WO2005020672A1

WO2005020672A1 - Method for mass production of wild ginseng roots by plant tissue culture

Info

Publication number: WO2005020672A1
Application number: PCT/KR2003/002548
Authority: WO
Inventors: Heon-Sik An; Tae-Seob Lee; Eun-Myeong Shin; Sun-Hwa Kim
Original assignee: Neo Bio Co., Ltd.
Priority date: 2003-09-02
Filing date: 2003-11-25
Publication date: 2005-03-10
Also published as: KR100506625B1; KR20050022834A; AU2003284754A1

Abstract

Disclosed is a method for mass production of tissue-cultured wild ginseng roots by tissue culture. Unlike a conventional method of mass-producing wild tissue-cultured wild ginseng roots by tissue culture, including selection of a raw material, sterilization of the raw material, induction of callus formation, culture and mass propagation, the method of the present invention is characterized by selecting a wild ginseng raw material by DNA fingerprint analysis and directly inducing root differentiation from the raw material without the induction of callus formation. Because of exclusion of callus formation induction, the present invention does not employ harmful growth regulators, such as 2,4-D. In addition, the present method remarkably increases the content of pharmaceutically active components that are generally contained in low concentrations in tissue-cultured wild ginseng roots.

Description

METHOD FOR MASS PRODUCTION OF WILD GINSENG ROOTS BY PLANT TISSUE CULTURE

Technical Field

The present invention relates to a method for mass production of tissue-cultured wild ginseng roots by tissue culture, and more particularly, to a method for mass production of tissue-cultured wild ginseng roots having increased content of ginsenosides that are physiologically active substances .

Background Art

Ginseng is an herb that belongs to the genus Panax of the family Araliaceae. The root of ginseng is used for medical purposes. Six or seven species in the Panax genus have been identified. Among them, three species, Korean ginseng, Chinese ginseng and American ginseng, are mainly cultivated and marketed worldwide. A ginseng species, referred to by the botanical name "Panax ginseng CA. Meyer" and distributed in Far East Asia, has been used as the most important material in Chinese herbal medicine. Ginseng exhibits surprising effects of treatment of several diseases and stimulation and recovery of one' s spirits. Based on these medical efficacies of ginseng, a broad range of studies has been conducted to identify pharmaceutically effective components and the pharmaceutical efficacy of ginseng. The representative efficacy experimentally proven until now is to maintain the homeostasis of the body's control functions. This efficacy results from a variety of activities of ginseng, including anti-fatigue and anti-stress activity, anti-diabetic activity, ability to control blood pressure, anticancer activity, and ability to reinforce brain functions. These activities are considered to result from ginseng' s immunoenhancing action. Major effective components of ginseng include saponins, sapogenins, polyacetylenes, pyrazine derivatives and maltol . In particular, wild ginseng is known to have higher contents of the effective components than field-grown ginseng, and contain substances beneficial to the body, which are not contained in field- grown ginseng. Field-grown ginseng is cultivated for a shorter period of time with an aim of increasing yields rather than improving the efficacy of ginseng, so that it harbors simplified genes. Thus, field-grown ginseng, even though it has an initial origin identical to wild ginseng and wild- simulated ginseng, derived from wild ginseng, has been found directly and indirectly to have greatly weakened effectiveness. In contrast, wild ginseng or wild-simulated ginseng having an origin very close to wild ginseng has been proven to have a complex gene structure, which provides amazing efficacies that have been made known by word of mouth for a long time. The amazing efficacies of wild ginseng and the difference between wild ginseng and field-grown ginseng have been demonstrated by modern analysis methods. However, to date, wild ginseng, which is superior in medical efficacy to field-grown ginseng and thus highly valuable, has been supplied to consumers through collection or cultivation, which is time-consuming and uneconomical and supplies wild ginseng in limited amounts. These problems associated with yield and efficiency can be solved by biotechnological methods. For example, Korean Pat. Application Nos. 2001-0003285 and 2001-0031029 disclose methods of mass-producing tissue- cultured wild ginseng roots using bioreactors. These methods allow the mass production of wild ginseng, but are problematic with respect to the quality of wild ginseng. That is, these methods essentially include a process inducing callus formation using a growth regulator harmful to the body, such as 2,4-D. 2,4-D is used as a defoliant, which is known not to be safe to the body. Also, these methods have another disadvantage of producing wild ginseng that has relatively low content of ginsenosides that are physiologically active substances. Disclosure of the Invention

