WO2007133054A1

WO2007133054A1 - A novel process for preparing black ginseng and the composition comprising the same

Info

Publication number: WO2007133054A1
Application number: PCT/KR2007/002418
Authority: WO
Inventors: Gyu Yong Song; Jee Hyun Lee; Yong Jin Park; Chang Seon Myung; Han Jin Oh; Hyun Joong Shin; Young Bae Seo
Original assignee: The Industry & Academic Cooperation In Chungnam National University (Iac)
Priority date: 2006-05-17
Filing date: 2007-05-17
Publication date: 2007-11-22
Also published as: KR100754253B1

Abstract

The present invention relates to a novel process for preparing black ginseng showing shorter time, easier to mass production than other conventional methods for preparing black ginseng and the inventive extract contains more abundant amount of ginseng saponin and showed potent anti-cancer effect.

Description

A NOVEL PROCESS FOR PREPARING BLACK GINSENG AND THE COMPOSITION COMPRISING THE SAME

Technical Field

[ 1 ] The present invention relates to a novel process for preparing black ginseng and the composition comprising the same.

[2]

Background Art

[3] It has been known that there are many genus of Panax genus plants belonged to

Araliaceae, for example, Panax ginseng distributed or cultivated in far-eastern Asia region, Panax quinquefolia in America and Canada, Panax notoginseng in China, Panax trifolia in eastern region of north America, Panax japonica in Japan, China and Nepal, Panax pseudoginseng in Nepal, Panax vietnamensis in Vietnam, Panax elegatior, Panax wangianus and Panax bipinratifidus etc

[4 J Hitherto, a ginseng has been widely known as a representative nutritive tonic agent.

Recently, various scientific studies on the chemical constituents and pharmacological effects of the ginseng have been reported so that the secret pharmacological effects are paid attention with modern scientific approaches. Until now, it has been known that the ginseng has various pharmacological effects such as prevention of aging, anti- arteriosclerosis, treatment of hyperlipidemia, treatment of hepatic insufficiency, improvement of liver function, protection of radiation injury, immune enhancement, improvement of cerebral function, anti-thrombotic, ahti-stress, anti-diabetic, antihypertensive, anti-tumor effects, etc

[5] It has been known that the main constituents of Panax genus plant are dammarane saponins. Ginsenosides Rb₁, Rb₂, Rc, Rd, Rgi and Re are main saponins of Panax ginseng. Their activities are different from each other in accordance with their chemical structures.

[6] There have been many attempts to process or modify Panax genus plants so as to increase their pharmacological potency, in particular, to modify the structure of gin- senoisides therein.

[7] For example, Korean Patent Publication No. 10-1996-017670 issued on May. 23,

1996, discloses a process for preparing a processed ginseng prepared by subjecting hot temperature treatment containing high contents of ginsenoside Rg ₃ and Rg₅ so as to obtaining processed ginseng having improved potency differing from original form of ginseng.

[8] In a while, the method for preparing modified or processed Chinese herbal medicine to reinforce its pharmacological activity, to attenuate its adverse response, to improve the stability or to change its medicinal effect, for example, nine times-steaming and nine times-sunbathing: %M%$ϊk), a method for preparing modified ginseng product which comprising the repeated steps of steaming crude drug with water and subsequently drying under sun nine times, which has been called as black ginseng , and already generally known and commonly used to Korean people.

[9] However, the conventionally used method for preparing black ginseng has several disadvantage such as long-term consuming production, limit to mass production etc

[ 10] Accordingly, the present inventors of the present invention have intensively studied to find more efficient method for preparing black ginseng than conventionally used method and finally, they have found that the new method for preparing black ginseng contains abundant pharmacologically active ginsenosides, for example, ginsenoside RbI, Rb2, Rc, Rd, Re, RgI, RkI, Rg5, Rg3, etc, which shows various favorable advantages such as short-term production, economic advantage etc and the extract of the black ginseng prepared from the method shows more potent anti-cancer activity than conventionally available ginseng products.

