KR20040092951A - Young antlers extracts and antlers extracts-based-drink having protective effect on cellular DNA damage - Google Patents

Abstract

PURPOSE: Provided are the hot water extract of young antlers and a drink containing the same. The cellular DNA which is treated with the extract or the drink containing the same has excellent resistance against stress from the outside. CONSTITUTION: The composition for protection of cellular DNA damage is characterized by containing the hot water extract of young antlers. It further contains hot water extracts of Angelica gigas, jujube and ginger. The extracts are effective for prevention of cancers.

Description

Young antlers extracts and antlers extracts-based-drink having protective effect on cellular DNA damage}

The present invention relates to an antler extract having a novel effect and a beverage containing the extract. More specifically, the present invention relates to a antler extract having a DNA damage protection effect and a beverage containing the extract.

Aging, smoking, environmental pollution and other factors increase the production of reactive oxygen species (ROS) with a wide range of detrimental activities in the body, exceeding the limits of the antioxidant system in our body and causing oxidative stress. As a result, it causes DNA damage, which is an early stage of cancer development, which causes cellular mutations to develop into cancer. Therefore, in order to prevent cancer, the oxidative stress accumulated in the body must be neutralized. There are two kinds of body antioxidant systems that neutralize the oxidative stress in the body. One of them is to increase antioxidant enzyme activity, and the other is to eat antioxidant foods or antioxidant vitamins to neutralize oxidative stress. Recent studies have shown that ingesting more antioxidant foods or vitamins is more efficient in the body than selectively increasing antioxidant enzyme activity.

Deer antler (cervi parvum cornu) is a medicinal herb that has been widely used as a tonic tonic for a long time by harvesting and processing fresh horns of deer. The results of animal experiments and clinical experiments showed that antler had growth and growth promotion effects, aging inhibitory effects, hematopoietic function, heart function improvement, anti-muscular fatigue effect and anti- analgesic effect in children.

Hypoxanthine in antler is known as one of the components of anti-aging, and it is known to act as an anti-aging agent by inhibiting the activity of monoamineoxidase B and also of glycolipids contained in the antler. Ganglioside (Ganglioside) is a glycolipid of polymers accumulated in the gray matter of the kidney, liver, spleen and brain, and is known to be effective in neurotoxicity related to neurotransmission disorders.

However, the protective effect of antler against DNA damage is still unknown.

For example, Korean Patent No. 10775 relates to a method of manufacturing a deer antler extract containing a large amount of active ingredient of antler in a short time, a method of manufacturing a dry antler extract fine powder from the deer antler extract, and a health food using the same. 95-16770 discloses a blood glucose reducing effect of antler lipids, and Korean Patent Publication No. 2002-84360 discloses the electron donating ability, SOD-like activity, and xanthine oxidase of herbal beverages including antler and other herbal medicines. Inhibitors of xanthine oxidase, tyrosinase, ACE (Angiotensin converting enzyme) inhibitor, osteoporosis prevention, hematopoiesis and heavy metal removal and tonic action are described.

An object of the present invention is to determine the DNA damage protection effect of the antler extract, and to provide a beverage containing the antler extract.

1 is a graph showing the DNA damage protection effect in% DNA for lymphocyte cells according to the treatment concentration of antler hydrothermal extract.

Figure 2 is a graph showing the DNA damage protection effect on lymphocytes according to the treatment concentration of antler hydrothermal extract in TL.

Figure 3 is a graph showing the effect of protecting the DNA damage to lymphocytes in accordance with the treatment concentration of the antler hydrothermal extract in TM.

Figure 4 is a graph showing the DNA damage protection effect in% DNA for lymphocyte cells of the beverage containing hot water extracts of Angelica, jujube and ginger in addition to antler.

Figure 5 is a graph showing the DNA damage protection effect on the lymphocyte cells of the beverage containing hot water extracts of Angelica, jujube and ginger in addition to antler.

Figure 6 is a graph showing the DNA damage protection effect on the lymphocyte cells of the beverage containing hot water extracts of Angelica, jujube and ginger in addition to antler.

