KR100972941B1 - Composition comprising an extract of black ginseng for preventing or treating hepatoma - Google Patents

Abstract

PURPOSE: A method for enhancing ginsenoside Rg3 of black ginseng extract is provided to ensure strong anti-cancer activity and to reduce bezoapyrene content. CONSTITUTION: A method for producing black ginseng extract comprises: a step of washing ginseng and separating the ginseng to ginseng body and fine root; a step of drying; a step of steaming at 80-90°C for 8-10 hours; a step of drying at 25-50°C for 3-5 hours; a step of repeating steaming step and drying step 5-12 times; and a step of extracting the black ginseng with a solvent. A pharmaceutical composition for preventing and treating liver cancer contains the black ginseng extract as an active ingredient.

Description

Composition comprising an extract of black ginseng for preventing or treating hepatoma

The present invention relates to a method for producing black ginseng by separating steamed ginseng, which is more than four years old, into the body and the rice, dried with white ginseng, and repeated several times.

More particularly, the present invention relates to a method for producing black ginseng having increased benzopyrene stability and Rg ₃ component and having a prophylactic or therapeutic effect on liver cancer, and a composition thereof.

Ginseng (人蔘, ginseng) is a perennial herb of the dicotyledon type Arboraceae, grows in deep mountain forests and grows as a medicinal plant. The root of ginseng is called ginseng because its shape is human. The shape of the roots varies with age, and harvest is done in 4-6 years. Ginseng as it is harvested is called ginseng, and dried ginseng is called white ginseng. Steaming red ginseng makes red ginseng, which is called red ginseng. When the red ginseng is steamed and dried several times, it becomes black ginseng. These red ginseng and black ginseng is known to improve the active ingredients beneficial to health compared to fresh ginseng by changing the pharmacological components through steaming and drying process. The main bioactive components of red ginseng and black ginseng are known as Ginsenoside-Rb ₁ , Rb ₂ , Rc, Rd, Re, Rg ₁ , Rg ₂ , Rg ₃ and Ro contained in saponin.

Black ginseng is known to be more enhanced ginsenoside Rg ₃ component than in particular red ginseng. Black ginseng is a newly developed product, and since it was recently developed, it is not listed as a regular food, but it contains ginsenoside Rg ₃ , which can be processed into pouches or concentrates and classified as red ginseng processed foods. Black ginseng has already been shown to be effective in boosting immunity, anticancer and sexual function. In addition, through the experience and research results of the present inventors, it was confirmed that it is effective in hepatitis, diabetes and cirrhosis.

The present inventors have developed a method for increasing the ginsenoside component of black ginseng through Korean Patent No. 529475 and again invented a method for enhancing Rg ₃ among the ginsenoside components. The present invention utilizes a new method that can further enhance the Rg ₃ component as compared to Korean Patent Publication No. 840003, which is known to be able to enhance ginsenoside Rg ₃ .

In addition, the method of the present invention proposes a method for producing black ginseng under conditions that can remove benzopyrene, which is known to be produced in the heating process during the manufacture of black ginseng, to below a safety standard. Benzopyrene is a yellow crystalline solid belonging to the group of polycyclic aromatic hydrocarbons (PAHs), and it is known that carbohydrates, proteins and lipids are produced as incomplete combustion when decomposed at temperatures between 300 and 600 ° C in food. Recently, it has also been reported to be produced in processed foods below 100 ° C. The International Agency for Research on Cancer (IARC) underwent a primary toxicity assessment in 1989 and re-evaluated in August 2006 to classify benzopyrene as the first group of human carcinogens. It is known that black ginseng production and benzopyrene reduction is disclosed in Korea Patent Publication No. 2009-93310. The method is a technique for controlling the drying temperature so that the benzopyrene is not increased, but the method of the present invention removes benzopyrene as the steaming process increases. The technique is different from the Korean Patent Publication No. 2009-93310.

In addition, the present inventors confirmed that the black ginseng extract of the present invention has a selectively excellent effect on liver cancer. Although black ginseng extract has an anticancer effect, it is known about the effects on cancer or oxidation-related diseases of colorectal cancer of Korean Patent Application Laid-Open No. 2006-83301 and 2008-106077 Korean Patent Publication No. 2006-83301. It is not about selective effects.

