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Neuropeltis acuminata (P. Beauv.): Investigation of the Chemical Variability and In Vitro Anti-inflammatory Activity of the Leaf Essential Oil from the Ivorian Species

The variability of chemical composition of the leaf essential oil (EO) from Neuropeltis acuminata, a climbing liana growing wild in Ivory Coast, was investigated for the first time. The in vitro anti-inflammatory activity was also evaluated. Thirty oil samples were isolated from leaves collected in three forests of the country and analyzed using a combination of Column Chromatography (CC), Gas Chromatography with Retention Indices (GC(FID)), Gas Chromatography-Mass Spectrometry (GC-MS), and 13Carbon-Nuclear Magnetic Resonance (13C-NMR). Fractionation by CC led to the first-time isolation from natural source of δ-cadinen-11-ol, whose structural elucidation by one dimension (1D) and 2D-NMR spectroscopy is reported here. Finally, 103 constituents accounting for 95.7 to 99.6% of the samples’ compositions were identified. As significant variations of the major constituents were observed, the 30 oil compositions were submitted to hierarchical cluster and principal components analyses. Five distinct groups were evidenced: Group I, dominated by (E)-β-caryophyllene, kessane, and δ-cadinene, while the main constituents of Group II were germacrene B, ledol, α-humulene, (E)-γ-bisabolen-12-ol, and γ-elemene. Group III exhibited guaiol, germacrene D, atractylone, (E)-γ-bisabolen-12-ol, δ-cadinene and bulnesol as main compounds. Group IV was dominated by (E)-nerolidol, guaiol, selina-4(15),7(11)-diene and bulnesol, whereas (E)-β-caryophyllene, α-humulene and α-muurolene were the prevalent compounds of Group V. As the harvest took place in the same dry season in the three forests, the observed chemical variability could be related to harvest sites, which includes climatic and pedologic factors, although genetic factors could not be excluded. The leaf oil sample S24 behaved as a high inhibitor of LipOXygenase (LOX) activity (half maximum Inhibitory Concentration, IC50: 0.059 ± 0.001 mg mL−1), suggesting an anti-inflammatory potential.

The 8 samples of Group I were harvested from station 2 of the Haut-Sassandra forest, while the 4 samples of Group II were collected in the Bossematié forest. Group III is constituted by the 9 samples from the Yapo-Abbé forest. Samples from Groups IV and V were respectively harvested at stations 3 and 4 of the Haut-Sassandra forest. As the harvest took place in the same dry season in the three forests, the observed chemical variability could be related to harvest sites, which include climatic and pedologic factors, then vegetative stage (young or old lianas), although genetics factors could not be excluded.   [a] Order of elution and percentages on apolar column (BP-1), except components with a hash (#), percentages calculated by combination of GC(FID) and 13

Evaluation of In Vitro Anti-Inflammatory Activity
The in vitro anti-inflammatory activity of N. acuminata leaf EO (S24) was evaluated by the LOX inhibition method. Indeed, LOXs are a nonheme iron-containing dioxygenases, which were responsible for the formation of biologically active metabolites. They were key enzymes in the biosynthesis of leukotrienes that were mediators of many disorders related with inflammatory processes such as arthritis, bronchial asthma, and cancer [33][34][35][36]. The discovery of novel LOX inhibitors appeared as crucial point because they would prevent overproduction of leukotrienes and thus could constituted new therapeutic tools for treating of human inflammation-related diseases.
The inhibition ability of soybean LOX by S24 was measured and considered as an indicator of its potential anti-inflammatory activity. Results of LOX inhibition tests were presented in Table 5. The N. acuminata leaf essential oil inhibited LOX activity and this inhibition increased with the concentration of the oil (15.20% at 0.0125 mg mL −1 up to 81.87% at 0.100 mg mL −1 ). The IC 50 values were calculated for S24 and for inhibitor NorDihydroGuaiaretic Acid (NDGA), a non-competitive inhibitor of lipoxygenase usually used as reference in anti-inflammatory assays (Table 5) [34][35][36]. The IC 50 value of S24 (0.059 ± 0.001 mg mL −1 ) was only 4.5-higher than IC 50 value of NDGA (0.013 ± 0.003 mg mL −1 ). This low ratio between the two IC 50 values (S24 vs. NDGA) allowed to consider the N. acuminata leaf essential oil as a high inhibitor of the LOX activity, suggesting an anti-inflammatory potential [37].

Gas Chromatography
Analyses were performed on a Clarus 500 PerkinElmer Chromatograph (PerkinElmer, Courtaboeuf, France), equipped with flame ionization detector (FID) and two fused-silica capillary columns (50 m × 0.22 mm, film thickness 0.25 µm), BP-1 (polydimethylsiloxane), and BP-20 (polyethylene glycol). The oven temperature was programmed from 60 • C to 220 • C at 2 • C/min and then held isothermal at 220 • C for 20 min; injector temperature: 250 • C; detector temperature: 250 • C; carrier gas: hydrogen (0.8 mL/min); split: 1/60; injected volume: 0.5 µL. Retention indices (RI) were calculated relative to the retention times of a series of n-alkanes (C8-C29) with linear interpolation ("Target Compounds" software from PerkinElmer, Courtaboeuf, France). The quantification of volatile compounds was obtained using Relative Response Factor (RFF), calculated according to the International Organization of the Flavor Industry (IOFI) [32]. The relative proportion of each compound (expressed in g/100 g) was calculated using the amount of EO and reference (Methyl octanoate), peak area and relative response factors.