FIG. 7 is a graph comparing% DNA as a protective effect of DNA damage on lymphocyte cells of diabetic patients and controls of beverages containing hot water extracts of Angelica, jujube and ginger in addition to antler before and after 20 days of intake of beverages. FIG. .

FIG. 8 is a graph comparing TL as a protective effect of DNA damage on lymphocyte cells of diabetic patients and controls of beverages containing hot water extracts of Angelica, jujube and ginger in addition to antler before and after 20 days of ingestion of beverages.

9 is a graph comparing the results of TM before and after 20 days of ingestion of the DNA damage protection effect on the lymphocyte cells of the diabetic group and the control group of the beverage containing hot water extracts of Angelica, jujube and ginger in addition to antler.

DNA damage protection effect of the antler extract of the present invention and the beverage containing the same was confirmed by treating the human lymphocytes with antler extract, treated with an oxidative stress-inducing substance and measuring the DNA damage of lymphocyte cells by Comet Assay method. .

Deer Antler extract for use in the present invention is prepared as follows. 100 parts by weight of domestic antler and about 7500 to 9000 parts by weight of water were added to an extraction container, and primary extraction was performed at a temperature of about 100 to 115 ° C. for 3.5 to 4.5 hours, followed by filtration to separate the filtrate and the residue. To 9000 parts by weight of water is added, followed by the second extraction through the same process as the first extraction, and then the filtrate is combined with the first extraction filtrate to obtain an extract. Extracts are centrifuged, lyophilized and sterilized. The freeze-dried antler extract was diluted with distilled water to a concentration of 0.2 g / ml to prepare a stock solution. The stock solution is centrifuged again to take the supernatant, and then filtered and sterilized with a Millipore filter (4 μm) to form a cell treatment solution.

The beverage containing the antler extract according to the present invention is prepared as follows. 100 parts by weight of antler, 60 to 70 parts by weight, jujube 170 to 180 parts by weight and 20 to 30 parts by weight of ginger are added to a pressure extraction vessel together with about 7500 to 9000 parts of water and 3.5 to 4.5 at a temperature of about 100 to 115 ° C. After primary extraction for a period of time, the filtrate was separated from the filtrate and the residue, and about 7500 to 9000 parts by weight of water was added again to the filtrate residue, followed by the same process as the first extraction, and then the filtrate was filtered first. In combination with a antler extract.

The cell treatment sample of the above beverage is prepared by the same method as the method of obtaining a cell treatment solution of the above antler extract.

Deer Antler extract containing beverage according to the present invention has an excellent DNA damage protection effect.

Hereinafter, the present invention will be described in detail through examples. However, the protection scope of the present invention is only limited by the appended claims, but is not limited by the present embodiment.

Preparation Example 1

Preparation of Deer Antler Extract

Around May-June 2002, 37.5 g of vacuum-freeze-dried deer antler was collected from a three-year-old elk deer at Cheongyang Deer Farm, Yangsa-ri, Bong-myeon, Cheongyang-gun, Chungcheongnam-do. After primary extraction for a period of time, the filtrate was separated from the filtrate and the residue, and 3 liters of water was added to the filtrate residue, followed by the same process as the first extraction, followed by secondary extraction, and the filtrate was combined with the first filtrate.

100 ml of the extract was centrifuged for 30 minutes at 4500 ° C. under a centrifugal force of 6500 × g (using Beckman J2-21, high performance centrifugator, U.S.A.), lyophilized and sterilized under reduced pressure. The freeze-dried antler extract was dissolved in distilled water at a concentration of 0.2 g / ml to prepare a stock solution. The stock solution was centrifuged again under the same conditions as above to take a supernatant, and then filtered and sterilized with a Millipore filter (4 μm) to prepare a cell treated sample.