It is an object of the present invention to provide a method for producing black ginseng body and rice ginseng, in which ginsenoside Rg ₃ is enhanced and benzopyrene is reduced by controlling steaming temperature and number of steaming using 4 g of ginseng or more.

Still another object of the present invention is to provide a method for producing black ginseng body and rice ginseng, which are effective in preventing or treating liver cancer.

The present invention is a method for producing black ginseng by repeating steaming and drying the ginseng,

(Step 1) washing the ginseng and separating the body and ginseng;

(Step 2) drying the washed ginseng body and the ginseng so that the moisture content is less than 14%;

(3 step) steaming the body and dried ginseng of dried ginseng at a temperature of 80 ~ 90 ℃ for 8 to 10 hours;

(Step 4) drying the steamed body and rice ginseng at a temperature of 25 ~ 50 ℃ for 3 to 5 hours;

(5 steps) repeating 5 to 12 times the steaming step of the three steps and the 25 ~ 50 ℃ drying step of the four steps in a row; And

(Step 6) drying the black ginseng body and black rice ginseng after repeated steaming and drying of step 5 at a temperature of 25-50 ° C. such that water is finally 10% or less;

It can be prepared including black ginseng.

Ginseng of the first step may be 4-6 years old ginseng.

When separating the body of the ginseng and the ginseng in the first step does not peel the epidermis to prevent the loss of saponin formed between the body and the epidermis of the ginseng.

The moisture of the ginseng dried in the second step is preferably about 5 ~ 14%.

The steaming temperature of the three steps is preferably 80 ~ 90 ℃, if less than 80 ℃ ginsenoside Rg ₃ The components are not increased much and benzopyrene is not easily removed. Even if it exceeds 90 ° C, Rg ₃ Benzopyrene is not removed by the safety regulations without increasing the content.

The total number of steaming after the completion of the process 5 to 3 is preferably 6 to 13 times by adding 5 to 12 additional steams to the first steam. If the total number of steams is less than six, the ginsenoside Rg ₃ component is not increased much and benzopyrene is not removed as well as the safety regulations. As the total number of steams increases, Rg ₃ increases but other ginsenosides fall off or disappear, so the number of steams and temperature must be well controlled.

In addition, the black ginseng body of the present invention and the pulverized extract or solvent (purified water or ethanol) extract of the ginseng may be used when preparing a health food or anti-cancer functional medicines, including tablets, capsules, pills, solutions, etc. .

In order to manufacture the black ginseng body and the extract of misam,

Grinding the dried black ginseng body or rice ginseng;

5 to 120 L of purified water having a temperature of 70 to 90 ° C. per 1 kg of the ground black ginseng body or rice ginseng is mixed with the ground black ginseng body or rice ginseng and stirred for 5 to 10 hours at a rotational speed of 50 to 70 rotations per minute. step;

Precipitating the stirred extract for 2 to 14 hours to separate the liquid and solid body; And

Filtering the extract to obtain a filtrate from which solids are removed;

It may include.

The extract of the black ginseng body and the rice ginseng may be prepared as a powder through lyophilization, hot air drying or spray drying, and may be added to pharmaceutical preparations or foods.

Ginseng as defined in the present invention is a perennial plant belonging to the genus Panax, Panax ginseng, Panax quinquefolia, Panax Notoginseng, Panax vietnamensis, Panax vietnamensis, Panax elegathior (Panax elegatior), Panax wangianus or Panax bipinratifidus, preferably Panax ginseng or Panax quinquefolia.

The pharmaceutical composition comprising the black ginseng body and the ginseng according to the present invention that can be used as an agent for treating cancer is freeze-dried, hot air-dried or spray-dried the extracts of the black ginseng body and the ginseng powder, granules, Oral formulations such as tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injectable solutions. Carriers, excipients, and diluents that may be included in compositions comprising black ginseng body and rice ginseng include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium Phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and such solid preparations may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin in the extract. The mixture is prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The dosage of the active ingredient will vary depending on the age, sex and weight of the subject to be treated, the particular disease or pathology to be treated, the severity of the disease or pathology, the route of administration and the judgment of the prescriber. Dosage determination based on these factors is within the level of skill in the art and generally dosages range from 0.001 mg / kg / day to approximately 2000 mg / kg / day. More preferred dosage is 1 mg / kg / day to 100 mg / kg / day. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

The pharmaceutical composition for preventing and treating liver cancer-related diseases of the present invention comprises 0.1 to 50% by weight of the black ginseng extract based on the total weight of the composition.

