Volatile Metabolites of Piper eriopodon (Miq.) C.DC. from Northern Region of Colombia and Assessment of In Vitro Bioactivities of the Leaf Essential Oil

Piper eriopodon is one of the Piper species found in the Sierra Nevada de Santa Marta, and the species has been reported with different compositions of their essential oils (EO). In this study, the volatile fractions/essential oil (by HS-SPME/SDE/MWHD-GC–MS/1H-NMR) of different parts from the plant were characterized, and assessments of the in vitro bio-properties of the leaf EO were conducted. The results indicated the following: (i) in the volatile fractions were β-caryophyllene (~23%)/myrcene (~20%) (inflorescences) and β-caryophyllene (~43%)/β-selinene (~20%) (leaves) using HS-SPME; myrcene (~31%)/β-pinene (~23%) (inflorescences), gibbilimbol B (~60%) (fruits) and gibbilimbol B (~46%)/β-caryophyllene (~11%) (leaves) through SDE; (ii) leaf EO contained gibbilimbol B (~72%), confirmed with 1H-NMR; (iii) the cytotoxic values (µg/mL) in erythrocytes/lymphocytes/Hep-2 were HC50: 115 ± 3 (eryth.), LC50: 71 ± 4 (lymph.) and LC50: 33 ± 2 (cell-line); (iv) the antibacterial susceptibilities (ϕ inh. zone, mm; 4–16 µg EO) were 22.5 ± 0.4–97 ± 4 (Staphylococcus aureus), 23 ± 2–77 ± 4 (Escherichia coli) and 17 ± 1–48 ± 3 (Listeria monocytogenes); (v) the TAA value was 2249 ± 130 mmol Trolox®/kg; (vi) the IC50 value was 13±1 µg/mL (AChE) with 20 ± 0–37 ± 6% repellency (2–4 h, Sitophilus zeamais). Thus, the EO of P. eriopodon leaves from northern Colombia could be a promising species for sustainable exploitation in the future due to its outstanding bioactivities.


Introduction
Colombia is the second richest country in the world in plant biodiversity (20,299 species), and registers 604 species of Piperaceae [1][2][3]. From this number, ca. 50 Piper spp. (four endemic and the others also found/disseminated in the rest of the Caribbean region and the Northern Andes) are distributed in the Sierra Nevada de Santa Marta (northern region of Colombia) [4].
One of them, Piper eriopodon (Miq) C.DC. (syn. Artanthe eriopoda Miq., P. leptophyllum C. DC.; common name: "cordoncillo") is a shrub (up to 4 m high) with erect inflorescences (length between 7-9 cm) and leaves that are scaly to the touch with a characteristic odor; it is native to Colombia, Venezuela and Ecuador [5][6][7]. However, there is little information in Colombia on traditional uses of the plant; even so, Saavedra Barrera [7] stated that the plant has analgesic, diuretic and antirheumatic properties, and is used as a treatment for kidney stones, bronchial conditions and as an antidote against snake bites. In the northern Colombian region, it is considered a weed.

