February 25, 2016

Dieugenol: An Ageing Product in Clove Basil Hydrosol

Research note - Alexis St-Gelais, M. Sc., chimiste

The clove basil (Ocimum gratissimum) comes for Africa. Upon distillation, it yields an essential oil containing mainly eugenol and β-caryophyllene.

Circle H Institute is an organization dedicated to furthering the knowledge about hydrosols. Its manager, Ann Harman, contacted us after performing clove basil hydrodistillation using a copper apparatus. After standing for several months, flaky crystals formed from the clove basil hydrosol, which could not be identified using standard mass spectral databases.

We were provided a few hundred milligrams of the flakes and were able to study them. The mass spectrum of the sample did not remind of any classical class of essential oils constituents (figure 1), but the GC run showed that the crystals were likely a single molecule of high purity. They were thus submitted to nuclear magnetic resonance (NMR) experiments to determine its structure.

Figure 1. 
NMR is the most important technique used for structural determination of molecules in chemistry. It uses intense magnetic fields to induce specific energetic excitation states in atoms. Upon turning off the magnet, the energy is allowed to be released as radiofrequencies, which are measured by a detector. Various specific experiments give the analyst information about specific types of atoms (chiefly hydrogen and carbon) and their connectivities. In the end, the spectroscopist obtains a set of spectra which, upon interpretation, can be used to deduce the molecular structure of the studied compound. The two basic NMR spectra (1H and APT-13C, for respectively hydrogen and carbon) are provided for the studied crystals on figure 2 and 3. Both of them indicate a very high purity of the molecule, which greatly simplifies interpretation as there are no interfering peaks.

Figure 2. 1H spectrum of deugenol, with zoomed section for the alcenic region. Numbering refers to figure 4.
Figure 3. APT (13C) spectrum of deugenol. Positive peaks indicate CH3 or CH groups, while negative peaks indicated CH2 or quaternary carbons. Numbering refers to figure 4.
At first glance, the spectra showed that the molecule comprised 10 carbons (each peak of the APT spectrum corresponds to a different atom) and 11 hydrogens (determined from the surface of each peak on the 1H spectrum). Without considering any complementary oxygen atom, this would only sum up to a molecular mass of 131, which is far less than the observed molecular mass of the mass spectrum, which is 326. This indicated that the molecule likely comprised two perfectly symmetric parts, so that each pair of equivalent atoms would yield a single signal. This way, with a mass of 326 daltons, the molecular formula of the compound could be deduced to be C20H22O4.

The following paragraph is more technical, and presented for the scientific reader.
Analyzis of the 1D spectra indicated the presence of two oxygenated methyls, four methylene, six methine and eight quaternary carbons. The DQF-COSY spectrum indicated that H-9, H-8 and H-7 were in the same spin system, with H-9 being a terminal alkene. This indicated an allyl moiety. H-7 showed HMBC correlations with C-1, C-2 and C-6, suggesting the C-7/C-1 bond. The HMBC correlations of meta doublets H-2 and H-6 completed an aromatic ring with C-3, C-4 and C-5. The H-10 methoxy group exhibits an HMBC correlation with C-3, while the alcoholic proton OH1 correlated with C-3, C-4 and quaternary C-5. This overall suggested a structure similar to eugenol, with quaternary C-5 bonded with itself to form a dimer. A summary of DQF-COSY and HMBC correlations is shown at figure 4.

Figure 4. 2D NMR correlations of deugenol. Bold lines: COSY correlations; arrows: HC HMBC correlations.
The resulting structure is an aryl-aryl dimer of eugenol, known as dieugenol. Spectral data excellently matched that of literature for this molecule, including the MS spectrum [1], which however is not included in standard MS databases. Although we still have to perform a time-dependant analyzis of clove basil hydrosol, it is likely that this only very weakly volatile compound is not present at all in the fresh hydrosol. The latter is an eugenol-rich media with a moderately acidic pH, and could undergo over time a photo- or oxygen-catalyzed radicalar dimerization to dieugenol. The use of copper apparatus might also imply that low concentrations of copper in the condensate catalyzes this reaction. Eventually, dieugenol concentration would reach a point at which it would outgrow it water solubility and begin to precipitate in a very pure form.

Dieugenol is known to occur in clove (Syzygium aromaticum, syn. Eugenia caryophyllata) [1] and nutmetg (Myristica fragrans) [2]. It usually is isolated by fractionation of non-volatile extracts, or produced by chemical synthesis, although up to now we have encountered no account of its formation in an aqueous medium.

Dieugenol. White crystals. 1H NMR (400 MHz, CDCl3) δ: 6.77 (d, J = 1.3, H-6), 6.74 (d, J = 1.3, H-2), 6.05 (s, OH), 6.00 (m, H-8), 5.11 (m, H-9), 3.93 (s, 10-OCH3), 3.38 (d, J = 6.55, H-7); 13C NMR (100 MHz, CDCl3) δ: 147.2 (C-3), 140.9 (C-4), 137.6 (C-8), 131.9 (C-1), 124.4 (C-5), 123.1 (C-6), 115.7 (C-9), 110.6 (C-2), 56.1 (10-OCH3), 40.0 (C-7); EI-MS m/z = 326, 297 (27), 327 (22), 165 (13), 244 (13), 115 (12), 229 (11), 152 (11).

Closing note: Laboratoire PhytoChemia is eager to pursue similar collaborations (for little to no cost) to widen knowledge about essential oils, hydrosols and derived products, as long as our time allows us to investigate the matter. If you have a research project idea that can lead to a publication, please contact us.

References

[1] M. Miyazawa and H. Masayoshi. Antimutagenic activity of phenylpropanoids from colve (Syzygium aromaticum), J. Agric. Food Chem. 2003, 51(22), 6413-6422.
[2] A. Isogai, S. Murakoshi, A. Suzuki and S. Tamura. Isolation from nutmeg of growth inhibitory substances to silkworm larvae, Agric. Biol. Chem., 1973, 37(4), 889-895.

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    1. It is our pleasure! Leading small or large research projects was one of my motivations in launching our laboratory.

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