한국해양대학교

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염생식물 Corydalis heterocarpa 와 Vitex rotundifolia 로부터 생리활성물질의 분리 및 구조결정

Title
염생식물 Corydalis heterocarpa 와 Vitex rotundifolia 로부터 생리활성물질의 분리 및 구조결정
Alternative Title
Isolation and Structural Elucidation of Bioactive Secondary Metabolites of the Halophytes Corydalis heterocarpa and Vitex rotundifolia
Author(s)
김유아
Publication Year
2009
Publisher
한국해양대학교 대학원
URI
http://kmou.dcollection.net/jsp/common/DcLoOrgPer.jsp?sItemId=000002175500
http://repository.kmou.ac.kr/handle/2014.oak/9734
Abstract
해양생물자원의 중요성에 대한 인식이 높아짐에 따라 해양생물을 이용한 새로운 생리활성물질의 개발에 관심이 증가되고 있으며, 그 중에서도 해수와 민물이 섞이는 독특한 환경에 서식하며 이에 적합한 적응기작을 발전시킨 염생식물이 새로운 신물질 자원으로서 주목 받고 있다.

염생식물의 일종인 염주괴불주머니 (Corydalis heterocarpa)와 순비기나무 (Vitex rotundifolia)는 한국의 각처 바닷가 모래 사장에서 자생하는 노란 꽃 및 자줏빛 꽃이 피는 식물로써, 조간대에서 매우 중요한 생태적 위치를 차지하고 있다. 또한 염주괴불주머니는 민간에서 오랫동안 산모의 진통이나 경련의 치료제로 사용되었으며, 순비기나무는 한방에서 그 열매를 만형자라 하여 두통, 안질, 귓병과 같은 질병의 치료에 사용되어 왔다. 염주괴불주머니의 경우, 같은 속의 다른 식물에서 다양한 이차 대사산물이 보고된 바 있으나, 지금까지 염주괴불주머니로부터 이차 대사산물의 분리나 유용한 생약학적 특성이 보고된 바 없다. 순비기나무는 다수의 연구에 의해 flavonoid류 및 terpenoid류의 화합물들이 분리된 바 있으며, 이들의 생리활성효과 또한 보고되었다.

따라서 본 연구에서는 염생식물 염주괴불주머니와 순비기나무에서 신규의 이차 대사물질을 분리하고, 분리된 화합물에 의한 항산화와 발암물질의 생성방지 및 생체방어 물질로서의 유용성을 검토하고자 하였다.

그 결과, 염주괴불주머니로부터 9종의 알려진 화합물 (1-3, 5, 7-11)과 2종의 신규 화합물 (4, 6)을 분리하여 총 11종의 순수한 화합물을 분리하였으며, 또한 순비기나무로부터 기지 화합물 14종 (12-19, 21, 22, 27-30)과 신규 화합물 5종 (20, 23-26)을 포함하여 총 19종의 이차 대사물질을 분리하였다. 분리된 화합물의 구조는 분광학적 자료의 해석과 문헌치의 비교에 의해 결정되었으며, 특히, 염주괴불주머니의 2종의 신규 화합물4와 6 은 각각 (2'S,7'S)-O-(2-methylbutanoyl)-columbianetin 과 (2'S)-columbianetin-3'-hydrogen sulfate 였으며, 순비기나무의 5종의 신규 화합물 20, 23-26은 차례로 (5S*,6R*,8R*,9R*,10S*)-6-acetoxy-9-hydroxy-16-hydroxy-13(14)-labden-16,15-olide, 3α-hydroxy pleuroziol, 9αH-manoyl oxide, abieta-11(12)-ene-9α,13α-endoperoxide 그리고 abieta-11(12)-ene-9β,13β-endoperoxide로 결정되었다.

분리된 화합물의 암세포 증식 억제 효과를 탐색하기 위한 기초 연구로서 AGS 인체 위암세포, HT-29 인체 결장암세포, HT1080 인체 섬유육종 세포, MCF-7 인체 유방암세포 및 U-937 인체 구성 림프종 세포의 증식에 미치는 영향을 MTT assay를 통해 관찰하였다. 그 결과, 전반적으로 다른 암세포에 비하여 AGS 인체 위암세포에서 화합물의 암세포 증식 억제 효과가 뛰어나 apoptosis와 관련이 있는 여러 요소의 mRNA 발현 정도를 AGS 인체 위암세포에서 검토하였다.

