KR101071851B1 - A Process for Preparing an Extract of Black Ginseng with Increased Rg3 and Rh2 Contents - Google Patents

Abstract

The present invention relates to a black ginseng and a method for producing the same, the content of Rg3 and Rh2 which is a ginseng saponin component having anticancer activity prepared by repeated steaming ginseng under the conditions of normal pressure and high pressure. In the method for producing black ginseng with increased contents of Rg3 and Rh2 of the present invention, the dried ginseng is steamed at 90 to 110 ° C. for 1 to 3 hours and then dried at 50 to 70 ° C. for 1 to 3 hours. In the method for repeatedly producing black ginseng, the first and sixth steaming steps are performed under a pressure of 5 to 15 atmospheres. When using the production method of the present invention, black ginseng black ginseng extract with increased content of Rg3 and Rh2, which are anti-cancer effect ginseng saponins, can be prepared, and thus will be widely used for the development of anticancer drugs and anti-cancer health foods. .

Black ginseng, black ginseng extract, high pressure, steaming, Rg3, Rh2, anticancer drug, anticancer health food

Description

A process for preparing an extract of black ginseng with increased rg3 and rh2 contents

The present invention relates to a black ginseng having an increased content of Rg3 and Rh2 and a method for producing the same. More specifically, the present invention relates to black ginseng and a method for producing the same, the content of Rg3 and Rh2, which is a ginseng saponin component having anticancer activity, prepared by repeated steaming ginseng under normal and high pressure conditions.

Ginseng is a herbal medicine that has long been known for its efficacy and effectiveness. The ginseng is used as it is, ginseng (水 蔘, fresh ginseng), or dried and prepared in the form of white ginseng (白 蔘, white ginseng) or steamed red ginseng (紅 蔘, red ginseng) used in the form of It became.

On the other hand, according to the processing method of ginseng, a new ingredient that does not exist in the ginseng is produced, it is possible to develop a new processed ginseng that has a superior effect on various physiological activities including antioxidant activity compared to ginseng (Okamura N., et al., Biol. Pharm. Bull., 17 (2), p. 270, 1994). In particular, red ginseng, which has been known to have been manufactured for about 1,000 years, undergoes secondary conversion during the steaming process, resulting in new active ingredients unique to red ginseng or red ginseng. .

According to the research so far, the active ingredient in ginseng is known as ginseng saponin (ginsenoside), which is an alcoholic OH of R1, R2 and R3 of triterpene (protopanaxadiol or protopanaxatriol). As a compound having a structure in which sugars such as glucose, rhamnose, xylose and arabinose are ester-bonded to the group, more than 30 species have been identified to date, and have anticancer activity, antidiabetic action, central nerve suppression effect, arteriosclerosis and hypertension. It is known to exhibit pharmacological activity effects such as prevention, liver function and hangover removal effect, anti-fatigue and anti-stress action, antioxidant activity, anti-inflammatory activity, protein synthesis promoting action, immune enhancing effect.

These ginseng saponins are generally known to exist in fresh ginseng, but some specific ginseng saponins are known to exist only in processed ginseng. Hideyuki Nakata, et al., Cancer Science, 89 (7): 733-740, 2005 YK Yue, et al., Biochemical Pharmacology, 72 (4): 437-445, 2006). For example, Rg2, Rg3, Rh1, Rh2 and the like are not present in ginseng saponins, but are known to exist in red ginseng, a processed ginseng produced by steaming and drying ginseng. Ginseng saponins present in the processed ginseng are superior to other ginseng saponins than other ginseng saponins such as cancer prevention, cancer cell growth inhibition, blood pressure lowering, neuroprotective neuroprotection, antithrombotic action and antioxidant activity. The research on the ginseng saponin which exists only in these processed ginseng is actively progressing.

Recently, many studies have been conducted to further increase the content of ginseng saponin present only in these processed ginseng, and as a result, processed ginseng such as taeguk ginseng and black ginseng have been developed. . In particular, black ginseng means ginseng that has changed to black color through several steaming and drying processes, and the ginseng saponin that is basically present during several growth and drying processes is not lost, and is present only in processed ginseng. Since the content of ginseng saponin is known to be enhanced, research on a method of more effectively producing such black ginseng has been actively conducted.

For example, in Korean Patent Registration No. 496418, the washed semi ginseng is first steamed at 60 to 70 ° C. for 1.5 to 2 hours, dried until the moisture content reaches 28%, and then the process is repeated again. After repeated several times, high-pressure steaming at a temperature of 135 ℃ in a high pressure steamer for about 2 to 3 hours, dried in a hot air rotary dryer to 60% moisture at 60 to 70 ℃, and then the process again After repeating, drying it to a final moisture content of 14% in a bulk dryer, there is disclosed a method for producing black rice, including the step of removing foreign matters, Korean Patent Publication No. 2003-0005089 after washing ginseng, A method for preparing black ginseng or black ginseng is disclosed that includes separating the body and the ginseng, drying the ginseng body and the ginseng to 14% or less, and then repeating the process seven or more times.

However, although black ginseng prepared using these methods generally contains a high content of ginseng saponin components, it is known that there is no obvious advantage when compared to red ginseng prepared by a simpler method. That is, in order to manufacture black ginseng, although the number of steaming and drying is increased or additional equipment such as a high pressure steamer is required than the manufacture of red ginseng, the content of the ginseng saponin component included in the finally prepared black ginseng is red ginseng Since it does not show much difference compared to that contained in, ginseng has no distinct advantage over red ginseng, and from an industrial point of view, black ginseng is considered to be less economical than red ginseng.