The extract of the present invention can be administered to mammals such as mice, livestock, humans, etc. by various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injections. Since the extract of the present invention has little toxicity and side effects, it is a drug that can be used safely even when taken for long periods of time.

In addition, the present invention provides a health functional food for cancer treatment improvement comprising the black ginseng and black rice ginseng according to the present invention is a food supplement acceptable food supplement. The health functional food of the present invention includes the form of tablets, capsules, pills or liquids, and the food to which the compound of the present invention can be added, for example, various foods, beverages, gums, teas, vitamin complexes, etc. And functional foods.

It was confirmed that ginsenoside Rg ₃ is significantly increased in the black ginseng prepared through the present invention than the black ginseng prepared in the prior art. In addition, it can be seen that the amount of ginsenoside Rg ₃ increases sequentially as the number of steaming increases, and the production of the amount is sensitive to temperature and drying conditions.

Benzopyrene, also known as a carcinogen, has been removed to below safety regulations by reducing its amount through repeated steaming at the temperature and steaming conditions of the present invention. In the early stage of steaming of the present invention, benzopyrene is detected to be 1.0 ppb or more, which is higher than the prescribed value, but it can be confirmed that the amount of benzopyrene decreases below the prescribed value through the steaming process. It is assumed that benzopyrene is produced from pesticides remaining in ginseng as a raw material, and the benzopyrene thus produced is removed through the repeated steaming and drying conditions of the present invention. In fact, benzopyrene was also detected in the ginseng before steaming, benzopyrene was detected only when black ginseng was prepared using the ginseng, and no benzopyrene was detected in ginseng from the beginning.

In addition, it was confirmed that the black ginseng with the increased ginsenoside Rg ₃ has an excellent anticancer effect against liver cancer. This has established a method for producing black ginseng that is effective in liver cancer-related diseases and safe from carcinogens.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the embodiments disclosed herein are provided so that the disclosure can be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

<Example 1: Preparation of black ginseng main body and rice ginseng and extracts using the same: steaming temperature 85 ℃, drying temperature 50 ℃, the total number of steaming 13 times>

Six years old Korean ginseng was collected and the soil or foreign matters were removed, washed with purified water, water was removed, and the fresh ginseng was separated into a body and rice ginseng (root). The body of separated ginseng and 10kg each of ginseng were naturally dried to have a water content of 14% or less, and then manufactured by ginseng. The dried ginseng was steamed for 10 hours at a temperature of 85 ℃, and then dried for 4 hours at a temperature of 50 ℃ in a dryer, and the same steaming and drying were repeated 12 times (total 13 steaming). ). Repeating the steaming and drying process, the dried white ginseng was changed to red ginseng and black ginseng to black ginseng, and the black ginseng body and rice were prepared, and the last drying process was completed until the water became less than 10%. Thereafter, the black ginseng body and the ginseng were ground to prepare an extract of the black ginseng body and the rice ginseng. The pulverized black ginseng body and rice ginseng were put into 100 l of purified water having a temperature of 80 ° C., respectively, and stirred for 8 hours at a rotational speed of 60 revolutions per minute, and then precipitated for 3 hours. The precipitated extract was filtered to remove solids, and the filtrate was lyophilized to finally prepare a black ginseng body and a ginseng extract as powder.

Example 2 Steaming Temperature

2-1. Steam temperature 80 ℃

The black ginseng body and the extract of the ginseng were prepared under the same conditions as in Example 1, but the steaming temperature was 80 ° C.

2-2. Steam temperature 90 ℃

The black ginseng body and the extract of the ginseng were prepared under the same conditions as in Example 1, but the steaming temperature was 90 ° C.

Example 3. Number of Steaming

3-1. 6 steaming (5 times steaming and drying)

The black ginseng body and the extract of the ginseng were prepared under the same conditions as in Example 1, but the total number of steaming was performed only six times.

