AU2019204739A1 - Hepatoprotective Composition Comprising Cabbage Extract - Google Patents

Abstract

The present invention relates to a hepatoprotective composition which may be used for the prevention or treatment 5 of liver disease and the improvement of liver function due to its hepatoprotective effect and, at the same time, may be used safely as a food material due to its non-toxicity. More specifically, the present invention relates to a hepatoprotective composition for preventing or treating liver 10 disease and improving liver function, which comprises a cabbage extract as an active ingredient. 18

Description

Hepatoprotective Composition Comprising Cabbage Extract

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to a hepatoprotective

composition which may be used for the prevention or treatment

of liver disease and the improvement of liver function due to

its hepatoprotective effect and, at the same time, can be used

safely as a food material due to its non-toxicity.

Description of the Prior Art

The liver is one of the important organs of the human

body, which is responsible for body's metabolism which

appropriately changes ingested foods into nutrients required by

various tissues and processes waste products remaining after

use in tissues. Specifically, the liver secretes bile, a

digestive fluid, metabolizes proteins, carbohydrates and fats,

stores glycogen, fat-soluble vitamins, and other substances,

synthesizes blood-clotting factors, removes wastes and toxins

from the blood, regulates blood volume, and destroys old red

blood cells.

Various factors, including mental stress, excessive intake

of fatty food or alcohol, viral infection, drugs or smoking,

exposure to harmful substances such as pollutants, and malnutrition, may cause hepatic dysfunction. In addition, hepatic dysfunction interferes with body's defense and detoxification mechanisms, causing abnormalities in the immune system and causing other diseases. Hepatic dysfunction causes various symptoms such as weakness, hypotension, frequent contusion and hemorrhage, delirium tremens, emotional dullness, electroencephalographic changes, and intraabdominal fluid accumulation.

Acetaminophen (APAP), a major component of Tylenol, is a

generic drug used worldwide for the treatment of fever and

pain. It is relatively safe when used at therapeutic

concentrations. However, when an excessive amount of APAP is

ingested it may cause side effects such as hepatic necrosis,

neurotoxin and cirrhosis, and can lead to death in severe

cases. Recently, it has been reported that when an excessive

amount of APAP is administered to a living body, it enters a

large amount of leukocytes, causing acute inflammation. In

other words, proinflammatory cytokines, such as interferon-y

(IFN-y), tumor necrosis factor-a (TNF-a), interleukin-1 and IL

6, which are well-known cytokines that cause inflammatory

responses, are produced in large amounts within 10 hours and

accelerate inflammatory responses. Furthermore, inflammatory

mediators, such as cyclooxygenase-2 (COX-2) and prostaglandin

E2 (PGE2), are induced by APAP and cause fatal damage to the

living body. It has been reported that patients with liver diseases caused by APAP reach about 10% and the toxicity of

APAP is more serious in children than adults and more serious

in alcohol eaters than non-alcohol eaters. NAC (N

acetylcysteine) is used for the treatment of hepatotoxicity

caused by APAP, but it has been reported that NAC is difficult

to administer orally due to its very unpleasant odor and taste

and the administration of NAC by intravenous injection can

cause anaphylactic shock.

In order to diagnose various liver-related diseases, it is

necessary to comprehensively perform several biochemical tests.

Several test items for this purpose are collectively referred

to as liver function tests. Major test items include AST

(aspartate aminotransferase), ALT (alanine aminotransferase),

ALP (alkaline phosphatase), GGT (gamma-glutamyltransferase),

and bilirubin. In addition to these test items, items such as

total protein, albumin, LDH (lactate dehydrogenase), ammonia

and the like may also be tested. Among them, AST (GOT) and ALT

(GPT) are enzymes present in hepatocytes and are released into

the blood mainly when hepatocytes are damaged. Thus, blood AST

and ALT levels can be used as markers of liver damage. In the

early stages of acute hepatocyte injury, the level of AST which

is present at a higher concentration in hepatocytes increases

compared than the level of ALT, but after 24 to 48 hours and in

chronic hepatocyte injury, the level of ALT with a longer half

life generally further increases. In alcoholic hepatitis, AST further increases.

