인삼 사포닌의 생화학적 연구 ( Ⅹ ) 한국산 인삼 사포닌의 지질대사에 미치는 영향

주충노,이희봉,김두식 ( Chung No Joo,Hee Bong Lee,Doo Sik Kim ) 생화학분자생물학회 1977 BMB Reports Vol.10 No.2

It was attempted to observe the effects of ginseng saponins, one of the major components of Korean ginseng roots, on fatty acid oxidation and synthesis in vitro as well as on lipid metabolism in vivo and the following results were obtained. 1. It was found that the ginseng saponins stimulated significantly both fatty acid oxidation by rat hepatic mitochondrial preparation and synthesis by the cytosol fraction. 2. Distribution of radioactivities of lipids in liver, brain, adipose tissue and blood serum of albino rats administered with the ginseng saponins prior to acetate -1, 2-^(14)C injection intraperitoneally have been investigated. 3. The high radioactivities of lipid in the above tissues of test group except blood serum appeared at about 30 min. to 60 min. after acetate injection while those of control group was observed at about two hours after suggesting that the ginseng saponins stimulated the biosynthesis of lipids in the above tissues. 4. Under the experimental conditions in this study, it was realized that biological half lives of hepatic cholesterol and fatty acid of test rat were found to be 19min. and 44min. while those of the corresponding lipids of control rat were 30min. and 70min. respectively suggesting that the ginseng saponins might stimulate the metabolism, both biosynthesis and degradation of cholesterol and fatty acid of the liver of this animal. 5. In the brain, the highest specific radioactivities of cholesterol and fatty acid of test group were observed at 15min. and 30min. after acetate-1, 2-^(14)C injection while those of the corresponding lipids of control group were found at 216min. and 156min. after the injection respectively suggesting that the ginseng saponins stimulated greatly the metabolism of brain lipids. 6. In the adipose tissue, the highest specific radioactivity of fatty acid of test group was observed at 61min. after acetate -1, 2-^(14)C injection while that of the control group appeared at 124 min. after the injection suggesting that the ginseng saponin stimulated Carbon-14 incorporation into fatty acid in this tissue. 7. It appeared that the high radioactivity of blood serum lipid of control group was observed at 216min. after acetate-1, 2-^(14)C injection but the radioactivity peak of test group was observed at 79 minutes after the injection of 1, 2-^(14)C-acetate signifying that the ginseng saponin affected not only lipid metabolism but also lipid transport in the animal body.

Biochemical Studies on the Absorption of Ginseng Saponin and Its Effect on Metabolism in Animal Body

주충노,구자현,이희봉,윤종복,변영숙,Joo, Chung-No,Koo, Ja-Hyun,Lee, Hee-Bong,Yoon, Jong-Bok,Byun, Yung-Sook 생화학분자생물학회 1982 한국생화학회지 Vol.15 No.3

인삼 뿌리의 절편을 효소원으로 사용하여 방사성 인삼 사포닌을 제조하였다. 1 mg의 인삼 사포닌(방사성 사포닌 1.58 ${\mu}Ci$함유)을 쥐에게 구경 투여하고, 3시간 후의 각 장기의 수용성 분획의 방사능을 조사한 결과 흡수된 동위원소 회수율이 약 17%토 추정되었으며 최소한 0.17mg의 사포닌이 흡수된 것으로 생각된다. 인삼 사포닌은 쥐 간의 미토콘드리아, 미크로좀 분획에 의한 인지질의 합성을 크게 촉진함이 관찰되었고, 과량의 주정을 투여하였을 때 미크로좀의 에탄올 산화계(MEOS)가 크게 활성화됨으로서 해독작용에 큰 효과가 있을 것으로 기대된다. 고 콜레스테롤 식이의 장기 투여로 유발한 고 콜레스테롤 혈증의 초기에서 인삼 사포닌은 대동맥 조직으로의 클레스테롤의 유입률을 저하시킴으로서 대동맥의 atheroma생성을 예방하는 효능이 있는 것으로 생각한다. Radioactive ginseng saponins were prepared using sliced raw ginseng roots enzyme source. The experimental results obtained from rats killed 3hrs. after the oral administration of 1mg of the saponin containing 1.58${\mu}Ci$ per rat, showed that the saponin absorbed at least 0.17mg (17%). It was observed that the biosynthesis of phospholipids by rat hepatic mitochondrial and/or microsomal fraction was stimulated significantly and the microsomal ethanol oxidizing system was activated greatly in the presence of suifiable amounts of the saponin. The oxidation of ethanol by MEOS in rats dosed with 2mg/day/rat for 8 weeks prior to oral feeding of excess amounts of ethanol was also found accelerated sufficiently. It was realized that the penetration rate of cholesterol from blood onto aorta tissue was diminished appreciably in ginseng administered rabbits fed with high cholesterol diet than control group suggesting that the saponin has some preventive effect against aortic atheroma formation.

