ABSTRACT
BACKGROUND: We recently reported that the ethyl acetate (EtOAc)-soluble fraction of the methanol extract of the root of Rhinacanthus nasutus showed tumor-specific non-apoptotic cytotoxicity and antiosteoclastogenic activity. In the present study, we investigated whether five rhinacanthins, mostly isolated from the EtOAc-soluble fraction of this plant, are responsible for these activities. MATERIALS AND METHODS: The cytotoxic activity was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) method. The 50% cytotoxic concentration (CC(50)) was determined by the dose-response curve. Tumor specificity (TS) was determined by the ratio of the mean CC(50) for normal cells to that of tumor cell lines. DNA fragmentation was assayed by agarose gel electrophoresis. Caspase-3 activation was monitored by substrate cleavage assay. Osteoclastogenesis was monitored by tartrate-resistant acid phosphatase (TRAP) activity in receptor activator of NF-κB ligand (RANKL)-stimulated bone marrow-derived macrophages. RESULTS: Among five rhinacanthins (rhinacanthin C, G, N and Q, and rhinacanthone), rhinacanthin C exhibited the highest tumor specificity (TS=15.2). Rhinacanthin C did not induce internucleosomal DNA fragmentation nor caspase-3 activation, suggesting non-apoptotic cell death. Rhinacanthin C most potently inhibited the RANKL-stimulated osteoclastogenesis. CONCLUSION: The present study suggests that rhinacanthin C may be responsible for the biological activity of the EtOAc-soluble fraction prepared from the methanolic extract of R. nasutus we previously reported on.
Subject(s)
Acanthaceae , Antineoplastic Agents/pharmacology , Phytotherapy , Plant Extracts/pharmacology , Plant Roots/chemistry , Acanthaceae/chemistry , Apoptosis/drug effects , Cell Line, Tumor , DNA Fragmentation/drug effects , Humans , Naphthoquinones/pharmacology , Osteoclasts/drug effectsABSTRACT
BACKGROUND: We recently reported that the MeOH extract of aerial parts and root of Rhinacanthus nasutus showed diverse biological activity, with most activity being concentrated into the EtOAc layer separated by sequential organic solvent extractions. In the present study, the EtOAc extracts were further separated by silica-gel column chromatography into five fractions (Frs. 1-5), and their cytotoxicity and apoptosis-inducing activity investigated. MATERIALS AND METHODS: Cytotoxic activity was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) method. The 50% cytotoxic concentration (CC(50)) was determined from the dose-response curve. Tumor specificity (TS) was determined by the ratio of the mean CC(50) for normal cells to the one for tumor cell lines. DNA fragmentation was assayed by agarose gel electrophoresis. Caspase-3/-7 activation was monitored by cleavage of substrates either spectrophotometrically or by western blot analysis. RESULTS: Among five fractions of the EtOAc extract, Fr. 1, eluted with CHCl(3)-MeOH (50:1), showed the highest tumor specificity (TS=3.3) as compared with other fractions eluted at higher concentrations of MeOH in CHCl(3) (TS=1.0-2.8). Fr. 1 did not induce internucleosomal DNA fragmentation or induced only marginal level of caspase-3 activity in either human promyelocytic leukemia HL-60 cells and human oral squamous cell carcinoma (OSCC) cell lines HSC-2. CONCLUSION: The present study suggests that hydrophobic substances of EtOAc extract show tumor specific cytotoxicity by inducing little or no apoptosis.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Carcinoma, Squamous Cell/pathology , Magnoliopsida/chemistry , Mouth Neoplasms/pathology , Plant Extracts/pharmacology , Acetates , Antineoplastic Agents, Phytogenic/isolation & purification , Caspase 3/metabolism , Cell Death/drug effects , Cell Line, Tumor/drug effects , Chromatography, Gel , DNA Fragmentation/drug effects , DNA, Neoplasm/drug effects , Drug Screening Assays, Antitumor , Enzyme Activation/drug effects , Epithelial Cells/drug effects , Fibroblasts/drug effects , HL-60 Cells/drug effects , HL-60 Cells/enzymology , Humans , Hydrophobic and Hydrophilic Interactions , Plant Roots/chemistry , Plant Stems/chemistry , SolventsABSTRACT
BACKGROUND: The MeOH extracts from aerial part and roots of Rhinacanthus nasutus were investigated for new biological activities. MATERIALS AND METHODS: The MeOH extract of the root was stepwise separated by organic solvents into n-hexane, EtOAc, n-BuOH and H(2)O layer fractions. Cytotoxic activity against human tumor and normal cells was determined by the MTT method. Nitric oxide (NO) was determined by the Griess method. Osteoclastogenesis was monitored by tartrate-resistant acid phosphatase (TRAP) activity. RESULTS: The MeOH extract of the root showed much higher tumor-specific cytotoxicity than that of the aerial part. The EtOAc fraction of the root showed the highest tumor-specific cytotoxicity, followed by the n-BuOH, n-hexane and H(2)O fractions. None of the four fractions protected the cells from the cytotoxicity of UV irradiation. The n-BuOH fraction not only stimulated NO production by mouse macrophage-like RAW264.7 cells, but also inhibited the lipopolysaccharide (LPS)-stimulated NO production. The EtOAc fraction inhibited the receptor activator for nuclear factor-κB ligand (RANKL)-stimulated osteoclastogenesis of the RAW264.7 cells most potently, followed by the n-hexane, n-BuOH and H(2)O fractions. The n-BuOH fraction slightly, but significantly stimulated osteoclastogenesis. CONCLUSION: Antitumor and macrophage/osteoclast-modulating substances are enriched in EtOAc and n-BuOH fractions of MeOH extract of R. nasutus roots.
