사리풀 (Hyoscyamus niger L.) 모상근의 Tropane Alkaloid 생성에 미치는 배지, 배양주기, Sucrose 및 Dextrose의 영향

최철희,김용해,양덕조 한국식물생명공학회 1999 식물생명공학회지 Vol.26 No.2

Agrobacterium tumefaciens $A_4$T에 의해 형질전환된 사리풀 (Hyoscyamus niger L.)의 모상근으로부터 tropane alkaloids (scopolamine, hyoscyamine)의 생산성 향상을 위한 최적배지, 배양기간 및 탄수화물의 영향을 조사하였다. Tropane alkaloids의 생산성이 우수한 모상근 (HNl8, HN57)의 최적 배지는 SH배지이며, 모상근의 생장, tropane alkaloids 함량 및 생산성에서 조사한 6가지 배지보다 높게 나타났다. Tropane alkaloids생산을 위한 최적 배양기간은 HNl8 clone이 6주, HN57 clone은 5주로 나타났으며, 배양기간에 따라 scopolamine의 함량은 계속적으로 증가하는데 반하여 hyoscyamine의 함량은 일정 수준을 유지하는 것으로 나타났다. 두 세포주는 sucrose농도가 7%까지 증가할수록 생장률이 증가하는 반면, tropane alkaloids 함량은 감소하였으며, tropane alkaloids생산성은 3% sucrose처리구에서 가장 높게 나타났다. HN18 clone의 경우 최적 sucrose(3%)및 dextrose (2%)에서 유사한 tropane alkaloids 생산성을 나타내었다. 하지만 HN57 clone은 sucrose 처리구에 비하여 dextrose 처리구에서 매우 낮은 tropane alkaloids생산성을 나타내었다. We have investigated the effect of culture conditions on tropane alkaloids (scopolamine, hyoscyamine) production in hairy root cultures of Hyoscyamus niger L. induced by Agrobacterium tumefaciens $A_4$T. SH medium was the best for tropane alkaloids production from the hairy root clones, HN18 and HN57. The optimum culture peroid was 5 weeks for HN18 clone and 6 weeks for HN57 clone, respectively. The optimum sucrose and dextrose concentrations in tropane alkaloids productivity were 3% and 2%, respectively. The growth of both HN18 and HN57 clones increased with as sucrose concentration increase up to 7% sucrose, but tropane alkaloid contents was significantly decreased. In the HN18 clone, the optimum concentration of sucrose for alkaloids productivity was 5% and those of dextrose was 2%. The productivity of tropane alkaloids for HN57 clone under dextrose treatments was quite a low level compared to sucrose treatments.

Eschscholtzia californica의 현탁 세포배양에서 질소원 조절에 의한 세포 성장 및 Benzo[c]phenanthridine Alkaloids 생산량 향상

이송은(Song-Eun Lee),이홍순(Hong Soon Rhee),손석영(Seok-Young SON),박종문(Jong Moon Park) 한국생물공학회 2009 KSBB Journal Vol.24 No.2

