KR20220001174A - A composition for improving liver function comprising immature citrus extract as effective component - Google Patents

Abstract

The present invention relates to a composition for improving liver health comprising a green tangerine extract as an active ingredient.

Description

Composition for improving liver function containing green tangerine extract as an active ingredient {A COMPOSITION FOR IMPROVING LIVER FUNCTION COMPRISING IMMATURE CITRUS EXTRACT AS EFFECTIVE COMPONENT}

The present invention relates to a composition for improving liver health comprising a green tangerine extract as an active ingredient, and can be used for various purposes such as medicines and food.

Not only fine dust, yellow dust, and other harmful substances, but also physical toxins such as waste products, active oxygen, and inflammation-inducing substances generated in the metabolic process, and toxins of the mind such as anger and stress are threatening the health of modern people.

In today's mass-produced livestock and livestock systems, a large amount of herbicides and pesticides are used for the mass production of grains, which are feed for livestock, antibiotics and growth promoters are administered in the breeding process, and various chemical additives are mixed in the processing process.

These chemicals are usually adipose-friendly, so they accumulate in the adipose tissue of livestock and can then accumulate again in the adipose tissue in the body that eats them.

As these health-threatening factors increase, interest in health is rapidly increasing, and recently, interest in so-called detox food that removes toxins from the body has rapidly increased.

Detox is an abbreviation of detoxification, which means removing toxins from the body.

On the other hand, the liver is the organ where metabolism in the body occurs most actively among the organs of the human body.

Since the ex vivo substance that enters the living body passes through the liver, the liver has a high risk of exposure to various toxic substances in addition to nutrients, and the probability of damage due to it is also very high.

Toxic substances that can affect the human body can be classified into toxic substances caused by environmental factors (exotoxins) and toxic substances produced in the body (endotoxins, endotoxins).

Humans are easily exposed to toxic substances such as food, air, paints, carpets, and dyes in modern society, and these toxic substances are mostly detoxified by the body's detoxification pathway, but when exposed for a long period of time, they accumulate in the body little by little.

Toxic substances enter the liver as water-soluble or fat-soluble substances, and water-soluble toxic substances are relatively easily metabolized and excreted by the kidneys, intestines, skin, and lungs. It may interfere with binding and adversely affect normal metabolic activity.

Since the liver has excellent regenerative ability, it recovers sufficiently from minor damage caused by toxic substances.

In addition, our body is always exposed to various toxic substances, so the liver is constantly suffering from detoxification. In addition to substances that cause liver toxicity, mental stress, excessive drinking, and smoking aggravate liver damage.

Liver disease is divided into viral liver disease, alcoholic liver disease, pharmacotoxic liver disease, fatty liver, autoimmune liver disease, metabolic liver disease and other liver diseases according to the cause of the disease.

Liver disease is detected only in a fairly advanced stage because there are no subjective symptoms in the early stages, so it is the leading cause of death not only in Korea but also in the world, but there are no effective therapeutic agents and diagnostic methods.

Despite the seriousness of liver disease, there is still no effective treatment method for liver disease, so there is an urgent need to develop a drug that can treat and prevent liver damage while maintaining the structure and function of the liver tissue.

As a liver function protective agent extracted from natural plants and applied in clinical practice, it is composed of isomers such as silybin, silydiamine, and silycristine extracted from a plant called Silybum marianum. There are silymarin preparations.

As such, despite numerous efforts to develop liver disease treatments from natural products, there are only a few cases currently being used as therapeutic agents or undergoing clinical trials.

Therefore, the present inventors confirmed that green tangerine extract can help improve liver health by affecting weight change, serum lipid metabolism and liver damage index analysis, histological analysis of liver tissue, and detoxification-related gene expression.

The present invention is to solve the problems of the prior art, and an object of the present invention is to provide a functional food product derived from green tangerine having excellent biosafety.

According to one aspect of the present invention, there is provided a food composition for improving liver health comprising a green tangerine extract or green tangerine fermented product as an active ingredient.

In one embodiment, the extract may be extracted with water, alcohol, ethyl acetate, acetone, nucleic acid, dichloromethane, or a mixed solvent thereof.

According to another aspect of the present invention, there is provided a food composition for improving liver damage, comprising a green tangerine extract or fermented green tangerine as an active ingredient.