3-2. 7 steaming (6 repeated steaming and drying)

The black ginseng body and the extract of the ginseng were prepared under the same conditions as in Example 1, but the total number of steaming was performed only seven times.

3-3. 8 steaming (7 times steaming and drying)

The black ginseng body and the extract of the ginseng were prepared under the same conditions as in Example 1, but the total number of steaming was performed only eight times.

3-4. 9 steaming (8 repeated steaming and drying)

The black ginseng body and the extract of the ginseng were prepared under the same conditions as in Example 1, but the total number of steaming was performed only 9 times.

3-5. 10 steaming (9 repeated steaming and drying)

The black ginseng body and the extract of the ginseng were prepared under the same conditions as in Example 1, but the total number of steaming was performed only 10 times.

3-6. 11 steaming cycles (10 times steaming and drying)

The black ginseng body and the extract were extracted under the same conditions as in Example 1, but the total number of steaming was performed only 11 times.

3-7. 12 steaming (11 repeated steaming and drying)

The black ginseng body and the extract of the ginseng were prepared under the same conditions as in Example 1, but the total number of steaming was performed 12 times.

<Example 4. Drying temperature 25 ℃>

The black ginseng body and the extract of the ginseng were prepared under the same conditions as in Example 1, but the drying temperature was 25 ° C.

Comparative Example 1: Steaming Temperature

1-1. Steam temperature 70 ℃

The black ginseng body and the extract of the ginseng were prepared under the same conditions as in Example 1, but the steaming temperature was 70 ° C.

1-2. Steam temperature 75 ℃

The black ginseng body and the rice extract were prepared under the same conditions as in Example 1, but the steaming temperature was 75 ° C.

1-3. Steam temperature 95 ℃

The black ginseng body and the rice extract were prepared under the same conditions as in Example 1, but the steaming temperature was 95 ° C.

Comparative Example 2: Total number of steaming

2-1. 1 steaming (no repeated steaming or drying)

The black ginseng body and the extract of the ginseng were prepared under the same conditions as in Example 1, but the total number of steaming was performed only once.

2-2. 2 steaming (1 time steaming and drying)

The black ginseng body and the extract of the ginseng were prepared under the same conditions as in Example 1, but the total number of steaming was performed twice.

2-3. 3 steaming (2 times steaming and drying)

The black ginseng body and the extract of the ginseng were prepared under the same conditions as in Example 1, but only three times of total steaming was performed.

2-4. 4 steaming (3 times steaming and drying)

The black ginseng body and the extract were extracted under the same conditions as in Example 1, but the total number of steaming was performed only four times.

2-5. 5 steaming (4 times steaming and drying)

The black ginseng body and the extract of the ginseng were prepared under the same conditions as in Example 1, but only five times of total steaming was performed.

2-6. 14 steaming (13 repeated steaming and drying)

The black ginseng body and the extract of the ginseng were prepared under the same conditions as in Example 1, but the total number of steaming was performed 14 times.

Comparative Example 3: Drying Temperature

3-1. 20 ℃

The black ginseng body and the extract of the ginseng were prepared under the same conditions as in Example 1, but drying was performed at 20 ° C.

3-2. 55 ℃

The black ginseng body and the extract of the ginseng were prepared under the same conditions as in Example 1, but drying was performed at 55 ° C.

Comparative Example 4: Preparation of Red and Red Ginseng Extracts

Six years old red ginseng root (red ginseng body) and red ginseng were distributed in the market, and red ginseng root and red ginseng extract were prepared using purified water at 80 ° C. in the same manner as the extraction method of Example 1.

Experimental Example 1: Component Analysis of Black Ginseng

The ginsenoside components of the black ginseng extract and the red ginseng extract of Comparative Example 4 prepared according to the methods of Examples 1 to 4 and Comparative Examples 1 to 3 were analyzed. Since ginsenoside components are known to be more in the root portion, black and red ginseng were used in this experiment, respectively.

The analysis of ginsenosides was carried out in accordance with Food Code [Chapter 4. Food Standards and Standards → 16.Ginseng Products → 16-10.Concentrated Red Ginseng (1) Red Ginseng Identification Test High-Speed Liquid Chromatography]. The senoside test method was used, and Table 1 is a table showing the analysis of ginsenoside content of black ginseng and red ginseng.

