KR100801902B1 - Composion for antimutagenic and anticancer containing the extracts of Zizyphus jujube seed - Google Patents

Abstract

The present invention relates to antimutagenic and cancer cell growth inhibitory active compositions containing jujube seed extract. More specifically, it relates to the antioxidant activity of the mutant containing the jujube seed water extract and the effect of inhibiting the growth of cancer cells, and can be usefully used as a component of prophylaxis and treatment or as a health food.

Jujube seed extract, antimutation, antioxidant, cancer cell suppression

Description

Antimutagenic and anticancer containing the extracts of Zizyphus jujube seed

The present invention relates to antimutagenic and anticancer active compositions containing jujube seed extract.

With the advancement of science, modern people can not escape from the pollution of harmful ingredients every day without knowing their own daily consumption of instant foods and processed foods containing all kinds of food additives. Very high. This has led to a number of changes in the form of the disease, especially the increasing incidence of adult diseases related to aging, including cancer. More than 85% of the known carcinogens are mutagens and less than 10% known to be non-carcinogenic are acting as mutagens, so there is a very high correlation between carcinogenesis and mutations, and mutations are very important in the early stages of cancer induction. It is known to work. Therefore, since a substance capable of inhibiting mutations may have an anti-carcinogenic effect, many efforts have been made to search for anti-mutagenic and anti-cancer substances from foods in our daily lives. Recently, efforts to search for bioactive substances in medicinal plants and herbal medicines, which have been known for a long time and have been used as folk remedies, have shown remarkable results. Among them, jujube has been widely used as a folk remedy by edible and various pharmacological effects for a long time, and especially has been used for chronic hepatitis, whole body weakness, lack of nutrition. In addition, it has been used a lot in our traditional foods such as porridge, tea, rice cake, medicinal rice, etc. Recently, it is also used in various processed forms such as jujube drink, liquid jujube tea, granulated jujube tea, jujube fructose, jujube syrup. Recently, the anti-inflammatory and periodontal tissue regeneration effects of jujube extract and the methanol extract of jujube inhibit liver necrosis and enzyme outflow of hepatocytes and improve liver resistance and hepatic function. In addition, the intestinal lactic acid bacteria growth and pH-lowering effect of jujube water extract, jujube water extract, ethylacetate fraction, β-glucuronidase and tryptophanase Inhibitory effects and colorectal cancer inhibitory effects have been reported. As a prior patent related to jujube extract, Korean Patent No. 0583051 “Cinnamon and Jujube Extract for Cancer Treatment and Prevention” relates to a pharmaceutical composition and a functional food for treating and preventing cancer, including cinnamon and jujube extract, respectively. It has been disclosed about the effect of inhibiting cell death and cancer cell migration by components and the increase of cancer cell growth inhibitory effect by mixing the components, Republic of Korea Publication No. 99-0039367 "composition for lowering blood pressure comprising jujube extract" Has disclosed a component of a high blood pressure treatment or health food that lowers blood pressure, including jujube extract. Jujube extract is known to treat and prevent cancer or hypertension, but research using jujube seed extract is incomplete.

The present invention has been made in view of the above point, an object of the present invention to provide a composition for preventing and treating anti-mutation and cancer cell growth inhibition containing jujube seed extract.

The above object of the present invention was achieved by measuring the antioxidant active radical scavenging ability, antimutagenicity and cancer cell proliferation inhibition after obtaining the jujube seed extract.

Hereinafter, the specific configuration of the present invention will be described in detail through examples and experimental examples.

The present invention comprises the steps of measuring the antioxidant active radical scavenging ability of the jujube seed extract using the water extract and ethanol extract as a sample of the powder sample of jujube seed; Anti-mutagenicity and cancer cell proliferation inhibitory effect and normal hepatocyte proliferation effect.

Jujube seed water extraction method and methanol extraction method in the present invention was used for hot water extraction.

Antioxidant scavenging activity of jujube seed extract in the present invention was measured by absorbing acetic acid and antioxidant active solution.

Antimutagenicity test of jujube seed extract in the present invention is a methylation agent (N-methyl-N'-nitro-N-nitrosoguanidine; MNNG) and nitronoline oxide (4-) as a mutagen of Salmonella typhimurium TA100 strain. Nitroquinoline-N-Oxide (4NQO) was confirmed as an inhibitory effect.

