Volatile compounds isolated by
A network of research groups has carried out a bioprospective study of Colombia’s vegetal biodiversity, with focus on aromatic plants. This chapter presents results on the chromatographic analysis of flower fragrances and essential oils obtained from vegetal material collected in botanical expeditions to various Colombian regions. Essential oils and flower fragrances are composed of volatile substances that differ greatly in polarity, functional groups, and relative amounts. The study of these complex mixtures requires special sampling and analysis techniques, described in this chapter. The large chemical diversity of the essential oil and flower fragrance constituents is a formidable characterization challenge. Typically, the number of essential oil components surpassed 50. It was rare to find an essential oil composition in which a single substance was present with a relative amount above 50%.
- essential oil
- flower scent
- gas chromatography
The Research Center for Agroindustrialization of Aromatic and Medicinal Tropical Vegetal Species, CENIVAM, is a multidisciplinary research network of groups from Colombian public and private universities that joined efforts to study Colombia’s agricultural biodiversity, with focus on aromatic plants. Under permit from Colombia’s Environment Ministry, botanical expeditions were organized to obtain vegetal material from different regions in the country. A primary taxonomical identification made by the researchers in the field was subsequently replaced by the assessment made at Colombia’s National Herbarium, where exsiccatae of all studied materials have been deposited. The vegetal material was dried, chopped, and either distilled or macerated, to obtain essential oils and extracts, respectively. Samples of these secondary metabolites were sent to several collaborating groups for the characterization of their biological activity. High-resolution chromatographic and mass spectrometric techniques were utilized in the chemical characterization of the essential oils and extracts. The combined knowledge of chemical composition and biological activity serves as the basis for the sustainable use of the biodiversity in the development of new consumer products for the cosmetics, hygiene, food, and pharmaceutical industries. Pilot essential oil production units have been implemented in some municipalities (Socorro, Sucre, and Barbosa) in the state of Santander. Farmers associations have been trained on good agricultural practices, post-harvest treatment of the vegetal material, and operation of stills designed at Universidad Industrial de Santander, UIS, for the rural essential oil extraction under either hydrodistillation or steam distillation. Thanks to these pilot units, farmers have begun production of essential oils of
This chapter presents results from essential oil and flower fragrance analysis. Flowers maintained in CENIVAM’s experimental garden were sampled both in vivo and in vitro to characterize their volatile compounds. The complex combination of volatile compounds emitted by flowers depends on the plant species, its habitat, phenological state, propagation strategy, time of day, circadian rhythm, climate, and many more variables.
2. Study of tropical flowers volatile compounds
The study of natural products includes a very interesting area: isolation and analysis of floral fragrances, which can be monitored both
Floral fragrances are complex mixtures, product of the metabolism of a flowering plant; they are composed of hundreds of molecules of different biochemical origin, with different physicochemical characteristics (polarity, volatility, and solubility); they contain various functional groups (hydrocarbons, alcohols, aldehydes, ketones, acids, esters, ethers, etc. ), and can be found in diverse concentrations (from parts per trillion, ppt, to parts per million, ppm). They are predominantly lipophilic substances, with molecular weight less than 300 Da, non-polar or moderately polar, and with high vapor pressure. The human nose can be more sensitive than a chromatographic detection system, to some floral fragrance substances, present at the trace level; therefore, it is necessary to carry out the extraction and concentration processes of the floral fragrance in such a way that its components are detectable and can be identified. This constitutes a very big analytical challenge. Nowadays, this challenge is solved by applying different strategies: headspace extraction techniques (
For the instrumental analysis of volatile fractions and extracts, gas chromatography (GC) is used in one-dimensional (1D) or two-dimensional (2D) versions (GCxGC), in capillary columns of different polarities, using universal detection systems (flame ionization detector (FID); mass selective detector (MSD), in
Many and very diverse compounds have been detected and identified in floral fragrances. More than 1700 have been recorded in a diverse group of flowers studied . The main families of chemical compounds found in floral scents include hydrocarbons (saturated, cyclic and olefinic); terpenes, basically, monoterpenoids and some sesquiterpenoids; benzenoids and phenylpropanoids, the oxygenated compounds of mixed nature,
The basic biological function of the floral fragrance is to promote or facilitate cross-pollination, which is a vital process in the life cycle of most angiosperm plants. The knowledge of the floral fragrance chemical composition is important to understand plant-insect interaction, the chemical strategies not only to attract the pollinators but also to deter the herbivores and to face the pathogens, to adapt to different abiotic stresses; to study the biochemical pathways of secondary metabolism in a plant, its adaptability and biological evolution. Also, it is of practical interest to know the floral composition as a source of inspiration to create new fragrances and odorous mixtures, which are used in the cosmetics, perfumes, personal hygiene products, or aromatherapy industries.
