KR100844991B1 - A composition comprising the extract of black ginseng leaf showing anti-cancer activity - Google Patents

Abstract

The present invention relates to a composition containing an extract extracted from the black trifoliate prepared using a method of manufacturing augmentation width or high temperature and high pressure, and this composition shows an excellent anticancer effect as a pharmaceutical composition and health functional food for anticancer Can be used.

Black leaf, mass production, gujeunggu, anticancer

Description

A composition comprising the extract of black ginseng leaf showing anti-cancer activity}

An object of the present invention is to provide an anticancer pharmaceutical composition and health functional food having an anticancer activity containing the extract extracted from the black trifolium prepared by using a method of producing agglomerate or high temperature and high pressure.

To capture the $ 3.5 billion global ginseng market, Korean bio venture companies are working hard to develop new global ginseng-related materials worth $ 3.5 billion annually. So far, the only company that has standardized ginseng as a functional food and is making a profit in the global market is “Pharmaton,” a company owned by Boehringer Ingelheim, Switzerland. National research on ginseng and commercialization of venture industry are underway in Korea.

Korean red ginseng has a historical record of about 1,000 years ago (there is a record of red ginseng in Goryeo-do, written by the Chinese Song Dynasty). have. In general, the numerical objectives of herbal medicines are known as: 1) reducing side effects such as toxicity, 2) altering the performance of herbal medicines, increasing the efficacy of drugs, 3) improving storage and storage, and 4) correcting taste and odor and coloring. In general, red ginseng undergoes a second ingredient conversion during an appropriate heat treatment process, thereby generating new active ingredients unique to red ginseng or fresh ginseng, and some active ingredients increase in content. As such, new ingredients that do not exist in ginseng are produced according to the processing method of ginseng, and new processed ginsengs having better effects on various physiological activities including anticancer activity can be developed compared to ginseng (Okamura N. et al. , Biol . Pharm . Bull. , 17 (2) , p270, 1994).

In China, it develops a method of mass-producing Rg3, a ginseng saponin, which is rarely present in white ginseng and is present in red ginseng, and is used for anticancer treatment under the trade name 'Shenyi'.

In Korea, the representative product of functional ginseng is 'Seonsam' developed by bio venture company Ginseng Science in 1998.It was developed as Sesamsamjeong in 2001 and recognized in domestic health functional food market. It has been established as a leader in functional ginseng (Keum YS et al., Antioxidant and anti-tumor promoting activities of the methanol extract of heat-processed ginseng, Cancer Lett ., 150 (1) , pp. 41-48, 2000).

In addition, 'Hwangsam EX' co-developed by Korea Research Institute of Biotechnology and Bio-Venture Company, 'Vitgin', 'Naengsam Farming Ginseng Farming Co.' Recently, various types of processed ginseng are being developed, such as Biomax, which was developed jointly with UPI in the US, Six Plus of CJ, and Yellow Ginseng of Ildong Pharm.

In recent years, as a result of efforts to further increase the active ingredient content of ginseng, black ginseng using the principle of gujeunggupo, the most representative method of using water and fire, has been developed. (Korean Patent Laid-Open Publication No. 2003-0005089; Manufacturing method of black ginseng and black rice ginseng), The laboratory has applied for a standardized manufacturing method of black ginseng that can be shortened in time and mass production compared to this method (Korea) Patent Application No. 10-2006-0044066; Novel black ginseng production method and composition containing the same).

Most of the studies on ginseng conducted so far have been conducted on ginseng roots, so most of the ginseng leaves have been discarded.

In this way, in the era of premium ginseng, the present inventors tried to overcome the limitation that research on ginseng and processed ginseng until now is mostly limited to roots. Patents have been applied for the development of black ginseng leaves, which are processed ginseng leaves manufactured using the principle, or standardized manufacturing methods of black ginseng leaves using high temperature and high pressure (Korean Patent Application No. 10-2006-0076232; Antioxidant composition containing the prepared black leaves and extracts thereof.

Accordingly, the present invention provides about 2-3 times more saponin content than ginseng roots (Yahara et al., Chem . Pharm ., Bull. , 24 (9) , p2204, 1976). To develop into economical raw materials, such as pharmaceuticals, health functional foods, food additives and cosmetics, by providing a method for producing black leaf lobe using the method of augmentation width and high temperature high pressure from ginseng leaf, It was confirmed that the extract extracted from the black trifoliate shows a strong anticancer effect, completed the present invention.

