WO2008075866A1

WO2008075866A1 - A composition comprising the processed extract of panax quinquefolium l. for the prevention and treatment of cancer

Info

Publication number: WO2008075866A1
Application number: PCT/KR2007/006597
Authority: WO
Inventors: Yong Jin Park; Gyu Yong Song
Original assignee: Yong Jin Park; Gyu Yong Song
Priority date: 2006-12-18
Filing date: 2007-12-17
Publication date: 2008-06-26

Abstract

The present invention relates to a preparation method of a processed of American black ginseng, the extract thereof, and the composition comprising the extract of Panax quinquefolium L. for the prevention and treatment of cancer.

Description

A COMPOSITION COMPRISING THE PROCESSED EXTRACT OF PANAX QUINQUEFOLIUM L. FOR THE PREVENTION AND TREATMENT OF CANCER

Technical Field

[1] The present invention relates to a preparation method of a processed American black ginseng, the extract thereof, and the composition comprising the extract of Panax quin- quefolium L. for the prevention and treatment of cancer.

[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]

[5] 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, anti-stress, anti-diabetic, antihypertensive, anti-tumor effects, etc.

[6]

[7] It has been known that the main constituents of Panax genus plant are darrmarane saponins. GinsenosidesRb , Rb , Rc, Rd, Rg and Re are main saponins of Panax ginseng. Their activities are different from each other in accordance with their chemical structures.

[8]

[9] 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- senosides therein.

[10] 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 time-sunbathing CfLJUΛBH) " a method for preparing modified 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 " and already generally known and commonly used to Korean people.

[11] The studies on black red ginseng, for example, Korean Patent No. 10-0754253 entitled to " novel method for preparing black ginseng and the composition comprising the same " discloses more efficient and mass-productive method for preparing black ginseng than those disclosed in already known preparation methods of black ginseng and so on, have been tried to reinforce the pharmacological potency of conventional ginseng product.

[12] However, there have been no disclosures or suggestions on the novel processing method for American ginseng and the extract thereof by modifying nine times- steaming and nine time-sunbathing

processing method and the composition comprising the extract prepared thereby showing potent anti-cancer activity in the above-described literatures or publication till now.

[13] Most of studies on the processing method of ginseng have been focused on Korean ginseng, however, present inventors have been tried to develop the new processing method for American ginseng (Panax quinquefolium L) having various commercial advantages such as cheaper cost and more abundant saponin content etc than Korean ginseng.

[14] There has been not reported or disclosed about a method for preparing American black ginseng showing anticancer activity and the composition comprising the same in any of above cited literatures, the disclosures of which are incorporated herein by reference.

[15]

[16] Accordingly, the present inventors of the present invention have intensively studied to find a processing method for preparing inventive American ginseng and the extract thereof and finally, they have found that the new processing method for preparing inventive extract of American black ginseng showing potent anti-cancer activity and abundant saponin content. [17]

[18] These and other objects of the present invention will become apparent from the detailed disclosure of the present invention provided hereinafter. [19]

Disclosure of Invention

Technical Problem [20] The present invention provides a method for preparing a process for preparing inventive American black ginseng and the extract thereof showing potent treating and preventing activity of cancer disease. [21] The present invention provides a pharmaceutical composition comprising a extract of

American black ginseng prepared from above described method as an active ingredient in an effective amount to treat and prevent cancer disease by anticancer activity. [22] The present invention also provides a use of above extract for the preparation of medicament to treat and prevent cancer disease by anticancer activity in mammal or human. [23]

Technical Solution [24] Accordingly, it is an object of the present invention to provide a process for preparing inventive American black ginseng modifying nine times-steaming and nine time-sunbathing (flWkfl^M) method, which comprises the step consisting of; washing

American ginseng, drying, steaming, heating and repeating said steps nine times. [25] [26] It is an another object of the present invention to provide a process for preparing the inventive American black ginseng using high-temperature and high pressure method, which comprises the step consisting of; washing the above-described American ginseng, drying, steaming, heating and repeating said steps twice under high temperature and high internal pressure. [27] It is the other object of the present invention to provide a pharmaceutical composition comprising the extract of American black ginseng prepared from the above-described method showing potent anti-cancer activity. [28] It is the other object of the present invention to provide a health care food composition comprising the extract of American black ginseng prepared from the above-described method showing potent anti-cancer activity. [29] The term " extract " disclosed herein comprises the extract which can be soluble in the polar solvent selected from water, methanol, ethanol, butanol or the mixture thereof, preferably, butanol, more preferably, the extract which shall be explained in detail hereinafter.

