May 19, 2016

Published Paper: Interesting and Rare Compounds from Bolivian Molle

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

Essential oils can sometimes be surprising. It so happens that well-known species that have been frequently studied yield, in particular conditions, peculiar volatile molecules that are otherwise not widely recognized to be characteristic of the plant. When such an observation is made, it is useful to publish the results, so that further occurences of these observations can stand on firm scientific ground and dissipate suspicions of contamination, misidentification or adulteration.

Figure 1. Schinus molle branch and fruits. The
resemblance of the latter with peppercorns gave
the plant its common, name, but it is unrelated
to black pepper. Source: Wikimedia commons.
In the course of a collaboration with Socodevi, a Canadian cooperative specialized in international economic development in 2014, we had to analyze samples from a Bolivian essential oil distillation facility operated by UNEC S. A., a company owned by local farmers to produce added-value products from their crops. The samples were distilled from locally picked Peruvian Peppertree, Schinus molle (figure 1).

Oil from both the leaves and fruits of Molle have been studied in numerous scientific papers (see our article for full list of references), as the plant is found throughout South America and has been planted widely elsewhere. What surprised us was the observation in the first oil that we analyzed of several rare sesquiterpenes. More batches were analyzed, and the same observations came back. We thus suggested to publish these results, which we did in Natural Product Communications recently (click here to read the abstract) [1].

Identification of the plants was confirmed by local agronomists, who are also coauthors of the study. Contamination of the plant material with others is also unlikely, given that the other species distilled by UNEC are oregano and thyme, which are devoid of the molecules that caught our interest. These were a set of oxygenated sesquiterpenes that are usually found in sweet flag, Acorus calamus, with names revolving around shyobunol and calamendiol. The whole set is depicted in figure 2.

Figure 2. Structures of the uncommon oxygenated sesquiterpenes observed in Bolivian Schinus molle essential oils.
Hints of their presence in Schinus molle were already present in literature. Indeed, a study conducted on a plant extract from a single tree grown in a botanical garden in Germany had previously lead to the isolation of preisocalamendiol [2], while shyobunol and dehydroxyisocalamendiol had been reported in essential oils of Molle in, respectively, Mexico and Saudi Arabia [3,4]. Never before though was the whole group of compounds observed at once in an essential oil from S. molle. The fact that the same observation was made on several batches produced months apart from each other further strenghtens our confidence in the results that a peculiar shyobunol/preisocalamendiol chemotype likely exists in Peruvian Peppertree, and that it can be found at the very least in Bolivia.

Figure 3. Acorus calamus, better known as
sweet flag. Source: Wikimedia commons.
Preisocalamendiol has been bolded out in figure 2 since it was one of the main constuents of the oils studied (5.6-11%). As I previously mentioned, these compounds are normally more characteristic of sweet flag, Acorus calamus (e. g. [5], see article for full references), a somewhat controversial plant. Sweet flag (figure 3) has a long history of use in aromatherapy from Asia to Europe, but has raised serious safety concerns. That is because the plant exists under three different forms, which are distinguished by the number of copies of each chromosome they bear (a phenomenon called ploidy). Diploid plants, which are native to some parts of North America, are not dangerous. However, triploid and especially tetraploid plants, which are more abundant, respectively produce an essential oil containing about 10% and 85-90% β-asarone [5], a toxic mutagenic molecule [6,7]. Sweet flag was once widely used as a flavouring agent, but is now banned by the FDA and Health Canada due to its asarone content.

Yet, sweet flag was used as a light sedative and tranquilizer. A recent paper studied the ability of several constituents of A. calamus oils to modulate the brain molecular target of several analgesic and anesthetic drugs, called GABAA. In this assay, β-asarone asarone was very efficient, but so were a few other compounds, including preisocalamendiol and shyobunone. This studied provided a good rationale for the tranquilizing properties of sweet flag [8], but this does not solve the safety concerns.

The fact that preisocalamendiol can be found in interesting concentrations in some S. molle essential oils, which is devoid of β-asarone, could point to putative tranquilizing properties with a favourable safety profile in aromatherapy, given the proper chemotype is employed. It might also in part explain why S. molle was sometimes used to treat rheumatisms, tooth pain or depression by natives. Of course, at this point, this is a mere hypothesis, and we cannot make any firm assessment of the efficiency of this oil in this regard.

