STUDY OF PTERIDOPHYTES THAT INHIBITS LYPOXYGENASE AND ISOLATION OF BIOACTIVE FROM BLECHNUM ORIENTALE FOLIUM

• Main Author: Rissyelly
• Format: Dissertations
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/74288
• Institution: Institut Teknologi Bandung

Pteridophytes or fern spikes are seedless vascular plant or flower that has existed since ancient times which is about three hundred million years ago during the Carboniferous era (era fern spikes). This plant has been used traditionally as food, medicines and ornaments. Some species of fern spikes used as a pain reliever, but not many studies that explore the anti-inflammatory activity of the fern spikes. This research explored chemical compounds that inhibit lypoxygenae activity as one of anti-inflammatory mechanisms. Lipoxygenase, an enzyme that plays a role in inflammation, as the activity can produce inflammation mediator such as leukotriene. This study aimed to explores lipoxydase inhibitor from fern sipkes plants based on activity guided method (lipoxygenase inhibition in vitro). Some endemic species from Indonesia in Pteridophyte were collected and characterized. Amount of 20 species collected and determined samples were: Selaginella sp, Histioptersi incise (Thunb.) J.Sm., Goniophlebium parsicifolium (Desv.) Presl, Nephrolepis sp Schott, Davallia trichomoides Blume, Odontosoria chinensis (L.) J.Smith, Dicranopteris linearis (Burn.f) Underw. Selliguea sp, Asplenium caudatum Forst, Pteridium aquilinum (L.) Kuhn, Pityrogramma calomelanos (L.), Blechnum orientale L., Oleandra nerriformis Cavanilles, Lycopodiella cernua (L.) Pic.Serm., Lycopodium complanatum L., Nephrolepis hisitula (G.Forst.) C.Presl, Adiantum raddianum C.Presl, Belvisia spicata (L.f) Mirb, Pteris vittata L., Hypolepis sp. Samples were extracted by maceration technique with ethanol 96 %. The ethanol extracts were then anaylzed their thin layer chromatography profile, tested their lipoxygenase inhibition activities in vitro. Results showed that Blechnum orientale species gave the best inhibtion activity wich was 74.22% with IC50 196.3 µg/mL. Total phenolic content was assessed using Folin Ciocalteu’s method of ethanolic extract B. orientale determined 28.11 ± 0.0064 (gGE/100), Gallic acid as reference compound with equation y = 0.0055x + 0.0028, R2 = 0.998. Total flavonoid content of ethanolic B. orientale was 0.537 ±0.002 (gQE/100), quercetin as reference compound with equation y = 0.0068x + 0.0533, R 2 = 0.995. Antioxidant activity of ethanolic extract B. orientale was measured by DPPH method, the extract showed strong activity with IC50 6.42 µg/mL, ascorbic acid was used as reference compound with IC50 3.40 µg/mL. The ethanolic extract of Blechnum orientale was then fractionated by liquid-liquid fractionation using n-hexane and ethyl acetate. Each fraction was tested its lipoxygenase inhibition activity. Ethyl acetate fraction showed the inhibition activity against lipoxygenase enzyme in vitro with IC50, 129.40. As much as 40 g of ethyl acetate was fractioned with coloumn chromatography using silica gel as stationary phase and n-hexane, ethyl acetate, and methanol with different composition. The fractions were further monitored by thin layer chromatography (TLC) with nhexane and ethyl acetate (8:2) as eluent, then visualized with spray reagent sulphuric acid in methanol 10%. An amount of 100 mg fraction was purified using radial chromatography with ethyl acetate and methanol yielded sub fraction A and B. Further purification was done with recrystallization method using ethyl acetate and methanol, yielded isolate 1 (12.1 mg) dan isolate 2 (11.9 mg). Isolated compounds were characterized using spectroscopy methods including UV, IR, LCMS, 13CNMR, 1HNMR, DEPT, COSY, HMBC, HMQC. Pure compounds were tested their lipoxygenase inhibitory activity by in vitro. According to spectroscopy data, it assumed that isolated compounds are quersetin 3-O-?-glucopyranose (isoquercitrine, C21H20O12) and kaempferol-3-O-?-D-glucopyranoside (astragalin, C21H20O11). Isolate 1 gave the lipoxygenase inhibition activity with IC50 33.91 µg/mL, compared to quercetin with IC50 20.95 µg/mL and zileuton with IC50 43.37 µg/mL.