In one embodiment, the liver damage may be liver fibrosis or liver cirrhosis.

In one embodiment, the green tangerine fermented product may be fermented with Saccharomyces cerevisiae.

According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating liver damage comprising a green tangerine extract or fermented green tangerine as an active ingredient.

In one embodiment, the liver damage may be liver fibrosis or liver cirrhosis.

In one embodiment, the green tangerine fermented product may be fermented with Saccharomyces cerevisiae.

Since the composition according to an aspect of the present invention contains a natural extract as an active ingredient, it has excellent biosafety as well as an effect of improving liver health, so it can be usefully used not only as a health functional food but also as a therapeutic use for liver disease.

It should be understood that the effects of the present invention are not limited to the above-described effects, and include all effects that can be inferred from the configuration of the invention described in the detailed description or claims of the present invention.

1 is a result of measuring changes in body weight and liver tissue in a liver fibrosis animal model according to administration of a green tangerine extract according to an embodiment of the present invention.
2 is a result of measuring changes in blood lipid content, ALT, and AST production in an animal model of liver fibrosis following administration of a green tangerine extract according to an embodiment of the present invention.
3 is a result of measuring malonedialdehyde content in an animal model of liver fibrosis following administration of green tangerine extract according to an embodiment of the present invention.
4 is a result of measuring the content and expression change of the cytochrome P450 family in an animal model of liver fibrosis following administration of a green tangerine extract according to an embodiment of the present invention.
5 is a result of measuring the expression change of the glutathione S-transferases family in an animal model of liver fibrosis according to administration of a green tangerine extract according to an embodiment of the present invention.
6 is a result of measuring the expression change of antioxidant enzymes in the liver fibrosis animal model according to the administration of green tangerine extract according to an embodiment of the present invention.
7 is a result of measuring the expression change of the inflammatory factor in the liver fibrosis animal model according to the administration of green tangerine extract according to an embodiment of the present invention.

The terms used in this specification have been selected as currently widely used general terms as possible while considering the functions in the present invention, but these may vary depending on the intention or precedent of a person skilled in the art, the emergence of new technology, and the like.

In addition, in a specific case, there is a term arbitrarily selected by the applicant, and in this case, the meaning will be described in detail in the description of the corresponding invention. Therefore, the term used in the present invention should be defined based on the meaning of the term and the overall content of the present invention, rather than the name of a simple term.

Unless defined otherwise, all terms used herein, including technical and scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms such as those defined in a commonly used dictionary should be interpreted as having a meaning consistent with the meaning in the context of the related art, and should not be interpreted in an ideal or excessively formal meaning unless explicitly defined in the present application. does not

Numerical ranges are inclusive of the values defined in that range. Every maximum numerical limitation given throughout this specification includes all lower numerical limitations as if the lower numerical limitation were expressly written. Every minimum numerical limitation given throughout this specification includes all higher numerical limitations as if the higher numerical limitation were expressly written. All numerical limitations given throughout this specification will include all numerical ranges that are better within the broader numerical limits, as if the narrower numerical limitations were expressly written.

Hereinafter, examples of the present invention will be described in detail, but it is obvious that the present invention is not limited by the following examples.

According to one aspect of the present invention, there is provided a food composition for improving liver health comprising a green tangerine extract or green tangerine fermented product as an active ingredient.

The “green tangerine” refers to an immature fruit of Citrus unshiu , which is in a green state before the peel is colored.

The immature fruit may refer to an extremely early raw onion citrus having a sugar content of less than 8 brix or an early and ordinary hot citrus citrus having a sugar content of less than 9 brix.

The "liver" plays a central role in the metabolism of substances and the body from outside the body, and is a living organ in which enzymatic reactions and energy metabolism occur continuously.

The green tangerine extract can improve liver health by protecting the liver from damage caused by various toxic substances.

The green tangerine extract can effectively improve liver health by inducing the expression of enzymes involved in liver detoxification.

The “improving liver health” refers to maintaining or improving liver function by protecting the liver from damage caused by toxic substances.

The "comprising as an active ingredient" means including an amount sufficient to provide a therapeutic or prophylactic effect on the subject to be administered, and since the green tangerine extract has excellent biosafety, a person skilled in the art can appropriately adjust the upper limit of the quantity can

The “extract” refers to a solvent in which the active ingredient contained in the extraction raw material is transferred by contacting the solvent and the extraction raw material under specific conditions. They can all be included regardless.