We look forward to establish such collaborations with other people, too. If any oil we analyze is original enough to be published, we will be pleased to write a joint manuscript, free of charge. Only this way can the knowledge of essential oils be brought further, for our collective enjoyment!

References

[1] A. St-Gelais, M. Mathieu, V. Levasseur, J. F. Ovando, R. Escamilla and H. Marceau. (2016) Preisocalamendiol, shyobunol and related oxygenated sesquiterpenes from Bolivian Schinus molle essential oil. Nat. Prod. Comm., 11(4), 547-550.
[2] D. M. Delvalle and G. Schwenker. (1987) Preisocalamenediol, a constituent of Schinus molle. Planta Medica, 53, 230.
[3] L. Guerra-Boone, R. Álvarez-Román, R. Salazar-Aranda, A. Torres-Cirio, V. M. Rivas-Galindo, N. Waksman de Torres, G. M. González González and A. L. Pérez-López. (2012) Chemical compositions and antimicrobial and antioxidant activities of the essential oils from Magnolia grandifloraChrysactinia mexicana, and Schinus molle found in northeast Mexico. Nat. Prod. Comm., 8, 135-138.
[4] E. Abdel-Sattar, A. A. Zaitoun, M. A. Farag, S. H. Gayed and F. M. H. Harraz. (2010) Chemical composition, insecticidal and insect repellent activity of
Schinus molle L. leaf and fruit essential oils against Trogoderma granarium and Tribolium castaneum. Nat. Prod. Res., 24, 226-235.
[5] F. X. Garneau, G. Collin, H. Gagnon, A. Bélanger, S. Lavoie, N. Savard and A. Pichette. (2008) Aromas from Quebec. I. Composition of the essential oil of the rhizomes of Acorus calamus L. J. Ess. Oil Res., 20, 250-254.
[6] G. Abel. (1987) Chromosome damaging effect on human lymphocytes by β-asarone. Planta Medica, 53, 251-253.
[7] W. Göggelmann and O. Schimmer. (1983) Mutagenicity testing of beta-asarone and commercial calamus drugs with Salmonella typhimurium. Mut. Res., 121, 191-194.
[8] J. Zaugg, E. Eickmeier, S. N. Ebrahimi, I. Baburin, S. Hering and M. Hamburger. (2011) Positive GABAA receptor modulators from Acorus calamus and structural analysis of (+)-dioxosarcoguaiacol by 1D and 2D NMR and molecular modeling. J. Nat. Prod., 74, 1437-1443.

April 26, 2016

New Chemist in our Scientific Team

Photo credit: http://www.myfunstudio.com/

We are pleased to introduce you Mr. Sylvain Mercier as our new analytical chemist specialized in natural product chemistry. During the past two years, Sylvain has been working as a formulation chemist in Les Produits Sanitaires Lépine where he developed new household, industrial and institutional soaps and cleaners.

Recipient of a research grant of the Natural Sciences and Engineering Research Council of Canada (NSERC) in 2009, this distinction gave him the chance to make his first steps in a chemistry research laboratory during his bachelor by working on the extraction of new bioactive molecules from a black spruce bark extract (Picea mariana). He also worked in the Université du Québec à Chicoutimi (UQAC) Forest Research laboratory as a research assistant for some of the most influential scientists in the domain of forest plant biology.

In 2013, Sylvain have graduated from UQAC with a Master in natural product chemistry. During his master, he had the opportunity to work on the extraction and caracterisation of new molecules found in balsam fir oleoresin (Canada balsam)His work has allowed him to collaborate by writting two scientific articles published in Beilstein Journal of Organic Chemistry1 and in Organic Letters2. Sylvain is also involved in the next generation of scientists by participating at a thematic weeks on chemistry for young people from 12 to 15 years old and by being judge for local and regional Expo-Sciences.

Originally from St-Félicien (Quebec, Canada), Sylvain is honored that a company like Phytochemia believes in his talent and gave him the opportunity to develop his full potential in his native region: “Wishing to be close to nature and live near my family, Phytochemia allows me to improve myself in an exceptional environment”.

Don’t hesitate to contact him for any questions, it will be a pleasure for him to answer to all of your interrogations.



References

(1)     Lavoie, S. et al. Lanostane- and cycloartane-type triterpenoids from Abies balsamea oleoresin. Beilstein J. Org. Chem. 9, 1333–1339 (2013).
(2)     Lavoie, S. et al. Abibalsamins A and B, Two New Tetraterpenoids from Abies balsamea Oleoresin. Org. Lett. 14, 1504–1507 (2012).

