사람피부섬유아세포에서 산화적 스트레스에 대한 항산화 활성을 가진 spermidine의 억제효과

In-Hwan Park(박인환),Moon-Moo Kim(김문무) 한국생명과학회 2011 생명과학회지 Vol.21 No.5

Spermidine은 spermine의 전구체로 역할을 하는 putrescine로부터 합성되는 polycation의 한 형태이다. 최근에 spermidine는 수명연장에 중요한 역할을 하는 하나의 polyamine으로 알려져 있다. Hydroxyl radical, superoxide 및 hydrogen peroxide와 같은 활성산소(ROS)는 노화뿐만 아니라 다양한 병원성 과정에서 관여한다고 보고되고 있다. DPPH radical, H₂O₂ 및 hydroxyl radical에 대한 spermidine의 소거활성과 산화적 스트레스와 관련된 DNA oxidation에 대한 보호 효과가 in-vitro에서 평가되었다. Spermidine은 500 μM 이상에서 DPPH radical와 H₂O₂에 대한 소거 효과를 보여주었다. 특히 활성산소종 중에서 spermidine는 hydroxyl radical에 대한 효과가 탁월하였다. 뿐만 아니라, spermidine는 500 μM에서 DNA oxidation에 대해서도 뚜렷한 보호 효과를 나타내었다. 더욱이 사람피부섬유아세포에서 superoxide dismutase와 같은 항산화 관련 단백질의 발현이 대조군과 비교 시 spermidine 존재 하에 증가하였다. 이상의 결과로부터 spermidine은 암, 노화 및 염증을 포함하는 활성산소와 관련 있는 질병들을 예방하기 위한 항산화제로 이용될 수 있을 것이다. Spermidine is a ubiquitous polycation that is synthesized from putrescine, which serves as a precursor of spermine. In recent years, spermidine was found to be a polyamine that plays an important role in longevity. Reactive oxygen species (ROS) such as hydroxyl radical, superoxide and hydrogen peroxide have been shown to be involved in various pathogenic processes as well as aging. The direct scavenging effect of spermidine on DPPH radical, H₂O₂ and hydroxyl radical, and its protective effect against DNA oxidation related to oxidative stress were evaluated in vitro. It was observed that spermidine exhibits scavenging activities on DPPH radical and H₂O₂ above 500 μM. Spermidine was especially effective in exerting a scavenging activity on hydroxyl radical. In addition, spermidine at 1000 μM showed a clear protective effect against DNA oxidation. Furthermore, the expression level of antioxidant enzymes such as superoxide dismutase in humam dermal fibroblasts increased in the presence of spermidine compared with blank group. These results suggest that spermidine can be used as an antioxidant to prevent ROS-related diseases including inflammation, cancer and aging.

Metabolic engineering of Saccharomyces cerevisiae for production of spermidine under optimal culture conditions

Kim, S.K.,Jo, J.H.,Park, Y.C.,Jin, Y.S.,Seo, J.H. IPC Science and Technology Press 2017 Enzyme and microbial technology Vol.101 No.-

<P>Spermidine is a polyamine compound exhibiting important biological activities, such as increasing lifes-pan, inflammation reduction, and plant growth control. As such, many applications of spermidine as a bio-modulating agent are anticipated. However, sustainable and scalable production of spermidine has not been achieved yet. Therefore, construction of a spermidine production system using Saccharomyces cerevisiae was attempted in this study. In order to secrete spermidine into fermentation broth, TPO1 coding for the polyamine transporter was overexpressed in an engineered S. cerevisiae strain capable of accumulating high concentrations of spermidine. Through optimization of fermentation conditions, the resulting strain (OS123/pTPO1) produced 63.6 mg/l spermidine with a yield of 1.3 mg spermidine/g glucose. However, we observed that spermidine production was repressed in the presence of glucose. To circumvent this problem, the genetic modifications for overproducing spermidine were introduced into an engineered S. cerevisiae capable of fermenting xylose. In a fed-batch fermentation using a mixture of glucose and xylose, the resulting strain (SR8 OS123/pTPo1) produced 224 mg/l spermidine with a yield of 2.2 mg spermidine/g sugars. These results suggest that engineered yeast constructed in this study can be employed for the production of spermidine. (C) 2017 Elsevier Inc. All rights reserved.</P>