[30] The term " American ginseng " disclosed herein comprises all the part of ginseng, for example, root, leaves, flower bud, stem, etc, preferably, root.

[31] Hereinafter, the inventive American black ginseng in the present invention is described in detail.

[32] The present invention provides a process for preparing inventive American black ginseng modifying nine times-steaming and nine time-sunbathing

method, comprising the step consisting of; washing American ginseng ranging from about 3 to 7 years old, preferably, from about 4 to 6 years old, drying at the temperature ranging from 30 to 7O⁰C, preferably, 40 to 6O⁰C, for the period ranging from 12 to 36 hours preferably, 20 to 28 hours, at the 1 ^s step; steaming the ginseng at the temperature ranging from 60 to 12O⁰C preferably, 85 to 105⁰C, for the period ranging from 1 to 5 hours, preferably, 2 to 4 hours, excepting pre-heating time at the 2° step; drying 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 the 3^r step; and repeating said steaming step and drying step nine times to afford inventive American black ginseng comprising less than 14% water content, which shall be designated as " BPQ-I " hereinafter.

[33]

[34] The present invention also provides a process for preparing inventive American black ginseng using high-temperature and high pressure method, which comprises the step consisting of; washing American ginseng ranging from about 3 to 7 years old, preferably, from about 4 to 6 years old, drying at the temperature ranging from 30 to 7O⁰C, preferably, 40 to 6O⁰C, for the period ranging from 12 to 36 hours preferably, 20 to 28 hours, at the I^s step; steaming the ginseng at the temperature ranging from 95 to 155⁰C, preferably 110 to 145⁰C, for the period ranging from 1 to 8 hours, preferably, from 2 to 6 hours excepting pre-heating time, under high pressured condition at the 2 " step; and drying at the temperature ranging from 40 to 8O⁰C, preferably, from 50 to 7O⁰C for the period ranging from 6 to 18 hours, preferably, from 9 to 15 hours at the 3 ^r step to afford inventive American black ginseng comprising less than 14% water content, which shall be designated as " BPQ-2 " hereinafter.

[35]

[36] Hereinafter, the inventive extract of American black ginseng in the present invention is described in detail.

[37]

[38] In a preferred embodiment, present invention also provide a process for preparing the

I^s inventive extract of American black ginseng comprising the step consisting of; pulverizing said BPQ-I American black ginseng and adding about 3 to 7-fold, preferably, about 4 to 6-fold volume of water to the black ginseng to be left alone for the period ranging from 3 to 7 hours at room temperature at the 1 ^s step; adding about 1 to 3-fold volume of water, lower alcohol such as methanol, butanol etc or the mixture thereof, preferably, water, methanol, ethanol, or the mixture thereof, 60-80% ethanol, added thereto to perform reflux extraction for the period ranging from 1 hours to 6 hours, preferably, about 3 hours at the 2° step; cooling, filtering the filtrate to obtain its filtrates and removing organic solvent to obtain the inventive extract of American black ginseng, which shall be designated as " BPQE-I " hereinafter.

[39] In a preferred embodiment, the present invention provide a process for preparing the

2° inventive extract of American black ginseng comprising the step consisting of; suspending said BPQE-I extract of black ginseng in distilled water and extracting said extract with non-polar organic solvent such as diethyl ether etc to remove non-polar substance in BPQE-I extract at the I^s step; adding water saturated butanol to remaining water layer at the 2° step; and extracting said water layer with butanol and removing butanol solvent to afford ginseng saponin- abundant butanol-soluble extract of the present invention, which shall be designated as " BPQBE-I " hereinafter.

[40] In a preferred embodiment, the present invention provide a process for preparing the

3^r inventive extract of American black ginseng comprising the step consisting of; pulverizing said BPQ-2 black ginseng and adding about 3 to 7-fold, preferably, about 4 to 6-fold volume of water to the ginseng to be left alone for the period ranging from 3 to 7 hours at room temperature at the 1 ^s step; adding about 1 to 3-fold volume of water, lower alcohol such as methanol, butanol etc or the mixture thereof, preferably, water, methanol, ethanol, or the mixture thereof, 60-80% ethanol, thereto to perform reflux extraction for the period ranging from 1 hours to 6 hours, preferably, about 3 hours at the 2° step; cooling, filtering the filtrate to obtain its filtrates and removing organic solvent to obtain the inventive extract of American black ginseng, which shall be designated as " BPQE-2 " hereinafter.

