Aerial parts of Hysterionica pinifolia were collected in Tandil, Buenos Aires province (west 59.1369, south 37.3286), Argentina, in March 2016 and were authenticated by Dr. Gustavo Giberti. A voucher specimen (BAF 858) was deposited in the Pharmacobotanical Museum of the Faculty of Pharmacy and Biochemistry, University of Buenos Aires, Argentina. The material was dried at room temperature and finely powdered.

The powered aerial parts (100 g) were extracted at room temperature by maceration using CH₂Cl₂ (1000 ml) for 1 h under continuous shaking. This procedure was repeated 5 times changing the solvent each time. These extracts were gathered. The plant residue was then extracted using the same volume of CH₃OH. After evaporation of the solvent, 4.6 and 8.1 g of each extract were obtained respectively.

A portion of the active CH₂Cl₂ extract (2.0 g) was fractionated by a preparative chromatography column (60 cm × 5 cm) using silica gel 60 MN (70 - 230 mesh, Macherey-Nagel, Germany), eluted with a gradient mixture of hexane (Hx)/ethyl acetate (EtOAc) (100:0 to 50:50, v/v), then EtOAc/CH₃OH (100:0 to 0:100, v/v). The chromatographic analyses were made by Thin Layer Chromatography (TLC) on silica gel F254 (MERCK-Germany), developed by different solvent systems and the spots were visualized under UV light at 254 and 366 nm. Four fractions (F1 to F4) were collected based on their TLC profiles. The insecticidal activity was found to be concentrated only in F2 (Table 4). Then, F2 (313 mg) was rechromatographed on an analytical column (45 cm × 3 cm) using silica gel 60 MN (70 - 230 mesh, Macherey-Nagel, Germany) and was eluted with gradient mixtures of Hx/ethyl ether (Et₂O) (100:0 to 50:50, v/v), then Et₂O/CH₃OH (100:0 to 0:100, v/v). Fifteen subfractions (F2-A to F2-O) were obtained. After the TLC analysis, the cromatographic profile of the subfractions F2-C and F2-E showed the main compounds present in the active fraction F2. For this reason, the subfractions F2-E and F2-C were selected for the identification of their bioactive compounds.

Volatile fraction was obtained by hydrodistillation of naturally air dried aerial parts during 3 h in a Clevenger-type trap [8]. The resulting volatile fraction was dried over anhydrous sodium sulfate and stored at 2˚C prior to GC-FID-MS analysis

The identification of the compounds in the subfractions F2-E and F2-C was conducted by GC-FID-MS, ¹H and ¹³C NMR 1D and 2D and MS-ESI. The identification of unknown compounds in complex mixtures using the combination of 1D and 2D NMR and MS, without the need for prior isolation, allows rapid identification [9] [10].

In order to identify the bioactive compounds, the subfractions F2-E and F2-C from the active CH₂Cl₂ extract of H. pinifolia were analyzed. The analysis was conducted by GC-FID-MS, ¹H and ¹³C NMR 1D and 2D experiments (COSY, TOCSY, HSQC, HMBC) and MS-ESI. The compounds were identified in the subfractions using the combination of 1D and 2D NMR and MS, without the need of prior isolation, wich allowed to a rapid identification [9] [10].

A new acetylenic alcohol was identified in subfraction F2-E. The molecular formula was assigned as C₁₁H₁₄O₁ on the basis of its MS-ESI analysis, which showed a protonated molecular ion peak at m/z 145.10 [M + H]⁺ (calcd for C₁₁H₁₄O₁, 162.23) (FigureS1). This molecular ion was obtained by dehydration of the alcohol functional group under electrospray ionization conditions [19]. In the ¹³C-NMR spectrum the signal of a carbon linked to a hydroxyl group at δ_C 72.11 ppm, the signals of alkenes carbons at δ_C 148.47 and 108.83 ppm and the signals of alkyl carbons at δ_C 75.02, 72.99, 65.19 and 84.42 ppm were observed. Aliphatic carbon signals δ_C 36.87, 21.52, 21.75 and 13.41 ppm were also observed (FigureS2). The ¹H-NMR spectrum showed signals of protons from alkenes with trans isomerism at δ_H 6.28 and 6.19 ppm (J = 15.9 Hz) and signals from protons bound to aliphatic carbons at δ_H 4.19, 2.32, 1.60, 1.56 and 1.02 ppm (FigureS3). The ¹H and ¹³C NMR spectra showed the signals corresponding to an acetylene skeleton. These compounds are characterized by the presence of two or more triple bonds [20]. With the aid of ¹H-¹H COSY, TOCSY, HSQC and HMBC spectroscopic data, all protons and carbons were fully assigned (Table 1, Figures S4-S7). Analysis of the ¹H-¹H COSY and TOCSY spectrum allowed to determine the connection between the protons and led to the establishment of two spin systems (H-1 to H2/H3/H4 and H9 to H10/H11) (Figure1). Furthermore, the HMBC correlations between C1/H3 and H4; C3/H1; C4/H1; C5/H4 and H9; C6/H3, H4 and H9; C7/H9; C8/H9 and H10; C9/H10 and H11; C10/H9 and H11 and C11/H9 and H10, confirm the structure of this new polyacetylene alcohol as (E)-undec-3-en-5,7-diyne-1-ol (1). This compound is reported for the first time in H. pinifolia and no reports have been found in other species.