UPLC를 이용한 동결건조 로열젤리 내 spermidine의 분석조건 확립 및 함량 분석

최홍민,김세건,김효영,우순옥,김선미,문효정,한상미 한국양봉학회 2022 韓國養蜂學會誌 Vol.37 No.1

Spermidine is one of biological amines with various functions such as anti-aging and life extension. This study aimed to analyze content of spermidine in freeze-dried royal jelly and establish an optimal analysis method. This assay was optimized to analyze spermidine in lyophilized royal jelly within 13 minutes using UPLC and evaluated the following performance parameters: linearity, limit of detection, limit of quantification, specificity, accuracy, and precision. The linear range of spermidine was 7.8~125 μg/mL with the correlation coefficient (R2)>0.9998. Limit of detection and limit of quantification were 0.63 and 1.93 μg/mL, respectively. Average recovery of spermidine ranged from 96.6% to 109.8%. The relative standard deviation (RSD) of the intra-day precision and inter-day precision for spermidine were 3.47~4.33% and 4.54~6.77%, respectively. Through this method, it was found that content of spermidine was 131.3±4.9 mg/kg in the freeze-dried royal jelly. This study revealed that this validated method was suitable for spermidine determination and it can be used as basic data in further studies on the bioactivity of spermidine.

Auranofin accelerates spermidine-induced apoptosis via reactive oxygen species generation and suppression of PI3K/Akt signaling pathway in hepatocellular carcinoma

황보현,김다혜,김민영,지선영,EunJin Bang,홍수현,최영현,정재헌 한국수산과학회 2023 Fisheries and Aquatic Sciences Vol.26 No.2

Auranofin is a US Food and Drug Administration (FDA)-approved anti-arthritis medication that functions as a thioredoxin reductase inhibitor. Spermidine, a polyamine present in marine algae, can exert various physiological functions. Herein, we examined the synergistic anticancer activity of auranofin and spermidine in hepatocellular carcinoma (HCC). Combined treatment with auranofin and spermidine suppressed cell viability more efficiently than either treatment alone in HCC Hep3B cells. The isobologram plotted by calculating the half maximal inhibitory concentration (IC50) values of each drug indicated that the two drugs exhibited a synergistic effect. Based on the analysis of annexin V and cell cycle distribution, auranofin and spermidine markedly induced apoptosis in Hep3B cells. Moreover, auranofin and spermidine increased mitochondria-mediated apoptosis by promoting mitochondrial membrane potential (Δψm) loss. Auranofin and spermidine significantly increased reactive oxygen species (ROS) production in Hep3B cells, and the blocking ROS suppressed apoptosis induced by spermidine and auranofin. In addition, auranofin and spermidine reduced the expression of phosphorylated phosphatidylinositol-3 kinase (PI3K) and protein kinase B (Akt), and PI3K inhibitor accelerated auranofin- and spermidine-induced apoptosis. Using ROS scavenger and PI3K inhibitor, we revealed that ROS acts upstream of auranofin- and spermidine-induced apoptosis. Collectively, our study suggests that combination treatment with auranofin and spermidine could afford synergistic anticancer activity via ROS overproduction and reduced PI3K/Akt signaling pathway.

어류병원성 세균 및 C2C12 근원세포에 대한 polyamine 계열 물질인 spermidine의 항균 및 항산화 활성

황보현,최은옥,김민영,지선영,홍수현,박철,차희재,김석만,김희수,황혜진,최영현 (사)한국해양바이오학회 2019 한국해양바이오학회지 Vol.11 No.2

