WO2006109979A9

WO2006109979A9 - Novel method for preparing processed ginseng to obtain increased amount of ginsenoside rg5

Info

Publication number: WO2006109979A9
Application number: PCT/KR2006/001324
Authority: WO
Inventors: You Sup Chung; Jin Hwa Choi
Original assignee: Biomedgenomics Co Ltd; You Sup Chung; Jin Hwa Choi
Priority date: 2005-04-12
Filing date: 2006-04-11
Publication date: 2007-05-10
Also published as: DE112006000924T5; CA2604064A1; CA2604064C; GB2441902B; GB2441902A; GB0719741D0; WO2006109979A1

Abstract

The present invention relates to a processing method for preparing pharmacologically potent ginseng product and the extract therefrom which could provide abundant amount ginsenoside Rg5 showing various pharmacological activities with applying selected range of pressure and temperature into the method and therefore the composition comprising the processed ginseng and the extract thereof of the present invention can be useful as a medicament or health care food in the prevention or treatment of various diseases, especially, cancer disease.

Description

NOVEL METHOD FOR PREPARING PROCESSED GINSENG TO OBTAIN INCREASED AMOUNT OF GINSENOSIDE RG5

Technical Field

[1] The present invention relates to a method for preparing novel processed ginseng to obtain increased amount of ginsenoside Rg5. More particularly, the present invention relates to a method for preparing processed ginseng product and the extract thereof which contains increased amount of ginsenoside Rg5 by treating ginseng under specific pressure and temperature range. Background Art

[2] It has been found that ginseng enforces non-specific resistance to psychological stress and shows maintaining effect on human homeostasis together with other potent pharmacological activities, i.e., alleviation of hypertension, reinforcing activity of insulin, blood glucose lowering activity, stimulating effect on liver RNA synthesis, protein, glucose and lipid metabolism or anticancer activity.

[3] 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] Three types of ginseng products have been commercially available i.e., four year's old, five year's and six year's old ginseng and six year's old ginseng product cultivated on autumn has been reported to most potent among them. Particularly, the husk of un- husked ginseng contains lots of saponin, which shows potent activity.

[6] Ginsenosidal saponins isolated from ginseng having dammarane skeleton linked to several saccharides are different from those isolated from the other plants. In particular, there have been reported that ginseng contains about 30 kinds of saponin ingredients, especially ginsenoside RbI, Rb2, Rc, Rd, Rg, Re etc as main components. Those saponin compounds shows various pharmacological activities and potency according to their chemical structure and ginsenoside Rg5 among them has been high- lightened as a medicine due to its potent immuno- potentiating activity as well as vasodilating activity, anti-cancer activity, neuronal cell protecting activity etc recently.

[7] At present, there have been tried to process conventional ginseng to obtain more potent efficacy or utility by way of changing the structure of ginseng saponin in the process.

[8]

[9] Korean Patent Registration No. 10-0192678, discloses a process for preparing a processed ginseng prepared by subjecting hot temperature treatment containing high contents of ginsenoside Rg5 so as to obtaining processed ginseng having improved potency differing from original form of ginseng. However, the processing method could not provide the information on the correlation between the content change of ginsenoside Rg5 and the change of temperature and internal pressure and the method requires toxic organic solvent such as butanol.

[10]

[11] The inventors of the present invention have intensively carried out the scientific investigation concerning new processing method to obtain safe and uniform ginseng product. As a result of the investigation, the inventors have found novel processing method for preparing pharmacologically potent ginseng product which have more increased content of ginsenoside Rg5 verified by comparing with conventional ginseng and the processed ginseng product disclosed in prior art and they have finally completed the present invention. Disclosure of Invention Technical Problem

[12] Accordingly, it is an object of the present invention to provide a processing method for preparing pharmacologically potent ginseng products which has increased content of ginsenoside Rg5. Technical Solution

[13] In accordance with the present invention, the present invention provides a processing method for preparing pharmacologically potent ginseng product and the extract therefrom which obtain maximized content of ginsenoside Rg5 characterized in treating ginseng with selected ranges of pressure and temperature.