The problem of black ginseng is that the content of ginseng saponin contained in black ginseng does not show a significant difference from that of red ginseng, and therefore, the content of ginseng saponin contained in black ginseng, especially the content of ginseng saponin present in processed ginseng If it can be significantly increased compared to that of the red ginseng, it is expected that the above-mentioned problem of the black ginseng can be solved, but the situation has not been reported yet.

The present inventors have diligently researched to develop a method for producing black ginseng with increased content of ginseng saponin components present only in processed ginseng, and when using a method of alternately repeating the steaming process at atmospheric pressure and the steaming process at high pressure. In the ginseng saponin components present only in processed ginseng, it was confirmed that black ginseng and black ginseng extracts having an increased anti-cancer effect of Rg3 and Rh2 could be prepared, thereby completing the present invention.

After all, the first object of the present invention is to provide a method for producing black ginseng with increased content of Rg3 and Rh2, which is a ginseng saponin component exhibiting an anticancer effect.

The second object of the present invention is to provide a black ginseng prepared by the above method, the content of Rg3 and Rh2, which is a ginseng saponin component having an anticancer effect, is increased.

It is a third object of the present invention to provide a method for preparing black ginseng extract from the black ginseng.

A fourth object of the present invention is to provide a black ginseng extract prepared by the above method, the content of Rg3 and Rh2, which is a ginseng saponin component exhibiting an anticancer effect.

A fifth object of the present invention is to provide a pharmaceutical composition for the prevention and treatment of cancer diseases comprising the black ginseng or black ginseng extract as an active ingredient.

A sixth object of the present invention is to provide an anticancer health functional food comprising the black ginseng or black ginseng extract.

When using the production method of the present invention, black ginseng and black ginseng extracts with increased content of Rg3 and Rh2, which are anti-cancer ginseng saponin components, can be prepared, and thus widely used in the development of anticancer drugs, anticancer agents, and anti-cancer health foods. Could be.

The inventors of the present invention, in order to find a method for producing black ginseng with enhanced content of ginseng saponin components present only in processed ginseng, according to the number of steaming processes, Rg2 which is a ginseng saponin component present only in processed ginseng included in black ginseng It was confirmed whether the content of Rg3, Rh1 or Rh2 is changed. As a result, when the number of steaming process increases, the contents of Rg2 and Rh1 contained in black ginseng did not change much, but the contents of Rg3 and Rh2 increased. However, when steaming more than 10 times, it was found that the contents of Rg3 and Rh2 tended to decrease, so the so-called "gujeungpo" method of steaming 9 times and drying 9 times was most preferable. I could see.

On the other hand, even using this 9-steaming process, the increase in the content of Rg3 and Rh2 contained in black ginseng was not remarkable, and therefore, it was intended to develop a method to significantly increase the content of Rg3 and Rh2 contained in black ginseng. Bar, when steaming at high pressure, the content of Rg3 and Rh2 contained in black ginseng was significantly increased than steaming at normal pressure, but when steaming at high pressure, benzopyrene, known as a carcinogen, was observed. It can be seen that the content of is increased rapidly.

The benzopyrene did not increase greatly until the process of steaming at high pressure was repeated twice, and thus, black ginseng was prepared by performing the process of steaming at high pressure twice and performing the process of steaming at atmospheric pressure seven times, and at atmospheric pressure. Rg3 and Rh2 content was significantly increased, and the content of benzopyrene was hardly increased, rather than performing nine times the steaming process. Therefore, the method of increasing the content of Rg3 and Rh2 in the most effective manner by setting appropriately the process of steaming at high pressure and the process of steaming at atmospheric pressure in order is considered as the first and sixth time. When the steaming process is performed, it can be seen that black ginseng having the highest content of Rg3 and Rh2 can be prepared.

On the other hand, in order to investigate the anticancer activity of the black ginseng prepared by the above method, the black ginseng extract obtained from the black ginseng prepared by the method of the present invention and the black ginseng prepared by the conventional method, respectively, and treated to the lung cancer cell line cultured under laboratory conditions And incubated. As a result, the black ginseng prepared by the method of the present invention lowered the survival rate of lung cancer cell lines more than the black ginseng prepared by the conventional method. Therefore, the black ginseng prepared by the method of the present invention is superior to the black ginseng prepared by the conventional method. It can be seen that the effect.

In addition, in order to confirm whether the anticancer activity of the black ginseng prepared by the method of the present invention is shown in vivo, each of the prepared black ginseng extracts is injected into the mouse together with the B6 melanoma cell line, which is a kind of metastatic cancer cells. After breeding, the lungs were removed and the number of melanoma colonies appearing on the surface of the lungs was counted. As a result, black ginseng prepared by the method of the present invention inhibited the metastatic activity of B16 melanoma cells injected externally more effectively than black ginseng prepared by the conventional method, further reducing the number of melanoma colonies appearing on the surface of the lung. It was found that the black ginseng prepared by the method of the present invention exhibited superior cancer prevention effects than the black ginseng prepared by the conventional method.

