WO2008147141A1

WO2008147141A1 - A method for preparing novel black-red ginseng and the extract therefrom and the composition comprising the extract isolated therefrom

Info

Publication number: WO2008147141A1
Application number: PCT/KR2008/003057
Authority: WO
Inventors: Yong Jin Park; Gyu Yong Song; Sang Kyum Kim; Eui Keom Kim; Woo Yong Song; Chung Yeol Lee
Original assignee: Yong Jin Park; Gyu Yong Song; Sang Kyum Kim; Eui Keom Kim; Woo Yong Song; Chung Yeol Lee
Priority date: 2007-05-31
Filing date: 2008-05-30
Publication date: 2008-12-04
Also published as: KR20080106077A; KR101082181B1

Abstract

The present invention relates the present invention relates to a novel process for preparing black-red ginseng and the extract isolated therefrom. The inventive extracts of the present invention showed more favorable advantages than the conventionally known red ginseng and black ginseng, for example, (1) the extract of black-red ginseng contains Rg2, Rg3, RhI, Rh2 and Rkl+Rg5, a trace component of red ginseng, as well as RbI, Rb2, Rc, Rd, Re and RgI, a trace component of black ginseng; (2) the inventive black red ginseng showed better final product having finely textured structure comparing with black ginseng with porous structure; (3) the extract of black-red ginseng contains no carcinogens such as benzopyrene, pyrane etc comparing with that of black ginseng; (4) the inventive black red ginseng showed more potent anti-cancer activity than that of red ginseng; (5) the inventive black-red ginseng showed more potent anti-oxidative activity than that of red ginseng, therefore, it can be used as a pharmaceutical composition and health functional food for treating and preventing cancer and hyper- oxidation-involved disease.

Description

A METHOD FOR PREPARING NOVEL BLACK-RED GINSENG

AND THE EXTRACT THEREFROM AND THE COMPOSITION

COMPRISING THE EXTRACT ISOLATED THEREFROM

Technical Field

[1] The present invention relates to a method for preparing novel black-red ginseng and the extract therefrom to obtain increased amount of ginsenosides with reduced carcinogenand the composition comprising the extract isolated therefrom.

[2]

Background Art

[3] Since long years ago, the preparation of red ginseng has been conventionally used as a representative processing method of fresh ginseng to (1) reduce the side effect of fresh ginseng, such as toxicity, (2) improve pharmacological potency or alter the medicinal indications of fresh ginseng, (3) improve the storage stability of fresh ginseng, (4) improve taste, flavor and color etc. Generally, red ginseng has been prepared by steaming a washed fresh ginseng at the temperature ranging from 94°C~100°C to produce novel and specific active ingredients or increase the level of effective ingredients such as ginsenoside Rg3, RgI, RgI, Rb2, Rc, Rd, and Re etc and subjecting the steamed ginseng to conventional drying process, which results in more potent and diverse pharmacological activity compared with fresh ginseng or un-husked ginseng (Okamura N et al., Biol. Pharm. Bull, 17(2). p270, 1994).

[4]

[5] There have been various attempts to develop new processing methods for preparing modified ginseng having more potent active ingredient with new active ingredients than conventionally used fresh ginseng and red ginseng till now. In an efforts to the developments, the method for preparing modified or processed ginseng 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 time- sunbathing C^Lϋ^HI) ",has been reported and the method for preparing modified black-colored ginseng product which comprises the repeated steps of steaming crude drug with water and subsequently drying under sun nine times, which has been called as "black ginseng" has been known to comprise diverseactive ingredients such as ginsenoside Rg3, RbI, Rb2, Rc, Rd, Re, Rf, Rg, RhI, and compound K etc (Korea Patent Registration No. 10-0543862). tbwever, it has lots of unfavorable problems: for example, unexpected carbonization during the process resulting in increased carcinogens such as benzopyrene etc;the significant loss of major ingredients in red ginseng or white ginseng, such as RgI, RbI, Rb2, Re, Rc etc, which results in difficulties in ingredient standardization; and the production of poor quality ginseng product caused by the denature of ginseng structure resulting in porous structure.

[6]

[7] IHbwever, there have been no disclosures or suggestions on the novel processing method for ginseng and the extract thereof by modifying nine times-steaming and nine time-sunbathing (fLM,^~ΑM$) processing method and the composition comprising the extract prepared thereby showing potent anti-cancer activity and potent anti-oxidant activity, in any of the above cited literatures, the disclosures of which are incorporated herein by reference.

[8]

[9] Accordingly, the present inventors of the present invention have intensively studied to find a novel processing method for preparing an inventive extract showing more favorable advantages than the conventionally known red ginseng and black ginseng and they have finally completed the present invention.

[10]

Disclosure of Invention Technical Problem

[11] The object of the present invention is to provide a novel method for preparing black- red ginseng and the extract isolated therefrom comprising abundant major ginsenoside saponins of red ginseng, such as RgI, RbI, Rb2, Rc, Rd, Re and the major ginsenoside saponins of black ginseng, such as Rg2, Rg3, RhI, Rh2, RkI, Rg5, which improved final ginseng product with no carcinogens such as benzopyrene, pyrane etc.

[12] The object of the present invention is to provide a composition comprising the extract of black-red ginseng prepared by the above^nentioned process as an active ingredient for preventing and treating cancer disease.

[13] The object of the present invention is to provide a composition comprising the extract of black-red ginseng prepared by the above^nentioned process as an active ingredient for preventing and treating hyper-oxidation involved diseases.

[14]

Technical Solution [15] Accordingly, it is an object of the present invention to provide a novel method for preparing black-red ginseng using by repeated drying and steaming process and the extract isolated therefrom comprising abundant major ginsenoside saponins of red ginseng, such as RgI, RbI, Rb2, Rc, Rd, Re etc and the major ginsenoside saponins of black ginseng, such as Rg2, Rg3, RhI, Rh2, RkI, Rg5, etc, which produce improved final ginseng product with no carcinogens such as benzopyrene.