[14] Specifically, the present invention provides a processing method for preparing pharmacologically potent ginseng product consisting of the steps comprising; adding about 1 to 3 times weight of water based on the weight of ginseng to five to seven years old ginseng material, preferably six year old ginseng material; and treating step

2 under the internal pressure ranging from 1.10 to 4.00 kgf/cm , preferably, 1.20 to 1.50 kgf/cm in case of un-husked ginseng or 2.30 to 3.00 kgf/cm in case of husked ginseng in the temperature ranging from about 70 to 150°C, preferably 110 to 130°C for a period ranging from 1 to 5 hrs, preferably, about 2 hrs to obtain purposed processed ginseng having large amount of ginsenoside Rg5. [15] [16] Through the above-described processing method in case of un-husked ginseng, the final ginseng product of the present invention contains much more amount of ginsenoside Rg5, about two to five times, specifically about 4.4 times than the processed ginseng prepared from the method disclosed in Korean Patent Registration No. 10-0192678.

[17] Through the above-described processing method in case of husked ginseng, the final ginseng product of the present invention contains much more amount of ginsenoside Rg5, about two to five times, specifically about 3.3 times than the processed ginseng prepared from the method disclosed in Korean Patent Registration No. 10-0192678.

[18]

[19] The present invention also provides a method for extracting the extract of processed ginseng consisting of the steps comprising: extracting processed ginseng material prepared from the above described step with the mixture of organic solvent, preferably, the mixture of methanol and methylene chloride, more preferably, mixture of methanol and methylene chloride with the mixed ratio ranging from 0.60: 1.40 to 1.20:0.80, more preferably, about l:l(v/v) mixture of methanol and methylene chloride with reflux extraction method in the period ranging from 1 hr to two days, preferably more than 1 hour; filtrating to obtain filtrate, concentrating the filtrate to remove remaining solvent and drying to obtain potent ginseng extract having large amount of ginsenoside Rg5.

[20]

[21] The "ginseng material" disclosed herein aged six years old is preferably used in the present invention since six years old ginseng showed higher amount of ginsenoside Rg5 than four years old ginseng, which was confirmed by following experiments prosecuted by the present inventors.

[22]

[23] The "ginseng material" disclosed herein comprise the leaf thereof which has been reported to be useless as well as root part of ginseng in the present invention since it is confirmed that the processed leaf of ginseng of the present invention showed equivalent amount of ginsenoside Rg5 to the root part of ginseng, which was confirmed by following experiments prosecuted by the present inventors.

[24]

[25] The ginseng thus processed or the extract thereof may be dried by a known manner to obtain a dried processed ginseng, for example, dried at a lower temperature, i.e., below 70°C for the period ranging from about 48 to 60 hrs or freeze-drying method and it may be further processed to be pulverized or powdered into the smaller size, preferably, the size ranging from about 50 to 200 micrometer by the method well- known in the art, if necessary, to prepare commercially available final product such as capsule, tablet etc using by pharmaceutically acceptable carrier or adjuvant.

[26]

[27] The inventive processed ginseng of the present invention contains abundant ginsenoside Rg5 showing potent pharmacological activity such as vasodilating activity, immuno-potentiating activity, anti-cancer activity, neuronal cell-protecting activity etc, anti-cancer activity in particular.

[28]

[29] Therefore, the present invention also provides a pharmaceutical composition comprising the ginseng extract prepared from the above-described processing method and a pharmaceutically acceptable carrier or adjuvants for the treatment or prevention of cancer disease and the method of the present invention can provide with maximized content of ginsenoside Rg5 by adopting selected ranges of pressure and temperature.