March 28, 2016

Synthetic Oranger and You

Alexis St-Gelais, M. Sc., Chimiste - Popularization

Essential oils are worth quite a lot of money. It so happens that, in order to boost profits, unscrupulous people alter oils in various ways. This "Adulterants and you" series is there to introduce you to some of the adulterations we encounter. This is because not all of them are necessarily obvious, nor bear the same level of risk for the final consumer.

One of our recent public reports for an adulterated bergamot oil mentions the presence of 1-acetonaphthone. This compound has to be presented along with its close analog, 2-acetonaphthone.

What are they?

These two siblings are quite similar molecules (Figure 1) which bear unequivocal names: 1-acetonaphthone (1) is also called oranger liquid, while 2-acetonaphthone (2) is known as oranger crystals. They are reportedly prepared since the late 60s by means of a classical chemical reaction, called Friedels-Craft acylation, to add an acetyl group to naphthalene [1], which is a very cheap and accessible raw material.


Figure 1. Structures of 1- and 2-acetonaphthones.
Oranger liquid, as its name suggests, shows as a yellow-brown liquid, while oranger crystals is a nearly white solid, and both are readily soluble in apolar media, such as essential oils. They are on sale at accessible prices: purchasing 25 kg of these at Vigon will cost respectively 27$/kg and 30$/kg.

What do they do as adulterants?

Both compounds feature a delicious, orange-like scent, over a significant period of time [2,3]. This has made them ingredients of choice in perfumes, cosmetics, households products [2,3], and food [4] for decades. Sadly, it also seems that it is used to mimic or boost the poor quality of batches of neroli or bergamot essential oils, at levels below 3%. From our experience, this occurs along with some other adulteration, such as addition of synthetic linalool and linalyl acetate.

Are they dangerous?

A volunteer was reportedly irritated during a patch test implying oranger crystal [1]. Otherwise, the oral toxic doses on animal models were extremely high, and unless anyone takes teaspoons of the pure compounds, there should be no major side effects. The compounds are even used as flavoring ingredients in food [4].


How do we detect them?

A basic GC-MS run will easily detect these ingredients, although we still have to purchase both isomers to be able to fully discriminate them. They exhibit pretty similar behaviors (mass spectrum and retention indexes), but would hardly be confounded with anything else.

Bottom of the line

Oranger liquid and oranger crystal are affordable orange-flavoring ingredients which are at times used to artifically boost neroli or bergamot oils. They sometimes can be mild irritants. Be cautious when one of those oils smell like orange-flavored candy!

References

[1] Opdyke, D. L. J. β-Methyl naphthyl ketone. In Monographs on Fragrance Raw Materials: A Collection of Monographs Originally Appearing in Food and Comestics Toxicology; Pergamon Press: Oxford (UK), 2013; p. 560
[2] The Good Scents Company. Alpha-naphthyl methyl ketone, [On line], page consulted on February 14, 2016, URL: http://www.thegoodscentscompany.com/data/rw1008371.html; and The Good Scents Company. Beta-naphthyl methyl ketone, [On line], page consulted on February 14, 2016, URL: http://www.thegoodscentscompany.com/data/rw1033571.html
[3] Givaudan. Oranger Crystals, [On Line], page consulted on February 14, 2016, URL: http://eindex.givaudan.com/eindex/displayMolecule.xhtml
[4] United States Pharmacopeia. Methyl β-Naphthyl Ketone. In Food Chemicals Codex; The United States Pharmacopoeial Convention: Rockville (MD), 2010;  p. 657

March 17, 2016

Quality Control 101. III. Is Carbon 14 Testing a Reliable Method to Detect Adulteration?

Alexis St-Gelais - Popularization


Our recent clarification about our usage of "synthetic" and "natural" to describe molecules shed light on one particular way to define synthetics, based on the origin of the building blocks (petroleum or fresh organic molecules from natural sources). We wanted to discuss this aspect further, since it is deeply tied to a trendy and highly specialized analytical technique, carbon 14 analyzis.