본 연구에서는 Eschscholtzia californica의 이차 대사산물인 benzo[c]phenanthridine alkaloids의 생산량을 향상시키기 위해 주요 영양성분인 질소원 농도를 조절하여 배지의 조성을 최적화하였다. 그 결과, 질산과 암모늄 이온의 초기 농도 비율은 세포 성장과 alkaloids 생산량 증대에 중요한 영향을 미치는 인자로서 작용함을 볼 수 있었다. 총 질소 농도를 standard MS배지의 총 질소원 농도와 동일하게 유지하고 (60 mM), 농도 비율 다양하게 조절했을 때, 세포 성장과 alkaloids 생산량은 질산과 암모늄 이온을 각각 단독으로 이용할 때 보다 효율적으로 증대함을 볼 수 있었다. 최대 성장 (9.84 g DCW/L)과 alkaloids 생산량 (60.72 mg/L)은 50:10의 비율에서 나타났으며, 농도 비율의 감소는 세포 성장을 억제하고 alkaloids 생산량을 감소 시켰다. 또한, 질산과 암모늄 이온이 세포 성장과 alkaloids 생산량에 미치는 영향력을 보다 명확히 확인하기 위해 질산 이온과 암모늄 이온 농도를 각각 조절한 결과, 암모늄 이온이 증가할수록 alkaloids 생산량은 비슷하나, 세포 성장은 감소하였으며, 또한, 질산 이온의 농도를 증가시킬수록 세포 성장은 비슷한 값을 나타내었지만, alkaloids 생산량은 다양한 차이를 나타내었다. 본 실험에서 세포성장과 alkaloids 생산량에 적합한 농도는 50:25 (mol/mol) 비율 에서 나타났으며, 본 실험을 통해 Eschscholtzia californica의 현탁 세포배양에서 질소원 농도의 조절을 통한 외부 환경 조절은 세포 성장과 alkaloids 향상에 매우 효과적이며, 소량의 암모늄 이온의 첨가 시 질산 이온 농도조절은 세포 성장을 억제하지 않으면서 alkaloids 생산량을 유도하는데 적합한 이온임을 확인하였다. The effect of nitrogen source on cell growth and benzo[c]phenanthridine alkaloids production by modifying NO₃-:NH₄+ ratio in cell suspension culture of Eschscholtzia californica was investigated. When total nitrogen concentration is maintained (60 mM), maximum benzo[c]phenanthridine alkaloids production is about 60.72 mg/L at 50:10 (mol/mol). This productivity was 3.8 times higher than that obtained when cells were grown instandard MS medium. The decrease of NO₃-:NH₄+ ratio at 60 mM of total nitrogen caused the decline of both growth and benzo[c]phenanthridine alkaloids production. Under the same concentration of NO₃- (50 mM), higher concentration of NH₄+ inhibited cell growth strongly but induced alkaloids production slightly. Also, under the same concentration of NH₄+ (25 mM), higher concentration of NO₃- induced alkaloids production strongly but high concentration of NO₃- (≥100 mM) interfered alkaloids instead. Maximum benzo[c]phenanthridine alkaloids production is about 62.71 mg/L at 50:25 (mol/mol). These results suggest that higher biomass and higher alkaloids production could be obtained by optimizing each nitrogen concentration as well as NO₃-:NH₄+ ratio in the culture medium. Nitrate and ammonium in culture medium have distinct role in the regulation of growth and alkaloids production; ammonium had a strong influence on growth while nitrate had an influence on alkaloids production.

Agrobacterium spp.에 의하여 형질전환된 독말풀(Datura stramonium var. tatula Torr.) 모상근의 성장과 tropane alkaloid의 생성

양덕조,강현미,이강섭,김용해,양덕춘 한국식물생명공학회 1997 식물생명공학회지 Vol.24 No.3

모상근의 성장을 위한 최적배지 및 tropane alkaloids함량을 위한 최적배지를 구명하기 위하여, 독말풀(Datura stramonium var. tatula Torr.)의 잎과 줄기에 Agrobacterium spp.spp.를 접종하였다. 접종된 균주 중 모상근의 유도에 효과적인 균주는 Agrobacterium tumefaciens $\textrm{A}_{4}$ T와 A rhizogenes ATCC 15834이었다. SH (Schenk and Hildebrandt, 1972) 액체배지에서 선발된 23개의 clone중 성장이 양호한 clone은 DTLA9 이었으며, tropane alkaloids의 함량이 양호한 clone은 DTLE6 이었다. 이들 두 종류의 세포주를 9종류의 서로 다른 액체배지에 각각 배양하여 성장 및 tropane alkaloids의 함량을 조사한 결과, 모상근의 성장은 두 세포주 모두 SH와 MS배지(SH > MS배지순)에서 다른 7개의 배지에서 보다 양호하였다. 한편, tropane alkaloids의 함량은 두 세포주 공히 GD와 B5 배지(GD > B5배지순)에서 다른 7개의 배지에서 보다 높게 나타났다. 따라서 SH배지에서 모상근을 성장시킨 후, GD배지에 옮겨 2단계 연속배양하면 tropane alkaloids의 함량을 높일 수 있을 것으로 확인되었다. In order to elucidate the optimum medium on the growth and tropane alkaloid production of hairy root, hairy root were induced by inoculating leaf and stem of Datura stramonium var. tatula Torr. with Agrobacterium spp. Both Agrobacterium tumefaciens $\textrm{A}_{4}$ T and A, rhizogenes ATCC 15834 among tested strains were effective on hairy root formation. Among 23 clones selected in SH (Schenk and Hildebrandt, 1972) liquid medium, DTLA9 clone was shown fast growth of hairy root and DTLE6 clone was shown high level production of tropane alkaloids. When both DTLA9 and DTLE6 clones were cultured in the GD (Gresshoff and Doy, 1972) medium, alkaloid production was higher than in 8 tested media. It was elucidated that optimum medium for root proliferation and for tropane alkaloid production is SH, GD medium, respectively.

