). Both diseases have a similar pathological spectrum ranging from simple steatosis, steatohepatitis, cirrhosis, and liver cancer, but when alcohol is the cause, it is called ALD, and when it shows histological damage similar to alcoholic liver disease without a history of alcohol abuse, it is called NAFLD. .

Fat accumulation in the liver is caused by increased fat inflow into the liver due to excessive intake of dietary fat or calories, increased lipolysis in adipose tissue, resulting in excessive influx of free fatty acids (FFAs) into the liver, and new substances in the liver. It is caused by disorders in several fat metabolic pathways, such as increased fat synthesis, decreased triglyceride outflow through very low density lipoprotein (VLDL) in the liver, and decreased fatty acid oxidation in the liver.

Oxidative stress also plays a major role in the development of liver disease. If oxidative stress continues with excessive accumulation of fat in the liver, an excessive inflammatory response occurs and the possibility of developing chronic liver diseases such as cirrhosis and liver cancer increases. In particular, in alcoholic liver disease, alcohol is oxidized by CYP2E1. ROS inhibits fatty acid oxidation in mitochondria and induces fat accumulation.

Meanwhile Panera penicilliloba is a shrub plant belonging to the legume family (Fabaceae), also called Bauhinia penicilliloba , and grows naturally in Thailand, Cambodia, Western Laos, and Vietnam. Locally, it is called Sieo daeng, and it is known to have been used for stomatitis in northeastern Thailand and diarrhea in Laos. Looking at literature data, it has been confirmed that the extract of Panera penicillova has immunomodulatory, antibacterial, and antioxidant effects, and as a result of toxicity evaluation in animal models, it has been confirmed that it does not show acute or chronic toxicity, but it is related to research on alcoholic and non-alcoholic ingredients. There is no literature to be found.

The present invention discloses the fatty liver disease improving activity of Panera penicillova extract, etc.

The purpose of the present invention is to provide a composition for improving fatty liver disease using Panera penicillova extract.

Other or specific purposes of the present invention will be presented below.

The present invention, as confirmed in the examples and experimental examples below, shows that Panera penicillova extract inhibits neutral fat accumulation, promotes neutral fat decomposition, and reduces oxidative stress in a cell test using HepG2 cells, which are human liver cancer cells. It was completed by confirming that it prevents damage to liver cells.

Considering the above, in one aspect, the present invention can be viewed as a composition for improving fatty liver disease containing Panera penicillova extract as an active ingredient, and in another aspect, Panera penicillova extract as an active ingredient. It can be understood as a composition for preventing liver damage or protecting the liver.

In this specification, “Panera penicillova extract” refers to the whole plant, leaf, branch, stem, fruit, root, or a mixture thereof of Panera penicillova as the extraction object, and water and a low-carbon solution of 1 to 4 carbon atoms as the extraction solvent. Alcohol (methanol, ethanol, butanol, etc.), methylene chloride, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,3 - Refers to an extract obtained by leaching using butylene glycol, propylene glycol, or a mixed solvent thereof, an extract obtained using a supercritical extraction solvent such as carbon dioxide, pentane, or a fraction obtained by fractionating the extract, and the extraction method refers to the active substance Considering the polarity, degree of extraction, and degree of preservation, any method such as cold immersion, reflux, heating, ultrasonic radiation, or supercritical extraction can be applied. In the case of a fractionated extract, the extract is suspended in a specific solvent and then mixed with a solvent of different polarity to form a fraction obtained by adsorbing the crude extract onto a column filled with silica gel and then mixed with a hydrophobic solvent, a hydrophilic solvent, or a mixture of these. It is meant to include fractions obtained using the mobile phase. In addition, the meaning of the extract includes concentrated liquid extract or solid extract from which the extraction solvent has been removed by methods such as freeze-drying, vacuum drying, hot air drying, and spray drying. Preferably, it refers to an extract obtained using water, an alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof as an extraction solvent, and more preferably, an extract obtained using a mixed solvent of water and an alcohol having 1 to 4 carbon atoms as an extraction solvent. do.

In addition, in this specification, “active ingredient” refers to an ingredient that exhibits the desired activity alone or can exhibit activity in combination with a carrier that is not active on its own.