[30]

[31] The term "cancer" disclosed herein comprises various cancers such as stomach cancer, liver cancer, lung cancer, cervical cancer, skin cancer, stomach cancer, or breast cancer, specifically skin cancer.

[32] The ginseng extract of the present invention has potent anticancer activity and therefore, the pharmaceutical composition of the present invention thus may be employed to treat or prevent various cancer diseases.

[33]

[34] The present invention also provides a use of ginseng extract prepared from above- described processing method for manufacture of medicines employed for treating or preventing various cancer such as stomach cancer, liver cancer, lung cancer in men, and cervical cancer, skin cancer, stomach cancer, breast cancer.

[35]

[36] In accordance with another aspect of the present invention, there is also provided an method of treating or preventing various cancer such as stomach cancer, liver cancer, lung cancer in men, and cervical cancer, stomach cancer, breast cancer, wherein the method comprises administering a therapeutically effective amount of the ginseng extract prepared from above-described processing method.

[37]

[38] The inventive composition may additionally comprise conventional carrier, adjuvants or diluents in accordance with a using method. It is preferable that said carrier is used as appropriate substance according to the usage and application method, but it is not limited. Appropriate diluents are listed in the written text of Remington's Pharmaceutical Science (Mack Publishing co, Easton PA).

[39] [40] Hereinafter, the following formulation methods and excipients are merely exemplary and in no way limit the invention.

[41]

[42] 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.

[43]

[44] For example, the compositions of the present invention can be dissolved in oils, propylene glycol or other solvents which 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 compounds of the present invention can be formulated in the form of ointments and creams.

[45]

[46] Pharmaceutical formulations containing present composition may be prepared in any form, such as oral dosage form (powder, latinor-pulves, granule, tablet, capsule, soft capsule, aqueous medicine, syrup, elixirs pill, solution, 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, injection).

[47]

[48] 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.

[49]

[50] 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 0.2-200mg/kg, preferably, 2 to 100mg/kg by weight/day of the inventive extract or compounds of the present invention. The dose may be administered in single or divided into several times per day. In terms of composition, the complex herbal composition should be present between 0.01 to 80% by weight, preferably 0.5 to 50% by weight based on the total weight of the composition.

[51]

[52] 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 intracerebroventricular injection.

[53]

[54] Accordingly, it is another object of the present invention to provide a health care food comprising above described extract prepared by the above processing method and a sitologically acceptable additive to prevent various diseases.

[55]

[56] Above described composition therein can be used for the prevention or alleviation of various cancer diseases. For the purpose of preventing or alleviating various cancer diseases, wherein, the amount of above described extract may generally range from about 0.1 to 15 w/w %, preferably 1 to 10 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 IOOD of the health beverage composition. The 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 intracerebroventricular injection in any form, such as oral dosage form (powder, latinor-pulves, granule, tablet, capsule, soft capsule, syrup, elixirs pill, solution, powder, sachet or granule).

[57]

[58] Providing that the health beverage composition of present invention contains the above described extract as an essential component in the indicated ratio, there is no particular limitation on the other liquid component, wherein the other component can be various deodorant or natural carbohydrate etc such as conventional beverage. Examples of aforementioned natural carbohydrate are monosaccharide such as glucose, fructose etc; 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 IOOD of present beverage composition.

[59]

[60] 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.

[61] Examples of addable food comprising aforementioned extract therein are various food, beverage, gum, vitamin complex, health care food and the like.

[62]

[63] 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.