It is an object of the present invention to provide an anticancer pharmaceutical composition and health functional food which exhibits anticancer activity containing an extract extracted from a black trifolium prepared using a method of producing acupoint or a high temperature and high pressure.

In order to achieve the above object, the present invention provides a pharmaceutical composition and health functional food for the prevention and treatment of cancer diseases containing the extract of the black trilobite prepared by the method of manufacturing augmentation width as an active ingredient.

In addition, the present invention provides a pharmaceutical composition and health functional food for the prevention and treatment of cancer diseases containing the extract of the black trilobite prepared by the method of high temperature and high pressure as an active ingredient.

The extract of the present invention provides a pharmaceutical composition and health functional food for preventing and treating cancer diseases extracted with a solvent selected from water and a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof.

Hereinafter, the present invention will be described in detail.

Black leaf extract of the present invention can be prepared through the following process.

  After washing three to seven years old, preferably four to six years old, ginseng leaves three times for 15 minutes with an ultrasonic cleaner, 12 to 36 hours at a temperature of 30 to 70 ° C, preferably 40 to 60 ° C, preferably The first step of drying for 20 to 28 hours; A second step of steaming the dried ginseng pulverized with a grinder into a steamer for 1 to 5 hours, preferably 2 to 4 hours at a temperature of 60 to 120 ° C., preferably 85 to 105 ° C. except for a preheating time; A third step of drying at a temperature of 40 to 80 ° C., preferably 50 to 70 ° C. for 6 to 18 hours, preferably 9 to 15 hours; The processed ginseng leaf (hereinafter referred to as BGL-1) of the present invention having a water content of 14% or less obtained by repeating the first to third steps 9 times may be prepared.

In addition, 3 to 7 years old, preferably 4 to 6 years old ginseng leaf of the present invention after washing three times for 3 minutes with an ultrasonic cleaner, 12 to 36 hours at a temperature of 30 to 70 ℃, preferably 40 to 60 ℃, Preferably the first step of drying for 20 to 28 hours; A second step of steaming the dried ginseng pulverized with a grinder under pressure at a temperature of 95 to 155 ° C., preferably 110 to 145 ° C. for 1 to 8 hours, preferably 2 to 6 hours; Processed ginseng leaf of the present invention having a moisture content of 14% or less obtained in the third step of drying at a temperature of 40 to 80 ° C., preferably 50 to 70 ° C. for 6 to 18 hours, preferably 9 to 15 hours. , BGL-2).

The method for obtaining the extract of BGL-1 prepared by the augmentation width production method will be described in detail, by adding water of about 2 to 7 times, preferably 2 to 5 times the mass of BGL-1 prepared by the preparation method. After being left for 3 to 7 hours at room temperature, 0.1 to 2 liters of water and a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, preferably ethanol or methanol, more preferably 80% methanol, preferably 0.5 to 1.5 L was added and refluxed three times for 3 hours. After the extracted solution was cooled to room temperature and filtered, a crude extract of BGL-1 of the present invention (hereinafter referred to as BGLE-1) obtained by removing methanol using a vacuum concentrator was obtained.

After suspending BGLE-1 in water, ether was added to remove the nonpolar substance, saturated saponin was added to the remaining aqueous layer four times to extract saponin present in ginseng, and then butanol was removed using a vacuum condenser to remove ginseng saponin. This butanol soluble extract of the present invention (hereinafter referred to as BGLBE-1) can be obtained.

In addition, the method for obtaining an extract of BGL-2 prepared by the high temperature and high pressure manufacturing method in detail, about 2 to 7 times, preferably 2 to 5 times the mass of water of BGL-2 prepared by the above production method After addition, the mixture was left at room temperature for 3 to 7 hours, and then 0.1 to 2 liters of water and a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, preferably ethanol or methanol, more preferably 80% methanol, preferably 0.5 To 1.5 L was added, and the mixture was extracted under reflux three times for three hours. After the extracted solution was cooled to room temperature and filtered, a crude extract of BGL-2 (hereinafter, referred to as BGLE-2) of the present invention obtained by removing methanol using a vacuum concentrator was obtained.