The results of Table 1 confirm that the ginsenoside Rg ₃ of black ginseng was significantly increased as the number of steaming was increased, and in particular, a large amount of ginsenoside Rg ₃ was increased in 8 or more times, and ginsenosides in 9 or more times. It was confirmed that the Rg ₃ content was maintained about the same. In addition, the steaming temperature of ginsenosides Rg ₃ and Rh ₁ was most increased between 80 and 90 ° C around 85 ° C, and when dried at too low a temperature (20 ° C), black ginseng was produced in poor quality. I could confirm it. In contrast, the ginsenosides Rg ₃ and Rh ₁ contents of red ginseng showed significantly lower values.

For reference, the total amount of irradiated black ginseng of Example 1 was analyzed to be 508.9 mg / g, and the irradiated amount of irradiated with the same amount of irradiated with red ginseng of Comparative Example 4 (72.1 mg / g) was increased during initial steaming. It was confirmed that the increase (not shown in the diagram).

Condition
Comparison of Ginsenoside Content According to Manufacturing Conditions (mg / g)
Rb1
Rb2
Rc
Rd
Re
Rg1
Rg3
Rh1
Example 1-1
12.2
6.0
5.6
8.2
0.7
0.0
146.8
9.5
Example 2-1
14.4
5.3
5.3
7.5
0.6
0.0
138.1
8.9
Example 2-2
13.2
5.2
5.7
7.7
0.6
0.0
137.2
9.1
Example 3-1
12.5
6.3
5.9
7.4
1.2
0.6
108.9
0.0
Example 3-2
15.2
7.5
6.7
8.6
1.6
0.0
118.4
0.0
Example 3-3
9.0
5.0
4.9
6.9
0.6
0.0
138.8
0.1
Example 3-4
10.7
5.6
5.3
7.5
0.7
0.0
143.3
0.4
Example 3-5
11.5
5.5
5.4
7.7
0.6
0.0
144.2
9.4
Example 3-6
9.5
5.8
5.1
7.4
0.5
0.0
145.1
9.8
Example 3-7
10.2
5.1
5.0
7.1
0.6
0.0
146.9
9.7
Example 4
11.2
5.0
5.9
7.2
0.9
0.0
142.1
8.8
Comparative Example 1-1
22.2
36.0
35.6
18.2
6.7
4.3
84.8
0.1
Comparative Example 1-2
22.2
8.0
10.6
18.2
4.7
3.1
70.8
0.2
Comparative Example 1-3
14.2
8.4
5.4
7.2
2.6
0.0
110.4
0.0
Comparative Example 2-1
120.3
63.0
60.8
26.3
64.2
15.0
5.4
0.0
Comparative Example 2-2
123.1
66.2
61.4
30.9
61.8
12.0
14.6
0.0
Comparative Example 2-3
97.9
57.7
52.4
25.2
38.1
10.7
22.6
0.0
Comparative Example 2-4
75.4
36.8
32.2
25.0
16.3
9.5
83.2
0.0
Comparative Example 2-5
20.8
10.8
9.1
10.5
1.9
1.4
106.1
0.0
Comparative Example 2-6
8.7
4.2
4.9
6.5
0.4
0.0
148.2
9.9
Comparative Example 3-1
20.2
7.0
12.6
25.2
16.7
10.1
90.8
0.2
Comparative Example 3-2
13.1
6.2
4.7
8.7
0.5
0.0
117.2
8.1
Comparative Example 4
4.3
2.6
2.5
1.1
1.0
2.1
21.4
4.7

Experimental Example 2 Analysis of Anticancer Effect

2-1. in-vitro anticancer effect experiment (cell experiment)

In order to confirm the anticancer effect of black ginseng, the cell survival rate of hepatic cancer cell HepG2 was measured using the black ginseng extract powder of Example 1 and the red ginseng extract powder of Comparative Example 4. Black ginseng was used as a representative of the extract of Example 1 having a high Rg ₃ content and black and red ginseng as in Experimental Example 1.