As a result, the manufacturing method of the black ginseng with the increased content of Rg3 and Rh2 of the present invention is to steam the dried ginseng for 1 to 3 hours at 90 to 110 ℃, and then to dry for 1 to 3 hours at 50 to 70 ℃ In the method for producing black ginseng by performing nine times, the first and sixth steaming step is performed under a pressure of 5 to 15 atm. In this case, the temperature and time conditions for steaming ginseng are generally the same as the temperature and time conditions used for steaming conventional ginseng, and the temperature and time conditions for drying steamed ginseng are also different from those used for normal ginseng drying. Mostly the same. In addition, when steaming twice under a pressure of 5 to 15 atm, the content of Rg3 and Rh2 was significantly increased, but the content of benzopyrene, which is known to be a carcinogen, did not increase. In this case, the content of benzopyrene was excessively increased, and the content of Rg3 and Rh2 did not increase when steaming under atmospheric pressure lower than 5 atm or steaming only once under high pressure.

According to another embodiment of the present invention, black ginseng prepared by the above method to increase the content of Rg3 and Rh2 is pulverized, and left to stand for 3 to 7 hours by adding 3 to 7 times (weight ratio) of water to the black ginseng, By extracting the lower alcohol having 1 to 4 carbon atoms as the extraction solvent, black ginseng extract with increased content of Rg3 and Rh2 can be prepared. At this time, the extraction is not particularly limited, but 5 to 15 times (volume ratio) of ethanol is added to the water, it is preferably extracted for 1 to 5 hours at 30 to 70 ℃, then filtered and the solvent is preferably carried out. .

According to another embodiment of the present invention, Rg3 and Rh2, which are contained in the black ginseng or black ginseng extract prepared in high amounts, have already been known for their safety and excellent anticancer activity (see Hideyuki Nakata, et al., Cancer Science, 89). (7): 733-740, 2005; YK Yue, et al., Biochemical Pharmacology, 72 (4): 437-445, 2006), black ginseng or black ginseng extract of the present invention having a higher content of Rg3 and Rh2 than conventional red ginseng It will be apparent that this safe and excellent anti-cancer activity, black ginseng or black ginseng extract of the present invention can be used as an active ingredient of the pharmaceutical composition for the prevention and treatment of cancer diseases. In this case, the cancer disease includes a general cancer disease, preferably gastric cancer, colon cancer, breast cancer, lung cancer, non-small cell lung cancer (non-small cell lung cancer), bone cancer (bone tumor), pancreatic cancer, liver cancer, skin cancer, head Or cervical cancer, skin or ocular melanoma, uterine cancer, ovarian cancer, colorectal cancer, small intestine cancer, rectal cancer, anal muscle cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hogkin's disease, esophageal cancer, small intestine Cancer, lymph gland cancer, bladder cancer, gallbladder cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocyte lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma , Renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, brain stem glioma or pituitary adenoma.

On the other hand, black ginseng or black ginseng extract contained as an active ingredient of the pharmaceutical composition of the present invention is a pharmaceutically acceptable binder (eg polyvinylpyrrolidone, hydroxypropyl cellulose), disintegrant (eg carboxymethyl cellulose calcium, starch) Sodium glycolate), diluents (e.g. corn starch, lactose, soybean oil, crystalline cellulose, mannitol), lubricants (e.g. magnesium stearate, talc), crudes (e.g. white sugar, fructose, sorbitol, aspartame), stabilizers (carboxymethyl Sodium cellulose, alpha or beta cyclodextrin, citric acid, white lead), preservatives (e.g. methyl paraoxybenzoate, propyl paraoxybenzoate, sodium benzoate) and flavorings (e.g. ethyl vanillin, masking flavor, menthol flavono, herbal flavor) It can be mixed with and prepared into pharmaceutical preparations such as tablets, capsules, soft capsules, solutions, ointments, injections and the like.

Acute Toxicity Test

Rg3 and Rh2 contained in black ginseng or black ginseng extract of the active ingredient of the pharmaceutical composition of the present invention has been proven anti-cancer activity and safety from the past, and red ginseng prepared by methods similar to ginseng and black ginseng, which are raw materials of black ginseng, has also been conventionally stable. As already proven, no separate acute toxicity studies were conducted for black ginseng.

Effective amount

Dosage of black ginseng or black ginseng extract contained as an active ingredient of the pharmaceutical composition of the present invention, depending on the age, sex, symptoms, administration method or purpose of the patient, in the case of black ginseng 20 to 200mg per kg body weight each day, black ginseng extract In the case of the body weight is preferably administered 0.2 to 2mg per kg each day, the dosage level for patients with specific symptoms is the weight, age, sex, health status, diet, time of administration, method of administration, excretion rate of the patient The dosage may be changed by those skilled in the art according to the severity of the disease.

According to another embodiment of the present invention, black ginseng or black ginseng extract with increased contents of Rg3 and Rh2, which have the anti-cancer effect, has little toxicity and side effects, and thus can be used safely for a long time. It can be used as an active ingredient in food. At this time, the form of the food is not particularly limited, but may be prepared by adding in the manufacture of a variety of foods, including wire, noodles, confectionery, beverages, meat, fish, herbs, steamed rice or rice, etc. The content of the electrical composition is not particularly limited thereto, but when the black ginseng or black ginseng extract is ingested in the form of powder, the black ginseng is preferably contained in an amount of 1 to 50% by weight based on the final weight of the food, and the black ginseng extract It is preferable to include 0.5 to 5% by weight relative to the final weight of the food, when the black ginseng or black ginseng extract is added to foods such as noodles, confectionery, beverages, meat, fish, herbs, steamed rice or rice In the black ginseng is preferably contained 1 to 10% by weight based on the final weight of the food, the black ginseng extract is 0.1 to 1 of the final weight of the food It is preferable to include in amount%.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example 1 Preparation of Black Ginseng and Component Analysis

Example 1-1 Preparation of Black Ginseng by Steaming and Drying

After purchasing 5-year-old ginseng produced and distributed in Geumsan, it was put in a screw washer and washed three times for a total of 10 minutes. In order to prevent the fresh ginseng from being damaged during steaming, it was left to dry at 40 ° C. for 24 hours. . Subsequently, the black ginseng was prepared by repeating the process of steaming at 100 ° C. for 2 hours and drying at 60 ° C. for 2 hours. The sample was taken every time the process was completed, and a total of 12 black ginseng samples were collected. Was collected.

