Studies in sesquiterpenes-XVIII. The proton magnetic resonance spectra of some sesquiterpenes and the structure of humulene

The nuclear magnetic resonance spectra of longifolene, zerumbone, humulene, and their hydroderivatives have been studied in order to gauge the potentialities of this new tool in the field of sesquiterpenes. On the basis of present study, it has been possible to unequivocally fix the positions of the ethylene linkages in humulene and thus provide a straightforward solution of this hitherto unsolved problem.

A stable rearrangement product of humulene-4,5-epoxide

Humulene-4,5-monoepoxide, 1, may rearrange to the cyclopropyl diol 2 during chromatography over silica. The rearrangement can be reversed with acid.

Studies in sesquiterpenes-XVI. Zerumbone, a monocyclic sesquiterpene ketone

The structure, previously assigned to zerumbone, has been found to be untenable. The ketone has been shown to be monocyclic containing three ethylenic linkages, and has been further correlated with humulene. Results from ozonolysis, and base-catalysed cleavage allowed the compound to be formulated as 2,6,9,9-tetramethyl-2,6,10-cyclo-undecatrien-1-one.

Caryophyllene-Rich Essential Oil of Didymocarpus tomentosa: Chemical Composition and Cytotoxic Activity

The essential oil from the leaves of Didymocarpus tomentosa was extracted by hydrodistillation and analyzed by GC/FID and GC/MS. Twenty five constituents amounting to 81.6% of the oil were identified. The leaf oil contained 78.7% sesquiterpenes and 2.9% monoterpenes. The leaf essential oil of D. tomentosa is a unique caryophyllene-rich natural source containing beta-caryophyllene, caryophyllene oxide, alpha-humulene and humulene oxide. The cytotoxic activity of the oil was determined by the BSLT using shrimp larva and the MTT assay using HeLa tumor cell line. The oil showed significant cytotoxic activity with LC50 and IC50 values of 12.26 and 11.4 mu g/mL, respectively. This is the first report on the chemical composition and cytotoxic activity of the essential oil of D. tomentosa.

Volatile terpenoids and C13 norisoprenoids in wines: development of rapid methods of analysis and evaluation of the sesquiterpenoids biological potential