Advantageous Effects

[64] The method for preparing processed ginseng according to present invention could provide abundant amount ginsenoside Rg5 showing various pharmacological activities with applying selected specific pressure and temperature into the method and the composition comprising the processed ginseng and the extract thereof can be useful as a medicament or health care food in the prevention or treatment of various diseases, cancer, particularly. Brief Description of the Drawings

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

[66]

[67] Fig. 1 shows the result of LC and Mass spectrum of ginseng extract (Gl-I);

[68] Fig. 2 shows the result of LC and Mass spectrum of ginseng extract (Gl-2);

[69] Fig. 3 shows the result of LC and Mass spectrum of ginseng extract (Gl-3);

[70] Fig. 4 shows the result of LC and Mass spectrum of ginseng extract (Gl-4);

[71] Fig. 5 shows the result of LC and Mass spectrum of ginseng extract (Gl-5); [72] Fig. 6 shows the result of LC and Mass spectrum of ginseng extract (Gl-6);

[73] Fig. 7 shows the result of LC and Mass spectrum of ginseng extract (Gl-7).

[74] Fig. 8 shows the result of LC and Mass spectrum of ginseng extract (G2-1);

[75] Fig. 9 shows the result of LC and Mass spectrum of ginseng extract (G2-2);

[76] Fig. 10 shows the result of LC and Mass spectrum of ginseng extract (G2-3).

[77] Fig. 11 shows the result of LC and Mass spectrum of ginseng extract (G3);

[78] Fig. 12 shows the result of LC and Mass spectrum of ginseng extract (G4);

[79] Fig. 13 shows the result of LC and Mass spectrum of ginseng extract (STD).

[80] Fig. 14 represents the anticancer activity of the ginseng extract.

Best Mode for Carrying Out the Invention

[81] 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. Mode for the Invention

[82] Comparative Example 1. Preparation of the processed ginseng according to the procedure disclosed in KR patent registration 10-0192678

[83]

[84] Non-dried and sliced lkg of Panax ginseng root was placed into crude drug extraction apparatus (2OL/ Kukje-kigong, Korea) and then was heated by steaming at 130°C for 2 hours. The steamed ginseng was dried at 50-60 °C, and pulverized into fine powder with the powder size ranging from 50-200 micrometer to obtain 195g of ginseng powder used as a comparative sample (designated as "STD" hereinafter).

[85]

[86] Example 1. Preparation of inventive un-husked ginseng extract

[87] 1-1. Preparation of ginseng extract (Gl-I)

[88] Air-dried and sliced 300g of six years old un-husked Panax ginseng root dipped into 60OmL of distilled water was placed into crude drug extraction apparatus (2OL/ Kukje-kigong, Korea) and then was heated maintaining the internal temperature ranging from 126°C to 130°C and internal pressure ranging from 1.4 to 1.5 kgf/cm for 2 hours. The processed ginseng was isolated from the water and dried with at 60-70 °C, and pulverized into fine powder with the powder size ranging from 50-200 micrometer to obtain 255g of ginseng powder (yield: 85.0%) used as a sample (designated as "Gl-I" hereinafter).

[89]

[90] 1-2. Preparation of ginseng extract (Gl-2)

[91] AU the procedure excepting modifying the internal temperature into the range from

103°C to 107°C and internal pressure into the range from 1.40 to 1.50 kgf/cm was similar to those disclosed in Example 1-1 to obtain 28Og of ginseng powder (yield:

93.3%) used as a sample (designated as "Gl-2" hereinafter). [92]

[93] 1-3. Preparation of ginseng extract (Gl-3)

[94] AU the procedure excepting modifying the internal temperature into the range from

110°C to 114°C and internal pressure into the range from 1.40 to 1.50 kgf/cm² was similar to those disclosed in Example 1-1 to obtain 26Og of ginseng powder (yield:

86.7%) used as a sample (designated as "Gl-3" hereinafter). [95]

[96] 1-4. Preparation of ginseng extract (Gl-4)

[97] AU the procedure excepting modifying the internal temperature into the range from

126°C to 130°C and internal pressure into the range from 1.85 to 2.00 kgf/cm was similar to those disclosed in Example 1-1 to obtain 245g of ginseng powder (yield: 81.7%) used as a sample (designated as "Gl-4" hereinafter).

