**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 *in vivo* and *ex vivo* [1]. The study of volatile secondary metabolites, emanated from flowers, is important in many areas of the biological and chemical sciences, in agriculture, for the pest control, in the study of plant-insect interactions, allelopathy, in analytical sciences (sample preparation and chromatography), in the pharmaceutical, in perfumes and cosmetics, flavors and fragrances industries, among others.

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 (*headspace*), distillation methods, extractive solvents, and an active surface, *i.e.*, adsorption/thermal desorption processes using adsorbents with different physical characteristics [2, 3].

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 *full scan* mode), or selective detection, e.g., selective nitrogen and phosphorus detector (NPD), flame photometric detector(FPD), to register nitrogenous or sulfur compounds, respectively, and very specific detection systems such as electroantennography, electronic nose, and so on [4]. High-resolution mass spectrometry detection systems (HR-TOF, Orbitrap) are nowadays an excellent alternative for exact mass determination (elemental composition analysis), selective and very sensitive detection of secondary metabolites in complex floral scent mixtures.

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. Highresolution 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 *Cymbopogon nardus*, *C*. *martinii*, and *Lippia origanoides*. New developments have started to extend the cultivation and essential oil production to additional species, such as *Cananga odorata*, *Pogostemon cablin*, *Vanilla planifolia*, *Lippia alba*, and *Rosmarinus officinalis*. 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,

The study of natural products includes a very interesting area: isolation and analysis of floral fragrances, which can be monitored both *in vivo* and *ex vivo* [1]. The study of volatile secondary metabolites, emanated from flowers, is important in many areas of the biological and chemical sciences, in agriculture, for the pest control, in the study of plant-insect interactions, allelopathy, in analytical sciences (sample preparation and chromatography), in the pharma-

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 (*headspace*), distillation methods, extractive solvents, and an active surface, *i.e.*, adsorption/thermal desorption processes using

ceutical, in perfumes and cosmetics, flavors and fragrances industries, among others.

time of day, circadian rhythm, climate, and many more variables.

**2. Study of tropical flowers volatile compounds**

60 Potential of Essential Oils

adsorbents with different physical characteristics [2, 3].

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 [5]. 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, *e.g.,* alcohols, aldehydes, ethers, esters (fatty acid derivatives), and substances that contain heteroatoms, such as sulfur or nitrogen.

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 the most frequent oxygenated monoterpenes one can find alcohols: linalool, geraniol, nerol, and their acetates; ketones: carvone, menthone, verbenone; and aldehydes: citral (geranial and neral), and their oxides. Another family in the floral fragrance is made up of hydrocarbons, aliphatics, C1 -C30 (more frequently, C13-C21 hydrocarbons, and olefinics, including some cycloparaffins). These substances, together with the fatty acids, C12-C22, are part of the wax protective layer that lines the petals of many flowers, a lot of fatty acid derivatives (alcohols, ketones, ethers, esters, and lactones) could predominate in the floral scents of some flowers. A distinctive odoriferous note in the floral fragrance is due to the presence in its mixtures of compounds that contain sulfur or nitrogen atoms, probably originating from the metabolism of amino acids; among these volatile secondary metabolites, there are compounds with nitro group, indoles, oximes, nitriles, anthranilates, and sulfides, among the most common.

We studied the chemical composition of volatile fractions of 30 tropical plants; their floral scents were monitored by *in vivo* solid-phase micro-extraction (SPME), exposing the fiber, coated with Carboxen/poly(dimethylsiloxane) (CAR/PDMS) or Carboxen/divinylbenzene/ poly(dimethylsiloxane) (CAR/DVB/PDMS) to the flower, mostly at its anthesis stage, for 20–30 minutes. The on-fiber collected volatiles immediately were desorbed into the injection port of a gas chromatograph coupled to mass spectrometer (GC-MS). **Table 1** contains the results of these analyses; the presence of diverse groups (monoterpenoids, sesquiterpenoids, benzenoids, oxygenated compounds, and fatty acid derivatives, as well as sulfur- and nitrogen-containing compounds, very characteristic for some plants (*Cananga odorata, Sansevieria guineensis, Erythroxylum coca, Moringa oleifera, Stapelia gigantea*) can be observed. The plants have been cultivated in the experimental plots of the Research Center CENIVAM (Bucaramanga, Colombia). Although diverse volatile fractions may contain common compounds, they are mostly different in their chemical composition.

**Species Family Chemical composition**

Acanthaceae **Monoterpenoids**: *trans*-β-ocimene, linalool. **Benzenoids**: methyl salicylate.

*n*-pentadecane.

nerol.

3-octanol, 1-octen-3-ol, lauryl acetate.

hexyl acetate, *cis*-3-hexenyl acetate.

eugenol, *trans*-cinnamaldehyde, methyl anisate,

pyrazine, methoxy-dimethyl pyrazine.

anthranilate, indole.

Aristolochiaceae **Sesquiterpenoids:** α-farnesene.

salicylate.

myristate.

*n*-pentadecane.

*cis, cis*-farnesol.

pentadecanal.

anthranilate.

**Oxygenated compounds**: acetaldehyde, 3-octanone, 1-octen-3-one,

The Expression of Biodiversity in the Secondary Metabolites of Aromatic Plants and Flowers…

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63

Annonaceae **Monoterpenoids:** α-pinene, β-pinene, β-myrcene, limonene, 1,8-cineole, *trans*-β-

elemene, *trans*-β-caryophyllene, *cis*-muurola-3,5-diene,

**Sulfur and nitrogen compounds:** dimethyl sulfide, benzyl nitrile. **Hydrocarbons:**

ocimene, terpinolene, *allo*-ocimene, linalool, α-terpineol, geranyl acetate, geraniol,

*trans*-muurola-3,5-diene, α-humulene, α-muurolene, γ-muurolene, germacrene D, α-farnesene, α-cadinene, γ-cadinene, δ-cadinene, cadina-1,4-diene, calamenene. **Benzenoids:** *p*-methyl anisole, benzaldehyde, methyl benzoate, ethyl benzoate, methyl salicylate, 3,4-dimethoxy-toluene, 2-ethyl phenyl acetate, anethole, benzyl acetate, *p-*cresol, cinnamyl acetate, methyl isoeugenol.  **Oxygenated compounds (fatty acid derivatives):** 2-methyl-butan-1-ol, tridecanal,

**Hydrocarbons:** heptadiene, tetradecadiene, methyl tridecane,

Asparagaceae **Monoterpenoids:** limonene, 1,8-cineole, carvone, α-terpineol. **Sesquiterpenoids:**

**Sulfur- and nitrogen-containing compounds:** methyl

**Sesquiterpenoids:** α-cubebene, α-ylangene, α-copaene, β-copaene, *trans*-β-

**Sulfur- and nitrogen-containing compounds:** phenyl acetonitrile, 4-methyl benzaldoxime, 2-phenyl-1-nitroethane, benzyl nitrile, methyl

*Thunbergia grandiflora*

*Cananga odorata*

*Aristolochia ringens*

*Sansevieria guineensis*

#### **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 *in vivo* (in the field) or *ex vivo*. The experimental setup for each purpose will be different. Some extraction techniques are not applicable in the field for *in vivo* flower monitoring. It is also important to have prior knowledge about the concentration of volatiles that the flower emits since the extraction and concentration system used will depend on the volatile fraction quantity. The bouquet of volatile substances produced by the flowers can have from 1 to more than 100 compounds but generally contains 20–60 different substances. The concentration of volatiles emitted ranges from low picogram levels to more than 30 μg/L [6].

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 [7]. The extraction methods of the flower volatile secondary metabolites can be divided into three large categories, namely: (I) Headspace techniques (*headspace*, HS) in static or dynamic modes; (II) Distillation techniques, among them steam distillation, water-steam distillation, hydro-distillation, hydro-distillation assisted by microwave radiation, and (III) Extractive techniques, using solvents of different nature, *e.g.,* fats (maceration, *enfleurage*, obtaining ointments), non-polar solvents (hydrocarbons, obtaining concretes), polar solvents (alcohols, obtaining absolutes), and supercritical fluids (mainly, CO2 ). Headspace techniques are used in two different "formats": *static headspace* (S-HS) and *dynamic headspace*, *e.g.,* purge and trap (P&T). Today, the most popular technique for the analysis of floral fragrances is solid-phase micro-extraction (SPME), operated in the *headspace*  mode (HS-SPME). This method of extraction on a polymeric adsorbent, which covers a fused silica fiber combines the high selectivity of extraction, which is achieved with the choice of the polymer, its chemical nature and thickness, and the optimization of sampling conditions (volumes of the material and headspace, temperatures, pre-equilibrium, and fiber exposure times, sample agitation modes, additives, etc.), with the concentration of analytes on the fiber. The extraction and simultaneous concentration of the sample are processes that distinguish the SPME technique and make it very advantageous compared to other methods [8].

We studied the chemical composition of volatile fractions of 30 tropical plants; their floral scents were monitored by *in vivo* solid-phase micro-extraction (SPME), exposing the fiber, coated with Carboxen/poly(dimethylsiloxane) (CAR/PDMS) or Carboxen/divinylbenzene/ poly(dimethylsiloxane) (CAR/DVB/PDMS) to the flower, mostly at its anthesis stage, for 20–30 minutes. The on-fiber collected volatiles immediately were desorbed into the injection port of a gas chromatograph coupled to mass spectrometer (GC-MS). **Table 1** contains the results of these analyses; the presence of diverse groups (monoterpenoids, sesquiterpenoids, benzenoids, oxygenated compounds, and fatty acid derivatives, as well as sulfur- and nitrogen-containing compounds, very characteristic for some plants (*Cananga odorata, Sansevieria guineensis, Erythroxylum coca, Moringa oleifera, Stapelia gigantea*) can be observed. The plants have been cultivated in the experimental plots of the Research Center CENIVAM (Bucaramanga, Colombia). Although diverse volatile fractions may contain common compounds, they are

Before proceeding to collect the volatile flowers, it is important to establish if their monitoring will be done *in vivo* (in the field) or *ex vivo*. The experimental setup for each purpose will be different. Some extraction techniques are not applicable in the field for *in vivo* flower monitoring. It is also important to have prior knowledge about the concentration of volatiles that the flower emits since the extraction and concentration system used will depend on the volatile fraction quantity. The bouquet of volatile substances produced by the flowers can have from 1 to more than 100 compounds but generally contains 20–60 different substances. The concentration of volatiles emitted ranges from low picogram levels to more than 30 μg/L [6].

