Discovery of anti-inflammatory compounds from ipomoea aquatica

국문 초록 (Abstract)

동남아시아 및 열대∙아열대 지역에서 널리 사용되어온 공심채(Ipomoea aquatica)는 항산화∙항염 등 다양한 효능에 기반하여 식용 및 약용 자원으로 활용되어 왔습니다. 최근 Ipomoea aquatica의 기능성에 관한 관심이 증가되며 관련 연구가 활발히 이루어지는 반면, 순수 분리 화합물 수준에서의 활성 연구는 미흡한 실정입니다.
본 연구에서는 Ipomoea aquatica의 생리활성 성분을 규명하고자 식물체의 메탄올 추출물을 hexane, CH2Cl2, EtOAc, n-BuOH로 용매 분획하고, GNPS 기반의 분자 네트워킹 분석을 통해 주요 대사산물의 구조적 유사성과 군집 분포를 확인하였습니다. 또한 각 분획물에 대해 NO 생성 억제 활성 기반 생리활성 스크리닝을 시행하였습니다. 그 결과, hexane, CH2Cl2, EtOAc, 분획에서 우수 생리활성 물질의 존재 가능성이 확인되어 추가 분리 연구를 진행하였고, 이로부터 총 8종의 화합물을 분리·정제하였습니다. 이 중 4종은 I. aquatica에서 최초로 보고되는 화합물임을 확인하였고, 분리된 화합물은 HR-ESIMS, ¹H-NMR 분석 및 문헌을 통해 구조를 규명하였습니다.
분리된 모든 화합물에 대해 항염증 활성을 평가한 결과, 일부 화합물이 농도 의존적으로 유의미한 항염효과를 나타냄을 확인하였습니다. 이에 따라 본 연구는 I. aquatica가 오랜 기간 약용 식물로 사용되어온 근거를 과학적으로 뒷받침하였으며, 공심채 유래 신규 화합물의 구조적 다양성을 제시함으로써, 향후 천연물 기반 항염제의 개발, 기능성 식품 소재화, 의약∙바이오 산업 응용에 활용 가능한 기초자료로 응용될 수 있음을 확인하였습니다.

다국어 초록 (Multilingual Abstract)

The Convolvulaceae family includes fast-growing climbing plants with trumpet-shaped flowers and simple leaves. Among them, Ipomoea aquatica (water spinach) is a semi-aquatic plant found in tropical and subtropical wetlands and is used in Vietnamese traditional medicine for its antioxidant and anti-inflammatory properties. Similarly, in Korea, I. aquatica has recently gained popularity as a food ingredient with potential anti-inflammatory effects. Despite its medicinal significance, phytochemical and biological studies on I. aquatica are limited. Therefore, this study was conducted to isolate and identify its bioactive compounds and evaluate their anti-inflammatory activities.
Dried leaves of I. aquatica were extracted with 80% aqueous methanol (MeOH), and the concentrated extract was subsequently solvent-partitioned into hexane-, dichloromethane (CH2Cl2)-, ethyl acetate (EtOAc)- and n- butanol (n-BuOH)-soluble fractions. Based on GNPS analysis and anti- inflammatory results, the hexane, CH2Cl2- and EtOAc-soluble fractions were selected as priority candidate fractions for subsequent isolation. Phytochemical analysis via column chromatography and HPLC purification led to the isolation and identification of Hexadecyl (E)-p-coumarate (1), Octadecyl (E)-p- coumarate (2), N-cis-feruloyltyramine (3), N-trans-p-coumaroyltyramine (4), N-trans-feruloyltyramine (5), (9S,10E,12S,13S,15Z)-9,12,13- trihydroxy-10,15-octadecadienoate (6), Kaempferol-3-O-glucoside (7), 5- O-caffeoylquinic acid (8). Their structures were determined by one- dimensional (1D) nuclear magnetic resonance (NMR) spectroscopy and high- resolution electrospray ionization mass spectrometry (HR-ESI-MS) analysis, together with comparison to previously reported literature data.
To evaluate anti-inflammatory activity, isolated compounds (1-8) were tested in lipopolysaccharide (LPS)-stimulated BV-2 cells, and compounds 1, 2, 6, and 7 significantly reduced NO production at 20 µ g/mL. These activities are consistent with reported NO inhibitory effects of unsaturated fatty acids such as α-linolenic acid and linoleic acid, and the flavonol glycoside kaempferol-3-O-β-rutinoside, supporting the anti-inflammatory potential and traditional medicinal use of I. aquatica.

목차 (Table of Contents)

• Chapter 1. Introduction １
• Chapter 2. Materials and methods ５
• 1. General experimental procedures ５
• 2. Isolation and purification ７
• 2.1 Plant material ７
• 2.2 Extraction and solvent partition ８
• 2.2.1 HR-ESIMSMS for molecular networking (GNPS) ９
• 2.3 Isolation of compounds from hexane-soluble fraction １0
• 2.4 Isolation of compounds from CH2Cl2-soluble fraction １２
• 2.5 Isolation of compounds from EtOAc-soluble fraction １５
• 3. Evaluation of anti-inflammatory activity １７
• 3.1 Cell culture and viability １７
• 3.2 Measurement of nitric oxide production １８
• Chapter 3. Results and discussion １９
• 1. Screening of anti-inflammatory activity of solvent-partitioned fractions １９
• 2. GNPS-based metabolite profiling of solvent-paritioned fractions ２１
• 3. Isolation and structural elucidation of compounds ２４
• 3.1 The structure of known compounds 1-8 ２６
• 4. Inhibitory activity on Nitric Oxide production ４２
• 4.1 Cytotoxicity of I. aquatica-derived compounds in BV-2 microglia ４２
• 4.2 Effects of I. aquatica-derived compounds on LPS-induced NO production ４３
• Chapter 4. Conclusion ４５
• Chapter 5. References ４６