KR910004950B1 - Novel cell line acg-1 for panax ginseng - Google Patents

Abstract

A process for culturing ginseng cell line ACG-1, KCTC 8973P (I) having much ginsenoside Rg1 of sapon in comprises (A) gaining the ginseng root, (B) removing remained strain by culturing (I) in Murashige-Skoog agar medium plus 3% sucrose and 0.1-3 mg/l 2,4-D for 4-5 wks 15-25 deg.C with or w/o light. Cell mass of (I) at 0.1 mg/l of 2,4-D is similar to in 3 mg/l 2,4-D, and with 1000 lux light, total saponin content is 9.97 mg/flask, esp. 6.09 g/g of ginsenoside.

Description

ACG-1 Cell Line and Preparation Method of Ginsenoside Rg1 Using the Same

The present invention relates to an ACG-1 cell line selected through ginseng tissue culture and a method for preparing ginsenoside Rg1 using the same.

Ginseng (Panax ginseng) is an expensive medicinal plant that has been used for a long time as a medicine, and contains saponin, polyacetylene, alkaloid, and phenol, which are drugs that show medicinal effects. Is being done intensively. In particular, recent research reports on saponins known as ginseng medicinal ingredients have revealed that ginsenoside Rg1 (Gin-senoside Rg1) among ginseng saponins has a specific effect on heart disease. It is known that Rg2, Rf, Re, Rb2, Rb1 and the like are contained in addition to the cenoside Rg1 (hereinafter referred to as "Rg1"). Their component distribution, ie, the ginsenoside pattern, is a factor that exemplifies the efficacy of ginseng. In general cultivated ginseng, the PT / PD ratio was about 1, and the triol ginsenoside and the diol ginsenoside were similarly contained. However, these ingredients do not act as the same drug for all diseases, and as described above, there are ingredients that selectively effect according to the disease, and the selective use of special ingredients may be a preferable method to increase the drug efficacy.

In the field of cell culture technology of ginseng, the mass production of saponin was enabled by the proliferation of ginseng cells through tissue culture, as disclosed in the patent application "ACG-2 cell line and a saponin production method using the same" with the present invention. have. Using such a tissue culture method, it is possible to mass-produce only a specific component, that is, to produce a large amount of a single component such as Rg1, which has been recognized as effective in heart disease as described above.

An object of the present invention is to provide a novel ginseng cell line containing a large amount of Rg1. Another object of the present invention to provide a method for producing a large amount of a single component of Rg1 using a novel ginseng cell line. These and other additional objects of the present invention will become more apparent from the following description.

The selection cell line, named ACG-1 herein, was inoculated and passaged by inoculating a cell line derived from the root of Korean ginseng into a solid incubator. In other words, the cell lines derived from the ginseng roots containing a lot of Rg1 were sucrose 3% by weight (based on MS-based medium) and 2,4-D 0.1mg / 1 in MS (Murashige-Skoog) base medium. Inoculated and incubated in the solid incubator added, but cultured for more than 4-5 weeks to continue subculture of the surviving cell line to obtain the cell line of the present invention ACG-1. The biologically pure culture of this cell line was deposited with the Korea Advanced Institute of Science and Technology on October 17, 1989 and was given accession number of KCTC 8473 P. The selected cell lines were used to further measure the effects of various nutrient media on cell line growth and ginsenoside Rg1 production, and to determine the most suitable and effective medium composition and fermentation conditions for this cell line.

[Characteristics of Cell Line]

As described above, the characteristics of the ACG-1 cell line (Accession No .: KCTC 8473 P) of the present invention selected and passaged in a solid incubator containing 2,4-D cell line organically derived from ginseng root are shown in Table I. For comparison, the ACG-1 cell line selected from the present invention and the general cell line of ginseng were incubated at 25 ° C. for 50 days in a solid incubator in which 3% by weight of sucrose and 2,4-D 0.1mg / 1 were added to the MS basic medium. Then, color, dry matter, saponin content and ginsenoside pattern were measured and compared with white ginseng, which is cultivated ginseng. In the ginsenoside pattern of Table I, the present invention shows that the Rg1 content of the ACG-1 cell line is 6.09 mg / g, which is much higher than that of general cell lines or white ginseng.