[H]

Disclosure of Invention Technical Problem

[ 12] The present invention provides a novel process for preparing black ginseng comprising the step consisting of; drying ginseng material at the temperature ranging from 20 to 60⁰C, for the period ranging from 12 to 36 hours at the 1^st step; steaming the fresh ginseng at the temperature ranging from 60 to 120⁰C for the period ranging from 3 to 9 hours, at the 2^nd step; drying at the temperature ranging from 40 to 80⁰C for the period ranging from 6 to 18 hours at the 3^rd step; steaming again at the temperature ranging from 85 to 135°C for the period ranging from 3 to 9 hours at the 4^th step; and drying again in the period ranging from 8 to 16 hours at the 5^th step.

[ 13] The present invention also provides a pharmaceutical composition comprising an extract of black ginseng prepared from the above-described process for treating and preventing cancer diseases.

[ 14] The present invention also provides a method of treating or preventing cancer diseases of human and mammals comprising administering an effective amount of an extract of black ginseng prepared from the above-described method with a pharmaceutically acceptable carrier thereof.

[ 15] The present invention also provides a health food composition comprising the above- described black ginseng or the extract thereof prepared from the above-described process for improving and preventing cancer diseases

[16]

Technical Solution

[17] Accordingly, it is an object of the present invention to provide a novel process for preparing black ginseng comprising the step consisting of; drying ginseng material at the temperature ranging from 20 to 60⁰C, for the period ranging from 12 to 36 hours at the 1^st step; steaming the fresh ginseng at the temperature ranging from 60 to 120⁰C for the period ranging from 3 to 9 hours, at the 2^nd step; drying at the temperature ranging from 40 to 80⁰C for the period ranging from 6 to 18 hours at the 3 ^rd step; steaming again at the temperature ranging from 85 to 135°C for the period ranging from 3 to 9 hours at the 4* step; and drying again in the period ranging from 8 to 16 hours at the 5^th step.

[18]

[19] Hereinafter, the present invention is described in detail.

[20] The inventive black ginseng can be prepared by following procedures.

[21 ] For example, washed fresh ginseng material ranging from 3 to 7 years old, preferably, five years old ginseng material is dried at the temperature ranging from 20 to 60⁰C, preferably, 35 to 55°C, for the period ranging from 12 to 36 hours, preferably, 20 to 28 hours at the 1^st step; subject to steaming at the temperature ranging from 60 to 120⁰C, preferably, 85 to 105 ⁰C, for the period ranging from 3 to 9 hours, preferably, 5 to 7 hours excluding preheating time at the 2^nd step; dried again at the temperature ranging from 40 to 80⁰C, preferably, 50 to 70⁰C, for the period ranging from 6 to 18 hours, preferably, 9 to 15 hours at the 3^rd step; subject to steaming again at the temperature ranging from 85 to 135°C, preferably, 95 to 125°C, for the period ranging from 3 to 9 hours, preferably, 5 to 7 hours at the 4* step; and dried for the period ranging from 8 to 16 hours, preferably, 10 to 14 hours at the 5* step to obtain inventive black ginseng of the present invention.

[22] The method of preparing black ginseng of the present invention could provide shorter time, easier to mass production than other existing methods for preparing black ginseng.

[23] In particular, the above-described process for preparing black ginseng could provide shorter time and easier to mass production than other existing methods for preparing black ginseng, i.e., nine times-steaming and nine times-sunbathing: tlMilM) disclosed in Korea Patent Registration Nos. 10-0529475 and 10-0543862, with producing the more abundant amount of ginseng saponins.

[24] The black ginseng product or the extract thereof of the present invention may be dried by the method well-known in the art, for example, dry in the shadow, lyo- philization etc The dried ginseng product may be cut into fine particles or powder, preferably, the particle having a particle size ranging from about 50 μm to 200 μxn with pulverizer and the powder can be formulated into pill, capsule, tablet and so on by adding pharmaceutically acceptable carriers or adjuvant well known in the art thereto.

[25]

[26] Additionally, the inventive extract of black ginseng of the present invention may be prepared by following procedures in detail.

[27] For example, the black ginseng product prepared by the above-described method is crushed to the powder and about 3 to 7-fold, preferably, 4 to 6-fold volume of water based on the volume of the powder is added thereto. The solution is left alone for the period ranging from 3 to 7 hours at room temperature and then about 1 to 3-fold volume of lower alcohol, preferably, methanol, based the volume of solution is added thereto to be subjected to reflux extraction for the period ranging from 1 hours to 6 hours, preferably, 3 to 4 hours, repeatedly. The extracted mixture is cooled to room temperature, filtered and the filtrates are subjected to evaporation with removing organic solvent to obtain the inventive extract of black ginseng.