After suspending BGLE-2 by adding water, ether was added to remove the nonpolar substance, and saturated saponin was added to the remaining aqueous layer to extract saponin present in ginseng four times, and then butanol was removed using a vacuum condenser to remove ginseng saponin. This butanol-soluble butanol soluble extract of the present invention (hereinafter referred to as BGLBE-2) can be obtained.

The black trifoliate extract prepared by the above manufacturing method is dried through a drying method common in the art such as shade drying and freeze drying, and then pulverized and powdered into fine particles, preferably a particle size of about 50 μm to 200 μm. It can be prepared into compositions such as pills, capsules, tablets, etc. using excipients or carriers well known in the art through the process.

The black ginseng leaf prepared by the above method contains about 3 to 5 times more saponin content than the ginseng root, and among these saponin components, Rg5 has anticancer effect and antidiabetic effect, and Rg3 is Since it exhibits various pharmacological activities such as antistress effect, hepatoprotective effect and antioxidant effect, the black trifoliate extract of the present invention may exhibit various physiological activities as described above.

Black ginseng leaf extract prepared by the production method of the present invention has a strong anti-cancer effect, characterized in that it exhibits a strong anti-cancer effect compared to the red ginseng and black ginseng extract.

Therefore, the present invention provides a pharmaceutical composition for preventing and treating cancer diseases and health functional foods containing the black trilobite extract prepared from the preparation method and the extraction method as an active ingredient.

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

Cancer diseases as defined herein include common cancer diseases, preferably gastric cancer, colon cancer, breast cancer, lung cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or ocular melanoma, uterine cancer, ovary Cancer, Colorectal Cancer, Small Intestine Cancer, Rectal Cancer, Anal Neoplasms, Fallopian Tube Carcinoma, Endometrial Carcinoma, Cervical Carcinoma, Vaginal Carcinoma, Vulvar Carcinoma, Hodgkin's Disease, Esophageal Cancer, Lymphatic Cancer, Gallbladder Cancer, Endocrine 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) A tumor, primary CNS lymphoma, spinal cord tumor, brain stem glioma or pituitary adenoma, more preferably cancer disease selected from kidney cancer, colon cancer, lung cancer or prostate cancer.

Pharmaceutical compositions comprising the extract of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Pharmaceutical compositions comprising extracts according to the invention, in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterile injectable solutions, respectively, according to conventional methods. Can be formulated and used. Carriers, excipients and diluents that may be included in the composition comprising the extract 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 include at least one excipient such as starch, calcium carbonate and sucrose in the extract. ) Or lactose, gelatin and the like are mixed. 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. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the extracts of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the preferred effect, the extract of the present invention is preferably administered at 0.2 to 200 mg / kg, preferably 2 to 100 mg / kg. 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 extract of the present invention can be administered to mammals such as mice, 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 intracerebroventricular injection.

Black ginseng leaf extract of the present invention can be used as a raw material that is more potent than the original ginseng, that is, the conventional ginseng has been used, that is, various pharmaceutical preparations, Chinese medicine, health food, food, tea, cosmetics.

Since the extract of the present invention has little toxicity and side effects, it can be used with confidence even for long-term use for the purpose of prevention or treatment.

The present invention provides a dietary supplement for the prevention and improvement of cancer diseases containing the black trifoliate extract as an active ingredient exhibiting a preventive effect of cancer diseases.

Foods to which the extract can be added include, for example, various foods, beverages, gums, teas, vitamin complexes, health functional foods, powders, granules, tablets, capsules or beverages.

It may also be added to foods or beverages for the purpose of preventing cancer diseases. At this time, the amount of the extract in the food or beverage may be added in 0.01 to 15% by weight of the total food weight, the health beverage composition may be added in a ratio of 0.02 to 5g, preferably 0.3 to 1g based on 100ml. .

The health functional beverage composition of the present invention is not particularly limited to other ingredients except for containing the extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. .

Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the extract of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the extracts of the present invention may contain flesh for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is usually selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the extract of the present invention.

Hereinafter, the present invention will be described in detail by the following Examples and Experimental Examples.