[98]

[99] 1-5. Preparation of ginseng extract fGl-5^*)

[100] AU the procedure excepting modifying the internal temperature into the range from

126°C to 130°C and internal pressure into the range from 2.30 to 2.50 kgf/cm was similar to those disclosed in Example 1-1 to obtain 24Og of ginseng powder (yield: 80.0%) used as a sample (designated as "Gl-5" hereinafter).

[101]

[102] 1-6. Preparation of ginseng extract (Gl-6)

[103] AU the procedure excepting modifying the internal temperature into the range from

126°C to 130°C and internal pressure into the range from 2.80 to 3.00 kgf/cm was similar to those disclosed in Example 1-1 to obtain 235g of ginseng powder (yield: 78.3%) used as a sample (designated as "Gl-6" hereinafter).

[104]

[105] 1-7. Preparation of ginseng extract (G 1-7)

[106] All the procedure excepting modifying the internal temperature into the range from

133°C to 137°C and internal pressure into the range from 3.30 to 3.50 kgf/cm was similar to those disclosed in Example 1-1 to obtain 162g of ginseng powder (yield:

54.0%) used as a sample (designated as "Gl-7" hereinafter). [107]

[108] Example 2. Preparation of inventive husked ginseng extract

[109]

[110] 2-1. Preparation of ginseng extract (G2-1^*)

[111] Air-dried and sUced 300g of six years old husked Panax ginseng root dipped into

60OmL of distilled water was placed into crude drug extraction apparatus (2OL/ Kukje- kigong, Korea) and then was heated maintaining the internal temperature ranging from 126°C to 130°C and internal pressure ranging from 2.80 to 3.00 kgf/cm² for 2 hours. The processed ginseng was isolated from the water and dried with at 60-70 °C, and pulverized into fine powder with the powder size ranging from 50-200 micrometer to obtain 249g of ginseng powder (yield: 83.0%) used as a sample (designated as "G2-1" hereinafter).

[112]

[113] 2-2. Preparation of ginseng extract fG2-2^*>

[114] All the procedure excepting modifying the internal temperature into the range from

126°C to 130°C and internal pressure into the range from 1.40 to 1.50 kgf/cm was similar to those disclosed in Example 2-1 to obtain 26Og of ginseng powder (yield: 86.7%) used as a sample (designated as "G2-2" hereinafter).

[115]

[116] 2-3. Preparation of ginseng extract fG2-3^*>

[117] AU the procedure excepting modifying the internal temperature into the range from

133°C to 137°C and internal pressure into the range from 3.30 to 3.50 kgf/cm was similar to those disclosed in Example 2-1 to obtain 22Og of ginseng powder (yield:

73.3%) used as a sample (designated as "G2-3" hereinafter). [118]

[119] Example 3. Preparation of inventive ginseng (G3)

[120] [121] AU the procedure excepting that 300g of four years old un-husked round Panax ginseng was used and the extraction condition of internal temperature and internal pressure were adjusted to 126°C to 130°C and 1.40 to 1.50 kgf/cm was similar to those disclosed in Example 1 to obtain 236g of ginseng powder (yield: 78.7%) used as a sample (designated as "G3" hereinafter). [122]

[123] Example 6. Preparation of inventive ginseng (G4)

[124] [125] AU the procedure excepting that 300g of six years old un-husked ginseng second grade was used and the extraction condition of internal temperature and internal pressure were adjusted to 126°C to 130°C and 1.40 to 1.50 kgf/cm was similar to those disclosed in Example 1 to obtain 25Og of ginseng powder (yield: 83.3%) used as a sample (designated as "G4" hereinafter).

