Extraction of Martinella obovata barkNaomi 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 Beck @ 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 CH2Cl2/MeOH (1:1)

Bark & solvent Sat @ rt overnight

Filtered out solids

Evaporated to drier conditions 6.09 g slimy residue

FractionationSolvent search

Round 1 HE/EtOAc (1:1) CH2Cl2/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

TLC Analysis – Hex/EtOAc (4:1)

Treated with vanillin stain

F1

F2

F4

F3

F6 F5

F5

Solvated fractions111 to 160 turnedfrom pale greento pale then darkyellow. The transitionoccurred at F6,making F6 visually difficult to classifyas either color.

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

Fraction 1 – NB1-6-2

NH

N

HO

O

NH

NH

HN

HN

NH

Martinellic acid

NH

N

O

O

NH

NH

HN

HN

NH

HN

HN

NH

Martinelline

Fraction 2 – NB1-6-3

NH

N

HO

O

NH

NH

HN

HN

NH

Martinellic acid

NH

N

O

O

NH

NH

HN

HN

NH

HN

HN

NH

Martinelline

Fraction 3 – NB1-6-4

NH

N

HO

O

NH

NH

HN

HN

NH

Martinellic acid

NH

N

O

O

NH

NH

HN

HN

NH

HN

HN

NH

Martinelline

Fraction 5 – NB1-6-6

NH

N

HO

O

NH

NH

HN

HN

NH

Martinellic acid

NH

N

O

O

NH

NH

HN

HN

NH

HN

HN

NH

Martinelline

Fraction 6 – NB1-6-7

NH

N

HO

O

NH

NH

HN

HN

NH

Martinellic acid

NH

N

O

O

NH

NH

HN

HN

NH

HN

HN

NH

Martinelline

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

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.