인삼 사포닌의 체내흡수 및 대사에 관한 생화학적 연구

주충노,구자현,이희봉,윤종복,변영숙 ( Chung No Joo,Ja Hyun Koo,Hee Bong Lee,Jong Bok Yoon,Yung Sook Byun ) 생화학분자생물학회 1982 BMB Reports Vol.15 No.3

Radioactive ginseng saponins were prepared using, : sliced raw ginseng roots enzyme source. The experimental results obtained from rats killed 3hrs. after the oral administration of 1 ㎎ of the saponin containing 1.58μCi per rat, showed that the` saponin absorbed at least 0.17㎎: (17%). It was observed that the biosynthesis of phospholipids by rat hepatic mitochondrial and/or microsomal fraction was stimulated significantly and the microsomal ethanol oxidizing system was activated greatly in the presence of suifiable amounts of the saponin. The oxidation of ethanol by MEOS in rats dosed with 2㎎/dad/rat for 8 weeks prior to oral feeding of excess amounts of ethanol was also found accelerated sufficiently. It was realized that the penetration rate of cholesterol from blood onto aorta tissue was diminished appreciably in ginseng administered rabbits fed with high cholesterol diet than control group suggesting that the saponin has some preventive effect against aortic atheroma formation.

Biochemical Studies on Ginseng Saponins(X) The Effect of Ginseng Saponins on Lipid Metabolism

주충노,이희봉,김두식,Joo, Chung-No,Lee, Hee-Bong,Kim, Doo-Sik 생화학분자생물학회 1977 한국생화학회지 Vol.10 No.2