Vitis vinifera L., the most widely cultivated fruit crop in the world, was the starting point for the development of this PhD thesis. This subject was exploited following on two actual trends: i) the development of rapid, simple, and high sensitive methodologies with minimal sample handling; and ii) the valuation of natural products as a source of compounds with potential health benefits. The target group of compounds under study were the volatile terpenoids (mono and sesquiterpenoids) and C13 norisoprenoids, since they may present biological impact, either from the sensorial point of view, as regards to the wine aroma, or by the beneficial properties for the human health. Two novel methodologies for quantification of C13 norisoprenoids in wines were developed. The first methodology, a rapid method, was based on the headspace solid-phase microextraction combined with gas chromatography-quadrupole mass spectrometry operating at selected ion monitoring mode (HS-SPME/GC-qMS-SIM), using GC conditions that allowed obtaining a C13 norisoprenoid volatile signature. It does not require any pre-treatment of the sample, and the C13 norisoprenoid composition of the wine was evaluated based on the chromatographic profile and specific m/z fragments, without complete chromatographic separation of its components. The second methodology, used as reference method, was based on the HS-SPME/GC-qMS-SIM, allowing the GC conditions for an adequate chromatographic resolution of wine components. For quantification purposes, external calibration curves were constructed with β-ionone, with regression coefficient (r2) of 0.9968 (RSD 12.51 %) and 0.9940 (RSD of 1.08 %) for the rapid method and for the reference method, respectively. Low detection limits (1.57 and 1.10 μg L-1) were observed. These methodologies were applied to seventeen white and red table wines. Two vitispirane isomers (158-1529 L-1) and 1,1,6-trimethyl-1,2-dihydronaphthalene (TDN) (6.42-39.45 μg L-1) were quantified. The data obtained for vitispirane isomers and TDN using the two methods were highly correlated (r2 of 0.9756 and 0.9630, respectively). A rapid methodology for the establishment of the varietal volatile profile of Vitis vinifera L. cv. 'Fernão-Pires' (FP) white wines by headspace solid-phase microextraction combined with comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry (HS-SPME/GCxGC-TOFMS) was developed. Monovarietal wines from different harvests, Appellations, and producers were analysed. The study was focused on the volatiles that seem to be significant to the varietal character, such as mono and sesquiterpenic compounds, and C13 norisoprenoids. Two-dimensional chromatographic spaces containing the varietal compounds using the m/z fragments 93, 121, 161, 175 and 204 were established as follows: 1tR = 255-575 s, 2tR = 0,424-1,840 s, for monoterpenoids, 1tR = 555-685 s, 2tR = 0,528-0,856 s, for C13 norisoprenoids, and 1tR = 695-950 s, 2tR = 0,520-0,960 s, for sesquiterpenic compounds. For the three chemical groups under study, from a total of 170 compounds, 45 were determined in all wines, allowing defining the "varietal volatile profile" of FP wine. Among these compounds, 15 were detected for the first time in FP wines. This study proposes a HS-SPME/GCxGC-TOFMS based methodology combined with classification-reference sample to be used for rapid assessment of varietal volatile profile of wines. This approach is very useful to eliminate the majority of the non-terpenic and non-C13 norisoprenic compounds, allowing the definition of a two-dimensional chromatographic space containing these compounds, simplifying the data compared to the original data, and reducing the time of analysis. The presence of sesquiterpenic compounds in Vitis vinifera L. related products, to which are assigned several biological properties, prompted us to investigate the antioxidant, antiproliferative and hepatoprotective activities of some sesquiterpenic compounds. Firstly, the antiradical capacity of trans,trans-farnesol, cis-nerolidol, α-humulene and guaiazulene was evaluated using chemical (DPPH• and hydroxyl radicals) and biological (Caco-2 cells) models. Guaiazulene (IC50= 0.73 mM) was the sesquiterpene with higher scavenger capacity against DPPH•, while trans,trans-farnesol (IC50= 1.81 mM) and cis-nerolidol (IC50= 1.48 mM) were more active towards hydroxyl radicals. All compounds, with the exception of α-humulene, at non-cytotoxic levels (≤ 1 mM), were able to protect Caco-2 cells from oxidative stress induced by tert-butyl hydroperoxide. The activity of the compounds under study was also evaluated as antiproliferative agents. Guaiazulene and cis-nerolidol were able to more effectively arrest the cell cycle in the S-phase than trans,trans-farnesol and α-humulene, being the last almost inactive. The relative hepatoprotection effect of fifteen sesquiterpenic compounds, presenting different chemical structures and commonly found in plants and plant-derived foods and beverages, was assessed. Endogenous lipid peroxidation and induced lipid peroxidation with tert-butyl hydroperoxide were evaluated in liver homogenates from Wistar rats. With the exception of α-humulene, all the sesquiterpenic compounds under study (1 mM) were effective in reducing the malonaldehyde levels in both endogenous and induced lipid peroxidation up to 35% and 70%, respectively. The developed 3D-QSAR models, relating the hepatoprotection activity with molecular properties, showed good fit (R2LOO > 0.819) with good prediction power (Q2 > 0.950 and SDEP < 2%) for both models. A network of effects associated with structural and chemical features of sesquiterpenic compounds such as shape, branching, symmetry, and presence of electronegative fragments, can modulate the hepatoprotective activity observed for these compounds. In conclusion, this study allowed the development of rapid and in-depth methods for the assessment of varietal volatile compounds that might have a positive impact on sensorial and health attributes related to Vitis vinifera L. These approaches can be extended to the analysis of other related food matrices, including grapes and musts, among others. In addition, the results of in vitro assays open a perspective for the promising use of the sesquiterpenic compounds, with similar chemical structures such as those studied in the present work, as antioxidants, hepatoprotective and antiproliferative agents, which meets the current challenges related to diseases of modern civilization.