[41] In a preferred embodiment, the present invention provide a process for preparing the

4 inventive extract of American black ginseng comprising the step consisting of; suspending said BPQE-2 extract of black ginseng in distilled water and extracting said extract with non-polar organic solvent such as diethyl ether etc to remove non-polar substance in BPQE-2 extract at the I^s step; adding water saturated butanol to remaining water layer at the 2° step; and extracting said water layer with butanol and removing butanol solvent to afford ginseng saponin- abundant butanol-soluble extract of the present invention, which shall be designated as " BPQBE-2 " hereinafter.

[42]

[43] The inventive extract of black ginseng of the present invention may be dried by the method well-known in the art, for example, dry in the shadow, lyophilization etc. The dried ginseng product may be cut into fine particles or powder, preferably, the particle having a particle size ranging from about 50 μmto 200 μmwith 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.

[44] It has been confirmed that the inventive extract of American black ginseng of the present invention contains more abundant amount of saponins in an amount ranging from about 12% to 16%, showing various pharmacological activities comparing than those of conventional ginseng.

[45] Accordingly, the inventive extract of American black ginseng prepared from the above-described preparation process in the present invention could show various pharmacological activities, particularly, more potent anti-cancer activity than those of conventionally available red ginseng and black ginseng being confirmed by following experimental examples.

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

[47] The term " Cancer disease " disclosed herein comprise nephritic or hydrouretic cancer, colonic cancer, prostatic cancer, lung cancer, arsenic cellular lung cancer, liver 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, breast cancer, endometrioma, cervical carcinoma, vaginal carcinoma, vulvul carcinoma, HodgMn's disease, esophageal cancer, enteric cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, smooth tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, lymphocytoma, cystic cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal medulla tumor, brain stem neuroglioma, hypophyseal adenomatosis and the like, preferably, nephritic or hydrouretic cancer, colonic cancer, prostatic cancer, or lung cancer, lung cancer, liver cancer.

[48] 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.

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

[50]

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

[52]

[53] The composition according to the present invention can 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, 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.

[54] 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.

[55] 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).

[56] The composition of the present invention 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.

[57] 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 lOOOmg/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.

[58] 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.

[59] Also, it is the other object of the present invention to provide a health care food composition comprising the extract of American black ginseng prepared from the above-described process for improving and preventing cancer diseases.

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

[61] The health care 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.

[62] Also, the present invention provide a health food beverage for the prevention and improvement of said 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 acids by weight and proper amount of sweetener and flavors.

[63] 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.

[64] Also, the extract of the present invention will be able to prevent and alleviate said 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.

[65] 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 100ml of the health beverage composition.

[66] Providing that the health beverage composition of present invention contains 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; disaccharide such as maltose, sucrose etc; conventional sugar such as dextrin, cy- clodextrin; 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.

[67] 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. [68] 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, α-tocopherol, rosemary extract, vitamin C, green tea extract, licorice root extract, chitosan, tannic acid, phytic acid etc.

Advantageous Effects

[69] As described in the present invention, the inventive extracts of American black ginseng prepared by inventive method show potent anticancer activity and abundant saponins, therefore, it can be used as the therapeutics for treating and preventing various cancer diseases.

[70]

Best Mode for Carrying Out the Invention

[71] 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.

[72] 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.

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

[74]

Mode for the Invention

[75] Comparison Example 1. Preparation of an extract of American ginseng

[76] 50g of American ginseng was cut into small pieces, mixed with 100ml of water and left alone for 5 hours at room temperature. If of 80% ethanol was added thereto to subject to reflux-extraction for 3 hours 3 times. The mixture was cooled into room temperature, subject to filtration with filter paper and the filtrate was evaporated with removing solvent by using rotary evaporator to obtain 18.46g of an extract of American ginseng.

