KR20090073304A - Methods for high yield production of majonoside-r2 by suspension cultures of vietnamese ngoc linh ginseng - Google Patents

Abstract

A method for manufacturing a Vietnamese Ngoc Linh ginseng cell is provided to increase the yield of majonoside-R2 by adding an elicitor. A method for manufacturing a Vietnamese Ngoc Linh ginseng cell improves the yield of majonoside-R2 by administering an elicitor. A method for manufacturing the Vietnamese Ngoc Linh ginseng cell is to induce a calus from a tissue of living ginseng and suspension-culturing. A calus induction medium is Murashige & Skoogs solid medium containing the NAA.

Description

Method for High Yield Production of Majonoside-R2 by Suspension Cultures of Vietnamese Ngoc Linh Ginseng}

The present invention relates to a method for producing Vietnamese Ngoc Linh ginseng cells with high yield of majoroside-R2 through Vietnam Ngoc Linh ginseng cell suspension culture. More specifically, Vietnamese Ngoc Linh ginseng was used to induce callus using MS (Murashige & Skoogs) medium for plant cell culture, to induce suspension culture using callus, The present invention relates to a Vietnamese Ngoc Linh ginseng produced by virtue of yield, and to producing Vietnamese Ngoc Linh ginseng cells which can be used as a therapeutic drug or an edible or cosmetic raw material.

The main medicinal bioactive substance of Vietnamese ginseng is known as saponin, ginsenoside. It contains various types of ginsenosides, of which major amounts of majoroside R2 are known. There have been few studies on major side R2 in Korea, and active research has been conducted for a long time in Japan. It is reported that the anti-stress effect and the anti-cancer activity are excellent as the representative efficacy of major side R2.

Vietnam Ngoc Linh ginseng is native to the Highland region, mainly in the central highlands of Vietnam. Vietnam has a high temperature and is not suitable for ginseng, but the Central Highland region maintains a high altitude and temperature suitable for ginseng. Currently, Ngoc Linh ginseng is not cultivated agriculturally and is naturally found in Vietnam because of its rareness. There is no reported data on the cultivation of Vietnamese ginseng in Korea. Therefore, in a situation where it is difficult to secure Vietnamese ginseng by agricultural cultivation, Vietnam Ngoc Linh ginseng cells using plant cell culture technology will be manufactured and secured. In addition, to increase the yield of major side-R2 of cultured ginseng cells to increase the efficiency of securing useful materials, and through this technology to secure foreign useful organisms or genetic resources.

Literature Information of the Prior Art

Huong et al., Majonoside-R2 and Stress-Induced Antinociception, Pharmacological Biochemistry and Behavior , 1995, Vol. 52, ISSN 0091-3057, pp. 427-432

[Reference 2] T. Konoshima et al., Cancer chemopreventive activity of majonoside-R2 from Vietnamese ginseng, Panax vietnamensis, Cancer Letters , 1999, Vol. 147, ISSN 0304-3835, pp. 11-16

Domestic use of Vietnamese ginseng is minimal. There may be several reasons, but the biggest reason is the rareness of the sample. It is difficult to secure samples because it is a rare medicine in Vietnam. In addition, even if a sample is obtained, the burden on the price may be very large. However, cultivating Vietnamese ginseng domestically cannot be easily attempted due to climate and technology problems. Therefore, using the Vietnamese Ngoc Linh ginseng to induce callus, cells containing cell majoroside R2 was prepared. However, since the major side-R2 content is not high, there are many aspects in terms of efficient production of useful materials.

Therefore, there is a constant need to develop a method for producing Vietnamese ginseng cells by using plant cell culture techniques to increase the yield of majoroside R2.

Therefore, the present inventors induce callus and induce suspension culture again using Vietnamese Ngoc Linh ginseng biological sample, to produce Vietnamese Ngoc Linh ginseng cells with high cell growth rate and high yield of majornoside-R2. The present invention has been completed.

As a result, the main object of the present invention is to increase the yield of majornoside-R2, and to contain a large amount of majoroside-R2, through the suspension cell culture method and the method of adding an illiter to Vietnam Ngoc Linh ginseng, which is highly rare. It is provided in the form of cells.

The present invention provides a cell prepared by a method of producing Vietnamese Ngoc Linh ginseng cells containing majoroside-R2 produced in high yield and cultured through the Vietnamese Ngoc Linh ginseng cell suspension culture.