Meanwhile, since the liver is an organ having a large

buffer capacity, liver disease symptoms generally do not appear

in the initial stage of the disease, and since the amount of

pain-feeling nerves in the liver is small, liver disease

symptoms are generally found after the disease has worsened

considerably. Liver cirrhosis or liver cancer is the last

stage to which various liver diseases commonly lead when

progressing to a chronic stage. The liver is an organ whose

initial care is very important. Accordingly, studies have been

conducted to provide hepatoprotective compositions comprising

natural substances that can be used safely, and examples

thereof include Korean Patent No. 10-0633851(Composition

comprising the extract of heat-treated Allium victorialis L.

var. platyphyllum for treating or preventing of liver disease

or liver protection) and Korean Patent No. 10-1106499(Food

composition with hepatoprotective effect containing the

peduncle extracts of Hovenia dulcis Thunb).

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a

pharmaceutical composition and a functional health food

composition, which have a beneficial effect on the human body

and which can be used safely for a long period of time without adverse side effects and also have excellent effects of protecting liver and improving liver function.

To achieve the above object, the present invention

provides a pharmaceutical composition for preventing or

treating liver disease, comprising a cabbage extract as an

active ingredient.

The cabbage extract of the present invention does not show

cytotoxicity, and thus can be used safely. In an experiment

performed on an animal model with liver injury induced by a

drug (APAP), it could be seen that the cabbage extract

exhibited the effects of protecting liver and improving liver

function by effectively reducing AST and ALT which are liver

injury markers, indicating that the cabbage extract is

effective as a pharmaceutical composition for preventing or

treating liver disease.

In particular, the cabbage extract was effective in

treating inflammation of the liver by reducing the expression

of the inflammatory mediators iNOS and COX-2 and the

proinflammatory cytokine IL-1B, which are involved in acute

inflammatory reactions accompanying liver injury.

In the present invention, an extract of cabbage heat

treated at a temperature of 100 to 1500C had a better liver

function-improving effect or anti-inflammatory effect than an

extract of raw cabbage. A preliminary experiment showed that

as the heat treatment temperature increased, the effect of the extract was better, but if the heat treatment temperature was excessively high, carbonization could occur. The heat treatment time is preferably 10 minutes to 12 hours in view of the size of a sample or the heat treatment temperature. It is natural that as the sample size increases and the heat treatment temperature decreases, the heat treatment time increases.

In an example of the present invention, the cabbage

extract was more effective against AST known to be more highly

expressed in cells in acute liver injury. Accordingly, the

pharmaceutical composition of the present invention is more

preferably used for the prevention or treatment of acute liver

diseases. Examples of these acute liver diseases include, but

are not limited to, drug-induced liver disease, alcoholic liver

disease, acute hepatitis, and fatty liver.

The cabbage extract may be prepared by water or organic

solvent extraction and/or fractionation of raw cabbage, dried

cabbage, frozen cabbage or heat-treated cabbage. For efficient

extraction, cabbage is more preferably extracted after it is

cut finely, crushed or milled. Extraction may be performed

using any of extraction methods that are used for extraction of

natural substances in the art. For example, extraction may be

performed either by juice extraction without using any solvent

or by a cold extraction, heat extraction or ultrasonic

extraction method that uses a solvent, but is not limited thereto. When extraction is performed using a solvent, the solvent is preferably water, a Cl to C4 alcohol, or a mixture thereof. In addition, in order to further concentrate the active ingredient having effects on liver protection and liver function improvement, the extract may also be fractionated. An extract obtained by juice extraction or an extraction method that uses a solvent may be used without further processing or be used after concentration or drying. It is a matter of course that a conventional drying method, such as spray drying, thermal drying or freeze drying, may be used.

For use as a Hepatoprotective pharmaceutical products,

the composition of the present invention may be prepared by a

known method in the pharmaceutical field and used alone or in

combination with a carrier, a forming agent, a diluent, etc.

