KR20160113065A - Composition for preventing, improving or treating liver injury comprising extract of citrus preicarp - Google Patents

Abstract

One example of the present invention provides a method for preparing an extract having high contents of nobiletin and tangeretin from citrus pericarps. The method for preparing a citrus pericarp extract according to the present invention can minimize the energy consumed during a condensation process and therefore is economical. In addition, the method uses water or lower alcohols as extraction or precipitation solvents and is therefore more suitable with respect to compliance with food standards. Moreover, nobiletin and tangeretin contained in the citrus pericarp extract according to the present invention can effectively suppress or ameliorate the liver damage caused by alcohol, drugs or cholestasis, through an organically synergistic action. Thus, the citrus pericarp extract according to the present invention can be used as a beneficial nutraceutical.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing, improving or treating liver damage, which comprises citrus peel extract,

The present invention relates to a method for preparing a citrus peel extract, and more particularly, to a method for producing an extract having a high content of polymethoxyplavoid from citrus peel. The present invention also relates to the use of citrus peel extracts as pharmaceuticals or as nutraceuticals.

When synthetic chemicals are used as foods or medicines, there have been constant problems such as excessive stimulation of the human body and various side effects. Recently, various foods and pharmaceutical compositions using natural products have been developed to reduce side effects and excessive stimulation of synthetic chemicals. Natural materials are not only less harmful but also highly appreciated by consumers, and their development value is increasing as a raw material for pharmaceuticals and cosmetics.

Citrus fruit is a lot of fruit produced in Korea, and citrus fruit (Citrus sunki hort. Ex Tanaka), which is a native citrus fruit, is cultivated in Jeju Island. For citrus fruits, vitamin C has been said to be synonymous. However, citrus has a variety of other functional materials that are beneficial to the body. Flavonoids, a polyphenolic compound with a small molecular weight, are a kind of this functional substance, and there exist about 60 kinds of citrus fruit. Most flavonoids have a hydroxyl group (-OH), which appears to be related to antioxidant functions. On the other hand, nobiletin belongs to a flavone group having many methoxy groups (CH3O-) among flavonoids, which is known to have higher biological activity than flavones having a hydroxy group. Polymethoxy flavonoids (PMF), found only in citrus fruits, include nobiletin, tangeretin, and sinensitin, which show the usefulness of citrus as a natural ingredient.

<Novyl Retin Chemical Structure>

<Anthrax Retinomical Structure>

<Synthetic Tin Chemical Structure>

Meanwhile, the polymethoxy flavonoid (PMF) is extracted or fractionated from the citrus peel. In order to increase the content of the polymethoxy flavonoid (PMF), most of the organic solvent such as acetic acid and hexane is used in the fractionation step. There is a limit to use as a medicine or a food material.

Anti-cancer, anti-inflammatory, immunomodulatory, neuroprotective, and memory-damaging effects (Li, RW, Theriault, AG, Au, K., Douglas, T., Casaschi, A., Kurowska, EM and Mukherjee, R., Citrus polymethoxylated flavones, lipid and glucose homeostasis and modulating adipocytokines in fructose-induced insulin resistant hamsters. : 365-373, 2006).

On the other hand, Korean Patent Laid-Open Publication No. 10-2015-0020001 (prior art 1) discloses that one of the hydroxyl group polymethoxy flavone compound and the pharmacological agent , Or a pharmaceutically acceptable carrier, in the form of a salt or a pharmaceutically acceptable salt thereof, or a salt or a pharmaceutically acceptable carrier thereof. Korean Patent Registration No. 10-0883356 (Prior Art 2) also discloses a pharmaceutical composition containing cinnensetin, anthraquine, 3,5,6,7,8, 3'4'-heptamethoxy flavone, or 5-hydroxy-6- , 7,8,3 ', 4'-pentamethoxy flavone as an active ingredient. However, in the prior art 1 and the prior art 2, only the fatty liver caused by the effect of polymethoxy flavonoid (PMF) caused by obesity or lipid metabolism abnormality is mentioned, but the liver damage caused by other factors is not mentioned . Also, in the prior art 3 (Satomi Onoue, et al., 2013. Physicochemical and biopharmaceutical characterization of amorphous solid dispersion of nobiletin, a citrus polymethoxylated flavone, with improved hepatoprotective effects. European Journal of Pharmaceutical Sciences, 49, 453-460. The effect of inhibiting liver damage of noviracin is disclosed. However, the prior art 3 does not mention an increase in the effect of inhibiting liver damage by the combination of novilectin and another polymethoxy flavonoid (PMF). Comprehensively, the prior art teaches in no way what the combined use of different polymethoxy flavonoids (PMF), in particular the combination of Novartin and anthrax with liver damage, caused by factors other than obesity or lipid metabolism abnormality It is not presented.

The present invention has been made under these circumstances, and an object of the present invention is to provide a method for producing an extract having a high content of a specific polymethoxyplavoid from a citrus peel in an economical and food-standard manner.

It is also a further object of the present invention to provide pharmaceutical or equivalent uses of citrus peel extracts containing specific polymethoxy flavonoids.

In order to solve one object of the present invention, there is provided a method for producing an extract of the present invention, comprising the steps of: adding a functional low alcohol having an alcohol concentration of 50 to 80% by weight to citrus peel and extracting it to obtain an extract containing the extract; Filtering the extract, concentrating the filtered extract to obtain a concentrated extract; And a step of adding a lower alcohol aqueous solution having a water or alcohol concentration of 30% by weight or less to the concentrated extract and allowing the extract to settle, and separating the extract to obtain a precipitate-like extract. do. In the method for preparing citrus peel extract according to an embodiment of the present invention, the lower alcohol is an alcohol having 1 to 4 carbon atoms. In addition, in the method for preparing citrus peel extract according to an embodiment of the present invention, the precipitate-type extract includes nobiletin and tangeretin.