[77] 14.86g of an extract of American ginseng was suspended in 400ml of water and mixed with 400ml of ether to remove non-polar substance. Remaining water layer was mixed with 400ml of saturated butanol to extract the saponin fraction in the ginseng and the butanol was evaporated by rotary evaporator to obtain 4.Og of a butanol- soluble extract of American ginseng comprising abundant amount of ginseng saponin (designated as " PQBE " hereinafter), which was further used as a test sample in following experiments.

[78]

[79] Example 1. Preparation of American black ginseng

[80]

[81] 1-1. Nine times-steaming and nine time-sunbathing method

[82]

[83] 5kg of five year's old American ginseng (Panax quinquefolium L.) purchased from

Daeduck Bio Co. Ltd. was washed with ultra-sonification washer for 15 mins three times, subjected to the 1 drying process at 5O⁰C for 24 hours and the 2 drying process at room temperature for 24 hours to obtain 2.23kg of dried American ginseng.

[84] 2.23Kg of the I^s dried American ginseng was subjected to steaming at 95⁰C for 3 hours excluding preheating period which is necessary time to require the leakage of steam from the steaming apparatus, generally, 30 mins. The internal temperature of the drying apparatus equipped with hot-wire was maintained to 6O⁰C with rotating its fan to subject to drying process for 12 hours and then the serial steaming and drying process was repeated nine times to obtain 2.02Kg of the darkened inventive American black ginseng comprising less than 14% water amount (BPQ-I).

[85]

[86] 1-2. High-temperature and high pressure method

[87]

[88] 5kg of five year's old American ginseng purchased from Daeduck Bio Co. Ltd. was washed with ultra-sonification washer for 15 mins three times, subjected to the 1 ^s drying process at 5O⁰C for 24 hours to obtain 2.23kg of dried American ginseng.

[89] 2.23Kg of the dried American ginseng was subjected to steaming maintaining

110-145⁰C for 4 hours under high pressure excluding preheating period which is necessary time to require the leakage of steam from the steaming apparatus, generally, 30 mins. The internal temperature of the drying apparatus equipped with hot-wire was maintained to 6O⁰C with rotating its fan to subject to drying process for 12 hours to obtain 2.05Kg of the inventive American black ginseng comprising less than 14% water amount (BPQ-2).

[90]

[91] Example 2. Preparation of the extract of American black ginseng [92] 2-1. crude extract of American black ginseng

[93] Each 50g of American black ginseng prepared from the methods disclosed in

Example 1-1 and 1-2 were cut into small pieces, 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 two kinds of inventive extract of American black ginseng [Example 1-1: BPQE- 1( 16.82g) and Example 1-2: BPQE-2 (16.7Og)].

[94]

[95] 2-2. butanol soluble extract of American black ginseng

[96] The two kinds of extract, i.e., BPQE- 1( 16.82g) and BPQE-2 (16.7Og) were suspended in 500ml of water and 500ml of ether was added thereto to remove the non- polar substance. 500ml of water saturated butanol was added to remaining water layer to extract the saponin fraction in ginseng four times and the extract was performed to evaporation with evaporator to remove butanol to obtain two kinds of inventive butanol soluble-extracts of American black ginseng comprising abundant amount of ginseng saponin [Example 1-1: BPQBE- l(4.07g) and Example 1-2: BPQBE-2 (4.12g)] , which were used as test samples in following experiments.

[97]

[98] Experimental Example 1. Component analysis of ginseng saponin

[99] In order to analyze the saponin component of inventive extracts of American black ginseng prepared in Example 2, each 20mg of butanol soluble- extract of American black ginseng (BPQBE-I and BPQBE-2) was dissolved in ImI of methanol, filtered with filter with the pore size of 0.45μm to analyze the component of the inventive extract of black ginseng by HPLC in accordance with the condition as shown in Table

1,

[100] At the result, it has been confirmed that Ig of the inventive extract of American black ginseng contains various pharmacologically active ginsenosides, i.e., RbI(O. l-0.4mg), Rb2 (1.7-3.1mg), Rc (1.4-2.5mg), Rd (1.2-1.7mg), Re(0.6-l.lmg), RgI (0.3-0.7mg), Rkl+Rg5 (121-145mg), Rg3$)+Rg3(R)(19-31mg) and Rh2 (34-41mg).