[16] It is an another object of the present invention to provide a novel method for preparing black-red ginseng using by high temperature and high pressure and the extract isolated therefrom comprising abundant major ginsenoside saponins of red ginseng, such as RgI, RbI, Rb2, Rc, Rd, Re etc and the major ginsenoside saponins of black ginseng, such as Rg2, Rg3, RhI, Rh2, RkI, Rg5, etc, which produce improved final ginseng product with no carcinogens such as benzopyrene.

[17]

[18] Specifically, the present invention provides a method for preparing a black-red ginseng comprising the steps consisting of; washing ginseng material and subjecting to the 1^st drying process at 1^st step; subject to steaming at the temperature ranging from 60 to 12O⁰C, for the period ranging from 1 to 8 hours, excluding preheating time at 2 ^nd step; subjecting to the 2^nd drying process at 3^rd step; and repeating said cycle consisting of the 1^st drying step, steaming step and the 2 ^nd drying step for 2 to 7 times, to afford inventive black-red ginseng comprising less than 14% water content, which shall be designated as "hs-1" or "hs-3" hereinafter.

[19] The present invention also provides a method for preparing a black-red ginseng comprising the steps consisting of; washing ginseng material and subjecting to the 1 ^st drying process at 1^st step; subject to the 1^st steaming process at the temperature ranging from 60 to 12O⁰C, for the period ranging from 3 to 9 hours, excluding preheating time at 2^nd step; subjecting to the 2^nd drying process at 3^rd step; subjecting to the 2^nd steaming process at the temperature ranging from 95 to 15O⁰C, under the pressure ranging from 0.05 to 0.45 MPa for the period ranging from 30 to 150 mins;and subjecting to the 3 ^rd drying process to afford inventive black-red ginseng comprising less than 14% water content, which shall be designated as "hs-2" or "hs-4" hereinafter.

[20]

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

[22] The inventive black-red ginseng of present invention can be prepared by repeating the process of steaming and drying by following procedures.

[23] For example, the inventive black-red ginseng may be prepared by the method comprising the steps consisting of; washing ginseng material ranging from 3 to 7 years old, preferably, 6 years old with sonication; subjecting to the 1^st drying process at the temperature ranging from 20 to 6O⁰C, preferably 35 to 55⁰C, for the period ranging from 12 to 36 hours, preferably, 20 to 28 hours at 1 ^st step; subjecting to steaming process at the temperature ranging from 60 to 12O⁰C, preferably, 75 to 100⁰C, for the period ranging from 1 to 8 hours, preferably, 2 to 6 hours excluding preheating time at 2^nd step; subjecting to the 2^nd drying process at the temperature ranging from 40 to 8O⁰C, preferably, 50 to 7O⁰C, for the period ranging from 6 to 18 hours, preferably, 9 to 15 hours at 3^rd step; repeating said cycle consistingof the 1^st drying step, steaming step and the 2^nd drying step for 2 to 7 times, preferably, 3 to 6 times, more preferably, 5 times to afford inventive black-red ginseng comprising less than 14% water content, which shall be designated as "hs-1" or "hs-3" hereinafter.

[24] Also the inventive black-red ginseng of present invention can be prepared by following procedures using by high temperature and high pressure.

[25] For example, the inventive black-red ginseng may be prepared by the method comprising the steps consisting of; washing ginseng material ranging from 3 to 7 years old, preferably, 6 years old with sonication; subjecting to the 1^st drying process at the temperature ranging from 20 to 6O⁰C, preferably 35 to 55⁰C, for the period ranging from 12 to 36 hours, preferably, 20 to 28 hours at 1 ^st step; subjecting to the 1^st steaming process at the temperature ranging from 60 to 11O⁰C, preferably, 85 to 100⁰C, for the period ranging from 3 to 9 hours, preferably, 5 to 7 hours excluding preheating time at 2^nd step; subjecting to the 2^nd drying process at the temperature ranging from 40 to 8O⁰C, preferably 50 to 7O⁰C, for the period ranging from 6 to 18 hours, preferably 9 to 15 hours at 3^rd step; subjecting to the 2^nd steaming process under the pressure ranging from 0.05 to 0.45 MPa, preferably, from 0.10 to 0.20 MPa, at the temperature ranging from 95 to 155⁰C, preferably, 110 to 145⁰C, for the period ranging from 30 to 150 minutes, preferably from 60 to 120 minutes at 4 * step; and subjecting to the 3^rd drying process with maintaining the internal temperature ranging from 40 to 8O⁰C, preferably 50 to 7O⁰C, for the period ranging from 8 to 16 hours, preferably, 10 to 14 hours to afford inventive black-red ginseng comprising less than 14% water content, which shall be designated as "hs-2" or "hs-4" hereinafter.

[26]

[27] The black-red ginseng of the present invention prepared as above^nentioned may be dried by the method well-known in the art, for example, drying in the shadow, lyophilization etc. The dried ginseng product may be cut into fine particles or powder, preferably, particles having a particle size ranging from about 50 to 200 micro meter 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.

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

[29] For example, the inventive extracts of black-red ginseng, may be prepared by the method comprising the steps consisting of; crushing the black-red ginseng powders prepared by the above-described methods, i.e., "hs-1", "hs-2", "hs-3" or "hs-4" to the powder; adding about 3 to 7-fold, preferably 4 to 6-fold volume of water based on the weight of the powder to be left alone for the period ranging from 3 to 7 hours at room temperature; adding about 1 to 20-fold, preferably 5 to 15 -fold volume of water, lower alcohol such as methanol, ethanol etc, and the mixture thereof, preferably, ethanol based on the volume of the solution thereto to subject extraction such as reflux extraction method etc for the period ranging from 1 to 6 hours, preferably, 3 to 4 hours, repeatedly for 3 times; cooling the extract to room temperature to subject to filtration; and removing remaining organic solvent to obtain the inventive respective extracts of black-red ginsengs, i.e., "ch-2", "ch-3", "ch-4" or "ch-5".