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 [7]. The extraction methods of the flower volatile secondary metabolites can be divided into three large categories, namely: (I) Headspace techniques (*headspace*, HS) in static or dynamic modes; (II) Distillation techniques, among them steam distillation, water-steam distillation, hydro-distillation, hydro-distillation assisted by microwave radiation, and (III) Extractive techniques, using solvents of different nature, *e.g.,* fats (maceration, *enfleurage*, obtaining ointments), non-polar solvents (hydrocarbons, obtaining concretes), polar solvents (alcohols, obtaining absolutes), and supercritical fluids (mainly,

). Headspace techniques are used in two different "formats": *static headspace* (S-HS) and *dynamic headspace*, *e.g.,* purge and trap (P&T). Today, the most popular technique for the analysis of floral fragrances is solid-phase micro-extraction (SPME), operated in the *headspace*  mode (HS-SPME). This method of extraction on a polymeric adsorbent, which covers a fused silica fiber combines the high selectivity of extraction, which is achieved with the choice of the polymer, its chemical nature and thickness, and the optimization of sampling conditions (volumes of the material and headspace, temperatures, pre-equilibrium, and fiber exposure times, sample agitation modes, additives, etc.), with the concentration of analytes on the fiber. The extraction and simultaneous concentration of the sample are processes that distinguish the

SPME technique and make it very advantageous compared to other methods [8].

mostly different in their chemical composition.

**2.1. Methods for floral fragrance isolation**

62 Potential of Essential Oils

CO2

Erythroxylaceae **Monoterpenoids:** β-myrcene, α-phellandrene, α-pinene, ∆3

**Sesquiterpenoids:** α-farnesene.

3-*cis*-hexenol, nonanol, bovolide.

6-methyl-2-pyridinecarboxaldehyde.

Fabaceae **Oxygenated compounds (fatty acid derivatives):** hexanol, 2-heptanone, 2-heptanol, 2-nonanol, 3-octanol.

*trans*-β-caryophyllene, α-humulene, *trans*-β-farnesene,

*cis*-3-hexenol, 3-octanol, *trans*-2-hexen-1-ol, 1-octen-3-ol.

**Benzenoids:** benzaldehyde, methyl salicylate.

*trans*-α-bergamotene, *trans*-β-caryophyllene,

Lauraceae **Monoterpenoids:** α-pinene, α-thujene, β-pinene, β-phellandrene,

*trans*-linalool oxide, *cis*-linalool oxide, linalool. **Sesquiterpenoids:** *trans*-β-caryophyllene. **Benzenoids:** benzeneacetaldehyde.

*trans*-2,4-hexadienal, *trans*-2-hexen-1-ol.

**Hydrocarbons:** tridecane.

Lamiaceae **Monoterpenoids:** perillene, linalool, perilla aldehyde.

Lamiaceae **Monoterpenoids:** Δ3

α-humulene.

β-phellandrene, *trans*-β-ocimene, *cis*-β-ocimene, *allo*-ocimene,

**compounds (fatty acid derivatives):** propan-2-one,

*neo-allo*-ocimene, 1,3,8-*p*-menthatriene, linalool, *cis*-linalool oxide, linalool.

The Expression of Biodiversity in the Secondary Metabolites of Aromatic Plants and Flowers…

**Benzenoids:** benzaldehyde, methyl benzoate, benzeneacetaldehyde, methyl salicylate, ethyl salicylate, benzyl alcohol, 2-phenyl ethanol. **Oxygenated**

2-methyl furane, butanal, propanol, butan-2-one, 2-methyl butanal, 3-methyl

*cis*-2-pentenol, 2-*trans*-hexanal, heptanal, 2-methyl-1-butanol, 3-methyl-1-butanol,

2-phenyl nitroethane, ecgonidine methyl ester, quinoline, benzene acetonitrile,

**Sesquiterpenoids:** α-cedrene, *trans*-α-bergamotene, *trans*-β-elemene, β-cedrene,

α-himachalene, α-selinene, β-selinene, *trans*, *trans*-α-farnesene, cuparene.

**Oxygenated compounds (fatty acid derivatives):** *trans*-2-hexenal,

**Oxygenated compounds (fatty acid derivatives):** 1-octen-3-ol.

β-myrcene, limonene, 1,8-cineole, 4,8-dimethyl-1,3,7-nonatiene,

**Oxygenated compounds (fatty acid derivatives):** acetaldehyde, butan-2-one, ethanol, pentan-3-one, 1-penten-3-ol, hexanal, 4-pentenal, 2-pentenal, 3-hexenal, 1-penten-3-ol, 3-methyl-1-butanol, *trans*-2-hexenal, hexanol, *cis*-3-hexenol, *trans,*

**Sulfur- and nitrogen-containing compounds:** benzeneacetonitrile, indole.

butanal, ethanol, 3-buten-2-one, pentanal, hexanal, 1-penten-3-ol,

**Sulfur- and nitrogen-containing compounds:** dimethyl sulfide, 2-methyl butane nitrile, 3-methyl butane nitrile, 1-methyl-1H-pyrrole,

65

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*Erythroxylum novogranatense*

*Brownea macrophylla*

*Perilla frutescens*

*Plectranthus amboinicus*

*Persea americana*

nerolidol.

Asparagaceae **Monoterpenoids:** limonene, *cis*-limonene oxide, *trans*-limonene oxide, linalool,

**Sulfur- and nitrogen-containing compounds:** benzyl nitrile.

**Sulfur- and nitrogen-containing compounds:** benzyl nitrile.

β-phellandrene, *trans*-β-ocimene, *cis*-β-ocimene, *allo*-ocimene,

octatetraene, *p-*cymenene, 2,6-dimethyl-1,3,5,7-octatetraene.

Arecaceae **Monoterpenoids:** limonene. 1,8-cineole, *trans*-β-ocimene, *cis*-β-ocimene, linalool

*p*-cymenene, linalool, limonen-4-ol, fenchol, α-terpineol.

bergamotene, *trans*-β-caryophyllene, *allo*-aromadendrene, *cis*-β-farnesene, α-humulene, α-selinene, β-selinene, γ-selinene, α-bulnesene, valencene, bicyclogermacrene, *trans, trans*-α-farnesene,

Convolvulaceae **Sesquiterpenoids:** α-cubebene, β- cubebene, α-copaene, β-copaene, *trans*-β-

5-diene, γ-muurolene, germacrene D, dauca-5,8-diene,

2-propenal, 2-methyl butanal, 3-methyl butanal, ethanol,

2-penthyl furane, pentanol, 3-octanone, 1-octen-3-one,

**Sulfur- and nitrogen-containing compounds:** dimethyl sulfide.

β-myrcene, α-phellandrene, β-phellandrene, α-terpinene, limonene, *cis*-β-ocimene, γ-terpinene, *trans*-β-ocimene, *allo*-ocimene, terpinolene,

**Sesquiterpenoids:** α-ylangene, *cis*-α-bergamotene, α-santalene, *trans*-β-

elemene, *trans*-β-caryophyllene, *trans*-muurola-3,5-diene, *trans*-muuro-4 (14),

α-selinene, β-selinene, bicyclogermacrene, δ-cadinene, γ-cadinene,

**Oxygenated compounds (fatty acid derivatives):** acetaldehyde, butanal,

1-penten-3-one, hexanal, *cis*-2-penten-1-al, 1-penten-3-ol, 3-methyl-1-butanol,

**Benzenoids:** methyl salicylate,2-phenyl ethanol.

*neo-allo*-ocimene, 1,3,8-*p*-menthatriene, *trans, trans*-2,6-dimethyl-1,3,5,7-

**Oxygenated compounds (fatty acid derivatives):** butanoic acid, pentanoic acid,

**Sulfur- and nitrogen-containing compounds:** dimethyl disulfide, dimethyl

**Benzenoids:** 2-phenyl ethanol, 3-hexenyl benzoate.

**Benzenoids:** 2-phenyl ethanol, 3-hexenyl benzoate.

*Plumeria rubra* Apocynaceae **Monoterpenoids:** limonene, *cis*-limonene oxide, *trans*-limonene oxide, linalool.

Apocynaceae **Monoterpenoids:** α-pinene, β-pinene, α-phellandrene, ∆3

**Benzenoids:** anisole, methoxy benzene.

trisulfide, methoxy phenyl oxime.

δ-amorphene, selina-3,7(11)-diene.

*cis*-calamenene, caryophyllene oxide. **Benzenoids:** 2-phenyl ethanol.

*cis*-2-pentenol, hexanol, *cis*-3-hexenol.

3-methyl hexanoic acid.

*Cannabis indica* Cannabaceae **Monoterpenoids:** α-pinene, α-thujene, camphene, β-pinene, Δ3

oxide, linalool.

**Sesquiterpenoids:** nerolidol.

*Polianthes tuberosa*

64 Potential of Essential Oils

*Stapelia gigantea*

*Veitchia merrillii*

*Ipomoea horsfalliae*

**Hydrocarbons:** tridecane.

*Vanilla planifolia*

*Vanilla pompona*

*Cattleya trianae* Orchidaceae **Sesquiterpenoids:** β-bourbonene, *trans*-β-caryophyllene.

benzyl alcohol, methyl cinnamate.