First, 1 × 10 ⁵ cells / ml cells were put into 96 well-plate at 37 ° C. using 100 µl cell culture medium (100 U / ml penicillin, 100 g / ml streptomycin, DMEM containing 10% bovine serum). Incubated for 24 hours in a 5% CO ₂ incubator. Test substance black ginseng extract and red ginseng extract were treated from the highest concentration of 1000 μg / ㎖ to 2 μg / ㎖ and incubated for 24 hours. After 200 μl of fresh medium was added to each well, MTT (thiazolyl blue, SIGMA co.) Was prepared at a concentration of 2 μg / ml, and 50 μl was added to react for 3 to 4 hours. After removing 220 μl of test substance for each well, only 30 μl of purple substance was added, and 150 μl of DMSO (dimethyl sulfoxide) was added and mixed well for 10 minutes to dissolve the precipitate. Then, the OD (optical density) value was measured at 540 nm with ELISA plate reader. Measured. All test results yielded corrected values for absorbance measured in wells in which the cells were not cultured. Table 2 is a table showing the results for the cell survival rate treated by liver cancer cells treated with black ginseng, Figure 2 is a graph showing this.

When confirming the results of Table 2 and Figure 2, the black ginseng extract of Example 1 of the present invention can confirm the 50% inhibitory activity against hepatocellular carcinoma proliferation at a concentration of 8μg / ㎖ or less, the red ginseng extract of Comparative Example 3 to the liver cancer cell proliferation It can be seen that 50% inhibition against the high concentration of 1000 μg / ㎖ or more. As a result, the black ginseng prepared by the production method of the present invention can be confirmed to have a selective and excellent anticancer effect on liver cancer cells.

Condition
Concentration (μg / ml)
0
2
4
8
16
32
63
125
250
500
1000
Example 1
Black Ginseng Extract
100%
80%
65%
45%
39%
30%
27%
10%
8%
6%
4%
Of Comparative Example 4
Red Ginseng Extract
100%
76%
70%
69%
68%
68%
65%
63%
58%
56%
54%

2-2. in vivo anticancer effect experiment (animal experiment)

HepG2 cells to be transplanted were placed in cell culture flasks using DMEM (Gibco BLR) cell culture medium containing 100 U / ml penicillin, 100 g / ml streptomycin, 10% bovine serum, and cultured in a 95% CO ₂ 37 ° C. cell incubator. . When the cells adhered, the cells were washed twice with HBSS (Hanks balanced salt solution, Gibco BRL) and collected with 0.2% trypsin. Experimental animals used 9-week old BALB / c nude mice (SLC inc., Shizuoka, Japan), each of which was 9 animals. Each extract was orally administered daily at 300 mg / kg and only purified water was administered to the control group. First, these nude mice were injected with 1 × 10 ⁷ HepG2 cells to obtain tumor lines, which were then passaged to the passage group. The tumor mass was recovered in three passages, and then the tumor mass was transplanted to eight passages. Tumor sections were made by cutting the outer areas where rapid division occurred to a certain size (3 × 3 × 3 mm) at the expense of animals containing tumors that grew rapidly with sufficient blood supply before tumor necrosis occurred. The tumor piece was placed at the end of the trocar and the end was reached to the trunk body side of the rear cell of the animal. The trocar was lightly and quickly rotated out of 360 ° to allow the tumor piece to settle in the desired location and the dissection site was disinfected. After 24 days after the initial administration, the experiment was terminated and the autopsy was performed to determine the weight of solid cancers extracted from individuals, and the actual volume was measured using digital plethysmomether.

Condition
Tumor Weight (g)
Tumor volume
Control
0.925 ± 0.136
0.843 ± 0.186
Black Ginseng Extract of Example 1
0.305 ± 0.239
0.315 ± 0.256
Red Ginseng Extract of Comparative Example 4
0.726 ± 0.215
0.636 ± 0.233

Through the results of Table 3, black ginseng extract of the present invention was confirmed that the ability to inhibit the weight and tumor volume of tumors in liver cancer animal model experiments than the red ginseng extract.