[30] Through various experiments executed by the present inventors, it has been verified that the inventive extracts of the present invention showed more favorable advantages than the conventionally known red ginseng and black ginseng, for example, (1) the extract of black-red ginseng contains Rg2, Rg3, RhI, Rh2 and Rkl+Rg5, a trace component of red ginseng, as well as RbI, Rb2, Rc, Rd, Re and RgI, a trace component of black ginseng, verified by saponin component analysis experiment; (2) the inventive black red ginseng showed better final product having finely textured structure comparing with black ginseng with porous structure, verified by tissue analysis experiment; (3) the extract of black-red ginseng contains no carcinogens such as benzopyrene, pyrane etc comparing with that of black ginseng, verified by detection test of carcinogen substances; (4) the inventive black red ginseng showed more potent anti-cancer activity than that of red ginseng, verified by cytotoxicity test against various cancer cell lines; (5) the inventive black red ginseng showed more potent anti- oxidative activity than that of red ginseng, verified by oxyradical scavenging activity and so on.

[31] 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 or ginseng budding flower 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 or ginseng budding flower with a form of medicine other than animal feed, a cosmetic or food in order to obtain more potent efficacy.

[32] The black-red ginseng and the extract thereof prepared by the above-described methods have characteristic in abundant amount of active ingredients which does not exist or exist only in a minimal amount in the conventionally available black ginseng or red ginseng.

[33] The inventive extract of black-red ginseng prepared by the above-described method has characteristic in having strong cell toxicity against cancer cell.

[34] Also, the inventive extract of black-red ginseng prepared by the above-described method has characteristic in having potent anti-oxidative activity.

[35] Accordingly, the present invention provides a pharmaceutical composition comprising the extract of black-red ginseng prepared by the above-described methods as an active ingredient for preventing and treating cancer disease.

[36] The present invention provides a pharmaceutical composition comprising the extract of black-red ginseng prepared by the above-described methods as an active ingredient for preventing and treating hyper-oxidation involved disease.

[37] The inventive composition for treating and preventing cancer disease or hyper- oxidation involved disease may comprise the above-described extract as 0.1 ~ 50 % by weight based on the total weight of the composition.

[38] The term "ginseng" disclosed herein comprises Panax ginseng, Panax quinquefolia,

Panax notoginseng, Panax vietnamensis, Panax elegatior, Panax wangianus or Panax Bipinratifidus , more preferably, Panax ginseng or Panax quinquefolia.

[39] The term "ginseng" disclosed herein comprises ginseng root, ginseng leaf or ginseng budding flower, more preferably ginseng root.

[40] The term "cancer disease" disclosed herein comprises general cancer diseases, preferably, hepatic cancer, breast cancer, stomach cancer, colon cancer, lung cancer, arsenic cellular lung cancer, bone cancer, pancreatic cancer, skin cancer, cephalic or cervical cancer, skin or endophthalmic melanoma, hysterocarcinoma, ovarian cancer, colonic cancer, small intestinal cancer, rectal cancer, perianal cancer, endometrioma, cervical carcinoma, vaginal carcinoma, vulvul carcinoma, tbdgMn'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 hy- drouretic cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal medulla tumor, brain stem neuroglioma or hypophyseal adenomatosis, more preferably, hepatic cancer or breast cancer.

[41] The term "hyper-oxidation involved disease disclosed herein comprises obesity, aging, diabetes, atherosclerosis, hyperlipemia, nephritic disease, gout, hypertension, liver disease, lung disease, thyroidism, athyria, chronic fatigue syndrome, heart disease, paralysis or degenerative disease of nervous system, preferably, obesity, aging, diabetes, atherosclerosis, hyperlipemia, nephritic disease or gout.

[42] The pharmaceutical composition of the present invention comprising the extract of black-red ginseng prepared by the above-described methods may additionally comprise conventional carrier, adjuvants or diluents in accordance with using a method well known in the art.

[43] The pharmaceutical composition comprising the extract of black-red ginseng prepared by the above-described methods in pharmaceutical dosage forms may be used in the form of their pharmaceutically acceptable salts, and also may be used alone or in appropriate association, as well as in combination with other pharmaceutically active compounds.

[44] The pharmaceutical composition comprising the extract of black-red ginseng prepared by the above-described methodsmay 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).

[45] The composition comprising the extract of black-red ginseng prepared by the above- described methodscan be provided as a pharmaceutical composition containing pharmaceutically acceptable 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, microcrystalline 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, emulsifiers, 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.

[46] For example, the extract of black-red ginseng prepared by the above-described methods 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.

[47] The desirable dose of the extract of black-red ginseng prepared by the above- described methodsvary 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. Fbwever, in order to obtain desirable effects, it is generally recommended to administer at the amount ranging 0.2 to 200 mg/kg, preferably, 2 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.

[48] The extract of black-red ginseng prepared by the above-described methods 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, intra-cutaneous, intrathecal, epidural or intra-cerebroventricular injection.

[49] It is an object of the present invention to provide a use of an extract of black-red ginseng prepared by the above-described methodsfor the preparation of therapeutic medicament for the treatment and prevention of cancer disease in human or mammal.

[50] It is an object of the present invention to provide a use of an extract of black-red ginseng prepared by the above-described methodsfor the preparation of therapeutic medicament for the treatment and prevention of hyper-oxidation involved disease in human or mammal.

[51]

[52] It is an object of the present invention to provide a method of treating or preventing cancer disease in a mammal or human comprising administering to said mammal or human an effective amount of extract of black-red ginseng prepared by the above- described methods, together with a pharmaceutically acceptable carrier thereof.