However, the following Examples and Experimental Examples are only illustrative of the present invention, and the content of the present invention is not limited by the following Experimental Examples.

Comparative Example  1. Preparation of Red Ginseng Extract

20 g of red ginseng purchased from Geumsan was taken and pulverized, 100 ml of water was added and allowed to stand at room temperature for 5 hours. Then, 1 liter of methanol was added and refluxed three times for 3 hours. The extracted solution was cooled to room temperature and filtered. Methanol was removed using a vacuum concentrator and 8.1 g of red ginseng extract was obtained. 200 g of water was added and suspended in 8.1 g of red ginseng extract, and then 200 mL of ether was added to remove the nonpolar material. 200 ml of saturated butanol was added to the remaining aqueous layer to extract saponins present in ginseng, and then, butanol was removed by a vacuum condenser to obtain 1.3 g of butanol extract containing a large amount of ginseng saponin (hereinafter referred to as RGRE-1). This RGRE-1 was used as a comparative sample of the anticancer experiment of the invention.

Comparative Example  2. Preparation of Black Ginseng Extract

Black ginseng extract (hereinafter referred to as BGRE-1) prepared by the method of preparing Korean Patent Application No. 10-2006-0044066 (a method for preparing a new black ginseng and a composition containing the same) was used as a comparative sample for anticancer experiments.

Comparative Example  3. Ginseng leaf  Preparation of Extract

      After collecting 5 years old ginseng leaves grown in Geumsan, washed twice with an ultrasonic cleaner for 15 minutes, 50 g of dried ginseng leaves were dried at 60 ° C for 10 hours, pulverized, 100 ml of water and left at room temperature for 5 hours. 1 L of 80% methanol (20% water) was added thereto, and the mixture was extracted under reflux three times for 3 hours. The extracted solution was cooled to room temperature, filtered, and methanol was removed using a reduced pressure concentrator to obtain 17.98 g of ginseng leaf extract.

17.98 g of this ginseng leaf extract was suspended in 500 ml of water, and then 500 ml of ether was added to remove the nonpolar material. 500 ml of saturated butanol was added to the remaining aqueous layer to extract saponin present in ginseng four times, and then, butanol was removed with a reduced pressure concentrator to extract butanol extract containing a large amount of ginseng saponin (crude saponin extract, hereinafter referred to as CGLE-1). ) 9.19 g was obtained. The above CGLE-1 was used as a comparative sample of the anticancer experiment.

Example  One. Black trilobite  Produce

1-1. To the gut width  By way

After purchasing 5-year-old ginseng leaf grown in Geumsan, it was put in an ultrasonic cleaner and washed repeatedly three times for 15 minutes. The washed ginseng leaves were first dried at 50 ° C. for 24 hours. The dried ginseng leaves were pulverized at a low temperature for 24 hours, placed in an Onggi used as a steamer, and steamed first at 95 ° C. for 3 hours except for the preheating time. The preheating time was typically about 30 minutes from the time the steam evaporated from the pot. After maintaining the internal temperature of the dryer with a built-in heating wire at 60 ℃, the ginseng leaves were evenly dried for 12 hours while rotating the fan. This steaming and drying process was repeated nine times to prepare BGL-1 of the present invention having a water content of 14% or less, wherein the green ginseng leaf turned black.

1-2. Method by high temperature and high pressure

After purchasing 5-year-old ginseng cultivated in Geumsan, it was put in an ultrasonic cleaner and washed three times in total for 3 minutes. The washed ginseng leaves were first dried at 50 ° C. for 24 hours and then pulverized, and further steamed at 110-145 ° C. for 4 hours under pressure. After maintaining the internal temperature of the dryer with a built-in heating wire at 60 ℃ the second ginseng leaf was evenly dried for 12 hours while rotating the fan to prepare a BGL-2 of the present invention having a moisture content of 14% or less.

Example  2. Black leaf  Preparation of Extract

2-1. Black leaf Crude extract  Produce

50 g of black trilobite prepared by the two methods of the amplification width and high temperature and high pressure of Example 1-1 and 1-2 were respectively added, and 100 ml of water was added and left at room temperature for 5 hours, followed by 80% methanol (20% water). 1L was added and refluxed three times for 3 hours, and the extracted solution was cooled to room temperature, filtered, and the crude black leaf extract obtained by removing methanol using a vacuum concentrator was obtained (BGLE-1; 17.85 g and BGLE-). 2; 18.12 g).