[28] Accordingly, it is an object of the present invention to provide a process for preparing an extract of black ginseng comprising the step consisting of; adding about 3 to 7-fold volume of water to the powdered black ginseng product prepared in the above-described step to be left alone for the period ranging from 3 to 7 hours at room temperature at the 1^st step; adding about 1 to 3-fold volume of lower alcohol thereto to perform reflux extraction for the period ranging from 1 hours to 6 hours at the 2^nd step; and cooling, filtering the filtrate to obtain its filtrates and removing organic solvent to obtain the inventive extract of black ginseng.

[29]

[30] The inventive process of the present invention can be applied to not only ginseng root but also the other parts of ginseng, for example, ginseng leaf which has been wasted or used as an animal feed, a cosmetic as a form of perfume or food. Therefore the inventive process of the present invention can recycle ginseng leaf with a form of medicine other than an animal feed, a cosmetic or food in order to obtain more potent efficacy.

[31] The term "black ginseng" or "the extract thereof disclosed herein has characteristic in containing novel active-ingredients which has not being existed in fresh ginseng, white ginseng or red ginseng and more abundant amount of active ingredients than those in conventionally available ginseng product.

[32] The above-described extract of black ginseng showed stronger cell toxicity in cancer cell line and stronger anti-cancer effect than the extract of conventionally available ginseng product, for example, red ginseng.

[33] Accordingly, it is an object of the present invention to provide a pharmaceutical composition comprising an extract of black ginseng prepared from the above-described process for treating and preventing cancer diseases.

[34] The inventive composition for treating and preventing cancer diseases may comprise the above-described extract as 0.1 ~ 50 % by weight based on the total weight of the composition.

[35] It is an the other object of the present invention to provide a method of treating or preventing cancer diseases of human and mammals comprising administering an effective amount of an extract of black ginseng prepared from the above-described method with a pharmaceutically acceptable carrier thereof.

[36]

[37] Additionally, the present invention also provide a use of the composition comprising an extract of blade ginseng prepared from the above-described method for the manufacture of a medicament for cancer diseases in a mammal, together with a pharmaceutically acceptable carrier thereof.

[38] The term cancer disease disclosed herein comprises various cancer diseases such as breast cancer, hepatic cancer, lung cancer, arsenic cellular lung cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, cephalic or cervical cancer, skin or en- dophthalmic melanoma, hysterocarcinoma, ovarian cancer, rectal cancer, stomach cancer, perianal cancer, colonic cancer, endometrioma, cervical carcinoma, vaginal carcinoma, vulvul carcinoma, Hodgkin's disease, esophageal cancer, enteric cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, smooth tissue sarcoma, urethral cancer, penile cancer, prostatic cancer, chronic or acute leukemia, lymphccytoma, cystic cancer, nephritic or hydrouretic cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal medulla tumor, brain stem neuroglioma and hypophyseal adenomatosis, preferably, breast cancer or hepatic cancer.

[39] The composition according to the present invention can be provided as a pharmaceutical composition containing pharmaceutically axeptable carriers, adjuvants or diluents, e.g., lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starches, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil. The formulations may additionally include fillers, anti-agglutinating agents, lubricating agents, wetting agents, flavoring agents, emulsifϊers, preservatives and the like. The compositions of the invention may be formulated so as to provide quick, sustained or delayed release of the active ingredient after their administration to a patient by employing any of the procedures well known in the art.

[40] For example, the compositions of the present invention can be dissolved in oils, propylene glycol or other solvents that are commonly used to produce an injection. Suitable examples of the carriers include physiological saline, polyethylene glycol, ethanol, vegetable oils, isopropyl myristate, etc, but are not limited to them. For topical administration, the extract of the present invention can be formulated in the form of ointments and creams.

[41 ] Pharmaceutical formulations containing present composition may be prepared in any form, such as oral dosage form (powder, tablet, capsule, soft capsule, aqueous medicine, syrup, elixirs pill, powder, sachet, granule), or topical preparation (cream, ointment, lotion, gel, balm, patch, paste, spray solution, aerosol and the like), or injectable preparation (solution, suspension, emulsion).