Example 1-2 : Preparation and Analysis of Black Ginseng Extract

Black ginseng extract was obtained from each of the collected black ginseng samples, and the contents of Rg2, Rg3, Rh1 and Rh2 present only in processed ginseng among the ginseng saponin components included in the black ginseng extract were measured.

Specifically, 20 g of each sample collected was crushed, and 100 ml of water was added thereto, and left for 5 hours at room temperature. Then, 1 L of ethanol was added and extracted at 50 ° C. for 3 hours, filtered, and then concentrated by applying to a vacuum concentrator, and lyophilized to obtain a solid black ginseng extract.

Then, 200 g of water was added to 3.2 g of each solid black ginseng extract, suspended, 200 mL of ether was added thereto, mixed vigorously, added to a separatory funnel, and allowed to stand at room temperature for 1 hour, after which a water layer was obtained. It was. 200 mL of saturated butanol was added to the aqueous layer to obtain a butanol extract fraction, and butanol was removed therefrom to obtain a solid sample.

1 mL of methanol was added to 20 mg of the analytical sample thus obtained, dissolved, filtered through a 0.45 μm filter, and then subjected to HPLC under the following conditions to determine the contents of Rg2, Rg3, Rh1, Rh2 and benzopyrene. See Table 1 and Table 2). Benzopyrene is a component that is detected from carbonized black ginseng during steaming, and is known as one of cancer causing substances. The content of benzopyrene was used as an indicator for confirming the occurrence of side effects caused by the steaming process.

HPLC analysis conditions column Shiseido C18 columm (5㎛, 1,504.6mm) Flow rate 1ml / min Detector ELSD-LT detector Solvent A; CH ₃ CN: H ₂ O: 5% CH ₃ COOH = 15: 80: 5 (v / v / v), B; CH ₃ CN: H ₂ O = 80:20 (v / v), 0 min (0% B), 0-10 min (30% B), 10-25 min (50% B), 25-40 (100 % B), 40-50 minutes (100% B), 50-55 minutes (0% B), 55-58 minutes (0% B) Temperature 40 ℃

Changes in the Contents of Ginseng Saponin in Different Steaming Units (mg / g) Steaming Rg2 Rg3 Rh1 Rh2 Benzopyrene One 0.06 0.13 0.06 0.22 0.6 ppb 2 0.06 1.43 0.07 1.43 0.6 ppb 3 0.07 3.47 0.07 5.57 0.6 ppb 4 0.08 5.69 0.08 7.68 0.6 ppb 5 0.08 8.03 0.09 10.11 0.6 ppb 6 0.08 10.16 0.09 13.14 0.6 ppb 7 0.09 11.63 0.11 16.54 0.7 ppb 8 0.11 13.11 0.11 19.11 0.7 ppb 9 0.15 14.55 0.13 23.40 0.7 ppb 10 0.14 13.70 0.10 18.44 0.7 ppb 11 0.09 11.06 0.08 15.24 0.7 ppb 12 0.08 10.38 0.07 13.69 0.7 ppb

As shown in Table 2, when the number of steaming process increases, the content of Rg2 and Rh1 contained in the black ginseng did not change much, but the content of Rg3 and Rh2 increased. However, when steaming more than 10 times, it was found that the contents of Rg3 and Rh2 tended to decrease, so the so-called "gujeungpo" method of steaming 9 times and drying 9 times was most preferable. I could see.

Example 2 Preparation of Black Ginseng Under High Pressure Conditions

As shown in the result of Example 1, it was found that even after 9 steaming, the increase in the content of Rg3 and Rh2 contained in the black ginseng was not significant. Thus, to increase the contents of Rg3 and Rh2 contained in the black ginseng, except that the steaming process is carried out under the condition of 10 atm, black ginseng is prepared in the same manner as in Example 1, and black ginseng extract is obtained therefrom. Next, the contents of Rg3, Rh2 and benzopyrene contained in the black ginseng extract were measured (see Table 3).

Changes in the content of Rg3, Rh2 and benzopyrene according to the number of steaming under high pressure (unit: mg / g) Steaming Rg3 Rh2 Benzopyrene One 0.26 0.44 0.8 ppb 2 2.43 2.43 0.9 ppb 3 6.47 10.57 2.2 ppb 4 10.69 14.68 3.5 ppb 5 16.03 20.11 4.9 ppb 6 20.16 26.14 6.5 ppb 7 22.63 32.54 8.3 ppb 8 26.11 38.11 9.8 ppb 9 28.55 46.40 10.2 ppb

As shown in Table 3, when steaming under high pressure conditions, the contents of Rg3 and Rh2 were significantly increased than steaming at normal pressure, but it was also found that the content of benzopyrene also increased rapidly. However, when the content of benzopyrene, which is known as a cancer causing substance, is increased, it cannot be used as an active ingredient of medicine, and this problem has to be solved.

In addition, while changing the pressure conditions, each black ginseng was prepared, and each black ginseng extract was obtained therefrom, and then the contents of Rg3, Rh2 and benzopyrene were measured.