한국산 인삼(Panax ginseng C.A. Meyer) 뿌리의 주요 성분 중의 하나인 인삼 사포닌류가 지방산의 산화와 합성에 미치는 영향(in vitro)과 아울러, 동위원소 $^{14}C$으로 표지된 acetate-1, 2-$^{14}C$을 쥐에 투여하여 지질대사에 미치는 영향(in vitro)을 관찰하고 다음과 같은 결과를 얻었다. 1. 인삼 사포닌은 쥐의 간 미토콘드리아에 의한 지방산 산화와 시토졸에 의한 지방 합성을 다같이 촉진하였다. 2. 쥐에게 인삼 사포닌을 투여하고 4시간 후 acetate-1, 2-$^{14}C$을 투여하여 적당 시간후(5분~240분)의 간장, 뇌, 지방 조직 및 혈청의 지질 방사능의 분포를 조사하였다. 3. 혈청을 제외한 다른 상기조직에서의 지질 방사능은 실험군에서는 30분에서 60분사이에 큰 방사성이 관찰되었고, 대조군의 경우는 2 시간 후에나 큰 방사성이 나타나는 것으로 미루어 보아 인삼 사포닌이 상기 조직에서 지방 합성을 크게 촉진한 것으로 생각된다. 4. 본 연구의 실험 조건하에서 측정된 간 조직의 콜레스테롤과 지방산의 생물학적 반감기는 대조군의 경우, 각각 30분과 70분인데 비하여 실험군은 19분과 44분이었다. 이와같은 실험 결과는 인삼 사포닌이 콜레스테롤과 지방산의 대사(분해와 합성)를 촉진하는 것으로 해석된다. 5. 뇌에서의 콜레스테롤과 지방산의 최고 방사능은 실험군의 경우 동위원소 주입 후 각각 15분과 30분이고 대조군의 경우는 각각 216분과 156분이었으며, 인삼 사포닌이 뇌의 지질대사를 크게 촉진하는 것으로 관찰되었다. 6. 지방조직에서는 실험군의 최고 방사능이 동위원소 주입 후 61분이었고 대조군은 124분이었으며, 인삼 사포닌의 동위원소의 지방산으로의 도입을 크게 촉진하였다. 7. 혈청 지질의 최고 방사능은 실험군의 경우 동위원소 주입 후 79분, 대조군은 216분이었으며, 인삼사포닌은 지질대사뿐 아니라 지질의 이동에도 크게 영향을 미치는 것으로 예측된다. It was attempted to observe the effects of ginseng saponins, one of the major components of Korean ginseng roots, on fatty acid oxidation and synthesis in vitro as well as on lipid metabolism in vivo and the following results were obtained. 1. It was found that the ginseng saponins stimulated significantly both fatty acid oxidation by rat hepatic mitochondrial preparation and synthesis by the cytosol fraction. 2. Distribution of radioactivities of lipids in liver, brain, adipose tissue and blood serum of albino rats administered with the ginseng saponins prior to acetate -1, 2-$^{14}C$ injection intraperitoneally have been investigated. 3. The high radioactivities of lipid in the above tissues of test group except blood serum appeared at about 30 min. to 60 min. after acetate injection while those of control group was observed at about two hours after suggesting that the ginseng saponins stimulated the biosynthesis of lipids in the above tissues. 4. Under the experimental conditions in this study, it was realized that biological half lives of hepatic cholesterol and fatty acid of test rat were found to be 19min. and 44min. while those of the corresponding lipids of control rat were 30min. and 70min. respectively suggesting that the ginseng saponins might stimulate the metabolism, both biosynthesis and degradation of cholesterol and fatty acid of the liver of this animal. 5. In the brain, the highest specific radioactivities of cholesterol and fatty acid of test group were observed at 15min. and 30min. after acetate-1, 2-$^{14}C$ injection while those of the corresponding lipids of control group were found at 216min. and 156min. after the injection respectively suggesting that the ginseng saponins stimulated greatly the metabolism of brain lipids. 6. In the adipose tissue, the highest specific radioactivity of fatty acid of test group was observed at 61min. after acetate-1, 2-$^{14}C$ injection while that of the control group appeared at 124 min. after the injection suggesting that the ginseng saponin stimulated Carbon-14 incorporation into fatty acid in this tissue. 7. It appeared that the high radioactivity of blood serum lipid of control group was observed at 216min. after acetate-1, 2-$^{14}C$ injection but the radioactivity peak of test group was observed at 79 minutes after the injection of 1, 2-$^{14}C$-acetate signifying that the ginseng saponin affected not only lipid metabolism but also lipid transport in the animal body.

인삼 사포닌의 체내대사 ( Ⅰ )

이희봉,주충노 ( Hee Bong Lee,Chung No Joo ) 생화학분자생물학회 1983 BMB Reports Vol.16 No.2

^(14)C-labelled ginseng saponins from ^(14)C-acetate were prepared using the root slices of panaxginseng C.A. Meyer as an enzyme source and the products were demonstrated to be identical with the saponins extracted from the roots by thin layer chromat ography, high performace liquid chromatography and autoradiography. The radioactivity of the liver was monitored on time course after the feeding of 1 ㎎ of ginseng saponin containing ^(14)-C-labelled saponin. The extract from the liver of rats killed at one hr. after the oral administration of 1 ㎎ of ginseng saponins containing the ^(14)C-saponins was analyzed found that 25 % of the radioactivity recovered was found as undissociated form of the saponin. It semed that the half life of the saponin might be 4∼6 hrs and the concentration of the saponin would be 10^(-5) % level in the liver.

