KR101706017B1 - Hepatoprotective composition comprising extracts of fruits of stauntonia hexaphylla for preventing alcoholic liver injury - Google Patents

Abstract

The present invention relates to a high-value-added functional pharmaceutical composition having liver protecting function against alcoholic liver damage which can be safely used without toxicity and side effects by using a mulberry fruit hot water extract of Korea as a natural resource. The pharmaceutical composition of the present invention is free from adverse effects on the human body and has excellent liver protection against alcoholic liver damage. Therefore, even when used for long time, it is safe to use alcoholic liver damage Can be used for liver protection.

Description

FIELD OF THE INVENTION The present invention relates to a pharmaceutical composition having liver protective function against alcoholic liver damage comprising mulberry fruit extract as an active ingredient. BACKGROUND OF THE INVENTION 1. Field of the Invention < RTI ID = 0.0 >

The present invention relates to a composition having liver protection against alcoholic liver damage. More particularly, the present invention relates to a composition for protecting liver against alcoholic liver damage which can be safely used without toxicity and side effects by using mulberry fruit extract as a natural raw material. ≪ / RTI >

Mullen (Stauntonia hexaphylla) is an evergreen vine plant with crossbred plants, buttercups, and the like. It is also called a mulberry tree. The mullen is male and female, and the main stem extends about 5 m. The leaves are alternate phyllotaxis of 5 to 7 small leaves. Leaves are thick, ovate or oval, with flat edges.

The petiole is about 6 to 8 cm long, and the petiole is about 3 cm long. Flowers bloom in May, yellowish white, and run on gun inflorescence. Small flower blooms of female flower become reddish brown in autumn, and there are many shrubs and are rough. The fruit is ovate or oval with a length of 5 cm to 10 cm in length, ripe in reddish brown in October and tastes better than flesh in flesh. Seeds are oval oval and black. The mullen is mainly distributed in Korea, Japan, Taiwan or China. In Korea, it grows well in valleys and forests in southern provinces such as Jeollanam-do, Gyeongsangnam-do and Chungcheongnam-do.

Drinking is indispensable for modern people. Excessive drinking temporarily causes hangover such as throbbing, headache, thirst, nausea, gastrointestinal disorder, diarrhea, etc., and chronic diseases such as fatty liver and cirrhosis Can cause. Headaches during hangover are due to the expansion of brain blood vessels. Thirst and dehydration are caused by the diuretic effect of ethanol, which is produced by the suppression of the secretion of vasopressin, an antidiuretic hormone secreted by ethanol in the pituitary gland. In addition, digestive organ abnormalities such as gastrointestinal disorders are caused by an increase in gastric acid secretion, and the main cause is toxicity of ethanol or acetaldehyde accumulated in hepatocytes.

In addition to the mechanism of ethanol degradation, toxicological studies of ethanol have been done in various ways. It has been reported that the toxicity of ethanol is not only observed neurologically but also genetically influenced (J. Caballeria, et al Life Sci., 41: 1021-1727, 1986). In recent years, many researches and experiments have been conducted on a number of substances that can reduce the toxicity of ethanol or inhibit the expression of toxins, and as a result, a variety of health supplements containing ingredients extracted from natural or herbal ingredients (Kim JJ, et al., Journal of the Korean Society of Agricultural Chemistry, 38 (6): 549-553, 1995).

A small amount of alcohol intake helps to relieve body fatigue and mental tension, stimulates the secretion of digestive juices to increase appetite, and recent studies have shown that proper alcohol intake can prevent cardiovascular diseases such as arteriosclerosis . However, excessive intake of alcohol or chronic alcohol impairs the gastrointestinal mucosa, causing problems with nutrient uptake and nutritional deficiencies, and harmful damage to all organs and tissues. Especially, it causes hepatocellular disorder which plays a pivotal role in alcohol metabolism, and causes alcoholic hepatitis, fatty liver and cirrhosis.