Specifically, except that the steaming process is carried out under the conditions of 1 atm, 5 atm, 10 atm, 15 atm or 20 atm, black ginseng was prepared in the same manner as in Example 1, and black ginseng extract was obtained therefrom. Next, the contents of Rg3, Rh2 and benzopyrene contained in the black ginseng extract were measured (see Tables 4a to 4c).

Changes in the content of Rg3 in different pressure conditions and steaming times (unit: mg / g) Steaming 1 atmosphere 5 atmospheres 10 atmospheres 15 atmospheres 20 atmospheres One 0.13 0.17 0.26 0.30 0.32 2 1.43 1.73 2.43 3.07 3.09 3 3.47 4.47 6.47 8.37 8.52 4 5.69 7.37 10.69 13.85 14.12 5 8.03 10.88 16.03 21.03 21.63 6 10.16 13.64 20.16 26.42 27.14 7 11.63 15.42 22.63 29.54 30.27 8 13.11 17.65 26.11 34.24 35.20 9 14.55 19.40 28.55 37.32 38.30

As shown in Table 4a, the content of Rg3 increased as the pressure and the number of steaming increased, but it was found that the rate of increase of the content of Rg3 was significantly reduced at a pressure of 20 atm.

Changes in Rh2 Content in Different Pressure Conditions and Steaming Cycles (Unit: mg / g) Steaming 1 atmosphere 5 atmospheres 10 atmospheres 15 atmospheres 20 atmospheres One 0.22 0.29 0.44 0.57 0.59 2 1.43 1.73 2.43 3.07 3.09 3 5.57 7.26 10.57 13.72 14.01 4 7.68 10.06 14.68 19.09 19.51 5 10.11 13.59 20.11 26.36 27.10 6 13.14 17.67 26.14 34.27 35.23 7 16.54 22.08 32.54 42.56 43.69 8 19.11 25.74 38.11 49.99 51.40 9 23.40 31.41 46.40 60.79 62.46

As shown in Table 4b, the Rh2 content was increased as the pressure and the number of steaming increases, but it was found that the increase rate of the Rh2 content was significantly reduced at a pressure of 20 atm.

Changes in Benzopyrene Content According to Different Pressure Conditions and Steaming Frequency (Unit: ppb) Steaming 1 atmosphere 5 atmospheres 10 atmospheres 15 atmospheres 20 atmospheres One 0.6 0.6 0.8 0.9 1.5 2 0.6 0.7 0.9 1.0 1.8 3 0.6 1.2 2.2 3.3 5.7 4 0.6 1.8 3.5 5.6 9.6 5 0.6 2.4 4.9 7.8 13.8 6 0.6 3.1 6.5 10.5 18.6 7 0.7 4.0 8.3 13.3 23.9 8 0.7 4.6 9.8 15.7 28.4 9 0.7 4.8 10.2 16.4 29.6

As shown in Table 4c, the content of benzopyrene increased rapidly as the pressure and the number of steaming increases, and unlike the case of Rg3 and Rh2, the increase rate of benzopyrene content does not decrease even at a pressure of 20 atm. In the case of steaming only once, the value exceeded the standard value of 1ppb, which is acceptable in food, and it was found that steaming under the condition of 20 atm is not preferable. In addition, when steamed at 1 atm, the contents of Rg3 and Rh2 were low.

From the above results, it was found that when the ginseng is steamed under high pressure, the pressure is preferably 5 to 15 atm.

On the other hand, as shown in Table 3 and Tables 4a to 4c, when steaming twice in a pressure condition of 5 to 15 atm did not significantly increase the content of benzopyrene, steamed twice at high pressure and 7 times at normal pressure It was confirmed how the content of Rg3, Rh2 and benzopyrene in the prepared black ginseng is changed. Specifically, the ginseng dried by the method of Example 1 was put into a steamer, steamed at 100 ° C. for 2 hours at a pressure of 10 atm, and then dried twice at 60 ° C. for 2 hours, under normal pressure. After steaming at 100 ° C. for 2 hours, drying was performed 7 times at 60 ° C. for 2 hours, black ginseng was prepared, and samples were taken at the completion of each process, and a total of 9 black ginseng samples were collected.

Then, by the method of Example 1, black ginseng extract was obtained from each sample collected, and the contents of Rg3, Rh2 and benzopyrene included in the black ginseng extract were measured (see Table 5).

Changes in the contents of Rg3, Rh2 and benzopyrene according to the number of steaming in two times of high pressure steam and seven times of normal pressure steaming (unit: mg / g) Steaming Rg3 Rh2 Benzopyrene 1 (high pressure) 0.26 0.44 0.8 ppb 2 (high pressure) 2.43 2.43 0.9 ppb 3 (atmospheric pressure) 4.47 7.57 0.9 ppb 4 (atmospheric pressure) 6.69 9.68 0.9 ppb 5 (atmospheric pressure) 8.53 12.11 0.9 ppb 6 (atmospheric pressure) 10.16 14.14 0.9 ppb 7 (atmospheric pressure) 11.63 16.54 0.9 ppb 8 (atmospheric pressure) 13.11 19.11 0.9 ppb 9 (atmospheric pressure) 14.55 23.40 0.9 ppb

As shown in Table 5, the contents of Rg3, Rh2 and benzopyrene increased when the high-pressure steaming was performed twice, but only the contents of Rg3 and Rh2 increased during atmospheric pressure steaming.