[53]

[54] It is an object of the present invention to provide a method of treating or preventing hyper-oxidation involved disease in a mammal or human comprising administering to said mammal or human an effective amount of extract of black-red ginseng prepared by the above-described methods, together with a pharmaceutically acceptable carrier thereof. [55]

[56] The extract of black-red ginseng prepared by the above-described methodsmay be used as conventionally used ginseng, for example formulation for various pharmaceutical compositions, oriental medication, health functional food, food, tea, cosmetic compositions etc. as a more effective ingredient than conventional used ginseng.

[57]

[58] Also, it is an object of the present invention to provide a health functional food comprising the extract of black-red ginseng prepared by the above-described methods for improving and preventing cancer disease.

[59]

[60] It is an object of the present invention to provide a health functional food comprising the extract of black-red ginseng prepared by the above-described methods for improving and preventing hyper-oxidation involved disease.

[61]

[62] The health functional 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.

[63] Also, the present invention provide a health food beverage for the prevention and improvement of cancer disease and hyper-oxidation involved disease by adding 0.01 to 15% the extract of black-red ginseng prepared by the above-described methods by weight. The present invention also provides a health beverage composition by adding 0.02 to 5 g, preferably 3 to 10 g on the ratio of 100 ml.

[64] Providing that the health beverage composition comprising the extract of black-red ginseng prepared by the above-described methodsas an active ingredient in the indicated ratio, there is no particular limitation on the other liquid components, 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; disaccharide 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 ml of present beverage composition.

[65] 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.

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

Advantageous Effects

[67] The present invention relates to a novel process for preparing black-red ginseng and an inventive extract isolated therefrom, which shows more favorable advantages than the conventionally known red ginseng and black ginseng, i. e., (1) the extract of black- red ginseng contains Rg2, Rg3, RhI, Rh2 and Rkl+Rg5, a trace component of red ginseng, as well as RbI, Rb2, Rc, Rd, Re and RgI, a trace component of black ginseng; (2) the inventive black red ginseng showed better final product having finely textured structure comparing with black ginseng with porous structure; (3) the extract of black-red ginseng contains no carcinogens such as benzopyrene, pyrane etc comparing with that of black ginseng, verified; (4) the inventive black red ginseng showed more potent anti-cancer activity than that of red ginseng; (5) the inventive black red ginseng showed more potent anti-oxidative activity than that of red ginseng and so on.

[68]

Brief Description of the Drawings

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

[70]

[71] Fig. 1 depicts the section of black ginseng prepared by the known nine time-steaming and nine time-sunbathing method;

[72] Fig. 2 depicts the section of black-red ginseng prepared by the inventive method of the present invention;

[73] Fig. 3 depicts the scavenging activity of 'ch-1' against peroxynitrite;

[74] Fig. 4 depicts the scavenging activity of 'ch-2' against peroxynitrite;

[75] Fig. 5 depicts the scavenging activity of 'ch-3' against peroxynitrite;

[76] Fig. 6 depicts the scavenging activity of 'ch-1' against peroxy radicals;

[77] Fig. 7 depicts the scavenging activity of 'ch-2' against peroxy radicals;

[78] Fig. 8 depicts the scavenging activity of 'ch-3' gainst peroxy radicals.

[79]

Best Mode for Carrying Out the Invention

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

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

[81]

Mode for the Invention

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

[83] 32.75 g of six years-old red ginseng procured from Korean Company (Korea

Ginseng Corp., Korea) was pulverized to powder, mixed with IL of 80% ethanol to subject to reflux-extraction for 4 hours 3 times. The mixture was cooled to room temperature, subject to filtration with filter paper and the filtrate was evaporated to remove the solvent by using rotary evaporator to obtain 9 g of an extract of red ginseng.

[84] 9 g of an extract of red ginseng was suspended in 300 ml of water and mixed with

300 ml of ethyl ether to remove non-polar substance. Remaining water layer was mixed with 300 ml of saturated butanol to extract the saponin fraction existing in the ginseng and butanol solvent was evaporated by rotary evaporator to obtain 1.25 g of an extract of red-ginseng (designated as "ch-1 hereinafter) comprising abundant amount of ginseng saponin, which was further used as a test sample in following experiments.

[85]

[86] Example 1. Preparation of black-red ginseng

[87] 1-1. Method using by repeating the process of steaming and drying (Panax ginseng )

[88] 2 kg of six years-oldfresh Panax ginseng (Samheongsa, Korea) cultivated in Kumsan

City of Korea was washed with sonicator for 3 mins 3 times, and subjected to the 1 ^st preliminary drying process at 4O⁰C for 24 hours. The ginseng was further dried at room temperature for 24 hours, subjected to the 1^st preliminary steaming process at 95⁰C for 6 hours and to the 2^nd drying process in drying apparatus equipped with hot-wire maintaining the internal temperature to 6O⁰C with rotating fan for 12 hours excluding preliminary time. The above steaming and drying process was repeated for 5 times to obtain 455 g of black-red ginseng of the present invention (designated as "hs-1" hereinafter) containing 13.8% moisture content, which was further used as a test sample in following experiments.

[89]

[90] 1-2. Method using by high temperature and high pressure ( Panax ginseng )

[91] 2 kg of six years oldfresh Panax ginseng (Samheongsa, Korea) cultivated in Kumsan of Korea was washed with sonicator for 3 mins 3 times and subjected to the 1 ^st drying process at 4O⁰C for 24 hours. The ginseng was further dried at room temperature for 24 hours, and subjected to the 1^st steaming process at 95⁰C for 6 hours excluding preheating period necessary to take the leakage of steam from the pottery, generally, 30 mins. The ginseng was subjected to the 2^nd drying process in a drying apparatus equipped with hot-wire maintaining internal temperature to 6O⁰C with rotating fan for 12 hours and then the 2^nd steaming process was performed at 118⁰C for 90 minutes under pressure (0.12MPa). Finally, the ginseng was subjected to the 3^rd drying process in a drying apparatus equipped with hot-wire maintaining internal temperature to 6O⁰C with rotating fan for 12 hours to obtain 473 g of the inventive black-red ginseng of the present invention (designated as "hs-2" hereinafter) containing 13.5% moisture content, which was further used as a test sample in following experiments.