In order to solve the other object of the present invention, one example of the present invention provides a pharmaceutical composition comprising citrus peel extract as an active ingredient. In the pharmaceutical composition according to an example of the present invention, the citrus peel extract includes nobiletin and tangeretin. In addition, the pharmaceutical composition according to one example of the present invention can be used for preventing or treating liver damage caused by alcohol, drug, abnormal bile secretion or bile congestion.

In order to solve the other object of the present invention, one example of the present invention provides a food composition comprising citrus peel extract as an active ingredient. In the food composition according to an example of the present invention, the citrus peel extract includes nobiletin and tangeretin. In addition, the food composition according to one embodiment of the present invention can be used for preventing or ameliorating liver damage caused by alcohol, drug, abnormal bile secretion or bile congestion.

When the process for producing citrus peel extract according to the present invention is used, an extract having a high content of nobiletin and anthraquinone (tangeretin) can be obtained. In addition, the process for preparing citrus peel extract according to the present invention is economical because energy consumption is minimized during the concentration process, and water or a lower alcohol is used as an extraction solvent or a precipitation solvent. In addition, the nobiletin and tangeretin contained in the citrus peel extract according to the present invention can effectively inhibit or ameliorate liver damage caused by alcohol, drug or bile secretion by organic synergism have. Accordingly, the citrus peel extract according to the present invention can be used as a useful pharmaceutical material.

FIG. 1 is a graph showing the effect of citrus peel extract on cell survival rate of liver damaged cells.
Fig. 2 is a photograph showing the effect of citrus peel extract on lipid accumulation of liver tissue in a model animal experiment in which liver injury was induced by alcohol.
FIG. 3 is a photograph showing the effect of citrus peel extract on cell death of liver tissue in a model animal experiment in which hepatic injury was induced by alcohol.
Fig. 4 is a photograph showing the effect of citrus peel extract on fibrosis of liver tissue in a model animal experiment in which chronic liver injury was induced by carbon tetrachloride. Fig.
FIG. 5 is a photograph showing the effect of citrus peel extract on lipid accumulation of liver tissue in a model animal experiment in which hepatic injury was induced by bile duct ligation (BDL).
FIG. 6 is a photograph showing the effect of citrus peel extract on fibrosis of liver tissue in a model animal experiment in which hepatic injury was induced by bile duct ligation.

The terms " pharmaceutically acceptable "and" pharmaceutically acceptable "in the present invention are intended to mean not significantly irritating the organism and not interfering with the biological activity and properties of the administered active substance.

As used herein, the term "prophylactic " means any act that inhibits the symptoms of a particular disease or delays its progress by administration of the composition of the present invention.

The term "treatment" as used herein refers to any action that improves or alleviates the symptoms of a particular disease upon administration of the composition of the present invention.

The term "improvement" as used in the present invention means all actions that at least reduce the degree of symptom associated with the condition being treated.

The term "administering" as used herein is meant to provide any desired composition of the invention to an individual by any suitable method. The term &quot; individual &quot; means any animal such as a human, a monkey, a dog, a goat, a pig, or a mouse having a disease in which symptoms of a specific disease can be improved by administering the composition of the present invention.

The term "pharmaceutically effective amount " as used herein means an amount sufficient to treat a disease at a reasonable benefit or risk ratio applicable to medical treatment, including the type of disease, severity, activity of the drug, Sensitivity, the time of administration, the route of administration and the rate of excretion, the duration of the treatment, factors including the drugs used concurrently, and other factors well known in the medical arts.

Hereinafter, the present invention will be described in detail.

An aspect of the present invention relates to a method for preparing an extract having a high content of a specific polymethoxyplavoid from citrus peels in an economical and food-standard manner. A method for producing citrus peel extract according to an embodiment of the present invention includes the steps of obtaining an extract containing an extract, obtaining a concentrated extract, and obtaining an extract in the form of a precipitate. Hereinafter, a method for preparing the citrus peel extract according to an embodiment of the present invention will be described for each step.

Step of obtaining an extract containing the extract

In the method for producing citrus peel extract according to an embodiment of the present invention, the step of obtaining the extract containing the extract comprises adding a functional lower alcohol to the citrus peel and extracting it. The extract is a concept that includes both components extracted from citrus rind, an extraction solvent, and citrus rind residue.

The type of the citrus fruits is not limited as long as they include perennial nobiletin and tangeretin. For example, when considering the amount of nobiletin and tangeretin contained in the perilla, the citrus fruits are selected from the group consisting of Citrus aurantium, Citrus depressa, Citrus tachibana, Citrus leucocarpa, Citrus tardiva, Citrus succosa, Citrus kinokuni, Citrus erythrosa, Citrus deliciosa, Citrus nobilis, Citrus reticulata, , Citrus tangerina, Citrus hanaya, Citrus nippokoreana, Citrus sunki hort. Ex Tanaka and Citrus hybrid Shiranuhi, and more preferably at least one selected from the group consisting of citrus fruits Citrus aurantium or Citrus sunki hort ex Tanaka is more preferable.

In addition, the alcohol concentration of the above-mentioned lower alcohol can be determined by extracting yields of polymethoxy flavonoids (PMF), especially nobiletin and anthraquinone, contained in citrus peels, It is preferably 50 to 80% by weight, more preferably 60 to 80% by weight, and most preferably 65 to 75% by weight. When the alcohol concentration of the functional lower alcohol is less than 50% by weight, the energy consumption and the concentration time are increased in the following concentration process, and the economical efficiency of the entire process may deteriorate. In addition, when the alcohol concentration of the functional lower alcohol exceeds 80% by weight, the extraction yield of polymethoxy flavonoids (PMF) may significantly decrease.