For example, the extract may include all of those obtained by extracting a component soluble in a solvent from a natural product using water or an organic solvent, and a specific component of a natural product, for example, extracting only a specific component such as oil.

The extract can be obtained by pulverizing the green tangerine after drying, or by various conventionally known extraction methods such as hot water extraction and ethanol extraction, and both the peel and fruit of the green tangerine can be used as extraction raw materials.

The extract may be extracted with water, alcohol, ethyl acetate, acetone, nucleic acid, dichloromethane, or a mixed solvent thereof, and preferably extracted with ethanol at a concentration of 50 to 90% (w/w).

Since the extraction ratio of the active ingredient included in the raw material may be different depending on the polarity of the solvent, it may be different in consideration of the selectivity of the physiologically active material according to the solvent.

The extract is extracted by conventional methods such as reflux circulation extraction, pressure extraction, ultrasonic extraction, etc. for about 1 to 24 hours with a solvent in a volume equivalent to 8 to 12 times the weight of the raw material by washing the raw material for extraction with water and drying and pulverizing it It can be prepared by filtration.

The extract may be obtained in a powder state by an additional process such as distillation under reduced pressure or freeze-drying.

The extract may also include an extract that has undergone a conventional purification process. For example, the extract is separated using an ultrafiltration membrane having a constant molecular weight cut-off value, and various purification methods are additionally performed, such as separation by various chromatography (prepared for separation according to size, charge, hydrophobicity or affinity). It may include a fraction obtained through

According to another aspect of the present invention, there is provided a food composition for improving liver damage or liver disease, comprising a green tangerine extract or fermented green tangerine as an active ingredient.

The “liver damage” may refer to a state in which liver function is reduced due to damage or damage to liver cells by toxic substances or the like.

The "liver disease" may occur because one or more of the functions performed by the liver has a problem and cannot normally perform metabolism, and preferably liver disease is hepatitis, hepatotoxicity, fatty liver, liver cirrhosis, liver fibrosis, and non-alcoholic liver. One or more may be selected from the group consisting of diseases, but the present invention is not limited thereto.

The “liver disease” may be caused by various causes such as alcohol, various drugs, toxic chemicals, hepatitis B and C viruses, cholestasis, autoimmunity, etc. can proceed with

The liver damage or liver disease may be liver fibrosis or liver cirrhosis.

The "liver fibrosis" may refer to a state in which damaged liver tissue is transformed into a fibrous tissue such as collagen, rather than being restored to normal hepatocytes, as part of a bioadaptation reaction accompanying chronic liver disease such as hepatitis.

The liver fibrosis is a bioadaptation reaction that occurs in the process of repairing tissue damage, but inevitably leads to a decrease in liver function in that the liver is replaced by a fibrous tissue that cannot perform any of the liver's intrinsic functions, such as metabolism and bile secretion in vivo. can be

The “liver cirrhosis” is the final state of chronic liver disease, and may refer to a state in which the structure of the liver is changed and hardened so that it cannot return to its original state.

The food composition for improving liver health may include fermented green tangerine as an active ingredient.

The green tangerine fermented product may be fermented with one or more selected from the group consisting of yeast, mushroom mycelium, lactic acid bacteria, and Bacillus genus fungi, preferably Saccharomyces cerevisiae ) It may be fermented with a strain. have.

The “fermentation” refers to a process in which a fermentation strain decomposes organic matter using an enzyme. Fermented metabolites not only contain various amino acids, organic acids, and antioxidants, but also particles become smaller and toxins such as heavy metals are decomposed during fermentation to improve absorption.

The fermented product is Saccharomyces cerevisiae It refers to a mixture of fermented products obtained by fermenting the strain on a medium containing an energy source such as sugar.

The fermented product may be prepared by a conventional microbial fermentation method. The fermentation method is apparent to those of ordinary skill in the art to which the present invention pertains.

The food composition may be used as a functional food related to liver damage or liver disease.

The health functional food means a food manufactured and processed using raw materials or ingredients useful for the human body in accordance with the Health Functional Food Act, and the functionality is to control nutrients or physiologically affect the structure and function of the human body. It may refer to ingestion for the purpose of obtaining useful effects for health purposes, such as action.