[42] The composition of the present invention in pharmaceutical dosage forms may be used in the form of their pharmaceutically axeptable salts, and also may be used alone or in appropriate association, as well as in combination with other pharmaceutically active compounds.

[43] The desirable dose of the inventive extract or composition varies depending on the condition and the weight of the subject, severity, drug form, route and period of administration, and may be chosen by those skilled in the art. However, in order to obtain desirable effects, it is generally recommended to administer at the amount ranging 0.1 to 1000mg/kg, preferably, 1 to 100 mg/kg by weight/day of the inventive extract of the present invention. The dose may be administered in single or divided into several times per day. In terms of composition, the amount of inventive extract should be present between 0.01 to 50% by weight, preferably 0.5 to 40% by weight based on the total weight of the composition.

[44] The pharmaceutical composition of present invention can be administered to a subject animal such as mammals (rat, mouse, domestic animals or human) via various routes. All modes of administration are contemplated, for example, administration can be made orally, rectally or by intravenous, intramuscular, subcutaneous, intracutaneous, intrathecal, epidural or intra-cerebroventricular injection.

[45] Also, it is an object of the present invention to provide a health food composition comprising the above-described blade ginseng or the extract thereof prepared from the above-described process for improving and preventing cancer diseases.

[46] The health food of the present invention comprises the above-described black ginseng or the extract thereof as 0.01 to 80 %, preferably, 1 to 50 % by weight based on the total weight of the composition.

[47] The health food of the present invention can be contained in health food, health beverage etc, and may be used as powder, granule, tablet, chewing tablet, capsule, beverage etc

[48] The health food of the present invention comprises the above-described black ginseng or the extract thereof as 0.01 to 80 %, preferably, 1 to 50 % by weight based on the total weight of the composition.

[49] The food additive of the present invention can be contained in health food, health beverage etc, and may be used as powder, granule, tablet, chewing tablet, capsule, beverage etc

[50] Also, the present invention provide a health food beverage for the prevention and improvement of cancer diseases by adding 0.01 to 80 % the above-described extract by weight, 0.001 to 5 % amino acids by weight, 0.001 to 2 % vitamins by weight, 0.001 to 20 % sugars by weight, 0.001 to 10 % organic adds by weight and proper amount of sweetener and flavors.

[51] To develop for health food, examples of addable food comprising the above- described extract of the present invention can be added to various food, beverage, gum, vitamin complex, health improving food and the like, and can be used as power, granule, tablet, chewing tablet, capsule or beverage etc

[52] Also, the extract of the present invention will be able to prevent and alleviate cancer disease by way of adding to child and infant food, such as modified milk powder, modified milk powder for growth period, modified food for growth period.

[53] The above-described composition therein can be added to food, additive or beverage, wherein, the amount of above described extract in food or beverage may generally range from about 0.1 to 80w/w %, preferably, 1 to 50 w/w % of total weight of food for the health food composition and 1 to 30 g, preferably, 3 to 10 g on the ratio of 100m£ of the health beverage composition.

[54] Providing that the health beverage composition of present invention contains the above-described extract as an essential component in the indicated ratio, there is no particular limitation on the other liquid component, wherein the other component can be various deodorant or natural carbohydrate etc such as conventional beverage. Examples of aforementioned natural carbohydrate are monosaccharide such as glucose, fructose etc; disacεharide such as maltose, sucrose etc; conventional sugar such as dextrin, cyclodextrin; and sugar alcohol such as xylitol, and erythritol etc As the other deodorant than aforementioned ones, natural deodorant such as taumatin, stevia extract such as levaudioside A, glycyrrhizin et al., and synthetic deodorant such as saccharin, aspartam et al., may be useful favorably. The amount of above described natural carbohydrate is generally ranges from about 1 to 20 g, preferably 5 to 12 g in the ratio of 100 mi of present beverage composition.