Chronic alcohol consumption promotes alcohol metabolism and increases oxygen consumption, leading to partial hypoxia and necrosis of liver tissue, or to accelerate the reaction of lipid peroxides by free radicals produced during alcohol metabolism, which may damage liver tissue have. In this case, the neutral lipid and cholesterol content of the liver tissue are greatly increased, and the active oxygen is increased to induce the drug metabolizing enzyme of the microsome in the liver cell, so that the medicines which are taken on a daily basis generate an excessive amount of free radicals, . Free radicals, which are not removed when the antioxidant enzyme activity is lowered, initiate a chain reaction that destroys unsaturated double bonds and generates aldehyde, dialdehyde, short-chain hydrocarbons, and promotes lipid peroxidation of each tissue. In normal physiological state, the free radical is balanced with the activity of antioxidant defense system, but when the free radical is generated in the body or the function of antioxidant defense system is decreased, Enemy stress increases.

        Overdose of alcohol can lead to various metabolic diseases and alcoholic liver diseases due to the intermediate metabolites produced during the alcohol or alcohol oxidation process. Alcohol is degraded by alcohol dehydrogenase, CYP450, catalase and metabolized to acetaldehyde. Acetaldehyde produced by excessive alcohol metabolism is involved in liver toxicity by producing acetaldehyde-protein by-products and lipid peroxides.

      The production of free radicals by alcohol occurs in the course of alcohol metabolism and acetaldehyde metabolism, or in the formation of free radicals by long-standing hydroxyethyl, resulting in lipid peroxidation (Pratt et al. 1990) . MDA, measured as the end product of lipid peroxidation, is widely used as a biochemical indicator of peroxidative damage in living tissue.

Therefore, it is important to reduce the oxidative stress in the body, which is increased by chronic alcohol consumption, and to prevent or prevent the diseases and harmful effects caused by alcohol by protecting and preventing the tissue from the toxicity of alcohol. The present invention provides a pharmaceutical composition having liver protecting function against alcoholic liver damage containing mulberry fruit extract which can be safely used without toxicity and side effects by using mulberry fruit as a natural raw material in order to utilize traditional plant resources of Korea I want to.

Korean Patent Laid-Open Publication No. 10-2011-0060002 relates to a composition for improving hangover, which comprises an appropriate ratio of post-fermented tea extract and chitooligosaccharide using a non-pathogenic microorganism, and contains an aldehyde dehydrogenase which degrades acetaldehyde, Compositions for the improvement of hangover, reduction of blood acetaldehyde concentration, hepatoprotection and improvement of hangover for fatigue recovery through synergistic effects on the activation of hypertension. Korean Patent Laid-Open Publication No. 10-2011-0033644 relates to a composition for relieving hangover which accelerates blood alcohol degradation rate and a method for producing the same, and more particularly to a composition for removing hangover which comprises an extract of Hovenia dulcis, Artemisia japonica, The present invention relates to a composition for use as a hangover detoxifying composition, and more particularly, to a composition for a hangover detoxifying composition, which shows a high synergistic effect as compared with the case where the respective components are separately used by mixing the components at appropriate ratios, And gastric inflammation. Korean Patent Laid-Open Publication No. 10-2010-0119150 relates to a composition for hangover decomposition containing a herbal medicine mixed extract and is characterized by containing an extract of Utila or an extract of Ufu, pomegranate and kidney as an active ingredient. From this, the activity of ADH and ALDH is promoted and the decomposition of alcohol and acetaldehyde is promoted, so that it can be usefully used as a hangover food and beverage. Korean Patent Publication No. 10-0372561 relates to a composition for hangover marrow containing an extract of natural herbal medicine and a health supplement containing the same as an effective ingredient and is characterized by containing at least one natural selected from the group consisting of red ginseng, red ginseng, And at least one natural herbal medicine extract selected from the group consisting of 40 to 80% by weight of the herbal medicine extract, 5 to 40% by weight of at least one natural herbal medicine extract selected from the group consisting of Rhizobium, Rhubarb, 5 to 40% by weight of the extract, 5 to 15% by weight of at least one natural herbal medicine extract selected from the group consisting of licorice, wormwood, and green tea, and a composition containing the same as an active ingredient ≪ / RTI > However, the above-mentioned prior arts have different materials and ingredients from the pharmaceutical composition for liver protection against alcoholic liver damage using the extract of mulberry fruit for the purpose of the present invention.

It is intended to provide a high value-added functional pharmaceutical composition having a liver protecting function against alcoholic liver damage which can be safely used without toxicity and side effects by using mulberry fruit extract as a natural resource of Korea.