The floral fragrances of diverse plants, despite of having a different smell, could contain many common compounds. Among these, the terpenoids are a large group: monoterpene (ocimenes, phellandrenes, carenes, terpinenes, limonene, and
We studied the chemical composition of volatile fractions of 30 tropical plants; their floral scents were monitored by
3-octanol, 1-octen-3-ol, lauryl acetate.
4-methyl benzaldoxime, 2-phenyl-1-nitroethane, benzyl nitrile, methyl anthranilate, indole.
α-bulnesene, valencene, bicyclogermacrene,
α-selinene, β-selinene, bicyclogermacrene, δ-cadinene, γ-cadinene,
2-methyl furane, butanal, propanol, butan-2-one, 2-methyl butanal, 3-methyl butanal, ethanol, 3-buten-2-one, pentanal, hexanal, 1-penten-3-ol,
2-methyl butane nitrile, 3-methyl butane nitrile, 1-methyl-1H-pyrrole,
2-phenyl nitroethane, ecgonidine methyl ester, quinoline, benzene acetonitrile, 6-methyl-2-pyridinecarboxaldehyde.
2-heptanone, 2-heptanol, 2-nonanol, 3-octanol.
α-himachalene, α-selinene, β-selinene,
β-myrcene, limonene, 1,8-cineole, 4,8-dimethyl-1,3,7-nonatiene,
3-octanone, hexyl acetate, octanal, 1-octen-3-one,
1-octen-3-ol, heptanol, octanol, nonanol,
β-pinene, sabinene, β-myrcene, α-phellandrene, α-terpinene, 2,3-dehydro-1,8-cineole, limonene, β-phellandrene, 1,8-cineole,
2-nonen-1-ol, 2,6-nonadien-1-ol, lauryl acetate.
4-ethyl resorcinol, 2-methoxy-4-methyl-1-(1-methylethyl) benzene, methyl benzoate, 3-hexen-1-yl benzoate, 1,2-dimethoxy benzene, 1,4-dimethoxy benzene, methyl salicylate,
4-methoxy phenol, benzyl benzoate.
2-propanone, 2-hydroxy acetic acid, propanol, 2-butanone, 1-penten-3-one,
2-pentyl furane, hexanol,
2-nonenal, octanol, nonanol.
1-pentadecene, 7-pentadecene, hexadecane, 3-hexadecene,
α-humulene, α-amorphene, germacrene D, α-muurolene,
1-penten-3-ol, 4-pentenyl acetate, 2-pentyl furane, 3-methyl-2-butenyl acetate, hexyl acetate,
benzyl alcohol, 2-phenyl ethanol, 4-methoxy benzaldehyde,
benzyl benzoate, benzyl salicylate.
1-penten-3-ol, 3-methyl-1-butanol, 2-pentyl furane, hexanol,
3-octanone, octanal, 1-octen-3-one,
2.1. Methods for floral fragrance isolation
Before proceeding to collect the volatile flowers, it is important to establish if their monitoring will be done
Some methods of collecting floral volatiles can have an automated design that allows monitoring for 24 hours or longer periods. However, most extraction techniques make a momentary capture, a “snapshot” of the floral volatiles emitted . The extraction methods of the flower volatile secondary metabolites can be divided into three large categories, namely: (I) Headspace techniques (
The headspace methods provide information on the chemical composition of the volatile fractions; distillation techniques, on essential oils, distillates or condensates while extractive methods (solvents, supercritical CO2), on the chemical composition of mixtures that may include substances of low-volatility, and higher molecular mass (> 400 Da), which in general are called extracts. The compositions of these mixtures can be differentiated not only quantitatively but also qualitatively. As mentioned above, in condensates and extracts will prevail “heavier” compounds, fatty acids, long-chain paraffinic hydrocarbons, their alcohols or aldehydes while in the volatile fractions, low-molecular-weight compounds are found, which eventually can “scape” during the distillation, in the depressurization stage (SFE-CO2) or during the concentration of the extracts.