Accordingly, the present invention has been made keeping in mind the above problems occurring in the prior art, and an object of the present invention is to provide a method capable of mass-producing high quality wild ginseng using a bioreactor, the method not employing a toxic substance such as 2,4-D by excluding a process of inducing callus formation. It is another object of the present invention to provide a method of preparing wild ginseng having high content of physiologically active substances.

Best Mode for Carrying Out the Invention

The present invention provides a method for the mass production of tissue-cultured wild ginseng roots using the tissue of wild ginseng grown in a wild forest habitat as a raw material, comprising: 1) selecting an individual wild ginseng harboring the unique genes of wild ginseng; 2) surface sterilizing the selected wild ginseng raw material; 3) directly inducing root differentiation from the surface sterilized raw material; 4) adapting resulting tissue-cultured wild ginseng roots to a liquid culture for mass production of the tissue-cultured wild ginseng roots; 5) preparing a seed culture of the tissue-cultured wild ginseng roots using a bioreactor; and 6) mass-producing the tissue-cultured wild ginseng roots using a bioreactor to increase the content of ginsenosides that are physiologically active substances. In order to obtain wild ginseng having high contents of physiologically active substances, a process for selecting an individual harboring the unique genes of wild ginseng is required. The most effective selection is achieved by DNA fingerprint analysis, which is described in detail by the present applicant in Korean Pat. Application No. 2002-36091 (titled "Method of identifying wild ginseng using DNA fingerprinting") . The selected individual wild ginseng is essentially sterilized by surface sterilization. Since plant tissues grow more slowly than microorganisms or fungi, a tissue sample should be cultivated under aseptic conditions . In the present invention, the selected wild ginseng is essentially sterilized before root differentiation induction. When a strong sterilization is carried out in order to reduce microbial or fungal contamination, plant tissue explants are destroyed, and thus, root differentiation is not induced. A weak sterilization increases microbial or fungal contamination. Thus, preferably, a weak sterilization is conducted with two-step sterilization using two different sterilizing agents. For example, wild ginseng roots are washed with distilled water to remove soil, and are primarily sterilized by being immersed in 70% ethanol for 30-120 seconds . After the wild ginseng roots are washed with sterile distilled water, they are secondarily sterilized by being immersed in a 2% hypochlorite solution for 1-10 minutes . More preferably, after the second sterilization, the sterilizing agent present on the surface of the wild ginseng roots is removed using an absorbing agent. When the surface sterilization of the raw material is carried out in this way, wild ginseng roots grow two times faster on a solid medium. The most characteristic feature of the present invention is to directly induce root differentiation from an aseptic raw material, thereby producing tissue-cultured wild ginseng roots. The direct induction of tissue-cultured wild ginseng roots from an aseptic raw material is carried out using WPM (Lloyd and McCown) medium or MS (Murashige and Skoog) medium. Herein, an auxin, indole 3-acetic acid (IAA), is used as a plant growth regulator in an amount of 0.1-10 pp , preferably 2-5 ppm, to induce tissue-cultured wild ginseng roots. IAA is superior to other plant growth regulators with respect to efficiency, growth rate and differentiation rate. Upon suspension culture, the tissue-cultured wild ginseng roots are propagated in a liquid medium for suspension culture, such as MS, WPM (Lloyd and McCown) or SH (Schenk and Hildebrandt) . The tissue-cultivated wild ginseng roots are inoculated in an amount of 8-12% based on the total volume of the medium, and preferably about 10% . When the medium is exchanged with a fresh one at 2-week intervals for a 4-week subculture, biomass increases more than three times. Tissue-cultured wild ginseng