Investigation of odors in the fragrance industry
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- Schilling, B., Kaiser, R., Natsch, A. et al. Chemoecology (2010) 20: 135. doi:10.1007/s00049-009-0035-5
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Scents form the basis for the fragrance industry and various research activities have been developed in different scientific disciplines all being linked by a common interest in odors and odor perception. In this paper, four different topics have been selected for a short discussion. Following a short overview on the history of perfumery, the first topic (Natural scents) is providing some insight into the investigation of natural scents and how this work has strongly stimulated fragrance creation as well as the quest to find new odoriferous substances for the perfumer’s palette. The second subject (Fragrance chemistry) gives a historical overview over the chemistry of fragrances and briefly describes the rational behind the synthesis and composition of new scents. Body odors and their biochemical formation concern the third topic (Body odor biochemistry) which describes our current understanding of this scientifically interesting field and how knowledge may find use to improve future deodorant products. The fourth subject (Olfactory mechanisms) deals with the biochemistry in the human nose when odorants are activating olfactory receptors and enzymes appear to rapidly metabolize the inhaled odorous stimuli. This review does not attempt to be comprehensive, but it describes selected successes in the fragrance industry and the motivation behind conducting various types of research. Ultimately, the activities are aiming to bring new ingredients onto the market and improve the quality of scented products but also to advance our understanding of the power of communication through fragrance.
KeywordsFragrance industryNatural scentsOdorantsBody odorsOlfactionP450
The history of perfumery dates back more than 4,000 years, when Mesopotamians used incense as the first form of perfume. Aromatics were kindled as incense to gods and ancestors. The incense from the fragrant cedar of Lebanon was one of the most preferred varieties, but the resinous woods of pine, cypress, and fir trees were also burned in public ceremonies and private devotions. Aromatic myrtle, juniper berries, and galbanum resin were also used in incense. The Egyptians further developed the art of perfumery not only as part of their religious rituals, but in applications of balms and ointments. Perfumed oils were applied to the skin for either cosmetic or medicinal purposes. The Greek also developed their own perfumery culture, and following Alexander the Great’s invasion of Egypt in the third century bc, the use of perfume and incense became even more widespread in Greece. Romans learned about perfumes from the Egyptians and the ancient Greeks. The Romans took perfumery use to extreme in the famous baths of Rome, and also indulged in the practice of applying perfume three times a day. Because of the empire’s extensive trade network, Rome’s perfumers had access to a large variety of aromatic raw materials, but the people of Rome loved the rose the most of all the flowers and spices. Linking the past and the present of the perfume industry are the Arabs when chemists invented the method to distill plant products. Italian workers perfected the craft in the fourteenth century and set up the first distillery in Modena. During the Renaissance, Venice and Florence were the capitals of the perfumes. When the Florentine noblewoman Caterina de’ Medici married the French king Henry II, she brought the arts and perfumery of Renaissance Italy with her. The perfume industry developed mostly around Grasse, in the South of France, where jasmine, rose, and lavender were grown. In the sixteenth century, it became fashionable to fragrance leather articles, and the glove making industry was allied with perfumery in a single guild in France. The eighteenth century saw a revolutionary advance in perfumery with the invention of eau de Cologne. Along with industry and the arts, perfumery underwent profound changes in the nineteenth century, when various perfume houses established themselves as independent businesses. Perfecting the process of solvent extraction allowed producing the fragrant materials from heat-sensitive flowers, such as jasmine, tuberose, narcissus, and mimosa. The development of modern chemistry laid the foundations of perfumery, as we know it today.
The concept of reconstituting natural scents using readily accessible, mostly synthetic ingredients allows creating olfactive schemes around the scents of flowers for which raw materials are not sufficiently available or which belong to endangered species. A modern fragrance is still likely to contain naturals, particular essential oils since they provide a balanced and rich odor perception. However, synthetic ingredients are vital in the formula to provide performance and signature for a new perfume.