The food composition may include normal food additives, and unless otherwise specified, the food additives are approved by the Ministry of Food and Drug Safety according to the general rules and general test methods of the Food Additives Code, according to the standards and standards for the item. suitability can be judged.

The items described in the Food Additives Codex include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid; Mixtures, such as an added alkali agent, a preservative agent, and a tar dye agent, are mentioned.

The food composition can be used in various ways, such as foods and beverages for liver damage or improvement of liver diseases, for example, various foods, beverages, gum, tea, vitamin complexes, health functional supplements, food additives, etc. .

In addition, the health functional food may be manufactured and processed into any one formulation selected from the group consisting of tablets, granules, powders, capsules, liquid solutions and pills for the purpose of improving liver damage or liver disease.

Specifically, the health functional food in tablet form is obtained by granulating a mixture with green tangerine extract, excipients, binders, disintegrants and other additives in a conventional manner, and then adding a lubricant to compression molding, or directly compression molding the mixture. can be manufactured. In addition, the health functional food in the form of tablets may contain a mating agent, etc., if necessary, and may be coated with a suitable skinning agent if necessary.

Among the health functional foods in the form of capsules, hard capsules can be prepared by filling conventional hard capsules with a mixture of green tangerine extract and additives such as excipients, or their granules or skinned granules, and soft capsules include green tangerine extract and excipients. It can be prepared by filling a mixture with additives, such as gelatin, in a capsule base. The soft capsules may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, if necessary.

The health functional food in the form of a ring can be prepared by molding a mixture of green tangerine extract, excipients, binders, disintegrants, etc. in an appropriate way, and if necessary, the skin is coated with sucrose or other suitable skinning agent, or starch, talc, or a suitable substance. It can also be dressed up as

The health functional food in the form of granules can be prepared in a granular form by a suitable method of a mixture of green tangerine extract, excipients, binders, disintegrants, etc., and may contain flavoring agents, flavoring agents, etc. as necessary.

In addition, the definitions of terms for the excipients, binders, disintegrants, lubricants, flavoring agents, and the like are described in documents known in the art and may include those having the same or similar functions.

According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating liver damage comprising a green tangerine extract or fermented green tangerine as an active ingredient.

The “prevention” refers to any action that reduces the frequency or extent of the occurrence of pathological phenomena. Prevention may be complete or partial. In this case, it may mean a phenomenon in which the symptoms caused by liver damage within the individual are reduced compared to the case where the composition is not used.

The "treatment" refers to any action that clinically intervenes to change the natural process of a subject or cell to be treated, and may be performed while a clinical pathology is in progress or to prevent it.

The desired therapeutic effect is to prevent the occurrence or recurrence of a disease, alleviate symptoms, reduce any direct or indirect pathological consequences of the disease, prevent metastasis, reduce the rate of disease progression, alleviating or temporarily ameliorating the condition, or improving the prognosis.

The composition may be administered in the form of oral delivery or parenteral delivery. The composition may be administered systemically or locally, and the administration may include oral administration and parenteral administration. The compositions may be formulated with a suitable amount of a pharmaceutically acceptable vehicle or carrier to provide an appropriate dosage form.

The composition may further include a carrier, excipient and diluent used in the preparation of the pharmaceutical composition. The carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil.

In addition, the composition can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, external preparations, suppositories, and sterile injection solutions.

A solid preparation for oral administration may include tablets, pills, powders, granules, capsules, etc., and the solid preparation includes at least one excipient in the green tangerine extract and its fractions, for example, starch, calcium carbonate, sucrose, It can be prepared by mixing lactose or gelatin. In addition to the above excipients, lubricants such as magnesium stearate and talc may be used.

Liquid formulations for oral administration may include suspensions, solutions, emulsions, syrups, etc., and various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. may be used in addition to simple diluents such as water and liquid paraffin.

Formulations for parenteral administration may be used as sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. The non-aqueous solvent and suspension agent may be propylene glycol, polyethylene glycol, vegetable oil such as olive oil, or injectable ester such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin oil, and glycerogelatin may be used.

The pharmaceutical composition may be administered to a subject in a pharmaceutically effective amount. The “pharmaceutically effective amount” means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is dependent on the patient's type, severity, drug activity, and drug Sensitivity, time of administration, route of administration and rate of excretion, duration of treatment, factors including concomitant drugs, and other factors well known in the medical field.