Preparation Example 2

Preparation of Drink Containing Deer Antler Extract

37.5 g of vacuum freeze dried antler, 25 g of Angelica jujube, 65 g of jujube and 10 g of ginger were added to a pressure extraction container with 3 L of water, and the same procedure as in Preparation Example 1 was carried out to prepare a beverage containing the antler extract and its cell treated sample. Was

Examples 1-4

Test of DNA Damage Protection Effect of Antler Extract

Isolation of Human Lymphocytes

Within 2 hours of blood collection, 100 μl of whole blood from a 26-year-old healthy non-smoking woman was used to culture RPMI-1640 medium containing 1 ml of 10% fetal bovine serum (FBS) (Roswell Park Memorial). 200 μl of the pre-dispensed solution after mixing withHisto Park(histopaque)-Poured carefully on -1077. Centrifugation of each test tube at 200 × g for 6 minutes causes histopark reagents to be placed in the lower layer due to the difference in specific gravity, with the upper RPMI layer and the middle opaque band (lymphocytes) as red blood cells or other blood. Separated into components. 2 ml of PBS (phosphate-buffered saline) was added to 200 µl of the band containing lymphocytes and centrifuged at 200 xg (Hanil table top multipurpose centrifuge, MF-550, Korea) for 6 minutes to remove supernatant. And again in RPMI-1640 containing 2 ml of 10% FBS. Lymphocyte cells were washed by mixing and centrifugation at 200 x g for 6 minutes to remove supernatant. The washed lymphocyte cells were placed in lymphocyte stock solution (40% RPMI-1640, 50% FBS, 10% DMSO (dimethyl sulfoxide) and stored in a freezer at -80 ° C (Examples 1-4, positive and negative controls 1-2). ).

Deer Antler Extract Treatment and Induction of Oxidative Stress

The number of cells contained in each test tube (1.5 ml eppendorf tube) of the lymphocytes prepared above was 10 to 20 × 104 cells. Was administered in serial dilution with PBS. 5 μg / ml for the five test tubes corresponding to Example 1, 50 μg / ml for the five test tubes corresponding to Example 2, 100 μg / ml for the five test tubes corresponding to Example 3, and Example 4 5 test tubes were mixed with antler extract treatment samples to a concentration of 250 ㎍ / ㎖ and 30 minutes at 4 ℃ The supernatant was removed after holding for a while. Thereafter, the positive control group and the test tube corresponding to Examples 1 to 4, H as an oxidative stress-inducing substance₂O₂1 ml (100 μM) was added, mixed and held at 4 ° C. for 5 minutes to induce DNA damage.

Evaluation of DNA Damage Protection Effect

DNA damage protection effect of the deer antler extract was evaluated by measuring the damage of lymphocyte DNA by the Comet assay (alkaline single cell gel electrophoresis) as follows.

First, cells of each test tube that caused oxidative stress in the stomach were suspended by mixing 75 μl of 0.75% low melting agarose (LMA) gel and fully frosted slide treated with 0.5% of Normal Melting Agarose (NMA). The suspension was evenly dispersed and then covered with a cover glass and refrigerated at 4 ° C. After the gel was solidified, the cover glass was peeled off and the slide was again prepared by applying it again with 75 μl of 0.75% LMA solution.

Thereafter, 10% DMSO and 1% Triton X-100 were mixed with pre-prepared cold alkali lysis buffer (2.5M NaCl, 100mM EDTA, 1% N-Lauroyl sarcosine, 10mM Tris). The slide prepared above was immersed in a solution and immersed in a low temperature dark room for 1 hour to release the DNA strands.

Lysis finished slides were arranged in an electrophoresis tank and unwinded with 4 ° C. cold buffer (300 mM NaOH, 10 mM Na 2 EDTA). After 40 minutes, electrophoresis was performed for 20 minutes under a voltage of 25 V / 300 ± 3 mA.

After electrophoresis, wash thoroughly with 0.4M Tris buffer (pH 7.5), stain the nuclei with 20 μl / ml ethidium bromide, observe them on a fluorescence microscope, and image each cell nucleus sent through a CCD camera. Was analyzed on a computer equipped with a Comet image analysis system. Two slides were made in each treatment group to measure the degree of DNA damage by H ₂ O ₂ and damage protection by antler treated samples of 50 cells.

Image analysis and statistical data processing

Microscopic analysis of the cells on the slides by Comet analysis revealed that the cells without DNA damage were generally rounded in the nucleus with the Komet 4.0 program. The planes are dragged toward the (+) pole, showing a tail dragged like a comet. The degree of damage to each image can be compared numerically with the Komet 4.0 program. Usually DNA damage is expressed through% DNA (in tail), tail length (TL), and tail moment (TM) indicators. % DNA (in tail) is the percentage of DNA in the tail that moves away from the nucleus and the greater the damage to the cell, the greater the value. The value of TL is the length of the tail drawn from the nucleus, which increases the length of the cell with a large amount of DNA damage. The TM value is the product of the length (TL) of DNA transferred from the nucleus and% DNA (in tail). Also, the greater the damage to the cell, the higher the TM value.

