KR910004950B1 - Novel cell line acg-1 for panax ginseng - Google Patents
Novel cell line acg-1 for panax ginseng Download PDFInfo
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Abstract
Description
본 발명은 인삼조직배양을 통하여 선발한 ACG-1 세포주 및 이를 이용한 진세노사이드 Rg1의 제조방법에 관한 것이다.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.
인삼(Panax ginseng)은 의약품으로 오랫동안 애용되어 온 고가의 약용식물로서 약효를 나타내는 물질인 사포닌(saponin), 폴리아세틸렌(polyacetylene), 알카로이드(alkaloid), 페놀(phenol) 등이 포함되어 있어 이에 관한 연구가 집중적으로 수행되고 있다. 특히 오래전부터 인삼 약효성분으로 알려진 사포닌에 관한 최근의 연구 보고에 의하면 인삼 사포닌 성분중 진세노사이드 Rg1(Gin-senoside Rg1)이 심장병 등에 특이한 약효가 있음이 밝혀진 바 있고, 인삼 사포닌 약효 성분중에는 상기 진세노사이드 Rg1(이하 "Rg1"이라 함)외에도 Rg2,Rf,Re,Rb2,Rb1 등이 함유되어 있는 것으로 알려져 있다. 이들의 성분 분포 즉, 진세노사이드 패턴(Ginsenoside Pattern)은 인삼의 약효를 예시하는 인자로서, 통상 상기 성분들을 트리올계(Rg1,Re,Rg2,Rf)와 디올계(Rd,Rc,Rb2,Rb1)로 대분하여 PT/PD비율로서 나타내고 있으며 일반 재배인삼의 경우는 PT/PD비율이 약 1로서 트리올계 진세노사이드와 디올계 진세노사이드가 비슷하게 들어있는 것으로 밝혀졌다. 그러나 이들 성분들이 모든 질병에 같은 약효로 작용하는 것은 아니고 상기한 바와 같이 질병에 따라 선택적으로 효과를 나타내는 성분이 있으며 특수성분의 선택사용은 약효를 높일 수 있는 바람직한 방법이 될 수도 있다.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.
인삼의 세포배양 기술분야에 있어서, 조직배양을 통한 인삼세포의 증식으로 사포닌의 대량생산이 가능하게 되었음은 본 발명과 함께 특허출원된 "ACG-2 세포주 및 이를 이용한 사포닌 생산방법"에서 공개된 바 있다. 이러한 조직배양방법을 이용하면 특정 성분만의 대량생산 즉, 상기한 바와 같이 심장병 등에 약효가 인정된 Rg1 등의 단일성분을 대량으로 생산하는 것이 가능하게 된다.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.
본 발명의 목적은 Rg1을 다량 함유하는 신규의 인삼 세포주를 제공하는 것이다. 본 발명의 또 하나의 목적은 신규의 인삼세포주를 이용하여 단일성분의 Rg1을 대량으로 생산하는 방법을 제공하는 것이다. 이러한 본 발명의 목적 및 기타의 부수적인 목적은 이하의 설명에서 좀더 명백해질 것이다.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.
본 명세서에서 ACG-1으로 명명되는 선발 세포주는 고려인삼의 뿌리로부터 유기된 세포주를 고체 배양기에 접종하여 계대 배양시킨 것이다. 즉, Rg1이 많이 포함된 인삼 뿌리의 동체에서 유기된 세포주를 M-S(Murashige-Skoog) 기본배지에 수크로스 3중량%(M-S 기본배지 기준-이하 동일하다)와 2,4-D 0.1mg/1를 첨가한 고체배양기에 접종하여 배양시키되, 4-5주 이상 배양시켜 살아 남는 세포주를 계속 계대 배양시키면 본 발명 세포주 ACG-1을 얻게 된다. 이 세포주의 생물학적으로 순수한 배양물은 1989. 10. 17자로 한국과학기술원에 기탁되어 KCTC 8473 P의 기탁번호를 부여받았다. 이렇게 선발된 세포주를 사용하여 각종 영양 배지가 세포주의 생육 및 진세노사이드 Rg1의 생성에 미치는 효과를 더욱 측정하고, 이 세포주에 대한 가장 적합하고 효과적인 배지조성과 발효조건을 결정하였다.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.
[세포주의 특성][Characteristics of Cell Line]
상기한 바와 같이 인삼 뿌리에서 유기시킨 세포주를 2,4-D를 함유하는 고체 배양기에서 계대 배양하여 선발한 본 발명의 ACG-1 세포주(기탁번호: KCTC 8473 P)의 특성은 표 Ⅰ과 같다. 비교을 위해, 본 발명에서 선발한 ACG-1 세포주와 인삼의 일반 세포주를 M-S 기본배지에 수크로스 3중량%와, 2,4-D 0.1mg/1를 첨가한 고체 배양기에서 25℃로 50일간 배양한 후 색, 건물중 및 사포닌 함량과 진세노사이드 패턴을 측정 분석하여 재배인삼인 백삼과 비교하였다. 표 Ⅰ의 진세노사이드의 패턴에 있어서, 본 발명은 ACG-1 세포주의 Rg1함량은 6.09mg/g으로서 일반 세포주나 백삼의 그것에 비해 월등히 높음을 알 수 있다.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.