[92]

[93] 1-3. Method using by repeating the process of steaming and drying (Panax quinquefolia )

[94] 1 Kg of five years-oldfresh Panax quinquefolia (DaedukBio Co., Korea) purchased in Kumsan City was prepared by the method of Example 1-1 to obtain the black-red ginseng 234 g of the present invention (designated as "hs-3" hereinafter) containing 13.7% moisture content, which was further used as a test sample in following experiments.

[95] [96] 1-4. Method using by high temperature and high pressure ( Panax quinquefolia

1

[97] 1 Kg of five years-oldfresh Panax quinquefolia (DaedukBio Co., Korea) purchased in Kumsan City was prepared by the method of Example 1-2 to obtain the black-red ginseng 231 g of the present invention (designated as "hs-4" hereinafter) containing 13.9% moisture content, which was further used as a test sample in following experiments.

[98]

[99] Example 2. Preparation of the extract of black-red ginseng

[100] 2- 1. Preparation of the extract of black-red ginseng ( Panax ginseng ) prepared by repeating the process of steaming and drying

[101] 23 g of "hs-1" prepared from the method disclosed in Example 1-1 was mixed with 100 ml of water and left alone for 5 hours at room temperature. 1 liter of 80% ethanol 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 ethanol to obtain 6 g of the crude extract of black-red ginseng. The crude extract of black-red ginseng was suspended in 500 ml of water and 500 ml of ether was added thereto to remove the non-polar substance. 500 ml of saturated butanol was added to the remaining water layer to extract the saponin fraction in ginseng 4 times. The extract was performed to evaporation with evaporator to remove butanol to obtain 1.56 g of an extract of black-red ginseng (designated as "ch-2" hereinafter) comprising abundant amount of ginseng saponin, which was further used as a test sample in following experiments.

[102]

[103] 2-2. Preparation of the extract of black-red ginseng ( Panax ginseng ) prepared by high temperature and high pressure

[104] 23 g of "hs-2" prepared from the method disclosed in Example 1-2 was mixed with 100 ml of water and left alone for 5 hours at room temperature. 1 liter of 80% ethanol 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 ethanol to obtain 5.5 g of the crude extract of black-red ginseng. The crude extract of black-red ginseng was suspended in 500 ml of water and 500 ml of ether was added thereto to remove the non-polar substance. 500ml of saturated butanol was added to remaining water layer to extract the saponin fraction in ginseng for 4 times. The extract was performed to evaporation with evaporator to remove butanol to obtain 2.12 g of an extract of black-red ginseng (designated as "ch- 3" hereinafter) comprising abundant amount of ginseng saponin, which was further used as a test sample in following experiments.

[105]

[106] 2-3. Preparation of the extract of black-red ginseng ( Panax quinquefolia ) prepared by repeating the process of steaming and drying

[107] 50 g of "hs-3" prepared from the method disclosed in Example 1-3 was mixed with 100 ml of water and left alone for 5 hours at room temperature. 1 liter of 80% ethanol 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 ethanol to obtain 16.7 g of the crude extract of black-red ginseng. The crude extract of black red ginseng was suspended in 500 ml of water and 500 ml of ether was added thereto to remove the non-polar substance. 500 ml of saturated butanol was added to the remaining water layer to extract the saponin fraction in ginseng 4 times. The extract was performed to evaporation with evaporator to remove butanol to obtain 4.56 g of an extract of black-red ginseng (designated as "ch-4" hereinafter) comprising abundant amount of ginseng saponin, which was further used as a test sample in following experiments.

[108]

[109] 2-4. Preparation of the extract of black-red ginseng ( Panax quinquefolia ) prepared by high temperature and high pressure

[110] 50 g of "hs-4" prepared from the method disclosed in Example 1-4 was mixed with 100 ml of water and left alone for 5 hours at room temperature. 1 liter of 80% ethanol 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 ethanol to obtain 17 g of the crude extract of black-red ginseng. The crude extract of black red ginseng was suspended in 500 ml of water and 500 ml of ether was added thereto to remove the non-polar substance. 500 ml of saturated butanol was added to the remaining water layer to extract the saponin fraction in ginseng 4 times. The extract was performed to evaporation with evaporator to remove butanol to obtain 4.98 g of an extract of black-red ginseng (designated as "ch-5" hereinafter) comprising abundant amount of ginseng saponin, which was further used as a test sample in following experiments.

[I l l]

[112] Experimental Example 1. Component analysis of black- red ginseng saponin [113] In order to analyze the saponin component of the extract "ch-1" prepared in Comparative Example 1, "ch-2" in Example 2-1, and "ch-3" in Example 2-2, 20 mg of each samples was dissolved in ImI of methanol, filtered with the filter having the pore size of 0.45 micro meter to analyze the component of the extracts by HPLC in accordance with the condition as shown in Table 1. The component of the extract was analyzed by referring to the data in the reference (Han sung tae et al., Archives of Pharmacol. Research, 49(6). pp490-494, 2005) and the result is shown in Table 2.

[114] [115] Table 1 [Table 1] [Table ]

[116] [117] Table 2

[Table 2] [Table ]

[118] [119] At the results, as shown in Table 2, the extract of red ginseng are found to contain RbI, Rb2, Rc, Rd, Re, and RgI, as a main component and Rg2, Rg3, RhI, Rh2, Rkl+Rg5 as a trace component. The extract of black ginseng are found to contain Rg2, Rg3, RhI, Rkl+Rg5 as a main component, which means that the main ginsenosides in the extract of red ginseng has been mostly disappeared during the extraction process.