The pharmaceutical composition may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Taking all of the above factors into consideration, it is preferable to administer an amount capable of obtaining the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

Through the following examples, the present invention will be described in more detail, but it is obvious that the present invention is not limited by the following examples.

Preparation Example 1: Preparation of green tangerine extract

20 times ethanol (50% v/v) was added to the crushed green mandarin orange, extracted with stirring for 24 hours, and filtered under reduced pressure.

The obtained liquid extract was concentrated (10 to 25 brix) with a rotary vacuum concentrator, and then the green tangerine extract was powdered using a freeze dryer to obtain it.

Preparation Example 2: Preparation of green tangerine fermented product

Purified water and sugar were mixed in a ratio of 90: 10 (w/w), and the yeast ( Saccharomyces cerevisiae ) was prepared by liquid culture.

Green mandarin oranges were washed and then crushed and dried in a dryer.

After mixing 9 kg of the above solution with 1 kg of dried green tangerine, 10% (w/w) yeast was added and fermented at 20° C. for 15 days to obtain a fermented green tangerine.

The obtained green tangerine fermented product was filtered and then materialized and used in the present invention.

Experimental Example 1: Manufacture of liver fibrosis animal model

For the experiment, male 5-week-old ICR mice were supplied from Central Lab. Ani. Inc, Korea.

The experimental animals were used in the experiment after acclimatization for 1 week by supplying sufficient solid feed and water until the day of the experiment, maintaining an environment of 22±2℃ temperature, 55±15% humidity, and a 12-hour light-dark cycle.

Experimental animals were divided into 10 animals per group, and normal control group (NC), induced control group (Dimethylnitrosamine; DMN), positive control group (Silymarin; 200 mg/kg), 50% green tangerine ethanol extract group (100 mg/kg, 300 mg/kg) ), fermented green tangerine 50% ethanol extract administered group (100 mg/kg, 300 mg/kg), there were 7 groups in total, and food and drinking water were freely ingested.

It has been approved by the Jeju National University Animal Care and Use Committee for ethical and scientific feasibility review and efficient management of animal experiments.

After acclimatizing the mice to the basic feed for 1 week, DMN (10 mg/kg) was administered intraperitoneally 3 times a week for 8 weeks. Forced intragastric administration.

After the end of the final experiment, blood was collected by weight measurement and cardiac puncture, and serum was separated, and blood chemistry and ELISA analysis were performed.

In addition, after liver tissue extraction, it was stored in a 10% formalin solution and used for Haemotoxylin and Eosin (H&E) staining. The liver tissue was pulverized and RNA was extracted, and detox-related genes were analyzed using real-time PCR.

Experimental Example 2: Weight change and histological analysis

DMN (10 mg/kg) was repeatedly administered intraperitoneally to mice 3 times a week for 8 weeks, and the positive control group and test substance were orally administered at the same time as the first DMN administration day.

After the end of the experiment, weight measurement and liver tissue extraction were fixed in 10% formalin solution and H&E staining was performed.

Referring to FIG. 1 , as a result of measuring the mouse body weight, compared with the DMN group (based on 100%), the silymarin-administered group had 108.68±3.93%, and the green tangerine ethanol extract group administered 104.47±3.05% (100 mg/kg), 109.21±5.44, respectively. % (300 mg/kg), fermented green tangerine ethanol extract administration group showed 105.79±4.61% (100 mg/kg) and 108.42±5.23% (300 mg/kg), respectively.

In addition, in the liver tissue H&E staining, in the DMN-treated group, hemorrhagic necrotic focus was observed, fibrous bands, fibrous network formation, septal collapse, and fibrosis were induced, while the symptoms were effectively alleviated in all other groups ( 1B).

Experimental Example 3: Blood lipid content, ALT, AST analysis

When the amount of triglyceride in blood increases, coronary artery disease occurs, which can induce insulin resistance by accumulating neutral lipids in peripheral tissues.

Neutral lipids accumulate in liver cells to cause fatty liver, and persistent fatty liver can increase the production of ALT and AST, which indicate liver damage.

Whether green tangerine extract inhibits blood lipid content and ALT and AST production was evaluated using ELISA.

