Isolation of Martinella obovata via flash column chromatography
ACS
2014
York College of Pennsylvania
Chemistry Program
a,bNaomi
bDavid
aKathleen
Bryner,
R. Singleton, PhD.,
M. Halligan, PhD.
a
b
York College of Pennsylvania, Department of Physical Sciences, Department of Biological Sciences
Introduction
The genus Martinella has been known to produce the alkaloids martinelline
(1) and martinellic acid (2). These metabolites have shown an affinity for
the bradykinin B1 and B2 receptors, and have a unique pyrroloquinoline
ring system, garnering much interest in the field of natural products
synthesis. Martinella is a member of the Bignoniaceae family, known to
have 104 genera and 860 species. The genus Martinella has only two
species: M. obovata (Figure 1) and M. iquitosensis1. According to the
literature, pyrroloquinoline alkaloids have been successfully isolated from
the root bark of M. iquitosensis and M. obovata has been used as eye
medication by the Amazon Tribes of Peru.1-4 The selected species for this
research, M. obovata, is distributed throughout the rainforests of Central
and South America.
Overview
The focus of this study was to isolate metabolites from M. obovata and
determine in particular if alkaloids 1 and 2 were present. To this end, a
simple solvent extraction was performed on M. obovata. Four distinct
metabolites were isolated via flash column chromatography (FCC) and
analyzed via Fourier transform infrared (FTIR) and nuclear magnetic
resonance (NMR) spectroscopy. Disk diffusion antibiotic sensitivity testing
was used to find susceptible bacterial strains and promising metabolites.
This activity was quantified via minimum inhibition concentration (MIC) in
the form of a microtiter assay. Metabolites were tested against a modest
range of bacterial strains, including gram positive and gram negative
strains, to gauge the bactericidal properties of each.
ACS
2014
York College of Pennsylvania
Chemistry Program
Table 1. Concentrations of NB1-6-7 and solvent carrier (EtOH or DMSO) as placed on 96-well round bottom plates. All wells
except foe column 12 received 50 μL of a bacterial strain, for a total of 100 μL liquid/well. Inhibition is indicated for E. coli α, P.
aeruginosa β, B. cereus γ, and S. aureus Δ.
Figure 1. The flower of Martinella obovata (left) compared to the experimental plant material, before (middle) and after
stripping bark (right).
E. coli
P. aeruginosa
B. cereus
S. aureus
NB1-6-7 in
EtOH
> 1 mg/mL
> 1 mg/mL
0.5 mg/mL
1 mg/mL
Different from
carrier?
no
no
yes
yes
NB1-6-7 in
DMSO
2.5 mg/mL
1.25 mg/mL
>10 mg/mL
2.5 mg/mL
Different from
carrier?
yes
no
no
yes
Results
IR for NB1-6-7
• The IR spectrum showed the absorption of O-H (3299.90 cm-1), C-H sp3
(2923.75 cm-1), C=O (1722.33 cm-1), C=C alkene (1624.59 cm-1) C=C
aromatic (1590.78 and 1498.82 cm-1), C=N (1573.47 cm-1) and C-N
(1346.79 cm-1) and C-O (1165.64 and 1073.93 cm-1).
1H, 13C APT, and H-C correlation NMR spectra (Figure 2) for NB1-6-7
• The four 13C signals at δC 128-135 correlated to four proton signals at δH
7.2-7.6 ppm; multiplicity not interpretable. The 13C signals at δC 101.7
and 107.5 correlated to the proton signals at δH 6.5 and 6.4 ppm (d, q,
respectively). The 13C signals at δC 61.3 and 69.3 correlated to proton
signals at δH 4.0 and 4.2 ppm (d, t, respectively).
Bactericidal Study
• Disk diffusion antibiotic sensitivity testing showed that NB1-6-1, NB1-66, and NB1-6-7 warranted further investigation.
• NB1-6-7 dissolved in 10.5% EtOH (1 mg/mL) inhibited S. aureus and B.
cereus at 0.5 mg/mL (Table 1).
