KR101837156B1 - Composition for improving liver function comprising ethyl acetate fraction of Diospyros lotus leaf extract as effective component - Google Patents

Abstract

The present invention relates to a composition for improving liver function comprising an ethyl acetate fraction of a gourd leaf extract as an active ingredient. The ethyl acetate fraction of the gourd leaf extract of the present invention is effective for improving liver function, Therefore, it is expected to be very useful in the functional food and pharmaceutical industry for improving liver function because it does not cause side effects and secures safety.

Description

[0001] The present invention relates to a composition for improving hepatic function comprising an ethyl acetate fraction of guacamole leaf extract as an active ingredient,

The present invention relates to a composition for improving liver function comprising an ethyl acetate fraction of a gomex leaf extract as an active ingredient.

Liver of human body organ is an active organ of various metabolism in vivo, and it is an important organ that regulates synthesis, metabolism, storage and redistribution of carbohydrate, protein or fat. The liver also plays a pivotal role in removing the body when it is exposed to toxic substances and is likely to be damaged by toxic substances. Damage of hepatocytes may impair the detoxifying function of removing toxic substances, causing abnormalities in the immune system and causing fatty liver, hepatitis, jaundice, liver cirrhosis, liver cancer and the like.

Acetaminophen (APAP; N-acetyl-p-aminophenol) is known to be safe when used in therapeutic doses and is widely used as analgesics and antipyretics. However, overdose can lead to serious and fatal hepatocellular toxicity. In the United States and Europe, APAP overdose is common, because APAP is classified as a common drug and can be easily taken without prescription, resulting in liver disease such as acute liver failure. APAP is metabolically activated by cytochrome P450 to form the active metabolite, N-acetyl-p-benzoquinoneimine (NAPQI), and NAPQI binds to the protein. At therapeutic doses, NAPQI forms an APAP-GSH conjugate by glutathione (GSH) and is effectively detoxified by excretion in the kidney. However, overdosage of APAP saturates the sulfate and glucuronide conjugation pathways, greatly increasing the amount and rate at which NAPQI is formed. NAPQI binds covalently to hepatocyte proteins to form 3- (cysteine-S-yl) -acetaminophen (APAP-Cys) adducts. Excess quantities of NAPQI also peroxynitrite proteins and oxidize macromolecules such as lipids, proteins, and DNA, leading to hepatocyte injury and necrosis.

Goomae ( Diospyros lotus L.) is a deciduous plant belonging to the persimmon family that naturally grows in our country and in Asia including China. In the field of traditional medicine, Goyom, which is mature fruit, has been used for calming, analgesic, convergence and constipation treatment. The biochemical components of Goyom are fatty acids, sugars, flavonoids, and nonvolatile materials. Recently, there have been reports of analgesic, anti-inflammatory, antipyretic and sedative effects of Goyom roots. There is a report on the antioxidant, anti-hemolytic, and kidney-protective activity of the goat seeds. There is a report on the antioxidant, antiallergic and anti-itch effect of goose leaves. However, studies on the improvement of liver function or hepatoprotection using gillium leaf fractions have been rarely conducted.

Korean Patent No. 1209574 discloses a pharmaceutical composition for improving liver function and a health functional food composition for improving liver function, which contains a compound isolated from the extracts as an active ingredient, and Korean Patent No. 1464337 Discloses a composition for anti-obesity containing a gomam extract as an active ingredient. However, a composition for improving liver function containing the ethyl acetate fraction of the gomule leaf extract of the present invention as an active ingredient has not been disclosed.

SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned needs, and it is an object of the present invention to provide a method for treating hepatocellular carcinoma which is effective for protecting liver cells from hepatotoxicity caused by administration of acetaminophen, The present invention has been completed.

The present invention to achieve the above object goyom (Diospyros The present invention provides a composition for improving liver function comprising an ethyl acetate fraction of a lotus leaf extract as an active ingredient.

In another aspect, the present invention goyom (Diospyros lotus ) leaf extract as an active ingredient. The present invention also provides a health functional food composition for improving liver function.