Incorporation of Foreign Gene with Ti Plasmid Vector System: (II) Expression of E. coli Thioredoxin Gene in Cultured Tobacco Cells.

이희봉,주충노,홍순주,김성완,임창진,김영명,Lee, Hee-Bong,Joo, Chung-No,Hong, Soon-Joo,Kim, Seong-Wan,Lim, Chang-Jin,Kim, Young-Myeong 생화학분자생물학회 1988 한국생화학회지 Vol.21 No.4

본 연구에서는 E. coli thioredoxin 유전자를 함유하고 있는 재조합된 plasmid pKDB3의 담배세포내로의 도입을 시도하고, 도입된 thioredoxin 유전자의 발현을 조사하였다. 담배(Nicotina tabacum cv Xanthi) 세포로의 재조합 plasmid pKDB3의 도입은 담배잎 절편과 재조합 DNA 및 helper Ti plasmid pTiBo 542를 함유하고 있는 Agrobacterium tumefaciens strain A281과의 cocultivation 방법을 이용하여 행하여졌으며, 형질전환된 담배세포는 항생물질 저항성 배지에서의 callus 형성 여부로 선별되었고, 선별된 형질전환된 calli는 shoot와 root 형성을 위해 적절한 MS agar 배지에서 계속 키워졌다. 이와 같이 형질전환된 담배세포에서 완전한 식물체로 재생된 담배잎에서의 E. coli thioredoxin 유전자의 발현을 조사한 결과 thioredoxin 활성이 형질전환된 담배세포가 정상세포에 비해 9배 정도 더 큰 것으로 나타났다. 이러한 일련의 결과들은 E. coli thioredoxin 유전자가 성공적으로 담배세포에 들어가서 높은 수준으로 발현됨을 보여주고 있다. This study was performed to observe the expression of E. coli thioredoxin gene incorporated in tobacco cells. The recombinant DNA used, pKDB3, had been constructed from a Ti plasmid vector pGA658 and a bacterial plasmid pCJF4 harboring E. coli thioredoxin gene, as described in the preceding paper (Lee et al., 1988). The leaf discs of plant (N. tabacum cv Xanthi) were transformed to kanamycin resistance by the cocultivation with Agrobacterium A281 containing plasmid pKDB3. Transformed leaf discs were cultured in MS agar medium with kanamycin for callus induction. Kanamycin-resistant tobacco calli were continuously grown in MS agar medium for shoot induction, and then in MS agar medium for root induction. Expression of E. coli thioredoxin gene in the plant tissue regenerated from transformed tobacco cells was confirmed by DTNB assay. The thioredoxin activity of transformed tobacco cells was much higher (about 9 times) than that of normal tobacco cells. Our results suggest that E. coli thioredoxin gene was successfully incorporated into tobacco cells, and the incorporated bacterial gene could be expressed at a high level.

Ti Plasmid Vector System 을 이용한 외래 유전자의 도입 : ( 1 ) A . tumefaciens 로의 E . coli Thioredonxin 유전자의 도입

이희봉,주충노,홍순주,김성완,임창진,김영명 ( Hee Bong Lee,Chung No Joo,Soon Joo Hong Seong Wan Kim,Chang Jin Lim,Young Myeong Kim ) 생화학분자생물학회 1988 BMB Reports Vol.21 No.4