The composition of the present invention may be formulated into

an oral or parenteral preparation and used as a preventive and

curative agent against liver diseases. The dosage of active

ingredients according to the present invention may be

determined appropriately depending on the drug absorption of

the active ingredients, dosage form, the patient's age, gender

and condition, and the severity of the disease. The

administration may be performed once or multiple times a day.

The general dosage is 0.001 mg/kg-day to 10 g/kg-day. The composition of the present invention can be used safely without toxicity or side effects, even in the case of long-term administration for the purpose of prevention.

The present invention also provides a functional health

food composition for protecting liver and improving liver

function, comprising a cabbage extract as an active ingredient.

The composition of the present invention may also be useful as

a composition for relieving hangover. As in the pharmaceutical

composition, it is more preferable to use an extract of cabbage

heat-treated at 100 to 1500C. The details of the cabbage

extract are as described above with respect to the

pharmaceutical composition.

The extract of cabbage may be added preferably in an

amount of 0.01 to 100 wt.% to the food composition of the

present invention. The effective dose of the health food

composition of the present invention can be used in accordance

with the effective dose of the pharmaceutical composition.

However, in the case of long-term ingestion for the purpose of

health and hygiene or for health care purposes of controlling

health, the effective dose may be lower than the lower limit of

the above-described range. Since the active ingredient shows

no problem in terms of safety, it may also be used in an amount

larger than the upper limit of the above-described range. The functional health food of the present invention includes any formulation like tablets, capsules, pills, liquid, etc. The examples of the food to which the composition of the present invention can be added may include any kind of foods, beverages, gums, teas, vitamin complexes, functional health foods, and so forth.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the results of evaluating the

cytotoxicity of a cabbage extract.

FIG. 2 is a graph showing the results of evaluating the

liver function improving activity (ALT) of a cabbage extract.

FIG. 3 is a graph showing the results of evaluating the

liver function improving activity (AST) of a cabbage extract.

FIG. 4 shows H & E staining images of liver tissue used to

evaluate the liver function improving effect of a cabbage

extract.

FIG. 5 is a graph showing the results of evaluating the

anti-inflammatory effect of a cabbage extract.

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, the present invention will be described in

detail with reference to the following examples, which are given for the illustrations of the present invention only and not construed to limit the scope of the present invention. The examples of the present invention are subjected to various changes and modification and provided for those skilled in the art to understand the prevent invention more completely.

Examples

Example 1: Preparation of Cabbage Extract

1) Preparation of Heat-Treated Cabbage Extract (HCE)

Cabbage purchased from an agricultural and marine

products market was washed, cut to a size of about 0.5 cm x 0.5

cm x 0.5 cm, and then freeze-dried. The freeze-dried sample

was placed in the inner chamber of a heat-treatment apparatus

(Jisco, Seoul, Korea), which was designed and manufactured to

be capable of resisting even a pressure of 10 kg/cm2 or higher.

Water was placed in the outer chamber, and the sample was heat

treated at a temperature of 140 to 1500C for 6 hours. The

apparatus could prevent the sample from coming into direct

contact with water and also prevent the carbonization of the

sample by direct heat transfer, due to water contained in the

outer chamber.

The heat-treated sample was cooled, and then crushed

using a crusher, and a 10-fold volume (v/v) of distilled water

was added, followed by extraction at 600C for 2 hours. The extract was filtered, and then freeze-dried before use. The yield of the freeze-dried extract was 1.4% (w/w) relative to the weight of the raw cabbage and 20% (w/w) relative to the weight of the dried cabbage.

2) Preparation of Cabbage Extract (CE)

An extract was prepared in the same manner as described

in Example 1, except the heat-treatment process was omitted.

The yield of the freeze-dried extract was 1.2% (w/w) relative

to the weight of the raw cabbage and 18% (w/w) relative to the

weight of the dried cabbage.