In another aspect, the present invention goyom (Diospyros The present invention also provides a pharmaceutical composition for improving liver function, comprising an ethyl acetate fraction of a lotus leaf extract as an active ingredient.

According to the present invention, the ethyl acetate fraction of guacamole leaf extract is effective for improving liver function, and since it is a natural material, it hardly causes side effects, so that safety can be ensured and it is very useful for functional food for improving liver function and pharmaceutical industry Can be used.

FIG. 1 is a graph showing the effect of ethyl acetate fraction of goat's leaf extract on APAP-treated mouse serum AST and ALT. control; Control group, APAP; Acetaminophen, EA; Ethyl acetate fraction, Hexane; Hexane fractions, MeOH; Methanol extract, SM; Silymarin, positive control. ** and *** indicate p <0.01 and p <0.001, respectively, between the ethyl acetate fraction treated group, the methanol extract treated group, and the hexane fraction treated group, respectively.
FIG. 2 is a graph showing the effect of the ethyl acetate fraction of goat's leaf extract on the peroxidation reaction of APAP-treated mouse liver. control; Control group, APAP; Acetaminophen, EA; Ethyl acetate fraction, Hexane; Hexane fractions, MeOH; Methanol extract, SM; Silymarin, positive control. * Indicates a significant difference of p <0.05 between the ethyl acetate fraction treated group, the methanol extract treated group and the hexane fraction treated group.
FIG. 3 is a graph showing the effect of ethyl acetate fraction of goat's leaf extract on SOD (superoxide dismutase) activity in APAP-treated mouse liver. control; Control group, APAP; Acetaminophen, EA; Ethyl acetate fraction, Hexane; Hexane fractions, MeOH; Methanol extract, SM; Silymarin, positive control. * And ** indicate p <0.05 and p <0.01, respectively, between the ethyl acetate fraction treated group and the hexane fraction treated group and the methanol extract treated group, respectively.
FIG. 4 is a graph showing the effect of ethyl acetate fraction of goat's leaf extract on the levels of TNF-.alpha. And IL-6 in APAP-treated mouse serum. control; Control group, APAP; Acetaminophen, EA; Ethyl acetate fraction, Hexane; Hexane fractions, MeOH; Methanol extract, SM; Silymarin, positive control. In the graph on the left, * and ** indicate p <0.05 and p <0.01, respectively, in the ethyl acetate fraction treated group, the hexane fraction treated group and the methanol extract treated group, respectively. In the right graph, ** indicates the ethyl acetate fraction P <0.01 compared with the treated group and the hexane fraction treated group and the methanol extract treated group.

In order to achieve the object of the present invention, the present invention goyom (Diospyros The present invention provides a composition for improving liver function comprising an ethyl acetate fraction of a lotus leaf extract as an active ingredient.

In for improving liver function according to the present invention composition, the goyom (Diospyros lotus ) leaf extract may be a methanol extract of gomum leaf. For example, it may be prepared by drying the gomule leaf blended for 4 to 6 minutes at a temperature of 35 to 45 ° C. for 8 to 16 hours and then extracting it with methanol. It does not.

The solvent extract may further comprise a step of filtering the extract to remove suspended solid particles. Ultrafiltration, freeze filtration, centrifugation or the like may be used, but the present invention is not limited thereto.

When the extract is concentrated, methods such as concentration under reduced pressure and reverse osmosis can be used. The post-concentration drying step includes, but is not limited to, freeze drying, vacuum drying, hot air drying, spray drying, vacuum drying, foam drying, high frequency drying, infrared drying and the like.

In one embodiment of the invention, liver function improvement or liver protection may be characterized as inhibiting hepatocyte injury induced by acetaminophen, but is not limited thereto.

In a composition according to an embodiment of the present invention, the composition may reduce alanine aminotransferase (ALT), asparate aminotransferase (AST), malondialdehyde (MDA), TNF-a or IL-6 levels.

In a composition according to an embodiment of the present invention, the composition may increase SOD activity.

The invention also goyom (Diospyros lotus ) leaf extract as an active ingredient. The present invention also provides a health functional food composition for improving liver function.