The chemical composition of the volatile fraction of flowers depends both on intrinsic (genetic) factors of the species and on extrinsic, environmental factors . The habitat, the environment where the plant grows, the conditions (temperature, humidity, light, type of soil, micronutrients, etc.) in which floral secondary metabolites are monitored, will affect the qualitative and quantitative composition of the volatile fraction emitted and collected. For this reason, it is very important during the collection of floral volatiles to maintain control, continually monitoring conditions. Many external factors will affect the production of flower volatiles. These include changes in temperature, humidity, increase or decrease in light energy, among others. Some stress conditions (water, light, and nutrition) can notably alter the generation of floral volatiles or even suppress their production .
Some aspects of the study of floral fragrances should contemplate the state of development of the flower . The flowers of the ylang-ylang tree (
The composition of the secondary metabolites in the floral emission also varies according to the part of the flower from which the volatiles are extracted. In the petals of the ylang-ylang flowers, oxygenated compounds (oxygenated monoterpenes, benzenoids, and phenylpropanoids) prevail while in the ovaries (central part of the flower, small and compact) monoterpene and sesquiterpene hydrocarbons abound . The relative percentage composition of the families of compounds present in the ylang-ylang flowers depends on the extraction method: steam distillation or simultaneous solvent distillation-extraction (SDE) allow mixtures of secondary metabolites to be obtained, rich in light oxygenated compounds (50–60%), and in heavy oxygenated compounds (18–20%) while extraction with supercritical fluid, SFE-CO2, isolates extracts, rich in aliphatic hydrocarbons (Cn > 20) and terpenes, nitrogen-containing compounds, and even some fatty acids (C14-C18).
The profile of volatile compounds emitted by the flower also depends on the time of day; the insects that pollinate it can be diurnal or nocturnal, and from this, the kinetics of emanation of fragrant compounds and the type of volatile emitted by the flower will also depend, which vary, for most of the flowers, with the time of the day (circadian rhythm), and according to the biological function they fulfill. For example, in ylang-ylang flowers, the amount of nitrogenous substances changes during the day: it is maximum at dawn, decreases afternoon, and increases again in the afternoon and evening hours.
The flower fragrance of
Notorious changes can be observed (Figure 1) in
In carrion flower
Distinct parts of the flower fulfill different biological roles in it; for example, to protect from herbivores or to attract pollinators, to call for natural enemies or to increase or diminish flower temperature or transpiration. Figure 3 shows chromatographic profiles (HS-SPME/GC/MS) of volatiles emitted from distinct parts of passion fruit (
2.2. Chromatographic analysis of floral fragrances
The substances that make up the volatile fraction isolated from flowers are of low-molecular-weight (<300 Da) and are mixtures of components with different polarity and concentration. Thanks to the volatile nature of these compounds, their analysis is done by gas chromatography (GC). Due to the complexity of some mixtures of volatiles isolated from flowers and the presence in them of isomeric substances (geometrical, positional, stereoisomers), it is recommended to make their analysis in capillary fused-silica columns, preferably long, of 50–60 m, with internal diameters (DI) of 0.25, 0.22, or 0.20 mm. The smaller internal diameters, although they allow to increase the resolution, eventually, can also compromise the sensitivity. Columns with the thickness of the stationary phase (df) equal to or greater than 0.25 μm are used, so that the shape of peaks, their separation and the sensitivity, necessary for their reproducible detection, are adequate.
Generally, for the injection of the sample (T° of the injector, usually, of 230–250°C), the split ratio of 1:30 can be used, but when the concentrations of some components of interest are low, it is convenient to inject in splitless mode. When the splitless injection mode is used, to decrease the “dispersion” or the widening of the peaks of very volatile substances, the injection can be done in the
For the analysis of the volatile fractions, the initial column temperature of 35–50°C would be advisable; the nature of the sample (volatile compounds) does not require that the final temperature of the column be high; 200–250°C will be sufficient to elute the most retained components. The heating speed of the column is a function of its length: the longer it is, the slower the column must be heated, 3–4°C/min, but if shorter columns are used,
For the analysis of the floral volatile fraction, two columns are used in combination: one with the polar stationary phase, poly(ethylene glycol) (e.g., INNOWAX, DB-WAX, HP-20 M, and others) and the other, with the non-polar stationary phase, poly (dimethyl siloxane) (HP-1, Ultra 1, DB-1, BP-1, and others) or 5% -phenyl poly (dimethyl siloxane) (HP-5, DB-5, Ultra 2, CPSil 5, BP-5, and others).