roots may be induced in a flask from a liquid suspension-cultured tissue. The secondary induction of tissue-cultured wild ginseng roots is carried out by inoculating only newly generated tissue- cultured roots, cut from the aseptic material differentiating tissue-cultured wild ginseng roots, or the whole tissue including newly generated roots, in a flask. Herein, 0.1-10 ppm of IAA as a growth regulator is added to a medium, such as WPM (Lloyd and McCown) , mB5 (modified Gamberg) , MS (Murashige and Skoog), 1/2 MS, B5 (Gamberg) , SH (Schenk and Hildebrandt), LP (Quoirin and Lepoivre) , Whit, GD (Gresshoff and Doy) , DKW (Driver and Kuniyuki) or DCR (Gupfa and Durzan) . Preferably, the culture medium is WPM medium that is supplemented with 2-5 ppm of IAA or IBA, most preferably 2-4 ppm of NAA. When the culture is carried out in a general airlift bioreactor, WPM medium is most preferable, and is essentially supplemented with about 0.5-5 ppm of IAA or IBA in order to induce tissue-cultured wild ginseng roots from the wild ginseng tissue. An inoculum is inoculated in an amount of about 0.1%-0.5% based on the total volume of the medium, and then cultured in a medium with an identical composition for about 4-6 weeks to effectively induce root differentiation. The culture of tissue-cultured wild ginseng roots using a bioreactor is scaled up in a stepwise manner. When tissue-cultured wild ginseng roots are cultured in a 5-L airlift bioreactor, the initial inoculum is used in an amount of about 0.5%. The same inoculum amount is applied to cultures using 10-L and 20-L bioreactors . The medium is preferably supplied on Day 7 to Day 10 after which tissue-cultured roots are aggregated and thus become immovable. Upon seed culture, the medium exchange is more effective in yield and production of high quality tissue-cultivated wild ginseng roots in a way that is carried out by completely exchanging the existing medium with fresh medium, than by additionally adding fresh medium to the existing medium. When tissue-cultivated wild ginseng roots are inoculated in a length of 1-3 cm, the total biomass in the final harvest greatly increases. When the culture is carried out in a bioreactor, since the culture is performed for about 45 days, compounds secreted by cells, especially growth regulators, in the culture should be removed using an absorption method. Most preferably, the medium is supplemented with activated charcoal at a preparation step. For fast growth, the culture is carried out at 25°C or lower, at which plant tissue or cell culture is generally carried out, and preferably 20-25°C. Culture at 26°C or higher, especially 28°C or higher, results in slow growth. When tissue-cultured wild ginseng roots are cultured in a large-scale bioreactor of 5 tons or greater, great quantities of air should be fed into the bioreactor in order to continuously stimulate the end region of the tissue-cultured roots during culture. This air injection is one of the major control factors of large-scale culture. Also, the contents of ginsenosides that are physiologically active substances may vary according to the aeration and hydraulic pressure. To further enhance the contents of ginsenosides that are physiologically active substances, crude saponins or ginsenosides extracted from ginseng may be added to the culture at a large-scale step. Preferably, the effective components are extracted from the upper ground parts of ginseng, which are generally not ingested by humans and are used only as feedstuff for farm animals. A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as the limit of the present invention.

EXAMPLE 1: Selection of raw material An individual wild ginseng was selected by a DNA fingerprinting method, Amplified Fragment Length Polymorphism (AFLP) -Silver Staining method, which is described by the present applicant in Korean Pat. Application No. 2002-36091 (titled "Method of identifying wild ginseng using DNA fingerprinting") . As a result, 80-year-old wild ginseng collected from Ganwon-do, Korea, was found to possess the unique complex genetic structure of wild ginseng. Tissue was induced from the selected individual and used as a raw material for the mass production of tissue-cultivated wild ginseng roots.