Body odor biochemistry
Finally yet importantly, body odors are also studied as potential semiochemicals. Ever since the first observation that mice differing in their major histocompatability complex (MHC) genes are more attracted to each other (Yamazaki et al. 1976) there has been an ongoing interest to understand the link between genes of the immune system and body odors. Several studies conducted at the Monell Chemical Senses Center by Yamazaki, Beauchamp and colleagues demonstrated that the genetics of the MHC locus and body odor formation are directly linked, and no bacterial metabolism is involved in the production of volatile semiochemicals contained in urine (Yamazaki et al. 1990). There are only few studies carried out with humans. One of them was conducted by Claus Wedekind of the University of Bern, Switzerland, who showed that women prefer the smell of T-shirts worn by men whose MHC genes are dissimilar from their own (Wedekind et al. 1995). Recently, strong analytical evidence has been provided that human genes are directly linked to at least some components of body odor. Fresh sweat of monozygotic twins was treated with the bacterial aminoacylase (AMRE), and the released sweat acid patterns of identical twins were significantly more similar to each other as compared to patterns of unrelated panelists (Kuhn and Natsch 2009). Whether or not the conscious or subconscious perception of human body odors is influencing our behavior, and in particular our preference for partner selection is still up to debate. In today’s society, body odors are offensive. We take all possible measures to prevent their formation and our odorous signature is in large parts defined by the type of fragranced products that we are applying. Nevertheless, there are numerous publications on the existence of human pheromones, however, a recent review has clearly pointed out that there is no evidence today that chemical structures of human pheromones have been identified yet (Wyatt 2009).
Whether the above-discussed odorants are natural or synthetic ingredients, perceived as pleasant or offensive, they all share a series of common features. They are low molecular weight chemicals, volatile, and they are targeting olfactory receptors in our nose. Of all our senses, smell is still the least understood. We still lack much information on how the brain is interpreting incoming signals originating from receptor activation, and how learning, exposure, and genetics are influencing the way these stimuli are shaping the olfactory percept. Research into the fundamentals of chemoreception in animals including humans has advanced dramatically during the last 18 years. The pioneering work of Linda Buck and Richard Axel which includes reporting of the first identification of the genes that encode olfactory receptors has opened a new scientific field towards deciphering the code of smell (Buck and Axel 1991). Nature equipped us with our nose to detect and discriminate thousands of distinct scents. Roughly, 3% of the human genes are dedicated to produce an array of olfactory receptors, the sensing elements to detect odorous stimuli. There is an olfactory region called “olfactory epithelium” in each of the two nasal passages, each about 1 square inch in size and harboring about 10 million primary sensory receptor cells. These olfactory sensory neurons are bipolar nerve cells having cilia protruding into the mucus that covers the olfactory epithelium. Olfactory receptor proteins, as well as other components of a whole biochemical cascade are present in the cilia to transduce the binding of an odorant molecule to one of its cognate receptors into an electric signal (action potential) that can be transmitted to the brain (Firestein 2001). Olfactory receptor genes are the largest gene family in the human genome with close to 1,000 genes that are scattered across all chromosomes except chromosome 20 and the male-specific Y chromosome (Glusman et al. 2001; Zozulya et al. 2001). The receptor proteins belong to a large family of G-protein-coupled receptors, which have seven transmembrane-spanning domains that to a large part are involved in the formation of the ligand-binding pocket. In humans, the majority of the receptor genes have been mutated into non-coding pseudogenes leaving us with close to 400 functional genes encoding receptor proteins. Interestingly, segregating pseudogenes have been identified, indicating that different people may have a slightly different number of pseudogenes on top of the occurrence of various alleles for each of the functional olfactory receptor genes (Menashe et al. 2003).