When the ethyl acetate fraction of the gourd leaf extract of the present invention is used as a food composition, the ethyl acetate fraction of the gourd leaf extract may be directly added or used together with other food or food ingredients, . The amount of the active ingredient to be mixed can be suitably determined according to its intended use (prevention, health or therapeutic treatment). Generally, the fraction of the present invention is added in an amount of not more than 15 parts by weight, preferably not more than 10 parts by weight, based on the raw material, in the production of food or beverage. However, in the case of long-term ingestion intended for health and hygiene purposes or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

There is no particular limitation on the kind of the food. As a specific example, the fraction can be used to produce a processed food having good storage properties while utilizing the characteristics of agricultural products, livestock products or aquatic products. Such processed foods include, for example, confectionery, drinks, liquor, fermented foods, canned foods, milk processed foods, meat processed foods, noodles and the like. The sweets include biscuits, pies, breads, candies, jellies, gums, cereals (including dinner utensils such as cereal flakes). Drinks include carbonated beverages, functional ionic beverages, juices (such as apples, pears, grapes, aloes, citrus fruits, peaches, carrots, tomato juices, etc.) and sikhye. The mainstream includes sake, whiskey, shochu, beer, liquor, and fruit wine. Fermented foods include soy sauce, miso, and kochujang. Canned products include canned products (for example, tuna, mackerel, sandwiches, canned fish, etc.), canned products (canned beef, pork, chicken and turkey canned products) and canned products (canned products such as corn, peach and canned pineapple). Milk processed foods include cheese, butter, yogurt and the like. Meat processed foods include pork cutlet, beef cutlet, chicken cutlet, sausage, sweet and sour pork, nuggets, nubucki, and the like. And noodles such as sealed packaging raw noodles. In addition, the composition may be used in retort food, soup and the like.

Further, the functional food, the health food or the health supplement food can be produced by using the fraction. The functional food, the health food or the health supplement food refers to a food that provides a biocontrol function including a physiologically active component in addition to the nutritional function. The fraction of the present invention is a product of the above various natural extracts Since the fraction contains the active ingredient, it can be used for the production of functional food, health food or health supplement food.

In another aspect, the present invention goyom (Diospyros The present invention also provides a pharmaceutical composition for improving liver function, comprising an ethyl acetate fraction of a lotus leaf extract as an active ingredient.

The pharmaceutical composition for improving liver function of the present invention may comprise suitable carriers, excipients and diluents conventionally used in the production of pharmaceutical compositions.

The pharmaceutical composition for improving liver function according to the present invention may be formulated in the form of powders, granules, tablets, capsules, oral preparations such as suspensions, emulsions, syrups and aerosols, external preparations, suppositories and sterilized injection solutions And can be used as formulations. Examples of carriers, excipients and diluents that can be included in the pharmaceutical composition for improving liver function include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Various compounds or mixtures including silicates, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, it is prepared by using one or more diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, lubricants, binders, surfactants and the like which are usually used. As the disintegrant, agar, starch, alginic acid or its sodium salt, anhydrous calcium monohydrogenphosphate, etc. may be used. As the lubricant, silica, talc, stearic acid or its magnesium salt or calcium salt, polyethylene glycol and the like may be used And examples of the binder include magnesium, aluminum, silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidine, and low-substituted hydroxypropylcellulose. In addition, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine, and the like can be used as a diluent. In some cases, a commonly known boiling mixture, an absorbent, a colorant, a flavoring agent and a sweetening agent can be used together. The composition may be sterilized or contain adjuvants such as preservatives, stabilizers, wettable or emulsifying accelerators, salts for controlling osmotic pressure, buffering agents and other therapeutically useful substances and may be prepared by conventional methods such as mixing, granulating or coating . &Lt; / RTI &gt;

The pharmaceutical composition for improving liver function of the present invention may be formulated into various oral or parenteral dosage forms described below, but is not limited thereto.

