Composition containing saponins of <i>Panax ginseng </i>as active ingredient

Abstract

Disclosed are uses of ginseng saponin, which contains at least 90% of a compound K, Rd, F2, and Rg3 as a main ingredient and has effects in extending the lifespan of the cell, promoting the cell differentiation, increasing the number of red blood cells, and reducing the triglycerides by extracting, heat-treating, and enzyme-converting the ginseng saponin to prepare active saponins Rd, F2, and Rg3 including a compound K.

Claims

1. A composition containing ginseng saponin as an active ingredient which is useful for extending the lifespan of cells and promoting differentiation of cells, wherein the ginseng saponin is prepared by: exposing the ginseng to steam under a pressure of 1.1 kg/cm.sup.2 while removing moisture at 121 C. for 10 minutes using an automatic temperature and humidity control device with a porous shelf (step S1); continuously injecting air into the ginseng subjected to the step S1 and cooling the ginseng to 90 C. or lower (step S2); repeating the steps S1 and S2 two times (step S3); cooling the ginseng to room temperature in an automatic temperature control device and pulverizing the ginseng to a particle size of 1 mm or less, after step S3 (step S4); extracting the saponin by adding 80% ethyl alcohol to the ginseng subjected to the step S4, and concentrating the extracted saponin (step S5); preparing a suspension by dissolving the concentrated saponin in ethanol and adding sterilized water, after step S5 (step S6); adding the suspension to and reacting the suspension with a pectinase enzyme solution drop by drop, after step S6 (step S7); precipitating the solution subjected to the reaction of step S7 by centrifugation (step S8); dissolving the generated precipitate in ethyl alcohol (step S9); suspending the dissolved precipitate by adding sterilized physiological saline solution (step (10); filtering the suspended precipitate (step S11); and resuspending the precipitate in sterile physiological saline solution (step S12).

2. The composition of claim 1, wherein when the composition is used in a method to extend the lifespan of the cells, the life span extension is suppressed when the cells are contacted by an effective amount of -galactosidase.

3. The composition of claim 1, wherein the promoting of the differentiation of cells is promoting of differentiation of stem cells.

4. A composition containing ginseng saponin as an active ingredient which is useful for reducing triglycerides and increasing the number of red blood cells, wherein the ginseng saponin is prepared by, exposing the ginseng to steam under a pressure of 1.1 kg/cm.sup.2 while removing moisture at 121 C. for 10 minutes using an automatic temperature and humidity control device with a porous shelf (step S1); continuously injecting air into the ginseng subjected to the step S1 and cooling the ginseng to 90 C. or lower (step S2); repeating the steps S1 and S2 two times (step S3); cooling the ginseng to room temperature in an automatic temperature control device and pulverizing the ginseng to a particle size of 1 mm or less, after step S3 (step S4); extracting the saponin by adding 80% ethyl alcohol to the ginseng subjected to the step S4, and concentrating the extracted saponin (step S5); preparing a suspension by dissolving the concentrated saponin in ethanol and adding sterilized water, after step S5 (step S6); adding the suspension to and reacting the suspension with a pectinase enzyme solution drop by drop, after step S6 (step S7); precipitating the solution subjected to the reaction of step S7 by centrifugation (step S8); dissolving the generated precipitate in ethyl alcohol (step S9); suspending the dissolved precipitate by adding sterilized physiological saline solution (step (10); filtering the suspended precipitate (step S11); and resuspending the precipitate in sterile physiological saline solution (step S12).

5. The composition of claim 1, wherein the ginseng saponin contains 90% or higher of protopanaxadiol-based saponin.

6. The composition of claim 5, wherein the protopanaxadiol-based saponin includes the compounds K, Rd, F2, and Rg3 which are active protopanaxadiols.

7. The composition of claim 4, wherein the ginseng saponin contains 90% or higher of protopanaxadiol-based saponin.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a side view of an automatic temperature-humidity control device.

(2) FIG. 2 is a PPT-based saponin structure and a PPD-based saponin structure.

(3) FIG. 3 is a diagram showing a process in which glucose is removed from ginseng saponin by an enzyme reaction (a diagram showing a process of preparing a compound K as active saponin by removing glucose from ginseng saponin Rb1 by an enzyme reaction).

(4) FIG. 4 is a diagram showing a process in which glucose is removed from a heat-treated saponin suspension.

(5) FIG. 5 is an analysis result of active saponin of CKS by HPLC (showing a result of a compound k of 52%, Rd of 34.4%, F2 of 2.69%, and Rg3 of 2% with respect to the total saponin content).

(6) FIG. 6 is an analysis result of a CKS solution by a particle size analyzer (it is analyzed that an average particle size is 1 m or less).

(7) FIG. 7 shows results of the blood and blood cells which are analyzed after extracting the blood by administering CKS to the abdominal cavity of 4 SD rats per group once a day for 20 months to 37 days (triglyceride (TG) shows a statistically significant result, a CKS-administered experimental group is reduced by about 55% as 250 mg/dl as compared with a control group showing average 450 mg/dl, and total hematocrit (Hct) is increased from 45% to 57.8%, a level of red blood cells (RBCs) is increased by about 30% from 7106/l to 9.12106 l).

