Amazon Rainforest Hidden Volatiles—Part I: Unveiling New Compounds from Acmella oleracea (L.) R.K. Jansen Essential Oil

Motivated by the culinary and ethnopharmacological use of Acmella oleracea (L.) R.K. Jansen, this study aimed to unveil new chemical compounds from its essential oil (EO). Acmella oleracea, known for its anesthetic and spicy properties, has been used in traditional medicine and cuisine, particularly in Northern Brazil. Through a detailed GC-MS analysis, 180 constituents were identified, including 12 tentatively identified long-chain α-keto esters of various acids. Additionally, 18 new esters were synthesized for structural verification. This research expands the known chemical diversity of A. oleracea EO, providing a basis for potential pharmacological applications. The identification of new natural products, including homologs and analogs of acmellonate, underscores the EO’s rich chemical profile and its potential for novel bioproduct development.


Introduction
The Amazon rainforest, known for its immense biodiversity, harbors countless plant species with significant pharmacological potential [1].One of these species is Acmella oleracea (L.) R.K. Jansen, an annual herb found in the Amazon and other tropical regions, used for its anesthetic and spicy properties in Northern Brazilian cuisine and traditional medicine [2].It has been widely cultivated for medicinal purposes besides horticultural and culinary.This plant's pharmacological properties include but are not limited to, antinociceptive, anti-inflammatory, antioxidant, immunomodulatory, antimicrobial, antiviral, and diuretic activity [1][2][3].The main biological activities of this plant are linked to (2E,6Z,8E)-N-isobutyl-2,6,8-decatrienamide (syn.spilanthol).
In the last decades, the essential oil (EO) of A. oleracea has been gaining more attention regarding its biological properties.Insecticidal activity against the southern house mosquito (Culex quinquefasciatus), the African cotton leafworm (Spodoptera littoralis), and the common housefly (Musca domestica) are of particular interest [3].Despite several studies on its EO, the chemical composition of A. oleracea remains incompletely understood [3][4][5][6][7][8].This study aims to reanalyze the EO composition of A. oleracea to discover new compounds and validate traditional uses.The identification of selected EO constituents required chromatographic fractionation of the full EO, followed by derivatization of the fractions by dimethyl disulfide, synthesis of certain major and minor components, and spectral (IR, MS, 1D-, and 2D-NMR) characterization of the EO fractions and synthesized constituents.By required chromatographic fractionation of the full EO, followed by derivatization of the fractions by dimethyl disulfide, synthesis of certain major and minor components, and spectral (IR, MS, 1D-, and 2D-NMR) characterization of the EO fractions and synthesized constituents.By identifying novel chemical constituents, we aim to contribute to the development of new bioproducts with potential pharmacological applications.
Unfortunately, these constituents could not be isolated from the EO sample due to their low relative abundance and the complexity of the EO.The 'dry-flash' chromatographic separation performed on SiO 2 of an EO portion, aimed to obtain pure EO constituents, resulted in fractions rich with such constituents (Table 1 and Figure S2).Thus, we focused our attention on the first eluting ester-containing fraction (F5 from Table 1), as it contained numerous minor esters that were hardly detectable in the initial GC-MS analyses of the unfractionated EO.The specific keto alcohols (1-hydroxyundecan-2-one, 1-hydroxydodecan-2-one, and 1-hydroxytridecan-2-one) needed to prepare the synthetic samples of esters for a direct comparison were commercially unavailable.For that reason, we followed an approach that included two parts: the synthesis of keto alcohols and the preparation of a small synthetic library of 18 esters (2-methylpropanoates, 2-methylbutanoates, 3-methylbutanoates, angelates, tiglates, and senecioates) via the Steglich procedure (Figure 2).All synthesized compounds represented new compounds at the time of the investigation.The creation of a small synthetic library enabled the identification of 12 new natural products.Notably, the identified esters, such as 2-oxoundecyl and 2-oxotridecyl derivatives, have not been previously reported.These findings enhance our understanding of the EO's chemical profile and suggest potential pharmacological applications, validating its traditional uses.