We compared the antibacterial activities of spermidine and astaxanthin against two gram-positive bacteria such as Streptococcus parauberis and S. iniae to find new antibacterial candidates. We also evaluated the preventive effects of spermidine against oxidative stress-induced cytotoxicity in C2C12 myoblasts. Our results indicated that spermidine has more significant antibacterial activities than astaxanthin against both two fish pathogenic bacteria as well as gram-negative bacteria Escherichia coli used as a control group. Minimum inhibitory concentration and minimum bactericidal concentration of spermidine were 0.25 mM and 1 mM against S. parauberis, 1 mM and 3 mM against S. iniae, and 0.5 mM and 1.5 mM against E. coli, respectively. In addition, the postantibiotic effect lasted from 7 h, 5 h and 6 h for S. parauberis, S. iniae and E. coli, respectively. The results also showed that the decreased C2C12 cell viability by H2O2 could be attributed to the induction of DNA damage and apoptosis accompanied by the increased production of reactive oxygen species, which was remarkably protected by spermidine. Additionally, the antioxidant effect of spermidine was associated with the activation of Nrf2 signaling pathway. According to the data, spermidine may be a potential lead compound which can be further optimized to discover novel antibacterial and antioxidant agents.

Anti-inflammatory effects of spermidine in lipopolysaccharide-stimulated BV2 microglial cells

Choi, Yung Hyun,Park, Hye Young BioMed Central 2012 JOURNAL OF BIOMEDICAL SCIENCE -BASEL- Vol.19 No.1

<P><B>Background</B></P><P>Spermidine, a naturally occurring polyamine, displays a wide variety of internal biological activities including cell growth and proliferation. However, the molecular mechanisms responsible for its anti-inflammatory activity have not yet been elucidated.</P><P><B>Methods</B></P><P>The anti-inflammatory properties of spermidine were studied using lipopolysaccharide (LPS)-stimulated murine BV2 microglia model. As inflammatory parameters, the production of nitric oxide (NO), prostaglandin E<SUB>2 </SUB>(PGE<SUB>2</SUB>), interleukin (IL)-6 and tumor necrosis factor (TNF)-α were evaluated. We also examined the spermidine's effect on the activity of nuclear factor-kappaB (NF-κB), and the phosphoinositide 3-kinase (PI3K)/Akt and mitogen-activated protein kinases (MAPKs) pathways.</P><P><B>Results</B></P><P>Pretreatment with spermidine prior to LPS treatment significantly inhibited excessive production of NO and PGE<SUB>2 </SUB>in a dose-dependent manner, and was associated with down-regulation of expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Spermidine treatment also attenuated the production of pro-inflammatory cytokines, including IL-6 and TNF-α, by suppressing their mRNA expressions. The mechanism underlying spermidine-mediated attenuation of inflammation in BV2 cells appeared to involve the suppression of translocation of NF-κB p65 subunit into the nucleus, and the phosphorylation of Akt and MAPKs.</P><P><B>Conclusions</B></P><P>The results indicate that spermidine appears to inhibit inflammation stimulated by LPS by blocking the NF-κB, PI3K/Akt and MAPKs signaling pathways in microglia.</P>