[55] The other components than aforementioned composition are various nutrients, a vitamin, a mineral or an electrolyte, synthetic flavoring agent, a coloring agent and improving agent in case of cheese chocolate et al., pectic acid and the salt thereof, alginic acid and the salt thereof, organic acid, protective colloidal adhesive, pH controlling agent, stabilizer, a preservative, glycerin, alcohol, carbonizing agent used in carbonate beverage et al. The other component than aforementioned ones may be fruit juice for preparing natural fruit juice, fruit juice beverage and vegetable beverage, wherein the component can be used independently or in combination. The ratio of the components is not so important but is generally range from about 0 to 20 w/w % per 100 w/w % present composition. Examples of addable food comprising aforementioned extract therein are various food, beverage, gum, vitamin complex, health improving food and the like.

[56] The inventive composition may additionally comprise one or more than one of organic acid, such as citric add, fumaric acid, adipic acid, lactic acid, malic acid; phosphate, such as phosphate, sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphate; natural antioxidants, such as polyphenol, catechin, α- tocopherol, rosemary extract, vitamin C, green tea extract, licorice root extract, chitosan, tannic acid, phytic acid etc

[57]

[58] It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions, use and preparations of the present invention without departing from the spirit or scope of the invention.

[59]

Advantageous Effects

[60] A novel process for preparing black ginseng of the present invention showed shorter time, easier to mass production than other conventional methods for preparing black ginseng and the inventive extract contains more abundant amount of ginseng saponin and showed potent anti-cancer effect.

[61 ]

Brief Description of the Drawings

[62] The above and other objects, features and other advantages of the present invention will more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which;

[63]

[64] Fig. 1 depicts the steaming apparatus for preparing the black ginseng of the present invention.

[65]

Best Mode for Carrying Out the Invention

[66] The present invention is more specifically explained by the following examples.

However, it should be understood that the present invention is not limited to these examples in any manner.

[67]

Mode for the Invention

[68] Comparison Example 1. Preparation of an extract of conventional red ginseng

[69] 2Og of red ginseng was cut into small pieces, mixed with 100ml of water and left alone for 5 hours at room temperature, lfof methanol was added thereto to be subjected to reflux-extraction for 3 hours 3 times. The mixture was cooled to room temperature, subject to filtration with filter paper and the filtrate was evaporated with removing methanol by using rotary evaporator to obtain 8.1g of an extract of red ginseng.

[70] 8.1 g of an extract of red ginseng was suspended in 200ml of water and mixed with

200ml of ether to remove non-polar substance. Remaining water layer was mixed with 200ml of saturated butanol to extract the saponin fraction in the ginseng and the butanol was evaporated by rotary evaporator to obtain 1.3g of a butanol-soluble extract of red-ginseng comprising abundant amount of ginseng saponin, which was further used as a test sample in following experiments.

[71]

[72] Example 1. Preparation of black ginseng

[73] Five years old fresh ginseng procured from Korea was washed with a screw washer for 10 min 3 times.

[74] The washed fresh ginseng was subjected to drying process with preliminary steaming at 40⁰C for 24 hours to prevent the rupture of fresh ginseng. The dried ginseng was poured into pottery as depicted in Fig. 1 and subjected to steaming at 95°C for 6 hours excluding preheating period which is a necessary time to provide the leakage of steam from the pottery, generally, 30 mins. The internal temperature of the drying apparatus equipped with hot-wire was maintained to 60°C with rotating its fan to be subjected to drying process for 12 hours and then the 2 ^nd steaming process was performed at 115°C for 6 hours. Finally, the steamed ginseng was subjected to the 3^rd drying process for 12 hours to obtain the inventive bla± ginseng of the present invention.

[75]

[76] Example 2. Preparation of the extract of black ginseng

[77] 2Og of black ginseng prepared from the method disclosed in Example 1 was cut into small pieces, mixed with 100 ml of water and left alone for 5 hours at room temperature. 1 liter of methanol was poured thereto to perform reflux extraction for 3 hours 3 times. The solution was cooled to room temperature, filtered with filter paper and the filtrate was evaporated with evaporator to remove methanol to obtain 7.4g of an inventive extract of black ginseng. The extract of black ginseng was suspended in 200ml of water and 200ml of ether was added thereto to remove its non-polar substance. 200ml of saturated butanol was added to remaining water layer to extract the saponin fraction in ginseng and the extract was performed to evaporation with evaporator to remove butanol solvent to obtain 1.4g of an inventive butanol soluble extract of black ginseng comprising abundant amount of ginseng saponin, which was used as a test sample in following experiments.