한국산 보성 덖음 녹차의 가공 및 저장중의 카테킨류의 변화

서봉순,서향순,Suh, Bong-Soon,Suh, Hyang-Soon 동아시아식생활학회 2007 동아시아식생활학회지 Vol.17 No.3

본 연구는 일상의 기호 음료이며 기능성 식품으로 각광을 받고 있는 녹차 중 수공 덖음 제다기법으로 제조한 보성 녹차의 제조 공정 및 저장 보관 중의 catechin 및 alkaloids의 함량 변화를 파악하고자 HPLC 와 UV-VIS Spectrophotometer 를 이용하여 실험하였다. 그 결과는 다음과 같다. 덖음차 제조 과정 중의 catechin, alkaloid 류의 변화를 조사한 결과, 차나무로부터 채취한 신선한 차잎은 EGCG 가 50.44 mg/g 으로 가장 많았으며 다음이 ECG 28.41 mg/g, CAF 25.13 mg/g, CG 가 1.26 mg/g, THB 1.24 mg/g, THP 0.10 mg/g 으로 소량 함유되어 있었다. Catechin, alkaloid류의 각 제조 과정 중의 함량 변화를 살펴보면 CG 를 제외한 모든 성분에서 제조 과정중의 유의미한 차이를 나타내었다(p<0.05, p<0.01, p<0.001). Catechin의 함량은 생잎일 때는 6.58 mg/g 이었으나 건조 후에는 증가하는 경향을 보였으며, GCG는 생잎일 때는 6.43 mg/g 이었으나 건조 후에는 3.61 mg/g 으로 급격하게 감소하였다. 또한 THB와 THP 역시 생잎일 때보다 건조한 후에 함량이 증가하는 것을 알 수 있었다. 또한, 저장 중 녹차 잎의 catechin 과 alkaloid 류의 함량 변화를 조사해 본 결과, $5^{\circ}C,\;25^{\circ}C$ 저장 중의 녹차 잎 모두에서 EGCG, ECG, CAF의 감소가 많았고, 1 년간 저장 시에는 $20{\sim}30%$나 감소하였으며, 저장 온도에 따른 catechin 및 alkaloid 류의 양적 차이는 거의 나타나지 않아 저장 온도에 따른 catechin 과 alkaloid 류의 함량 변화는 거의 없는 것을 알 수 있었다. This study investigated changes in the catechin and alkaloid contents of Bosung green tea during different manufacturing processes and storage periods, using HPLC and UV-VIS spectrophotometer analysis. For changes in the catechin and alkaloid contents by roasting technique, we found that EGCG, CG and GCG decreased just slightly by processes performed before roasting, rather than after roasting. In addition, theobromine, caffeine, and ECG changed minimally throughout all the processes. For changes in the catechin and purine alkaloid contents of the green tea leaves during storage, EGCG, ECG, and CAF decreased considerably in the green tea stored at temperatures of $5^{\circ}C$ and $25^{\circ}C$, and they decreased by $20{\sim}30%$ after storage for 1 year. However, a quantitative difference was hardly observed in the catechin and alkaloid contents regardless of storage temperature.