NMR Spectral Characterization of the New Esters
The obtained esters and starting keto alcohols were subjected, beside MS and IR measurements, to a battery of 1D-( 1 H and 13 C, including 1 H spectra with homonuclear and 13 C spectra without heteronuclear decoupling, as well as DEPT90 and DEPT135) and 2D-(gradient NOESY, HSQC, and HMBC) NMR experiments.The spectral data and assignments are summarized in the Experimental Section and Supplementary Materials (Figures S3-S43), whereas a numbering scheme of C atoms is presented in Figure 3.The assignment of signals is discussed in detail for one of the selected new natural products with the highest relative amount in the EO-2-oxotridecyl senecioate (3f).Both 1 H and 13 C NMR spectra of the esters from the same subfamily of compounds (1a-1f) differed only in the signals of the atoms from the acid moieties, whereas the opposite was true when comparing the spectra of the esters of different alcohols (1-3) and the same acid (e.g., compounds 1f, 2f, and 3f).The 1 H and 13 C NMR spectra of compound 3f contained the expected number of signals (Supplementary Materials Figures S34 and S35).A singlet at 4.66 ppm was assigned to the methylene group at position 1 (Figure 3).The HSQC spectrum (Supplementary Materials Figure S40) enabled the assignation of the 13 C NMR signal of the carbon atom from the same methylene group (C-1) at 67.36 ppm.The HMBC spectrum (Supplementary Materials Figure S41) showed a correlation between C-1 protons and two 13 C NMR signals at 204.85 and 165.59 ppm that were assigned to C-1′ and C-2 carbon atoms from the keto and ester groups, respectively.Additional HMBC correlations of the C-2 carbon atom with a triplet at 2.43 ppm (J = 7.5 Hz), followed by analysis of the HSQC correlations, allowed the assignation of the signals for the C-3 and H-3 atoms at 38.87 ppm and 2.43 ppm, respectively.Using the same approach, based on HSQC, HMBC, DEPT90, and DEPT135 spectra, signals at 1.61 (quintet, J = 7.0 Hz), 1.33-1.21(16H, overlapping peaks), and 0.87 (pseudo triplet, J = 7.0 Hz) were assigned to C-4, C-5-C-12, and C-13 protons, respectively.This approach only allowed additional assignations of the C-4 and C-13 carbon atoms (23.32 and 14.13, respectively) from the alkyl chain, whereas signals at 31.91, 29.60, 29.44, 29.35, 29.34, 29.17

NMR Spectral Characterization of the New Esters
The obtained esters and starting keto alcohols were subjected, beside MS and IR measurements, to a battery of 1D-( 1 H and 13 C, including 1 H spectra with homonuclear and 13 C spectra without heteronuclear decoupling, as well as DEPT90 and DEPT135) and 2D-(gradient NOESY, HSQC, and HMBC) NMR experiments.The spectral data and assignments are summarized in the Experimental Section and Supplementary Materials (Figures S3-S43), whereas a numbering scheme of C atoms is presented in Figure 3.