(8) FIG. 8 shows a result of observing a doubling time of mesenchymal stem cells (MSCs) in an experiment using human tooth-derived stem cells (as a result, shows the smallest population stem cell doubling time (PDT) at a concentration of 10 ng/ml).

(9) FIG. 9 shows a result of measuring a PDT for human-derived leukocyte cancer cells in an experiment using human tooth-derived stem cells (shows the longest time at a concentration of 10 ng/ml to make a growth speed of the leukocyte cancer cells slow.

(10) FIG. 10 shows a content of -galactosidase in cells, which is evaluated using human tooth-derived stem cells.

DETAILED DESCRIPTION OF THE INVENTION

(11) The Applicant will hereinafter describe the technical solutions in detail. The detailed description of known technology, which is considered to be unnecessarily obscured by the gist of the present invention, will be omitted.

(12) In the present invention, ginseng is heat-treated using an automatic temperature and humidity control device, and the ginseng refers to any one or at least two of roots of wild ginseng, wood-cultivated ginseng, cultivated roots, water culture ginseng, Panax quinquefolius L., Panax notoginseng, or red ginseng. First, when describing a structure of the automatic temperature and humidity control device through FIG. 1, steam in a high temperature and high pressure steam chamber is sent to an automatic temperature and humidity control chamber through a valve, in which active ginseng is manufactured and dried by continuous operation. When a steam injection automatic valve is opened, an air valve automatically opens, and when the steam is filled in the automatic temperature and humidity control chamber and the air is removed, the air valve closes automatically due to automatic recognition, and when the temperature and the pressure reach activating points, the steam injection automatic valve is closed and maintained for 10 minutes. After 10 minutes, the air valve is automatically opened and the pressure drops. When the temperature reaches 90 C., the air valve closes again and the steam injection valve opens. When the steam is filled in the automatic temperature and humidity control chamber and the air is removed, the air valve automatically closes due to automatic recognition, and when the temperature and the pressure reach activating points, the steam injection automatic valve is closed and maintained for 10 minutes. The above operation is repeated one more time, and the ginseng is heat-treated total three times and activated.

(13) In the present invention, the active ingredient was prepared by heat treatment. Specifically, the heat treatment was performed at 121 C. and 1.1 kg/cm.sup.2 for 10 minutes and repeated total three times (prepared as heated saponin, hereinafter referred to as HS). The heated saponin was pulverized by a pulverizer and suspended in 80% pharmacopoeia ethanol (alternatively, 80% ethyl alcohol) to extract a saponin ingredient. This is because the extraction of PPD in a 80% ethanol solution is better than PPT. The saponin ingredient extracted above was concentrated by evaporation, which was suspended in water and used as a raw material for PPD active production. The active ingredient was prepared by decomposing the sugar chain of saponin by injecting a suspension material into an enzyme solution by a certain amount using a peristaltic pump (preparation of PPD-based saponin containing compound K, hereinafter referred to as CKS). When a reaction material is not added to the enzyme in a certain amount, the yield is low and the reaction does not occur due to an aggregation phenomenon. Accordingly, in the present invention, the PPD-based active ingredient containing the compound k was prepared by injecting the reaction material into the enzyme by a predetermined amount by using a tube peristaltic pump. The prepared active ingredient was collected by centrifugation and then resuspended in ethyl alcohol and filtered to concentrate a filtrate.

(14) Hereinafter, the present invention will be described in detail with reference to Examples and drawings.

Example 1

(15) Ginseng containing wood-cultivated ginseng were placed on a porous shelf for 10 minutes at 121 C. by using an automatic temperature and humidity control device which was filled with steam from which air is removed and exposed to the stream under a pressure of 1.1 kg/cm.sup.2 and heat-treated while moisture dropped down to remove a large amount of moisture. The heat-treated ginseng were cooled to 90 C. or lower while the air was injected by opening a ventilation valve and the same operation was further performed two times again.

Example 2

(16) Active ginseng prepared in Example 1 were finely pulverized into particles having a size of 1 mm or less and dried, added with 80% ethyl alcohol, and then sufficiently heated to extract active saponin. Specifically, 60 g of the prepared active ginseng were pulverized and added with 80% ethyl alcohol to extract the active saponin and filtrated by a particle filter of about 500 m to remove a solid and collect an ethyl alcohol solution (hereinafter, referred to as a heat-treated saponin ethanol solution or HS ethanol solution).

(17) The heat-treated saponin ethanol solution was concentrated using a rotary evaporator (hereinafter, referred to as heat-treated saponin concentrate or HS concentrate. The heat-treated saponin concentrate was dissolved in a small amount of ethyl alcohol (ethanol) and added with sterilized distilled water to prepare a suspension (hereinafter, referred to as a heat-treated saponin suspension, concentration of 0.23 w/v %). The heat-treated saponin suspension was kept at 50 C. or higher in order to prevent the generation of harmful bacteria due to contamination.