[120] In a while, the black-red ginseng prepared by the inventive method of the present invention are found to contain not only abundant amount of Rg2, Rg3, RhI, Rh2, and Rkl+Rg5, a trace component in red ginseng, but also abundant amount of RbI, Rb2, Rc, Rd, Re, and RgI, a trace component in black ginseng. Accordingly, it has been confirmed that the black-red ginseng of the present invention is the higher quality product containing abundant amount of the saponins present in red ginseng and black ginseng as a trace component, of which data may be useful information for the stan- dardization of black-red ginseng in commercialization. [121] [122] Experimental Example 2. Tissue analysis of black- red ginseng

[123] In order to analyze the tissue of inventive black-red ginseng, the section of black red ginseng was compared with that of black ginseng prepared by already known process of nine time-steaming and nine time-sunbathing method ( See Fig. 1 and Fig. 2).

[124] At the result, the tissue of black ginseng was decomposed and denatured to a vesicular structure during the repeated process of steaming and drying step. In a contrary, "hs-1" of Example 1-1 and "hs-2" of Example 1-2 was found to have a fine structure, not a vesicular structure.

[125]

[126] Experimental Example 3. Detection of Carcinogen

[127] The detection test of the carcinogen in "hs-1" of Example 1-1, "hs-2" of Example

1-2, "hs-3" of Example 1-3 and "hs-4" of Example 1-4 was performed at Drug Misuse Institute in Kongju University ( See Table 3).

[128]

[129] Table 3

[Table 3] [Table ]

[130] [131] At the result, as shown in Table 3, the black-red ginseng prepared by inventive processing method was found to have no carcinogens consisting of 14 types including benzopyrene.

[132] [133] Experimental Example 4. Cell Toxicity test [134] In order to determine the cell toxicity of "ch-2", "ch-3", "ch-4" and "ch-5", 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).

[135] MCF-I (human breast carcinoma cells line, ATCC, USA) and SK- Hep- 1 (Human hepatic adenocarcinoma cell line, ATCC, USA) were cultured at RPMI 1640 medium supplemented with 10% FBS at 37⁰C in 5% CO₂ incubator for 48 hours. After culturing the cells, the cells were isolated from the culture container using by trypsin- EDTA buffer, and IxIO⁴ cells were distributed to 96 well plates to incubate for 24 hours. The serially diluted test sample prepared in "ch-1" of Comparison Example 1, "ch-2" of Example 2-1 and "ch-3" of Example 2-2, were added to the plates in the concentration of 100 micro liter per each well to incubate for 48 hours. 10 micro liter of MTT (5 mg/ml) was added thereto, incubated for 4 hours, and washed with PBS (phosphate-buffered solution). 100 micro liter of DMSO was added thereto and the plates were left alone for 20 min at room temperature to determine their absorbance at 570 nm by ELISA. The death rate of the cells was calculated by setting the vale of negative control group treated with only solvent to 100% according to following Math Figure 1 and the result is shown in Table 4.

[136] [137] MathFigure 1 [Math.l]

Cell Toxicity (%) = [1-(TZC)] x 100%

T : OD values of the samples

C : OD value of the negative control group

[138] [139] Table 4 [Table 4] [Table ]

[140]

[141] At the result, as shown in Table 4, "ch-1" of Comparative Example 1 showed cell toxicity (IC₅₀) at 75.51 μg/ml against MCF-7, while "ch-2" of Example 2-1 and "ch-3" of Example 2-2 showed 20.29 μg/ml and 21.54 μg/ml respectively, which showed ap- proximately 3-fold stronger cell toxicity comparing with that of "ch-1". Moreover, "ch- 2" and "ch-3" showed approximately 3-fold stronger cell toxicity against SK- Hep- 1 comparing with that of "ch-1". On the other hand, "ch-4" of Example 2-3 and "ch-5" of Example 2-4 showed cell toxicity at 30.53 and 25.71 μg/ml respectively, which showed approximately 2.5-fold stronger cell toxicity comparing with that of "ch-1". Moreover, "ch-4" of Example 2-3 or "ch-5" of Example 2-4 showed approximately 2.5-fold stronger cell toxicity comparing with that of "ch-1". Accordingly, the inventive extract of black-red ginseng has been confirmed to show approximately 2.5 to 3-fold stronger anti-cancer activity than the conventional red ginseng extract.

[142]

[143] Experimental Example 5. Total Oxyradical Scavenging Capacity (TOSC) assay

[144] To determine the anti-oxidative activity of the extracts, i.e., "ch-2" prepared in Example 2-1 and "ch-3" in Example 2-2, the radical scavenging assay using TOSC method was performed according to the procedure disclosed in the literature as follows (Winston et al., Free Radic. Biol Med., Feb, 24(31 pp480-493. 1998; Regoli and Winston, Toxicol. Appl. Pharmacol., Apr 15, 156(2). pp96-105, 1999).

[145] 20 mM of 2,2'-azobisamidinopropane (ABAP, Sigma, USA) was spontaneously decomposed at 35⁰C to induce a peroxy radical, and 70 μM of SIN-I (Sigma, USA) was spontaneously decomposed to induce a peroxynitrite. Both of induced radicals were reacted with 0.2 mM α-keto-γ^nethiolbutyric acid (KMBA) to produce ethylene gas to be used in detecting the radical scavenging activity of test samples.