The lower alcohol may be selected from alcohols having 1 to 4 carbon atoms (for example, methanol, ethanol, propanol, isopropanol, isopropanol, butanol and the like) in consideration of extraction yield, distribution of polymethoxy flavonoids (PMF) Propanol, and butanol), and more preferably ethanol.

In addition, in the step of obtaining the extract containing the extract, the amount of the lower alcohol to be added is preferably 5 to 20 times, more preferably 8 to 20 times the weight of the citrus peel in view of uniform extraction and economy of the whole process And most preferably from 8 times to 15 times.

In the step of obtaining the extract containing the extract, the extraction temperature is preferably 10 to 30 ° C, more preferably 15 to 25 ° C, in view of extraction yield and economical efficiency of the whole process.

In the step of obtaining the extract containing the extract, the extraction time is preferably 2 to 10 days, more preferably 3 to 7 days, in view of extraction yield and economical efficiency of the whole process.

Step of obtaining a concentrated extract

In the method for producing a citrus peel extract according to an embodiment of the present invention, the step of obtaining a concentrated extract comprises filtering an extract containing the extract and concentrating the filtered extract.

In the method for preparing the citrus peel extract according to an embodiment of the present invention, the means for filtering the extract containing the extract is not limited to a specific type as long as it is used in the field of food or extraction, and examples thereof include filter cloth, filter paper, Filters, membranes, and the like. The extract containing the extract is separated into an extract and a filter cake which are filtered by filtration. The filtered extract is then concentrated to a predetermined concentration through atmospheric pressure concentration or reduced pressure concentration.

Step of obtaining an extract in the form of a precipitate

In the method for producing a citrus peel extract according to an embodiment of the present invention, the step of obtaining a precipitate-like extract comprises adding water or a lower alcohol aqueous solution to the concentrated extract to precipitate the extract to separate the precipitated extract do.

In the step of obtaining the above precipitate type extract, the alcohol concentration of the lower alcohol aqueous solution is not more than 30% by weight (for example, 1 to 30% by weight), considering the precipitation rate of the concentrated extract, the precipitation yield, It is preferably 5 to 25% by weight, more preferably 10 to 25% by weight.

The lower alcohol may be an alcohol having a carbon number of 1 to 4 in consideration of the precipitation rate, the precipitation yield, the purity of the precipitate, the distribution of the polymethoxy flavonoids (PMF) contained in the precipitate, , Methanol, ethanol, propanol, and butanol), and more preferably ethanol.

In addition, in the step of obtaining the above precipitate type extract, the addition amount of water or a lower alcohol aqueous solution is preferably 3 to 12 times, more preferably 4 to 4 times as much as the weight of the concentrated extract in consideration of the precipitation rate, precipitation yield, To 10 times, and most preferably from 4 times to 8 times.

In the process for preparing citrus peel extract according to an embodiment of the present invention, the precipitate-type extract includes nobiletin and tangeretin, and may further include other polymethoxy flavonoids (PMF) have. In addition, the content of nobiletin in the above-mentioned precipitate-type extract is preferably 25 to 30% by weight, based on the dry weight of the extract in the form of a precipitate. In addition, the content of tangeretin in the above-mentioned precipitate type extract is preferably 20 to 25% by weight, based on the dry weight of the extract in the form of a precipitate. The content of polymethoxy flavonoids (PMF) in combination with nobiletin and tangeretin in the above-mentioned precipitate-type extract is preferably 45 to 55%, based on the dry weight of the extract of the precipitate form, Weight%. However, the above-mentioned precipitate-type extract may contain polymethoxy flavonoids (PMF) other than nobiletin and tangeretin, so that all of the polymethoxy flavonoids Polymethoxy flavonoids, PMF) may be greater than 55% by weight, for example 55 to 65% by weight, based on the dry weight of the extract in the form of a precipitate. In addition, the weight ratio of nobiletin to tangeretin in the precipitate-type extract may vary depending on the kind of citrus used as an extraction raw material, the type of extraction solvent, the type of precipitation solvent, the extraction temperature, and the like , 1: 0.5 to 1: 1, and more preferably 1: 0.7 to 1: 0.9 in view of the organic interaction and synergistic effect of nobiletin and anthraquine on liver damage suppression and liver function improvement.

In the method for preparing citrus peel extract according to an embodiment of the present invention, the precipitated extract is separated from the precipitating solvent and impurities by various separation means such as centrifugation. Subsequently, the separated precipitate-type extract is converted to a solid phase extract, such as a powder or granule form, through a drying process.

Another aspect of the invention relates to the medicinal use or equivalent use of citrus peel extracts (for example as a health functional food). The present invention relates to the use of citrus peel extracts as an active ingredient to prevent liver damage caused by alcohol, drugs, bile secretion abnormalities or bile congestion. Lt; RTI ID = 0.0 &gt; and / or &lt; / RTI &gt;

In the composition of the present invention, the citrus peel extract comprises nobiletin and tangeretin, and is preferably produced by the above-described method for producing citrus peel extract. In addition, the weight ratio of nobiletin to tangeretin present in the citrus peel extract in the composition of the present invention is not limited to the organic interaction and synergistic effect of nobiletin and anthraquinone on liver injury suppression or liver function improvement It is preferably 1: 0.5 to 1: 1, more preferably 1: 0.7 to 1: 0.9. In addition, the composition of the present invention preferably contains 25-30% by weight of nobiletin and 20-25% by weight of anhydrous tangerine based on the dry weight of the citrus peel extract.