Flavour characterization of fresh and processed pennywort (Centella asiatica L.) juices

The flavour characteristics of fresh and processed pennywort juices treated by pasteurization, sterilization and high pressure processing (HPP) were investigated by using solid-phase micro-extraction combined with gas chromatography-mass spectrometry. Sesquiterpene hydrocarbons comprised the major class of volatile components present and the juices had a characteristic smell due to the presence of volatile compounds including β-caryophyllene, humulene, E-β-farnesene, α-copaene, alloaromadendrene and β-elemene. All processing operations caused a reduction in the total volatile concentration, but HPP caused more volatile acyclic alcohols, aldehydes and oxygenated monoterpenoids to be retained than pasteurization and sterilization. Ketones were not present in fresh pennywort juice, but 2-butanone and 3-nonen-2-one were generated in all processed juices, and 2-nonanone and 2-hexanone were present in pasteurized and sterilized juices. Other chemical changes including isomerization were also reduced by HPP compared to pasteurization, and sterilization.

Flavor characterization of sugar-added pennywort (Centella asiatica L.) juices treated with ultra-high pressure and thermal processes

The flavor characteristics of pennywort juices with added sugar treated by ultra-high pressure, pasteurization, and sterilization were investigated using solid phase microextraction combined with gas chromatography-mass spectrometry. It was found that sesquiterpene hydrocarbons comprised the major class of volatile components present and the juices had a characteristic aroma due to the presence of volatiles including beta-caryophyllene and humulene and alpha-copaene. In comparison with heated juices, HPP-treated samples could retain more volatile compounds such as linalool and geraniol similar to those present in fresh juice, whereas some volatiles such as alpha-terpinene and ketone class were apparently formed by thermal treatment. All processing operations produced juice that was not significantly different in the concentration of total volatiles. Practical Application: Pennywort juice is considered a nutraceutical drink for health benefits. Therefore, to preserve all aroma and active components in this juice, a nonthermal process such as ultra-high pressure should be a more appropriate technique for retention of its nutritive values than pasteurization and sterilization.

Headspace solid-phase microextraction combined with mass spectrometry as a powerful analytical tool for profiling the terpenoid metabolomic pattern of hop-essential oil derived from Saaz variety

Hop(HumuluslupulusL.,Cannabaceaefamily)isprizedforitsessentialoilcontents,usedin beer production and, more recently, in biological and pharmacological applications. In this work,a methodinvolvingheadspace solid-phase microextractionand gas chromatography– mass spectrometry was developed and optimized to establish the terpenoid (monoterpenes and sesquiterpenes) metabolomic pattern of hop-essential oil derived from Saaz variety as a mean to explore this matrix as a powerful biological source for newer, more selective, biodegradable and naturally produced antimicrobial and antioxidant compounds. Different parameters affecting terpenoid metabolites extraction by headspace solid-phase microextraction were considered and optimized: type of fiber coatings, extraction temperature, extraction time, ionic strength, and sample agitation. In the optimized method, analytes were extracted for 30 min at 40 C in the sample headspace with a 50/30 m divinylbenzene/carboxen/polydimethylsiloxane coating fiber. The methodology allowed the identification of a total of 27 terpenoid metabolites, representing 92.5% of the total Saaz hop-essential oil volatile terpenoid composition. The headspace composition was dominated by monoterpenes (56.1%, 13 compounds), sesquiterpenes (34.9%, 10), oxygenated monoterpenes (1.41%, 3), and hemiterpenes (0.04%, 1) some of which can probably contribute to the hop of Saaz variety aroma. Mass spectrometry analysis revealed that the main metabolites are the monoterpene -myrcene (53.0±1.1% of the total volatile fraction), and the cyclic sesquiterpenes, -humulene (16.6 ± 0.8%), and -caryophyllene (14.7 ± 0.4%), which together represent about 80% of the total volatile fraction from the hop-essential oil. Thesefindingssuggestthatthismatrixcanbeexploredasapowerfulbiosourceofterpenoid metabolites.