[78]

[79] Experimental Example 1. Component analysis of black ginseng saponin

[80] In order to analyze the saponin component of black ginseng prepared in Example 2,

20mg of butanol soluble extract of black ginseng was dissolved in ImI of methanol, filtered with filter with the pore size of 0.45 μm to analyze the component of the inventive extract of black ginseng by HPLC in accordance with the condition as shown in Table 1.

[81] At the result, it has been confirmed that Ig of the extract of black ginseng contains 2.3mg of Rbl, 2.1mg of Rb2, 1.3mg of Rc, 1.4mg of Rd, 0.5mg of Re, 1.2mg of Rgl, 3.7mg of RkI, 4.7mg of Rg5, 5.4mg of Rg3(S) and 4.5mg of Rg3(R), which showed that the inventive black ginseng contains more abundant mount of ginseng saponin than the conventionally available red ginseng prepared in Comparison Example 1.

[82] [83] Table 1 [Table 1 ] [Table ]

[84] [85] Experimental Example 2. Determination of anti-cancer activity [86] 2-1. Cell toxicity test [87] In order to examine the cell toxicity of the inventive extract prepared in Example 2, MTT assay was performed according to the procedure disclosed in the literature (Rubinstein, L.V. 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, pp.2418, 1989).

[88] MCF- I (human breast carcinoma cells line, ATCC, USA), HT- 1080 (Human fibrosarcoma cell line for Caucasian human, ATCC, USA) and Hepa 1C1C7 (Murine Hepatoma cell line, ATCC, USA) were cultured at RPMI 1640 medium supplemented with 10% FBS at 37°C in 5% CO₂ incubator. After culturing the cells, the cells were isolated from the culture container using by trypsin-EDTA buffer, and 1 xlO⁴ cells was distributed in 96 well plates to incubate for 24 hours. The serially diluted test sample prepared in Comparison Example 1 and Example 1 was added to the plates in the concentration of 100 μi per each well. The plates was incubated for 48 hours and 10 βt of MTT (5mg/ml) was added thereto, incubated for 4 hours, and washed with PBS (phosphate-buffered solution). 100 μl of DMSO was added thereto and the plates were left alone for 20 min at room temperature to determine their absorbance at 570nm by ELISA. The death rate was calculated by setting the vale of negative control group treated with only solvent to 100% and the result was shown in Table 2.

[89] At the result, it has been confirmed that the inventive extract of black ginseng showed more potent cell toxicity than the extract of red ginseng prepared in Comparison Example 1.

[90] [91] Table 2 [Table 2] [Table ]

[92] [93] 2-2. Determination of anti-cancer activity in mouse [94] Four weeks-old male BDF-mice weighing from 20 to 23g (Jung Ang laboratory animal) was used and bred in the place maintaining the temperature to 24°C with freely accessible to water and antibiotic-free animal feed. Each group consists of five mice. Subculturing cell line (Lewis lung carcinoma, 1x10 ⁶ cells/mouse) in 10% FBS medium containing RPMI 1640 was transplanted to the left armpit of the mice. In accordance with the procedure disclosed in the literature (Kim Y. et al., Deoxypodo- phyllotoxin; the cytotoxic and antiangiogenic component from Pulsatilla koreana, Planta Med., 68(3). pp.271-274, 2003). From 24 hours after the transplantation, the test samples prepared in Comparison Example 1 and Example 1 had been peritoneally administrated for 2 weeks with checking the change of body weight. The tumor size of each group was determined when the tumor volume of control group is reached to about 2 an³. The size of tumor was calculated according to following Math Figure 1 and the anti-cancer activity of the test samples was determined by using following Math Figure 2.

[95]

[96] MathFigure 1

[Math.l]

T. V. (tumor volume, mm³) = L (mm) X S² (mm²)/2

L: Long length

S: Short length

T. V.: Tumor Volume , mm³

[97]

[98] MathFigure 2

[Math.2]

The anti-cancer activity (%) = Mean T. V. of control group - Mean T. V. of treated group / Mean T. V. of control group X 100

L: Long length

S: Short length

T. V.: Tumor Volume, mm³

[99] [100] As shown in Table 3, taxol, a potent anti-cancer agent used as a control group showed potent anti-cancer activity (38.9%) and the anti-cancer activity of inventive extract of black ginseng was 35.4% whereas that of the extract of red ginseng prepared in Comparison Example 1 was 23.3%. Accordingly, it has been confirmed that the inventive extract of black ginseng showed more potent anti-cancer activity than the extract of red ginseng.