Experimental Example 3: Benzopyrene Detection Analysis

In this experiment, black ginseng and red ginseng extracts were used in the same manner as in Experiment 1 and Experiment 2. The benzopyrene analysis method is a black ginseng body prepared in Examples 1 to 4 and Comparative Examples 1 to 3 according to the benzopyrene test method of the health functional food (so-called black ginseng) of December 31, 2007 4 g of the extract was extracted and concentrated with dichloromethane, dissolved in hexane, passed through a prolysyl cartridge, dried, and dissolved in acetonitrile, and analyzed by high performance liquid chromatography (High Chromatograph). 3-methylcollanthene was used as an internal standard, and a fluorescence detector was used as a detector. In addition, the column was used in the reverse phase distribution type (C18 Supelco LC-PAH 4.6mm × 250mm I.D.) and the analysis conditions are shown in Table 4 below and are shown in Table 5 repeated five times. Benzopyrene is less than 1.0 ppb below safety regulations.

Operating
Condition
Injection volume
10 μl
Column temp.
35 ℃
Mobile phase
Acetonitrile: H ₂ O = 8: 2
Detector wavelength
Excitation wavelength 296nm, Measurement wavelength 404nm
Flow rate
1.0ml / min

Condition

Detection amount of benzopyrene according to repeated measurement of benzopyrene (ppb)
first
second
third
fourth
Fifth
Example 1
0.57
0.60
0.59
0.51
0.65
Example 2-1
0.59
0.59
0.55
0.53
0.62
Example 2-2
0.62
0.65
0.68
0.52
0.68
Example 3-1
0.74
0.76
0.77
0.69
0.88
Example 3-2
0.73
0.72
0.73
0.59
0.74
Example 3-3
0.61
0.69
0.71
0.59
0.73
Example 3-4
0.60
0.66
0.70
0.58
0.74
Example 3-5
0.59
0.65
0.69
0.57
0.75
Example 3-6
0.52
0.57
0.54
0.50
0.55
Example 3-7
0.51
0.52
0.51
0.48
0.53
Example 4
0.55
0.58
0.59
0.58
0.60
Comparative Example 1-1
1.52
1.42
1.34
1.42
1.32
Comparative Example 1-2
1.32
1.51
1.22
1.32
1.14
Comparative Example 1-3
2.01
1.92
1.88
1.95
1.84
Comparative Example 2-1
1.87
0.89
1.87
1.43
1.05
Comparative Example 2-2
1.64
0.89
1.64
0.89
0.89
Comparative Example 2-3
1.27
0.79
1.27
0.79
1.07
Comparative Example 2-4
1.62
0.72
1.62
0.72
0.79
Comparative Example 2-5
0.75
0.77
0.75
0.77
0.94
Comparative Example 2-6
0.48
0.50
0.49
0.46
0.50
Comparative Example 3-1
0.75
0.78
0.62
0.77
0.79
Comparative Example 3-2
1.23
0.89
1.25
0.99
1.05

From the results of Table 5, in the production of black ginseng of the present invention, it was confirmed that the amount of benzopyrene was increased in the initial steaming condition, but the amount of benzopyrene was gradually decreased as the number of steaming was increased. Since it is defined as safe, the black ginseng prepared in Examples 1 to 4 was confirmed to be a safe product. In addition, when the temperature of drying black ginseng is higher than the conditions of the present invention, benzopyrene is not completely reduced to 1.0 ppb or less. As a result, it can be seen that the method of the present invention can remove benzopyrene, which may occur in the black ginseng manufacturing process.

For reference, the amount of benzopyrene was also detected in ginseng before steaming, but as expected, the amount of benzopyrene was detected as 1.02 ppb. Because of this, clean ginseng that does not detect benzopyrene was steamed separately before steaming. However, black ginseng steamed using ginseng that does not detect benzopyrene was confirmed that benzopyrene was not detected unlike before (as shown in the diagram). Not).

The black ginseng produced using the present invention may be added to medicines or foods and used as a cancer prevention agent, a cancer treatment agent, or the like. As a specific example, the present invention provides a high anticancer drug and a method for preparing a functional food containing a black ginseng extract using black ginseng body and rice ginseng.

<Use Example 1: Preparation of Pharmaceuticals>

1-1. Manufacture of powder

Black ginseng extract powder of Example 1 ... 300 mg

Lactose ......................................... 100 mg

Talc ......................................... 10 mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

1-2. Manufacture of tablets

Black ginseng extract powder of Example 1 ... 300 mg

Corn starch ..................... 100 mg

Lactose ......................................... 100 mg

Magnesium Stearate ......................................... 2 mg

After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.