Essential oil of the Pothomorphe peltata (L.) Miq.

The essential oils of leaves of the Pothomorphe peltata (L.) Miq., collected in vegetative and reproductive periods, showed yields of 0.10-0.20%. Analysis of the oil by GC/MS revealed that the predominant components were those of the sesquiterpene class. The major constituents were beta-caryophyllene (39.5-68.0%), germacrene D (6.2-13.3%), (E)-nerolidol (4.8-11.1%) and alpha-humulene (2.1-6.5%).

Composição fitoquímica dos óleos essenciais de folhas de Lippia alba (Mill.)N.E.Br. em diferentes épocas de colheita e partes do ramo

This work aimed to determine the best harvest time for biomass production, yield and essential oil composition considering the seasonal variation (spring, summer, autumn and winter) on different plant parts (apical, medial and basal). Essential oils were extracted by hydro-distillation with a Clevenger apparatus for both fresh and dry mass obtained in field and lab conditions respectively. The extracted essential oils were analyzed by GS/MS (Shimadzu, QP-5000). The chemical components were identified by comparing their mass spectrum to the patterns filed in the MS computer memory (Wiley,139,Lib.), to the literature references, and by co-injection with authentic standards. Applying phyto-chemical tests on fresh and dry mass, the chemical component percentages of essential oils were calculated and identified as follows: citral (neral and geranial), myrcene, caryophylene and elemene.

Composição do óleo essencial de rubim (Leonurus sibiricus L. - Lamiaceae)

The essential oils are found in a great number of Lamiaceae family species, but few researches were carried out on this subject. This work had as a goal to identify the Leonurus sibiricus L. (rubim) essential oil composition. Rubim leaves and flowers in infusion are able to avoid vomit, diarrhea, and are also indicated in cold, cough, bronchitis and rheumatisms cases. In order to know what are the phytochemical compositions involved, it was used the gas chromatography techniques with mass spectrometry (GC-MS) as methodology. The results showed 70% volatility compound by trans-cariophylene, alpha-humulene and germacrene-D. Other substances like gamacadinene, beta-bourborene and alpha-copaene were found like compounds of this essential oil species.

Determination of disease biomarkers in Eucalyptus by comprehensive two-dimensional gas chromatography and multivariate data analysis

In this paper is reported the use of the chromatographic profiles of volatiles to determine disease markers in plants - in this case, leaves of Eucalyptus globulus contaminated by the necrotroph fungus Teratosphaeria nubilosa. The volatile fraction was isolated by headspace solid phase microextraction (HS-SPME) and analyzed by comprehensive two-dimensional gas chromatography-fast quadrupole mass spectrometry (GC. ×. GC-qMS). For the correlation between the metabolic profile described by the chromatograms and the presence of the infection, unfolded-partial least squares discriminant analysis (U-PLS-DA) with orthogonal signal correction (OSC) were employed. The proposed method was checked to be independent of factors such as the age of the harvested plants. The manipulation of the mathematical model obtained also resulted in graphic representations similar to real chromatograms, which allowed the tentative identification of more than 40 compounds potentially useful as disease biomarkers for this plant/pathogen pair. The proposed methodology can be considered as highly reliable, since the diagnosis is based on the whole chromatographic profile rather than in the detection of a single analyte. © 2013 Elsevier B.V..

Estudo farmacognóstico e avaliação das atividades antibacteriana e imunomoduladora de Melampodium divaricatum (Rich. In Pers) DC. (Asteraceae)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Caracterização da composição química dos óleos essenciais de Lychnophora pinaster Mart. em função da sazonalidade

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Variabilidade genética em uma população de Cordia verbenacea DC. para características agronômicas e fitoquímicas

Pós-graduação em Agronomia (Horticultura) - FCA