NMR Spectral Characterization of the New Esters
The obtained esters and starting keto alcohols were subjected, beside MS and IR measurements, to a battery of 1D-( 1 H and 13 C, including 1 H spectra with homonuclear and 13 C spectra without heteronuclear decoupling, as well as DEPT90 and DEPT135) and 2D-(gradient NOESY, HSQC, and HMBC) NMR experiments.The spectral data and assignments are summarized in the Experimental Section and Supplementary Materials (Figures S3-S43), whereas a numbering scheme of C atoms is presented in Figure 3.The assignment of signals is discussed in detail for one of the selected new natural products with the highest relative amount in the EO-2-oxotridecyl senecioate (3f).Both 1 H and 13 C NMR spectra of the esters from the same subfamily of compounds (1a-1f) differed only in the signals of the atoms from the acid moieties, whereas the opposite was true when comparing the spectra of the esters of different alcohols (1-3) and the same acid (e.g., compounds 1f, 2f, and 3f).The 1 H and 13 C NMR spectra of compound 3f contained the expected number of signals (Supplementary Materials Figures S34 and S35).A singlet at 4.66 ppm was assigned to the methylene group at position 1 (Figure 3).The HSQC spectrum (Supplementary Materials Figure S40) enabled the assignation of the 13 C NMR signal of the carbon atom from the same methylene group (C-1) at 67.36 ppm.The HMBC spectrum (Supplementary Materials Figure S41) showed a correlation between C-1 protons and two 13   The assignment of signals is discussed in detail for one of the selected new natural products with the highest relative amount in the EO-2-oxotridecyl senecioate (3f).Both 1 H and 13 C NMR spectra of the esters from the same subfamily of compounds (1a-1f) differed only in the signals of the atoms from the acid moieties, whereas the opposite was true when comparing the spectra of the esters of different alcohols (1-3) and the same acid (e.g., compounds 1f, 2f, and 3f).The 1 H and 13 C NMR spectra of compound 3f contained the expected number of signals (Supplementary Materials Figures S34 and S35).A singlet at 4.66 ppm was assigned to the methylene group at position 1 (Figure 3).The HSQC spectrum (Supplementary Materials Figure S40) enabled the assignation of the 13 C NMR signal of the carbon atom from the same methylene group (C-1) at 67.36 ppm.The HMBC spectrum (Supplementary Materials Figure S41) showed a correlation between C-1 protons and two 13 C NMR signals at 204.85 and 165.59 ppm that were assigned to C-1 ′ and C-2 carbon atoms from the keto and ester groups, respectively.Additional HMBC correlations of the C-2 carbon atom with a triplet at 2.43 ppm (J = 7.5 Hz), followed by analysis of the HSQC correlations, allowed the assignation of the signals for the C-3 and H-3 atoms at 38.87 ppm and 2.43 ppm, respectively.Using the same approach, based on HSQC, HMBC, DEPT90, and DEPT135 spectra, signals at 1.61 (quintet, J = 7.0 Hz), 1.33-1.21(16H, overlapping peaks), and 0.87 (pseudo triplet, J = 7.0 Hz) were assigned to C-4, C-5-C-12, and C-13 protons, respectively.This approach only allowed additional assignations of the C-4 and C-13 carbon atoms (23.32 and 14.13, respectively) from the alkyl chain, whereas signals at 31.91, 29.60, 29.44, 29.35, 29.34, 29.17, and 22.40 ppm originated from C-5-C-12 atoms.Assignation of the acidic part in the 2-oxotridecyl senecioate and other synthesized esters was based on a previous analysis by Radulović et al. [9].
The presence of only one peak before and after DMDS derivatization pointed to a single specific isomer, either (E) or (Z) of the 2-oxoundec-7-en-1-yl, 2-oxotridec-6-en-1-yl, and 2-oxotridec-7-en-1-yl esters [12].In analogy with the double-bond configuration of The observed correlation of RI data presented in Figure 5 also suggested that the remaining detected ester was (7Z,9E)-2-oxoundeca-7,9-dien-1-yl 2-methylpropanoate (RI = 1815).At the time of the investigation, except for acmellonate, all other identified esters of the diastereoisomeric 2-oxoundeca-7,9-dien-1-ols (12 compounds) represented new compounds and newly identified natural products (Figure 6).The observed correlation of RI data presented in Figure 5 also suggested that the remaining detected ester was (7Z,9E)-2-oxoundeca-7,9-dien-1-yl 2-methylpropanoate (RI = 1815).At the time of the investigation, except for acmellonate, all other identified esters of the diastereoisomeric 2-oxoundeca-7,9-dien-1-ols (12 compounds) represented new compounds and newly identified natural products (Figure 6).A GC-MS analysis of the essential oil and essential-oil fraction 'rich' with esters before and after derivatization with DMDS revealed the presence of an additional group of related constituents that eluted slightly faster compared to the n-chain saturated keto esters.Reaction with DMDS implied the existence of a non-conjugated double-bond somewhere in the keto alcohol moiety.For instance, the peak at 27.30 min with the molecular ion at m/z 266 and typical MS fragmentation of 2-pentenoates (Figure S47), eluting 8 RI units faster than 2-oxoundecyl senecioate, was tentatively identified as a 2-oxoundecen-1yl senecioate.Additionally, the appearance of DMDS adducts in the chromatogram of the derivatized fraction F5 (Figure S48), having an appropriate molecular weight (at m/z 360, which comes from 266 + 94), confirmed our assumption.The MS of the DMDS adduct displayed a fragment ion at m/z 103 (CH3CH2CH2CHSCH3 + ) that implied the position of the double-bond at position 7 (Figure S48), i.e., 2-oxoundec-7-en-1-yl senecioate.Since other double-bond positions would give rise to a specific fragment ion at m/z CH3(CH2)nCHSCH3, we inspected PICs of the derivatized fractions for their presence (Figure S49) and found peaks of ten additional DMDS adducts of esters of 2-oxoundec-7-en-1-ol, 2-oxotridec-6-en-1-ol, and 2-oxotridec-7-en-1-ol.All of the mentioned esters represented new natural products as well as new compounds in general.