• NB1-6-7 dissolved in 24.875% DMSO inhibited E. coli and S. aureus at
2.5 mg/mL, however, 24.875% DMSO itself did not inhibit these strains.
• The DMSO carrier alone at 99.5% inhibited all bacteria growth.
Discussion
• Comparison of the 1H NMR spectra of NB1-6-7 and martinellic acid (2)
indicated a strong resemblance.4 Some signals were absent or shifted,
thus further research would need to be conducted to determine whether
NB1-6-7 is in fact 2 or a structurally similar compound. HPLC purification
followed by LC-MS analysis may prove helpful in this regard.
• According to the bactericidal study, B. cereus was not susceptible to
dilute EtOH carrier alone, but with NB1-6-7 showed inhibition at 0.5
mg/mL. S. aureus was not inhibited with dilute EtOH carrier alone, but
with NB1-6-7 showed inhibition at 1 mg/mL. S. aureus, P. aeruginosa
and E. coli were inhibited by concentrations of DMSO up to 49.75%, and
continued to show inhibition with DMSO (24.875%) with 2.5 mg/mL of
NB1-6-7
• A consistent pattern of no disk inhibition for any bacteria with NB1-6-2
led to exclusion of that compound from further investigation.
Conclusion
Although 1 and 2 were not confirmed to be present in this extraction of M.
obovata, there is evidence for the presence of a pyrroloquinoline ring
system in NB1-6-7. Bactericidal properties were confirmed for NB1-6-7
against S. aureus, P. aeruginosa and B. cereus in EtOH, and against E. coli
and S. aureus in DMSO. TLC stains yield a single distinct spot, but a future
study would involve purification via high performance liquid
chromatography to ensure the activity is of only one metabolite.
Materials & Methods
Plant Material
• Martinella obovata was collected from the Amazon region of Peru in
1975 by Albert Sneden of Virginia Commonwealth University. In August
2006, a portion of this collection was donated to us by Dr. John Beck at
Sweet Briar College.
Extraction/Separation
• The scraped bark (37.3 g) was milled briefly in CH2Cl2/MeOH (1:1) and
extracted over 24 h at rt., resulting in 6.09 g residue after evaporation of
solvent.
• The residue was subjected to silica gel flash CC (Hex/EtOAc).
Structural Characterization
• IR spectra were obtained using a PerkinElmer Spectrum 100 FT-IR
spectrometer.
• 1H, 13C APT, and H-C correlation spectra were obtained on a Bruker 300
MHz spectrometer.
Bactericidal Study
• Metabolites (10 mg/mL) were dissolved in EtOH (95%) and DMSO
(99.5%) and used to prepare sterile disks and wells
• Bacterial strains used included Escherichia coli, Pseudomonas
aeruginosa, Bacillus cereus, and Staphylococcus aureus.
• Microbial susceptibility to compound NB1-6-7 dissolved in either EtOH
or DMSO was performed by preparing a 2-fold dilution series of stock
compounds in sterile liquid media, and adding a constant amount of
exponential phase bacteria. Inhibition of growth was assessed following
incubation.
References
(1) Arevalo et. al. Nat. Prod. Comm., 2011, 6:7, 957.
(2) Gentry. Ann. Missouri Bot. Gard., 1992, 79, 53.
(3) Gentry and Cook. J. Ethnopharmacol., 1984, 11, 337.
(4) Witherup et. al. JACS, 1995, 117, 6682.
Acknowledgements
Additional thanks for special funding support from
York College of Pennsylvania:
Student Senate, Department of Physical Sciences, and Chemistry Society;
Gaussian, Inc.;
Travel Grants from the American Chemical Society
and the Southeastern PA Section of the ACS
YCP
6
39
88.906
15
12.011
30.974
Chemistry Society
Figure 2. C-H NMR spectrum showing seven roughly correlated C-H signals. Expanded is a highly resolved proton signal
selection.