2-2. Black leaf Butanol  Preparation of Soluble Extracts

500 ml of water was added to the black tricotyledonous extracts (BGLE-1 and BGLE-2) obtained in Example 2-1 and suspended, and 500 ml of ether was added to remove the nonpolar material. 500 ml of saturated butanol was added to the remaining aqueous layer, followed by extraction of saponin present in ginseng four times, followed by removing butanol with a reduced pressure concentrator, to obtain a butanol soluble extract (crude saponin extract) containing a large amount of ginseng saponin (augmentation method); 9.16g, hereinafter referred to as BGLBE-1 and high temperature and high pressure method; 9.35g, hereinafter referred to as BGLBE-2). This black trifoliate crude saponin extract was used as a sample for saponin analysis and anticancer experiment.

Example  3. Black leaf  Saponin ingredient analysis

In order to analyze the components of ginseng saponin present in the black trifoliate extract, 20 mg of the black trifoliate butanol extract obtained in Example 2 was dissolved in 1 ml of methanol, and the filtrate was filtered with a 0.45 μm filter. The components were analyzed at.

Flow rate 1ml / min Detector ELSD-LT detector  Solvent A; CH 3 CN: H 2 O: 5% CH 3 COOH = 15: 80: 5, B; CH3CN: H2O = 80:20 0 min (0% B), 0-10 min (30% B), 10-25 min (50% B), 25-40 (100% B), 40-50 min (100 % B), 50-55 minutes (0% B), 55-58 minutes (0% B) Temperature 40 degrees

As a result of analyzing the components, Rb1 0.2-0.5mg, Rb2 1.4-2.1mg, Rc 1.3-2.0mg, Rd 2.1-2.5mg, Re 1.0-1.5mg, Rg1 0.4-0.9 mg, Rk1 + Rg5 112-134 mg, Rg3 (S) + Rg3 (R) was confirmed that the 17-26 mg and Rh2 30-38 mg were present.

In particular, Rg5, which is produced specifically by steaming, has an anti-anxiety effect (Cha HY et al., Biol Pharm Bull., 28 (9) , pp1621-1625, 2006), brain function improvement effect (Kang JS et al., Arch Pharm Res. , 28 (3) , pp335-342, 2005), antidiabetic effect (Kordella T et al., Diabetes Forecast, 57 (1) , pRG5-8, 2004), Hepatitis C inhibitory effect (Neuberger J et al., The Royal College of Physicians of London and the British Society of Gastroenterology ., 49 , Suppl 1 : I1-21, 2001), etc., Rh2 has anticancer effects (Guns ES., J Urol , 175 (5) , p1926-1931, 2006), brain function improvement effect (Rhim H et al., Eur J Pharmacol . 536 (1-2 ), pp69-77, 2006), protective effect of hepatocytes (Kim DH et bal., Biol Pharm Bull , 28 (10) , pp1992-1994, 2005), etc., Rg3 has antistress effect (Chung BC et al., Pharmacol Res. , 54 (1) , pp46-49, 2006), anticancer effect (Huang C. et al., Sichuan Da Xue Xue Bao Yi Xue Ban, 37 (1) , pp60-62, 2006), hepatoprotective effect (Kim DH et al., Biol Pharm Bull., 28 (10) , pp1992-1994, 2005), antioxidant effect (Sun YX et al. , J Agric Food Chem ., 51 (9) , pp2555-2558, 2003), Angiogenesis Inhibitory Effect (Qui H et al., Zhonghua Wai Ke Za Zhi , 40 (8 ), pp606-608, 2002), vasorelaxation (Schini-Kerth VB et al., Eur J Pharmacol . , 367 (1) , pp51-57, 1999), platelet aggregation inhibitory activity (Lee HK et al., Biol Pharm Bull., 21 (1) , pp79-80, 1998), etc., are known to exhibit various physiological activities. Therefore, black saponins containing a large amount of these saponins can expect various physiological activities as described above.