In the method for producing Vietnamese Ngoc Linh ginseng cells, the cell culture was promoted by suspension culture method, and the yield of majoroside-R2 was increased by 3-5 times using an illister and the like, thereby improving economic efficiency. The cells thus prepared show the superiority of the present invention because they can be used as a therapeutic drug or an edible or cosmetic raw material.

Vietnam Ngoc Linh ginseng plant used in the present invention was collected in the highland region of central Vietnam, the scientific name is Panax vietnamensis , the age of 10-15 years old, induce callus from their roots and leaves, using a stable callus cell line Suspension culture was induced. The suspension cultured cells thus obtained were confirmed to contain majoroside-R2, and each of the inventions described below was carried out.

The cell culture method used in the present invention is a known suspension culture method. Suspension culture uses a liquid medium and agitated culture at a gyrotory shaker at 120-150 rpm to increase cell growth rate faster than callus solid culture, thereby producing useful materials by cell biomass production or plant cell culture. It is a suitable method.

Suspension cultures of the invention were possible after successful callus induction. Callus from Vietnamese Ngoc Linh ginseng was derived as follows. The leaves and stems sterilized by surface sterilization with sodium hypochlorite and ethyl alcohol are cut to an appropriate size and cultivated aseptically on a solid culture medium. After a few weeks of culture, callus cells are formed at the cutting surface of the roots and leaves and begin to grow. The callus is passaged continuously to establish a stable cell line. MS (Murashige & Skoogs) medium was the most suitable medium used for inducing callus cells. 0.5-1.0% of agar is added to the MS medium and used as a solid medium. Phytohormone auxin is added as a growth regulator, but it is preferable to use 1-naphthalene acetic acid (NAA) instead of 2,4-D, which is seriously harmful to humans. In addition, when the sucrose concentration is maintained at 30 g / L or less in the medium, cell growth is good.

Suspension cultures were induced using stable callus cell lines. Callus was added to a sterilized liquid medium without agar, and then stirred gently for one day in a gyrotory shaker, followed by an adaptation period, followed by induction of suspension culture with stirring at a speed of 120-150 rpm. Suspension cultures using a liquid medium and cultured with agitation have a faster cell growth rate than callus solid cultures, so that subcultures are performed about every 10 days. However, since the majornoside-R2 content (yield) of the suspension cultured cells is not high, the invention to increase it was performed as follows.

Plant cells have a self-defence ability to kill invading bacteria by increasing the production of secondary metabolites in response to invasion by external pathogens. However, when pathogens are added to plant cells in culture, the production of secondary metabolites may initially increase, but the whole cell is contaminated by the added bacteria, causing the whole cell to die. However, if you put a pathogen-like material that acts as a pathogen and does not contaminate the cultured cells themselves, you can increase the production of secondary metabolites without killing the cultured cells. Such materials are called Elicitors and are currently used for the production of Shikonin, Sanguinarine, etc. (Cell Culture and Somatic Cell Genetics of Plants Vol. 4, p153-196, 1987). As such, the illisters are used in various plant cell culture systems, but the applied illisters are not consistent with each other in each plant cell culture. In general, there is no one-liquidator that is commonly applied to all plant cell culture systems, and one-liquidter that is suitable for each system is used as an experiment (Journal of Fermentation and Bioengineering, Vol. 173 (5), p380-385, 1992). ).

Yields were significantly improved by applying an illister to majornoside-R2 production in Ngoc Linh ginseng suspension cultured cell culture in Vietnam. By adding various kinds of illiitters to the suspension culture and investigating whether the major noside-R2 production is increased, the present inventors have found an illitter that drastically increases the production of major noside-R2 and completed the present invention.

Preferably, the illisitor according to the present invention is a cell wall extraction illiter of yeast or mold, and the production of majoroside-R2 is more than threefold increased when the illisitter is administered into the cells in culture. Jasmonic acid, an intermediate medium for cellular transduction, was also effective in improving majoroside-R2 yield, and the yield of majoroside-R2 when administered at 500 μM concentration in suspension culture. This increased 5.5 times.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

< Example  1>: Vietnam Ngoc Linh  Ginseng cells suspension  culture

Callus was induced using Vietnamese Ngoc Linh ginseng biological samples collected from the highland of central Vietnam and cell lines were established through stable culture. Biological samples were donated by Professor Quang of the National Defense University of Vietnam. The scientific name of Vietnamese Ngoc Linh ginseng collected is Panax Roots and leaves with vietnamensis and 10-15 year old growth were used for callus induction.