Referring to FIG. 2 , compared with the DMN group (based on 100%), the mouse blood neutral lipid content was 65.25±10.02% in the silymarin-administered group, and 90.76±12.92% (100 mg/kg), 86.99±16.21 in the green tangerine ethanol extract-administered group, respectively. % (300 mg/kg), the fermented green tangerine ethanol extract administration group showed 84.08±9.80% (100 mg/kg) and 73.63±10.70% (300 mg/kg), respectively (FIG. 2A.).

Compared to the DMN group (based on 100%), the ALT content in the mouse blood was 58.06±13.80% in the silymarin group, 87.49±10.93% (100 mg/kg), and 90.30±8.38% (300 mg/kg) in the green tangerine ethanol extract group, respectively. , fermented green tangerine ethanol extract administration group showed 69.11±4.09% (100 mg/kg) and 64.84±7.50% (300 mg/kg), respectively.

Compared to the DMN group (based on 100%), the AST content in the blood was 61.85±7.05% in the silymarin group, 77.35±5.49% (100 mg/kg), and 81.96±6.88% (300 mg/kg) in the green tangerine ethanol extract group, respectively. The fermented green tangerine ethanol extract administration group showed 74.36±4.04% (100 mg/kg) and 67.62±6.55% (300 mg/kg), respectively ( FIG. 2B ).

The above results suggest that green tangerine extract can reduce blood lipid content and inhibit the occurrence of fatty liver.

Experimental Example 4: Analysis of malonedialdehyde in blood

Lipid peroxide is the most representative substance for liver damage caused by various toxic compounds or drugs.

The production of lipid peroxide may be caused by an increase in intracellular oxidative stress, that is, an increase in free radical production and a decrease in antioxidant defense.

Malonedialdehyde (MDA), which is a representative marker of lipid peroxide, can be measured through Thiobarbituric Acid Reactive Substance (TBARS).

In the case of the lipid peroxidation screen method, the degree of lipid peroxidation can be evaluated by measuring the MDA-TBA adduct formed by the reaction of MDA with TBA (thiobarbituric acid).

Referring to FIG. 3 , compared with the DMN group (based on 100%), the mouse blood MDA content was 78.40±8.22% in the silymarin-administered group, and 74.96±3.50% (100 mg/kg), 84.25±6.83% in the green tangerine ethanol extract-administered group, respectively. (300 mg/kg), the fermented green tangerine ethanol extract administration group showed 86.27±12.37% (100 mg/kg) and 79.61±4.63% (300 mg/kg), respectively.

The above results suggest that green tangerine extract can reduce the occurrence of lipid peroxide and inhibit the occurrence of liver damage.

Experimental Example 5: CYP85 (Cytochrome P450 family) analysis

The most important process of liver detoxification pathways can be classified into three stages.

In Phase I, mainly cytochrome P450s are involved, producing more water-soluble toxic substances during detoxification and converting them into less toxic molecules. This allows toxic substances to easily enter the blood and be excreted in the urine via the kidneys.

The liver detoxification function can be measured by analyzing the content and expression level of the cytochrome P450 family in mouse blood or liver tissue using ELISA and real-time PCR.

4, as a result of measuring the CYP2E1 content in the mouse blood by ELISA, compared to the DMN group (based on 100%), the silymarin-administered group was 47.22±7.22%, and the green tangerine ethanol extract-administered group was 83.77±3.35% (100 mg/kg), respectively. , 72.59±8.01% (300 mg/kg), and fermented green tangerine ethanol extract administration group showed 77.66±5.15% (100 mg/kg) and 76.98±6.90% (300 mg/kg), respectively (FIG. 4A).

In addition, as a result of measuring CYP1A1 gene expression in mouse liver tissue, compared to the DMN group (based on 100%), the silymarin-administered group received 90.95±5.17%, and the green tangerine ethanol extract group administered 94.98±0.66% (100 mg/kg), 96.84± 0.39% (300 mg/kg), the fermented green tangerine ethanol extract group showed 90.33±2.35% (100 mg/kg) and 92.57±1.33% (300 mg/kg), respectively, and in the case of CYP1A2 gene expression, the DMN group (100 %), the silymarin-administered group received 86.24±4.35%, the green tangerine ethanol extract group administered 88.88±5.08% (100 mg/kg), 96.88±3.09% (300 mg/kg), and the fermented green tangerine ethanol extract group administered 94.71, respectively. ±1.00% (100 mg/kg), 91.98±4.40% (300 mg/kg) were shown ( FIG. 4B ).

