Structure Elucidation and Mechanisms of Anti-Cancer Steroidal Saponins in Solanum nigrum Leaves = Solanum nigrum 잎의 스테로이드 사포닌 구조 규명 및 항암 작용 기전

다국어 초록 (Multilingual Abstract)

This research examines the anticancer potential of steroidal saponins derived from Solanum nigrum leaves. It aims to develop new therapies to address challenges such as drug resistance and toxicity in standard cancer treatments. By focusing on the bioactive potential of these compounds, this study isolates steroidal saponins from S. nigrum and investigates their therapeutic effects.
To optimize steroidal saponin extraction, various solvent systems were tested, and multivariate analysis of compound intensities across extracts revealed that 70% aqueous organic solvents efficiently extracted furostanol-type saponins, while 30% aqueous organic solvents favored spirostane-type saponins. The 70% acetone extract was further subjected to sequential separation by liquid-liquid extraction, column chromatography with macroporous and silica gel resins, and preparative liquid chromatography guided by bioactivity fractionation to investigate the anticancer activity of furostanol-type saponins. This approach yielded five compounds (SN1–SN5). Among these, SN1 and SN2 were identified as solamargine and protodioscin through comparison with authentic standards, while SN3, SN4, and SN5 were characterized by MS and NMR as (25S)-uttroside B, (25R)-uttroside B, and (25S)-26-O-β-D-glucopyranosyl-22-hydroxyfurostan-3-O-α-D-rhamnopyranosyl-(1→2)-[α-D-xylopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→3)]-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside, with SN3 and SN5 being reported here for the first time. The anticancer potential of the isolated saponins was further investigated, with a particular focus on SN5. In HeLa cell assays, SN5 exhibited notable effects by inhibiting cell proliferation and migration, inducing G2/M phase cell cycle arrest, and promoting apoptosis. A metabolomic analysis was conducted to elucidate the underlying mechanisms of SN5’s anticancer activity. Results indicated that SN5 impacted arginine metabolism pathways, suggesting a metabolic mechanism through which SN5 exerts its anticancer effects. Collectively, these findings highlight the therapeutic potential of steroidal saponins from S. nigrum, especially in the context of cervical cancer, and underscore the importance of further research to fully explore their mechanisms of action.

목차 (Table of Contents)

• Literature review 1
• ChapterⅠ. Effects of Solvents on Phytochemical Yield and Biological Activity of Solanum nigrum Leaf Extracts 6
• Abstract 6
• 1. Introduction 7
• 2. Material and Methods 9
• 2.1. Chemical and plant material 9
• 2.2. Cell culture 9
• 2.3. Preparation of S.nigrum leaves extract using various solvent 9
• 2.4. Determination of phytochemical 10
• 2.4.1. Total of phenolic content(TPC) 10
• 2.4.2. Total of flavonoid content(TFC) 10
• 2.4.3. Total of steroidal saponin content(TSSC) 10
• 2.5. Antioxidant activity 11
• 2.5.1. DPPH radical scavenging activity 11
• 2.5.2. ABTS radical scavenging activity 11
• 2.6. CCK-8 assay 12
• 2.7. UPLC-QTOF MS/MS analysis 12
• 2.8. Statistical analysis 13
• 3. Results and Discussion 14
• 3.1. Phytochemical content and extraction yield using different solvent 14
• 3.2. Evaluation of antioxidant and cytotoxicity activity using different solvent 18
• 3.2.1 Antioxidant activity 18
• 3.2.2 Cytotoxicity activity 21
• 3.3. Characterization of phytochemicals in S.nigrum extracts 23
• 3.4. Multivariate analysis 31
• 4. Conclusion 38
• Chapter Ⅱ. Metabolomics-Based Insights into the Anti-Cancer Effects of Steroidal saponin from Solanum nigrum 39
• Abstract 39
• 1. Introduction 40
• 2. Materials and Methods 42
• 2.1. Chemical and reagent 42
• 2.2. Preparation of steroidal saponin from Solanum nigrum using bioactivity-guided
• fractionation 42
• 2.3. Cell culture 43
• 2.4. Cell viability assay 43
• 2.5. Colony formation assay· 43
• 2.6. Wound healing assay 44
• 2.7. Cell cycle analysis 44
• 2.8. Analysis of cell apoptosis using Annexin V-FITC/PI staining. 44
• 2.9. Determination of sugar component using GC-MS 45
• 2.10. Intracellular metabolite profiling 45
• 2.10.1. Sample preparation 45
• 2.10.2. UPLC-QTOF/MS analysis 46
• 2.10.3. Data processing 47
• 2.11. Statistical analysis 48
• 3. Results and discussion 49
• 3.1. Bioactivity-guided fractionation and purification of anti-cancer compound 49
• 3.2. Selected compound identification and structural elucidation 53
• 3.3. Effect of SN5 on HeLa cell line. 66
• 3.4. Metabolomic and pathway analysis of SN5 treated HeLa cell line. 73
• 4. Conclusion 81
• General Discussion 82
• Reference 84
• Abstract in Korean 96