Cyclohexylamine과 Polyamine이 대두 자엽의 부정근 형성에 미치는 영향

한태진,조형일 한국식물생명공학회 1994 식물생명공학회지 Vol.21 No.4

Cryclohexylarnine(CHA)에 의한 부정근 억제가 spermidine 과 spermine에 의한 부정근 형성 회복에 미치는 영향을 알아 보고자 대두 자엽 절편을 이용하여 조직내의 polyamine함량을 조사하였다. 부정근 형성 배지에 CHA를 처리한 결과 $10^{-2}$ M CHA 처리시에는 spermidine이나 spermine을 혼합처리함에도 불구하고 부정근이 형성되지 않았다. 또한 2.5 $\times$ $10^{-3}$ M CHA 단독처리시에는 부정근 형성이 억제되었으나, $10^{-4}$ M spermine 혼합처리시에는 부정근 형성능이 회복되었다. Putrescine 함량은 부정근이 형성돠지 않았던 $10^{-2}$ M CHA 단독 처리구 및 $10^{-2}$ M CHA와 $10^{-3}$ M Spermine 혼합처리구에서는 배양 3일 이후 대조구보다 감소하였지만 부정근이 다소 형성되었고, 부정근 회복능이 가장 높았던 2.5 $\times$ $10^{-3}$ M CHA와 $10^{-4}$ M spermine 혼합처리구에서는 대조구보다 높은 함량을 나타내었다. Spermidine 함량은 모든 처리구에서 대조구보다 낮아서 함량이 낮을수록 부정근 형성이 억제되었으며, spermine의 함량은 대조구에 비하여 모든 처리구에서 급격히 증가되어 대조구에 비하여 함량이 높을 수록 부정근 형성이 억제되었다. In order to study the effect of cyclohexylamine (CHA) and polyamines during adventitious root formation, the correlation of adventitious root formation with endogenous polyamine content was investigated in inoculated soybean cotyledons at the rooting medium supplemented with CHA, spermidine and spermine. Adventitious root formation was inhibited in medium containing 10$^{-3}$ -10$^{-2}$ M CHA. Adventitious root formation was not formed in treatment with 10$^{-2}$ M CHA plus polyamine. It was inhibited in 2.5 $\times$ 10$^{-3}$ M CHA alone and reverved in 2.5 $\times$ 10$^{-3}$ M CHA plus 10$^{-4}$ M spermine. Putrescine content was lower in 10$^{-2}$ M CHA and 10$^{-2}$ M CHAplus 10$^{-4}$ M spermine treatment than in control after 3 days of incubation. However, it was higher in 2.5 $\times$ 10$^{-3}$ M CHA and 2.5 $\times$ 10$^{-3}$ M CHA plus 10$^{-4}$ M spermine treatment than in control. Spermine content was higher in all CHA treatments than in control, while spermidine content was lower than in control.

薄荷 懸濁培養에서 培地 濃度와 spermidine의 細胞生長, 精油含量 및 精油成分에 미치는 影響

Man Young Chung(鄭萬永),Young Am Chae(蔡永岩) 한국육종학회 1994 한국육종학회지 Vol.26 No.3

This experiment was carried out to know the effects of medium strength and spermidine on cell growth, essential oil production and its composition in Mentha piperita cell suspension cultures. The results indicated that half strength of modified Lin-Staba medium(potassium phosphate 170㎎/ℓ, sucrose 20g/ℓ, ratio of ammonium nitrogen to nitrate=1 : 3) was the most effective on cell growth and oil production. But menthol content was higher in full strength of modified Lin-Staba medium. Concentration of 10㎎/ℓ spermidine showed positive effect on both cell growth and essential oil production. Menthol and menthone content were increased more than two times compared with the control when 10㎎/ℓ spermidine was added.

Effects of Polyamines on Shoot and Root Development in Arabidopsis Seedlings and Carnation Cultures

Jang, Su Jin,Choi, Yu Jin,Park, Ky Young 한국식물학회 2002 Journal of Plant Biology Vol.45 No.4

Polyamines, a class of aliphatic amines, are active in the growth and development of bacteria, animals, and plants. To better understand their physiological role in plants, we used spermidine and the inhibitor of SAMDC to analyze the developmental patterns of roots and shoots from Arabidopsis seedlings and in-vitro carnation shoot cultures. We also monitored mRNA levels of the polyamine biosynthetic gene after adding various phytohormones to the growing media. Treating wild-type Arabidopsis seedlings with polyamine resulted in greater growth after four weeks; 1 mM spermidine increased root lengths by 39% and seedling weights by 44%. Spermidine was more effective in enhancing root growth in the det2 mutants than in the wild-type plants. In contrast, MGBG, an irreversible inhibitor of SAMDC activity, resulted in root lengths that were 29% of the controls, as well as seedling weights that were only 77% of normal. In our carnation cultures, shoot-growth rates were severely reduced by both spermidine and MGBG; root growth was also markedly inhibited by treatment with spermidine. Transcripts of ADC and SAMDC were significantly greater in cultures treated with kinetin and IAA, with levels being higher with the former. Previous research has shown that sensitivity and the appropriate cellular content for polyamines vary among plant species, and may also be organ- or tissue-specific. Polyamines may play an important developmental role because of their potentially stimulatory effects on phytohormonal signaling and/or synergism.