In order to achieve the object of the present invention, the present invention provides a pharmaceutical composition having liver protecting function against alcoholic liver damage comprising mulberry fruit extract or nonpolar soluble extract as an active ingredient.

The mullen fruit crude extract may be obtained by extracting at least one selected from the group consisting of water, methanol, ethanol, propanol, isopropanol, butanol or a mixed solvent thereof as an extracting solvent, The extract may be fractionated by using any one selected from the group consisting of hexane, chloroform, dichloromethane and ethyl acetate as a nonpolar solvent as a fraction solvent.

Furthermore, a drink may be provided as a food having liver protection against alcoholic liver damage including the above extract as an active ingredient, and the extract may contain 0.01 to 100 wt% of the total composition having hepatic protection against alcoholic liver damage %. ≪ / RTI >

Since the pharmaceutical composition having the liver protecting function against alcoholic liver damage of the present invention is excellent in the liver protecting function against alcoholic liver damage while having no adverse effect on the human body, May be useful for liver protection against safe alcoholic liver damage without side effects.

Fig. 1 is a photograph showing mulberry fruit.
Fig. 2 shows the extraction and fractionation diagram of the mullen fruit hot water extract.
Fig. 3 shows the results of cell viability measurement for mullen fruit hot water extract.
FIG. 4 shows the results of measurement of MDA production in liver tissue of acute alcoholic liver damaged mice for alum fruit extract.
FIG. 5 shows the results of measurement of MDA production in liver tissue of chronic alcoholic liver injured mice against mullen fruit hot water extract (200 mg / kg).
6 shows the results of measurement of TBAR (MDA) production in hepatic tissue of chronic alcoholic liver injured mice against mullen fruit hot water extract (10, 50, 100, and 200 mg / kg).
FIG. 7 shows GOT activity measurement results of chronic alcoholic liver injured mice on mullen fruit hot water extract (10, 50, 100, and 200 mg / kg).
Figure 8 shows the results of measuring GPT activity in chronic alcoholic liver injured mice for mullen fruit hot water extract (10, 50, 100, and 200 mg / kg).
Figure 9 shows the results of measurement of ALDH activity in liver tissue for mullen fruit hot water extract (10, 50, 100, 200 mg / kg).

In the present invention, a pharmaceutical composition having liver protecting function against alcoholic liver damage containing mulberry fruit leaf extract which effectively acts on liver protection against alcoholic liver damage containing mulberry fruit leaf extract or nonpolar soluble extract as an active ingredient / RTI >

Korean Patent Application No. 10-2011-0082498 filed by the present inventors discloses a composition for protecting the liver comprising 0.001 wt% to 99 wt% of mullen fruit hot water extract. The present invention relates to a composition for preventing liver damage caused by hepatotoxic drugs such as carbon tetrachloride (CCl4) or acetaminophen (APAP) used in the present invention, From the point of view, the use and action mechanism of the liver protective composition against liver injury induced by alcohol as in the present invention are different from each other.

Specifically, in the case of liver protection against liver damage induced by hepatotoxic drugs such as carbon tetrachloride (CCl4) or acetaminophen (APAP), inhibition of lipid peroxidation, GOT in serum. The inhibition of mtDNA expression by cytochrome P450, and the increase of expression of Nrf-2 and HO-1 are set as the evaluation items.

On the other hand, in the case of hangover resolution, inhibition of lipid peroxidation, GOT in serum. We investigated whether the decrease in blood alcohol concentration, the activity of alcohol dehydrogenase (ADH activity), and the increase of aldehyde dehydrogenase activity (ALDH activity), which are major biomarkers of alcohol metabolism including inhibition of GPT increase,

As in the present invention, in the case of liver protection against acute liver injury and chronic liver injury induced by ethanol, lipid peroxidation in liver tissues by alcohol metabolism is set as the most important evaluation item for efficacy, The effect of ALDH enzyme activity, which is an enzyme that degrades aldehyde, which is a toxic substance in liver tissue produced by ethanol administration, by GOT, GPT, And that the inhibition of lipid peroxidation in liver injury induced by APAP and CCl4 has another mechanism. Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings.