In addition, the alcohol causing the liver damage in the composition of the present invention includes a mainstream containing ethanol. In addition, the drug is not limited in its kind, as long as it contains a component that is difficult to detoxify in the liver or induces the death of liver cells. For example, the drug is a therapeutic agent for hyperthyroidism (Prophylthiouracil component), a drug for athlete's foot (Isoniazid series), antibiotics (amoxacillin series), Chinese medicine and the like. In addition, the bile secretion abnormality is a concept that includes both excessive bile secretion, too little bile secreted, or disturbed bile flow, and is preferably cholestatic secretion. In addition, in the composition of the present invention, the liver damage includes hepatocellular necrosis, fatty liver, hepatic disorder, hepatitis, liver fibrosis, or liver cirrhosis, for example, alcoholic fatty liver disease, chronic chronic infection with alcohol, toxic liver injury and biliary cirrhosis.

The composition of the present invention may be formulated into a pharmaceutical composition, a food additive, a food composition (in particular, a functional food composition), a feed additive or the like depending on the intended use or aspects thereof. The content of the citrus peel extract, May be adjusted in various ranges depending on the specific form of the composition, the purpose of use, and the manner of use.

The content of the citrus peel extract as an active ingredient in the pharmaceutical composition according to the present invention is not particularly limited and may be, for example, 0.01 to 99% by weight, preferably 0.5 to 50% by weight, more preferably 1 To 30% by weight. In addition, the pharmaceutical composition according to the present invention may further contain, in addition to the active ingredient, an additive such as a pharmaceutically acceptable carrier, excipient or diluent. Examples of carriers, excipients and diluents that can be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, the pharmaceutical composition of the present invention may contain at least one known active ingredient having an effect of inhibiting liver damage or improving liver function, in addition to a citrus peel extract. The pharmaceutical composition of the present invention can be formulated into a formulation for oral administration or parenteral administration by a conventional method, and can be formulated into a pharmaceutical composition such as a filler, an extender, a binder, a wetting agent, a disintegrant, Diluents or excipients. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose ), Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate talc may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions and syrups. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like. Further, it can be suitably formulated according to each disease or ingredient, using appropriate methods in the art or by the method disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA. The pharmaceutical composition of the present invention may be administered orally or parenterally to a mammal including a human according to a desired method. Examples of the parenteral administration method include external dermal application, intraperitoneal injection, intramuscular injection, subcutaneous injection, intravenous injection, Intravenous injection or intra-thoracic injection. The dosage of the pharmaceutical composition of the present invention is not limited as long as it is a pharmacologically effective amount and is not limited as long as it depends on the body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, Varies. The typical daily dose of the pharmaceutical composition of the present invention is not particularly limited, and is, for example, 1 to 3000 mg / kg, preferably 10 to 2000 mg / kg, based on the active ingredient, 20 to 1000 mg / kg. In addition, the pharmaceutical composition of the present invention can be administered once a day or divided into several times.

The content of the citrus peel extract as an active ingredient in the food composition according to the present invention is 0.01 to 50% by weight, preferably 0.1 to 25% by weight, more preferably 0.5 to 10% by weight based on the total weight of the composition, But is not limited thereto. The food composition of the present invention may be in the form of a pill, a powder, a granule, an infusion, a tablet, a capsule, or a liquid preparation. Examples of the food include meat, sausage, bread, chocolate, candy, snack, Other noodles, gums, dairy products including ice cream, various soups, drinks, tea, functional water, drinks, alcoholic beverages and vitamin complexes. In addition to the active ingredient, the food composition of the present invention may contain a pharmaceutically acceptable carrier, various flavors or natural carbohydrates as an additional ingredient. In addition, the food composition of the present invention can be used as a food composition containing various nutrients, vitamins, electrolytes, flavors, colorants, pectic acids and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, , A carbonating agent used in carbonated drinks, and the like. In addition, the food composition of the present invention may contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The above-mentioned natural carbohydrates are sugar alcohols such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and xylitol, sorbitol and erythritol. Natural flavors such as tau Martin and stevia extract, and synthetic flavors such as saccharin and aspartame may be used as the flavor.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the following examples are intended to clearly illustrate the technical features of the present invention and do not limit the scope of protection of the present invention.

One. Citrus pericarp  Preparation of extract

Production Example 1

To 20 g of citrus aurantium crust powder was added 200 ml of a functional ethanol (ethanol concentration: 70% by weight) and the mixture was extracted at room temperature (about 20 ± 5 ° C) for 3 days. Thereafter, the extract was filtered to remove the insoluble residue. Then, the filtered extract was concentrated at room temperature (about 20 ± 5 ° C) and under reduced pressure. Then, water was added to the concentrated extract at a weight corresponding to about 5 times of the concentrated extract, and the mixture was allowed to stand for about 24 hours to precipitate the extract. Thereafter, the mixture was centrifuged at 3000 rpm for about 3 minutes to obtain an extract in the form of a precipitate. Thereafter, the precipitate-type extract was dried to obtain a solid citrus peel extract.

Comparative Production Example 1

To 20 g of citrus aurantium crust powder was added 200 ml of a functional ethanol (ethanol concentration: 70% by weight) and the mixture was extracted at room temperature (about 20 ± 5 ° C) for 3 days. Thereafter, the extract was filtered to remove the insoluble residue. Then, the filtered extract was concentrated at room temperature (about 20 ± 5 ° C) and under reduced pressure. Thereafter, the concentrated extract was lyophilized to obtain a solid citrus peel extract.