*cis-*carveol, *trans-*carveol, carvacrol,

butanol, hexanol, octanol, nonanol.

**Benzenoids:** 4-methyl phenol.

*Passiflora edulis* Passifloraceae **Monoterpenoids:** *cis*-β-ocimene*, trans*-β-ocimene, *p*-cymene,

*cis*-calamenene.

benzene, eugenol,

Orchidaceae **Monoterpenoids:** *cis*-geranyl acetone.

**Hydrocarbons:** *trans*-2,4-undecadiene.

**Hydrocarbons:** *trans*-2-methyl-2-pentene.

*trans-*2,6-dimethyl-1,3,5,7-octatetraene.

4-methoxy phenol, benzyl benzoate.

**Benzenoids:** benzaldehyde, methyl benzoate, methyl salicylate, 2-phenyl ethanol,

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67

**Oxygenated compounds (fatty acid derivatives):** nonanal, octanal.

The Expression of Biodiversity in the Secondary Metabolites of Aromatic Plants and Flowers…

*cis*-β-ocimene, *trans*-β-ocimene, *p*-cymene, terpinolene, *allo*-ocimene, 1,3,8-*p*-menthatriene, perillene, *cis*-limonene oxide, *trans*-limonene oxide, *cis*-salvene, *p*-cymenene, *cis*-*epoxy*-ocimene, myrtenal, terpinene-4-ol, *cis*dihydrocarvone, *trans-*dihydrocarvone, limonen-4-ol, piperitone, borneol, 2,6-dimethyl-1,5,7-octatriene, carvone, dihydrocarveol, nerol, geraniol, *cis-*

**Oxygenated compounds (fatty acid derivatives):** 1-(furan-2-yl) pentan-2-one,

**Oxygenated compounds (fatty acid derivatives):** *cis-*3,7-dimethyl octa-2,6-dienal, nonanal, 6-methyl-5-hepten-2-one, hexanol, *cis-*3-hexen-1-ol, 2-ethylhexan-1-ol.  **Sulfur- and nitrogen-containing compounds:** indole.

**Sulfur- and nitrogen-containing compounds:** methoxy phenyl oxime.

Orchidaceae **Monoterpenoids:** α-pinene, β-pinene, β-myrcene, limonene, 1,8-cineole,

dihydrocarvone oxide, *trans*-dihydrocarvone oxide,

**Benzenoids:** 2-phenyl ethanol, 4-methyl phenol.

**Sesquiterpenoids:** α-patchoulene, *trans*-salvene, guaiacol.

Malvaceae **Monoterpenoids:** α-pinene, camphene, β-pinene, sabinene, β-myrcene,

1,3,8-*p*-menthatriene, bornyl acetate.

*cis*-calamenene.

1-penten-3-ol.

Melastomataceae **Monoterpenoids:** italicene, *cis*-thujopsene. **Sesquiterpenoids:** β-cedrene.

*Moringa oleifera* Moringaceae **Monoterpenoids:** α-pinene, camphene, 4,8-dimethyl-1,7-nonadiene,

cinnamate, *trans*-cinnamyl acetate.

3-buten-3-ol, 3-methyl-butan-1-ol,

benzylnitrile, benzyl iso-thiocyanate.

*cis*-nerolidol.

isopentyl butyrate.

Orchidaceae **Monoterpenoids:** α-pinene, β-myrcene, limonene, *cis*-β-ocimene,

octanol, nonyl acetate, 2,6-nonadienal, nonanol, 2-nonen-1-ol, 2,6-nonadien-1-ol, lauryl acetate.

α-phellandrene, β-phellandrene, α-terpinene, limonene, *cis-*β-ocimene, *trans*-β-ocimene, γ-terpinene, *p*-cymene, *allo*-ocimene, *neo-allo*-ocimene,

**Oxygenated compounds (fatty acid derivatives):** acetaldehyde, ethanol,

**Sesquiterpenoids:** α-cubebene, α-copaene, *trans*-β-caryophyllene, aromadendrene, γ-gurjunene, α-bulnesene, α-humulene, γ-muurolene,

viridiflorene, α-muurolene, bicyclogermacrene, γ-cadinene,

**Sulfur- and nitrogen-containing compounds:** dimethyl sulfide.

**Oxygenated compounds (fatty acid derivatives):** hexanal, heptanal,

1-octen-3-ol, heptanol, octanol, nonanol, *cis*-3-nonen-1-ol, 2-undecanone.

β-pinene, sabinene, β-myrcene, α-phellandrene, α-terpinene, 2,3-dehydro-1,8-cineole, limonene, β-phellandrene, 1,8-cineole, *cis*-β-ocimene, γ-terpinene, *trans*-β-ocimene, *p*-cymene, terpinolene, *p*-cymenene, thujone, *cis*-sabinene hydrate, *cis*-linalool oxide, terpinene-4-ol, linalool, *trans*-sabinene hydrate, α-terpineol, citronellol, nerol, *trans*-β-ionone. **Sesquiterpenoids:** *cis*-β-farnesene, α-farnesene, *trans*-nerolidol. **Benzenoids:** benzaldehyde, methyl benzoate, benzyl acetate, benzyl alcohol, *cis*-methyl cinnamate, *trans*-methyl cinnamate, *trans*-ethyl

**Oxygenated compounds (fatty acid derivatives):** acetaldehyde, ethanol, 2-ethyl furane, pentan-3-one, 1-penten-3-one, butyl acetate, hexanal, 3-methyl-2-butenal,

*n*-hexyl acetate, octanal, *cis*-3-hexenyl acetate, *trans*-3-hexenyl acetate, 6-methyl-5-penten-2-one, heptyl acetate, 2-heptenyl acetate, 1-octen-3-ol, heptanol, octanal,

**Sulfur- and nitrogen-containing compounds:** carbonyl sulfide, carbon disulfide,

thiocyanate, 1-butyl iso-thiocyanate, 2-butyl iso-thiocyanate, benzeneacetonitrile,

**Benzenoids:** benzaldehyde, benzyl acetate, methyl salicylate, 2-phenyl ethanol. **Oxygenated compounds (fatty acid derivatives):** 3-octanone, isopentyl acetate,

*trans*-2-hexenal, 2-penten-1-yl acetate, 2-pentyl furane, octan-3-one,

dimethyl sulfide, *sec*-butyl nitrile, 3-methyl butanenitrile, 2-propyl iso-

*trans*-β-ocimene *p*-cymene, linalool oxide, linalool, terpinene-4-ol. **Sesquiterpenoids:** α-cubebene, β-cubebene, α-copaene, aromadendrene, α-muurolene, γ-muurolene, germacrene D, *trans, trans*-α-farnesene, γ-cadinene,

5-heptene-2-one, hexanol, *cis*-3-hexenol, 3-octanol, nonanal,

3-octanone, hexyl acetate, octanal, 1-octen-3-one, *cis*-3-hexenyl acetate, 6-methyl-

**Benzenoids:** *p*-methyl anisole, benzaldehyde.

*Gossypium barbadense*

66 Potential of Essential Oils

*Medinilla myriantha*

*Cattleya mendelii*

*Datura metel* Solanaceae **Monoterpenoids:** linalool.

*Petrea volubilis* Verbenaceae **Monoterpenoids:** β-myrcene, ∆3

geraniol, geranial.

benzyl benzoate, benzyl salicylate.

*cis*-3-hexenol, 4-methyl hexanol.

*trans, trans*-α-farnesene.

*cis*-3-hexenyl benzoate.

nonane.

**Table 1.** Volatile compounds isolated by *in vivo* HS-SPME from 30 tropical flowers, grown in Colombia.

*cis*-3-hexen-1-ol, hexanol, nonanal, decanal.

**Hydrocarbons:** *n*-dodecane, *n*-pentadecane.

α-copaene, *trans*-β-caryophyllene, farnesol,

pentanoate, octanol, 1-nonen-3-ol, nonanol, *cis*-3-hexenyl angelate, *cis*-3-nonen-1-ol. **Hydrocarbons:** 3-methyl pentadecane.

Zingiberaceae **Monoterpenoids:** α-pinene, β-pinene, β-myrcene, 1,8-cineole,

*trans, trans*-α-farnesene, *trans, trans-*farnesol.

Solanaceae **Monoterpenoids:** α-thujene, α-pinene, 6-methyl hept-5-en-2-one, sabinene, β-pinene, β-myrcene, α-terpinene, limonene, 1,8-cineole,

> **Sesquiterpenoids:** *trans*-β-caryophyllene, *trans*-β-farnesene, α-terpineol, *trans, trans-*farnesol, farnesal, *trans*-nerolidol.  **Sesquiterpenoids:**

3-octanone, octanal, 1-octen-3-one, *cis*-2-penten-1-ol, hexanol, 3-penten-2-ol, *cis*-3-hexenol, 3-octanol, *trans*-2-hexen-1-ol, hexyl 2-methyl butanoate, 1-octen-3-ol, *cis*-3-hexenyl butanoate, *cis*-3-hexenyl 2-methyl butanoate, *cis*-3-hexenyl

*cis*-β-ocimene, *trans*-β-ocimene, *allo*-ocimene, γ-terpinene, terpinolene, linalool, *trans-p*-2-menthen-1-ol, terpinene-4-ol, α-terpineol, *cis-*jasmone. **Sesquiterpenoids:** *trans*-β-caryophyllene, caryophyllene oxide,

**Benzenoids:** methyl benzoate, 2-methylbutyl benzoate, benzyl benzoate.

**Sulfur- and nitrogen-containing compounds:** 1-nitro-2-methyl butane, *cis*-2 methyl butyl aldoxime, *trans*-2-methyl butyl aldoxime, 3-methyl butyl aldoxime, *trans*-3-methyl butyl aldoxime, phenyl acetonitrile, indole. **Hydrocarbons:**

**Oxygenated compounds (fatty acid derivatives):** *cis*-3-hexenol.

**Sulfur- and nitrogen-containing compounds:** Indole.

*cis*-β-ocimene, *trans*-β-ocimene, *p*-cymene, α-terpinolene, *allo*-ocimene, citronellal, *p*-cymenene, *cis*-sabinene hydrate, linalool, terpinen-4-ol, citronellol, neral,

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69

The Expression of Biodiversity in the Secondary Metabolites of Aromatic Plants and Flowers…

*Brugmansia suaveolens*

*Hedychium coronarium*

Rubiaceae **Monoterpenoids:** β-myrcene, limonene, *cis*-β-ocimene, *trans*-β-ocimene,

**(fatty acid derivatives):** *trans*-2-hexenal,

*trans*-2-octen-1-ol, *cis*-3-nonen-1-ol.