The presence of only one peak before and after DMDS derivatization pointed to a single specific isomer, either (E) or (Z) of the 2-oxoundec-7-en-1-yl, 2-oxotridec-6-en-1-yl, and 2-oxotridec-7-en-1-yl esters [12].In analogy with the double-bond configuration of A GC-MS analysis of the essential oil and essential-oil fraction 'rich' with esters before and after derivatization with DMDS revealed the presence of an additional group of related constituents that eluted slightly faster compared to the n-chain saturated keto esters.Reaction with DMDS implied the existence of a non-conjugated double-bond somewhere in the keto alcohol moiety.For instance, the peak at 27.30 min with the molecular ion at m/z 266 and typical MS fragmentation of 2-pentenoates (Figure S47), eluting 8 RI units faster than 2-oxoundecyl senecioate, was tentatively identified as a 2-oxoundecen-1-yl senecioate.Additionally, the appearance of DMDS adducts in the chromatogram of the derivatized fraction F5 (Figure S48), having an appropriate molecular weight (at m/z 360, which comes from 266 + 94), confirmed our assumption.The MS of the DMDS adduct displayed a fragment ion at m/z 103 (CH 3 CH 2 CH 2 CHSCH 3 + ) that implied the position of the doublebond at position 7 (Figure S48), i.e., 2-oxoundec-7-en-1-yl senecioate.Since other doublebond positions would give rise to a specific fragment ion at m/z CH 3 (CH 2 ) n CHSCH 3 , we inspected PICs of the derivatized fractions for their presence (Figure S49) and found peaks of ten additional DMDS adducts of esters of 2-oxoundec-7-en-1-ol, 2-oxotridec-6-en-1-ol, and 2-oxotridec-7-en-1-ol.All of the mentioned esters represented new natural products as well as new compounds in general.The presence of only one peak before and after DMDS derivatization pointed to a single specific isomer, either (E) or (Z) of the 2-oxoundec-7-en-1-yl, 2-oxotridec-6-en-1-yl, and 2-oxotridec-7-en-1-yl esters [12].In analogy with the double-bond configuration of one of the major EO constituents, acmellonate, we expected Z configuration at position 7.The difference in the RI values for the internal alkenes with the different positions of the double-bond and the same configuration (e.g., (6Z)-tetradec-6-ene and (7Z)-tetradec-7-ene) was ca. 2 RI units, whereas the difference between alkenes with the same position of the double-bond but with the different configurations of the double-bond was higher than 5 units (e.g., difference in the RI data for (6Z)-tetradec-6-ene and (6E)-tetradec-6-ene is 7 units) [13].The difference in the RI data for the detected 2-oxotridec-7-en-1-yl and 2oxotridec-6-en-1-yl esters suggested different configurations of the double-bond in position 6 compared to the counterparts with the double-bond in position 7.However, the exact double-bond configuration requires additional research that includes synthesis and spectral characterization of stereoisomerically pure compounds.
An additional group of EO constituents that caught our attention was a series of compounds with a similar mass spectral fragmentation pattern, one of them an already well-known natural product, (2E,6Z,8E)-N-isobutyldeca-2,6,8-trienamide (syn.spilanthol) [3].Chromatographic separation of the EO sample yielded a polar fraction (F7 from Table 1), containing more than 50% of spilanthol and related compounds.A comparison of the obtained NMR data of fraction F7 (Figures S51 and S52) with the literature data confirmed the identity of spilanthol [14,15].The specific MS fragmentation pattern of the four additionally detected constituents (base peak at m/z 81 and intense peaks at 141, 126, and 98) indicated analogous unsaturated amides [3].Unfortunately, based on the differences in their RI values and the mentioned similarity in the MS fragmentation pattern (Figure S50), we can only conclude that the detected constituents represent regio-isomers and/or stereoisomers of N-isobutyldeca-2,6,8-trienamide.Some of the identified isomers were already detected as constituents of A. oleracea ethanolic extract, however, without determining the configurations of the double-bonds [16].
The generated PIC chromatogram of fraction F7 that showed changes in the m/z 81 ion current allowed us to detect six spilanthol-related amides.The presence of the ion peak at m/z 155 (C 9 H 17 NO + ), compared to m/z 141 (C 8 H 15 NO + ) from spilanthol, suggested that the detected constituents could be isomeric N-pentyl amides of spilanthic acid (Figure S53).A comparison of the RI data of the detected constituents and ones for spilanthol suggested that one of the constituents, at RI = 2014 (Table 1), represents (2E,6Z,8E)-N-(2-methylbutyl)deca-2,6,8-trienamide (syn.homospilanthol) [17], whereas other constituents could represent regio-isomers and/or stereoisomers of homospilanthol.The identification of acmellonate analogs and spilanthol isomers further highlights the EO's complexity and potential for developing new insecticidal and antimicrobial agents.