Example 2: Evaluation of Cytotoxicity

To measure cytotoxicity, RAW 264.7 cells were dispensed

in each well of a 96-well plate at a concentration of 5 x 101

cells/100 pL. The RAW 264.7 cells were obtained from the

Korean Cell Line Bank and cultured with Dulbecco's Modified

Eagle's Medium (DMEM) (supplemented with 5% fetal bovine serum

(FBS) and 100 IU/mL penicillin and 100 pg/mL streptomycin

(P/S)) at 37 0 C under 5% C02. Each of the cabbage extract and

heat-treated cabbage extract prepared in Example 1 was added to

the dispensed cells at a concentration of 125, 250, 500 or

1,000 pg/ml and incubated for 24 hours, and then the viability

of the cells was measured by MTT assay.

FIG. 1 is a graph showing the results of measuring the

cell viability. As can be seen therein, both the cabbage extract and the heat-treated cabbage extract showed no cytotoxicity, indicating that these extracts can be used safely.

Example 3: Evaluation of Hepatoprotective Effect of

Cabbage Extract

6-8-week-old male BALB/c mice (weighing 19 to 22 g)

purchased from Dongyang Biotechnology (Gyeonggi-do, South

Korea) were acclimated in a pathogen-free facility under

constant condition (temperature: 21 ± 20C; relative humidity:

60 ± 10%; 12-hr light/12-hr dark cycle) before use. During the

experimental period, the animals were allowed to access water

and food ad libitum. The animals were divided into the

following groups, each consisting of 6 animals: a normal group

(untreated group), a negative control group (treated with APAP

alone), a positive control group (treated with APAP + NAC), and

a sample-administered group (treated with APAP + cabbage

extract).

The positive control group was orally administered with

NAC, known to have the effect of treating APAP-induced

hepatotoxicity, at a daily dose of 75 mg/kg for 7 days, and the

sample-administered group was orally administered with 500

mg/kg of the heat-treated cabbage extract, prepared in Example

1, at a daily dose of 500 mg/kg. At 2 hours after the last

oral administration of NAC or the sample to the positive

control group and the sample-administered group, APAP for

inducing hepatotoxicity was intravenously administered to all the groups excluding the normal group at a concentration of 400 mg/kg.

24 Hours after administration of the sample, blood was

collected through the tail vein, and ALT and AST levels in the

collected blood were measured by an assay kit (Asan

Pharmaceutical) using a substrate-enzyme reaction. FIGS. 2 and

3 show the results of measuring ALT and AST levels,

respectively, in the blood. As can be seen therein, the

cabbage extract is effective against APAP-induced liver injury.

In particular, the cabbage extract had a significant effect

against the expression of AST, suggesting that the cabbage

extract is more effective against acute liver injury.

After blood collection, the mice were sacrificed, and the

liver was dissected out, embedded in paraffin, and then stained

with H & E. FIG. 5 shows staining images of the liver tissue.

In FIG. 5, A indicates the normal group; B indicates the

negative control group; C indicates the positive control group;

and D indicates the sample-administered group. As can be seen

in FIG. 5, serious hepatocyte injury induced by APAP was

observed, but this injury was significantly healed by NAC or

the cabbage extract.

Example 4: Evaluation of Anti-Inflammatory Effect of

Cabbage Extract

Since liver injury generally causes acute inflammation,

whether the cabbage extract would have an anti-inflammatory effect was examined. To this end, RAW 264.7 cells were dispensed in each well of a 6-well plate at a concentration of

5 x 105 cells/100 pL, and then treated with 0.1 pg/ml of LPS.

At 30 minutes after LPS treatment, the cells were treated with

1 mg/ml of the cabbage extract (CE) or the heat-treated cabbage

extract (HCE) and incubated for 18 hours. Next, RNA was

extracted using TRIzol reagent (Invitrogen) according to the

manufacturer's manual. Thereafter, the expression levels of

proinflammatory mediators and cytokines were measured by qRT

PCR using the primers shown in Table 1 below. The results of

the measurement are shown in FIG. 5.