1. Production of mullen fruit hot water extract and fractions

Figure 2 shows the process of obtaining mulberry fruit extract and fractions by organic solvent. 2.1 kg of mulberry fruit was washed with distilled water, 40 L of distilled water was added, and the mixture was heated and extracted at 100 캜 for 3 hours with an electric chemical booster. The extracted solution was filtered with 400 mesh filter cloth and then concentrated with a rotary evaporator under reduced pressure. After the filtration, the same amount of distilled water is again used for the remaining residue, which is further extracted twice, filtered and concentrated under reduced pressure. The concentrated hot water extract was lyophilized in a freeze dryer. 148g (7.05%) of mullen fruit hot water extract was obtained.

2. Preparation of polar and non-polar solvent-soluble fractions of mullen fruit hot water extract

The extracts of mullen fruit hot water extract prepared as shown in FIG. 2 were fractionated using an organic solvent. The production of polar and nonpolar solvent soluble fractions of mulberry fruit Forty grams of mullen fruit hot water extract was completely dissolved in 1 L of distilled water, and the water layer and the hexane layer were separated by adding 1 L of hexane (Hexane) to the fractional funnel. This process was repeated three times.

Chloroform, ethyl acetate and butanol were sequentially added through the same procedure to obtain fractions. Each of the fractions thus obtained was filtered with a vacuum filtration apparatus and concentrated. The extract was lyophilized to remove the solvent completely This test was used for this experiment.

2.1. Hexane-soluble fraction separation

Forty grams of mullen fruit hot water extract was completely dissolved in lL of distilled water. Then, the mixture was placed in a separating funnel and 1 liter of hexane was added to separate the hexane insoluble layer (water layer) and the hexane soluble layer. Again, the hexane insoluble fraction and the soluble fraction were collected by repeating the same process three times for the hexane insoluble layer (water layer).

2.2. Isolation of Soluble Fraction from Chloroform

The hexane insoluble fraction (aqueous layer) was mixed with 5 L of chloroform, and then the chloroform-soluble fraction and the insoluble fraction were separated. The chloroform-insoluble fraction and the soluble fraction were collected by repeating the same process three times for the chloroform insoluble layer (water layer).

2.3. Ethyl acetate soluble fractionation

After adding 5 L of ethyl acetate to the chloroform insoluble fraction (aqueous layer), the ethyl acetate soluble fraction and the insoluble fraction were separated. The ethyl acetate insoluble layer (water layer) was subjected to the same process three times to collect the ethyl acetate insoluble fraction and the soluble fraction Respectively.

2.4. Butanol soluble fraction fractionation

The butanol insoluble fraction and the insoluble fraction were separated by adding 5 L of butanol to the ethyl acetate insoluble fraction (water layer), and the butanol insoluble fraction was repeated three times to collect the butanol insoluble fraction and the soluble fraction.

2.5 Acquisition of hot water extract and fractions of mulberry fruit

The hexane-soluble fraction, the chloroform-soluble fraction, the ethyl acetate-soluble fraction and the butanol-soluble fraction were concentrated under reduced pressure and then lyophilized to obtain 0.1 g of a hexane fraction, 0.6 g of a chloroform fraction, 2 g of an ethyl acetate fraction and 15 g of a butanol fraction in 40 g of mulberry fruit hot water extract And used as a sample.

3. Cytotoxicity test of mullen fruit hot water extract (by MTT assay)

To measure cytotoxicity of the mullen fruit hot water extract prepared in Example 1, mouse macrophage RAW264.7 cells were purchased from ATCC. (Dulbecco's modified Eagle's medium / Nutrient Mixture Ham's F12), fetal bovine serum (FBS), L-glutamine and penicillin-streptomycin used in the cell culture were purchased from Gibco / BRL (USA).

The RAW264.7 cells were cultured in a DMEM / F12 medium supplemented with 10% FBS, 1% penicillin streptomycin and 1% L-glutamine, and incubated at 37 ° C in a humidified CO2 incubator (5% CO2 / 95% air ). After the cells were cultured to about 80% of the culture dish, the cells were washed with PBS (pH 7.4), washed, and subcultured by treatment with 0.25% trypsin and 2.56 mmol / L EDTA. The medium was changed every 2 days.