Nuclear Magnetic Resonance
All spectra were recorded on a Bruker AVANCE 400 Fourier transform spectrometer (Bruker, Wissembourg, France) operating at 400.132 MHz for 1 H and 100.623 MHz for 13 C, equipped with a 5 mm probe. Solvents used were CDCl 3 and C 6 D 6 , with all shifts referred to internal TMS. The 1 H-NMR spectra were recorded with the following parameters: pulse width (PW), 4.3 µs; relaxation delay 1 s and acquisition time 2.6 s for 32 K data table with a spectral width (SW) of 6000 Hz. 13 C-NMR spectra of the oil samples and fractions of CC were recorded with the following parameters: pulse width = 4 µs (flip angle 45 • ); relaxation delay D1 = 0.1 s, acquisition time = 2.7 s for 128 K data table with a spectral width of 25,000 Hz (250 ppm); CPD mode decoupling; digital resolution = 0.183 Hz/pt. The number of accumulated scans was 3000 for each sample or fraction (40 mg, when available, in 0.5 mL of CDCl 3 or C 6 D 6 ). For the 2D spectra, sequences from Bruker Topspin TM (Bruker, Wissembourg, France) library (DEPT, COSY, HMBC and NOESY) and Gradient-enhanced sequences were used. 1D and 2D Spectra were processed via MestreNOVA software (version 12.0.0-20080, Mestrelab, Santiago de Compostela, Spain).

Identification of Individual Components
Identification of the individual components was based on (i) comparison of their GC retention indices on apolar and polar columns, with those of reference compounds [24,38]; (ii) computer search using digital libraries of mass spectral data [38][39][40]; (iii) 13 C-NMR spectroscopy following a computerized method developed in our laboratory using a homemade software by comparison of the chemical shift values in EO or fraction spectrum with those of reference spectra compiled in the laboratory-built library [15,17]. In the investigated samples, individual components were identified by 13 C-NMR at contents as low as 0.4-0.5%. A few compounds were identified by comparison with literature data.

Statistical Analysis
Data of the 30 investigated samples of N. acuminata were submitted to hierarchical cluster analysis (HCA) and principal component analysis (PCA) using XLSTAT software (Addinsoft, Paris, France) [41]. Only constituents in a concentration higher than 1.0% were used as variables for the PCA analysis.

In Vitro Anti-Inflammatory Capacity of Neuropeltis acuminata Leaf Essential Oil
The in vitro anti-inflammatory capacity of N. acuminata leaf EO (S24) was conducted by in vitro lipoxygenase inhibition assay [42][43][44]. Lipoxidase type I-B (Lipoxygenase, LOX, EC 1.13.11.12) from soybean purchased from Sigma-Aldrich (Saint-Quentin-Fallavier, France) was used for the in vitro analysis for LOX inhibitory activity. It was determined by continuously monitoring the formation of conjugated dienes of the 13-hydroperoxides of linoleic acid at 234 nm using a spectrophotometric method [42][43][44].
The LOX solution was prepared by dissolving around 5.7 units mL −1 of LOX in PBS (Phosphate Buffer Solution; 1 unit corresponding to 1 µmol of hydroperoxide formed per min). The S24 sample diluted in dimethyl-sulfoxide (DMSO) was used as inhibitor solution for LOX inhibition activity assay. Five concentrations were tested: 0.0125, 0.0250, 0.0500, 0.0800 and 0.1000 mg/mL.
The LOX inhibition assays were performed as previously described [44]. Briefly, 10 µL of LOX solution and 10 µL of inhibitor solution were mixed in 970 µL of boric acid buffer (50 mM; pH 9.0) and incubating them briefly at room temperature. The enzymatic reaction started by addition of 10 µL of substrate solution (Linoleic acid, 25 mM) and the reaction rate was recorded for 30 s at 234 nm. One measurement was carried out in the absence of inhibitor solution and another with DMSO mixed with distilled water (99.85% DMSO in distilled water) in order to evaluate a possible inhibitory effect of DMSO. LOX activity was not affected by DMSO and the measurement of the LOX activity without inhibitor solution was considered as control (100% enzymatic reaction). All measurements were performed in triplicate. The percentage of LOX inhibition was calculated according to the equation: Inhibition % = (V control − V S24 ) × 100/V control V control is the activity of LOX in absence of inhibitor solution and V S24 is the activity of LOX in presence of inhibitor solution [45]. The IC 50 was calculated by the concentration of S24 in DMSO inhibiting 50% of LOX activity.
N. acuminata leaf EO is a complex mixture characterized by a preeminence of sesquiterpenes (87.1-98.1%) exhibiting various skeletons and a tremendous chemical variability. Contents of heat-sensitive compounds such as germacrene B, furanodiene and their corresponding rearranged products, γ-elemene and curzerene were determined by combination of GC(FID) and 13 C-NMR data. This combination of techniques ensured a correct qualitative and quantitative analysis of the thermolabile compounds.
Statistical analysis exhibited five distinct chemical groups. Groups I, II, and V were characterized by high contents of sesquiterpene hydrocarbons (respectively, M = 58.6, 55.8, and 75.3%, vs. 34.5, 30.7, and 20.6% of oxygenated sesquiterpenes) whereas groups III and IV were dominated by oxygenated sesquiterpenes-rich oils (respectively, M = 49.1 and 67.7%, vs. 42.1 and 27.0% of sesquiterpene hydrocarbons). The observed chemical variability could be related to climatic and pedologic factors.
Concerning the anti-inflammatory activity, the low ratio between the two values of IC 50 (EO vs. NDGA) makes it possible to consider the essential oil as a high inhibitor of the LOX activity.