The solid preparation for oral administration of the pharmaceutical composition for improving liver function includes a tablet, a pill, a powder, a granule or a light / soft capsule, and the solid preparation may contain at least one excipient such as starch Calcium carbonate, sucrose, lactose, gelatin, or the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral administration include suspensions, solutions, emulsions or syrups, and various excipients such as wetting agents, sweetening agents, fragrances or preservatives may be included in addition to water and liquid paraffin, which are simple diluents commonly used .

The pharmaceutical composition for improving liver function may be administered parenterally, and parenteral administration may be performed by injecting subcutaneous injection, intravenous injection, intramuscular injection, or intra-thoracic injection. In this case, in order to formulate the composition for parenteral administration, the neck, the hot-water extract or the pharmaceutically acceptable salt thereof may be mixed with a stabilizer or a buffer in water to prepare a solution or suspension, which is then formulated into unit dosage forms of ampoules or vials can do.

Desirable dosages of the fractions of the present invention will vary depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the fraction of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably 0.001 to 100 mg / kg per day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Materials and methods

1. Guillaume Leaf Extract and Fraction  Ready

The goryom ( D. lotus L.) used in the experiment was collected in June 2014 at Gugyeong-myeon, Jinan-gun, Jeollabuk-do. Goyom Leaf was sympathized by professor Kim Hong-joon of Woosuk University School of Oriental Medicine and herbal medicine. The goat leaf specimen (# 2014-07-08) is kept in the Health Management Laboratory of the College of Alternative Medicine of Jeonju University. The collected leaves were immediately washed with distilled water, steamed for 5 minutes, and dried in a dryer at 40 ° C for 12 hours. 400 g of dried goat leaves were inoculated with 4,000 ml of methanol and allowed to stand for 24 hours. The extract was filtered using a 0.45 μm filter, concentrated under reduced pressure to remove organic solvent, and finally lyophilized to obtain 50.3 g of a methanol extract. After that, 50 g was taken and re-dissolved with warmed tertiary distilled water. After that, n-hexane, chloroform, ethyl acetate, butanol, The fractionation process was carried out and the yield (%) was measured.

2. Experimental animals

Seven weeks old male Balb / c mice were purchased from Central Experimental Animal Co. (Korea) and used. The experimental animals were fed with solid feed and water freely during the whole experimental period. The animals were kept in a controlled environment of temperature 22 ± 2 ℃, humidity 55 ± 5%, day and night (12 hours) Was carried out in accordance with the experimental conditions of FIG.

3. Experimental design

Mice were divided into 6 groups, each group consisting of 6 mice: Group 1, control (saline); Group 2, APAP 300 mg / kg; Group 3, 50 mg / kg APAP + EA (ethyl acetate) fraction; Group 4, 50 mg / kg APAP + Hexane fraction; Group 5, 50 mg / kg of APAP + MeOH (methanol extract); Group 6 (positive control), APAP + silymarin 50 mg / kg. Methanol extract, EA fraction, hexane fraction and silymarin of goose 's leaf were dissolved in saline and APAP was dissolved in 1% ethanol and Tween - 20 solution. Groups 1 and 2 were injected with saline and Groups 3 to 6 were injected intraperitoneally with EA fraction, hexane fraction, methanol extract or silymarin daily for 7 days prior to APAP injection. One hour after the last injection, APAP was intraperitoneally injected into groups 2 to 6 at a dose of 300 mg / kg, and then fasted for 16 hours and sacrificed. Mice were anesthetized with ether and the heart was punctured to collect blood. Blood samples were left at room temperature for 30 minutes and then centrifuged at 2,000g for 15 minutes at 4 ° C to separate the serum. Liver samples were collected, washed with cold saline, rapidly frozen in liquid nitrogen, and stored at -80 ° C.

4. ALT  And AST  Level analysis

The liver enzymes ALT (alanine aminotransferase) and AST (asparate aminotransferase) activity were measured using the AST and ALT assay kit (ASAN Pharmaceutical., Korea) according to the manufacturer's protocol.

5. Fat peroxidation reaction

The lipid peroxidation reaction was measured using an MDA ELISA kit (Cell Biolabs., USA) according to the manufacturer's protocol.