Experimental Example  1. Measurement of anticancer activity

1-1. Intracellular  Cytotoxicity Measurement

Rubinstein, LV et al., Correlation of screening data generated with a tetrazolium assay (MTT) versus a protein assay (SRB) against a broad panel of human tumor cell lines, Proceedings of the American Association for Cancer Research , 30 , p.2418, 1989), cytotoxicity was measured. The cell lines used for cytotoxicity measurement were human renal tumor cell line (ACHN), human colon tumor cell line (HCT 15), human prostate tumor cell line (PC-3), and human lung tumor cell line (NCI-H23). RPMI 1640 with% FBS was incubated in a 5% CO ₂ , 37 ° C. incubator. After culturing the cells, cells were separated from the culture vessel using trypsin-EDTA, 1 × 104 cells were dispensed into 96-well plates, cultured for 24 hours, and the samples obtained in Comparative Examples 1 to 2 were prepared. Subsequent dilutions were made at each concentration and 100 μl was dispensed per well. After 48 hours of incubation, 10 μl of MTT (5 mg / ml) was added thereto, followed by 4 hours of incubation, and washed with PBS (phosphate-buffered solution). 100 μl of DMSO was added to the washed plate, and the plate was left at room temperature for 20 minutes. The absorbance was measured at 570 nm, and the mortality was calculated using 100% of the non-treated control group.

sample IC ₅₀ (μg / ml) ACHN NCI-H23 HCT-15 PC-3 RGRE-1 > 100 > 100 > 100 > 100 BGRE-1 60.3 ± 1.26 ^* > 100 60.3 ± 2.65 ^* 90.8 ± 5.09 CGLE-1 > 100 > 100 > 100 > 100 BGLBE-1 68.7 ± 5.37 ^* 99.6 ± 4.96 80.4 ± 6.34 66.4 ± 2.90 ^* BGLBE-2 66.6 ± 3.65 ^* 99.8 ± 3.65 75.6 ± 4.38 ^* 63.3 ± 2.65 ^* Adriamycin 0.1 ± 0.05 ^* 0.1 ± 0.05 ^* 0.1 ± 0.04 ^* 0.3 ± 0.05 ^{* *} Significant difference compared to control group (p <0.05)

As a result, red ginseng extract (RGRE-1) and ginseng leaf extract (CGLE-1) did not show cytotoxicity against the four cancer cells used in the experiment. However, the black ginseng leaf extract (gut amplification method; BGLBE-1 and high temperature and high pressure method; BGLBE-2) processed the ginseng leaf showed the strongest cytotoxicity against the four types of cancer cells used in the experiment. In other words, renal cancer cells (ACHN) and colon cancer cells (HCT-15) showed almost the same cytotoxicity as that of black ginseng extract (BGRE-1), and to lung cancer cells (NCI-H23) and prostate cancer cells (PC-3). It showed strong cytotoxicity compared to black ginseng extract.

Experimental Example  2. Acute Toxicity Test

Acute toxicity test was performed using 6-week-old specific pathogen-free (SPF) SD rats. Two animals of each group were orally administered to the black trilobite extract of the present invention at a dose of 1 g / kg. After administration of the test substance, the mortality, clinical symptoms, and weight changes of the animals were observed, hematological and hematological examinations were performed.

As a result, there were no clinical symptoms or deaths in all animals treated with the test substance, and no toxic changes were observed in weight change, blood test, blood biochemical test and autopsy findings. As a result, the extract of the present invention did not show a change in toxicity even in rats up to 1 g / kg, respectively, the minimum lethal dose (LD ₅₀ ) was determined to be a safe substance of more than 1 g / kg.

One example of the formulation of the pharmaceutical composition comprising the extract of the present invention, but the present invention is not intended to limit it, but is intended to explain in detail.

Formulation example  One. Powder  Produce

BGLBE-1 20 mg

Lactose 100 mg

Talc 10 mg

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

Formulation example  2. Preparation of Tablets

BGLBE-1 10 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

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

Formulation example  3. Manufacture of capsule

BGLBE-1 10 mg

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

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

Formulation example  4. Preparation of Injectables

BGLBE-1 10 mg

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

_{Na 2 HPO 4 · 12H 2 O} 26 mg

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

Formulation example  5. Liquid  Produce

BGLBE-2 20 mg

10 g of isomerized sugar

5 g of mannitol

Purified water

According to the conventional method of preparing a liquid solution, each component is added and dissolved in purified water, lemon flavor is added to the mixture, and then the above ingredients are mixed, purified water is added to adjust the total amount to 100 ml, and then filled in a brown bottle. The solution is prepared by sterilization.