The cultured cells were divided into 48 wells at a density of 50,000 cells / well and further cultured for 24 hours. After 24 hours, the control group treated with LPS alone without any treatment, LPS and the mullen fruit extract of Example 1 were cultured in DMSO, which was confirmed to have no significant effect on cell survival, After the culture was further cultured for 24 hours, the culture solution was removed and the number of living cells was measured by MTT assay.

The MTT analysis was performed as follows. First, after removing the cell culture medium, DMEM / F12 medium containing 1 mg / ml of MTT was treated with 1 ml per well, followed by further incubation for 4 hours in a 37 ° C humidified CO2 incubator. After the medium was removed, the tetrazolium bromide salt was removed and 200 μl of DMSO was added to dissolve the formazan crystals formed in each well. The absorbance was measured at 540 nm wavelength in a microplate reader (BIO-RAD) Respectively. The results of the treatment with the mulberry fruit extract are shown in FIG. 3 as an average value of the measured values by performing the same experiment three times in the same manner.

As shown in FIG. 3, even when the mullen fruit hot water extract prepared in Example 1 was treated at various concentrations, specifically, at concentrations ranging from 10 μg / ml to 200 μg / ml for 24 hours, no sample As a result, it was confirmed that the treatment of the mullen fruit hot water extract prepared in Example 1 at various concentrations showed no significant effect on cell proliferation. Therefore, it was confirmed from the above results that the extract of Mulberry fruit was not cytotoxic up to 200 μg / ml.

4. Experimental animals, breeding

A male rat (ICR mouse, male) weighing 22-24 g and 4 weeks of age was purchased from Samutoco as an experimental animal for the liver protection against alcoholic liver damage of mullen fruit hot water extract, (control), and 5 mice per group. The animals were housed in a 12 hour cycle of contrast medium at a temperature of 22 ° C and 2 ° C and a relative humidity of 65 ° C and 5% for 7 days. Solid feed (Samyang feed) and water were supplied sufficiently.

4.1 Alcohol induced acute and chronic liver injury induced animal model experiment and sample treatment

The whole experimental group was divided into two groups of acute and chronic experiment. After 10 days, 50, 100, and 200 mg / kg samples of mullen fruit extract were administered at 1 / (50, 100 and 200 mg / kg) samples were administered to the chronic experimental group in which 5 g / kg EtOH (50% with saline) After 30 minutes, the mice were divided into an acute experimental group in which 5 g / kg EtOH (50% with saline) of inducer was administered.

After the final administration of each sample and the inducer, the animals were fasted and water-saved. After 18 hours, the animals were lavaged under ether anesthesia and blood was collected from the abdominal aorta and centrifuged at 1,500 rpm for 10 minutes to separate the serum. Liver tissues were harvested and stored at -70 ° C before use in the experiment.

5. Experimental results of liver protection against alcoholic liver damage of mullen fruit hot water extract

5.1 Evaluation of lipid peroxidation inhibitory effect in hepatic tissue of alcoholic acute liver injury-induced mice of mulberry fruit hot water extract

Figure 4 shows the results of measurement of MDA production in hepatic tissue of acute alcoholic liver injured mice to mullen fruit hot water extract (50, 100 and 200 mg / kg). Transfer 0.2 g of liver tissue from the alcoholic acute hepatic injury-induced mice to the 1.5 ml EP tube. 1 ml of PBS buffer is placed in a homogenizer glass tube and the liver tissue is homogenized.

72% TCA 195 ul (final conc. 10%) is added and centrifuged at 4 ° C and 12,000 rpm for 15 minutes. The supernatant is transferred to a new EP tube and centrifuged once more. Transfer 0.6 ml of supernatant to a new EP tube. Add 272ul (final conc. 0.25%) of 0.8% TBA to the supernatant. Boiling at 95 ° C. Control When the color changes to pink, stop the reaction on ice. Transfer 100 ul of the supernatant to a 96-well plate and measure the amount of MDA (malondialdehyde) produced by absorbance at 532 nm.

As shown in Fig. 4, in the case of the normal group not treated with ethanol, a low MDA secretion amount was confirmed, and in the case of the control group (acute) caused by ethanol, lipid peroxidation was induced, and the content of MDA was remarkably increased . In the experimental group treated with mullen fruit hot water extract (50, 100 and 200 mg / kg), the amount of MDA secreted was 31.9% (50 mg / kg) compared with the control group kg), 29.7% (100 mg / kg), and 29.9% (200 mg / kg) lower than the positive control group. In the positive control group administered with the positive control substance (YM: 200 mg / kg), the amount of MDA secretion was 41.7% lower than that of the control group (100% standard).