세포 현탁배양에 의한 현호색 알칼로이드의 생산

장정인,지형준,신승원,Chang, Jung-In,Chi, Hyung-Joon,Shin, Seung-Won 한국생약학회 1996 생약학회지 Vol.27 No.4

In previous paper, we described the induced callus of Corydalis remota contains a significant amount of alkaloids. This study describes an optimal condition to maximize alkaloid production. The suspension cultures maintained alkaloid production ability after fifth subculture and a small amount of alkaloid seemed to be released out of cells. The yields of alkaloid by cultured cells was varied depending on the concentrations of NAA, carbon sources and phosphate ion and depending on the vitamin combinations and concentrations. Biosynthetic precursor and an elicitor treatment also affected the total alkaloid yield of the cultures. The optimal conditions for alkaloid production were as follows: 1) MS basal salt containing 30 g/l of glucose, 1.0 mg/l of NAA, and vitamins of LS medium should be used. 2) The culture should be treated with tyrosine 20 mg/l, and yeast extract 1.5 ml/l after the culture reached a stationary phase of growth. Five alkaloids were isolated from the cultures and they were characterized. The spectral data unambiguously revealed that the isolated compounds were dihydrosanguinarine, protopine. tetrahydropalmatine, allocyptopine and ambinine, respectively.

한국산 야부끼다종 차엽으로 만든 홍차 제조과정 중의 catechins, theaflavins, alkaloids 함량 변화에 관한 연구

최석현 ( Suk Hyun Choi ) 한국식생활문화학회 2009 韓國食生活文化學會誌 Vol.24 No.3

본 연구에 사용한 홍차는 한국산 야부끼다종 차잎을 이용해 위조처리(withering) 유념처리(rolling, roll breaking) 및 발효처리(fermentation)를 거쳐 제조하였으며 홍차 제조 과정 중의 차잎에 포함되어 있는 catechin 및 theaflavin, alkaloid의 각 성분을 분석하고 최적의 홍차제조 조건을 조사한 것이다. 1) 생차잎의 수분함량의 평균은 70.85%였지만 최종단계의 홍차제품 수분함량은 3.07%로 생잎의 1/23로 감소하였다. 2) HPLC로 분석한 결과 차잎에는 7종류의 catechin, 4종류의 theaflavin 그리고 3종류의 alkaloid가 동정되었다. 3) 홍차의 제조과정 중의 catechin, theaflavin 및 alkaloid의 변동을 조사한 결과 발효과정 중에 EGCG, ECG, EGC, EC가 급격히 감소·변화하여 홍차의 특성을 결정하는 theaflavin이 생성됨을 알 수 있었다. 특히 발효가 시작되고 1~2시간 후에 theaflavin이 최대로 생성되는 것으로 보아 본 실험제조의 조건하에서는 발효처리를 1~2시간 하는 것이 최적의 홍차를 만드는 제조법이라 사료된다. In this study, we examined the composition of catechins, theaflavins and alkaloids in leaves during processing to fermented black tea, which is produced by withering, roll breaking, and fermentation of Korean Yabukida tea plant. In addition, we determined the optimal conditions for the production of fermented black tea. The average moisture content in fresh leaves was 70.85%, which dropped to 3.07% in fermented black tea at the last stage of production. When the leaves were analyzed by HPLC, seven types of catechins, four types of theaflavins and three types of alkaloids were identified. The levels of catechins, theaflavins, and alkaloids were then evaluated after being processed into fermented tea. From these experiments, we found that the level of theaflavins, which determines the property of the tea, increased during fermentation. This effect resulted from the change in EGCG, ECG, EGC, EC during the process of fermentation. We also found that the maximal amount of theaflavins was created after 1-2 hours of fermentation. Thus, our results imply that the best condition for producing fermented black tea would be to ferment for 1-2 hours.

