Supplementary Methods
Olive Oil Ratings
The degree of throat irritation from each of 10 commercial Greek, Italian, and U.S. extra
virgin olive oils (Falconero, Laudemio, Frantoio, Calonna, Spitiko, Horio, Lucini, Caroli,
Sitia, Olio Santo) was quantified by 17 volunteers. Each subject was tested only 2 times
per day with two different olive oil samples with 1-2 hours separating each test, since the
perceived irritation intensity appears to be sensitive to short inter-trial intervals. Subjects
wore nose clips to eliminate olfactory cues. Tasting consisted of placing approximately
3.5 ml of olive oil in the mouth, holding it for 3 seconds and then swallowing it in two
aliquots so as to insure the throat would be stimulated. After 45 sec passed, subjects were
asked to rate the peak throat irritation intensity using a general labeled magnitude scale, a
sensory scale developed to generate magnitude estimation-like ratio-quality data1,2. Each
subject was tested twice with all 10 oils presented in random order.
Supplementary figure S1. Pharyngeal irritation caused by (–)oleocanthal in different
oils. Solid red line, least-squares regression (r=0.9) for ten extra-virgin olive oils (circles)
evoking different degrees of irritation; dashed blue line, least-squares regression of
synthetic (–)oleocanthal at different concentrations (triangles) in non-irritating corn oil;
(oleocanthal concentrations are scaled logarithmically; irritation ratings by 17 and 10
observers, respectively, on a general-labelled magnitude scale). Participants provided
informed consent on a form approved by an Institutional Review Board.
Evaluation of Synthetic (-)-Oleocanthal
Ten subjects were tested with non-irritating commercial corn oils presented neat and
mixed with either synthesized (-)oleocanthal or the bitter agent sucrose octaacetate
(SOA) (Sigma-Aldrich). The addition of SOA enabled forced-choice trials to be
conducted without revealing to subjects the identity of the irritating samples due to
bitterness or other non-irritating cues. (-)Oleocanthal was tested at the highest
concentration identified in the ten rated oils, 200 microgram/ml, and at one half and
whole log steps more dilute 63.25 and 20 microgram /ml. SOA was added to the corn oil
(4 X 10-4, 1 X 10-4, 5 x 10-5 M) to intensity match the irritation of the three levels of ()oleocanthal. Subjects participated in two-alternative forced-choice (2AFC) trials (four
trials at every concentration for each subject) and in intensity ratings sessions (four
ratings per each oil). For the 2AFC trials subjects were presented with two 3.0 ml corn
oil samples with matching intensities of SOA and (-)oleocanthal in ascending order, and
were required to sample oils as described above. While blind to stimulus position,
subjects were asked two questions on each trial, “Which of the two oils was more
irritating in the throat?” and “Which one was more bitter?” At the 20 microgram/ml and
5x10-5 M level most subjects reported on some trials that the same oil was both the more
irritating and the more bitter of the two. This demonstrates that participants were willing
to select the one oil as stronger on both traits within a trial. Subjects performed at chance
when selecting among two unadulterated corn oils, when the correct choice was
randomly assigned prior to testing. At 20 microgram/ml subjects were correct 24 out of
40 trials, indicating that this concentration is near detection threshold levels in corn oil.
The other two concentrations were correct 39/40 and 40/40 trials. For the intensity rating
trials subjects were presented with all eight oils in ascending order, counterbalanced for
stimulus order and asked to rate the throat irritation and bitterness of every oil on a
general labeled magnitude scale (described above).
Oleocanthal Isolation
To isolate and purify oleocanthal, we extracted the irritant from olive oil with
methanol/water (80/20, v/v) using a modification of an existing procedure3. The phenolic
extract was separated into 15 fractions with reversed-phase HPLC. Only one fraction was
identified as irritating by human observers. To obtain pure material, we pre-fractionated
the olive oil phenolic extract on a C18 solid phase extraction cartridge. Retention
information about the throat-irritating principal from the HPLC method allowed us to
separate it from the majority of the other co-extracted phenolic compounds using
methanol and water solvent mixtures at three different ratios of eluting solvents. HPLC
analysis of the throat-irritating fraction revealed the presence of several unresolved
compounds. A new HPLC gradient was thus developed and only one well-resolved peak
was throat-irritating. A detailed NMR (1D and 2D) analysis was conducted with this
material. Although 1H-NMR spectra indicated the presence of minor impurities, the
structure of the major compound was readily identified to be 2-(4-hydroxyphenyl) ethyl,
4-formyl-3-(2-oxoethyl)-4-hexenoic acid ester, the deacetoxy-dialdehydic ligstroside
aglycone, confirming a previous isolation4 and sensory identification3.