In the present invention, the proliferative effect of cancer cells and normal cells of jujube seed extract was confirmed by cytotoxicity by MTT (3- [4,5-dimerthylthiazol-2,5-diphenyl tetrazolium bromide; MTT) measurement.

Example 1 : Extract preparation of jujube seed

10 times distilled water was added to the jujube seed powder, followed by hot water extraction at 90-100 ° C., repeated extraction three times for 8 hours, followed by filtration, and 10 times of 99% methanol to the jujube seed powder. After extracting three times for 8 hours in a water bath equipped with a cooler and filtered. The filtrates were concentrated under reduced pressure and used as a water extract and a methanol extract.

Experimental Example 1 : DPPH Radical  Scavenging Action

Antioxidant activity (1,1-diphenyl-2- picrylhydrazyl; DPPH) radical scavenging activity of jujube seed water extract and methanol extract was measured in 0.1 M acetate buffer (pH 5.5) and 0.2 mM DPPH (1,1-diphenyl-2- After adding picryhydrazyl) and shaking for 10 seconds, the mixture was reacted at 37 ° C for 30 minutes, and then the reaction solution was measured at 517 nm using a spectrophotometer (Spectronic GENESYS 5, MILTON ROY).

Experimental results showed that the antioxidant activity of 84% at 0.5mg / mL and 89% at 0.25mg / mL in jujube seed water extract was shown in Table 1, and 91.7% at 1mg / mL and 0.75mg / mL in methanol extract. It showed 88.3% of activity. Jujube seed water extract showed higher antioxidant scavenging activity than methanol extract.

<Table 1> Antioxidant Activity Scavenging Activity of Jujube Seed Water Extract and Methanol Extract

Autumn water Concentration (mg / mL) Antioxidant Rate (%) Water extract One 72.8 0.75 77.8 0.5 84.0 0.25 89.0 0.01 55.3 Methanol extract One 91.7 0.75 88.3 0.5 70.0 0.25 34.5 0.01 -

Experimental Example 2 Antimutagenicity test

Antimutagenicity test of jujube seed water extract and methanol extract was carried out in a sterile tube with 50 μL of sample, 50 μL of mutagen, 0.2M buffer, Salmonella of jujube seed extract itself. typhimurium ) 100 μl of TA100 strain was added and pre-cultured at 37 ° C. for 30 minutes. This was mixed with 2 mL of top agar and evenly spread on minimal glucose agar plates. After 48 hours of incubation at 37 ° C. the number of revertant strains on the medium was counted. Three minimum plate media were used for one sample, and the inhibitory effect on the mutagen was expressed by the inhibition rate (%) for the mutagen material.

As a result, as shown in Tables 2 to 3, Salmonella showed antimutagenic effects of water extracts of jujube seed on mutant organism methyl chemistry and nitronoline oxide, which are substances that directly induce damage to DNA in cells. ( Salmonella typhimurium ) The antimutagenic effect of jujube seed water extract against MNNG in TA100 strain showed a high inhibitory effect of 75.2% at the concentration of 10% water extract. As a result, the jujube seed water extract is Salmonella typhimurium ) showed inhibition of MNNG mutation in TA100 strain. In addition, the antimutagenic effect on 4-NQO showed an inhibitory effect of 72.2%, 67.2% at 5%, 2.5% of sample concentration. As a result, it was confirmed that the jujube seed water extract had a high antimutagenic effect.

<Table 2> Antimutagenic Effects of Methyl Chemical Agents from Jujube Seed Water Extracts

Sample processing Revertants / plate Salmonella Inhibition Rate (%) Control (no treatment) 126 ± 4.24 4NQO 1495 ± 53.24 4NQO + Water Extract 10% water extract 618 ± 27.25 64.0 5% water extract 507 ± 15.14 72.1 2.5% water extract 575 ± 22.17 67.2

Experiment 3 : Inhibition of cancer cell proliferation and measurement of normal cell proliferation activity