On the other hand, when comparing the results of Table 5 with the results of Table 2, the content of Rg3 and Rh2 increases each time steaming is performed, but showed a similar aspect to each other, but the content of the contents of Rg3 and Rh2 disclosed in Table 5 The increase level was superior to that of Table 2. In addition, the increased levels of the contents of Rg3 and Rh2 disclosed in Table 5 can be confirmed that the superiority of those of Table 2 not only during high pressure steaming but also during normal pressure steaming, when mixing the high pressure steaming step and the normal pressure steaming step, Rg3 and It can be seen that the content of Rh2 can be significantly increased.

Example 3 Optimization of High Pressure Steam and Normal Pressure Steam Stages

From the results of Example 2, it can be seen that when mixing the high pressure steaming step and the normal pressure steaming step, the contents of Rg3 and Rh2 can be significantly increased, so that the optimum contents of Rg3 and Rh2 can be maximized. We tried to establish steam conditions.

First, it was confirmed that the high-pressure steaming can be performed up to two times from the results of Example 2. As shown in Table 6 below, each of the experimental groups was set in the order of allocating two high-pressure steaming processes among the nine steaming processes. . At this time, the high-pressure steaming was performed by steaming for 2 hours at 10 atm and 100 ℃.

Experimental group setting according to the order of high pressure steaming process Experimental group Autoclave sequence Experimental group Autoclave sequence Experimental group Autoclave sequence 12
13
14
15
16
17
18
19
23
24
25
26 Once and twice
1 and 3 times
1 and 4 times
1 and 5 times
1 and 6 times
1 and 7 times
1 and 8 times
1 and 9 times
2 times and 3 times
2 and 4 times
2 and 5 times
2 and 6 times 27
28
29
34
35
36
37
38
39
45
46
47 2 and 7 times
2 and 8 times
2 times and 9 times
3 times and 4 times
3 and 5 times
3 times and 6 times
3 times and 7 times
3 times and 8 times
3 times and 9 times
4 and 8 times
4 and 6 times
4 and 7 times 48
49
56
57
58
59
67
68
69
78
79
89 4 and 8 times
4 and 9 times
5 and 6 times
5 and 7 times
5 and 8 times
5 and 9 times
6 and 7 times
6 and 8 times
6 and 9 times
7 and 8 times
7 and 9 times
8 and 9 times

Except for steaming at high pressure and normal pressure by the method of each experimental group, a total of 36 black ginseng and each black ginseng extract was prepared by the method of Example 1, and Rg3, Rh2 and benzopyrene contained in the respective black ginseng extracts Was measured (see Tables 7a to 7c).

Rg3 content in mg / g according to the order of high-pressure steaming process Experimental group Rg3 content Experimental group Rg3 content Experimental group Rg3 content 12
13
14
15
16
17
18
19
23
24
25
26 14.55
15.53
15.78
17.43
26.41
24.20
23.11
21.43
13.67
13.92
14.94
24.57 27
28
29
34
35
36
37
38
39
45
46
47 23.69
22.87
21.33
12.42
13.51
23.87
22.67
21.37
20.34
12.37
22.40
21.04 48
49
56
57
58
59
67
68
69
78
79
89 19.96
18.41
21.69
20.38
18.22
16.44
18.54
15.17
14.67
13.87
13.24
12.11

As shown in Table 7a, when steamed at high pressure in the first and sixth times (Experimental group 16), it was found that the content of Rg3 is the best.

Content of Rh2 in the order of allocation of autoclave (unit: mg / g) Experimental group Rh2 content Experimental group Rh2 content Experimental group Rh2 content 12
13
14
15
16
17
18
19
23
24
25
26 23.40
25.34
27.94
29.35
45.47
43.28
41.22
39.17
24.28
26.32
28.47
42.70 27
28
29
34
35
36
37
38
39
45
46
47 41.32
40.06
37.34
25.18
27.55
40.53
39.98
38.67
35.29
26.14
39.67
38.42 48
49
56
57
58
59
67
68
69
78
79
89 36.41
33.18
37.94
37.08
34.20
30.87
35.74
32.64
38.64
30.87
36.50
33.24

As shown in Table 7b, when steamed at high pressure in the first and sixth times (Experimental group 16), it was found that the content of Rh2 is the best.

Benzopyrene content according to the order of high pressure steaming process (unit: ppb) Experimental group Benzopyrene Content Experimental group Benzopyrene Content Experimental group Benzopyrene Content 12
13
14
15
16
17
18
19
23
24
25
26 1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0 27
28
29
34
35
36
37
38
39
45
46
47 1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0 48
49
56
57
58
59
67
68
69
78
79
89 1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0

As shown in Table 7c, the content of benzopyrene did not change in all cases.

As a result, when the results of Tables 7a to 7c are summarized, the contents of Rg3 and Rh2 are the best in the case of high-pressure steaming in the first and sixth times (Experimental group 16), and the content of benzopyrene does not increase. As a result, it was found that black ginseng maximizing the contents of Rg3 and Rh2 could be produced when steaming at high pressure in the first and sixth times and steaming the rest at normal pressure.

Example 4 Anticancer Effect of Black Ginseng

In order to confirm whether the black ginseng and black ginseng extract prepared by the method of the present invention show relatively superior anticancer activity than the black ginseng and black ginseng extract prepared by the conventional method, the black ginseng prepared by the method of the present invention and the conventional method Each black ginseng extract was obtained from black ginseng and treated to lung cancer cells to compare their anticancer activity.