Table 1

Gene Primer sequence

GAPDH F: 5'-CACTCACGGCAAATTCAACGGCAC-3 S EQ ID NO:1 R: '-GACTCCACGACATACTCAGCAC-3' SEQ ID NO:2

iNOS F: 5'-CCCTTCCGAAGTTTCTGGCAGCAGC-3' SEQ ID NO:3 R: 5'-GGCTGTCAGAGCCTCGTGGCTTTGG-3' SEQ ID NO:4

COX-2 F: 5'-CACTACATCCTGACCCACTT-3' SEQ ID NO:5 R: 5'-ATGCTCCTGCTTGAGTATGT-3' SEQ ID NO:6

TNF-a F: 5'-TTGACCTCAGCGCTGAGTTG-3' SEQ ID NO:7 R: 5'-CCTGTAGCCCACGTCGTAGC-3' SEQ ID NO:8

IL-10 F: 5'-CTGTGGAGAAGCTGTGGCAG-3' SEQ ID NO:9 R: 5'-GGGATCCACACTCTCCAGCr-3' SEQ ID NO:10

IL-6 F: 5'-GTACTCCAGAAGACCAGAGG-3' SEQ ID NO:11 R: S'-TGCTGGTGACAACCACGGCC-3' SEQ ID NO:12

As can be seen in FIG. 5, the cabbage extract inhibited

the expression of the proinflammatory mediators iNOS and COX-2,

which was increased by stimulation with LPS, and the expression

inhibitory effect of the heat-treated cabbage extract was

better than that of the raw cabbage extract.

As described above, the composition of the present

invention is prepared using cabbage that has long been used as

a food material, and thus it can be used safely in the human

body. In addition, it has excellent effects on liver

protection and liver function improvement, and thus may be

useful as a pharmaceutical composition for preventing or

treating liver disease and as a functional health food

composition for protecting liver and improving liver function.

Sequence Listing 02 Jul 2019

<110> SEOWON UNIVERSITY INSTITUTE OF INDUSTRY-ACADEMY COLLABORATION

<120> Hepatoprotective Composition Comprising Cabbage Extract

<130>

<160> 12 2019204739

<170> KoPatentIn 3.0

<210> 1 <211> 24 <212> DNA <213> Artificial Sequence

<220> <223> primer

<400> cactcacggc aaattcaacg gcac 24

<210> 2 <211> 22 <212> DNA <213> Artificial Sequence

<220> <223> primer

<400> 2 gactccacga catactcagc ac 22

<210> 3 <211> 25 <212> DNA <213> Artificial Sequence

<220> <223> primer

<400> 3 cccttccgaa gtttctggca gcagc 25

<210> 4 <211> 25 <212> DNA <213> Artificial Sequence

<220> <223> primer

<400> 4 ggctgtcaga gcctcgtggc tttgg 25

<210> 5 <211> 20 <212> DNA <213> Artificial Sequence

<220> 2019204739

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<210> 6 <211> 20 <212> DNA <213> Artificial Sequence

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<220> <223> primer

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<211> 20 02 Jul 2019

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<210> 10 <211> 20 <212> DNA <213> Artificial Sequence

<220> <223> primer

<400> 10 gggatccaca ctctccagct 20

<210> 11 <211> 20 <212> DNA <213> Artificial Sequence

<220> <223> primer

<400> 11 gtactccaga agaccagagg 20

<210> 12 <211> 20 <212> DNA <213> Artificial Sequence

<220> <223> primer

<400> 12 tgctggtgac aaccacggcc 20

Claims (1)

WHAT IS CLAIMED IS:

1. A pharmaceutical composition for preventing or

treating liver disease, comprising a cabbage extract as an

active ingredient.

2. The pharmaceutical composition of claim 1, wherein

the cabbage extract is obtained by extracting cabbage after

heat-treating the cabbage at a temperature of 100 to 150°C.

3. The pharmaceutical composition of claim 1 or 2,

wherein the liver disease is one or more selected from the

group consisting of drug-induced liver disease, alcoholic liver

disease, acute hepatitis, and fatty liver.

4. The pharmaceutical composition of claim 1 or 2,

wherein the extract is obtained by extraction with water, a Cl

to C4 alcohol, or a mixture thereof.

5. A functional health food composition for protecting

liver and improving liver function, comprising a cabbage

extract as an active ingredient.

6. The functional health food composition of claim 5,

wherein the cabbage extract is obtained by extracting cabbage after heat-treating the cabbage at a temperature of 100 to

1500C.

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