Extraction of Martinella obovata bark Naomi Bryner

Overview - Genus  Bignoniaceae family  120 genera, 800 species  Found in Central and South America  Tropical rainforests  Versatile plant  Horticulture, food, crafts, timber, dyes, rituals, and medicine

Overview - Species  Eye medication  13 ethnolinguistic groups  8 S. American countries  Amazon Indian tribes  ‘yuquilla’  From fleshy root bark  Stripped, pounded, and strained  Applications for curing conjunctivitis  Martinelline  Martinellic acid

Research goals  Investigate processes for pre-extraction treatment of root bark  Develop a protocol for extraction  Perform FCC and identify useful fractions via TLC  Isolate martinelline and martinellic acid  Characterize the compounds via spectroscopic methods

Pre-extraction treatment  Dr. Halligan’s provisions  John SBC  Stripping bark from twigs and branches  Tedious & timely  37.3 g bark material obtained  Milling the plant material  Blender & solvent

Extraction  Merck Research Labs 1995  CH 2 Cl 2 /MeOH (1:1)  Bark & solvent  rt overnight  Filtered out solids  Evaporated to drier conditions  6.09 g slimy residue

Fractionation Solvent search  Round 1  HE/EtOAc (1:1)  CH 2 Cl 2 /MeOH (95:5)  Round 2  HE/EtOAc (2:1)  HE/EtOAc (4:1)  Round 3  HE/EtOAc (8:1) FCC  Column  Length – 30 cm  Diameter – 6 cm  Silica gel – 18 cm  Sand – 1 cm  Gradient (Hex/EtOAc)  Tubes 1-20 (8:1)  Tubes (4:1)  Tubes (1:1)  Tubes EtOAc

TLC Analysis – Hex/EtOAc (4:1) Treated with vanillin stain F1 F2 F4 F3 F6F5 Solvated fractions 111 to 160 turned from pale green to pale then dark yellow. The transition occurred at F6, making F6 visually difficult to classify as either color.

Fraction summary  F1: Fractions 3-12  Blue/black on TLC plate Rf = 0.69  Oily yellow, g  F2: Fractions  Pink on TLC plate Rf = 0.33  Pale green powder, g  F3: Fractions  Pink/red on TLC plate Rf = 0.13  Thin yellow film, g  F4: Fractions  Discard – same Rf as F5 & F6 Much fainter on TLC plate  Solvated fraction pale in color  F6: Fractions  Pink/red on TLC plate Rf = 0.10  Dark green powder, g  F5: Fractions  Pink/red on TLC plate Rf = 0.08  Yellow/tan powder, g

Fraction 1 – NB1-6-2

Fraction 2 – NB1-6-3

Fraction 3 – NB1-6-4

Fraction 5 – NB1-6-6

Fraction 6 – NB1-6-7

Final thoughts Conclusions  IR analysis was inconclusive  NMR study was cut short  F5 & F6 comparison  May be multiple compounds present  May apply to more fractions Future Work  HPLC to complete isolation  UV-vis  Identify!  LC-MS  Complete NMR  Antibacterial properties  Potency  Killing mechanism

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