분리된 모든 화합물에 대한 인체 암세포 증식 억제 효과를 검토한 결과, 염주괴불주머니의 신규화합물 6과 순비기나무의 화합물 14가 가장 활성이 뛰어났으며, 이들 화합물의 암세포 증식 억제 효과가 pro-/anti-apoptotic Bax/Bcl-2 유전자의 발현 조절 및 종양 억제 유전자 p53 및 p21 유전자의 up-regulation에 의한 것으로 사료되었다.

화합물의 항산화 활성을 탐색하기 위한 일환으로 세포내 NO, ROS, GSH 및 산화적 스트레스와 관련된 iNOS와 COX-2 유전자의 mRNA 발현 정도를 macrophage Raw 264.7 세포에서 확인하였다.

세포내 NO의 함량은 Griess reaction을 바탕으로 확인하였으며, 세포내 ROS와 GSH의 발생은 각각 형광물질인2',7'-dichlorofluorescin diacetate (DCFH-DA)와 monobromobimane을 이용하여 측정하였다. 또한, LPS의 자극에 의하여 산화적 스트레스가 유도된 Raw 264.7 세포의 iNOS와 COX-2 유전자의 발현은 RT-PCR에 의하여 확인되었다.

산화적 스트레스가 유도된 Raw 264.7 세포에 염주괴불주머니와 순비기나무에서 분리된 화합물을 농도별로 처리한 결과, 농도의존적으로 다수의 화합물이 항산화 활성을 지님을 확인하였으며, 각각의 측정 방법에 따라 화합물의 활성을 나타내는 정도의 차이가 있었다. 특히, 염주괴불주머니에서 분리된 화합물 2-4와 순비기나무에서 분리된 화합물 16-18및 신규화합물23과 24가 우수한 항산화 활성을 나타내는 것으로 확인되었다.

본 연구를 통해 염생식물의 일종인 염주괴불주머니와 순비기나무의 암세포 증식 억제 효과 및 다양한 항산화 활성 검색을 통해 암 예방 및 항산화 기능성 소재로써의 개발 가능성을 확인할 수 있었다. 특히, 각각의 연구에서 우수한 활성을 보인 화합물에 대한 집중적인 연구를 통해 새로운 생리활성물질의 개발이 기대된다.
Stan et al., 2008).

In the present study, Corydalis heterocarpa and Vitex rotundifolia, which are salt-tolerant plants and which have been used traditionally as a folk medicine, were investigated for their bioactive constituents.

The investigation of these two salt marsh plants provided eleven compounds, including two new compounds, from C. heterocarpa and nineteen compounds, including five new compounds, from V. rotundifolia. The chemical structures of the two new compounds 4 and 6 from C. heterocarpa were established as (2'S,7'S)-O-(2-methylbutanoyl)-columbianetin and (2'S)-columbianetin-3'-hydrogen sulfate. Five new compounds 20 and 23-26 from V. rotundifolia were determined as (5S*,6R*,8R*,9R*,10S*)-6-acetoxy-9-hydroxy-16-hydroxy-13(14)-labden-16,15-olide, 3α-hydroxy pleuroziol, 9αH-manoyl oxide, abieta-11(12)-ene-9α,13α-endoperoxide, and abieta-11(12)-ene-9β,13β-endoperoxide. The NMR spectral data of the new compounds were completely assigned by a combination of 1D/2D NMR experiments including ¹H-¹H gDQCOSY, TOCSY, DEPT, gHMQC and gHMBC correlations.

The nine known compounds from C. heterocarpa were identified as (2'S)-columbianetin (1), libanoridin (2), cnidiadin (3), (2'S)-columbianetin-O-β-D-glucopyranoside (5), isopimpinellin (7), hyunganol Ⅱ (8), cnidimol A (9), cnidimoside A (10) and 2,3-dihydro-4-hydroxy-7-methyl-2-isopropenyl-5H-furo[3,2g][1] benzopyran-5-one (11).

The fourteen known compounds from V. rotundifolia were identified as luteolin (12), quercetin 3,7-dimethyl ether (13), vitexicarpin (14), artemetin (15), vitetrifolin F (16), vitetrifolin E (17), vitetrifolin D (18), vitexilactone (19), (5S*,6R*,8R*,9R*,10S*)-6-acetoxy-9-hydroxy-15-methoxy-13(14)-labden-16,15-olide (21), aurantiamide (22), abieta-9(11):12(13)-di-α-epoxide (27), ferruginol (28), β-amyrenone (29) and α-amyrenone (30) by a combination of spectroscopic analysis and by comparison with the data reported in the literature.