From this, it was confirmed that the extract of Mulberry fruit extract inhibited MDA production, that is, the lipid peroxidation activity inhibitory effect.

5.2 Measurement of lipid peroxidation inhibitory effect in liver tissue of alcoholic chronic liver injury induced mullen fruit hot water extract

FIG. 5 shows the results of measurement of MDA production in liver tissue of chronic alcoholic liver injured mice against mullen fruit hot water extract (200 mg / kg). Transfer 0.2 g of liver tissue from the alcoholic chronic liver injury-induced mice to the 1.5 ml EP tube. 1 ml of PBS buffer is placed in a homogenizer glass tube and the liver tissue is homogenized.

72% TCA 195 ul (final conc. 10%) is added and centrifuged at 4 ° C and 12,000 rpm for 15 minutes. The supernatant is transferred to a new EP tube and centrifuged once more. Transfer 0.6 ml of supernatant to a new EP tube. Add 272ul (final conc. 0.25%) of 0.8% TBA to the supernatant. Boiling at 95 ° C. Control When the color changes to pink, stop the reaction on ice. Transfer 100 ul of the supernatant to a 96-well plate and measure the amount of MDA (malondialdehyde) produced by absorbance at 532 nm.

As shown in FIG. 5, in the case of the normal group not treated with ethanol, a low MDA secretion amount was confirmed, and in the case of the ethanol-induced control group, lipid peroxidation was induced, and the content of MDA was remarkably increased. In addition, in the experimental group treated with mullen fruit leaf hydrothermal extract (200 mg / kg), the amount of MDA secreted was lower than that of the control group in spite of treatment with ethanol.

From these results, it was confirmed that the hot water extract of mullen fruit inhibited the MDA production, that is, the lipid peroxidation activity inhibitory effect.

5.3 Inhibition of lipid peroxidation in hepatic tissues of alcoholic chronic liver injury-induced mice of mullen berries hydrothermal extract

Figure 6 shows the results of measurement of MDA production in liver tissues of chronic alcoholic liver injured mice against mullen fruit hot water extract (10, 50, 100, and 200 mg / kg). Transfer 0.2 g of liver tissue from the alcoholic chronic liver injury-induced mice to the 1.5 ml EP tube. 1 ml of PBS buffer is placed in a homogenizer glass tube and the liver tissue is homogenized.

72% TCA 195 ul (final conc. 10%) is added and centrifuged at 4 ° C and 12,000 rpm for 15 minutes. The supernatant is transferred to a new EP tube and centrifuged once more. Transfer 0.6 ml of supernatant to a new EP tube. Add 272ul (final conc. 0.25%) of 0.8% TBA to the supernatant. Boiling at 95 ° C. Control When the color changes to pink, stop the reaction on ice. Transfer 100 ul of the supernatant to a 96-well plate and measure the amount of MDA (malondialdehyde) produced by absorbance at 532 nm.

As shown in FIG. 6, in the case of the normal group not treated with ethanol, a low MDA secretion amount was confirmed, and in the case of the ethanol-induced control group, lipid peroxidation was induced, and the content of MDA was remarkably increased. In addition, in the experimental group treated with mullen fruit leaf hydrothermal extract (10, 50, 100, and 200 mg / kg), the amount of MDA secreted was lower than the control group in spite of treatment with ethanol.

In particular, MDA production was 57.4% (50 mg / kg) and 54.3% (100%) in the experimental group administered with 50 mg / kg, 100 mg / kg and 200 mg / kg of mullen fruit hot water extract, 100 mg / kg) and 59.1% (200 mg / kg).

In the experimental group administered 50 mg / kg, 100 mg / kg and 200 mg / kg of mullen fruit hot water extract, the MDA production inhibitory effect was 142.6%, 145.7%, and 140.9% higher than the control group (100% This is the result of confirming that there is.

From these results, it was confirmed that the hot water extract of mullen fruit inhibited the MDA production, that is, the lipid peroxidation activity inhibitory effect.

