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Bioactivities and chemical constituents of several ferns in Malaysia, with emphasis on Blechnum orientale Linn.
thesisposted on 22.03.2017, 01:34 by Lai, How Yee
In the preliminary study to find new and natural sources of antioxidant, tyrosinase inhibition and antibacterial compounds, methanolic extracts from fifteen fern species were investigated. The total phenolic content (TPC) was determined using the Folin-Ciocalteau method and the antioxidant capacities were measured using the DPPH radical scavenging, ferric reducing power (FRP), metal ion chelating power and β–carotene bleaching (BCB) assays. Five fern species that possessed strong DPPH radical scavenging activity, FRP and BCB activity were Blechnum orientale L., Cyathea latebrosa (Wall. Ex. Hook) Copel, Cibotium barometz (L.) J. Sm., Drynaria quercifolia (L.) J. Sm. and Dicranopteris linearis (Burm.) Underwood var. linearis. Strong ion chelating activity was shown by Pteris vittata L. and Pteris venulosa Bl. Moderate tyrosinase inhibition activity was found in B. orientale and C. latebrosa, while antibacterial activities were exhibited by B. orientale, D. linearis and C. barometz. Five solvent extracts of B. orientale (BO) and C. barometz (CB) were prepared by suspending the respective methanolic crude extracts in water and successively extracting with petroleum ether, chloroform, ethyl acetate and butanol. HPLC and LC-MS analyses identified rutin, chlorogenic acid, kaempferol, caffeic acid, apigenin-7,4-diglucoside, luteolin-7-rutinoside and luteolin triglycoside in BO, while rutin and esculin were found in CB. Bioactivities investigations showed that the ethyl acetate- (BOeaf), butanol- (BObutf) and water- (BOwatf) soluble extracts possessed strong radical scavenging (IC50 8.6-13.0 µg/mL), antibacterial activity against Bacillus cereus, Micrococcus luteus, methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA) and Stapylococcus epidermidis (minimum bactericidal concentration MBC 15.6-250 µg/mL) and selective cytotoxic activity against human colon cancer cells HT29 (IC50 27.5-42.9 µg/mL). Tyrosinase inhibition activities were strongest in BOeaf (70% at 2.5 mg/mL) and BOwatf (51%). The water extract (BOwatf) was found to be safe (i.e. low toxicity) for oral consumption following an oral acute toxicity study. The traditional usage of BO in wound healing was verified in an in vivo study using the water extract, as revealed by the significant wound size reduction, mean epithelisation time and the increased collagen synthesis in the 2% extract-treated group, compared with those of the vehicle-group. A polymeric proanthocyanidin (condensed tannins) and a flavan-3-ol were isolated from the active solvent extracts of BO. Spectroscopic analyses showed that the proanthocyanidin was a heterogeneous polymer possessing epicatechin subunits with 2-12 degree of polymerization and co-existing with minor units of epiafzelechin and epigallocatechin at C4-C8 interflavanoid linkages. Bioactivity studies recorded strong radical scavenging activity (IC50 5.6 ± 0.1 µg/mL), bactericidal activity and selective cytotoxicity towards colon cancer cells HT29 (IC50 7.0 ± 0.3 µg/mL). Further studies were conducted to assess the cytotoxic potential and apoptosis induction effects on HT29. Cell death was found to be time and dose dependent. The induction of apoptosis was confirmed using fluorescence microscopy, FITC-Annexin V staining coupled with flow cytometry, caspase colorimetric assays and western blot analyses. The molecular mechanism underlying the apoptosis in the extract-treated HT29 cells involved caspases -3, -6 and -8. These results provided the first evidence of the apoptotic induction capability of the proanthocyanidins of B. orientale via an extrinsic-mediated pathway. The flavan-3-ol isolated was characterized as 4β-carboxymethyl-(-)-epicatechin which possessed antioxidant potential (IC50 14.66 ± 0.29 µM) comparable to that of ascorbic acid. The overall results indicate the promising potential of the proanthocyanidins of B. orientale as a natural therapeutic source to treat oxidative related medical health problems, for the chemotherapy of colon cancer and a source of bactericidal agent.