[101] Table 1 [Table 1] [Table ]

[1(E] [103] Reference Example 1. Cell Culture [104] ACHN (human renal tumor cell line), HCT (human colon tumor cell line), PC-3 (human prostate tumor cell line) and NCI-H23 (human lung tumor cell line) cells were grown on the culture dish having 100mm diameter (TPP Co., Ltd., Switzerland) in RPMI 1640 (Gibco BRL Co., Ltd., USA), supplemented with 10% fetal bovine serum which was inactivated at 37⁰C in 5% CO and 95% air condition in a humidified

2 incubator.

[105] [106] Experimental Example 1. Cell cytotoxicity determination [107] The cell cytotoxicity of the extracts of American black ginseng in ACHN (human renal tumor cell line), HCT (human colon tumor cell line), PC-3 (human prostate tumor cell line) and NCI-H23 (human lung tumor cell line) cells was determined by (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay method. (Rubinstein L. V. et al, Correlation of screening data generated with tetrazolium assay (MTT) vs protein assay (SRB) against a broad panel of human tumor cell lines, Proceedings of the American Association for Cancer Research, 30, p2418, 1989).

[108] Cells on 10mm culture dish were treated with 500 microliter of trypsin-EDTA (Gibco BRL Co., Ltd., U.S.A.) to separate from the dish and 10 ml of fresh medium was added thereto so as to neutralize trypsin-EDTA solution and obtain cell suspension.

4

[109] 1 X 10 cells/well were seeded in 96- well plate and after 24 hours incubation. The cells were treated with 100 microliter/well of various concentrations of the extracts prepared in Comparative Example 1 and Example 2 and incubated for another 48 hours. After 48 hours, the medium was discarded and 10 microliter of MTT solution (5mg/ml suspended in medium, Sigma Co., U.S.A.) diluted with PBS buffer adjusted the concentration to 5mg/ml was added to each cells to incubate at 37⁰C for 4 hours. The medium was washed with PBS to remove remaining MTT reagent and 100 ul of DMSO (Dimethy Sulfoxide) was dropped into each well. The plate was incubated for 20 minutes at room temperature and then UV absorbance the samples was measured by microplate reader (ELISA reader, DENLEY Co., Japan) to calculate the cell viability at 570nm.

[HO] The cell viability rate of negative control group treated with only medium was calculated as a 100% and those of other sample and positive control were calculated. [111] As a positive control group, lOOmicroliter/well of various concentrations of adriamycin, i.e., 0, 3.125, 6.25, 12.5, 25 were seeded onto 96 well pates, incubated for 48 hours and treated with diluted MTT in the concentration of 5mg/ml.

[112] The measured absorbance showing the reduced amount of existing cellular enzyme in MTT was proportioned to survival cell density. The concentration of sample required to inhibit the survival of cancer cell by 50% was expressed IC values. [113] At the result, the group treated with the extract in Comparative Example (PQBE) showed no cytotoxic effects on four kinds of cell lines. However the groups treated with inventive extracts in Example 2, i.e., BPQBE-I and BPQBE-2, showed very strong cytotoxic effect on the four kinds of tumor cell lines ( See.Table 2)

[114] Table 2 [Table 2] [Table ]

[115] [116] Experimental Example 2. Inhibitory effect on the volumetric increase of cancer cell using by Lewis lung carcinoma cell [117] To determine the inhibitory effect on the volumetric increase of cancer cell, following experiment was performed by modifying the procedure disclosed in the literature (Teruhiro et al., Cancer Res., 56, pp2809-14, 1996).

[118] Each group consists of is five 4- weeks aged male BDF-I mice weighing 20-23g procured from Chungang Experimental Animal Center (Korea) and the mice had bred under temperature-controlled room maintaining at 24⁰C, with freely accessible to water and mice feed (antibiotic-free).

[119] Lewis lung carcinoma cells (1 x 10 cells/well) which had been sub-cultured in vivo of experimental mouse and adjusted the concentration to 1 x 10 cells/well, was injected into the left armpit of BDF-I male mice. After 24 hours, adriamycin in the concentration of 0.5 and 1 mg/mouse, and the extracts prepared in Comparative Example 1, and Examples in the concentration of 50 and 100 ul/mouse were injected into the mice intraperitoneally. The injection of samples was maintained for 2 weeks until the tumor volume of non-treatment group used as control group become to 2 an . After 2 weeks, the tumor volume was calculated by using following Math Figure 1 and 2 to measure the inhibition rate of tumor volume.