Comparative Preparation Example 2

To 20 g of citrus aurantium crust powder was added 200 ml of a functional ethanol (ethanol concentration: 50% by weight), and the mixture was extracted at room temperature (about 20 ± 5 ° C) for 3 days. Thereafter, the extract was filtered to remove the insoluble residue. Then, the filtered extract was concentrated at room temperature (about 20 ± 5 ° C) and under reduced pressure. Thereafter, the concentrated extract was lyophilized to obtain a solid citrus peel extract.

Comparative Preparation Example 3

200 ml of 100% ethanol was added to 20 g of citrus aurantium crust powder and the mixture was extracted at room temperature (about 20 ± 5 ° C) for 3 days. Thereafter, the extract was filtered to remove the insoluble residue. Then, the filtered extract was concentrated at room temperature (about 20 ± 5 ° C) and under reduced pressure. Thereafter, the concentrated extract was lyophilized to obtain a solid citrus peel extract.

Comparative Preparation Example 4

To 20 g of citrus aurantium crust powder was added 200 ml of water and extracted on a water bath at about 60 캜 for 3 days. Thereafter, the extract was filtered to remove the insoluble residue. Then, the filtered extract was concentrated at room temperature (about 20 ± 5 ° C) and under reduced pressure. Thereafter, the concentrated extract was lyophilized to obtain a solid citrus peel extract.

2. Citrus pericarp  Polymethoxy flavonoids contained in the extract ( Polymethoxy  flavonoids, PMF ) Content analysis

The contents of nobiletin and tangeretin contained in the solid citrus peel extracts prepared in Preparation Example 1 and Comparative Preparation Examples 1 to 4 were analyzed using high performance liquid chromatography (HPLC) . High-performance liquid chromatography (HPLC) The analytical conditions are as follows.

* Analytical sample: Citrus peel extract is prepared by dissolving in methanol to a concentration of 10 mg / ml.

* Two-component mobile phase: A solution of distilled water and methanol in a volume ratio of 50:50 was used as the first phase, 100% methanol was used as the second phase, and the volume ratio of the first phase to the second phase was 100: 0 Using a linear gradient solvent composition method, starting at 0: 100 over 50 minutes and then being held for 10 minutes.

* Detector: UV detector fixed at a wavelength of 215 nm

Thereafter, nobiletin and tangeretin standard reagents (Wako, JP) were analyzed by concentration and standard calibration curves were prepared based on peak area values. Then, the contents of nobiletin and tangeretin in the analytical sample were calculated using a standard calibration curve.

The contents of nobiletin and tangeretin contained in citrus peel extracts are shown in Table 1 below based on the dry weight of citrus peel extracts.

Citrus peel extract PMF content (%, based on dry weight of citrus peel extract) Novyletin Anthrax Retin Nobilreten + Angora Retin Production Example 1 27.5 22 49.5 Comparative Preparation Example 1 10.3 7.8 18.1 Comparative Preparation Example 2 12.9 9.7 22.6 Comparative Production Example 3 1.1 0.7 1.8 Comparative Production Example 4 10.7 7.5 18.2

As shown in Table 1, the citrus peel extracts prepared in Preparation Example 1 showed higher contents of nobiletin and tangeretin than those of other citrus peel extracts. On the other hand, when 100% ethanol was used as the extraction solvent, the content of nobiletin and the content of tangeretin in the citrus peel extract were remarkably decreased (see the result of Comparative Production Example 3), and hot water was used as the extraction solvent Nobiletin and tangeretin contents of citrus peel extracts were similar to those of 70% ethanol but the enrichment time and energy consumption were increased. Respectively.

3. Citrus through liver damage cell experiments pericarp  Evaluation of efficacy of extract

(1) Experimental method

HepG2 cells were cultured in DMEM medium containing 10% FBS. Thereafter, HepG2 cells were inoculated to a 96-well plate in an amount of 1 × 10 ⁴ , and after 24 hours, samples (Novyl Retin, Anzan Retin, citrus peel extract of Preparation Example 1, citrus peel extract of Comparative Preparation Example 1) 30 ㎍ / ㎖. After 6 hours, 2% ethanol and sample were treated simultaneously. On the other hand, the normal group was not treated with the sample and ethanol, and the negative control group was treated with only ethanol. After 24 hours of ethanol and sample treatment, 20 ml of MTT reagent was added to each well and incubated for 4 hours. Then, the medium was removed from each well, and 150 ml of DMSO solution was added to the cells to dissolve the cells. Then, the absorbance at 570 nm was measured using an ELISA reader, and the cell viability was calculated.

(2) Experimental results

FIG. 1 is a graph showing the effect of citrus peel extract on cell survival rate of liver damaged cells. In Fig. 1, " control "on the X axis represents the normal group without treatment with the sample and ethanol," EtOH " represents the negative control group treated only with ethanol, "EtOH + N" represents the experimental group treated with novirretin and ethanol , "EtOH + T" represents an experimental group treated with anthraquinone and ethanol, "70% EtOH" represents an experimental group treated with citrus peel extract prepared in Comparative Preparation Example 1 and ethanol, and " The experimental group treated with citrus peel extract and ethanol produced is shown. In Fig. 1, the value of the Y-axis represents the cell survival rate (%). In FIG. 1, the cell viability was expressed as a relative value with respect to the normal group as 100. As shown in FIG. 1, the citrus peel extract prepared in Preparation Example 1 effectively inhibited alcohol-induced hepatocyte apoptosis compared to the citrus peel extract prepared in Comparative Preparation Example 1, based on the same treatment concentration. On the other hand, the total content of Novartin and anthraquinone contained in the citrus peel extract prepared in Preparation Example 1 is about 50% by weight. Therefore, when the citrus peel extract is treated with the same concentrations of pure Novartin or pure anthrax, The effect was expected to decline. However, due to the synergistic effect of Novartin and anthraquine contained in the citrus peel extract and the effect of other polymethoxy flavonoids (PMF) contained in the citrus peel extract, the citrus peel extracts prepared in Preparation Example 1 were subjected to the same treatment The inhibitory effect on hepatocyte apoptosis was similar or superior to that of pure Novartin or pure anthrax.