*Coffea arabica* Rubiaceae **Monoterpenoids:** α-pinene, β-myrcene, limonene, 1,8-cineole,

2-pentyl furane,

2-nonenal, octanol, nonanol.

7-hexadecene, heptadecane.

*cis*-3-nonen-1-ol.

**Hydrocarbons:** dodecane.

**Sesquiterpenoids:** α-*cis*-bergamotene, β-elemene, β-copaene,

2-pentyl furane, hexanol, *cis*-3-hexenol, 2-hexen-1-ol, 2-octenal,

**Hydrocarbons:** tridecane, tetradecane, hexadecane.

*p*-cymenene, linalool oxide, nerol oxide, linalool, neral.

6-methyl-5-hepten-2-one, heptanol, 2,4-heptadienal, decanal,

**Sulfur- and nitrogen-containing compounds:** dimethyl sulfide,

**Hydrocarbons:** tridecane, tetradecane, pentadecane, 1-pentadecene, 7-pentadecene, hexadecane, 3-hexadecene,

Rubiaceae **Monoterpenoids:** *trans*-β-ocimene, γ-terpinene, *allo-*ocimene, linalool. **Sesquiterpenoids:** α-cubebene, β-bourbonene, β-copaene, *trans*-β-elemene, *trans*-β-caryophyllene, caryophyllene oxide, α-humulene, α-amorphene, germacrene D, α-muurolene, *trans*, *trans*-α-farnesene, γ-cadinene, cubebene, *cis*-calamene.

> 2-phenyl ethanol, isopentyl benzoate, 2-methoxy-phenol, *trans*-methyl cinnamate, *cis*-3-hexyl benzoate, eugenol.

hexyl acetate, *cis*-3-hexyl acetate, *trans*-2-heptanal,

3-methyl butyl aldoxime, benzyl nitrile.

**Sulfur- and nitrogen-containing compounds:** benzyl nitrile, indole.

*allo*-ocimene, *p*-cymene, linalool oxide, linalool, nerol, geraniol, cyperene.

*trans*-β-caryophyllene, *cis*-muurola-3,5-diene, α-humulene, β-farnesene, germacrene D, α-muurolene, α-selinene, α-farnesene, α-cadinene,

δ-cadinene, γ-cadinene, *cis*-calamenene, *epi*-α-muurolol, α-cadinol. **Benzenoids:** benzaldehyde, methyl salicylate, 2-phenyl ethanol. **Oxygenated compounds**

*cis*-β-ocimene, γ-terpinene, *trans*-β-ocimene, *p*-cymene, *p*-cymen-8-ol, perillene,

**Benzenoids:** methyl benzoate, phenyl acetaldehyde, ethyl benzoate. **Oxygenated compounds (fatty acid derivatives):** propanal, 2-methyl propanal, 2-propenal, propanol, 2-butanone, 2-methyl-butenal, 3-methyl butanal, 2-pentanal,

3-pentanone, 1-penten-3-one, hexanal, *tert*-butyl alcohol, *trans*-2-methyl-2-butenal, *cis*-2-penten-1-al, 2-methyl-2-hexenone, heptanal, 2-methyl-1-butanol, 2-hexenal,

2-hexanol, 2-octanone, *cis*-2-penten-1-ol, *trans*-2-heptenal, 5-methyl-2-hexanol,

*o*-methyl oxime 2-propanone, 2-methyl butyl nitrile, 3-methyl butyl aldoxime.

**Benzenoids:** benzaldehyde, methyl benzoate, phenyl acetaldehyde, ethyl benzoate, benzyl acetate, methyl salicylate, benzyl alcohol, benzyl isovalerate,

1-penten-3-ol, 4-pentenyl acetate, 2-pentyl furane, 3-methyl-2-butenyl acetate,

*trans*-2-hexenyl acetate, hexanol, *cis*-3-hexenol, nonanal, *trans*-2-octenal, 1-octen-3-ol, octyl acetate, *trans, trans*-2,4-heptadienal, octanol, nonyl acetate, nonanol,

**Sulfur- and nitrogen-containing compounds:** *cis*-3-methyl butyl aldoxime, *trans*-

**Oxygenated compounds (fatty acid derivatives):** isopentyl acetate,

*Gardenia augusta*

68 Potential of Essential Oils

*Posoqueria longiflora*

**Table 1.** Volatile compounds isolated by *in vivo* HS-SPME from 30 tropical flowers, grown in Colombia.

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.

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,

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

The flower fragrance of *Brugmansia suaveolens* (Solanaceae family) follows a clear circadian rhythm: the emission of volatiles increases in the evening and reaches its maximum at nine o'clock at night; then, the volatile emission slowly begins to decrease; in the morning and during the day, the flowers almost do not smell, although they attract massively the bees. It is interesting to note that some flowers change their fragrance after they have been pollinated;

Notorious changes can be observed (**Figure 1**) in *Vanilla pompona* (Orchidaceae family) volatile

In carrion flower *Stapelia gigantea* (Apocynaceae family), which emitted fetid, nasty, and badly smelling volatiles (dimethyl disulfide, dimethyl trisulfide, butanoic acid, 3-methyl butanoic acid, hexanoic acid), the number of volatiles diminished after the oviposition of the green

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

**Figure 1.** Chromatographic profiles (GC–MS, EI, 70 eV, DB-WAX column, 60 m) of *Vanilla planifolia* flower volatiles, isolated by *in vivo* HS-SPME (CAR/PDMS) at: (**A**) 7 a.m. before pollination and (**B**) 7 p.m. after pollination. Main compounds found in vanilla floral scent: (**a**) β-myrcene; (**b**) limonene; (**c**) *trans*-epoxy myrcene; (**d**) *trans*-limonene oxide; (**e**) *trans-*dihydrocarvone; (**f**) carvone; (**g**) *trans*-carvone oxide; (**h**) *trans*-carveol; (**i**) *p*-methyl phenol; and (**j**) *diepoxy*

containing compounds, and even some fatty acids (C14-C18).

increases again in the afternoon and evening hours.

bottle fly (*Lucilia sericata*) had occurred (**Figure 2**).

limonene. Internal standard (ISTD) – *n*-tetradecane.

this happens with the flowers of some orchids (*Ophrys sphegodes*) [14].

fraction isolated by HS-SPME from flowers after their pollination.

, isolates extracts, rich in aliphatic hydrocarbons (Cn > 20) and terpenes, nitrogen-

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SFE-CO2

The chemical composition of the volatile fraction of flowers depends both on intrinsic (genetic) factors of the species and on extrinsic, environmental factors [9]. 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 [10].

Some aspects of the study of floral fragrances should contemplate the state of development of the flower [11]. The flowers of the ylang-ylang tree (*Cananga odorata* Hook Fil. and Thomson, genuine form, Annonaceae family) are important raw material for obtaining essential and absolute oils, which are valuable ingredients in many perfumes, soaps, shampoos, and lotions. In the tree, the flower remains several weeks while it matures, it starts as a very small, green flower, which then increases in size, staying several days green, and then turns yellow, large, with brown spots. In the same tree, it is common to find flowers in different degrees of maturation, along with the fruits that carry seeds, through which this plant species spread. In the ylang-ylang tree, the composition of flower volatiles varies markedly with its state of maturity. In small, green flowers, 10 times fewer components are recorded, than in a yellow, mature flower. In the mature, yellow and fully developed ylang-ylang flowers, 16–4 times more lightoxygenated substances are found (*p*-methylanisole, benzyl alcohol, 1,8-cineole, methyl and ethyl benzoate and salicylate, linalool, nerol, geraniol, benzyl acetate, anethole, cinnamyl acetate, and others) and heavier oxygenated substances (sesquiterpenols, farnesal, farnesol, nerolidol and their acetates, cedrol, benzyl benzoate and benzyl salicylate, others), than in green, small flowers that begin their development. The nitrogen-containing compounds, phenyl acetonitrile, 4-methylbenzaldoxime, indole, 2-phenyl-1-nitroethane, and methyl anthranilate, only appear in mature, large and yellow flowers [12].

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 [13]. 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, nitrogencontaining compounds, and even some fatty acids (C14-C18).