[146] "ch-1" of Comparative Example 1, "ch-2" of Example 2-1 and "ch-3" of Example

2-2, were serially diluted to 0.0125, 0.025, 0.05 and 0.1 mg/ml respectively with water to the extent that the final volume of the solution has reached to 1 ml. The reaction was performed in 10 ml volume of sealed container sealed with rubber septum, and 200 micro liter of internal gas in the head-space was collected to subject gas chromatography (GC) according to following condition using by head-space technique method ( See Table 5). For the quantitative determination of the amount of ethylene gas of vials, 150 micro liter of internal gas was injected into the injector using by gas- tight injector. The value of TOSC was calculated according to following Math Figure 2 ( See Table 6 and Table 7).

[147]

[148] Table 5 [Table 5] [Table ]

[149] [150] MathFigure 2 [Math.2]

TOSC = 100 - (JSA/JCA x 100)

JSA = integrated area from the curve of the sample reaction

JCA = iiiteajated area from the curve of the control reaction

[151] [152] The sample with no oxyradical scavenging capacity will obtain JSA/JCA = 1, TOSC = 0. In other words, JSA → 0, the value of TOSC is close to 100. The value of TOSC is compared with that of the control group, of which value is regarded as being not interfered by the sensitivity of the instrument, reagent, and other reaction conditions.

[153] [154] Table 6

[Table 6]

[Table ]

Scavenging activity of Peroxynitrite

[155] [156] Table 7 [Table 7] [Table ] Scavenging activity of Peroxyradical

[157] [158] At the result, as shown in Table 6 and Table 7, the TOSC values against peroxy radicals of "ch-2" in Example 2-1 and "ch-3" in Example 2-2 showed approximately 817 to 826 TOSC/mg, which are approximately 2.5-fold higher compared with that of "ch-1" prepared in Comparative Example 1 (approximately 336 TOSC/mg). Moreover, the TOSC values against peroxynitrite of "ch-2" in Example 2-1 and "ch-3" in Example 2-2 were 731 and 782 TOSC/mg respectively, which was approximately 2.1 -fold higher compared with that of "ch-1" in Comparative Example 1 (approximately 377 TOSC/mg). Accordingly, the inventive extract of black-red ginseng has been confirmed to show potent anti-oxidative activity against peroxy radicals and peroxynitrite, in particular, approximately 2 to 3-fold higher anti-oxidative activity compared with that of conventional red ginseng.

[159]

[160] Experimental Example 6. Acute toxicity test

[161] The acute toxicity test was performed by administrating inventive extract of black- red ginseng of Example 2 to 6- weeks aged SPF Sprague-Dawley rats. 1 g/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.

[162] 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 1 g/kg of inventive extract.

[163] Accordingly, it has been confirmed that the inventive extract prepared in the present invention was potent and safe substance showing LD₅₀ (more than 1 g/kg) in oral administration.

[164]

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

[166]

[167] Preparation of powder

[168] "hs-1" of Example 1-1 20 mg

[169] Lactose 100 mg

[170] Talc 10 mg [171] Powder preparation was prepared by mixing above components and filling sealed package. [172]

[173] Preparation of tablet

[174] "hs-2"of Example 1-2 10 mg

[175] Corn Starch 100 mg

[176] Lactose 100 mg

[177] Magnesium Stearate 2 mg

[178] Tablet preparation was prepared by mixing above components and entabletting. [179]

[180] Preparation of capsule

[181] "ch-2"of Example 2-1 10 mg

[182] Corn Starch 3 mg

[183] Lactose 14.8 mg

[184] Magnesium Stearate 0.2 mg

[185] Capsule preparation was prepared by mixing above components and filling gelatin capsule by conventional gelatin preparation method. [186]

[187] Preparation of injection

[188] "ch-3"of Example 2-2 10 mg

[189] Mannitol 180 mg

[190] Distilled water for injection 2974 mg

[191] Na₂HPO₄, 12H₂O 26 mg

[192] Injection preparation was prepared by dissolving the components in 2 mi ample and sterilizing by conventional injection preparation method. [193]

[194] Preparation of liquid

[195] "hs-4" ofExample 1-4 20 g

[196] Isomerized Sugar 1O g

[197] Mannitol 5 g

[198] Distilled water optimum amount

[199] Liquid preparation was prepared by dissolving active component, filling all the components and sterilizing by conventional liquid preparation method. [200]

[201] Preparation of health functional food [202] "ch-4"of Example 2-3 1000 mg

[203] Vitamin mixture optimum amount

[204] Vitamin A acetate 70mg

[205] Vitamin E LOmg

[206] Vitamin B ₁ 0.13mg

[207] Vitamin B ₂ 0.15mg

[208] Vitamin B6 0.5mg

[209] Vitamin B 12 0.2mg

[210] Vitamin C lOmg

[211] Biotin lOmg

[212] Amide nicotinic acid 1.7mg

[213] Folic acid 50mg

[214] Calcium pantothenic acid 0.5mg

[215] Mineral mixture optimum amount

[216] Ferrous sulfate 1.75mg

[217] Zinc oxide 0.82mg

[218] Magnesium carbonate 25.3mg

[219] Monopotassium phosphate 15mg

[220] Dicalcium phosphate 55mg

[221 ] Potassium citrate 90mg

[222] Calcium carbonate lOOrrg

[223] Magnesium chloride 24.8mg

[224] The above -mentioned vitamin and mineral mixture 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. [225]

[226] Preparation of health beverage

[227] "ch-5"of Example 2-4 100 mg

[228] Vitamin C 15 g

[229] Vitamin E (powder) 100 g

[230] Ferrous Lactate 19.75 g

[231] Zinc oxide 3.5 g

[232] Nicotinic acid amide 3.5 g

[233] Vitamin A 0.2 g

[234] Vitamin B j 0.25 g [235] Vitamin B ₂0.3 g

[236] Distilled water optimum amount

[237] Health beverage preparation was prepared by dissolving active component, mixing, stirred at 85°C for 1 hour, filtered and then filling all the components in 2 L ample and sterilizing by conventional health beverage preparation method.

[238] 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.