3. Experimental animal model in which liver damage was induced by alcohol. pericarp  Evaluation of efficacy of extract

(1) Experimental method

6-7 week old male C57BL / 6 mice were used for the experiment after being purified for 1 week. During the purification process, the mice were housed at a temperature of 22 ° C, humidity of 55%, a light-dark cycle of 12 hours, and feeding and drinking water were provided freely. The mice that had undergone the purification process were divided into 6 groups of 5 mice each. Thereafter, distilled water was orally administered to the normal group in an amount of 5 ml / kg once a day for 3 days, and then the ethanol treatment was not performed. In the case of ethanol alone treatment, distilled water was orally administered at a dose of 5 ml / kg once a day for 3 days, and after 3 hours, a 20% (v / v) aqueous ethanol solution was administered at intervals of 12 hours three times at a dose of 5 g / , And the experiment was terminated after 6 hours. In the 'ethanol + silymarin' treatment group, silymarin was orally administered in an amount of 200 mg / kg once a day for 3 days, and then treated in the same manner as the ethanol treatment group. In addition, the citrus peel extracts prepared in Preparation Example 1 were orally administered to the 'ethanol + extract (50)' group in an amount of 50 mg / kg once a day for 3 days, and then treated in the same manner as the ethanol treatment group. In addition, citrus peel extracts prepared in Preparation Example 1 were orally administered at a dose of 100 mg / kg once a day for 3 days to the 'ethanol + extract (100)' treatment group, and then treated in the same manner as the ethanol treatment group. In addition, citrus peel extracts prepared in Preparation Example 1 were orally administered at a dose of 200 mg / kg once a day for 3 days to the ethanol / extract (200) treatment group, and then treated in the same manner as the ethanol treatment group. After the experiment, autopsy was performed. Autopsy was carried out after anesthetizing the mice using carbon dioxide. After that, blood was collected from the posterior vena cava and liver tissues were either fixed with formalin or quenched with liquid nitrogen for later testing.

(2) Experimental results

1) Blood analysis result

The collected blood was centrifuged to prepare analytical samples and the activity of alanine aminotransferase (ALT) and aspartate aminotransferase (ALT) and the activity of TG (aspartate aminotransferase) were measured using a blood analyzer (model: FuJk Dri-Chem 4000i; manufactured by Fujifilm, triglyceride) was measured. Table 2 shows blood analysis results.

Experiment Group ALT (U / L) AST (U / L) TG (mg / dL) Normal group 13.6 62.2 95.2 Ethanol alone treatment group 101.6 208.8 184.6 Treatment with 'ethanol + silymarin' 22.2 103.6 136.8 Treatment with 'ethanol + extract (50)' 77.4 121.8 184.4 'Ethanol + extract (100)' treated group 48.8 79.0 174.4 Treatment with 'ethanol + extract (200)' 21.2 71.2 148.0

As shown in Table 2, the citrus peel extract prepared in Preparation Example 1 of the present invention is used as an adjuvant treatment for liver damage such as toxic liver disease, chronic hepatitis, and cirrhosis in a model animal experiment in which hepatic injury is induced by alcohol Similar to that of silymarin.

2) Analysis of fat accumulation level in liver tissue

The liver tissue was fixed with a 10% phosphate buffered formalin solution and sliced to a thickness of about 5 μm to prepare specimens. The specimens were then stained with an oil red O solution and analyzed for levels of fat accumulation in liver tissue. Fig. 2 is a photograph showing the effect of citrus peel extract on lipid accumulation of liver tissue in a model animal experiment in which liver injury was induced by alcohol. 2 represents the normal group, "B" represents the ethanol alone treatment group, "C" represents the ethanol + silymarin treatment group, and "D" represents the ethanol + ) ',' E 'represents' ethanol + extract (100)' treated group, and 'F' represents treated group of 'ethanol + extract (200)'. As shown in FIG. 2, the citrus peel extract prepared in Preparation Example 1 of the present invention showed an inhibitory effect on lipid accumulation in liver tissues similar to silymarin.

3) Analysis of cell death level in liver tissue

The liver tissue was fixed with a 10% phosphate buffered formalin solution and sliced to a thickness of about 5 μm to prepare specimens. Then, the degree of apoptosis of liver tissue was measured by the TUNEL assay using a cell death detection kit (model: ApopTag Peroxidase In Situ Apoptosis Detection Kit; manufactured by Milipore, USA). FIG. 3 is a photograph showing the effect of citrus peel extract on cell death of liver tissue in a model animal experiment in which hepatic injury was induced by alcohol. In FIG. 3, " Normal "on the X axis represents the normal group," 0 "represents ethanol alone treatment group," 50 " represents &quot; ethanol + extract (50) (100) ', and' 200 'represents the' ethanol + extract (200) 'treatment group. As shown in FIG. 3, the citrus peel extract prepared in Preparation Example 1 of the present invention inhibited cell death of liver tissue in a concentration-dependent manner.