The headspace methods provide information on the chemical composition of the volatile fractions; distillation techniques, on essential oils, distillates or condensates while extractive

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

The chemical composition of the volatile fraction of flowers depends both on intrinsic (genetic) factors of the species and on extrinsic, environmental factors [9]. 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

Some aspects of the study of floral fragrances should contemplate the state of development of the flower [11]. The flowers of the ylang-ylang tree (*Cananga odorata* Hook Fil. and Thomson, genuine form, Annonaceae family) are important raw material for obtaining essential and absolute oils, which are valuable ingredients in many perfumes, soaps, shampoos, and lotions. In the tree, the flower remains several weeks while it matures, it starts as a very small, green flower, which then increases in size, staying several days green, and then turns yellow, large, with brown spots. In the same tree, it is common to find flowers in different degrees of maturation, along with the fruits that carry seeds, through which this plant species spread. In the ylang-ylang tree, the composition of flower volatiles varies markedly with its state of maturity. In small, green flowers, 10 times fewer components are recorded, than in a yellow, mature flower. In the mature, yellow and fully developed ylang-ylang flowers, 16–4 times more lightoxygenated substances are found (*p*-methylanisole, benzyl alcohol, 1,8-cineole, methyl and ethyl benzoate and salicylate, linalool, nerol, geraniol, benzyl acetate, anethole, cinnamyl acetate, and others) and heavier oxygenated substances (sesquiterpenols, farnesal, farnesol, nerolidol and their acetates, cedrol, benzyl benzoate and benzyl salicylate, others), than in green, small flowers that begin their development. The nitrogen-containing compounds, phenyl acetonitrile, 4-methylbenzaldoxime, indole, 2-phenyl-1-nitroethane, and methyl anthranilate, only appear in

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 [13]. The relative percentage composition

), on the chemical composition of mixtures that may

)

methods (solvents, supercritical CO2

70 Potential of Essential Oils

or during the concentration of the extracts.

floral volatiles or even suppress their production [10].

mature, large and yellow flowers [12].

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 *Brugmansia suaveolens* (Solanaceae family) follows a clear circadian rhythm: the emission of volatiles increases in the evening and reaches its maximum at nine o'clock at night; then, the volatile emission slowly begins to decrease; in the morning and during the day, the flowers almost do not smell, although they attract massively the bees. It is interesting to note that some flowers change their fragrance after they have been pollinated; this happens with the flowers of some orchids (*Ophrys sphegodes*) [14].

Notorious changes can be observed (**Figure 1**) in *Vanilla pompona* (Orchidaceae family) volatile fraction isolated by HS-SPME from flowers after their pollination.

In carrion flower *Stapelia gigantea* (Apocynaceae family), which emitted fetid, nasty, and badly smelling volatiles (dimethyl disulfide, dimethyl trisulfide, butanoic acid, 3-methyl butanoic acid, hexanoic acid), the number of volatiles diminished after the oviposition of the green bottle fly (*Lucilia sericata*) had occurred (**Figure 2**).

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

**Figure 1.** Chromatographic profiles (GC–MS, EI, 70 eV, DB-WAX column, 60 m) of *Vanilla planifolia* flower volatiles, isolated by *in vivo* HS-SPME (CAR/PDMS) at: (**A**) 7 a.m. before pollination and (**B**) 7 p.m. after pollination. Main compounds found in vanilla floral scent: (**a**) β-myrcene; (**b**) limonene; (**c**) *trans*-epoxy myrcene; (**d**) *trans*-limonene oxide; (**e**) *trans-*dihydrocarvone; (**f**) carvone; (**g**) *trans*-carvone oxide; (**h**) *trans*-carveol; (**i**) *p*-methyl phenol; and (**j**) *diepoxy* limonene. Internal standard (ISTD) – *n*-tetradecane.

**Figure 2.** Chromatographic profiles (GC–MS, EI, 70 eV, DB-WAX column, 60 m) of volatiles isolated from *Stapelia gigantea* carrion flower, by *in vivo* HS-SPME (CAR/PDMS): (**A**) during oviposition and (**B**) after oviposition of the green bottle fly *Lucilia sericata* (Insecta: Diptera: Calliphoridae). Main compounds found in carrion flower odor are as follows: (**a**) dimethyl disulfide; (**b**) (3*E-*, 5*E-*)-2,6-dimethyl-1,3,5,7-octatetraene; (**c**) *trans*-β-ocimene; (**d**) dimethyl trisulfide; (**e**) 2,6-dimethyl-1,3,5,7-octatetraene (isomer); (**f**) butanoic acid; (**g**) 3-methyl butanoic acid; (**h**) methoxy phenyl oxime; and (**i**) hexanoic acid.

flower temperature or transpiration. **Figure 3** shows chromatographic profiles (HS-SPME/ GC/MS) of volatiles emitted from distinct parts of passion fruit (*Passiflora edulis*) flower, where volatile compounds differ qualitatively or quantitatively; some of these volatile metabolites are unique to each part of the flower.

#### **2.2. Chromatographic analysis of floral fragrances**

The substances that make up the volatile fraction isolated from flowers are of low-molecularweight (<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.

injection can be done in the *pulsed splitless* mode; it is when the inlet pressure of the carrier gas, during the transfer of the sample by the liner, increases by 2–3 times. Considering the presence of some thermolabile substances (esthers, oximes), the *on-column* injection or the temperature-programmed injection (PTV, programmed-temperature vaporizer), could be a

**Figure 3.** Chromatographic profiles (GC–MS, EI, 70 eV, DB-WAX column, 60 m) of volatiles, obtained by HS-SPME (CAR/PDMS) from distinct parts of passion fruit flower (*Passiflora edulis*). Each part of the flower (sepals, petals, nectaries, stamens or carpels) possesses a "diagnostic", unique volatile compounds (\*), found only in this part of the flower.

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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

suitable alternative.

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 The Expression of Biodiversity in the Secondary Metabolites of Aromatic Plants and Flowers… http://dx.doi.org/10.5772/intechopen.78001 73

flower temperature or transpiration. **Figure 3** shows chromatographic profiles (HS-SPME/ GC/MS) of volatiles emitted from distinct parts of passion fruit (*Passiflora edulis*) flower, where volatile compounds differ qualitatively or quantitatively; some of these volatile metabolites

**Figure 2.** Chromatographic profiles (GC–MS, EI, 70 eV, DB-WAX column, 60 m) of volatiles isolated from *Stapelia gigantea* carrion flower, by *in vivo* HS-SPME (CAR/PDMS): (**A**) during oviposition and (**B**) after oviposition of the green bottle fly *Lucilia sericata* (Insecta: Diptera: Calliphoridae). Main compounds found in carrion flower odor are as follows: (**a**) dimethyl disulfide; (**b**) (3*E-*, 5*E-*)-2,6-dimethyl-1,3,5,7-octatetraene; (**c**) *trans*-β-ocimene; (**d**) dimethyl trisulfide; (**e**) 2,6-dimethyl-1,3,5,7-octatetraene (isomer); (**f**) butanoic acid; (**g**) 3-methyl butanoic acid; (**h**) methoxy phenyl oxime;

The substances that make up the volatile fraction isolated from flowers are of low-molecularweight (<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 sensitiv-

are used, so that the shape of peaks, their separation and the sensitivity, necessary for their

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

) equal to or greater than 0.25 μm

are unique to each part of the flower.

and (**i**) hexanoic acid.

72 Potential of Essential Oils

reproducible detection, are adequate.

**2.2. Chromatographic analysis of floral fragrances**

ity. Columns with the thickness of the stationary phase (df

**Figure 3.** Chromatographic profiles (GC–MS, EI, 70 eV, DB-WAX column, 60 m) of volatiles, obtained by HS-SPME (CAR/PDMS) from distinct parts of passion fruit flower (*Passiflora edulis*). Each part of the flower (sepals, petals, nectaries, stamens or carpels) possesses a "diagnostic", unique volatile compounds (\*), found only in this part of the flower.

injection can be done in the *pulsed splitless* mode; it is when the inlet pressure of the carrier gas, during the transfer of the sample by the liner, increases by 2–3 times. Considering the presence of some thermolabile substances (esthers, oximes), the *on-column* injection or the temperature-programmed injection (PTV, programmed-temperature vaporizer), could be a suitable alternative.

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, *e.g.,* 30 m, one could increase the temperature of the column more rapidly, at a rate of 5–10°C/min. Of course, the process of programming the temperature in the column is optimizable, depending on the complexity of the mixture of substances to be analyzed (number of components, isomerism or structural similarity), its nature (polarity, molecular weight), the column dimensions (L, DI), the type of the stationary phase (polarity), and its thickness (df ).

(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

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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

The mass (*m/z*) of fragments (ions or ion-radicals) and their relative abundances, which make up the fragmentation pattern, are the guide to differentiate the structures. The linear retention indexes (LRI) measured experimentally in the polar and non-polar columns are compared with those recorded in the literature or in databases [21–24]. Probably, the greatest progress in the analysis of complex mixtures, such as essential oils or volatile fractions isolated from flowers, has been done with the introduction of comprehensive chromatography GC × GC [25–27]. The use of two orthogonal columns (non-polar and polar) linked through a modulator, and the use of a low-resolution (quadrupole, linear time-of-flight time, and TOF) or high-resolution (HR TOF) mass spectrometers, allow to have a complete picture on the number, and quantity of components in a mixture since some analytes can co-elute in one of the columns typically used in one-dimensional chromatography; but the use of two orthogonal

**Figure 4.** GCxGC chromatogram (TIC) of *Cannabis indica* female flower volatiles, obtained with high-resolution time-offlight analyzer (HRTOF-MS) and cryogenic dual jet/loop modulator.1D – First column: Rxi-5MS, 30 m, L, 250 μm, DI,

terpenoids, *that is,* monoterpenoids and sesquiterpenoids are clearly distinguished in the chromatogram. Compounds

. Modulation time: 5 s. Two groups of

. 2D – Second column: Rxi-17Sil MS, 2 m, L, 250 μm, DI, 0,25 μm, df

co-eluted in the 1D column are separated in the 2D column (areas of co-eluted peaks are encircled).

sulfur compounds, very common in floral fragrances.

they are isomers.

0,25 μm, df

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 (*Jasminum grandiflorum*) and other flowers (*Osmanthus fragrans*, *Boronia megastigma*) contain a mixture of methyl jasmonate stereoisomers. Wilfred König reported the separation of all isomers by means of preparative gas chromatography in which he used columns packed with cyclodextrins [15]. This allowed to confirm the estimate made by Acree and Barnard, that the methyl (+)-*epi*-jasmonate isomer has an odor threshold about 500 times lower than that of the major isomer, methyl (−)-jasmonate [16].