[표 Ⅰ]TABLE I
*g/flask ** mg/g* g / flask ** mg / g
[생육조건][Growing conditions]
ACG-1 세포주는 조절된 조건하에서 적절한 수성영양 배지로 배양시킴으로써 생육이 진행된다. 배지는 동화가능한 탄소, 질소 및 무기염의 공급원을 함유하는데 수크로스와 2,4-D가 M-S 기본배지에 첨가되어 본 발명의 가장 적절한 배지조성으로 사용된다. 이러한 생육조건 위한 배지조성은 선발 세포주의 생육면에서 뿐 아니라 본 발명의 목적상, 생성 세포주의 진세노사이드 패턴을 고려함이 필수적이다. 또 한가지 생육 조건에서 고려되어야 할 중요한 인자는 온도와 명암조건이다. 본 발명 세포주인 ACG-1 세포주는 광(光), 암(暗)배양시 그 생장에는 별 영향이 없었으나 Rg1의 함량은 광배양시에 증가하고 있음을 확인할 수 있었다. 본 발명 ACG-1 세포주의 배지조성은 M-S 기본배지에 수크로스 1-5중량%, 2,4-D 0.05-5mg/1가 바람직하며, 가장 바람직하기로는 수크로스 1-5중량% 2,4-D 0.1-3mg/1이다. 배양온도는 15-25℃, 광처리하에 배양시킨다. 본 명세서에 기술한 영양배지는 사용할 수 있는 광범위한 종류의 배지의 단순한 예에 불과하며 이것으로 제한되지는 않는다. 이하 본 발명을 실시예를 통하여 상세히 설명한다. 생성된 세포주로부터 아래의 방법에 의해 사포닌의 진세노사이드 패턴을 측정 분석하고 그 결과를 표 Ⅱ에 표시하였다.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.
[진세노사이드 패턴 측정방법]Ginsenoside Pattern Measurement Method
수확된 시료를 60℃에서 건조시켜 정확히 1g을 취한 후 메탄올, 에테르, 클로로포름 및 부탄올 순으로 용매 추출하여 사포닌을 추출한다. 다음 이 추출액을 증류수로 3회 세척하고 농축하여 분석시료로 하였다.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.
이렇게 농축된 사포닌을 HPLC용 메탄올로 용해시킨 다음 HPLC로 진세노사이드 함량 및 패턴을 분석하였다.This concentrated saponin was dissolved in methanol for HPLC and then analyzed for ginsenoside content and pattern by HPLC.
[실시예 1]Example 1
M-S기본배지에 수크로스를 3중량%, 2,4-D를 0.1mg/1 첨가한 배지를 35ml씩 분주한 고체배양기에 선발 세포주 ACG-1을 접종하여 25℃에서 1,000룩스 광을 조사하며 배양하여 50일 후에 세포조직을 수확하고 진세노사이드 패턴을 측정 분석하였다.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.
[실시예 2]Example 2
실시예 1의 2,4-D 첨가량 0.1mg/1 대신에 3mg/1을 첨가한 배지를 사용하는 외에는 실시예 1과 동일한 방법으로 세포조직을 수확하고 진세노사이드 패턴을 측정분석하였다.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.
[실시예 3]Example 3
실시예 1의 1,000룩스 광을 조사하는 대신에 암상태에서 ACG-1세포주를 배양하는 외에는 실시예 1과 동일한 방법으로 세포조직을 수확하고 진세노사이드 패턴을 측정분석하였다.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.
[비교예 1]Comparative Example 1
실시예 1의 ACG-1세포주를 사용하는 대신에 일반 세포주를 사용하여 실시예 1과 동일한 방법으로 일반 세포조직을 수확하고 진세노사이드 패턴을 측정분석하였다.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.
[비교예 2]Comparative Example 2
비교예 1의 2,4-D 첨가량 0.1mg/1 대신에 3mg/1을 첨가한 배지를 사용하는 외에는 비교예 1과 동일한 방법으로 일반 세포조직을 수확하고 진세노사이드 패턴을 측정분석하였다.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.
[표 Ⅱ]TABLE II
표 Ⅱ에 나타난 바와 같이, 본 발명의 ACG-1 세포주는 일반 세포주에 비해 같은 생육조건에서 Rg1의 생성능이 6 내지 8배에 달한다.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.
2,4-D 농도에 따른 ACG-1 세포주의 Rg1 생산량은 고농도에서(1.33mg/g)보다 저농도에서 (6.09mg/g) 약 4.6배 더 생산됨을 알 수 있고 광 배양시가 암 배양시보다 약 1.7배 더 많이 생성됨을 알 수 있다. 이와 같이, 본 발명 ACG-1 세포주는 심장병 등에 약효가 있는 Rg1을 다량 함유하는 신규의 세포주로서 본 발명 방법에 따라 조직배양할 경우 단일성분 순품으로 Rg1의 대량생산이 가능하다.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.
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KR1019890016000A KR910004950B1 (en) | 1989-11-04 | 1989-11-04 | Novel cell line acg-1 for panax ginseng |
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