Analytical Characterization of Aza-Indole Alkaloids in the Biosynthesis of Catharanthus Roseus

( Hyang Yeol Lee ) 한국유화학회 2012 한국응용과학기술학회지 Vol.29 No.2

Aza-indoles are important pharmacophores that have similar size and biological properties of indole. Here we have synthesized 4- and 7-azaindole tryptamines and showed that they are successfully incorporated in the biosynthesis of monoterepene indole alkaloids (MIAs) to form novel azaindole alkaloids by enzymatic reactions of strictosidine synthase(STR) and strictosidine glucosidase(SDG) monitored by UPLC/MS. By using HPLC equipped with a HPLC photo diode array(PDA) detector, each of the UV spectra of azaindole alkaloids was obtained and characterized. When hydrophilicity of azaindole alkaloids was compared, 4-azaindole alkaloids were more hydrophilic than 7-azaindole alkaloids.

General Fragmentations of Alkaloids in Electrospray Ionization Tandem Mass Spectrometry

심희정,이지예,김병주,홍종기 사단법인 한국질량분석학회 2013 Mass spectrometry letters Vol.4 No.4

Various types of alkaloids observed in several herbal medicines were analyzed by electrospray ionization tandemmass spectrometry in positive ion mode. In the present study, MS/MS spectralpatterns were investigated for eight-types of alkaloids(aporpine, protoberberine, tetrahydroprotoberberine, benzylisoquinoline, protopine, phthalide, morpine, and bisbenzylisoquinoline). For aporpine- and protoberberine-type alkaloids, main fragmentations occurred at substituted groups on rigid ringstructures, not showing ring fusion. Interesting fragmentations due to iminolization and retro-Diels-Alder (RDA) reaction wereobserved in MS/MS spectra of protopine- and tetrahydroprotobereberine-type alkaloids. Also, several types of fragmentationssuch as inductive cleavage and α-cleavage, or bond cleavage between two ring structures were observed depending on theirstructural characteristics. These fragmentation patterns are expected to allow instant classification of the specific alkaloid type invarious MS/MS spectra of alkaloids.

General Fragmentations of Alkaloids in Electrospray Ionization Tandem Mass Spectrometry

Hee Jung Shim,Ji Ye Lee,Byungjoo Kim,Jongki Hong 한국질량분석학회 2013 Mass spectrometry letters Vol.4 No.4

Various types of alkaloids observed in several herbal medicines were analyzed by electrospray ionization tandem mass spectrometry in positive ion mode. In the present study, MS/MS spectralpatterns were investigated for eight-types of alkaloids (aporpine, protoberberine, tetrahydroprotoberberine, benzylisoquinoline, protopine, phthalide, morpine, and bisbenzylisoquinoline). For aporpine- and protoberberine-type alkaloids, main fragmentations occurred at substituted groups on rigid ring structures, not showing ring fusion. Interesting fragmentations due to iminolization and retro-Diels-Alder (RDA) reaction were observed in MS/MS spectra of protopine- and tetrahydroprotobereberine-type alkaloids. Also, several types of fragmentations such as inductive cleavage and α-cleavage, or bond cleavage between two ring structures were observed depending on their structural characteristics. These fragmentation patterns are expected to allow instant classification of the specific alkaloid type in various MS/MS spectra of alkaloids.

General Fragmentations of Alkaloids in Electrospray Ionization Tandem Mass Spectrometry

Shim, Hee Jung,Lee, Ji Ye,Kim, Byungjoo,Hong, Jongki Korean Society for Mass Spectrometry 2013 Mass spectrometry letters Vol.4 No.4

Various types of alkaloids observed in several herbal medicines were analyzed by electrospray ionization tandem mass spectrometry in positive ion mode. In the present study, MS/MS spectralpatterns were investigated for eight-types of alkaloids (aporpine, protoberberine, tetrahydroprotoberberine, benzylisoquinoline, protopine, phthalide, morpine, and bisbenzylisoquinoline). For aporpine- and protoberberine-type alkaloids, main fragmentations occurred at substituted groups on rigid ring structures, not showing ring fusion. Interesting fragmentations due to iminolization and retro-Diels-Alder (RDA) reaction were observed in MS/MS spectra of protopine- and tetrahydroprotobereberine-type alkaloids. Also, several types of fragmentations such as inductive cleavage and ${\alpha}$-cleavage, or bond cleavage between two ring structures were observed depending on their structural characteristics. These fragmentation patterns are expected to allow instant classification of the specific alkaloid type in various MS/MS spectra of alkaloids.