The aldehydes and lilac alcohols are oxygenated monoterpenes found in plant species of many families. Lamiaceae (*Origanum vulgare)* [17], Orchidaceae (*Platanthera* sp.) [18], Rosaceae (*Prunus padus)* [19], and Rubiaceae (*Cephalanthus occidentalis*) are some examples of these families. Each of the lilac molecules has three chiral carbons, which gives rise to eight stereoisomers of the aldehyde and eight stereoisomers of the lilac alcohol. Dötterl and colleagues [20] managed to separate all the isomers of the aldehyde and seven isomers of the lilac alcohol, by means of a two-dimensional gas chromatography system in which a 30 m-capillary column with stationary phase of 5%-phenyl poly (dimethyl siloxane) was bound by means of a T-valve to a mass selective detector, and another capillary column of 30 m with a stationary phase formed by phenyl-poly (dimethyl siloxane) (70%) and a cyclodextrin derivative (30%). Each column had an independent oven. This system was modified to convert it into micropreparative chromatography. The output of the second column was connected to a flow divider that allowed to directing a part of the effluent toward an FID, and the other part toward a stirring bar covered with PDMS, to absorb the separated analytes. This modification allowed to collecting the isomers that were then used in electroantennography experiments in which antennas of different insects were used to examine if there was any selective response for any of the isomers. It was found that the antennae of the *Hadena bicruris* moth responded to the eight isomers of aldehyde lilac, but they were more sensitive to some isomers than to others.

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.

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, *e.g.,* 30 m, one could increase the temperature of the column more rapidly, at a rate of 5–10°C/min. Of course, the process of programming the temperature in the column is optimizable, depending on the complexity of the mixture of substances to be analyzed (number of components, isomerism or structural similarity), its nature (polarity, molecular weight), the column dimensions

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,

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 (*Jasminum grandiflorum*) and other flowers (*Osmanthus fragrans*, *Boronia megastigma*) contain a mixture of methyl jasmonate stereoisomers. Wilfred König reported the separation of all isomers by means of preparative gas chromatography in which he used columns packed with cyclodextrins [15]. This allowed to confirm the estimate made by Acree and Barnard, that the methyl (+)-*epi*-jasmonate isomer has an odor threshold about 500 times lower than that of the major isomer, methyl (−)-jasmonate [16].

The aldehydes and lilac alcohols are oxygenated monoterpenes found in plant species of many families. Lamiaceae (*Origanum vulgare)* [17], Orchidaceae (*Platanthera* sp.) [18], Rosaceae (*Prunus padus)* [19], and Rubiaceae (*Cephalanthus occidentalis*) are some examples of these families. Each of the lilac molecules has three chiral carbons, which gives rise to eight stereoisomers of the aldehyde and eight stereoisomers of the lilac alcohol. Dötterl and colleagues [20] managed to separate all the isomers of the aldehyde and seven isomers of the lilac alcohol, by means of a two-dimensional gas chromatography system in which a 30 m-capillary column with stationary phase of 5%-phenyl poly (dimethyl siloxane) was bound by means of a T-valve to a mass selective detector, and another capillary column of 30 m with a stationary phase formed by phenyl-poly (dimethyl siloxane) (70%) and a cyclodextrin derivative (30%). Each column had an independent oven. This system was modified to convert it into micropreparative chromatography. The output of the second column was connected to a flow divider that allowed to directing a part of the effluent toward an FID, and the other part toward a stirring bar covered with PDMS, to absorb the separated analytes. This modification allowed to collecting the isomers that were then used in electroantennography experiments in which antennas of different insects were used to examine if there was any selective response for any of the isomers. It was found that the antennae of the *Hadena bicruris* moth responded to the eight isomers of aldehyde lilac, but they were more sensitive to some isomers than to others. 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

).

(L, DI), the type of the stationary phase (polarity), and its thickness (df

CPSil 5, BP-5, and others).

74 Potential of Essential Oils

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 (*m/z*) of fragments (ions or ion-radicals) and their relative abundances, which make up the fragmentation pattern, are the guide to differentiate the structures. The linear retention indexes (LRI) measured experimentally in the polar and non-polar columns are compared with those recorded in the literature or in databases [21–24]. Probably, the greatest progress in the analysis of complex mixtures, such as essential oils or volatile fractions isolated from flowers, has been done with the introduction of comprehensive chromatography GC × GC [25–27]. The use of two orthogonal columns (non-polar and polar) linked through a modulator, and the use of a low-resolution (quadrupole, linear time-of-flight time, and TOF) or high-resolution (HR TOF) mass spectrometers, allow to have a complete picture on the number, and quantity of components in a mixture since some analytes can co-elute in one of the columns typically used in one-dimensional chromatography; but the use of two orthogonal

**Figure 4.** GCxGC chromatogram (TIC) of *Cannabis indica* female flower volatiles, obtained with high-resolution time-offlight analyzer (HRTOF-MS) and cryogenic dual jet/loop modulator.1D – First column: Rxi-5MS, 30 m, L, 250 μm, DI, 0,25 μm, df . 2D – Second column: Rxi-17Sil MS, 2 m, L, 250 μm, DI, 0,25 μm, df . Modulation time: 5 s. Two groups of terpenoids, *that is,* monoterpenoids and sesquiterpenoids are clearly distinguished in the chromatogram. Compounds co-eluted in the 1D column are separated in the 2D column (areas of co-eluted peaks are encircled).

columns avoids this problem. In addition, GCxGC allows "classifying" the substances by families, such as can be observed in **Figure 4**, which shows two groups of substances, monoterpenes and sesquiterpenes, in the scent emitted by *Cannabis* female flowers.

**Species Composition**

*Condylidium cuatrecasasii*

*Ichthyothere terminalis*

*Parthenium hysterophorus*

*Verbesina centroboyaca*

**Boraginaceae family**

**Burseraceae family**

**Chloranthaceae family**

*Protium heptaphyllum*

*Hedyosmum racemosum*

1,3,5-triene (2%).

limonene (3%).

squalene (6%).

β-sesquiphellandrene (4%).

(14%).

Δ2

(3%).

oxide (6%).

limonene (12%).

ocimene (2%).

valerianol (12%).

α-pinene (6%).

germacrene D (3%).

(9%).

ocimene (12%), spathulenol (5%).

*trans-*tagetone (3%), *cis*-tagetone (3%).

*Bidens reptans p-*Cymene (3%), β-copaene (3%), germacrene D (3%), caryophyllene oxide (3%), 1-phenyl-hepta-

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*Calea glomerata* α-Zingiberene (27%), germacrene D (11%), *trans-*β-caryophyllene (7%), *ar*-curcumene (5%),

*Calea prunifolia* 1,8-Cineole (4%), borneol (6%), *trans-*β-farnesene (3%), *ar*-curcumene (16%), α-zingiberene

*Chromolaena pellia* Caryophyllene oxide (5%), β-amyrin (6%), germacrene D (3%), *trans*-β-caryophyllene (1%),

*Conyza bonariensis* 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%).

*Simsia fruticulosa* α-Thujene (11%), α-pinene (12%), β-myrcene (4%), α-copaene (4%), spathulenol (4%). *Stevia aff. Lucida* α-Pinene (25%), camphene (16%), β-pinene (11%), α-phellandrene (12%), *p-*cymene (5%),

germacrene D-4-ol (4%), caryophyllene oxide (4%), guaiol (4%). *Tagetes caracasana cis-*β-Ocimene (12%), dihydrotagetone (16%), *allo*-ocimene (2%), *cis*-tagetone (58%), *trans*-

*Tagetes heterocarpha cis-*β-Ocimene (3%), dihydrotagetone (13%), *cis*-tagetone (16%), *cis*-β-ocimene (6%), *trans*-

*Tagetes zipaquirensis* β-Myrcene (5%), *trans-*β-ocimene (12%), dihydrotagetone (42%), 6,7-epoxy myrcene (13%),

*Wedelia calycina* Germacrene D (15%), β-phellandrene (14%), β-pinene (14%), α-pinene (20%), α-phellandrene

*Cordia curassavica* α-Copaene (17%), *trans*-β-caryophyllene (22%), germacrene D (18%), *trans*-β-guaiene (8%),

*Stevia ovata trans-*β-Caryophyllene(10%), germacrene D (8%), bicyclogermacrene (5%), *trans-*nerolidol (19%),

α-Pinene (4%), sabinene (40%), β-pinene (3%), terpinen-4-ol (7%), *trans-*β-caryophyllene (3%)

*trans*-β-Caryophyllene (12%), limonene (12%), germacrene B (6%), *p*-cymene (6%), caryophyllene

β-Myrcene (8%), α-humulene (4%), germacrene D (7%), germacrene D-4-ol (4%), hinesol (4%),

*trans*-β-Caryophyllene (20%), α-humulene (9%), γ-cadinene (13%), caryophyllene oxide (20%),

α-Pinene (6%), sabinene (21%), β-pinene (9%), 1,8-cineole (6%), *trans*-4-thujanol (5%).

*Calea sessiliflora* α-Zingiberene (35%), germacrene D (17%), *ar*-curcumene (13%), viridiflorol (3%),


*Article* **Analysis of Vancomycin-Resistant Enterococci in Hemato-Oncological Patients**

#### **Kristýna Hricová, Tat'ána Štosová, Pavla Kuˇcová, Kateˇrina Fišerová \*, Jan Bardo ˇn and Milan Koláˇr**

Received: 28 September 2020; Accepted: 6 November 2020; Published: 7 November 2020

Department of Microbiology, University Hospital Olomouc and Faculty of Medicine and Dentistry, Palacký University Olomouc, 77900 Olomouc, Czech Republic<EMAIL_ADDRESS>(K.H<EMAIL_ADDRESS>(T.Š<EMAIL_ADDRESS>(P.K<EMAIL_ADDRESS>(J.B<EMAIL_ADDRESS>(M.K.)