Figure 1: 14C formation (source)
Carbon (which is mainly constituted of Carbon-12, the most stable and abundant isotope) is the building block of life on Earth. Most of the carbon found in living creatures originate from plants, which fix it from atmospheric carbon dioxide through photosynthesis. This carbon is turned into sugars, then processed further into an impressive variety of molecules within plants themselves, and by whatever feeds on the plants or decaying matter of plant origin (animals, fungi, bacteria, and so on). Thus, "fresh" carbon is picked by plants from the atmosphere. And that atmosphere is a quite exposed place: among others, it is significantly irradiated from outer space, where one finds a plethora of radiations. These include some cosmic rays that create a cascade of reactions producing neutrons. These will collide with Nitrogen-14 atoms, turning them into Carbon-14. Since the Earth's atmosphere exposure to radiations is quite constant, the proportion of Nitrogen-14 that turns to Carbon-14 in the atmosphere is very steady. Variation do occur though: among others, the nuclear testings of the 50's increased the ratio by a lot, and more recently the generalized burning of fossil fuel diluted the Carbon-14 with a massive amount of Carbon-12.

Still, the amount of Carbon-14 of the biosphere as a whole is either constant or known for various timeframes. As molecules comprise a great number of carbons, and that living beings are made of astounding numbers of molecules, statistical rules apply and any given natural molecule will contain roughly the same proportion of Carbon-14 atoms amongst the dominant Carbon-12. So analyzing a bit of your skin, an herbal tea or a natural essential oil will yield the same result - a ratio of Carbon-14 closely matching that known to be found in any living being of the same era.

Figure 2: The "compact" AMS used at the University of Georgia
 Bigger model do exist, see here. (source)
Carbon 14 is a radioactive isotope of carbon, meaning that it will slowly decay over time. It will emit β radiation and transform into a stable Nitrogen-14 atom. The Carbon-14 has a half-life of about 5700 years, which mean that almost all of the Carbon-14 will be gone after about 10 000 years, provided there is no contact of the whole carbon reserve with any more radiation. Petroleum, forming from dead beings decaying under thick layers of soil over the course of hundreds of thousands years, is well shielded from these. As such, it contains no Carbon-14.

So, in analyzing for example benzene, a subproduct of petroleum distillation, for its Carbon-14 content, one can quickly conclude its fossil fuel origin. Carbon-14 is detected by highly specialized mass spectrometric equipment, way beyond GC-MS, and much more sensitive, since the isotope is only found in minute quantities overall. The equipement called AMS or Accelerated Mass Spectrometry is a cross between a mass spectrometer (MS) and a particle accelerator (figure 2). These instrument are currently room sized and are generally owned by universities or public research centers due to their price.

The question is, is that a reliable method to detect adulteration by synthetics in essential oils?

The answer will depend on the definition you are using to define synthetics. Using the petro-centered definition of synthetic material presented in our last post, Carbon-14 is great: it can tell with excellent certitude whether a sample contains petro-derived carbon or not. And when it comes from petroleum, obviously, something was added to the essential oil, which should only be derived from the plant's photosynthetic carbon.

But that does not make the method reliable at all for bulk detection! That is because using other, broader definitions of synthetic molecules, one can perfectly imagine compounds that are purely synthetic (not even found in natural beings at all) but that would pass with no trouble the Carbon-14 testing, because all of their building blocks would come from nature.

Take a look back at the linalool synthetized from beta-pinene: all of the carbon atoms in the final molecule (10 carbons) were present in the starting material (also 10 carbons), and the latter is obtained from conifers. A regular amount of Carbon-14 would be found, and the costly Carbon-14 analyzis would not reveal any adulteration, whereas gas chromatography would probably cast light on the problem.

One could also think of isobornyl cyclohexanol, which we spoke of earlier on these pages. The molecule is obtained from camphene and guaiacol. The former is derived from alpha-pinene, and thus perfectly natural. The latter can be obtained from petroleum derivatives, but also from natural sources. If natural-sourced guaiacol is used, isobornyl cyclohexanol (a purely synthetic molecule with no natural equivalent known) will go entirely unnoticed on Carbon-14 testing.

In conclusion, a Carbon-14 results showing a deviation from normality will always be sign of adulteration or contamination. But results not showing this deviation will not necessarly mean that it is not adulterated.

All in all, Carbon-14 is great to confirm suspicions of adulteration with petroleum-derived material. However, it certainly is not a frontline adulteration detection method, since it can completely omit obvious adulterations that can be detected by chromatographic techniques.