In addition, as used herein, “fatty liver disease” includes simple steatosis, fatty liver, and steatohepatitis accompanied by inflammation, and particularly non-alcoholic fatty liver disease.

Also, as used herein, “improvement of fatty liver disease” includes prevention and treatment of fatty liver disease, and inhibition of fat accumulation in liver tissue.

Also, in this specification, “prevention of liver damage” means suppressing or recovering from damage such as death of liver cells caused by heavy metals, carbon tetrachloride, reactive oxygen species, etc. that cause liver cell toxicity.

The composition for improving fatty liver disease or preventing liver damage of the present invention may be included in any amount (effective amount) depending on the use, formulation, purpose of formulation, etc., as long as the active ingredient can exhibit fatty liver improvement activity, liver damage prevention activity, etc. There is, a typical effective amount will be determined within the range of 0.001% by weight to 20.0% by weight based on the total weight of the composition. Here, the “effective amount” refers to the intended functional and pharmacological effects, such as the effect of improving fatty liver and preventing liver damage, when the composition of the present invention is administered to mammals, preferably humans, to which it is applied during the administration period according to the suggestions of medical experts, etc. It refers to the amount of the active ingredient included in the composition of the present invention, which can represent. Such effective amounts can be determined experimentally within the scope of the ordinary ability of those skilled in the art.

In addition to the active ingredients, the composition of the present invention provides convenience of taking or ingestion by increasing or reinforcing the fatty liver improvement effect or by adding similar activities such as anti-obesity activity, blood pressure control activity, blood lipid improvement activity, and fatigue improvement activity. In order to improve the composition, any compounds or natural extracts whose safety has already been proven in the art and known to have the corresponding activity may be additionally included.

These compounds or extracts include compounds, extracts, and pharmaceuticals listed in compendial documents such as the Pharmacopoeia of each country (in Korea, “Korea Pharmacopoeia”) and each country’s Code of Health Functional Foods (in Korea, it is “Standards and Specifications for Health Functional Foods” notified by the Ministry of Food and Drug Safety). The laws of each country that govern the manufacture and sale of compounds or extracts and health functional foods that have received product approval in accordance with the laws of each country (in Korea, this is the Pharmaceutical Affairs Act) (in Korea, it is the “Health Functional Foods Act”) 」) includes compounds or extracts whose functionality has been recognized.

For example, according to the Korean 「Health Functional Foods Act」, Garcinia cambogia peel extract, conjugated linolenic acid (free fatty acid), conjugated linolenic acid (triglyceride), green tea extract, chitosan, and Lactobacillus gasseri are recognized as functional for 'body fat reduction'. BNR17 ( Lactobacillus gasseri BNR17), L-carnitine tartrate, green mate extract, green coffee bean extract, perilla leaf extract, soybean germ extract, etc. complex, ginseng leaf alcohol extract powder, lactoferrin (milk purified protein), lemon balm extract mixed powder , mate thermal water extract, seaweed and other complex extracts (xanthigen), fermented vinegar pomegranate complex, pu-erh tea extract, soybean peptide complex, vegetable oil diglyceride, wild mango seed extract, medium chain fatty acid (MCFA)-containing oil, Complex extracts such as coleus forskohlli extract, chitooligosaccharide, finger root extract powder, hibiscus, etc., GABA-containing powder derived from L-glutamic acid recognized for its functionality in 'blood pressure regulation', katsuobushi oligopeptide, Natto bacteria culture powder, Seomoktae ( Peptide complex, salmon peptide, olive leaf extract, sardine peptide, casein hydrolyzate, coenzyme Q10, grape seed enzymatically decomposed extract powder, Haitai oligopeptide, etc., DHA concentrated oil and globin singer recognized for their functionality in ‘improving blood neutral fat’ Decomposition products, indigestible maltodextrin, bamboo leaf extract, vegetable oil diglyceride, refined sardine fish oil, refined squid oil, etc., L-arabinose, nopal extract, cinnamon extract powder, guava leaf extract, Indigestible maltodextrin, freeze-dried silkworm powder, hemp spirit extract, banaba leaf extract, leaf extract, etc., fermentation-generated amino acid complex recognized for its functionality as 'fatigue improvement', Hovenia vulgaris fruit extract, rhodiola extract, etc., as 'anti-stress' These compounds or extracts include L-theanine, asiaganda extract, milk protein hydrolyzate, and ginseng leaf extract, which are recognized for their functionality.