**\*** Correspondence<EMAIL_ADDRESS>Tel.: +420-585-639-511

**Abstract:** Enterococci are important bacterial pathogens, and their significance is even greater in the case of vancomycin-resistant enterococci (VRE). The study analyzed the presence of VRE in the gastrointestinal tract (GIT) of hemato-oncological patients. Active screening using selective agars yielded VRE for phenotypic and genotypic analyses. Isolated strains were identified with MALDI-TOF MS, (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry) their susceptibility to antibiotics was tested, and resistance genes (*vanA*, *vanB*, *vanC-1*, *vanC2-C3*) and genes encoding virulence factors (*asa1*, *gelE*, *cylA*, *esp*, *hyl*) were detected. Pulsed-field gel electrophoresis was used to assess the relationship of the isolated strains. Over a period of three years, 103 VanA-type VRE were identified in 1405 hemato-oncological patients. The most frequently detected virulence factor was extracellular surface protein (84%), followed by hyaluronidase (40%). Unique restriction profiles were observed in 33% of strains; clonality was detected in 67% of isolates. The study found that 7% of hemato-oncological patients carried VRE in their GIT. In all cases, the species identified was *Enterococcus faecium*. No clone persisted for the entire 3-year study period. However, genetically different clusters were observed for shorter periods of time, no longer than eight months, with identical VRE spreading among patients.

**Keywords:** VRE; GIT; hemato-oncological patients; clonality

#### **1. Introduction**

In today's medicine, important bacterial pathogens are enterococci. As etiological agents, they are responsible for community as well as healthcare-associated infections (HAIs), particularly in patients with prolonged hospital stays, severe underlying disease, or previous broad-spectrum antibiotic therapy [1]. Sievert et al. reported that enterococci belong to the most common bacterial pathogens causing HAIs in the USA [2]. According to a 2017 U.S. study, enterococci were implicated in 14% of HAIs in the country [3]. Using results from a large multicenter study involving 15 centers in the Czech Republic, Koláˇr et al. documented that 9% of bloodstream infections were due to enterococci [4]. Herkel et al. found that enterococci accounted for 5% of all bacterial pathogens causing healthcare-associated pneumonia [5]. In these cases, they originated in the upper gastrointestinal tract (GIT), with pneumonia developing as a result of gastric content regurgitation and subsequent microaspiration [6]. Since approximately the 1970s, a vast majority of enterococcal HAIs have been caused by *Enterococcus faecium* and *Enterococcus faecalis* [7].

Treatment of enterococcal infections is considerably influenced not only by their natural resistance to many antibacterial agents, but also by acquired resistance, representing a factor limiting antibiotic therapy. A therapeutic challenge and real threat to patients are vancomycin-resistant enterococci (VRE). The most widespread VRE type is the VanA phenotype, determined by the D-alanyl-D-lactate structure encoded by the *vanA* gene that is very often located on transposon Tn1546 [8–11]. The first description of VRE comes from the UK and was published in 1988 [12]. In the Czech Republic, VRE were first detected in hemato-oncological patients at the University Hospital Olomouc in 1997 [13].

At present, a serious public issue is the presence of multidrug-resistant bacteria including VRE in the GIT as components of the normal microflora. The clinical significance of multidrug-resistant bacteria in the GIT was documented by Koláˇr et al. In their study, an ESBL-positive strain of *Klebsiella pneumoniae* and an AmpC-positive strain of *Enterobacter cloacae*, isolated from rectal swabs and thus being part of gastrointestinal bacterial flora, were found to be identical with strains causing bacterial infections, namely urinary tract infection and bloodstream infection [14]. Arias and Murray reported translocation of enterococci across the GIT wall into the bloodstream with subsequent endocarditis [7]. This fact is of utmost importance in VRE that may be part of GIT microflora, with the length of hospital stay or interventions such as hemodialysis, transplantation, and application of artificial materials playing an important role [15–17]. Heisel et al. showed that the proportion of patients with VRE colonization of the GIT changed from 3% before hospital admission to 59% during their hospital stay [18]. Another important factor promoting the presence of VRE in the GIT is the selection pressure of antibiotics. Koláˇr et al. documented the positive selection pressure of third-generation cephalosporins and glycopeptides with regard to the presence of VRE [19]. Paterson et al. found that after treatment with piperacillin-tazobactam or cefepime, 26% and 31%, respectively, of patients primarily tested negative for VRE were colonized with these bacteria [20].

Data from the EARS-Net provide information on the current prevalence of VRE in the Czech Republic [21]. The percentage of vancomycin-resistant strains of *E. faecium* ranges from 4% to 13% [22–24]. However, this prominent European database only considers blood isolates and cannot provide more accurate comprehensive data on the prevalence of VRE in both the community and hospital human populations including GIT carriage. In the Czech Republic, such data were last published by Koláˇr et al. in 2006 [19,25]. In their study on the prevalence of VRE in hemato-oncological patients in a Czech university hospital, these strains accounted for 5% of all isolated enterococci. The most frequent strains were VanA-type *E. faecium* (78%) and VanB-type *E. faecalis* (10%). The fact that VRE were most frequently isolated from rectal swabs (55%) illustrates the significance of their carriage in the GIT [19].

A key role in the etiopathogenesis of infections caused by enterococci may be played by selected virulence factors. These are essential for biofilm formation and subsequent colonization [26]. The presence of selected virulence factors in VRE was studied by Yang et al. For *E. faecium*, the most frequent gene was *esp*, encoding extracellular surface protein, detected in 90% of strains. Additionally present (28%) was the *hyl* (hyaluronidase) gene. The combination of these two genes (*esp* + *hyl*) was noted in 26% of isolates. Genes encoding aggregation substance (*asa1*), cytolysin (*cylA*), or gelatinase (*gelE*) were not detected in *E. faecium*, but only in *E. faecalis* strains, which were considerably less frequently detected in the study [27].

The present study aimed to determine the presence of VRE in the GIT of hemato-oncological patients in the University Hospital Olomouc and to carry out their genetic analysis including the assessment of selected virulence factors.

#### **2. Results**

Over a 3-year study period, stool samples and perianal swabs obtained from 1405 subjects were analyzed and a total of 103 VRE were isolated, suggesting that 7.3% of hemato-oncological patients carried VRE in their GIT. In all cases, the species identified was *E. faecium*.

The results of VRE susceptibility testing showed very high susceptibility to linezolid (100%) and tigecycline (96%) and, conversely, 100% resistance to ampicillin and teicoplanin (Table 1).

**Table 1.** Susceptibility of vancomycin-resistant enterococci (VRE) to selected antibiotics (percentages).

Legend: AMP—ampicillin, TIG—tigecycline, TET—tetracycline, TEI—teicoplanin, FUR—nitrofurantoin, LNZ—linezolid.

In all isolated VRE, the *vanA* gene was present. Strains with VanB, VanC-1, and VanC2-C3 phenotypes were not detected.

Analysis of the selected virulence factors showed that extracellular surface protein (*esp*) was most frequent. It was present in 84% of isolates, either alone (53%) or in combination with hyaluronidase (*esp* + *hyl*) in 31% of isolates. Hyaluronidase (*hyl*) was found in 40% of isolates (alone in 9%). No virulence factor was present in 7% of isolates.

Based on the pulsed-field gel electrophoresis (PFGE) results, a dendrogram was produced, as shown in Figure 1. Among the 103 VRE, a total of 69 strains were identified and distributed into 18 groups (A–R) based on their similarity (tolerance 1.5; cut-off, 95%). Thus, the overall clonality was 67%. The remaining 34 strains (33%) had unique restriction profiles. Within individual clonal groups, isolates showed 98% similarity. Clone G was the largest cluster, containing 12 strains. Genotypes A, E, F, H, I, J, O, P, and Q were relatively small, all containing two strains. Clones D, N, and R contained three strains each. Five strains in Clone M were isolated from patients staying in the same ward over a period of one month. Analysis of the time–spatial relationships of these patients confirmed transmission of a single VRE clone, but failed to identify the index patient or other source of the outbreak. Clone G consisted of 12 strains isolated from patients over a period of eight months. Clone K consisted of eight strains, all of which were isolated over a period of three months, namely September–November 2017. Similarly, Clone C consisted of eight strains, isolated over four months (September–December 2018). Clone B contained five strains isolated over three months (January–March 2017) and clone L consisted of fours strains obtained over six months in 2018. The timeline of the prevalence of VRE clones throughout the study duration is shown in Figure 2.

*Antibiotics* **2020**, *9*, x FOR PEER REVIEW 4 of 10

**Figure 1.** Pulsed-field gel electrophoresis (PFGE) dendrogram of 103 vancomycin-resistant *E. faecium* isolates. The letters represent 18 clonal groups of VRE (A–R) based on their similarity.

**Figure 2.** Timeline of the prevalence of VRE clones throughout the study duration. The letters represent 18 clonal groups of VRE (A–R) based on their similarity.

#### **3. Discussion**

Infections caused by VRE are often preceded by gastrointestinal colonization with these significant bacterial pathogens. A meta-analysis of 45 studies comprising over 8000 patients with malignancies showed approximately 20% colonization of the GIT with VRE [28]. In a 2019 study on GIT colonization with resistant bacteria in patients with acute myeloid leukemia, Ballo et al. even detected a much higher rate of 74% VRE colonization [29]. In the same year, another study found that VRE-colonized patients were as much as 24 times more likely to develop bloodstream infections caused by these strains [30]. Of importance is also the increase in the prevalence of VRE in the GIT of nursing facility patients from 10% to 29%, with MRSA detection rates declining from 37% to 13% over the 2003–2016 period [31]. In the present study, VRE were detected in the GIT of only 7% of hemato-oncological patients. This may be explained by very low prevalence of VRE (below 1%, unpublished data) in the GIT of people living in the community in the region from which the monitored patients come. Another important reason is the implementation of bacteriological surveillance in hemato-oncological patients including active search for bacterial strains with dangerous resistance phenotypes (e.g., VRE, ESBL-, AmpC- and KPC-positive enterobacteria) in the microflora of the upper respiratory tract and GIT. In the case of positive detection, barrier measures were strictly observed in these patients.