Enantioselective gas chromatography takes advantage of the fact that the enantiomers have different retention times when compounds that can form adducts are inserted in the stationary phase whose stability is a function of the three-dimensional (3D) form of the analyte. The cyclodextrins with their cone geometry with cavity of different size have turned out to be very effective chiral agents, constituting inclusion complexes that allow the discrimination of isomers according to their shape. The fragrances of jasmine (
The aldehydes and lilac alcohols are oxygenated monoterpenes found in plant species of many families. Lamiaceae (
The most common detection system for comparative analysis and for the quantification of compounds in the volatile fractions isolated from flowers is the flame ionization detector (FID), a simple, robust system with an acceptable sensitivity, and a wide dynamic range. Selective detectors, such as the nitrogen and phosphorus detector (NPD) and the flame photometric detector (FPD), are very useful tools for the selective detection of nitrogenous and sulfur compounds, very common in floral fragrances.
The most important and widely used detection system in the analysis of volatile mixtures is the mass selective detector; its combination with capillary gas chromatography (GC-MS) is a perfect instrument to achieve separation and identification (presumptive or confirmatory) of components present in a mixture. The ionization mode most used for the analysis of volatile substances is the impact with electrons (EI) of 70 eV-energy. The EI mass spectra contain a lot of information because in the spectrum signals of numerous ionized fragments appear, which form a unique combination that allows to differentiate one molecule from the other, even if they are isomers.
The mass (
3. Essential oil composition
Essential oils were obtained by steam distillation. High-resolution gas chromatography coupled to mass spectrometry was used for component identification. Relative amounts of essential oil constituents were calculated from the peak areas of chromatograms obtained with gas chromatography with flame ionization detection. Linear retention indices were determined on polar (Carbowax) and non-polar (DB-5) capillary chromatographic columns. Tentative compound identification was based on the comparison of retention indices with published values, and the comparison of mass spectra with those of databases [21, 22, 23, 24]. Table 2 presents the main constituents found in the gas chromatographic analysis of essential oils isolated from plant material collected in botanical expeditions carried out by CENIVAM.
|α-Ylangene (10%), |
|β-Bourbonene (4%), β-elemene (10%), |
|α-Pinene (3%), |
|α-Pinene (7%), |
|Thymol (26%), carvacrol (37%), δ-cadinene (1%).|
|Chrysanthenone (14%), β-cubebene (4%), 2-ethyldien-6-methyl-heptadienal (4%), γ-curcumene (19%), |
|γ-Curcumene (14%), |
|α-Pinene (2%), |
|α-Eudesmol (17%), squalene (1%), spathulenol (1%), caryophyllene oxide (1%).|
|α-Pinene (3%), limonene (8%), kessane (4%), viridiflorol (4%), |
|α-Zingiberene (27%), germacrene D (11%), |
|1,8-Cineole (4%), borneol (6%), |
|α-Zingiberene (35%), germacrene D (17%), |
|Caryophyllene oxide (5%), β-amyrin (6%), germacrene D (3%), |
|Δ2-Carene (7%), Δ3-carene (37%), β-phellandrene (3%), |
|α-Pinene (8%), β-pinene (7%), α-phellandrene (4%), cyclofenchene (4%), isoelemicin (4%), caryophyllene oxide (5%), 1,3,5-trimethoxy-3-methyl-propenyl benzene (4%).|
|α-Pinene (4%), sabinene (40%), β-pinene (3%), terpinen-4-ol (7%), |
|α-Thujene (11%), α-pinene (12%), β-myrcene (4%), α-copaene (4%), spathulenol (4%).|
|α-Pinene (25%), camphene (16%), β-pinene (11%), α-phellandrene (12%), |
|β-Myrcene (5%), |
|β-Myrcene (8%), α-humulene (4%), germacrene D (7%), germacrene D-4-ol (4%), hinesol (4%), valerianol (12%).|
|Germacrene D (15%), β-phellandrene (14%), β-pinene (14%), α-pinene (20%), α-phellandrene (9%).|
|α-Copaene (17%), |
|α-Pinene (6%), sabinene (21%), β-pinene (9%), 1,8-cineole (6%), |