[239]

Industrial Applicability

[240] As shown in the above, the present invention relates to a novel process for preparing black-red ginseng and the extract isolated therefrom, the inventive extracts of the present invention showed more favorable advantages than the conventionally known red ginseng and black ginseng, for example, (1) the extract of black-red ginseng contains Rg2, Rg3, RhI, Rh2 and Rkl+Rg5, a trace component of red ginseng, as well as RbI, Rb2, Rc, Rd, Re and RgI, a trace component of black ginseng; (2) the inventive black red ginseng showed better final product having finely textured structure comparing with black ginseng with porous structure; (3) the extract of black- red ginseng contains no carcinogens such as benzopyrene, pyrane etc comparing with that of black ginseng; (4) the inventive black red ginseng showed more potent anticancer activity than that of red ginseng; (5) the inventive black-red ginseng showed more potent anti-oxidative activity than that of red ginseng, therefore, it can be used as a pharmaceutical composition and health functional food for treating and preventing cancer and hyper-oxidation-involved disease.

Claims

[1] A method for preparing a black-red ginseng comprising the steps consisting of; washing ginseng material and subjecting to the 1 ^st drying process at 1^st step; subject to steaming at the temperature ranging from 60 to 12O⁰C, for the period ranging from 1 to 8 hours, excluding preheating time at 2 ^nd step; subjecting to the 2^nd drying process at 3^rd step;and repeating said cycle consisting of the 1^st drying step, steaming step and the 2 ^nd drying step for 2 to 7 times, to afford inventive black-red ginseng comprising less than 14% water content.

[2] A method for preparing a black-red ginseng comprising the steps consisting of; washing ginseng material and subjecting to the 1 ^st drying process at 1^st step; subject to the 1^st steaming process at the temperature ranging from 60 to 12O⁰C, for the period ranging from 3 to 9 hours, excluding preheating time at 2 ^nd step; subjecting to the 2^nd drying process at 3^rd step;subjecting to the 2^nd steaming process at the temperature ranging from 95 to 155⁰C, under the pressure ranging from 0.05 to 0.45 MPa for the period ranging from 30 to 150 mins; and subjecting to the 3^rd drying process to afford inventive black-red ginseng comprising less than 14% water content.

[3] The method according to claim 1 or claim 2, wherein said ginseng is selected from Panax ginseng, Panax quinquefolia, Panax noto ginseng, Panax viet- namensis, Panax elegatior, Panax wangianus or Panax Bipinratifidus.

[4] The method according to claim 1 or claim 2, wherein said ginseng comprises ginseng root, ginseng leaf or ginseng budding flower.

[5] A method for preparing an extract of black-red ginseng comprising the steps consisting of; crushing the black-red ginseng powders prepared by the method as set forth in claim 1 or 2 to the powder; adding about 3 to 7-fold volume of water based on the volume of the powder to be left alone for the period ranging from 3 to 7 hours at room temperature; adding about 1 to 20-fold volume of water, lower alcohol based on the volume of the solution thereto to subject extraction for the period ranging from 1 to 6 hours, for 3 times; cooling the extract to room temperature to subject to filtration; and removing remaining organic solvent to obtain the inventive extract of black-red ginseng.

[6] A pharmaceutical composition comprising the extract of black red ginseng prepared by the methods as set forth in claim 5 as an active ingredient for preventing and treating cancer disease.

[7] The pharmaceutical composition according to claim 6, wherein said cancer disease is selected from hepatic cancer, breast cancer, stomach cancer, colon cancer, lung cancer, arsenic cellular lung cancer, bone cancer, pancreatic cancer, sMn cancer, cephalic or cervical cancer, sMn or endophthalmic melanoma, hyste- rocarcinoma, ovarian cancer, colonic cancer, small intestinal cancer, rectal cancer, perianal cancer, endometrioma, cervical carcinoma, vaginal carcinoma, vulvul carcinoma, IHbdgkin 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 or hypophyseal adenomatosis.

[8] A pharmaceutical composition comprising the extract of black red ginseng prepared by the method as set forth in claim 5 as an active ingredient for preventing and treating hyper-oxidation involved disease.

[9] The pharmaceutical composition according to claim 8 wherein said hyper- oxidation involved disease is selected from obesity, aging, diabetes, atherosclerosis, hyperlipemia, nephritic disease, gout, hypertension, liver disease, lung disease, thyroidism, athyria, chronic fatigue syndrome, heart disease, paralysis or degenerative disease of nervous system.

[10] A method of treating or preventing cancer diseases of human and mammals comprising administering an effective amount of the extract of black red ginseng prepared by the method as set forth in claim 5 with a pharmaceutically acceptable carrier thereof.

[11] A use of the composition comprising the extract of black-red ginseng prepared by the method as set forth in claim 5 for the manufacture of a medicament for in a mammal, together with a pharmaceutically acceptable carrier thereof.

[12] A method of treating or preventing hyper-oxidation involved disease of human and mammals comprising administering an effective amount of the extract of black-red ginseng prepared by the method as set forth in claim 5 with a pharmaceutically acceptable carrier thereof.

[13] A use of the composition comprising the extract of black-red ginseng prepared by the method as set forth in claim 5 for the manufacture of a medicament for hyper-oxidation involved diseases in a mammal, together with a pharma- ceutically acceptable carrier thereof.

[14] A health functional food comprising the black-red ginseng prepared by the process as set forth in claim 1 or claim 2 or the extract of black-red ginseng prepared by the process as set forth in claim 5 as an active ingredient for preventing and improving cancer disease.

[15] A health functional food comprising the black-red ginseng prepared by the process as set forth in claim 1 or claim 2 or the extract of black-red ginseng prepared by the process as set forth in claim 5 as an active ingredient for preventing and improving hyper-oxidation involved diseases.

[16] The health functional food according to claim 14 or claim 15, the food is provided as a form of powder, granule, tablet, capsule or beverage type.