MTT measurements of cell proliferation and toxicity were examined for the effect of jujube seed extract on cancer and normal cell growth. Inhibitory effect of jujube seed extract on hepatocellular carcinoma Hep G2 (hepatoma cell, human) and colon cancer cell HT-29 (colon carcinoma cell, human), hematological cancer U937, promyelocytic leukocyte HL60 and human Confirmed. First, remove the medium from the cultured cells, mix lightly with the addition of the buffer solution, remove the buffer again, incubate for 5 minutes at 37 ° C by adding 0.25% trypsin-EDTA, and check whether the cells were completely separated from the bottom of the culture dish. After observation, the culture medium was added and mixed well, and then the cell number was adjusted to 1 × 10 5 cells / mL. Samples were dissolved in DMSO (dimethysulfoxide) at appropriate concentrations and filtered through a 0.22 μm membrane filter. Each cell prepared in a 96-well plate was dispensed 198 μl and incubated for 24 hours at 37 ℃, 5% CO2. 2 μl of each concentration sample was added thereto, followed by incubation for 48 hours, and then MTT (0.5 mg / mL) solution was added to reduce MTT by enzymatic action of viable cancer cells. After 4 hours, the supernatant was removed by an aspirator by carefully treating the forazan formed on the bottom of the well to prevent scattering. 200 μl of DMSO was added to each well to dissolve the formazon, and then the O.D. Absorbance was measured at 540 nm.

The experimental results are as shown in Tables 4 to 6. HepG2, a hepatocarcinoma cell, showed 56.3% and 58% of inhibitory effect at 1mg / mL and 0.5mg / mL, respectively, and HT29, a colon cancer cell, at 7mg% and 66.7% at 0.5mg / mL, respectively. Showed a high inhibitory effect. In human blood cancer cell U937, the inhibitory effect was 70.1% and 70.7% at the concentration of 1mg / mL and 0.5mg / mL, respectively, and 59.5 at the concentration of 1mg / mL and 0.5mg / mL, respectively, in the proliferation of HL60. %, 58.3% showed an inhibitory effect. Chang, a normal human hepatocyte, showed cell proliferation effects of 15.5% and 16.9% at concentrations of 1 mg / mL and 0.5 mg / mL, respectively. As a result, jujube seed extract had a growth inhibitory effect on cancer cells and a cell proliferation effect on normal hepatocytes.

Table 4 Inhibitory Effects of Jujube Seed Water Extracts on Hepatocellular Carcinoma and Colon Cancer Cells

Sample processing (mg / mL) HepG2 Cell Proliferation (O.D.) Inhibition Rate (%) Control 0.245 ± 0.151 Water extract concentration 0.5 0.103 ± 0.076 58.0 Water Extract Concentration 1 0.107 ± 0.022 56.3 HT29 Control 0.456 ± 0.039 Water extract concentration 0.5 0.152 ± 0.041 66.7 Water Extract Concentration 1 0.131 ± 0.039 71.3

<Table 5> Inhibitory Effects of Jujube Seed Water Extracts on Hematological Cancer Cells and Promyelocytic Leukocytes

Sample processing (mg / mL) U937 Cell Proliferation (O.D.) Inhibition Rate (%) Control 0.321 ± 0.151 Water extract concentration 0.5 0.094 ± 0.076 70.7 Water Extract Concentration 1 0.096 ± 0.022 70.1 HL60 Control 0.247 ± 0.039 Water extract concentration 0.5 0.103 ± 0.041 58.3 Water Extract Concentration 1 0.100 ± 0.039 59.5

<Table 6> Effect of Jujube Seed Water Extract on the Growth of Normal Hepatocytes

Sample processing (mg / mL) Chang Cell Proliferation (O.D.) Growth rate (%) Control 0.942 ± 0.089 Water extract concentration 0.5 1.088 ± 0.121 15.5 Water Extract Concentration 1 1.110 ± 0.139 16.9

As described in the above Examples and Experimental Examples, the present invention is a water extract extracted from 90 ~ 100 ℃ in jujube or methanol extract using 10 times 99% methanol of the sample is the antioxidant scavenging effect and anti-mutagenic direct mutagens It has an excellent effect on the inhibition of mutations against the organism methyl chemical agent and nitronoline oxide.

In addition, the present invention, since jujube seed water extract exhibits the effect of inhibiting the proliferation of human liver cancer cells, colon cancer cells, blood cancer cells and promyelocytic leukocytes and normal hepatocyte proliferation, it is a very useful invention for functional food manufacturing and pharmaceutical industry.

Claims (2)

Antimutagenic and anticancer composition comprising jujube seed water extract or methanol extract as an active ingredient. The antimutagenic and anticancer composition according to claim 1, wherein the cancer is liver cancer, colon cancer or hematological cancer.