[120] MathFigure 1 [Math.l]

l umor volume * cm^' ) = -

[121] L (an) : Length of the tumor [122] W² (an ²) : Width of the tumor [123] MathFigure 2 [Math.2]

J β

I] i h i hi to c){ of tin ri o r * v I u 11 Ϊ f- ϊ ^{t :}ό ⁾ = - — ; — ^* H)O

[124] A: Tumor volume (cm ) of control group

[125] B: Tumor volume (an ) of sample treatment group

[126]

[127] At the result, it was confirmed that the groups treated with the extract of Example 2 (Inhibition rate: 39.05%) and adriamycin (Inhibition rate: 76.40%) showed stronger inhibition of the volumetric growth of Lewis lung carcinoma cells in dose dependent manner compared with the groups treated with the extract of Comparative Example 1 (Inhibition rate: 15.87%) ( See Table 3).

[128] Table 3 [Table 3] [Table ]

[129] [130] Experimental Example 3. Toxicity test [131] [132] Methods (I) [133] The acute toxicity tests on ICR mice (mean body weight 25±5g) and Sprague- Dawley rats (235+1Og, Jung-Ang Lab Animal Inc.) were performed using the extract of the Example 1. Four group consisting of 10 mice or rats was administrated orally in- traperitoneally with 250mg/kg, 500mg/kg, lOOOmg/kg and 5000mg/kg of test sample or solvents (0.2 m-6, i.p.) respectively and observed for 2 weeks.

[134] [135] Methods (2^s) [136] The acute toxicity tests on ICR mice and Sprague-Dawley rats were performed using the extract of the Example 2. Four group consisting of 10 mice or rats was administrated intraperitoneally with 25mg/kg, 250mg/kg, 500mg/kg and 725mg/kg of test sample or solvents (0.2 ml, i.p.), respectively and observed for 24 hours.

[137] [138] Results [139] There were no treatment-related effects on mortality, clinical signs, body weight changes and gross findings in any group or either gender. These results suggested that the extract prepared in the present invention were potent and safe.

[140] [141] 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. [142]

[143] Preparation of powder

[144] BPQBE-2 of Example 2 50mg

[145] Lactose lOOmg

[146] Talc lOmg

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

[148]

[149] Preparation of tablet

[150] BPQBE-2 of Example 2 50mg

[151] Corn Starch lOOmg

[152] Lactose lOOmg

[153] Magnesium Stearate 2mg

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

[155]

[156] Preparation of capsule

[157] BPQBE-2 of Example 2 50mg

[158] Corn starch lOOmg

[159] Lactose lOOmg

[160] Magnesium Stearate 2mg

[161] Tablet preparation was prepared by mixing above components and filling gelatin capsule by conventional gelatin preparation method.

[162]

[163] Preparation of injection

[164] BPQBE-2 of Example 2 50mg

[165] Distilled water for injection optimum amount

[ 166] PH controller optimum amount

[167] Injection preparation was prepared by dissolving active component, controlling pH to about 7.5 and then filling all the components in 2ml ample and sterilizing by conventional injection preparation method.

[168]

[169] Preparation of liquid

[170] BPQBE-2 of Example 2 0.1 ~80g

[171] Sugar 5~10g

[172] Citric acid 0.05-0.3% [173] Caramel 0.005-0.02%

[174] Vitamin C 0.1-1%

[175] Distilled water 79-94%

[176] CO 2 gas 0.5-0.82%

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

[178]

[179] 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.

[180]

Industrial Applicability

[181] As described in the present invention, the inventive extracts of American black ginseng prepared by inventive method show potent anticancer activity and abundant saponins, therefore, it can be used as the therapeutics for treating and preventing various cancer diseases.

Claims

[1] A process for preparing inventive American black ginseng modifying nine times- steaming and nine time-sunbathing (flWkfl^M) method, which comprises the step consisting of; washing American ginseng ranging from about 3 to 7 years old, drying at the temperature ranging from 30 to 7O⁰C, for the period ranging from 12 to 36 hours, at the 1 ^s step; steaming the ginseng at the temperature ranging from 60 to 12O⁰C, for the period ranging from 1 to 5 hours, excepting pre-heating time at the 2ⁿ step; drying at the temperature ranging from 40 to 8O⁰C for the period ranging from 6 to 18 hours at the 3 step; and repeating said steaming step and drying step nine times to afford inventive BPQ- 1 American black ginseng comprising less than 14% water content (BPQ-I).