TABLE I

* g / flask ** mg / g

[Growing conditions]

ACG-1 cell lines grow by incubating in an appropriate aqueous nutrient medium under controlled conditions. The medium contains a source of assimilable carbon, nitrogen and inorganic salts in which sucrose and 2,4-D are added to the M-S base medium and used as the most suitable medium composition of the present invention. It is essential to consider the ginsenoside pattern of the production cell line for the purpose of the present invention as well as for the purpose of the present invention, as well as the medium composition for such growth conditions. Another important factor to be considered in growth conditions is temperature and contrast. ACG-1 cell line of the present invention did not affect the growth of the light (light), cancer (cultivation), but it was confirmed that the content of Rg1 is increased at the time of photoculture. The medium composition of the ACG-1 cell line of the present invention is preferably 1-5% by weight sucrose, 2,4-D 0.05-5mg / 1 in MS basic medium, and most preferably 1-5% by weight sucrose 2,4 -D 0.1-3 mg / 1. Incubation temperature is 15-25 ° C., incubation under light treatment. The nutrient mediums described herein are merely examples of, but are not limited to, the wide variety of media that can be used. Hereinafter, the present invention will be described in detail through examples. The ginsenoside pattern of saponin was measured and analyzed from the generated cell line by the following method, and the result is shown in Table II.

Ginsenoside Pattern Measurement Method

The harvested sample is dried at 60 ° C., exactly 1 g is taken, and the solvent is extracted in the order of methanol, ether, chloroform and butanol to extract saponin. Next, the extract was washed three times with distilled water and concentrated to prepare an assay sample.

This concentrated saponin was dissolved in methanol for HPLC and then analyzed for ginsenoside content and pattern by HPLC.

Example 1

Inoculate the cell line ACG-1 into a solid incubator in which 35 ml of medium containing 3% by weight of sucrose and 0.1 mg / 1 of 2,4-D was added to the MS basic medium and inoculated with 1,000 lux light at 25 ° C. After 50 days, the tissue was harvested and analyzed by ginsenoside pattern measurement.

Example 2

Cell tissues were harvested and ginsenoside patterns were measured and analyzed in the same manner as in Example 1, except that 3 mg / 1 medium was added instead of 0.1 mg / 1 of 2,4-D.

Example 3

Instead of irradiating the 1000 lux light of Example 1 except for culturing the ACG-1 cell line in the cancer state, the tissue was harvested in the same manner as in Example 1 and ginsenoside pattern was measured and analyzed.

Comparative Example 1

Instead of using the ACG-1 cell line of Example 1 using a normal cell line in the same manner as in Example 1 and harvesting the normal cell tissue ginsenoside pattern was analyzed.

Comparative Example 2

General cell tissues were harvested and ginsenoside patterns were measured and analyzed in the same manner as in Comparative Example 1, except that 3 mg / 1 medium was added instead of 0.1 mg / 1 of 2,4-D added in Comparative Example 1.

TABLE II

As shown in Table II, the ACG-1 cell line of the present invention reaches 6 to 8 times the ability to produce Rg1 under the same growth conditions as the general cell line.

Rg1 production of ACG-1 cell line according to 2,4-D concentration was about 4.6 times higher at low concentration (6.09mg / g) than at high concentration (1.33mg / g). It can be seen that about 1.7 times more is produced. As described above, the ACG-1 cell line of the present invention is a novel cell line containing a large amount of Rg1, which is medicinal for cardiac diseases, etc., and when mass-cultured according to the method of the present invention, mass production of Rg1 is possible as a single component pure product.

Claims (3)

ACG of Accession No. KCTC 8473 P (Deposit date: Oct. 17, 1989, Depositary institution: Korea Advanced Institute of Science and Technology) selected by subcultured cell line derived from Panax ginseng tissue in medium containing 2,4-D -1 cell line. ACG-1 cell line of Accession No. KCTC 8473 P selected by subcultured cell line derived from Panax ginseng tissue in medium containing 2,4-D was cultured: Sucrose 1-5 weight in MS basic medium %, 2,4-D in the incubator containing 0.05-5mg / 1, Ginsenoside Rg1 characterized in that the photoincubation at a culture temperature of 15 ° C-25 ° C, from which ginsenoside Rg1 is recovered. Manufacturing method. The method for producing ginsenoside Rg1 according to claim 2, wherein the content of 2, 4-D in the medium composition is 0.1-3 mg / 1.