4. Carbon tetrachloride ( CCl4 ) Induced by chronic liver injury, pericarp  Evaluation of efficacy of extract

(1) Experimental method

6-7 week old male ICR mice were used for the experiment after being purified for 1 week. During the purification process, the mice were housed at a temperature of 22 ° C, humidity of 55%, a light-dark cycle of 12 hours, and feeding and drinking water were provided freely. The mice that had undergone the purification process were divided into 5 groups of 6 mice. Then, 20% of the vehicle except for the normal group was intravenously injected with vehicle (olive oil) at a dose of 1 ml / kg for a total of 4 weeks twice a week to induce liver toxicity. Thereafter, the normal group and the carbon tetrachloride alone treatment group were orally administered once a day for 4 weeks with distilled water, and the experiment was terminated after 6 hours. Silymarin was orally administered in the amount of 200 mg / kg once a day for 4 weeks in the group of 'carbon tetrachloride + silymarin', and the experiment was terminated after 6 hours. In addition, citrus peel extracts prepared in Preparation Example 1 were orally administered to the group treated with carbon tetrachloride + extract (50) orally in an amount of 50 mg / kg once a day for 4 weeks, and the experiment was completed after 6 hours. In addition, the citrus peel extracts prepared in Preparation Example 1 were orally administered at a dose of 200 mg / kg once a day for 4 weeks to the group treated with the carbon tetrachloride + extract (200), and the experiment was completed after 6 hours. After the experiment, autopsy was performed. Autopsy was carried out after anesthetizing the mice using carbon dioxide. After that, blood was collected from the posterior vena cava and liver tissues were either fixed with formalin or quenched with liquid nitrogen for later testing.

(2) Experimental results

1) Blood analysis result

The collected blood was centrifuged to prepare analytical samples and the activity of ALT (alanine aminotransferase) and AST (aspartate aminotransferase) was measured using a blood analyzer (model: FuJk Dri-Chem 4000i; manufactured by Fujifilm, . Table 3 shows blood analysis results.

Experiment Group ALT (U / L) AST (U / L) Normal group 23 85 Carbon tetrachloride alone treatment group 234 211 Treatment with 'carbon tetrachloride + silymarin' 24 97 Treatment with 'carbon tetrachloride + extract (50)' 87 184 'Carbon tetrachloride + extract (200)' treated group 65 144

As shown in Table 3, the citrus peel extract prepared in Preparation Example 1 of the present invention inhibited liver damage in a concentration-dependent manner.

2) Analysis of fibrosis level of liver tissue

The liver tissue was fixed with a 10% phosphate buffered formalin solution and sliced to a thickness of about 5 μm to prepare specimens. Then, the specimens were stained with Haematoxylin and eosin (H & E) solution and the level of fibrosis of the liver tissue was analyzed. Fig. 4 is a photograph showing the effect of citrus peel extract on fibrosis of liver tissue in a model animal experiment in which chronic liver injury was induced by carbon tetrachloride. Fig. 4 represents the normal group, "B" represents the group treated with carbon tetrachloride alone, "C" represents the group treated with the carbon tetrachloride + silymarin, and "D" represents the carbon tetrachloride + ) ', And' E 'represents the group treated with' carbon tetrachloride + extract (200) '. As shown in FIG. 4, the citrus peel extract prepared in Preparation Example 1 of the present invention inhibited fibrosis of the liver tissue in a concentration-dependent manner.

5. Bile duct ligation (BDL)  A model animal study in which liver damage was induced by citrus pericarp  Evaluation of efficacy of extract

(1) Experimental method

6-7 week old male ICR mice were used for the experiment after being purified for 1 week. During the purification process, the mice were housed at a temperature of 22 ° C, humidity of 55%, a light-dark cycle of 12 hours, and feeding and drinking water were provided freely. Seven mice were divided into five groups. All groups were then anesthetized with zoletil. After the midline incision, only exploratory laparotomy was performed for the normal subjects, and then sutured. On the other hand, for the rest of the group except for the normal group, trabeculae were obtained after trabeculation to secure the common bile duct, and the common bile duct was severed after double ligation. After recovery from the surgery, the normal group and bile duct ligation alone were treated with distilled water once per day for 4 weeks, and the experiment was terminated after 3 hours. Silymarin was orally administered to the bile duct ligation + silymarin group in an amount of 200 mg / kg once a day for 4 weeks, and the experiment was terminated after 3 hours. In addition, the citrus peel extracts prepared in Preparation Example 1 were orally administered in an amount of 50 mg / kg once a day for 4 weeks to the group treated with 'Biliary Ligation + Extract (50)', and the experiment was terminated after 3 hours. In addition, the citrus peel extracts prepared in Preparation Example 1 were orally administered in an amount of 200 mg / kg once a day for 4 weeks to the group treated with 'Bile Duct Ligation + Extract (200)', and the experiment was terminated after 3 hours. After the experiment, autopsy was performed. Autopsy was carried out after anesthetizing the mice using carbon dioxide. After that, blood was collected from the posterior vena cava and liver tissues were either fixed with formalin or quenched with liquid nitrogen for later testing.

(2) Experimental results

1) Blood analysis result

The collected blood was centrifuged to prepare analytical samples and analyzed using ALT (alanine aminotransferase), aspartate aminotransferase (AST), and GGT (gamma-alanine aminotransferase) using a blood analyzer (model: FuJk Dri-Chem 4000i; manufactured by Fujifilm, glutamyl transferase activity and total cholesterol (TCHO) and total bilirubin (TBIL) content were measured. Table 4 shows blood analysis results.

Experiment Group ALT (U / L) AST (U / L) GGT (U / L) TCHO (mg / dL) TBIL (mg / dL) Normal group 23.0 84.8 11.1 139.0 1.14 BDL alone treatment group 507.7 604.0 31.5 239.7 17.85 'BDL + Silymarin' treated group 375.2 452.2 22.5 134.1 15.5 Treatment with 'BDL + extract (50)' 320.2 418.6 32.6 296.0 18.1 'BDL + extract (200)' treated group 310.4 391.6 19.4 174.8 14.6

* BDL: Ligation of the bile duct

As shown in Table 4, the citrus peel extract prepared in Preparation Example 1 of the present invention exhibited similar or better liver injury inhibitory effects to silymarin in a model animal experiment in which liver injury was induced by bile duct ligation (BDL) Or liver function improvement.