EXAMPLE 2: Surface sterilization of the raw material and direct induction of root differentiation to generate tissue-cultivated wild ginseng roots

Roots, obtained from the selected wild ginseng, were washed with tap water containing a few drops of a surfactant, Tween-20, several times to primarily remove soil and microorganisms. The wild ginseng roots were immersed in 70% ethanol for 1 min and washed with sterile distilled water. Subsequently, for second sterilization, the wild ginseng roots were immersed in a 2% hypochlorite solution for 5 min, and the sterilizing agent on the surface of the roots was removed using an absorbing agent. The surface sterilized roots were cut into four parts, inoculated in a medium supplemented with a plant growth factor, and observed for root differentiation. An MS medium was used, and an auxin, IBA, as the plant growth factor, was added to the medium in 0.5 ppm increments from 0.5 to 10 ppm (mg/L) . When subculture was carried out three times or more at 2-week intervals, rapid growth and differentiation of the roots was observed.

EXAMPLE 3: Propagation of tissue-cultured wild ginseng roots by suspension culture

When tissue-cultured wild ginseng roots were induced from the wild ginseng tissue, the tissue containing the generated tissue-cultured roots was suspended in a liquid culture medium and subcultured at about one-month intervals. For continuous subculture, the end portions of the tissue, where root growth occurs, were stimulated using surgical knives or forceps upon subculture, and abscission was minimized by unraveling tangled tissue using forceps. For liquid suspension culture, the use of the initial tissue inoculum in an amount of 10% based on the total medium volume was found to be effective, and biomass increased more than three times over a 4-week culture period. When MS, WPM (Lloyd and McCown), and SH (Schenk and Hildebrandt) media were used in the liquid suspension culture, no major difference was observed between the media in biomass increase of tissue-cultivated wild ginseng roots, but the WPM medium was found to be most effective. In order to adapt tissue-cultivated wild ginseng roots to liquid culture, the culture was scaled up stepwise to 5-L, 10-L and 20-L airlift bioreactors . Then, for secondary induction of tissue-cultivated wild ginseng roots, tissue-cultivated wild ginseng roots grown in the same bioreactor were used in comparative tests.

EXAMPLE 4 : Induction of secondary lateral roots from tissue-cultivated wild ginseng roots obtained by suspension culture The tissue-cultivated wild ginseng roots obtained by suspension culture were not found to increase in number when cultured in a flask or an airlift bioreactor, but secondary growth in roots was obviously observed. Among culture media used, WPM medium was most effective. In order to induce secondary lateral roots from the tissue-cultivated wild ginseng roots, the medium was supplemented with about 3 ppm of IAA, and the tissue- cultivated wild ginseng roots were inoculated in a density of about 7% based on the total medium volume and maintained in the same medium for a 5-week culture period.

EXAMPLE 5 : Culture of tissue-cultivated wild ginseng roots using small-scale bioreactors

The tissue-cultivated wild ginseng roots directly induced from the wild ginseng tissue were about 1-10 cm long and about 20-40 in number per piece of the wild ginseng tissue. To determine conditions optical for continuous production of the obtained tissue-cultured roots in small-scale bioreactors, the effect of the size of the tissue-cultivated roots on root growth was investigated. As a result, when the tissue-cultivated wild ginseng roots were inoculated in a length of 0.5 cm to 2.0 cm, a great increase in biomass was observed. In a 5-L bioreactor, a proper amount of the initial inoculum was about 5%, and it was much more effective when the medium was supplied in a manner of being exchanged at intervals of 7 to 10 days after which tissue-cultured roots had aggregated, than when the medium was added regardless of the aggregation of roots or upon batch culture. After four weeks, the tissue-cultured wild ginseng roots grown in the 5-L bioreactor were scaled up to a 20-L bioreactor. Upon this subculture, the harvested tissue- cultured roots were inoculated after being cut to a length of 0.5-2.0 cm using forceps and knives.