In this part of study on the incorporation of foreign gene into plant cells, a derivative of Ti plasmid vector (pGA658), containing E. coli thioredoxin gene, was prepared and introduced into Agrobacterium tumefaciens. A recombinant plasmid, pKDB3, was constructed by transferring HindIII-BamHI DNA fragment of pCJF4, including E. coli thioredoxin gene, into HindIII-BgIII restriction sites of plasmid pGA658. By doing this, E. coli thioredoxin gene is expected to express from nos promoter of pGA658 after the incorporation into plant cells. The structure of DNAs isolated from kanamycin-resistant E. coli transformants was convinced by restriction mapping. As a preceding step before incorporation into plant cells, the recombinant plasmid pKDB3 was transformed into A. tumefaciens by freeze-thaw procedure. In Agrobacterium transformants, the expression of E. coli thioredoxin gene was positively observed, and this suggested the stable existence of the E. coli gene.

Metabolism of Ginseng Saponin in Animal Body (I)

이희봉,주충노,Lee, Hee-Bong,Joo, Chung-No 생화학분자생물학회 1983 한국생화학회지 Vol.16 No.2

인삼(Panax ginseng C.A. Meyer) 뿌리 절편을 효소원으로 사용하여 $^{14}C$-acetate로부터 제조한 방사성 인삼 사포닌을 Thin layer chromatography, High performance liquid chromatography, Autoradiography 등으로 확인하였다. 1 mg의 인삼 사포닌을 쥐에게 구경 투여하고 1시간 후, 간장으로부터의 추출물을 분석한 결과, 회수된 방사능의 약 25%가 분해되지 않은 사포닌 상태로 존재하고 있음을 확인하였다. 또한 인삼 사포닌 투여 (구경)후 시간에 따른 방사능의 변화 양상을 초사한 결과 간에서의 사포닌의 반감기는 4~6시간, 인삼 사포닌의 농도는 $10^{-5}%$ 수준으로 예측되었다. $^{14}C$-labelled ginseng saponins from $^{14}C$-acetate were prepared using the root slices of panaxginseng C.A. Meyer as an enzyme source and the products were demonstrated to be identical with the saponins extracted from the roots by thin layer chromatography, high performace liquid chromatography and autoradiography. The radioactivity of the liver was monitored on time course after the feeding of 1 mg of ginseng saponin containing $^{14}C$-labelled saponin. The extract from the liver of rats killed at one hr. after the oral administration of 1 mg of ginseng saponins containing the $^{14}C$-saponins was analyzed found that 25% of the radioactivity recovered was found as undissociated form of the saponin. It semed that the half life of the saponin might be 4~6 hrs and the concentration of the saponin would be $10^{-5}%$ level in the liver.

The Incorporation of Eucaryotic Gene Using Ti Plasmid Vector: I. The Introduction of Yeast Homoserine Dehydrogenase Gene into Agrobacterium tumefaciens

이희봉,주충노,홍순주,김성완,임창진,김영명,Lee, Hee-Bong,Joo, Chung-No,Hong, Sun-Joo,Kim, Seong-Wan,Lim, Chang-Jin,Kim, Young-Myeong 생화학분자생물학회 1989 한국생화학회지 Vol.22 No.1