The major compound, cis lachnophyllum methyl ester (2), was identified in subfraction F2-C by GC-FID-MS (63.0%, rt: 45.45 min) and ¹H and ¹³C NMR 1D and 2D (Table 2, Figures S8-S11). In the ¹³C-NMR spectrum, signals of a carbonyl carbon at δ_C 166.8 ppm and of a methyl carbon at δ_C 51.6 ppm were observed, indicating the presence of an ester group. The alkene carbon signals at δ_C 130.7 and 122.5 ppm and the alkyl carbon signals δ_C70.8, 86.6, 65.2 and 90.1 ppm indicated the presence of one double bond and two triple bonds in the molecule. Signals of aliphatic carbons δ_C 21.60, 21.79 and 13.5 ppm were also observed (FigureS8). The ¹H-NMR spectrum showed signals of protons of alkenes with cis isomerism at δ_H 6.23 and 6.19 ppm (J = 11.4 Hz), a singlet at δ_H 3.80 ppm corresponding to the methyl protons of the ester group and the signals of the protons linked to aliphatic carbons at δ_H 2.37, 1.61 and 1.03 ppm (FigureS9). Like in compound 1 the ¹H and ¹³C NMR spectra showed the signals corresponding to an acetylene skeleton. Additionally, the heteronuclear correlation spectra HSQC (FigureS10) and HMBC allowed to fully assign the protons and carbons in the molecule (Table 2, Figure1). The HMBC correlations between C1/H2, H3 and H11; C3/H2; C4/H2, H3 and H8; C5/H2 and H8; C6/H2, H3 and H8; C7/H8 and H9; C8/H9 and H10; C9/H8 and H10 and C10/H8 and H9 (FigureS11) confirm the structure of this polyacetylene ester as cis lachnophyllum methyl ester. The results of NMR analysis agreed with the data described in the literature [21]. This compound was previously described by Bohlmann et al. [2] for this species.

The compounds eucalyptol (3), benzoic aldehyde (4) and benzyl alcohol (5) were identified in fractions F2-E and F2-C by GC-FID-MS. Eucalyptol (rt: 6.51 min) 3.50% in F2-C and 4.90% in F2-E, benzoic aldehyde (rt: 14.2 min) 4.90% in F2-C and 30.0% in F2-E and benzyl alcohol (rt: 28.69 min) 2.50% in F2-C and 13.5 % in F2-E. These compounds are reported for first time in H. pinifolia.

^a Data recorded at 125 MHz. ^bData recorded at 600 MHz.

^a Data recorded at 125 MHz. ^bData recorded at 600 MHz.

Acetylenic compounds have also been found in essential oils [22]. For this reason, the volatile fraction of H. pinifolia was obtained and analyzed. Forty-six components were identified by GC-FID-MS and these constituted the 84.2% of the volatile fraction.

The principal compounds identified were cis lachnophyllum methyl ester (38.4%) as the major constituent, trans-β-ocimene (17.5%), γ-curcumene (12.5%), ar-curmumene (4.5%), β-caryophyllene (1.9%) and geranylacetone (1.3%) (Table 3).

The insecticidal activity was evaluated on T. castaneum larvae from a laboratory strain susceptible to all insecticides. The CH₂Cl₂ extract exhibited only larval mortality with EC₅₀ value of 15.7 mg/ml (CI = 14.9 - 16.6 mg/ml) and no sublethal effects were observed (Table 4). These promising results led to a bioguided fractionation in order to identify the active compounds present in the CH₂Cl₂ extract. For that reason, the activity of the four fractions (F1 to F4) of the CH₂Cl₂ extract was evaluated. Only the fraction F2 showed lethal effects with 95% of larval mortality at 25.0 mg/ml and no sublethal effects were observed (Table 4). The mortality observed in F2 fraction was comparable to that obtained with the crude CH₂Cl₂ extract (92.5%) at the same concentration of 25.0 mg/mL (Table 4). These results showed that the insecticidal activity of the crude CH₂Cl₂ extract would be concentrated in the F2 fraction. The CH₃OH extract did not present lethal or sublethal effects (Table 4).

The high larval mortality obtained with the CH₂Cl₂ extract and the fraction F2 was very interesting due to the extensive losses in stored grains caused by the voracity of T. castaneum larvae in their different instars [23]. In addition, adults of T. castaneum also cause an important damage. Therefore, the insecticidal activity of the CH₂Cl₂ extract on T. castaneum adults was evaluated. Mortality and immobility were initially observed and, at the end of the trial, 100% mortality only at 50.0 mg/ml was obtained. In the control group, no mobility alteration or mortality was exhibited.

T: trace, less than 0.05%. ^aLinear retention index on non polar column, experimentally determined using homologous series of C8-C24 alkanes. ^bLinear retention index on polar column experimentally determined using homologous series of C8-C24 alkanes.

Different letters indicate significant differences (p < 0.05).

The insecticidal activity showed with the CH₂Cl₂ extract and its F2 fraction could be attributed to the identified compounds. The major compound cis lachnophyllum methyl ester has reported larvicidal, nematicidal, and antifeedant activities on various pests [24] [25] [26] as well as other acetylene alcohols [27]. Additionally, the compounds eucalyptol, benzoic aldehyde, and benzyl alcohol have also reported insecticidal activity [28] [29] [30]. The cis lachnophyllum methyl ester was also the major compound in the volatile fraction. Therefore it is expected that this volatile fraction presents insecticidal activity such as the CH₂Cl₂ extract and the F2 fraction.