One or more of these compounds or extracts may be included in the composition of the present invention along with the active ingredient.

The composition of the present invention can be regarded as a food composition in a specific aspect.

The food composition of the present invention can be manufactured in any form, for example, beverages such as tea, juice, carbonated drinks, and electrolyte drinks, processed oils such as milk and yogurt, gums, rice cakes, Korean snacks, bread, It can be manufactured into foods such as snacks and noodles, and health functional food preparations such as tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jellies, bars, etc. In addition, the food composition of the present invention can have any product classification in terms of legal and functional classification as long as it complies with the enforcement laws and regulations at the time of manufacture and distribution. For example, it is a health functional food according to the Korean 「Act on Health Functional Foods」, or confectionery, pulses, tea, etc. according to each food type according to the food code of the Korean 「Food Sanitation Act」 (“Food Standards and Specifications” notified by the Ministry of Food and Drug Safety). These may be beverages, special purpose foods, etc.

The food composition of the present invention may contain food additives in addition to the active ingredients. Food additives can generally be understood as substances that are added to, mixed with, or infiltrated into food when manufacturing, processing, or preserving food. Since they are consumed daily and for a long period of time with food, their safety must be guaranteed. In the Food Additives Code of Laws of each country that regulates the manufacturing and distribution of food (in Korea, it is the Food Sanitation Act), food additives with guaranteed safety are limited in terms of ingredients or functions. In the Korea Food Additive Code (Ministry of Food and Drug Safety Notification “Food Additive Standards and Specifications”), food additives are classified into chemical synthetics, natural additives, and mixed preparations in terms of composition. These food additives are classified as sweeteners and flavoring agents in terms of function. , preservatives, emulsifiers, acidulants, thickeners, etc.

Sweeteners are used to impart an appropriate sweetness to foods, and both natural and synthetic ones can be used in the food composition of the present invention. Preferably, a natural sweetener is used, and natural sweeteners include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose, and maltose.

Flavoring agents are used to improve taste or aroma, and both natural and synthetic ones can be used. Preferably, natural products are used. When using natural products, they can serve the purpose of enhancing nutrition in addition to flavor. Natural flavoring agents may be obtained from apples, lemons, tangerines, grapes, strawberries, peaches, etc., or may be obtained from green tea leaves, coriander leaves, bamboo leaves, cinnamon, chrysanthemum leaves, jasmine, etc. You can also use things obtained from ginseng (red ginseng), bamboo shoots, aloe vera, and ginkgo nuts. Natural flavors can be liquid concentrates or solid extracts. In some cases, synthetic flavoring agents may be used, and as synthetic flavoring agents, esters, alcohols, aldehydes, terpenes, etc. may be used.

Preservatives include calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), etc., and emulsifiers include acacia gum, carboxymethyl cellulose, xanthan gum, and pectin. etc. may be used, and as acidulants, acidic acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, etc. may be used. In addition to improving taste, acidulants may be added to ensure that the food composition has an appropriate acidity for the purpose of suppressing the growth of microorganisms. As a thickening agent, a suspending agent, settling agent, gel forming agent, bulking agent, etc. may be used.

In addition to the food additives described above, the food composition of the present invention may contain bioactive substances or minerals known in the art and whose safety is guaranteed as food additives for the purpose of supplementing and reinforcing functionality and nutrition.

Such physiologically active substances include catechins contained in green tea, vitamins such as vitamin B1, vitamin C, vitamin E, and vitamin B12, tocopherol, dibenzoylthiamine, etc., and minerals include calcium preparations such as calcium citrate and magnesium stearate. Magnesium preparations such as iron citrate, iron preparations such as chromium chloride, potassium iodine, selenium, germanium, vanadium, zinc, etc. are included.

The food composition of the present invention may contain the above-described food additives in an appropriate amount to achieve the purpose of addition depending on the product type.

Regarding other food additives that may be included in the food composition of the present invention, reference may be made to the food code or food additive code according to the laws of each country.