[101] [102] Table 3 [Table 3] [Table ]

[103]

[104] Experimental Example 3. Acute toxicity test

[105] The acute toxicity test was performed by administrating the inventive extract to 6-weeks aged SPF Sprague-Dawley rats.

[106] lg/kg of inventive extract was orally administrated to each group consisting of 2 rats. After administrating the extract, all the clinical changes i.e., mortality, clinical signs, body weight changes was observed and blood test such as hematological test and hematological biochemistry test was performed. The abnormal changes of abdominal organ and thoracic organ were observed after autopsy.

[ 107] There did not show any changes in mortality, clinical signs, body weight changes and gross findings in any group or either gender. Furthermore, there did not show any toxicity in test group treated with lg/kg of inventive extract.

[108] Accordingly, it has been confirmed that the inventive extract prepared in the present invention was potent and safe substance showing LD₅₀ (more than lg/kg) in oral administration.

[109]

[HO]

[11 1] Hereinafter, the formulating methods and kinds of exάpients will be described, but the present invention is not limited to them. The representative preparation examples were described as follows.

[1 12]

[1 13]

[1 14] Preparation of powder

[1 15] Black ginseng of Example 1 20mg

[116] Lactose lOOmg

[117] Talc lOmg

[118] Powder preparation was prepared by mixing above components and filling sealed package.

[119]

[120] Preparation of tablet

[121 ] Black ginseng of Example 1 lOmg

[122] Corn Starch lOOmg

[123] Lactose lOOmg

[124] Magnesium Stearate 2mg

[ 125] Tablet preparation was prepared by mixing above components and entabletting. [126]

[127] Preparation of capsule

[ 128] Extract of black ginseng of Example 2 lOmg

[129] Corn Starch 3mg

[130] Lactose 14.8mg

[131] Magnesium Stearate 0.2mg

[132] Capsule preparation was prepared by mixing above components and filling gelatin capsule by conventional gelatin preparation method. [133]

[ 134] Preparation of injection

[ 135] Extract of black ginseng of Example 2 lOmg

[136] Mannitol 180mg

[ 137] Distilled water for injection 2974mg

[138] Na₂HPO₄, 12H₂O 26mg

[139] Injection preparation was prepared by dissolving the components in 2m# ample and sterilizing by conventional injection preparation method. [140]

[141] Preparation of health care food

[ 142] Extract of black ginseng of Example 2 lOOOmg

[ 143] Vitamin mixture optimum amount

[ 144] Vitamin A acetate 70mg

[145] Vitamin E 1.Omg

[146] Vitamin B, 0.13mg

[147] Vitamin B₂ 0.15mg

[148] Vitamin B6 0.5mg

[149] Vitamin B 12 0.2mg

[150] Vitamin C lOmg

[151] Biotin lOmg

[152] Amide nicotinic acid 1.7mg

[153] Folic aad 50mg

[ 154] Calcium pantothenic acid 0.5mg

[155] Mineral mixture optimum amount

[156] Ferrous sulfate 1.75mg

[ 157] Znc oxide O.82mg

[ 158] Magnesium carbonate 25.3mg [159] Monopotassium phosphate 15mg

[ 160] Dkalάum phosphate 55mg

[161] Potassium citrate 90mg

[162] Calcium carbonate lOOmg

[163] Magnesium chloride 24.8mg

[164] The above mentioned vitamin and mineral mixture may be varied in may ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention.

[165]

[166] Preparation of health beverage

[ 167] Extract of black ginseng of example 2 lOOOmg

[168] Citric add lOOOmg

[ 169] Oligosaxharide 10Og

[ 170] Apricot concentration 2g

[171] Taurine Ig

[172] Concentrated plum solution 2g

[ 173] Distilled water 900m£

[174] Health beverage preparation was prepared by dissolving active component, mixing, stirred at 85 ⁰C for 1 hour, filtered and then filling all the components in 2000mβ ample and sterilizing by conventional health beverage preparation method.