|Methyleugenol (69%), |
|α-Terpinene (21%), |
|6-Methyl-5-hepten-2-one (5%), α-cubebene (3%), α-copaene (3%), methyl citronellate (3%), α-muurolene (3%).|
|α-Copaene (5%), |
|Caryophyllene oxide (2%), |
|Germacrene D (22%), |
|β-Elemene (6%), |
|β-Bourbonene (8%), |
|Sabinene (2%), α-phellandrene (2%), fenchone (7%), |
|α-Pinene (8%), β-pinene (7%), limonene (10%), 1,8-cineole (8%), |
|β-Pinene (13%), α-copaene (4%), |
|α-Pinene (5%), β-pinene (10%), |
|α-Pinene (3%), β-pinene (10%), Δ3-carene (5%), limonene (21%), |
|α-Pinene (8%), 3-octenol (9%), limonene (19%), |
|α-Copaene (5%), |
|Menthone (7%), pulegone (6%), |
|Piperitone epoxide (59%), piperitone oxide (13%), |
|Linalool (23%), estragole (63%), |
|Methyleugenol (54%), 1,8-cineole (3%), |
|Eugenol (22%), β-elemene (23%), |
|Perilla ketone (48%), 1-octen-3-ol (32%), linalool (6%), 3-octanone (5%), 3-octanol (3%).|
|Carvacrol (13%), |
|Limonene (2%), |
|1,8-Cineole (20%), α-terpineol (6%), |
|1,8-Cineole (16%), terpinen-4-ol (11%), α-terpineol (13%), |
|Safrol (9%), myristicin (5%).|
|α-Pinene (9%), α-phellandrene (14%), |
|α-Pinene (20%), β-pinene (32%), limonene (4%), β-elemene (4%), |
|α-Pinene (11%), β-pinene (5%), α-phellandrene (6%), 1,8-cineole (18%), linalool (15%), |
|Apiol (27%), dillapiol (1%), |
|α-Phellandrene (11%), limonene (8%), β-elemene (4%), |
|β-Phellandrene (22%), germacrene D (12%), |
|Drima-7,9(11)-diene (23%), valencene (6%), β-selinene (6%), viridiflorene (7%), dihydrokaranone (15%).|
|α-Phellandrene (6%), |
|α-Pinene (5%), sabinene (11%), 1,8-cineole (14%), |
|β-Phellandrene (4%), |
|Limonene (30%), carvone (50%), piperitone (3%), piperitenone (6%), bicyclosesquiphellandrene (4%).|
|γ-Terpinene (6%), |
4. Biological activity of essential oils
One of the most important research lines of the CENIVAM Project is related to the study of different biological activities of essential oils. Bioactivity assays against
Different CENIVAM groups, among them, in the Research Center for Biomolecules, CIBIMOL [35, 36], Environmental and Computational Chemistry of the University of Cartagena [37, 38], Chemistry and Biology of the Universidad del Norte, in Barranquilla  measured the antioxidant activity of essential oils by different techniques, e.g., lipid oxidation, measurement of secondary end products of lipoxidation, thiobarbituric acid reactive substances (TBARS) test, and free radical trapping tests (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid, ABTS + and 2,2-diphenyl-1-picrylhydrazyl, DPPH). Grosso modo, about 40% of the analyzed samples were active in different tests of antioxidant activity.
The study of the cytotoxic activity (acute toxicity, LC50) of the essential oils was carried out by several groups. Cecilia Mesa et al. examined the activity against
Insect repellency is an interesting biological activity that leads to rather soon implementation of essential oils as active ingredients of commercial products. Olivero et al. have examined the potential application of essential oils to repel insects of importance to food storage [46, 47, 48, 49]. Another application of insect repellence is the prevention of diseases for which
The assays of the anti-genotoxic and chemopreventive activity carried out at the CIBIMOL-UIS group demonstrated a DNA protective effect of the essential oils of several chemotypes of
Several bacterial strains have been employed in assays of essential oil antibacterial activity [57, 58]. Due to their carvacrol and thymol content,
This work was supported by the “Patrimonio Autonomo Fondo Nacional de Financiamiento para la Ciencia, la Tecnologia y la Innovacion, Francisco Jose de Caldas,” Grants RC-0572–2012, RC-245-2011, RC-432-2004. The “Ministerio de Ambiente y Desarrollo Sostenible” of Colombia supported the present project through access permits to genetic resources and derivatives for scientific research (Agreement No101, Resolution No 0812).
Conflict of interest
The authors declare that they have no conflict of interest with this chapter contents.