EXAMPLE 6: Large-scale (5 tons or higher) culture of tissue-cultured wild ginseng roots

The tissue-cultured wild ginseng roots were transported to a large-scale bioreactor using an aseptic transport system and propagated therein for about 45 days. During propagation, an increase in biomass results not from the regeneration of tissue from the tissue-cultured wild ginseng roots but from secondary growth and third growth of lateral roots, growth in thickness, and the like. It is most important in large-scale culture of tissue-cultured wild ginseng roots that tissue-cultured wild ginseng root tissue should be under a minimum of hydraulic pressure. To do this, a window is installed at the bioreactor to monitor that the amount of culture material and culture medium increases proportionally during the initial stage, thereby allowing direct evaluation of the effect of culture temperature and hydraulic pressure on growth of the end portions of the tissue-cultured wild ginseng roots. When temporal growth inhibition by culture temperature and hydraulic pressure is maintained for a long period of time, the root growth is stopped. Therefore, the maintenance of the culture temperature and hydraulic pressure within proper ranges is a very important factor.

EXAMPLE 7: Large-scale (5 tons or higher) culture I of tissue-cultured wild ginseng roots for enhancement of effective component content

Tissue-cultivated wild ginseng produced according to a general wild ginseng culture method such as the method of Example 6 has relatively low ginsenoside content and flavor compared to a wild ginseng raw material. To improve the ginsenoside content and flavor, ginsenosides were extracted with 70% ethanol from the upper ground parts (leaves, stems, etc.) of ginseng (including wild-simulated ginseng), concentrated, and adjusted to have a moisture content of 99%. Then, a ginsenoside extract from ginseng leaves was added to a culture medium in an amount of 1-5 ppm from the middle stage of the scale-up culture. The tissue-cultured wild ginseng roots thus obtained were found to have markedly increased contents of ginsenosides that are physiologically active components . These results are summarized in Table 1, below.

TABLE 1

EXAMPLE 8: Large-scale (5 tons or higher) culture II of tissue-cultured wild ginseng roots for enhancement of effective component content

To improve the content of physiologically active components in tissue-cultured wild ginseng, total crude saponin components were extracted from ginseng, concentrated, and adjusted to have a moisture content of 99%. Then, the crude saponin extract from ginseng leaves was added to a culture medium in an amount of 0.1-0.5 ppm from the middle stage of the scale-up culture. When treated with the crude saponin extract, tissue- cultured wild ginseng roots were found to have significantly increased content of ginsenosides that are physiologically active components . These results are summarized in Table 2, below.

TABLE 2

I weight) | | | | ^" (Note : Treatment with 0.5 ppm or higher of crude saponin resulted in a significant increase in ginsenoside content but was meaningless due to the high cost . )

Industrial Applicability As apparent from the results of examples, because of exclusion of the callus formation induction, the present invention does not employ a growth regulator such as 2,4-D, which has been proven not to be safe. In addition, the present invention remarkably increases the contents of pharmaceutically active components that are generally contained in low concentrations in tissue-cultured wild ginseng roots .

Claims

1. A method of mass-producing tissue-cultivated wild ginseng roots, comprising selecting a wild ginseng raw material by a DNA fingerprinting method and directly inducing root differentiation from the raw material to generate tissue-cultivated wild ginseng roots, the method excluding induction of callus formation in a base process including raw material selection, sterilization of the raw material, induction of callus formation, culture and mass propagation.

2. The method of mass-producing tissue-cultivated wild ginseng roots according to claim 1, wherein the root differentiation is directly induced from the raw material using a WPM or MS medium.

3. The method of mass-producing tissue-cultivated wild ginseng roots according to claim 1, wherein the root differentiation is directly induced from the raw material using a medium supplemented with 0.1-10 ppm of IAA or IBA.

4. The method of mass-producing tissue-cultivated wild ginseng roots according to claim 1, wherein the raw material is sterilized in two steps using two different sterilizing agents.

5. The method of mass-producing tissue-cultivated wild ginseng roots according to claim 4, wherein the sterilizing agents are removed from a surface of the raw material using an absorbing agent after the two-step sterilization.

6. The method of mass-producing tissue-cultivated wild ginseng roots according to claim 1, wherein the mass propagation is carried out using a medium supplemented with a ginseng extract containing crude saponins or ginsenosides .