A Simple, Sensitive, and Specific HPLC Analysis of Tissue Polyamines using FNBT Derivatization: Its Application on the Study of Polyamine Metabolism in Regenerating Rat Liver

최상현,김형건,박홍익,전보권,Choi, Sang-Hyun,Kim, Hyung-Gun,Park, Hong-Ik,Chun, Boe-Gwun The Korean Society of Pharmacology 1988 대한약리학잡지 Vol.24 No.2

복잡한 시료-전처리과정을 배제하여 간편하고 선택적이며 예민한 polyamine 측정방법으로써 4-fluoro-3-nitrobenzotrifluoride (FNBT)유도체 및 단일이동상을 이용하는 역상의 고속액체 크로마토 그래피 (HPLC) 방법과 아울러 이 방법을 이용한 흰쥐의 재생성간내 polyamine대사의 변동에 관한 관찰-결과를 요약하면 다음과 같다. 1. 간내-polyamine은 0.4M perchloric acid에 추출한 후 Spragg와 Hutchings의 방법에 따라서 FNBT로 N-2'-nitro-4'-trifluoromethylphenyl polyamine (NTP-polyamine) 유도체를 만들고 이를HPLC로 분석 하였다. 2. HPLC-분석은 ERC ODS-1161 (3 um, $6{\times}100$ mm) column에서 분당 1.2ml 속도의 단일-이동상(acetonitril 80%의 물)으로 분리 하며 파장 242 nm로 검출-정량하였다. 3. HPLC-분석은 시료당 약20분이 소요되었는데, 크로마토그래피의 계치들에 있어서 각polyamine의 capacity factor는 putrescine: 4.12, spermidine: 9.25, 및 spermine: 18.75로써 완전한 분리도를 보였으며, 검량한계는 10 picomole이하의 높은 예민도를 나타내었고, 시료처리-과정의 회수율은 약 12.1-88.5%로써 비교적 안정하였다. 4. Higgins와 Anderson의 방법에 따라서 흰쥐의 간을 부분-절제한 결과 $70.4{\pm}1.99%$가 절제되고 $29.7{\pm}1.45%$가 남게 되었으며, 수술후 48시간 후에 남은 간은 생리식염수-처치군 및 methylglyoxal bis (guanylhydrazone) dihydrochloride (MGBG)-처치군에서 각각 수술 전 간의$52.1%$ 및 $53.3%$로 회복되었다. 5. 정상 간의 polyamine 함량은 각각 putrescine: $158.7{\pm}14.1$, spermidine: $829.7{\pm}36.4$, 및 spermine: $875.6{\pm}42.1$ nanomole/g wet liver이었고 ; 부분-절제하고 남은 간의 polyamine의 수술후변동에 있어서, 생리식염수-처치군의 putrescine과 spermine은 6시간까지의 유의한 증가를 보인다음 48시간에 정상치로 회복되었으나 spermidine은 48시간까지도 계속 증가하여 $2004.9{\pm}170.4nanomole/g$ wet liver가 되었다. MGBG-처치군의 spermidine 및 spermine 함량은 생리식염수군의 변동과 유사하였으나 putrescine의 함량은 6시간의 증가 후 계속 증가하여 48시간에는 $1806.5{\pm}159.4$ nanomole/g wet liver가 되었다. 이상의 성적으로 미루어 볼 때, 위의 단일 이등상의 역상-MPLC방법은 생체의 polyamine 정량분석을 위한 간편하고 예민하며 매우 선택적인 것으로 사료된다. A simple and selective isocratic HPLC method for the analysis of tissue polyamine contents is described and applied on the study of the changes of the hepatic polyamine contents after partial hepatectomy in male rats. The hepatic polyamines are extracted with 0.4 M perchloric acid containing 2 mM disodium EDTA, and then the extract is redissolved in 100 ul of 1 M sodium carbonate and incubated with 300 ul of FNBT-dimethylsulfoxide (1: 100) mixture. The N-2'-nitro-4'-trifluoromethylphenyl drivatives of polyamines are separated through a ERC-ODS column in an isocratic mode with an acetonitrile-water (80:20) mobile phase within 20 min. per a sample, while monitoring the effluent at 242 nm. This improved method which could detect subnanogram of each polyamines is highly specific and reproducible as evidenced by the application of it on the study of the changes of polyamine contents in the regenerating rat liver after partial hepatectomy.