1. Extraction of Martinella obovata bark Naomi Bryner

2. 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

3. 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

4. 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

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

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

7. 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 21-138 (4:1)  Tubes 139-156 (1:1)  Tubes 157-160 EtOAc

8. 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.

9. Fraction summary  F1: Fractions 3-12  Blue/black on TLC plate Rf = 0.69  Oily yellow, 0.9508 g  F2: Fractions 35-49  Pink on TLC plate Rf = 0.33  Pale green powder, 1.0286 g  F3: Fractions 73-84  Pink/red on TLC plate Rf = 0.13  Thin yellow film, 0.0471 g  F4: Fractions 111-125  Discard – same Rf as F5 & F6 Much fainter on TLC plate  Solvated fraction pale in color  F6: Fractions 126-130  Pink/red on TLC plate Rf = 0.10  Dark green powder, 0.2404 g  F5: Fractions 131-160  Pink/red on TLC plate Rf = 0.08  Yellow/tan powder, 0.9731 g

10. Fraction 1 – NB1-6-2

11. Fraction 2 – NB1-6-3

12. Fraction 3 – NB1-6-4

13. Fraction 5 – NB1-6-6

14. Fraction 6 – NB1-6-7

15. 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

16. References  (1) Arevalo, C.; Ruiz, I.; Piccinelli, A.; Campone, L.; Rastrelli, L. Phenolic derivatives from the leaves of Martinella obovata (Bignoniaceae). Natural Product Communications, 2011, 6:7, 957-960.  (2) Gentry, A. H. A synopsis of Bignoniaceae ethnobotany and economic botany. Annals of the Missouri Botanical Garden, 1992, 79, 53-64.  (3) Gentry, A.H.; Cook, K. Martinella (Bignoniaceae): A widely used eye medicine of South America. Journal of Ethnopharmacology, 1984, 11, 337-343.  (4) Witherup, K.; Ransom, R.; Graham, A.; Bernard, A.; Salvatore, M.; Lumma, W.; Anderson, P.; Pitzenberger, S.; Varga, S. Martinelline and martinellic acid, novel G-protein linked receptor antagonists from the tropical plant Martinella iquitosensis (Bignoniaceae). Journal of the American Chemical Society, 1995, 117, 6682-6685.  (5) Ma, D.; Xia, C.; Jiang, J.; Zhang, J. First Total Synthesis of Martinellic Acid, a Naturally Occurring Bradykinin Receptor Antagonist. Organic Letters, 2001, 3:14, 2189-2191.  (6) Zhang, Z.; Zhang, Q.; Yan, Z.; Liu, Q. One-Step Synthesis of the Tricyclic Core of Martinellic Acid from 2-(Cyanomethyl)-3-oxo-N-arylbutanamides. Journal of Organic Chemistry, 2007, 72, 9808-9810.  (7) Ma, D.; Xia, C.; Jiang, J.; Zhang, J.; Tang, W. Aromatic Nucleophilic Substitution or CuI-Catalyzed Coupling Route to Martinellic Acid. Journal of Organic Chemistry, 2003, 68, 442-451.  (8) Davies, S.; Fletcher, Ai.; Lee, J.; Lorkin, T.; Roberts, P.; Thomson, J. Asymmetric Synthesis of (-)- Martinellic Acid. Organic Letters, 2013, 15:8, 2050-2053.  (9) Powell, D.; Batey, R. Total Synthesis of the Alkaloids Martinelline and Martinellic Acid via a Hetero Diels-Alder Multicomponent Coupling Reaction. Organic Letters, 2002, 4:17, 2913-2916.  (10) Yee Ng, Pui.; Masse, C.; Shaw, J. Cycloaddition Reactions of Imines with 3-Thiosuccinic Anhydrides: Synthesis of the Tricyclic Core of Martinellic Acid. Organic Letters, 2006, 8:18, 3999-4002.  (11) Shirai, A.; Miyata, O.; Tohnai, N.; Miyata, M.; Procter, D.; Sucunza, D.; Naito, T. Total Synthesis of (-)- Martinellic Acid via Radical Addition-Cyclization-Elimination Reaction. Journal of Organic Chemistry, 2008, 73, 4464-4475.  (12) Lovely, C.; Mahmud, H. An approach to the pyrroloquinoline core of martinelline and martinellic acid. Tetrahedron Letters, 1999, 40, 2079-2082.