[ 175] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

[176]

Industrial Applicability

[177] As described in the above, a novel process for preparing black ginseng of the present invention showed shorter time, easier to mass production than other conventional methods for preparing black ginseng and the inventive extract contains more abundant amount of ginseng saponin and showed potent anti-cancer effect. Therefore, inventive extract of black ginseng can be used as a medicament or functional food for treatment or invention of cancer diseases.

Claims

[1 ] A process for preparing black ginseng comprising the step consisting of; drying ginseng material at the temperature ranging from 20 to 60⁰C, for the period ranging from 12 to 36 hours at the 1^st step; steaming the fresh ginseng at the temperature ranging from 60 to 120⁰C for the period ranging from 3 to 9 hours, at the 2^nd step; drying at the temperature ranging from 40 to 80⁰C for the period ranging from 6 to 18 hours at the 3^rd step; steaming again at the temperature ranging from 85 to 135°C for the period ranging from 3 to 9 hours at the 4* step; and drying again in the period ranging from 8 to 16 hours at the 5^th step.

[2] The process according to claim 1, wherein said ginseng material is 3 to 7 years old ginseng.

[3] The process according to claim 1 , wherein said drying process at the 1 ^st step is performed at the temperature ranges from 35 to 55°C for the period ranging from 20 to 28 hours.

[4] The process according to claim 1, wherein said steaming process at the 2 ^nd step is performed at the temperature ranges from 85 to 105 ⁰C for the period ranging from 5 to 7 hours excluding preheating time.

[5] The process according to claim 1, wherein said drying process at the 3 ^rf step is performed at the temperature ranges from 50 to 70⁰C for the period ranging from 9 to 15 hours.

[6] The process according to claim 1, wherein said steaming process at the 4 * step is performed at the temperature ranges from 95 to 125°C for the period ranging from 5 to 7 hours .

[7] The process according to claim 1, wherein said drying process at the 5 ^th step is performed for the period ranging from 10 to 14 hours.

[8] A process for preparing an extract of black ginseng comprising the step consisting of; adding about 3 to 7-fold volume of water to the powdered black ginseng product prepared by the process as set forth in claim 1 to be left alone for the period ranging from 3 to 7 hours at room temperature at the 1^st step; adding about 1 to 3-fold volume of lower alcohol thereto to perform reflux extraction for the period ranging from 1 hours to 6 hours at the 2 ^nd step; and cooling, filtering the filtrate to obtain its filtrates and removing organic solvent to obtain the inventive extract of black ginseng.

[9] A pharmaceutical composition comprising the extract of black ginseng prepared by the process as set forth in claim 8 for preventing and treating cancer disease.

[10] The pharmaceutical composition according to claim 9, wherein said cancer disease is selected from lung cancer, arsenic cellular lung cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, cephalic or cervical cancer, skin or endophthalmic melanoma, hysterocarcinoma, ovarian cancer, rectal cancer, stomach cancer, perianal cancer, colonic cancer, breast cancer, endometrioma, cervical carcinoma, vaginal carcinoma, vulvul carcinoma, Hodgkin's disease, esophageal cancer, enteric cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, smooth tissue sarcoma, urethral cancer, penile cancer, prostatic cancer, chronic or acute leukemia, lymphocytoma, cystic cancer, nephritic or hydrouretic cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal medulla tumor, brain stem neuroglioma and hypophyseal adenomatosis.

[1 1] A method of treating or preventing cancer diseases of human and mammals comprising administering an effective amount of an extract of black ginseng prepared from the method as set forth in claim 8 with a pharmaceutically acceptable carrier thereof.

[12] A use of the composition comprising an extract of black ginseng prepared from the method as set forth in claim 8 for the manufacture of a medicament for cancer diseases in a mammal, together with a pharmaceutically acceptable carrier thereof.

[13] A health care food comprising the black ginseng as set forth in claim 1 or the extract of black ginseng of claim 8 for prevention and alleviation of cancer diseases.

[14] The health care food according to claim 13 wherein said health food is provided as powder, granule, tablet, capsule or beverage type.