효모에 있어서 threonine과 methionine 생합성에 관여하는 효소들 중 하나인 homoserine dehydrogenase가 식물체내로 도입된 후, 그 역할을 조사하고자 Ti plasmid에서 유도된 pGA658 vector와 homoserine dehydrogenase 유전자 함유 DNA의 재조합을 시도하였다. 그 결과 pGA658 vector의 nos promoter 다음에 효모의 homoserine dehydrogenase 유전자를 함유한 DNA 절편이 방향이 반대로 삽입된 재조합 plasmids가 성공적으로 만들어졌다. 형질전환된 E. coli와 Agrovbacterium tumefaciens는 적절한 항생물질 함유배지에서 저항성을 나타내었으며, 그들의 plasmid를 분리하여 여러가지 제한효소를 이용한 크기를 조사해 보니, 예상되는 크기와 일치하는 것을 관찰할 수 있었다. 또한 형질전환된 E. coli와 A. tumefaciens내에서의 yeast homoserine dehydrogenase 유전자 발현을 조사하기 위해 그 효소의 활성을 조사해 본 결과, 형질전환된 E. coli에서는 대조군에 비해 약간의 증가를 나타내었으나, 형질전환된 A. tumefaciens에서는 변화가 없었다. DNA recombination using plasmid pGA658, a Ti plasmid vector, was tried to study the role of yeast homoserine dehydrogenase after introducing its gene into plant cells. Two recombinant plasmids with opposite orientation of the gene DNA under nos promoter of vector pGA658, pKDB1 and pKDB2, were constructed by the aid of two intermediate vectors, pUC7 and pUC119. Transformed E. coli and Agrobacterium tumefaciens were confirmed by the resistance to appropriate antibiotics and by sizing DNA restriction fragments after plasmid isolation. Assay of homoserine dehydrogenase activity showed that a little increase in its activity was detected in transformed E. coli, but no increase in transformed Agrobacterium tumefaciens, compared with its untransformed strain.

Incorportation of Foreign Gene with Ti Plasmid Vector System: (I) Introduction of E. coli Thioredoxin Gene into A. tumefaciens.

이희봉,주충노,홍순주,김성완,임창진,김영명,Lee, Hee-Bong,Joo, Chung-No,Hong, Soon-Joo,Kim, Seong-Wan,Lim, Chang-Jin,kim, Young-Myeong 생화학분자생물학회 1988 한국생화학회지 Vol.21 No.4

외래 유전자를 도입함으로써 식물체에 새로운 유전정보를 부여하려는 노력이 다각적으로 수행되어 왔으며 최근에는 넓은 숙주범위를 갖는 Ti plasmid를 vector로 이용한 외래 유전자의 도입이 활발히 진행되고 있다. 본 연구에서는 Ti plasmid vector의 일종인 pGA658을 이용하여 광합성 조절 등 여러가지 기능을 갖고 있는 것으로 알려져 있는 thioredoxin 유전자를 식물체내로 도입하여 식물체내에서의 그 역할을 조사할 목적으로 DNA 재조합을 시도하였다. E. coli thioredoxin 유전자를 함유하는 pCJF 4의 Hind III-BamHI DNA fragment를 pGA658의 Hind III-Bgl II site에 삽입시키고 E. coli에 transformation 한 후 형질전환체의 확인은 항생물질 marker에 대한 저항성으로, 재조합 DNA의 유전자 지도는 여러가지 제한효소를 이용한 절편의 크기 확인으로, 그리고 도입 유전자의 발현은 효소 활성 측정에 의 하여 확인하였다. 이렇게 확인된 재조합된 plasmid pKDB3를 담배세포로 도입하기 위한 전 단계로서 freeze-thaw 방법으로 Agrobacterium에 transformation 한 후 항생물질 저항성과 제한효소를 이용한 절편의 크기 확인 및 효소 활성 측정에 의하여 pKDB3가 도입되어 안정하게 유지됨을 확인할 수 있었다. In this part of study on the incorporation of foreign gene into plant cells, a derivative of Ti plasmid vector (pGA658), containing E. coli thioredoxin gene, was prepared and introduced into Agrobacterium tumefaciens. A recombinant plasmid, pKDB3, was constructed by transferring HindlII-BamHI DNA fragment of pCJF4, including E. coli thioredoxin gene, into HindIII-BglII restriction sites of plasmid pGA658. By doing this, E. coli thioredoxin gene is expected to express from nos promoter of pGA658 after the incorporation into plant cells. The structure of DNAs isolated from kanamycin-resistant E. coli transformants was convinced by restriction mapping. As a preceding step before incorporation into plant cells, the recombinant plasmid pKDB3 was transformed into A. tumefaciens by freeze-thaw procedure. In Agrobacterium transformants, the expression of E. coli thioredoxin gene was positively observed, and this suggested the stable existence of the E. coli gene.