The composition of the present invention may be considered a pharmaceutical composition in other specific embodiments.

The pharmaceutical composition of the present invention contains a pharmaceutically acceptable carrier in addition to the active ingredient and can be prepared into an oral formulation or a parenteral formulation depending on the route of administration by a conventional method known in the art. Here, “pharmaceutically acceptable” means that it does not inhibit the activity of the active ingredient and does not have toxicity beyond what is acceptable for the subject of application (prescription).

When the pharmaceutical composition of the present invention is prepared as an oral dosage form, it can be prepared as powder, granules, tablets, pills, dragees, capsules, solutions, gels, syrups, suspensions, and wafers using a suitable carrier according to methods known in the art. It can be manufactured in a dosage form such as: At this time, examples of suitable pharmaceutically acceptable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol, starches such as corn starch, potato starch, and wheat starch, cellulose, methylcellulose, ethylcellulose, Cellulose such as sodium carboxymethylcellulose and hydroxypropylmethylcellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable. Yu, etc. can be mentioned. In the case of formulation, if necessary, diluents and/or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants may be included in the formulation.

When the pharmaceutical composition of the present invention is prepared as a parenteral formulation, it can be formulated in the form of eye drops, injections, transdermal administration, nasal inhalation, or suppositories along with a suitable carrier according to methods known in the art. When formulating as eye drops, suitable carriers include sterile water, saline solution, and isotonic solutions such as 5% dextrose. If necessary, benzalkonium chloride, methylparaben, ethylparaben, etc. can be added for preservative purposes. When formulated as an injection, suitable carriers include sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof, preferably Ringer's solution, phosphate buffered saline (PBS) containing triethanol amine, or sterile for injection. Isotonic solutions such as water or 5% dextrose can be used. When formulated for transdermal administration, it can be formulated in the form of ointments, creams, lotions, gels, external solutions, paste preparations, linear preparations, and aerol preparations. In the case of nasal inhalation, it can be formulated in the form of an aerosol spray using suitable propellants such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, and carbon dioxide. When formulated as a suppository, the base is Wethepsol ( witepsol), Tween 61, polyethylene glycols, cocoa fat, laurel paper, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, sorbitan fatty acid esters, etc. can be used.

Regarding the specific formulation of pharmaceutical compositions, it is known in the art, and references can be made to, for example, Remington's Pharmaceutical Sciences (19th ed., 1995). The above documents are considered a part of this specification.

The preferred dosage of the pharmaceutical composition of the present invention is in the range of 0.001 mg/kg to 10 g/kg per day, preferably 0.001 mg/kg to 1 g, depending on the patient's condition, weight, gender, age, patient's severity, and administration route. It may be in the /kg range. Administration can be done once a day or divided into several times. These dosages should not be construed as limiting the scope of the invention in any respect.

As described above, according to the present invention, a composition for improving fatty liver disease using Panera penicillova extract can be provided. The composition for improving fatty liver disease of the present invention can be commercialized as functional foods, drugs, etc.

Figure 1 shows the results of evaluating the degree of toxicity of Panera penicillova extract on HepG2 cells, human liver cancer cells, in terms of cell survival rate.
Figure 2 shows the results of evaluating the degree to which fat accumulation was suppressed by treatment with Panera penicillova extract in HepG2 cells in which fat accumulation was induced with free fatty acids (FFAs).
Figure 3 shows the results of evaluating the degree of triglyceride decomposition according to treatment with Panera penicillova extract in HepG2 cells in which fat accumulation was induced with free fatty acids (FFAs).
Figure 4 shows the results of evaluating the extent to which cell damage is recovered following treatment with Panera penicillova extract in HepG2 cells induced by oxidative stress.

Hereinafter, the present invention will be described with reference to examples and experimental examples. However, the scope of the present invention is not limited to these examples and experimental examples.

<Example> Evaluation of fatty liver improvement activity and liver protection activity of Panera penicillova extract

1. Preparation of Panera penicillova extract

Methanol (1.5 L) was added to Panera penicillova whole plant dried powder (200 g) and extracted 30 times with an ultrasonic extractor (15 minutes of extraction, 2 hours of standing, 10 times per day). The extract was filtered using non-fluorescent cotton and concentrated using a rotary concentrator and a vacuum centrifugal concentrator to obtain a solid Phanera penicilliloba extract (PPE).

