Anti-oxidative and Anti-tumor Effects of Isoflavones Isolated from Leaves of Maackia fauriei

국문 초록 (Abstract)

솔비나무(Maackia fauriei)는 제주도 한라산 일대에 분포하는 우리나라 특산의 콩과 식물로서, 그 꽃은 고혈압과 뇌일혈, 자궁출혈 등의 출혈성질환 치료목적으로 민간적으로 사용되었다. 최근 연구를 통해 솔비나무의 수피와 목부에서 콩과 식물의 특징인 이소플라본 화합물과 플라보노이드 화합물이 발견되었으며, 또한 이 식물에서 분리한 렉틴이 암세포 증식 억제 효과를 가지고 있음이 보고되었다. 본 연구에서는 항종양 효과를 기반으로 이 식물의 잎으로부터 이소플라본 화합물들을 분리하여 그 화학 구조를 규명하였으며, 이들 화합물에 대한 항산화 및 항종양 효과를 검토하였다.
먼저 유효성분의 추적 결과, 솔비나무 잎의 80% 아세톤 추출물로부터 5종의 이소플라본 화합물 [genistein (1), pratensein (2), genistin (3), 2'-hydroxygenistein-7-O-β-D-glucopyranoside (4), 2,3-dehydrokievitone (5)]과 그 유도체인 3종의 테로카판류 화합물 [medicarpin (6), maackiain (7), 4-hydroxy maackiain (8)], 그리고 한 종의 플라보노이드 [isoquercitrin (9)]를 분리하였다. 이후 분리된 화합물의 항산화 및 항종양 효과를 검토해본 결과, 플라보노이드 (9)와 한 종의 테로카판 화합물 (8)에서 우수한 DPPH radical 소거능과 NBT superoxide 소거능이 관찰되었으며, 이소플라본과 테로카판류 화합물들 (1-8)의 전립선 암세포주 (LNCaP, PC-3)와 부인과 종양 (HeLa, OVCAR-3)에 대한 세포독성이 관찰되었다. 특히, 프레닐 (prenyl)기를 가지는 이소플라본인 5번 화합물은 flowcytometry를 이용하여 annexin V / PI dual staining 한 결과 남성 전립선암 세포주 및 여성 부인과암 세포주의 apoptosis를 효과적으로 유도함을 확인 할 수 있었다.
이상과 같은 결과를 종합하여 솔비나무 추출물 또는 솔비나무 유래 isoflavone은 남성 및 여성종양 치료를 위한 천연물신약으로 개발될 수 있을 것으로 사료된다.

다국어 초록 (Multilingual Abstract)

Maackia faurei (Fabaceae) is native to Korea and endemic to Halla Mountain in Jeju Island. The flower of M. fauriei has been traditionally used for hypertension, apoplexy, hemostasis, vaginal bleeding and dystocia, and the bark of this plant has been used as natural dyes. In a recent study, several biological activities including cytotoxic activity of lectin of this plant were reported. However, the study about the phytochemical constituents and biological activities of leaves of M. fauriei was not reported yet.
Activity guided isolation of 80% acetone extract of leaves of Maackia fauriei yielded five isoflavones (1-5) [genistein (1), pratensein (2), genistin (3), 2'-hydroxygenistein-7-O-β-D-glucopyranoside (4) and 2,3-dehydrokievitone (5)], three pterocarpans (6-8) [medicarpin (6), maackiain (7) and 4-hydroxy maackiain (8)] and one flavonol [isoquercitrin (9)]. The structures were elucidated by the analysis NMR spectroscopy.
In order to evaluate their anti-oxidative effects, DPPH radical scavenging activity and NBT superoxide scavenging activity were measured. In general, isoflavones showed weaker activity than a flavonol (9), but a pterocarpan which has a hydroxyl group at the C-4 position of A-ring (8) showed exceptionally strong anti-oxidative activity.
And the results of measurement of the cytotoxicity of 80% acetone extract of leaves of M. fauriei and its isolated compounds, isoflavones and its derivatives (pterocarpans) generally showed the potent cytotoxic activity against all of four human cancer cell lines in genital system. Especially, compound 5 which had a prenyl group at C-8 position of the A-ring showed strong cytotoxic activity and induced apoptosis efficiently in both male and female genital cancer cells.
These results suggested that the leaves of M. fauriei and its isoflavones can be developed as anti-tumor agents.

목차 (Table of Contents)

• - PART A - 1
• Isolation and Structure Elucidation of Compounds from Leaves of Maackia fauriei 1
• 1. Introduction 1
• 1-1. Scope and Purpose 2
• 2. Material and Methods 4
• 2-1. Plant material 4
• 2-2. General experimental procedure 4
• 2-3. Extraction and Isolation 5
• 3. Results 18
• 3-1. Compound 1 8
• 3-2. Compound 2 8
• 3-3. Compound 3 9
• 3-4. Compound 4 9
• 3-5. Compound 5 10
• 3-6. Compound 6 10
• 3-7. Compound 7 11
• 3-8. Compound 8 11
• 3-9. Compound 9 12
• 4. Discussions 13
• 4-1. Compound 1 13
• 4-2. Compound 2 15
• 4-3. Compound 3 17
• 4-4. Compound 4 19
• 4-5. Compound 5 21
• 4-6. Compound 6 23
• 4-7. Compound 7 25
• 4-8. Compound 8 27
• 4-9. Compound 9 29
• 5. Summary 31
• - PART B - 33
• Anti-oxidative and Anti-tumor Activities of Compounds and extract from Leaves of Maackia fauriei 33
• 1. Introduction 33
• 1-1. Anti-oxidative activity 34
• 1-2. Anti-tumor activity 36
• 1-3. Screening of Korean native plants and selection of the leaves of M. fauriei 38
• 2. Material and Methods 40
• 2-1. Anti-oxidative activity 40
• 2-1-1. Measurement of DPPH radical scavenging activity 40
• 2-1-2. Measurement of NBT/Superoxide scavenging activity 41
• 2-2. Anti-tumor activity 42
• 2-2-1. Cell culture 42
• 2-2-2. MTT assay 43
• 2-2-3. Annexin V / PI double staining 44
• 2-3. Statistical analysis 45
• 3. Results and discussion 46
• 3-1. Anti-oxidative activity 46
• 3-1-1. DPPH radical scavenging activity 46
• 3-1-2. NBT/Superoxide scavenging activity 50
• 3-2. Anti-tumor activity 54
• 3-2-1. Cytotoxic activity against cancer cells 54
• 3-2-2. Flowcytometry for apoptosis using annexin V / PI staining 66
• 4. Conclusion 69
• References 70
• Appendix 74
• 국문초록 102