Example 4-1 : Obtaining Black Ginseng and Black Ginseng Extract

After purchasing 5-year-old ginseng produced and distributed in Geumsan, it was put in a screw washer and washed three times for a total of 10 minutes. In order to prevent the fresh ginseng from being damaged during steaming, it was left to dry at 40 ° C. for 24 hours. . Subsequently, it was put into a steamer and steamed at 100 ° C. for 2 hours, and dried at 60 ° C. for 2 hours to prepare black ginseng nine times. At this time, the first and sixth steaming process was performed under a pressure of 10 atm.

100 g of each sample was crushed, and 500 ml of water was added thereto, and the mixture was left at room temperature for 5 hours, 5 L of methanol was added thereto, extracted at 60 ° C. for 4 hours, filtered, and concentrated by applying to a vacuum concentrator. And lyophilized to obtain a solid black ginseng extract (test group), respectively.

On the other hand, the ginseng was left to dry at 40 ℃ for 24 hours in a steamer, steamed for 2 hours at 100 ℃ and atmospheric pressure, and then dried for 6 hours at 60 ℃ repeated 9 times to prepare a conventional black ginseng Then, this was extracted in the same manner as described above to obtain a solid black ginseng extract (comparative experimental group).

Example 4-2 Anticancer Effects of Black Ginseng Extract against Lung Cancer Cell Lines

In order to confirm the anticancer effect of black ginseng extract in laboratory conditions, the cultured lung cancer cell line was treated with black ginseng extract, and its cancer treatment effect was confirmed.

First, human lung cancer cells (A549) obtained from the American Type Culture Collection (ATCC) were prepared using RPMI culture medium (GIBCO / Invitrogen, USA) (10% (v / v) fetal bovine serum, 100 units / ml penicillin and 2.5 × 10 ⁵ cells per well of 6-well plates in 100 μg / ml streptomycin) were aliquoted and incubated for 24 hours under conditions of 37 ° C., 5% (v / v) CO ₂ .

Then, each RPMI culture medium in which each black ginseng extract obtained in Example 4-1 was dissolved at a concentration of 100 mg / ml was added to the cultured lung cancer cell line by 1 to 10 ml per well. After 5 days, the number of surviving lung cancer cells was counted, and the counted lung cancer cell numbers were converted into the ratio (survival rate) to the initial lung cancer cell numbers, and the survival rates were compared for each experimental group (see Table 8). ). At this time, lung cancer cell line without black ginseng extract was used as a control.

Survival Rate of Lung Cancer Cell Lines According to the Treatment Rate of Black Ginseng Extract (Unit:%) Containing Black Ginseng Extract
Throughput of RPMI Culture Medium Experimental group Comparative experiment group Control (0ml)
1ml
2ml
3 ml
4ml
5ml
6ml
7ml
8ml
9 ml
10 ml 130
67
63
58
50
38
32
25
21
17
15 130
71
67
66
63
59
54
50
48
46
42

As shown in Table 8, when the black ginseng extract was not added to the lung cancer cell line (control), after 5 days, the cell number of the lung cancer cell line increased by about 30%, but the black ginseng extract of the present invention or the conventional black ginseng extract When treated, it was confirmed that the cell number of the lung cancer cell line was drastically reduced in proportion to the throughput of the black ginseng extract. In addition, when comparing the survival rate of lung cancer cell lines treated with the black ginseng extract (experimental group) of the present invention or the conventional black ginseng extract (comparison experimental group), the experimental group further reduces the survival rate of lung cancer cell line than the comparison experiment group, the throughput of the black ginseng extract increased The more the experimental group reduced the survival rate of lung cancer cell line significantly more than the comparative group.

The black ginseng extract of the present invention was estimated to be due to the increased content of Rg3 and Rh2 compared to the conventional black ginseng extract, from which the black ginseng and black ginseng extract of the present invention is a conventional black ginseng and in vitro conditions (in vitro) It was found that the anticancer activity was higher than that of the black ginseng extract.

Example 4-3 Anticancer Effect of Black Ginseng Extract on Metastasis of Cancer Cells

In order to confirm the anticancer effect of black ginseng extract in vivo, metastatic cancer cells and black ginseng extract were injected into the mouse at the same time to confirm its cancer treatment effect and cancer prevention effect.

First, B16 melanoma cells, metastatic cancer cells, were unicellularized with 0.02% EDTA and carefully resuspended in serum free RPMI-1640 medium at a concentration of 7.5 × 10 ⁵ / ml. Subsequently, serum-free RPMI-1640 medium (0.1-1 ml) and serum-free RPMI- in which the black ginseng extract of the experimental group or the comparative group obtained in 1 ml of the B16 melanoma cell suspension was dissolved at a concentration of 100 mg / ml. 1640 medium was mixed to obtain a total of 3 ml of the mixed solution, and 500 μl of this was injected into the tail vein of C57BL / 6 mice (Charles River, Japan) and bred for 14 days. After 14 days, each of the mice was killed to remove the lungs, and the number of melanoma colonies present on the surface of the lungs was counted using an anatomical microscope (see Table 9). At this time, C57BL / 6 mice injected with only B16 melanoma cell suspension without black ginseng extract were used as a control.