The biological sample was cut to about 10 cm in length, washed, and then sterilized in a 0.5% sodium hypochloride solution containing 0.1% of Tween-20 and stirred for 20 minutes. During this period, ultrasonic sonication was performed for 30 seconds to remove microbubbles on the surface of the biological sample. It is washed once with sterile water and then sterilized again in a 70% ethanol solution for 1 minute, and then the sterilized and washed biological sample is cut to about 0.5 cm in size and mixed with agar in MS medium. The culture was started by transferring to the medium. The main components of the medium were 3% sucrose, NAA 1 mg / L and kinetin 0.5 mg / L based on MS composition. All manipulations were performed aseptically on a sterile operating table, and callus was induced from 2-3 weeks after incubation at 25 ° C dark conditions. Callus was well derived from various biological samples, and the excellent ones were transferred to fresh medium every month to establish a cell line.

Suspension cultures were induced using stable callus cell lines. Callus was added to a sterilized liquid medium without agar in the medium, and gently stirred for one day in a gyrotory shaker to undergo an adaptation period, followed by induction of suspension culture with stirring at a speed of 120-150 rpm. Suspension cultures using a liquid medium and cultured with agitation have a faster cell growth rate than callus solid cultures, so that subcultures are performed about every 10 days.

< Example  2>: Suspension Culture  Cellular Ginsenoside  content

Vietnam Ngoc Linh ginseng cells suspended in Example 1 was dried, and extracted with methanol to analyze the content of majoroside-R2. At this time, HPLC was used as an analytical method, and C-18 was used as an analytical column. The mobile phase was used by varying the water and acetonitrile (MeCN) composition to a gradient, the first 30 minutes slowly increasing the concentration of MeCN from 18% to 19%, and then increasing the concentration of MeCN to 35% for 5 minutes. The analysis was then increased to 55% for 25 minutes. The Majornoside-R2 standard was donated from Professor Quang of Vietnam National University of Defense Medicine.

HPLC analysis showed that the majoroside-R2 content in the cultured cells was 5.1 mg / g cells, which was based on dry weight. The majoroside-R2 content of suspension cultured cells was slightly lower than that of callus cells, which is thought to be due to the shortening of the culture period. It was confirmed that major noside-R2 could be produced through suspension culture of Ngoc Linh ginseng in Vietnam.

< Example  3>: Major North Side - R2  Yield improvement

The content or production yield of majornoside-R2 of Vietnamese Ngoc Linh ginseng cells suspended in Example 1 is not very high. In order to increase the yield was applied an illister. Under the same culture conditions as in Example 1, various kinds of illisters were added, and cultured cells were harvested after one week, and the majoroside-R2 produced in the same manner as in Example 2 was analyzed and shown in Table 1. . The applied illisters were yeast extract, mold extract and jasmonic acid.

Increasing Majornoside-R2 Yield by Ilyciter Addition Illiitter Concentration Major side-R2 content of cultured cells without addition Major side-R2 content of cultured cells added Yield increase Yeast extract 150 ㎍ / g cell 5.1 mg / g cell 21.6 mg / g cell 4.2 times Mold Extract 100 ㎍ / g cell 5.1 mg / g cell 15.8 mg / g cell 3.1 times Jasmon Mountain 500 μM 5.1 mg / g cell 28.3 mg / g cell 5.5 times

Claims (3)

Vietnam Ngoc Linh ginseng suspension culture, to improve the yield of majoroside-R2 by administering an illiitter, Vietnam Ngoc Linh ginseng cell production method. According to claim 1, Ngoc Linh ginseng cell production of Vietnam Ngoc Linh ginseng, characterized in that the induction of the callus in the callus induced Murashige & Skoogs solid medium containing NAA, suspension culture Way. The method for preparing Vietnamese Ngoc Linh ginseng cells according to claim 1, wherein the yeast extract and the fungus extract are administered at a concentration of 50-500 micrograms per gram of cells, and 50-1000 micromoles of jasmonic acid as an eliminator.