The above results suggest that green tangerine extract can further enhance liver detoxification function by being involved in detoxification pathways.

Experimental Example 6: glutathione S-transferases family analysis

In the second phase (Phase II) of the liver detoxification pathways, the intermediate metabolites generated in the phase I process are finally converted into water-soluble ones through a detoxifying mechanism such as conjugation and can be discharged outside the body.

Glutathione-S-transferases (GST) are representative enzymes involved in the detoxification of Phase II, and the types of GST distribution are different for each tissue.

There are 4 types of GST present in the cytoplasm (α, μ, πθ types), and GST-α and GST-μ exist in the liver, and GST-α accounts for more than 75%.

Therefore, the detoxification function of the liver was evaluated by analyzing the expression level of the GST family in the mouse liver tissue using real-time PCR.

Referring to FIG. 5 , in the case of GST-α1 gene expression in mouse liver tissue, compared to the DMN group (based on 100%), the silymarin-administered group had 162.74±20.74%, and the green tangerine ethanol extract-administered group had 103.14±7.14% (100 mg/kg), respectively. ), 98.94±28.25% (300 mg/kg), and fermented green tangerine ethanol extract administration group showed 131.98±11.46% (100 mg/kg) and 117.63±33.57% (300 mg/kg), respectively.

In the case of GST-α2 gene expression, 239.40±8.38% in the silymarin group, 90.63±15.06% (100 mg/kg), and 144.13±29.35% (300 mg/kg) in the silymarin-administered group, respectively, compared to the DMN group (100% standard). kg) and fermented green mandarin orange ethanol extract administration group showed 197.61±17.07% (100 mg/kg) and 166.74±6.46% (300 mg/kg), respectively.

In the case of GST-α3 gene expression, compared with the DMN group (based on 100%), the silymarin-administered group received 132.91±2.94%, and the green tangerine ethanol extract group received 96.26±11.53% (100 mg/kg) and 98.77±4.73% (300 mg), respectively. /kg) and fermented green tangerine ethanol extract administration group showed 86.72±8.41% (100 mg/kg) and 92.69±13.93% (300 mg/kg), respectively.

In the case of GST-α4 gene expression, compared to the DMN group (based on 100%), the silymarin-administered group received 150.72±8.80%, and the green tangerine ethanol extract group administered 138.00±7.78% (100 mg/kg) and 127.74±5.76% (300 mg/kg), respectively. kg) and fermented green tangerine ethanol extract administration group showed 148.64±19.61% (100 mg/kg) and 195.85±15.73% (300 mg/kg), respectively.

In the case of GST-μ1 gene expression, compared with the DMN group (based on 100%), the silymarin-administered group received 261.57±10.04%, and the green tangerine ethanol extract group administered 212.05±7.18% (100 mg/kg) and 163.21±15.65% (300 mg/kg), respectively. kg) and fermented green tangerine ethanol extract administration group showed 180.37±14.00% (100 mg/kg) and 240.19±9.26% (300 mg/kg), respectively.

In the case of GST-μ2 gene expression, compared to the DMN group (based on 100%), the silymarin-administered group received 108.63±13.16%, and the green tangerine ethanol extract group administered 106.55±6.98% (100 mg/kg) and 100.41±10.22% (300 mg/kg), respectively. kg), the fermented green tangerine ethanol extract administration group showed 96.86±1.77% (100 mg/kg) and 104.95±9.43% (300 mg/kg), respectively (FIG. 5).

The above results suggest that green tangerine extract can further enhance liver detoxification function by being involved in detoxification pathways.

Experimental Example 7: Antioxidant enzyme expression analysis

Heme oxygenase-1 (HO-1) is known as an antioxidant enzyme that decomposes heme present in cells, and byproducts produced during heme decomposition perform various roles in cells.

Decomposition of heme by HO-1 produces Fe(II), Bilirubin, and CO. Fe(II) has a cytoprotection effect, Bilirubin has an antioxidant effect, and CO suppresses inflammatory response and apoptosis.

NQOl (NAD(P)H: quinone oxidoreductase 1) is an enzyme that detoxifies by removing active quinones or reactive oxygen species produced by metabolic activation of Phase I.