Plant Material
Leaves and inflorescences of Acmella oleracea (L.) R. K. Jansen were collected in April 2019 from the district of Fazendinha (S 0 • 02 ′ 30.40 ′′ /W 5106 ′ 37.5 ′′ ), Macapá City, Amapá State, Brazil.A voucher specimen was deposited in the Herbarium of the Institute of Scientific and Technological Research of Amapá-IEPA under the identification number HAMAB-020058.The identity of the plant material was confirmed by a trained botanist, the custodian of the mentioned herbarium.The request for permission to access the material was registered with the Genetic Heritage Management Council (SISGEN) under the number A7C63F0.

Hydro-Distillation and Chromatographic Fractionation of A. oleracea Essential Oil
The fresh leaves and inflorescences (three batches, ca.300 g each) were crushed and submitted to hydro-distillation in a modified Clevenger-type apparatus for 2 h with the addition of 5 mL of hexane.The obtained EO were separated by extraction and dried with anhydrous sodium sulfate.The solvent was evaporated under a gentle stream of nitrogen at room temperature and stored at around 8 • C before analysis by GC-MS.A portion of the EO (300 mg) was subjected to dry-flash chromatography, resulting in 7 different fractions in total (the mass of the fractions was 24, 41, 33, 1, 5, 26, and 30 mg).A gradient of hexane-diethyl ether, from 100:0 to 0:100 (v/v), was employed for the chromatography, and the mentioned fractions were immediately analyzed by GC and GC-MS upon solvent removal in vacuo.