There has been an ongoing interest both from academia and industry to further decipher the olfactory code, to determine what receptors are activated by the standard fragrance and flavor ingredients, and to see whether these characterization patterns can help design new odorants as well as to find out whether a better understanding of peripheral events may help explain the perception of mixtures. Various research groups have reported encouraging results on response patterns of human olfactory receptors to odorants (Wetzel et al. 1999; Spehr et al. 2003; Sanz et al. 2005; Schmiedeberg et al. 2007; Keller et al. 2007; Saito et al. 2009). In all these studies, receptor genes have been expressed in mammalian cell lines partially engineered with a suitable biochemical cascade that allows detecting receptor activation by measuring fluorescent or luminescent signals at high intensity. Human olfactory receptors were also characterized using other host expression systems, namely Xenopus oocytes and baculovirus Sf9 insect cells (Wetzel et al. 1999; Matarazzo et al. 2005; Menashe et al. 2007). Correlating olfactory receptor activation patterns from high-throughput receptor screening platforms with odor descriptors (quality) and their olfactive thresholds (sensitivity), is a challenge, but a concerted approach of biologists, fragrance chemists, psychophysicists, and perfumery experts will eventually provide a tool to predict odor quality of fragrance materials. In addition, it may open the gate for ligand docking studies using receptor homology modeling and for the design of novel odorant molecules. Knowing that some odorants are activating certain human olfactory receptors but block others (Spehr et al. 2003; Sanz et al. 2005) is opening the way to a more scientific approach towards understanding the perception of mixtures.
A couple of historical publications indicated that biotransformation enzymes in the nose could have an impact on olfaction (Dahl 1991). The initial hypothesis that enzymatic activities are involved in the nature of the sensation of smell was proposed by a chemist from Harvard University more than 50 years ago (Kistiakowsky 1950). The first evidence of in-nose metabolism of a volatile compound was provided at the sixth international symposium of olfaction and taste (Hornung and Mozell 1977). The scientists observed that upon channeling tritium-labeled octane through a frog’s nose, some of the labeled material became water-soluble and speculated that the compound was somehow transformed at the olfactory receptor site. In 1982, it was shown that the fragrance material heliotropin (piperonal) inhibits rat nasal P450 activity, and the author postulated that part of the effectiveness of heliotropin may result from prolonging the residency time of other odorants in the nasal cavity by inhibiting their enzymatic oxidation (Dahl 1982). In recent years, interest in the role of nasal metabolism in olfaction has gained momentum again. Of the about 60 human P450 genes, about one dozen is expressed in the human olfactory mucosa. Other biotransformation genes are also strongly expressed in this neuroepithelium (Ding and Dahl 2003; Ding and Kaminsky 2003; Zhang et al. 2005). In particular, CYP2A13 has been identified to be specifically expressed in the human respiratory tract, predominantly in the olfactory mucosa (Su et al. 2000). When testing standard fragrance ingredients with CYP2A13, a surprisingly large number of odorants were identified as substrates of this enzyme (45–54) (Fig. 13). Examples include O-demethylation of 2-methoxyacetophenone (45) to produce 2-hydroxyacetophenone (46), hydroxylation of coumarin (29) to produce 7-hydroxycoumarin (47), N-demethylation of methyl-N-methylanthranilate (48) to produce methylanthranilate (49), epoxidation of delta-3-carene (50) to delta-3-carene-epoxide (51) and oxidation and rearrangement of (R)-(+)-pulegone (52) to (R)-(+)-menthofuran (53) and further oxidation to mintlactone (54). The metabolism of 52 to 53 and 54 has also been described to be catalyzed by human liver P450 enzymes (Khojasteh-Bakht et al. 1999).
The results suggest that in-nose biotransformation of odorants can modify the quality and perhaps also the quantity of compounds reaching the olfactory mucosa Those events may have to be taken into consideration when interpreting data derived from receptor high-throughput screening campaigns. Ideally, an in vitro screen could be designed, where metabolic interfaces mimicking the possible biotransformation reactions in the olfactory mucosa are included in the receptor activity screen. Such information may be important to fully understand structure-odor-relationships and structure–activity relationships and ultimately provide new means to design novel fragrance ingredients.
We would like to thank all the co-workers at Givaudan for their valuable contributions in the described research areas. We are particularly grateful to the reviewers for numerous constructive comments on the manuscript.