1-3. Manufacture of capsule

Black ginseng extract powder of Example 1 ... 300 mg

Corn starch ..................... 100 mg

Lactose ......................................... 100 mg

Magnesium Stearate ........................... 2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

<Use Example 2: Production Example of Health Food>

2-1. Manufacture of health food

Black rice ginseng extract powder of Example 1 .. 200 mg

Vitamin Blend ...........

Vitamin A Acetate ............... 70 μg

Vitamin E ..................................... 1.0 mg

Vitamin B1 ......................................... 0.13 mg

Vitamin C ......................................... 10 mg

Biotin ......................................... 10 μg

Nicotinamide ......................................... 1.7mg

Folic acid ........................... 50 ㎍

Calcium Pantothenate ..................... 0.5 mg

Ferrous Sulfate ............... 1.75 mg

Zinc Oxide ............... 0.82 mg

Magnesium Carbonate ......................................... 25.3 mg

Potassium monophosphate ............... 15 mg

Dicalcium Phosphate Dibasic ......................................... 55 mg

Potassium Citrate ............... 90 mg

Calcium Carbonate ......................... 100 mg

Magnesium Chloride ......................................... 24.8 mg

The composition ratio of the above-mentioned vitamin and mineral mixtures is composed of relatively suitable ingredients for health foods as a preferred formulation, but the formulation ratio may be arbitrarily modified, and the above ingredients may be mixed according to a general health food manufacturing method. The granules may be prepared and used for preparing a health food composition according to a conventional method.

2-2. Manufacture of healthy drinks

Black rice ginseng extract powder of Example 1 ... 500 mg

Citric Acid ......................................... 100 mg

Oligosaccharide ......................................... 100 g

Plum concentrate ..................................... 2 g

Taurine ......................................... 1 g

Purified water is added to the whole ........... 900 ㎖

After mixing the above components according to a conventional healthy beverage production method, and then stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and then refrigerated and stored in the present invention For the preparation of healthy beverage compositions.

Although the composition ratio is a composition suitable for a preferred beverage in a preferred formulation example, the compounding ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and use purpose.

1 is a flowchart illustrating a process of preparing black ginseng according to the method of the first preferred embodiment.

Figure 2 is a graph showing the results of the MTT assay for the cell survival rate of hepatic cancer cell HepG2 of Experimental Example 2-1.

Claims (6)

(Step 1) washing the ginseng and separating the body and ginseng; (Step 2) drying the washed ginseng body and the ginseng so that the moisture content is less than 14%; (3 step) steaming the body and dried ginseng of dried ginseng at a temperature of 80 ~ 90 ℃ for 8 to 10 hours; (Step 4) drying the body and rice ginseng of steamed ginseng in step 3 for 3 to 5 hours at 25 to 50 ° C .; (5 steps) repeating the steaming step of the three steps and the drying step of the fourth step 5 to 12 times in succession; (Step 6) drying the black ginseng body and the rice ginseng, which has been repeatedly steamed and dried in step 5, to finally have water of 10% or less at 25 to 50 ° C .; And (7 step) to obtain a black ginseng extract obtained by solvent extraction of the black ginseng of step 6 and then dried to remove the black ginseng extract; A method of producing black ginseng extract, characterized in that the Rg ₃ content is 130mg / g or more and the benzopyrene detection amount is 1.0ppb or less. The method of claim 1, The ginseng of the first step is a production method of black ginseng, characterized in that 4 to 6 years old ginseng. The method of claim 2, The ginseng is a perennial plant belonging to the genus Panax, Panax ginseng, Panax quinquefolia, Panax ginseng, Panax notoginseng, Panax vietnamensis, Panax elegatior. , Panax wanianus (Panax wangianus) or Panax bipinratifidus (Panax bipinratifidus) manufacturing method characterized in that it comprises a. delete Liver cancer prevention and improvement health functional food comprising the black ginseng extract of claim 1 as an active ingredient. A pharmaceutical composition for preventing or treating liver cancer comprising the black ginseng extract of claim 1 as an active ingredient.

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