Component Identification
Essential oil constituents were identified by comparison of their linear retention indices (relative to the mentioned homologous series of n-alkanes on a DB-5MS column) with literature values, their mass spectra with those of authentic standards, as well as those from Wiley 11, NIST17, MassFinder 2.3, and a homemade MS library, with the spectra corresponding to pure substances, NMR analysis of isolated compounds, and wherever possible, by co-injection with an authentic sample.Additionally, a sample of the selected chromatographic fraction was subjected to derivatization reactions with dimethyl disulfide (DMDS), described in detail below, and afterwards to additional GC-MS analyses.

Gas Chromatography-Mass Spectrometry (GC-MS) Analyses
The GC-MS analyses (three repetitions) of the obtained samples were carried out using a Hewlett-Packard 6890N gas chromatograph equipped with a fused-silica capillary column (DB-5MS (5% diphenylpolysiloxane and 95% dimethylpolysiloxane, 30 m × 0.25 mm, film thickness 0.25 µm) and coupled with a 5975B mass-selective detector from the same company.The injector and interface were operated at 250 • C and 300 • C or 320 • C, respectively.Two temperature programs were used.Program 1 was used for the EO sample and EO fractions: the oven temperature was raised from 70 • C to 290 • C at a heating rate of 5 • C/min, and the program ended with an isothermal period of 10 min.Program 2 was for DMDS derivatized samples: oven temperature was raised from 70 to 315 • C at a heating rate of 5 • C/min, and then isothermally held for 30 min.As a carrier, gas helium at 1.0 mL/min was used.The samples, 1.0 µL of the Et 2 O solutions of the esters, were injected in a pulsed split mode (the flow was 1.5 mL/min for the first 0.5 min and then set to 1.0 mL/min throughout the remainder of the analysis; split ratio 40:1).MS conditions were as follows: ionization voltage of 70 eV, acquisition mass range of 35-650, and scan time of 0.32 s.The linear retention indices were determined relative to the retention times of C 7 -C 33 n-alkanes [18].

NMR Measurements
The 1 H (including 1 H NMR-selective homonuclear decoupling experiments), 13 C (with and without heteronuclear decoupling) nuclear magnetic resonance (NMR) spectra, distortion less enhancement by polarization transfer (DEPT90 and DEPT135), and 2D (NOESY, and gradient 1 H-1 H COSY, HSQC, and HMBC) NMR spectra were recorded on a Bruker Avance III 400 MHz NMR spectrometer (Fällanden, Switzerland; 1 H at 400 MHz, 13 C at 101 MHz) equipped with a 5-11 mm dual 13 C/ 1 H probe head.All NMR spectra were measured at 25 • C in CDCl 3 with tetramethylsilane (TMS) as an internal standard.Chemical shifts were reported in ppm (δ) and referenced to TMS (δ H = 0 ppm) in 1 H NMR spectra and/or to solvent protons (deuterated chloroform: δ H = 7.26 ppm and δ C = 77.16ppm) in 13 C and heteronuclear 2D spectra.The samples were dissolved in 1 mL of the solvent, and 0.7 mL of the solutions were transferred into a 5 mm Wilmad, 528-TR-7 NMR tube.The acquired NMR experiments, both 1D and 2D, were recorded using standard Bruker built-in pulse sequences.