Formulation example  6. Manufacture of healthy food

BGLBE-2 1000 mg

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

0.2 μg of vitamin B12

Vitamin C 10 mg

10 μg biotin

Nicotinic Acid 1.7 mg

50 μg folic acid

Calcium Pantothenate 0.5mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixtures is mixed with a component suitable for a health food in a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method. The granules may be prepared and used for preparing a health food composition according to a conventional method.

Formulation example  7. Manufacture of health drinks

BGLBE-2 100 mg

15 g of vitamin C

100 g of vitamin E (powder)

Iron lactate 19.75 g

3.5 g of zinc oxide

Nicotinamide 3.5 g

0.2 g of vitamin A

0.25 g of vitamin B1

0.3 g of vitamin B2

Water quantification

After mixing the above components in accordance with the conventional healthy beverage manufacturing method, and 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 refrigerated Used to prepare the healthy beverage composition of the invention. Although the composition ratio is mixed with a component suitable for a favorite beverage in a preferred embodiment, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and usage.

As described above, the present invention relates to a composition containing the extract extracted from the black trifoliate prepared using a method of manufacturing augmentation width or high temperature and high pressure, the composition of the present invention shows a strong anticancer effect It can be usefully used as a composition and health functional food.

Claims (6)

A first step of washing 4 to 6 years old ginseng leaves three times with an ultrasonic cleaner for 15 minutes and then drying at a temperature of 40 to 60 ° C. for 20 to 28 hours; Putting the dried ginseng leaf ground in a steamer into a steamer for steaming at a temperature of 85 to 105 ° C. for 2 to 4 hours except for a preheating time; A third step of drying at a temperature of 50 to 70 ° C. for 9 to 15 hours; Pharmaceutical composition for the prevention and treatment of kidney cancer, colon cancer or prostate cancer containing water and methanol of black trilobite having 14% or less of water obtained by repeating the first to third steps as an active ingredient or a soluble extract of butanol as an active ingredient . A first step of washing 4 to 6 years old ginseng leaves three times with an ultrasonic cleaner for 3 minutes and then drying at a temperature of 40 to 60 ° C. for 20 to 28 hours; A second step of steaming the dried ginseng pulverized with a grinder for 2 to 6 hours at a temperature of 110 to 145 ° C under pressure; Kidney cancer, colon cancer or prostate cancer containing as an active ingredient water and methanol, or butanol soluble extract of black trilobite having a water content of 14% or less, obtained by the third step of drying at a temperature of 50 to 70 ° C. for 9 to 15 hours. Pharmaceutical compositions for the prevention and treatment of delete delete A first step of washing 4 to 6 years old ginseng leaves three times with an ultrasonic cleaner for 15 minutes and then drying at a temperature of 40 to 60 ° C. for 20 to 28 hours; Putting the dried ginseng leaf ground in a steamer into a steamer for steaming at a temperature of 85 to 105 ° C. for 2 to 4 hours except for a preheating time; A third step of drying at a temperature of 50 to 70 ° C. for 9 to 15 hours; The health functional food for improvement of kidney cancer, colon cancer or prostate cancer containing water and methanol of black trilobite having less than 14% water obtained by repeating the first to third steps 9 times or soluble extract of butanol as an active ingredient. A first step of washing 4 to 6 years old ginseng leaves three times with an ultrasonic cleaner for 3 minutes and then drying at a temperature of 40 to 60 ° C. for 20 to 28 hours; A second step of steaming the dried ginseng pulverized with a grinder for 2 to 6 hours at a temperature of 110 to 145 ° C under pressure; Kidney cancer, colon cancer or prostate cancer containing as an active ingredient water and methanol, or butanol soluble extract of black trilobite having a water content of 14% or less, obtained by the third step of drying at a temperature of 50 to 70 ° C. for 9 to 15 hours. Health functional food for improvement.