Quantification of Oleocanthal in Olive Oils
The amount of oleocanthal in each of the ten extra virgin oils was quantified. The
compound was extracted from small amounts of each of the 10 oils (1 g) by hexaneacetonitrile (liquid-liquid) extraction. The solvent extract was analyzed by reversed-phase
HPLC with UV detection at 278nm. Oleocanthal was chromatographically separated
from the other extracted compounds with an elution gradient of acetonitrile and water.
All analyses were done in duplicate using solutions of pure, previously-isolated
oleocanthal as the external standard. We also used synthesized oleocanthal as a standard
to confirm these methods. Overall, the reproducibility was high (RSD = 4.7%), recovery
was good (> 95%), the calibration curve was linear (r2 = 0.999) and the limit of
quantitation was < 1 ppm.
De Novo Synthesis of Oleocanthal
Both enantiomers of oleocanthal were synthesized in13 steps. The synthesis is described
in detail in a paper by ABSIII and QH, “Synthesis of (+)- and (-)-Oleocanthal (a.k.a.
Deacetoxy Ligstroside Aglycon)” which has been submitted for publication and can not
be presented here. We also measured the optical rotation of the oleocanthals and
identified the natural enantiomer to be levorotary.
Anti-inflammatory Assays
We chose to evaluate inhibition of cyclooxygenase (COX) and lipoxygenase (LOX), two
enzymes central to arachidonic acid-based inflammatory processes5,6. Ibuprofen is a
potent COX-1 and COX-2 inhibitor but does not inhibit lipoxygenase7,8. The
concentration dependence of oleocanthal for inhibition of ovine COX-1, human
recombinant COX-2 and soybean 15-lipoxygenase activities was measured using
commercially available kits (Cayman Chemicals). Indomethacin was used as a positive
(inhibitory) control in the cyclooxygenase assays and nor-dihydroguaiaretic acid
(NDGA) and caffeic acid were used as positive (inhibitory) controls in the lipoxygenase
assays; for comparison, ibuprofen was tested along with oleocanthal in these assays. Both
enantiomers of oleocanthal, exhibited a dose-dependent inhibition of COX-1 and COX-2
activities, with no effect on lipoxygenase activity, much as observed with ibuprofen. The
calculated IC50 (least squares regression analysis of inhibition vs. concentration) for (-)oleocanthal was 23 M and 28 M for COX-1 and COX-2, respectively. The IC50 for
(+)-oleocanthal was 25 M and 40 M for COX-1 and COX-2, respectively. The percent
inhibition of COX-1 and COX-2 by indomethacin presented in Table 1 was determined
by monitoring oxygen consumption with an Oxytherm Electrode Unit by Hansatech for
COX enzyme activity in a Cayman Chemicals free enzyme assay.
Supplemental References
S1.
S2.
Green, B. G., Shaffer, G. S. & Gilmore, M. M. Derivation and evaluation of a
semantic scale of oral sensation magnitude with apparent ratio properties.
Chemical Senses 18, 683-702 (1993).
Green, B. G. et al. Evaluating the 'Labeled Magnitude Scale' for measuring
sensations of taste and smell. Chemical Senses 21, 323-324 (1996).
S3.
S4.
S5.
S6.
S7.
S8.
Andrewes, P., Busch, J. L. H. C., De Joode, T., Groenewegen, A. & Alexandre,
H. Sensory properties of virgin olive oil polyphenols: Identification of deacetoxyligostride aglycon as a key contributor to pungency. Journal of Agricultural and
Food Chemistry 51, 1415-1420 (2003).
Montedoro, G. F. et al. Simple and hydrolyzable compounds in virgin olive oil. 3.
Spectroscopic characterization of the secoridoid derivatives. Journal of
Agricultural and Food Chemistry 41, 2228-2234 (1993).
Smith, W. L. Prostanoid biosynthesis and mechanisms of action. American
Journal of Physiology: Renal, Fluid, and Electrolyte Physiology 263, F181-F191
(1992).
Vila, L. Cyclooxygenase and 5-lipoxygenase pathways in the vessel wall: role in
atherosclerosis. Medical Research Reviews 24, 399-424 (2004).
Vane, J. R. & Botting, R. M. New insights into the mode of action of antiinflammatory drugs. Inflammatory Research 44, 1-10 (1995).
Abramson, S. R. & Weismann, G. The mechanisms of action of non-steroidal
anti-inflammatory drugs. Arthritis and Rheumatology 32, 1-9 (1989).