A study by Pudová et al. showed that the GIT is an important source of enterococci causing healthcare-associated pneumonia [6]. If VRE were implicated, the situation would be much more serious due to potential failure of initial antibiotic therapy. Therefore, these dangerous bacteria need to be detected in the GIT of patients, especially immunocompromised ones. All VRE isolated in our study belong to the VanA phenotype of *E. faecium* strains. In a 2006 study conducted in the same department, genetic analysis of VRE revealed that in hemato-oncological patients, the most frequent source was the GIT (55%) and the strains were mostly VanA-positive *E. faecium* (78%) and VanB-positive *E. faecalis* (10%) [19].

In Europe, the most frequent VRE phenotype is VanA [32]. This was confirmed by the results of genetic analysis of VRE in the present study showing that the *vanA* gene was present in 100% of the tested isolates while the *vanB*, *vanC-1*, and *vanC2*/*C3* genes were completely absent. This is consistent with results from a 2018 Serbian study by Jovanovi´c et al. investigating VRE isolated from the oropharynx and stools of hemato-oncological patients. They also showed 100% presence of the *vanA* gene and 0% presence of the *vanB* gene [33].

Virulence factors are likely to play an important role in the etiopathogenesis of enterococcal infections [34,35]. These factors contribute to colonization, biofilm formation, and the production of enzymes potentially increasing the severity of developing infections [7]. In the present study, selected virulence factors were identified in 93% of isolated VRE. The presence of the most frequent virulence factor (*esp*) was lower than that in a similar 2018 study [36]. While Marchi et al. reported 97% prevalence of the *esp* gene in VRE, there were 84% of *esp*-positive VRE in hemato-oncological patients in the present study. Molecular analysis of virulence factors in VRE isolated from oncological patients is available in a 2013 Mexican study. Compared to our study, the *esp* gene was less frequent, being present in 50% of VRE isolates. The *hyl* gene was detected in 17% of cases compared with 40% in the present study. The *esp* + *hyl* gene combination was equally present in both studies, namely in

31% in the present study and in 33% in the study by Ochoa et al. [37]. Comparison of the presence of virulence factors between individual VRE clones did not show significant differences.

Even though VRE are widespread globally, their epidemiology varies by region. The spread of VRE between various hospitals was illustrated by a study from Ireland that had long faced high VRE prevalence rates, reaching approximately 45% [24,38]. Recurring VRE outbreaks remained an unresolved problem in the healthcare system. Only adequately selected molecular typing methods may confirm or rule out epidemiologically related cases. If a new outbreak or merely increased rate of VRE is reasonably suspected, the clonal relationship of strains needs to be analyzed to reveal the source or route of transmission of the infection. In the present study, the clonal relationship of strains was assessed with PFGE. Despite certain limitations, PFGE continues to be considered a gold-standard method as its results may be quantified with high discriminatory ability and reproducibility [39,40]. In the present study, molecular typing results confirmed clonal spread of identical strains in 67% of tested VRE isolates. This confirms the possibility of colonization in staff members and subsequent transmission to hospitalized patients via various environmental vectors. Apparently, the hands of the staff members are the most important one. The literature also mentions wastewater in a commode as an appropriate vehicle for VRE survival, enabling spread in the form of aerosol or by contact with hands or clothes [41]. With regard to the finding of unique profiles in 33% of analyzed VRE, their endogenous origin from the GIT is to be admitted, with the following selection caused by broad-spectrum antibiotic therapy.

Analysis of the restriction profiles of individual clones yielded clonal groups containing two to 12 strains obtained from various patients. The study results suggest that no single epidemic clone was spreading at the hemato-oncology department for a longer period of time and that individual epidemic clones were present for limited time intervals of one to eight months. It can be assumed that the time of occurrence of individual VRE clones was mainly conditioned by the length of hospital stay. There were no changes in the environmental parameters during the study (e.g., remodeling of the rooms or new ventilation).

#### **4. Materials and Methods**

Between 1 January 2016 and 31 December 2018, stool samples and perianal swabs were collected from patients staying at the Department of Hemato-Oncology, University Hospital Olomouc to isolate enterococci using standard procedures. All samples were inoculated onto both blood agar plates and Brilliance VRE Agar chromogenic screening plates (Oxoid, Brno, Czech Republic). Species identification of isolates was carried out with MALDI-TOF MS (Biotyper Microflex, Bruker Daltonics, Billerica, MA, USA). From each patient, only one isolate identified as the first one was included. Strains were stored in cryotubes at −80 ◦C (Cryobank B, ITEST, Hradec Králové, Czech Republic). Stool samples and perianal swabs were collected only as part of standard clinical care.

Susceptibility to antibiotics (ampicillin, tigecycline, tetracycline, teicoplanin, nitrofurantoin, and linezolid) was tested using the microdilution method as recommended by the EUCAST [42]. *Staphylococcus aureus* ATCC 29213 and *Enterococcus faecalis* ATCC 29212 reference strains were used for quality control.

DNA for genetic analysis was isolated with a Qiagen Kit (DNeasy Blood and Tissue Kit, Qiagen, Hilden, Germany). A PCR (polymerase chain reaction) method was used to detect glycopeptide resistance genes (*vanA*, *vanB*, *vanC-1*, *vanC2-C3*) with primers as specified in Table 2 [43]. The amplification conditions were as follows: initial denaturation at 95 ◦C for 7 min, followed by 35 denaturation cycles at 95 ◦C/30 s, annealing at 62 ◦C/30 s, extension at 72 ◦C/60 s, and final extension at 72 ◦C for 7 min. Selected virulence factors were identified with multiplex PCR [44]. The presence of genes encoding gelatinase (*gelE*), aggregation substance (*asa1*), hyaluronidase (*hyl*), cytolysin (*cylA*), and extracellular surface protein (*esp*) was investigated using the primers listed in Table 2. The reaction was run under the following conditions: initial denaturation at 95 ◦C for 15 min, followed by 30 denaturation cycles at 95 ◦C/60 s, annealing at 62 ◦C/60 s, extension at 72 ◦C/60 s, and final extension at 72 ◦C for 10 min. The final products were separated in 1.5% agarose gel at 110 V for 60 min using SYBR Safe DNA Gel Stain (Thermo Fisher Scientific, Waltham, MA, USA). Visualization was carried out under UV light with a UV transilluminator (Transluminator DiscoveryTM, UltraLum, Claremont, CA, USA).

**Table 2.** PCR primers and products for detecting *van* genes and virulence factor genes.

Molecular typing of all isolated VRE was performed using PFGE. Intact DNA was obtained according to a previously described protocol [45]. Restriction cleavage of blocks with DNA was carried out in a *SmaI* restriction enzyme solution (Takara Biotechnology, Kyoto, Japan) containing 5 µL of *SmaI* restriction buffer, 5 µL of 0.1% BSA, 50 µL of deionized water, and 10U of *SmaI*. The blocks were incubated at 30 ◦C overnight. Restriction fragments were separated in 1.2% (*w*/*v*) agarose gel in the CHEF-DRII system (Bio-Rad, Hercules, CA, USA). The parameters selected for analysis were as follows: 24 h, voltage 6 V·cm−1 , and pulse times 2–35 s. The restriction fragments were assessed with GelCompare, version 2.0 (Applied Maths, Kortrijk, Belgium). The similarity coefficient was calculated using the Dice algorithm (based on the Dice coefficient of similarity of macrorestriction profiles set at 2%). Individual clusters were analyzed with the UPGMA (unweighted pair group method with arithmetic mean) algorithm and the results were interpreted using criteria defined by Tenover et al. [46].

#### **5. Conclusions**

The results of PFGE showed no significant outbreak of clonal spread of VRE, but confirmed repeated smaller outbreaks. As seen from the constructed timeline, there was no single hospital strain with an unchanged genetic profile to repeatedly occur over the three-year study period. During that time, a total of 18 clonal groups were identified. In 2016, an identical VRE clone detected in 12 patients persisted for as long as eight months. The year 2017 seems to be most diverse, with only 1-month persistence of a clone replaced by a genetically different clone being noted in four cases. The number of clone groups in the last year was comparable to that in 2017 as it only decreased by one group. However, identical strains persisted for a longer time in various patients and no clone persisted for less than three months. Moreover, the study revealed the presence of genetically unique isolates accounting for 33% of all VRE. One of the risk factors for colonization is the long-term hospital stay of patients sharing the same room combined with the ability of enterococci to persist in the hospital environment [19]. It is those small groups of clones identified in two or three patients in the present study that suggest their spread among patients hospitalized at the same time. The potential routes of transmission are the hands of both patients and healthcare professionals or even family members visiting patients. Considering the selection pressure of antibiotics used, the immune status of hemato-oncological patients and chemotherapy, if administered, the likely source of VRE is their GIT.

**Author Contributions:** Conceptualization, M.K., K.H., and J.B.; Methodology, M.K., K.H., and T.Š.; Validation, T.Š. and P.K.; Investigation, K.H., T.Š., and P.K.; Resources, M.K., K.H., and J.B.; Data curation, K.H.; Writing—original draft preparation, K.H. and M.K.; Writing—review and editing, M.K., K.H., J.B. and K.F.; Visualization, K.H. and K.F.; Supervision, M.K.; Project administration, M.K.; Funding acquisition, J.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Czech Health Research Council (project no. NV18-05-00340).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

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# **Advances and Trends in Mathematical Modelling, Control and Identification of Vibrating Systems**

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