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Research and Creative Experience for
Undergraduates Program (RCEU)
Summer 2018

Analysis of Interactions Between Caffeine
and Berberine Anti-Cancer Drug in Aqueous
Solution Using NMR Spectroscopy
Bethany Kregger, Charity Yarbrough, Grace Springer, and Dr.
Bernhard Vogler, Department of Chemistry, UAH
Background Methods
• Cancer is a disease of uncontrolled cell proliferation. DNA replication • Samples were prepared using serial dilutions of caffeine and berberine in
occurs more frequently due to loss of cell cycle control mechanisms, D2O with 4,4-Dimethyl-4-silapentane-1-sulfonic acid (DSS) reference
leading to tumor propagation. standard, providing a range of concentrations for each sample set.
• Berberine is a planar, aromatic compound under investigation as a • Caffeine samples and Berberine samples were each tested at 25̊ C and
treatment for certain cancers. Berberine may induce cancer cell apoptosis 35 ̊C and compared to mixtures of the two compounds together at the
by intercalating into the bases of the cell’s DNA, thus inhibiting replication same temperatures.
and suppressing tumor growth. • 1HNMR experiments were run on a Varian Unity INOVA® 500MHz NMR.
• An NMR stack of chemical shift behavior was prepared for each sample
using MestreNova analysis software. Peak chemical shifts were analyzed
Figure 1: Structure of Berberine. for potential interaction using Ka calculations on MestreNova and
Number labels represent peak positions Microsoft Excel.
on berberine’s 1HNMR spectrum.

• Caffeine is also a planar molecule which may interfere with the
effectiveness of anti-cancer intercalating agents such as berberine by
directly binding with the drug and preventing it from targeting the DNA of
cancer cells.

Figure 2: Structure of Caffeine.
Number labels represent peak positions
on caffeine’s 1HNMR spectrum.

• NMR Spectroscopy was used to analyze the chemical shifts of aqueous
samples of caffeine and berberine molecules individually and in mixture to
determine association constants (Ka) and derive points of interaction
between the two molecules. Understanding these interactions will
contribute to understanding effectiveness of Berberine as an anticancer
drug and caffeine’s role in inhibiting the effects of berberine in fighting
cancer.

Figure 4: Methods of Chemical Analysis. Berberine 5mM sample set in NMR capillary tubes,
Berberine 5mM original solution, and 1HNMR spectrum of Berberine 5mM Sample 1 at 25 degrees
(top left); Bethany Kregger placing sample in Varian Unity INOVA® 500MHz NMR (bottom left); Data
Scatterplot of Berberine 5mM Peak 5 Chemical Shift using experimental values at 25 degrees and
trendline values to determine Ka (top right); Graph of Change in Ka value from Berberine 5mM
Standard sample set at 25 degrees to other relevant sample sets, where increase indicates that Ka is
higher in Berberine 5mM sample set and decrease indicates that Ka is higher in compared sample set
(bottom right).
Key Findings
• Berberine’s distinctly higher peak 3 Ka in standard than in mixture indicates that
peak 3 may be more involved in self-stacking than in interacting with caffeine.
• Berberine’s increased Ka values at peaks 1, 6, and 7 in mixture with caffeine
indicate that they may be involved in stacking with caffeine.
• In higher temperatures, there is a trend toward increase in Ka values of
berberine peaks, indicating that berberine may be more likely to self-associate
at body temperature than to interact with caffeine.
• In higher concentrations, berberine peaks tends to increase in Ka value,
Figure 3: NMR Stack of Chemical Shift Behavior using MestreNova Analysis Software. Samples indicating that berberine is more likely to self-associate than to repel like
range from high concentration (top of each stack) to low concentration (bottom of each stack) for
each sample set. Peak 3 of caffeine in Caffeine Standard sample set at 25 degrees (top left); Peak 3
molecules.
of caffeine in Berberine/Caffeine Mixture sample set at 25 degrees (bottom left); Peak 5 of berberine • At lower temperatures, increased Ka values indicate that berberine peak 1 as
in Berberine 5mM Standard sample set at 25 degrees (top right); Peak 5 of berberine in well as peaks 5, 6, 7, and 11 may be involved in stacking with caffeine, while at
Caffeine/Berberine Mixture sample set at 25 degrees (bottom right). higher temperatures berberine peaks 1, 6, 7, 8, and 10 all show the opposite
trend and instead peaks 3, 5, and especially 9 may become more involved in
Acknowledgements stacking behavior, whether with caffeine or in self-stacking.
• Overall, it might be inferred that at lower temperatures, multiple berberine peaks
Bethany Kregger thanks Dr. Bernhard Vogler, David Cook, and the RCEU staff for (1, 5, 6, 7, 8, and 11) may play a role in associating with all caffeine peaks -
guidance and mentorship, the Department of Chemistry at UAH for hosting RCEU especially caffeine’s peak 4 – and in self-association. However, as temperatures
2018, and the UAH Office of the Provost, the UAH Office of the Vice President for increase to conditions similar to body temperature, berberine may dissociate
Research and Economic Development, and the Alabama Space Grant Consortium from caffeine and increase in self-association at peaks 3, 5, and 9 while caffeine
for sponsorship. may increase in self-association at peak 4.
References • Further studies may benefit from measurements using different reference
1. Knott, E. (2014) Caffeine Modulation of Anticancer Drug Activity Against Topoisomerase II Thesis submitted to the
Department of Chemistry and the School of Graduate Studies at the University of Alabama in Huntsville.
standards for comparison, as there was some indication that the molecules
2. Redmon, W. (2017) Interactions of Caffeine with Aromatic Anticancer Drugs in Aqueous Solutions Studied by Nuclear being studied may have had some level of interaction with the DSS reference
Magnetic Resonance Spectroscopy Thesis submitted to the Department of Chemistry and the School of Graduate
Studies at the University of Alabama in Huntsville.
standard.