Synthesis of 1-hydroxyundecan-2-one, 1-hydroxydodecan-2-one, and 1-hydroxytridecan-2-one
A solution of diisopropylamine (8.33 mmol; 1.1 eq) in freshly dried THF (10 mL) was vigorously stirred under nitrogen in the bath with the cooled acetone, and n-BuLi (3.60 mL) was added dropwise through the septum and left for 10 min at −78 • C and an additional 10 min at 0 • C.Then, a solution of the 2-tridecanone (7.57mmol; 1.5 g) in the THF (10 mL) was added dropwise through the septum, and 10 min later, trimethylsilyl chloride (1.05 mL; 1.1 eq) and stirred at ambient temperature overnight.The solvent was evaporated, and the remaining slurry was diluted in CH 2 Cl 2 (10 mL), and m-CPBA (2.05 g in 10 mL CH 2 Cl 2 ) was added dropwise and stirred at ambient temperature overnight.A solution of the sulfuric acid at 15 mL (10%, v/v) was added and stirred for 2 h.The organic layers were separated, dried over anhydrous MgSO 4 , and concentrated under reduced pressure to yield a crude mixture that was further fractionated by dry-flash chromatography on SiO 2 , using mixtures of the increasing polarity of hexane and Et 2 O as the eluent to yield pure 1-hydroxytridecan-2-one (1.03 g).Using the same synthetic procedure, 2-undecanone and 2-dodecanone, 1-hydroxyundecan-2-one, and 1-hydroxydodecan-2-one were synthesized.The spectral data (NMR and MS) and assignments of 1 H and 13 C signals for the synthesized compounds are provided below and in the Supplementary Materials (Figures S3-S43).

Dimethyl Disulfide (DMDS) Derivatization
A portion of the EO fraction was dissolved in DMDS (0.25 mL per mg of the sample), and a solution (0.05 mL per mg of the sample) of iodine in diethyl ether (60 mg/mL) was added.The mixture was stirred at room temperature overnight.Then, diethyl ether was added, and the obtained mixture was washed with 10% aq.Na 2 S 2 O 3 , dried over anhydrous MgSO 4 , and evaporated to dryness.The residue was taken up in Et 2 O and directly analyzed by GC-MS.

Conclusions
This study successfully identified 180 constituents in the EO of Acmella oleracea, including 12 new natural products and various acmellonate analogs.These findings not only enhanced our understanding of the chemical diversity of A. oleracea EO but also underscored its potential for pharmacological applications.The identification of new esters and spilanthol isomers suggests potential for developing novel bioproducts, aligning with traditional uses and paving the way for future research in drug development.
, and 22.40 ppm originated from C-5-C-12 atoms.Assignation of the acidic part in the 2-oxotridecyl senecioate and other synthesized esters was based on a previous analysis by Radulović et al. [9].2.3.Identification of the Isomers/Homologs/Analogs of Acmellonate
C NMR signals at 204.85 and 165.59 ppm that were assigned to C-1′ and C-2 carbon atoms from the keto and ester groups, respectively.Additional HMBC correlations of the C-2 carbon atom with a triplet at 2.43 ppm (J = 7.5 Hz), followed by analysis of the HSQC correlations, allowed the assignation of the signals for the C-3 and H-3 atoms at 38.87 ppm and 2.43 ppm, respectively.Using the same approach, based on HSQC, HMBC, DEPT90, and DEPT135 spectra, signals at 1.61 (quintet, J = 7.0 Hz), 1.33-1.21(16H, overlapping peaks), and 0.87 (pseudo triplet, J = 7.0 Hz) were assigned to C-4, C-5-C-12, and C-13 protons, respectively.This approach only allowed additional assignations of the C-4 and C-13 carbon atoms (23.32 and 14.13, respectively) from the alkyl chain, whereas signals at 31.91, 29.60, 29.44, 29.35, 29.34, 29.17, and 22.40 ppm originated from C-5-C-12 atoms.Assignation of the acidic part in the 2-oxotridecyl senecioate and other synthesized esters was based on a previous analysis by Radulović et al. [9].2.3.Identification of the Isomers/Homologs/Analogs of Acmellonate

Table 1 .
Chemical composition of the essential oil and essential oil fractions of Acmella oleracea (L.) R.K. Jansen from Pará-Brazil.