[126]

[127] Experimental Example 1. Analysis of component and content

[128]

[129] Sample preparation [130] [131] 20ml of methanol and 20ml of methylene chloride were added to each 2g of the samples prepared from Comparative Example and Examples and subjected to reflux extraction for 60mins. The solution was cooled, filtrated to remove insoluble material and the supernatant was concentrated to obtain their residue. The weight of each final sample extract was 0.183g (Gl-I), 0.110 (Gl-2), 0.160 (Gl-3), 0.154g (Gl-4), 0.131g (Gl-5), 0.192g (Gl-6), 0.079g (Gl-7), 0.148g (G2-1), 0.070 (G2-2), 0.061g (G2-3), 0.079g (G3), 0.083g (G4) and 0.124g (STD) respectively.

[132] [133] LC Mass Analysis [134] [135] The solvent mixture of methanol and methylene chloride (1:1) was added to test samples prepared in step 1-1 in an amount of 18.3 ml (Gl-I), 11.0ml (Gl-2), 16.0ml (Gl-3), 15.4 ml (Gl-4), 13.1ml (Gl-5), 19.2ml (Gl-6), 7.9ml (Gl-7), 14.8mL(G2-l), 7.0ml (G2-2), 6.1ml (G2-3), 7.9 ml (G3), 8.3ml (G4) and 12.4 ml (STD) respectively to the extent that the final sample concentration of each samples was adjusted to identical with each other. 3ml of each sample was subjected to micro-centrifugation with a speed of 1300 rpm for 15 mins. 5 microliter of each supernatant was injected to LC Mass apparatus under the condition shown in Table 1 and the result was shown in Figs. 1 to 13.

[136] Table 1

[137] The resulting comparison with each sample was shown in Table 2. [138] As can be seen in Table 1, it is confirmed that the amount of ginsenoside Rg5 prepared in Examples are about 4.4 and 3.3 fold higher than that in Comparative Example.

[139] Accordingly, it is confirmed that the preparation method of the present invention is superior to the method disclosed in the prior art. [140] Table 2

[141] Additionally, the present inventors have been investigated to find optimum extraction condition, especially, the mixture ratio of extracting solvent system by varying the mixture volume ratio of methanol and methylene chloride within the range from 0.60: 1.40 to 1.20:0.80 and finally found that most efficient extraction condition among them is the solvent mixture of methanol and methylene chloride (1 : 1) of which electric conductivity constant is 20.75.

[142] Accordingly, we have confirmed that most efficient extraction solvent system to obtain abundant ginsesnoside Rg5 is the solvent mixture of methanol and methylene chloride (1:1) to obtain abundant ginsenoside Rg5.

[143] [144] Experimental Example 2: Determination of anticancer activity [145] [146] The anticancer activity of the ginseng extract containing abundant ginsenoside Rg5 prepared in Experimental Example 1 was determined by following experiments.

[147] [148] Sample Preparation [149] The inventive ginseng extract extracted with methanol and methylene chloride solvent mixture (1:1) prepared by the method according to the procedure disclosed in Example 2-1 was used as a test sample (designated as "GMM" hereinafter) by dissolving in distilled water (5mg/ml). [150]

[151] Test animals

[152] Four weeks-old C57BL/6 mice weighing 10-15g bred under identical circumstance were classified into two groups, i.e., 80 mice as test groups and 20 mice as control group. Test groups were treated with B 16 melanoma cell line to occur cancer and control groups were not treated. The test groups were further classified into two groups, i.e., test group (1) treated with only distilled water and test group (2) treated with equivalent amount of inventive GMM extract at 5^th week and the control groups were also classified into two groups, i.e., control group (1) treated with only distilled water and control group (2) treated with equivalent amount of inventive extract at 5^th week.

[153]

[154] Test Procedure

[155] Four weeks-old C57BL/6 mice weighing 10-15g bred under identical circumstance were freely accessible to drink the solution comprising inventive extract prepared by above step in control group (2) and test group (2) after the delactation of the mice (four weeks after the birth). B 16 melanoma cell was injected into the abdomen of mice sub- cutaneously in cancer occurred groups.

[156] B 16 melanoma cells were sub-cultured in CO incubator for 1 week and the floated cells diluted RMPI 1640 medium comprising 10% fetal calf serum adjusted to IxIO⁶ cells/ml were injected by 0.1ml.