2. Cytotoxicity evaluation

MTT assay was performed to determine whether Panera penicillova extract exhibits hepatocellular toxicity. After human liver cancer cells (HepG2 cells) were cultured in a 96-well plate, Panera penicillova extract, which is a sample of the example, was treated at various concentrations (10 to 400 μg/mL) and cultured for 2 hours. After treating with MTT reagent and reacting for 4 hours, the absorbance of the reactant was measured at 540 nm using a spectrophotometer, and the cell survival rate was calculated as a ratio compared to the untreated sample.

The results are shown in Figure 1. The results in Figure 1 show that Panera penicillova extract does not exhibit cytotoxicity up to the maximum treatment concentration of 400 μg/mL.

3. Inhibitory effect of fat accumulation in hepatocytes

(1) Lipid staining method

Oil red O staining method was used to confirm the efficacy of Panera penicillova extract in inhibiting lipid accumulation in hepatocytes. After culturing HepG2 cells in a 12-well plate, 10 μM of silibinin as a positive control and/or Panera penicillova extract of the example were treated at various concentrations (1 to 100 μg/mL) and cultured for 2 hours. . Fat accumulation in hepatocytes was induced by treatment with 0.5mM free fatty acid mixture (FFAs mix), which is a mixture of palmitic acid and oleic acid in a ratio of 1:2. After staining lipids in hepatocytes using Oil red O reagent, the absorbance of the reactant was measured at 492 nm using a spectrophotometer, and the degree of fat accumulation was quantified as a ratio compared to the untreated group.

The results are shown in Figure 2, and the results in Figure 2 show that the Panera penicillova extract of the example inhibits lipid accumulation in hepatocytes at the level of the positive control.

(2) Glycerol quantitative method

To confirm the effectiveness of Panera penicillova extract in suppressing lipid accumulation in hepatocytes, the amount of glycerol released outside the cells was measured. HepG2 cells were cultured in a 12-well plate, then treated with 10 μM of positive control silibinin and/or PPE at various concentrations (1 to 200 μg/mL) and cultured for 2 hours. Fat accumulation in hepatocytes was induced by treatment with 0.5mM FFAs mix. The concentration of free glycerol in the cell culture medium was measured using an assay kit and is shown in Figure 3.

Referring to FIG. 3, it was confirmed that the Panera penicillova extract of the example decomposes fat accumulated in hepatocytes and induces the release of glycerol.

4. Prevention of liver damage or evaluation of hepatoprotective effect

In order to evaluate the liver damage prevention or liver protection effect of Panera penicillova extract, the inhibitory effect on hepatocellular toxicity caused by oxidative stress was confirmed through MTT assay. After culturing HepG2 cells in a 96-well plate, they were treated with PPE at different concentrations (0.5 to 200 μg/mL) and incubated for 1 hour. Cytotoxicity due to oxidative stress was induced by treatment with 100 μM hydrogen peroxide (H ₂ O ₂ ). After treating with MTT reagent and reacting for 4 hours, the absorbance of the reactant was measured at 540 nm using a spectrophotometer, and the cell survival rate was calculated as a ratio compared to the untreated sample.

The results are shown in Figure 4. Referring to FIG. 4, it was confirmed that Panera penicillova extract inhibits hepatocellular toxicity caused by H ₂ O ₂ in a concentration-dependent manner.

Claims (9)

A composition for improving fatty liver comprising Panera penicillova extract as an active ingredient.
According to paragraph 1,
A composition characterized in that the extract is obtained by extraction with water, alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof.
According to paragraph 1,
A composition wherein the fatty liver disease is non-alcoholic fatty liver disease.
According to claim 1 or 2,
The composition is a food composition.
According to claim 1 or 2,
The composition is a pharmaceutical composition.
A composition for preventing liver damage or protecting the liver containing Panera penicillova extract as an active ingredient.
According to clause 5,
A composition characterized in that the extract is obtained by extraction with water, alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof.
According to clause 6 or 7,
The composition is a food composition.
According to clause 6 or 7,
The composition is a pharmaceutical composition.