A graph showing the DNA damage protection effect of the antler extract is shown in FIGS.

1 shows that the DNA damage was significantly reduced from 50 μg / mL compared to the positive control treated with H ₂ O ₂ by showing% DNA (in tail). Figure 2 shows the TL value in the lymphocytes treated with the antler sample, the tail length was reduced compared to the positive control according to the increase in the concentration of antler extract, it can be seen that DNA damage is reduced. Finally, Figure 3 shows the TM. As can be seen from the graph, as the antler concentrations increased to 5, 50, 100, and 250 μg / mL, the degree of DNA damage decreased to 68.3, 48.4, 51.9, and 48.3% of the positive control. Taken together, the results of the treatment of antler extract have a high protection against human lymphocyte DNA damage.

Example 5

Test of DNA Damage Protection Effect of Drink Containing Deer Antler Extract

Lymphocytes were isolated and washed in the same manner as in Examples 1 to 4 above to prepare lymphocyte cells. The prepared lymphocyte cells were treated with antler beverage samples in lymphocyte cells contained in each of five test tubes at a concentration of 250 µg / ml, which was a proper concentration having no protective effect against DNA damage of cells but preliminary experiments. The antler beverage treatment method and the induction and comet analysis of oxidative stress are the same as in Examples 1 to 4.

The test results are shown in FIGS. 4 to 6.

Figure 1 shows the% DNA (in tail), showing a 28.3% DNA damage protection effect in the cells treated with antler compared to the positive control (positive control) treated with H ₂ O ₂ , 2 is a graph showing the TL in the lymphocytes treated with antler drink, the length of the tail is reduced by about 40% compared to the positive control it can be seen that the DNA damage is reduced. Finally, Figure 3 shows the TM, and as can be seen from the graph, the degree of DNA damage in the cell group treated with the antler beverage was observed to be 55% lower than that of the positive control group. Taken together, the results showed that the treatment of antler drinks had a high protective effect on human lymphocyte DNA damage.

Example 6

Test of the DNA Damage Protection Effect of Diabetic Patients Containing Antler Extract

The following patient groups and control subjects were selected for the test (Example 9 and Control 3) to evaluate the protective effect of lymphocyte DNA damage on diabetic patients of the beverage containing antler.

Insulin-independent diabetic patients and healthy individuals without a history of diabetes were selected as patient and control groups, respectively. Their general characteristics such as their age, height and weight, smoking status, exercise status, vitamin nutrient intake, alcohol consumption, WHR (waist-hip ratio), body mass index (BMI) and nutrient intake are shown in Table 1 below. It was.

Each subject of the patient group and the control group was ingested 100 ml of antler drink prepared in the preparation example twice a day for 20 days, and was instructed to maintain the dietary pattern before ingestion during the experiment. Blood was collected before and 20 days after the drink intake, and lymphocytes were isolated and washed in the same manner as in Examples 1 to 4, followed by a Comet assay. Test results are shown in FIGS. 7 to 9.

Figure 7 shows the degree of damage of cells due to oxidative stress of lymphocytes isolated from blood collected before and after ingestion of antler drink in% DNA, which showed a significant decrease in DNA damage after ingestion of antler drink in both patient and control groups. Is showing.

8 shows the change in TL under the same conditions as in FIG. 7, and FIG. 9 shows the change in the TM value under the same conditions, which shows that the antler drink of the present invention has a protective activity against DNA damage.

The present invention provides a beverage comprising an antler extract having excellent protection against DNA damage.

The antler hot water extract of the present invention and the beverage containing the extract are expected to be useful as cancer prevention substances.

Claims (2)

A composition for protecting DNA damage containing hot water extract of antler. The composition for protecting DNA damage according to claim 1, further comprising hot water extracts of Angelica, jujube and ginger in addition to antler.