[157]

[158] The cancer growth rate and survival period was determined by the size of cancer every five days after the injection and the survival period of each group. The cancer growth rate was compared with the weight of cancer and the result was calculated by the following math figure 1.

[159]

[160] MathFigure 1 _τhcWcφ^β_^ongLcπgth- ^rheLenSthOβ^ongLength(mm)srheLength^

[161] Test Result

[162] As can be seen in Fig. 14, 55% mice occurred cancer in test group (1) while 25% mice in test group (2) at 1 week. At the 12^th day and 17^th day, 80% and 95% mice occurred cancer in test group (1) respectively while 35% and 42.5% in test group (2) respectively. At the 24^th day, 100% mice occurred cancer in test group (1) while 52.5% in test group (2).

[163] As can be seen in Table 3, there showed no significant difference between the mean cancer weight at 1 week after the injection of cancer cell line (mean weight of cancer: O.lg) however, the difference of mean weight of cancer between the test groups was gradually increased, i.e., the mean weight of cancer in test group (1) and (2) was 0.4g and 0.3g at the 12^th day, 1.8+0.3g and 1.5+0.2g at the 17^th day, 5.1+0.2g and 3.3+l.Og at the 22^nd day and 22.4+5.3g and 9.8+2.1g at the 57^th day respectively.

[164] [165] Additionally, there showed no dead mice in test group (1) and (2) at 12^th day while one mouse was died in test group (1) at 17 day. 85% mice in test group (2) were survived for more than 42 days after the injection of cancer cell and about 50% mice in test group (1) were survived. At 60 day, all the mice in test group (1) were died while 60% mice in test group (2) were survived.

[166] Table 3

[167] Accordingly, it is confirmed that the ginseng extract prepared by inventive method in the present invention showed potent anti-cancer activity since the method of the present invention could provide with abundant ginsenoside Rg5 showing potent anticancer activity.

[168] [169] Experimental Example 3: Toxicity test [170] [171] Methods [172] The acute toxicity tests on SD mice (mean body weight 25+5g) and Sprague- Dawley rats (235+1 Og) were performed by using inventive extract. Each group consisting of 3 mice or rats was administrated orally with lg/kg of test sample or solvents (0.2 D, Lp.), respectively and observed for 24 hrs.

[173] [174] Results [175] There were no treatment-related effects on mortality, clinical signs, body weight changes and gross findings in any group or either gender and it has been confirmed that the LD of orally administrated inventive extract was more than lg/kg. These results suggested that the inventive extract prepared in the present invention were potent and safe.

[176] [177] 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.

[178]

[179] Preparation of powder

[180] Dried powder (Gl-I) of Example 1 20mg

[181] Lactose lOOmg

[182] Talc lOmg

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

[184]

[185] Preparation of tablet

[186] Dried powder (Gl-I) of Example 1 lOmg

[187] Corn Starch lOOmg

[188] Lactose lOOmg

[189] Magnesium Stearate 2mg

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

[191]

[192] Preparation of capsule

[193] Dried powder (Gl-I) of Example 1 lOmg

[194] Crystalline cellulose lOOmg

[195] Lactose lOOmg

[196] Magnesium Stearate 2mg

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

[198]

[199] Preparation of injection

[200] Extract (G2- 1 ) of Example 2 1 Omg

[201] Distilled water for injection optimum amount

[202] PH controller optimum amount

[203] 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.