2) Analysis of fat accumulation level in liver tissue

The liver tissue was fixed with a 10% phosphate buffered formalin solution and sliced to a thickness of about 5 μm to prepare specimens. The specimens were then stained with an oil red O solution and analyzed for levels of fat accumulation in liver tissue. FIG. 5 is a photograph showing the effect of citrus peel extract on lipid accumulation of liver tissue in a model animal experiment in which hepatic injury was induced by bile duct ligation (BDL). In FIG. 5, " A "represents the normal group," B "represents the bile duct ligation alone group," C " represents the &quot; Extract (200) '. As shown in FIG. 5, the citrus peel extract prepared in Preparation Example 1 of the present invention inhibited lipid accumulation of liver tissue in a concentration-dependent manner.

3) Analysis of fibrosis level of liver tissue

The liver tissue was fixed with a 10% phosphate buffered formalin solution and sliced to a thickness of about 5 μm to prepare specimens. Then, the specimens were stained with Haematoxylin and eosin (H & E) solution and the level of fibrosis of the liver tissue was analyzed. FIG. 6 is a photograph showing the effect of citrus peel extract on fibrosis of liver tissue in a model animal experiment in which hepatic injury was induced by bile duct ligation. In FIG. 6, " A "represents the normal group," B "represents the group treated with bile duct ligation alone," C " Extract (200) '. As shown in FIG. 6, the citrus peel extract prepared in Preparation Example 1 of the present invention inhibited fibrosis of the liver tissue in a concentration-dependent manner.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. Therefore, the scope of the present invention should be construed as including all embodiments falling within the scope of the appended claims.

Claims (12)

Citrus peel extract as an active ingredient,
Wherein the citrus peel extract comprises nobiletin and tangeretin,
Wherein the content of nobiletin is 25-30 wt% and the content of tangeretin is 20-25 wt% based on the dry weight of the citrus peel extract,
Wherein the weight ratio of nobiletin to tangeretin present in the citrus peel extract is from 1: 0.5 to 1: 1, characterized in that the liver damage caused by alcohol, drug, Lt; RTI ID = 0.0 &gt; and / or &lt; / RTI &gt;
The pharmaceutical composition according to claim 1, wherein the citrus peel extract is prepared by the following steps:
(a) adding a functional lower alcohol having a concentration of 50 to 80% by weight of a lower alcohol having 1 to 4 carbon atoms to citrus rind and extracting to obtain an extract containing the extract;
(b) filtering the extract, and concentrating the filtered extract to obtain a concentrated extract; And
(c) adding water to the concentrated extract, allowing the extract to settle, and separating the extract to obtain an extract in the form of a precipitate.
The method of claim 2, wherein the citrus fruits are selected from the group consisting of Citrus aurantium, Citrus depressa, Citrus tachibana, Citrus leiocarpa, Citrus tardiva, Citrus succosa, Citrus kinokuni, Citrus erythrosa, Citrus deliciosa, Citrus nobilis, Citrus reticulata, Citrus tangerina, Citrus hanaya, Citrus nippokoreana, ), Citrus sunki hort (ex Tanaka), and citrus hybrid (Shiranuhi).
[3] The pharmaceutical composition according to claim 2, wherein the lower alcohol in step (a) is ethanol, and the amount of the lower alcohol is 5 to 20 times the weight of the citrus peel.
The pharmaceutical composition according to claim 2, wherein the extraction temperature in step (a) is 10 to 30 ° C and the extraction time is 2 to 10 days.
The pharmaceutical composition according to claim 2, wherein the amount of water added in step (c) is 3 to 12 times the weight of the concentrated extract.
Citrus peel extract as an active ingredient,
Wherein the citrus peel extract comprises nobiletin and tangeretin,
Wherein the content of nobiletin is 25-30 wt% and the content of tangeretin is 20-25 wt% based on the dry weight of the citrus peel extract,
Wherein the weight ratio of nobiletin to tangeretin present in the citrus peel extract is from 1: 0.5 to 1: 1, characterized in that the liver damage caused by alcohol, drug, &Lt; / RTI &gt;
8. The food composition according to claim 7, wherein the citrus peel extract is prepared by the following steps:
(a) adding a functional lower alcohol having a concentration of 50 to 80% by weight of a lower alcohol having 1 to 4 carbon atoms to citrus rind and extracting to obtain an extract containing the extract;
(b) filtering the extract, and concentrating the filtered extract to obtain a concentrated extract; And
(c) adding water to the concentrated extract, allowing the extract to settle, and separating the extract to obtain an extract in the form of a precipitate.
The method of claim 8, wherein the citrus fruits are selected from the group consisting of Citrus aurantium, Citrus depressa, Citrus tachibana, Citrus leiocarpa, Citrus tardiva, Citrus succosa, Citrus kinokuni, Citrus erythrosa, Citrus deliciosa, Citrus nobilis, Citrus reticulata, Citrus tangerina, Citrus hanaya, Citrus nippokoreana, ), Citrus sunki hort (ex Tanaka), and citrus hybrid (Shiranuhi).
The food composition according to claim 8, wherein the lower alcohol is ethanol in step (a), and the amount of the lower alcohol is 5 to 20 times the weight of the citrus peel.
The food composition according to claim 8, wherein the extraction temperature in step (a) is 10 to 30 ° C and the extraction time is 2 to 10 days.
The food composition according to claim 8, wherein the amount of water added in step (c) is 3 to 12 times the weight of the concentrated extract.

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