Number of Lung Melanoma Colonies According to the Processing Capacity of Black Ginseng Extract (Unit: dogs) Black Ginseng Extract Throughput Experimental group Comparative experiment group Control (0ml)
1ml
2ml
3 ml
4ml
5ml
6ml
7ml
8ml
9 ml
10 ml 144 ± 40
137 ± 28
123 ± 25
118 ± 23
100 ± 21
88 ± 18
62 ± 13
55 ± 11
41 ± 8
37 ± 6
25 ± 2 144 ± 40
139 ± 31
130 ± 27
121 ± 25
112 ± 23
103 ± 19
94 ± 18
85 ± 15
76 ± 12
67 ± 8
55 ± 4

As shown in Table 9, when only the B16 melanoma cell suspension was injected without the black ginseng extract (control), after 14 days, the number of lung melanoma colonies was counted as about 144, but the black ginseng extract of the present invention (experimental group) or Injecting the conventional black ginseng extract (comparative experimental group) with B16 melanoma cell suspension reduced the number of lung melanoma colonies in proportion to the amount of black ginseng extract injected, so the black ginseng extract not only shows cancer treatment but also prevents cancer. Confirmed. In addition, when comparing the number of lung melanoma colonies in the mice injected with the black ginseng extract (experimental group) of the present invention and the conventional black ginseng extract (comparison experimental group), the experimental group further reduces the number of lung melanoma colonies than the comparison experiment group, As the amount of black ginseng extract increased, the experimental group significantly decreased the number of lung melanoma colonies than the comparative group.

The black ginseng extract of the present invention was estimated to be due to the increased content of Rg3 and Rh2 compared to the conventional black ginseng extract, inhibiting the metastatic activity of B16 melanoma cells injected from the outside, from this black ginseng and black ginseng of the present invention The extract was found to be superior to the conventional black ginseng and black ginseng extract in terms of cancer prevention effect as well as cancer treatment effect in laboratory conditions (in vitro) and in vivo (in vivo).

Example 5 Preparation of Anticancer Functional Health Foods

The black ginseng extract of the present invention was added during the preparation of a conventional food, and various anti-cancer health functional foods having a prevention and improvement effect of cancer disease were prepared as follows.

Example 5-1 Preparation of Black Ginseng Extract

After purchasing 5-year-old ginseng produced and distributed in Geumsan, it was put in a screw washer and washed three times for a total of 10 minutes. In order to prevent the fresh ginseng from being damaged during steaming, it was left to dry at 40 ° C. for 24 hours. . Subsequently, it was put into a steamer and steamed at 100 ° C. for 2 hours, and dried at 60 ° C. for 2 hours to prepare black ginseng nine times. At this time, the first and sixth steaming process was performed under a pressure of 15 atm.

500 g of each sample was crushed, 2.5 liters of water was added thereto, and the mixture was left at room temperature for 5 hours, 15 liters of methanol was added thereto, extracted at 70 DEG C for 5 hours, filtered, and concentrated under reduced pressure. And lyophilized to prepare a solid black ginseng extract.

Example 5-2 Preparation of Anticancer Noodles

5 g of black ginseng extract obtained in Example 5-1, 715 g of wheat flour, 80 g of eggs, and 200 g of water were mixed to prepare a dough, and a cotton pad was prepared by a conventional noodle manufacturing method to prepare anti-cancer noodles.

Example 5-3 Preparation of Anticancer Biscuits

5 g of black ginseng extract obtained in Example 5-1, 715 g of wheat flour, 200 g of milk, and 80 g of eggs were mixed to prepare a dough, and an anticancer biscuit was prepared by a conventional biscuit manufacturing method.

Example 5-4 Preparation of Anticancer Beverage

5 g of black ginseng extract obtained in Example 5-1, 10 mg of fruit flavor, 100 g of fruit juice, 10 mg of sodium benzoate and 900 ml of water were mixed to prepare an anticancer beverage.

Example 5-5 Preparation of Anticancer Meat Seasoning

2 g of black ginseng extract obtained in Example 5-1, 100 g of pear juice, 100 g of radish juice, 300 g of red pepper paste, a small amount of salt, garlic, pepper, onion and ginger were mixed to prepare an anti-cancer meat seasoning.

Example 5-6 Preparation of Anticancer Bean Sprouts

5 g of black ginseng extract obtained in Example 5-1, 150 g of red pepper powder, a small amount of salt, garlic, pepper, onion and ginger were mixed to prepare a seasoning for bean sprouts, 200 g of the prepared seasoning and 800 g of bean sprouts were mixed. , In a usual manner, to prepare anti-cancer sprouts.

Example 5-7 Preparation of Anticancer Miso Stew

2 g of black ginseng extract obtained in Example 5-1, 600 g of soybean paste, a small amount of salt, garlic, pepper, onion, and ginger were mixed, put into 500 ml of water, and cooked in a conventional manner to prepare an anti-cancer miso crab.

Example 5-8 Preparation of Anticancer Multigrain Rice

2 g of black ginseng extract obtained in Example 5-1, 50 g of black soybean, 50 g of sorghum, 50 g of millet rice, and 348 g of white rice were mixed and cooked in a conventional manner to prepare anticancer grain rice.

Claims (13)

delete delete (Iii) The dried ginseng is steamed at 90 to 110 ° C. for 1 to 3 hours, and then dried 9 times at 50 to 70 ° C. for 1 to 3 hours, but the first and sixth steaming steps are performed. Preparing black ginseng having an increased content of Rg 3 and Rh 2 by performing the same at a pressure of 5 to 15 atmospheres; And, (Ii) pulverizing the prepared black ginseng, adding 3 to 7 times (weight ratio) of water to black ginseng and leaving it for 3 to 7 hours, and then 5 to 15 times (volume ratio) of water having 1 to 4 carbon atoms. Adding a lower alcohol, extraction for 1 to 5 hours at 30 to 70 ℃, and filtration to remove the solvent, a method for producing black ginseng extract. delete delete delete delete delete delete delete delete delete delete