[2] The process according to claim 1, said process further comprises the step consisting of; pulverizing said BPQ-I American black ginseng and adding about 3 to 7-fold volume of water to the black ginseng to be left alone for the period ranging from 3 to 7 hours at room temperature at the 1 ^s step; adding about 1 to 3-fold volume of water, lower alcohol such as methanol, butanol etc or the mixture thereof, added thereto to perform reflux extraction for the period ranging from 1 hours to 6 hours at the 2ⁿ step; cooling, filtering the filtrate to obtain its filtrates and removing organic solvent to obtain the inventive extract of American black ginseng (BPQE-I).

[3] The process according to claim 1, said process further comprises the step consisting of; suspending said BPQE-I extract of black ginseng in distilled water and extracting said extract with non-polar organic solvent such as diethyl ether etc to remove non-polar substance in BPQE-I extract at the 1 step; adding saturated butanol to remaining water layer at the 2ⁿ step; and extracting said water layer with butanol and removing butanol solvent to afford saponin- abundant butanol- soluble extract (BPQBE-I).

[4] A process for preparing the inventive American black ginseng using high- temperature and high pressure method, which comprises the step consisting of; washing American ginseng ranging from about 3 to 7 years old, drying at the temperature ranging from 30 to 7O⁰C, for the period ranging from 12 to 36 hours, at the I^s step; steaming the ginseng at the temperature ranging from 95 to 155⁰C, for the period ranging from 1 to 8 hours excepting pre-heating time, under high pressured condition at the 2 step; and drying at the temperature ranging from 40 to 8O⁰C for the period ranging from 6 to 18 hours at the 3^r step to afford inventive BPQ-2 American black ginseng comprising less than 14% water content(BPQ-2).

[5] The process according to claim 4, said process further comprises the step consisting of; pulverizing said BPQ-2 black ginseng and adding about 3 to 7-fold volume of water to the ginseng to be left alone for the period ranging from 3 to 7 hours at room temperature at the 1 ^s step; adding about 1 to 3-fold volume of water, lower alcohol such as methanol, butanol etc or the mixture thereof, hereto to perform reflux extraction for the period ranging from 1 hours to 6 hours at the 2° step; cooling, filtering the filtrate to obtain its filtrates and removing organic solvent to obtain the inventive extract of American black ginseng(BPQE-2).

[6] The process according to claim 4, said process further comprises the step consisting of; suspending said BPQE-2 extract of black ginseng in distilled water and extracting said extract with non-polar organic solvent such as diethyl ether etc to remove non-polar substance in BPQE-2 extract at the I^s step; adding saturated butanol to remaining water layer at the 2° step; and extracting said water layer with butanol and removing butanol solvent to afford ginseng saponin-abundant butanol-soluble extract of the present invention (BPQBE-2).

[7] A pharmaceutical composition comprising a extract of American black ginseng showing anti-cancer activity prepared from the process as set forth in claim 3 or claim 6 for treating and preventing cancer diseases.

[8] The pharmaceutical composition according to claim 7 wherein said cancer disease is selected from nephritic or hydrouretic cancer, colonic cancer, prostatic cancer, lung cancer, arsenic cellular lung cancer, liver 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, breast cancer, endometrioma, cervical carcinoma, vaginal carcinoma, vulvul carcinoma, HodgMn's disease, esophageal cancer, enteric cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, smooth tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, lymphocytoma, cystic cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal medulla tumor or brain stem neuroglioma, hypophyseal adenomatosis.

[9] A use of a composition comprising an extract of American black ginseng prepared from the process as set forth in claim 3 or 6 for the manufacture of a medicament for cancer diseases in a mammal in need thereof, together with a pharmaceutically acceptable carrier thereof.

[10] A process of treating or preventing cancer diseases of human and marrmals comprising administering an effective amount of an extract of American black ginseng prepared from the process as set forth in claim 3 or 6 with a pharmaceutically acceptable carrier thereof.

[11] A health care food composition comprising the extract of American black ginseng prepared from the process as set forth in claim 3 or 6 for improving and preventing cancer diseases.