Potential inhibitory activity of the isolated compounds against human cancer cells was evaluated by MTT assay and mRNA expression of several factors related to apoptosis. In comparative analysis, the AGS human cancer cell line was the most sensitive to the antiproliferative effect of all of the isolated compounds.

Among compounds isolated from C. heterocarpa, compounds 2, 5, 6 and 11 showed strong growth inhibitory effects against human cancer cells in a dose-dependent manner (p < 0.05). The antiproliferative effect of new compound 6 was the highest among all compounds tested. These effects may be caused by modulation of apoptosis through the Bax and Bcl-2 dependent pathway and may also be related to the significant up-regulation of p53 and p21 seen in response to treatment with salt marsh plant secondary metabolites.

The antiproliferative effects of compounds 12, 14 and 15 isolated from V. rotundifolia were compared with the control by MTT assay. Among the tested compounds, 14 exhibited the highest inhibitory activity in the AGS cells. At the gene expression levels, it strongly down-regulated Bcl-2 level and up-regulated p53 and p21 levels.

The antioxidant activity of the compounds was evaluated by four different activity tests, including measurement of intracellular NO, ROS, GSH, and mRNA expression of iNOS and COX-2. Compounds 1-6 isolated from C. heterocarpa obviously decreased production of NO and ROS, increased production of GSH, and inhibited both iNOS and COX-2 expressions under LPS-stimulated condition. Compounds 2-4 in particular consistently exhibited higher antioxidant effects compared to the other compounds.

In the cases of compounds 12, 14-19 and 23-26 isolated from V. rotundifolia, compounds 16-19 remarkably suppressed NO production while compounds 16-18 and 23 showed strong ROS-scavenging effects. However, there were no significant effects on GSH levels. The expression levels of iNOS and COX-2 were down- regulated by compounds 16-19, 23 and 24. On the basis of these above results, these compounds may be useful candidates as antioxidant sources for treatment of oxidative damage caused by reactive species in macrophage cells.

Although further studies are needed to confirm these observations, the current study suggests that compounds isolated from C. heterocarpa and V. rotundifolia may be used to reduce human cancer risk, and that their antiproliferative effects are partly related to induction of several genes related to apoptosis, including Bax, Bcl-2, p53, and p21. In addition, these compounds may be used to retard oxidative destruction by reactive species and thus reduce the risk of many diseases that involve oxidative stress, such as aging, cancer, diabetes mellitus, neurological disorders, renal disorders, and hypertension.

Therefore, the two salt marsh plants examined here, C. heterocarpa and V. rotundifolia, may be beneficial natural sources of valuable chemopreventive agents and may also contribute to the reduction of risks of cancer and other oxidation-related diseases.
Gomes et al., 2005). Therefore, oxidative stress by ROS/RNS may be the direct or indirect cause of many human diseases such as aging, cancer, atherosclerosis, and inflammation.

For these reasons, interest has considerably increased in searching for naturally occurring chemopreventive agents to replace synthetic materials, many of which are prohibited due to their carcinogenicity (Sasaki et al., 2002). Therefore, natural chemopreventive agents isolated from plants and marine algae represent the most useful and as yet unmined source of material for disease prevention (Park and Pezzuto, 2002
r, 2001
Stan et al., 2008). Oxygen free radicals, or reactive oxygen species (ROS) and reactive nitrogen species (RNS), are products of normal cellular metabolism. ROS and RNS are well recognized for playing a dual role as both deleterious and beneficial species (Valko et al., 2007). Usually, antioxidants defend against the harmful effects of these naturally occurring free radicals. However, excessive production of ROS/RNS may lead to oxidative stress, which damages normal cellular function and ultimately induces apoptosis or necrosis (Nordberg and Arné
Lee and Seo, 2006
Seo et al., 2004
Lee et al., 2004
Jung et al., 2004
In recent years, there has been much interest and research into the influence of natural products on several diseases, including aging-related illness, cancer, neurodegenerative disease, and cardiovascular disease. Recent epidemiological studies have suggested that dietary pattern is commonly associated with the risk of chronic diseases (Hu, 2002). This association may be attributed to the presence of natural chemo-preventers, such as vitamins, polyphenols, and flavonoids, which prevent free radical damage (Park and Pezzuto, 2002
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