5.4 GOT / GPT assay for hot-water extract of mulberry fruit in alcohol-induced chronic liver injury-induced mouse model

FIG. 7 shows the results of measurement of GOT activity in serum of chronic alcoholic liver-injured mice against mullen fruit hot water extract (10, 50, 100, and 200 mg / kg) , and 200 mg / kg) in chronic alcoholic liver injured mice. Blood was collected from the abdominal aorta of the anesthetized mouse, and the collected blood was centrifuged at 3,000 rpm for 5 minutes to obtain serum.

L-aspartic acid and a-ketoglutaric acid for GOT and L-alanine and a-ketoglutaric acid for GPT were used to measure the activity of transaminase (GOT, GPT) in the serum. The diagnostic kit prepared by the Reitman-Frankel method, which quantifies the color of hydrazine produced by the action of 2,4-dinitrophenyl hydrazine under alkaline conditions, is obtained by reacting these substrates with 37 캜 for 39 minutes. , Korea) was used to measure the absorbance at 505 nm.

The unit of activity is expressed as Karmen unit per ml of serum. As shown in FIGS. 7 and 8, when 10 mg / kg of mulberry fruit hydrolyzate was administered at 10, 50, 100 and 200 mg / kg, the inhibitory effect of GOT and GPT .

Therefore, it was confirmed that the hot water extract of mullen fruit did not affect liver damage and inhibited the increase of GOT and GPT activity due to chronic alcohol consumption.

5.5 Measurement of ALDH activity in liver tissues for hot water extract of mulberry fruit

After the last blood collection from the femoral artery (4h), the liver was extracted from the alcohol-treated rats and homogenized using a homogenizer at 4 ° C with 0.25M sucrose. This was centrifuged at 600 × g for 15 minutes to obtain a supernatant from which the nuclei and unmarshaled portions were removed. The supernatant was further centrifuged at 10,000 g for 20 minutes to obtain supernatant. The separated supernatant was mixed with ALDH assay buffer, developer and coenzyme NAD +, and the amount of NADH produced by reacting at 37 ° C for 30 minutes at intervals of 10 minutes was measured at 450 nm (ALDH activity colorimetric assay kit, # 731 -100; BioVision), and the activity unit is expressed in nmole as the amount of NADH produced per minute of the protein 1 mg, and the graphical output is expressed as a relative value (relative,%) to the value of the control group (ethanol treated group).

As shown in FIG. 9, ALDH activity activity in liver tissues was significantly higher in ethanol-treated group than in the control group treated with mullen fruit extract (10, 50, 100, and 200 mg / kg) Of the total number of patients.

Specifically, ALDH activity was 91.1% (10 mg / kg) compared with the control group (70.9%), which was administered with 10 mg / kg, 50 mg / kg, 100 mg / kg and 200 mg / kg of mullen fruit hot water extract ), 97.3% (50mg / kg), 99.7% (100mg / kg), and 106.7% (200mg / kg), the ALDH activity recovered close to the normal group (100%).

In particular, ALDH activity in the experimental group treated with mullen fruit hot water extract was 120.5% (10 mg / kg), 137.2% (50 mg / kg) and 140.6% (100 mg) compared with the control group / kg) and 150.5% (200 mg / kg), respectively.

From this result, it was confirmed that the extract of mulberry fruit was injected at a certain ratio with respect to the body weight of the experimental animals, and thus it was confirmed that the ethanol extract had an effect of promoting aldehyde decomposition, The compositions according to the present invention can be used to protect and prevent liver tissue from alcoholic liver damage caused by alcohol ingestion.

As a pharmaceutical composition having liver protecting function against alcoholic liver damage, Mullen fruit extract has excellent effects on liver protection against alcoholic liver damage without adverse effect on human body. Therefore, long-term administration It can be used for protecting liver against safe alcoholic liver damage without side effects and it can be expected to reduce the manufacturing production cost by replacing it with a plant inhabited by nature, and to effect import substitution and export through industrialization.

Claims (4)

Wherein the crude extract is extracted with water, ethanol, or a mixed solvent thereof as an extraction solvent. The pharmaceutical composition having liver protecting function against alcoholic liver damage
The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition having liver protecting function against alcoholic liver damage is contained in an amount of 50-200 mg per kg of body weight. delete delete

Images