(18) Separately from the heat-treated saponin suspension, an aqueous solution (2.4% pectinase solution) containing pectinase (trademark name of Rapidase, DSM food, Netherlands) was kept at a temperature of 50 C. using an automatic temperature device (hereinafter referred to as an enzyme solution), and the heat-treated saponin suspension was continuously injected to the enzyme solution by one drop. An injection speed of the heat-treated saponin suspension was 50 ml/min. More specifically, as shown in FIG. 4, the heat-treated saponin suspension was injected into the enzyme solution by a predetermined amount (50 ml/min) using a tube peristaltic pump and the prepared ingredients were collected by centrifugation and then suspended with ethyl ethanol again and filtered to concentrate a filtrate to finally secure a concentrate. 1 to 1.4 g of the final concentrate was prepared from 60 g of wood-cultivated ginseng having the moisture content of 30% and among them, the content of a compound k (CK) was highest of 52%, the content of Rd was 34.4%, the contend of F2 was 2.7%, and the content of Rg3 was 2% (see FIGS. 5 and 6).

(19) TABLE-US-00001 TABLE 1 Saponin compound K F2 Rd Rg3 Content 52% 2.69% 34.4 2%

(20) The final concentrate is hereinafter referred to as CKS because the PPD-based active ingredients such as CK, Rd, F2, and Rg3 occupy 91% or higher and the content of the compound k (CK) is highest as 52%.

Example 3

(21) Preparation and Analysis of CKS Suspension

(22) The prepared CKS was weighted, added and dissolved with ethyl alcohol having 10 times weight, and then mixed and suspended with a sterile physiological saline containing 0.01% polysorbate 80. The suspension prepared above was analyzed using a particle size analyzer (Malvern nano ZS-902). As the analyzed result, the average size of the particles was 785.5 nm and the size was less than 1 m.

Example 4

(23) Animal Administration Experiment

(24) Male SD rats grown in an animal room for up to 19 months were used for the experiment and were administered for a total of 37 days from 20 months. A total of 10 SD rats were divided into two groups of 5 SD rats. In a control group, 1 ml of sterile physiological saline was administered to the abdominal cavity once per day, and in an experimental group as a CKS-administered group, CKS was administered to the abdominal cavity at a concentration of 1.25 mg/kg once per day and an aseptic CKS suspension was used. One rat in the experimental group that had natural cancer during the experiment was eliminated from the experiment. At day 38, the blood was collected and used for analysis. For the blood analysis, data were obtained by Green Cross Labs Co., Ltd., a specialized blood analysis agency.

(25) As the analyzed result, the red blood cells (RBCs) were increased to 30% and hematocrit (Hct) was increased from 45% to 57.8%. In addition, there was no change in other hemoglobin (Hb), platelet, total cholesterol (T-cho), high-density cholesterol (HDL), and low-density cholesterol (LDL) (see FIG. 7).

Example 5

(26) The human wisdom tooth was pulled to extract stem cells. The number of cells was counted by adding CKS for each concentration while stem cells were cultured, and a generation time (or doubling time) was calculated based on the count. In the control group (Con), the generation time was averaged 46 hours and was highest as average 30 hours at a concentration of 10 ng/ml (see FIG. 8). In addition, the inhibitory effect on cancer cells was observed, and the generation time was observed after culturing a human-derived lung cancer cell line A549, and then an average generation time of the control group Con was 20 hours, whereas in the case of using long/ml of CKS, it could be seen that the average generation time was increased to average 25 hours. The average generation time was increased even at other concentrations, but the highest generation time was shown at 10 ng/ml (see FIG. 9).

Example 6

(27) The human wisdom tooth was pulled to extract stem cells. While the stem cells were cultured, the content analysis for -galactosidase was performed by adding long/ml of CKS. The suppression of the expression of -galactosidase in the cells means extending the lifespan of the cell. The content analysis for -galactosidase using mesenchymal stem cells, human tooth-derived stem cells, was performed by ELISA which was an enzyme immunity measuring method. As a result, in the case of treating CKS, the content of -galactosidase was reduced in all of three persons in wisdom tooth-derived mesenchymal stem cells (Dental MSCs) isolated from three persons. That is, in a first subject Den1, the content of -galactosidase was reduced by 16%, in a second subject Den2, the content of -galactosidase was reduced by 10%, and in a third subject Den3, the content of -galactosidase was reduced by 12%, as compared with the control group Con. The content of -galactosidase of average 12.7% was reduced, which was related with the lifespan extension of the cells.

(28) The present invention has industrial applicability to provide a pharmaceutical composition which contains ginseng saponin as an active ingredient by preparing the ginseng saponin as an active ingredient by removing a sugar attached to the active ingredient to be useful to increase the number of red blood cells and reduce triglycerides and to provide a pharmaceutical composition which contains ginseng saponin as an active ingredient which overcomes the problem on persistence of harmful organic solvents to be useful to increase the number of red blood cells, reduce triglycerides, differentiate the stem cells, and extend the lifespan of the cell.