6. Liver SOD  Activity analysis

Liver superoxide dismutase (SOD) activity was measured using a commercially available assay kit (Cayman Chemical., USA) according to the manufacturer's protocol.

7. Serum TNF -α and IL -6 level analysis

Quantification of TNF-α and IL-6 was performed using an ELISA kit (R & D Systems, USA) according to the manufacturer's protocol.

8. Statistical Analysis

All data were expressed as mean ± SD. Statistical analysis was performed with Student's t test, with a significance level of p <0.05.

Example  1: Goose leaf Fraction APAP  In the treated mouse serum AST Wow ALT  Level analysis

Since both AST and ALT show high blood levels after liver injury, methods of measuring AST and ALT are often used as methods to detect liver damage. The level of AST and ALT induced by APAP (Sigma-Aldrich, USA) was assayed by measuring the levels of AST and ALT in serum after pretreatment with Gomex leaf extract and each fraction. The levels of AST and ALT in serum were significantly increased during APAP treatment. AST and ALT levels were decreased in the group treated with goat leaf extract (MeOH) and hexane fraction (Hexane), similar to the positive control group, silamarin treated group (SM). The ethyl acetate fraction treated with guacamole leaf (EA) was significantly lower than the methanol extract or hexane fraction treated group (FIG. 1). Therefore, it was confirmed that the ethyl acetate fraction of Goyom leaf had a more effective protective effect against liver damage than the Gomex leaf extract or other fractions.

Example 2: APAP  Treated mice ( liver )

The concentration of malondialdehyde (MDA) was measured to determine the hepatic peroxidation reaction (Fig. 2). Treatment with goat leaf extract (MeOH) and hexane fraction (Hexane) inhibited the increase of MDA concentration by APAP similar to the positive control group, silamarin treatment group (SM). The MDA concentration of the Gomex leaf treated ethyl acetate fraction (EA) was lower than that of the methanol extract or hexane fraction treated group. Therefore, it was confirmed that the ethyl acetate fraction of Goyom leaf was superior to the Gomex leaf extract or other fractions in improving liver function.

Example 3: APAP  Treated mice ( liver ) My SOD Activity analysis

SOD activity in the liver was analyzed to determine the effect of the goat leaf fraction on oxidative stress in APAP treated mice. As shown in FIG. 3, the SOD activity, which is an antioxidative enzyme, was significantly decreased in the APAP-treated group, but the treatment with the goat leaf extract (MeOH) and the hexane fraction (Hexane) Compared with the control group. Therefore, it was found that the ethyl acetate fraction of Goyom leaf had an antioxidative activity superior to that of Goyom leaf extract or other fractions.

Example 4: APAP  Analysis of inflammatory responses in treated mice

The inflammatory mediators TNF-α and IL-6 levels were investigated to explain the protective mechanism of the gomoma leaf extract against liver injury. The levels of TNF-α and IL-6 in serum were significantly increased in the APAP-treated group compared to the control group, but not in the APAP-treated group but in the gomoma leaf extract (MeOH) and the hexane fraction Hexane) treated group (Fig. 4). The silymarin treated group showed similar results to the goat leaf extract treated group.

Claims (6)

delete delete delete delete Diatomaceous earth extract of Diospyros lotus leaves contains the ethyl acetate fraction as an active ingredient and reduces ALT (alanine aminotransferase), AST (asparate aminotransferase), MDA (malondialdehyde), TNF-α and IL- superoxide dismutase) activity, characterized in that it is a composition for improving liver function,
The ethylacetate fraction of the methanol extract of gomum leaf
(a) washing collected gomule leaf with distilled water, steam-steamed for 5 minutes, and dried in a dryer at 40 ° C for 12 hours;
(b) 4,000 ml of methanol was injected into 400 g of the dried gomule leaf, allowed to stand for 24 hours, extracted, filtered, concentrated under reduced pressure to remove the organic solvent, and lyophilized to obtain a methanol extract; And
(c) re-dissolving the obtained methanol extract using warmed distilled water, and then adding ethyl acetate to the separated fraction to prepare a composition for improving liver function. delete

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