[204]

[205] Preparation of liquid

[206] Extract (G2- 1 ) of Example 2 0.1 ~S

[207] Sugar 5~10g

[208] Citric acid 0.05-0.3% [209] Caramel 0.005-0.02%

[210] Vitamin C 0.1-1%

[211] Distilled water 79-94%

[212] CO₂ gas 0.5-0.82%

[213] Liquid preparation was prepared by dissolving active component, filling all the components and sterilizing by conventional liquid preparation method. [214]

[215] Preparation of health care food

[216] Extract (G2- 1 ) of Example 1 1 OOOmg

[217] Vitamin mixture optimum amount

[218] Vitamin A acetate 70mg

[219] Vitamin E l.Omg

[220] Vitamin B 0.13mg

[221] Vitamin B 0.15mg

[222] Vitamin B 0.5mg

6

[223] Vitamin B 0.2mg

[224] Vitamin C lOmg

[225] Biotin lOmg

[226] Amide nicotinic acid 1.7mg

[227] Folic acid 50mg

[228] Calcium pantothenic acid 0.5mg

[229] Mineral mixture optimum amount

[230] Ferrous sulfate 1.75mg

[231] Zinc oxide 0.82mg

[232] Magnesium carbonate 25.3mg

[233] Monopotassium phosphate 15mg

[234] Dicalcium phosphate 55mg

[235] Potassium citrate 90mg

[236] Calcium carbonate lOOmg

[237] Magnesium chloride 24.8mg

[238] 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. [239]

[240] Preparation of health beverage

[241 ] Extract (G2- 1 ) of Example 2 1 OOOmg

[242] Citric acid lOOOmg

[243] Oligosaccharide lOOg [244] Apricot concentration 2g

[245] Taurine Ig

[246] Distilled water 900D

[247] 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 IOOOD ample and sterilizing by conventional health beverage preparation method.

[248]

[249] 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.

[250]

Industrial Applicability

[251] The method for preparing processed ginseng according to present invention could provide abundant amount ginsenoside Rg5 showing various pharmacological activities with applying selected specific pressure and temperature into the method and the composition comprising the processed ginseng and the extract thereof can be useful as a medicament or health care food in the prevention or treatment of various diseases, cancer, particularly.

Claims

[1] A method for preparing pharmacologically potent ginseng product consisting of the steps comprising; adding about 1 to 3 times volume of water based on the weight of ginseng to five to seven years old ginseng material and treating step under the internal pressure ranging from 1.20 to 1.50 kgf/cm in case of un- husked ginseng or 2.30 to 3.00 kgf/cm in case of husked ginseng in the temperature ranging from about 110 to 130°C, for a period ranging from 1 to 5 hrs to obtain purposed processed ginseng having large amount of ginsenoside Rg5.

[2] The method according to claim 1, said ginseng material is six years old un- husked ginseng or husked ginseng.

[3] A method for extracting the extract of processed ginseng consisting of the steps comprising: extracting processed ginseng material prepared from the method as set forth in claim 1 with organic solvent mixture mixed with methanol and methylene chloride with reflux extraction method in the period ranging from 1 hr to two days; filtrating to obtain filtrate, concentrating the filtrate to remove remaining solvent and drying to obtain potent ginseng extract containing abundant amount of ginsenoside Rg5.

[4] The method according to claim 3, said solvent mixture is solvent mixture of methanol and methylene chloride with the mixed volume ratio ranging from 0.60:1.40 to 1.20:0.80.

[5] A pharmaceutical composition comprising the ginseng extract prepared from the method as set forth in claim 1 or 3 and a pharmaceutically acceptable carrier or adjuvants for the treatment or prevention of cancer disease.

[6] The pharmaceutical composition according to claim 5, wherein said composition is provided as powder, latinor-pulves, granule, tablet, capsule, soft capsule, aqueous medicine, syrup, elixirs pill, solution, powder, sachet, granule, cream, ointment, lotion, gel, balm, patch, paste, spray solution, aerosol or injection.

[7] A health care food comprising the extract prepared by the method as set forth in claim 1 or 3 showing potent anti-cancer activity and a sitologically acceptable food additive.

[8] The health care food according to claim 7, wherein said health care food is provided as a tablet, capsule, powder, latinor-pulves, or granule.