NQOl expression is increased by oxidative stress and performs a cellular defense function against oxidative damage in cells.

Therefore, the expression levels of antioxidant enzymes HO-1 and NQO1 in mouse liver tissue were analyzed using real-time PCR.

6, the HO-1 gene expression in the mouse liver tissue was 162.57±12.79% in the silymarin-administered group and 143.16±11.72% (100 mg/kg) in the silymarin-administered group, respectively, compared to the DMN group (100% standard). , 149.46±1.32% (300 mg/kg), and fermented green tangerine ethanol extract administration group showed 184.39±3.45% (100 mg/kg) and 195.52±13.15% (300 mg/kg), respectively.

In the case of NQO1 gene expression, compared to the DMN group (based on 100%), the silymarin-administered group received 184.22±13.10%, and the green tangerine ethanol extract group received 161.90±22.22% (100 mg/kg) and 141.41±13.56% (300 mg/kg), respectively. , fermented green tangerine ethanol extract administration group showed 190.78±12.62% (100 mg/kg) and 189.00±12.28% (300 mg/kg), respectively.

The above results suggest that green tangerine extract can effectively alleviate liver damage by enhancing the expression of an enzyme that performs a defense function against oxidative damage in cells.

Experimental Example 8: Inflammatory factor expression analysis

In the early stages of liver damage, various causes such as viruses, alcohol, drugs, and toxic substances can act.

Free radicals, cytokines such as IL-6 (interleukin-6), and inflammation-related factors such as COX-2 (cyclooxygenase-2) and iNOS (nitric oxide synthase) can be generated by virus or liver toxic metabolism. May cause liver toxicity.

To evaluate whether green tangerine extract affects liver toxicity, the expression levels of IL-6, COX-2, and iNOS in mouse liver tissue were analyzed using real-time PCR.

Referring to FIG. 7 , the IL-6 gene expression in the mouse liver tissue was 63.34±5.04% in the silymarin-administered group and 67.46±5.39% (100 mg/kg) in the silymarin-administered group, respectively, compared to the DMN group (100% standard). , 70.46±4.41% (300 mg/kg), and fermented green tangerine ethanol extract administration group showed 71.61±9.43% (100 mg/kg) and 70.94±4.76% (300 mg/kg), respectively.

In the case of COX-2 gene expression, compared to the DMN group (based on 100%), the silymarin-administered group received 67.26±4.08%, and the green tangerine ethanol extract group administered 85.01±4.63% (100 mg/kg) and 86.22±6.87% (300 mg/kg), respectively. kg) and fermented green tangerine ethanol extract administration group showed 89.52±4.73% (100 mg/kg) and 86.90±7.49% (300 mg/kg), respectively.

In the case of iNOS gene expression, compared to the DMN group (based on 100%), the silymarin group treated 41.40±0.75%, and the green tangerine ethanol extract group treated 71.50±5.31% (100 mg/kg) and 73.80±5.49% (300 mg/kg), respectively. , the fermented green tangerine ethanol extract administration group showed 62.36±3.75% (100 mg/kg) and 68.11±2.04% (300 mg/kg), respectively ( FIG. 7 ).

The above results suggest that green tangerine extract can reduce the occurrence of factors causing liver toxicity.

The description of the present invention described above is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. For example, each component described as a single type may be implemented in a dispersed form, and likewise components described as distributed may also be implemented in a combined form.

The scope of the present invention is indicated by the following claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included in the scope of the present invention.

Claims (8)

A food composition for improving liver health, comprising a green tangerine extract or fermented green tangerine as an active ingredient. According to claim 1,
The extract is a food composition extracted with water, alcohol, ethyl acetate, acetone, nucleic acid, dichloromethane, or a mixed solvent thereof. A food composition for improving liver damage, comprising green tangerine extract or fermented green tangerine as an active ingredient. 4. The method of claim 3,
The liver damage is liver fibrosis or liver cirrhosis food composition. According to claim 1,
The green mandarin fermented product is Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) Food composition that is fermented with a strain. A pharmaceutical composition for preventing or treating liver damage, comprising green tangerine extract or green tangerine fermented product as an active ingredient. 7. The method of claim 6,
wherein the liver damage is liver fibrosis or liver cirrhosis. 7. The method of claim 6,
The green tangerine fermented product is Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) A pharmaceutical composition that is fermented with a strain.

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