Identity of Plant
The botanical sample was identified as Piper eriopodon (Miq.) C.DC., and the leaf EO was a yellowish liquid (at room temperature) which became solid at 4 • C; the EO yield was 0.08%.
As an additional process to corroborate the presence of gibbilimbol B and other terpene constituents in the P. eriopodon leaves and inflorescences, ethyl acetate extracts were obtained from these parts and analyzed using GC-MS; the chemical compositions are included in Table 1. Accordingly, gibbilimbol B (~70%) and β-caryophyllene (~7%) were the majority constituents found in the inflorescence extract (composition similar to leaf EO), while β-caryophyllene (~19%), phytol (~11%) and gibbilimbol B/β-selinene (~10% each) were for the leaf extract.   Otherwise, considering the biological prospects of the EO, the results obtained were reported in Table 2. According to the table, the cytotoxicity on erythrocytes, reported as the 50% hemolytic concentration (CH50-as a measure (approximation) of the skin-irritant capacity of a substance), established that the EO was moderately hemolytic (100 µg/mL < HC50 < 1000 µg/mL); whereas, the EO was cytotoxic (LC50 < 100 µg/mL) against lymphocytes/Hep-2 line, being more effective on the cell line than on lymphocytes, and showing a selectivity index of 2.2 on the Hep-2 cells. As per the F test, there were significant differences between each of the control substances and the EO for the tested cells (p ≤ 0.0001), as well as amongst the cell types.
A remarkable bioproperty demonstrated by P. eriopodon EO was its high efficacy for inhibiting bacterial growth. Thereby, the three tested strains (S. aureus, E. coli and L. monocytogenes) were susceptible to the different evaluated amounts (4-16 µg) of the EO. The effect of the tested amount (µg) of EO on the inhibition of radial growth for each bacterial species is shown in Figure 1, and from this, it could be observed that the effect on the inhibition of bacterial growth (for all of them) was dose dependent in an exponential mode. It is worth highlighting that the lowest amount (4 µg) of tested EO was capable of inhibiting equal to or higher than the positive control (ϕ inh. zone of EO ≥ ϕ inh. zone of control antibiotic). Nonetheless, the descending order of bacterial susceptibility (from highest to lowest) towards the EO was S. aureus > E. coli > L. monocytogenes. The Anova results for these data showed that the general effect of EO on the evaluated bacterial strains was similar [there were no significant differences, [p: 0.2357, F (2.1194) < Otherwise, considering the biological prospects of the EO, the results obtained were reported in Table 2. According to the table, the cytotoxicity on erythrocytes, reported as the 50% hemolytic concentration (CH 50 -as a measure (approximation) of the skin-irritant capacity of a substance), established that the EO was moderately hemolytic (100 µg/mL < HC 50 < 1000 µg/mL); whereas, the EO was cytotoxic (LC 50 < 100 µg/mL) against lymphocytes/Hep-2 line, being more effective on the cell line than on lymphocytes, and showing a selectivity index of 2.2 on the Hep-2 cells. As per the F test, there were significant differences between each of the control substances and the EO for the tested cells (p ≤ 0.0001), as well as amongst the cell types. A remarkable bioproperty demonstrated by P. eriopodon EO was its high efficacy for inhibiting bacterial growth. Thereby, the three tested strains (S. aureus, E. coli and L. monocytogenes) were susceptible to the different evaluated amounts (4-16 µg) of the EO. The effect of the tested amount (µg) of EO on the inhibition of radial growth for each bacterial species is shown in Figure 1, and from this, it could be observed that the effect on the inhibition of bacterial growth (for all of them) was dose dependent in an exponential mode. It is worth highlighting that the lowest amount (4 µg) of tested EO was capable of inhibiting equal to or higher than the positive control (φ inh. zone of EO ≥ φ inh. zone of control antibiotic). Nonetheless, the descending order of bacterial susceptibility (from highest to lowest) towards the EO was S. aureus > E. coli > L. monocytogenes. The Anova results for these data showed that the general effect of EO on the evaluated bacterial strains was similar [there were no significant differences, [p: 0.2357, F (2.1194) < F crit (6.9443)], whilst the effect of the different amounts of EO was significant [p: 0.01, F (16.053) > F crit (6.9443)] on each strain, all of the above evidenced by the trends shown in Figure 1. In turn, the same Anova demonstrated that the lowest amount (4 µg) of the EO evaluated on strains had no differences [p: 0.1468-0.2394, F (3.18-5.35) < F crit (18.5-19)] with the control antibiotic (4-30 µg) when compared, indicating that the EO and control had similar antibacterial effectiveness under the conditions of this assay. In contrast, the other EO amounts tested (8-16 µg), pursuant to the Anova, were significantly different (p: 0.002-0.02, F (52.88) > F crit (18.5)]) in relation to the standard antibiotic.
Then, the repellent effect against Sitophilus zeamais (Coleoptera: Curculionidae) and the in vitro inhibition of the acetylcholinesterase enzyme (AChE) were also evaluated for this EO. As a result, the degree of repellency for the EO was moderate (~1.6-2.9 ratios, compared to and favoring the "control" standard), and the inhibitory effect of EO on AChE was significant (p: 0.0004, F: 0.0004), although it did not exceed the value obtained for the positive control.
Finally, according to the TAA value (2249 ± 130 mmol Trolox ® /kg) of the EO evaluated, the reactivity of the EO towards the ABTS +• radical-cation, as a measure of its antioxidant capacity, was slightly higher (ratio 1.04) than that of the "control" antioxidant (BHA); however, there was not a significant difference (p F : 0.1902, p t-s :0.07/0.140, F: 0.235) between these values, and therefore, the radical-scavenging capacity of P. eriopodon EO was comparable to the BHA.

Discussion
As a starting point for the discussion, the EO yield regarding the consulted literature showed significant differences; e.g., Castañeda [12] and Ustáriz Fajardo et al. [14] reported EO yields of 0.16% and 0.19%, respectively, values that were equal to or greater than double when compared with that of this research (0.08%). Nonetheless, Ustáriz Fajardo et al. isolated the EO from leaves/stems of the plant, while the other authors obtained it from the leaves. Even with this fact, the differences could also be attributed to some environmental factors such as climate conditions (rainy or dry season-drought stress), soil type (organic matter and mineral contents) and location (latitude, longitude, relative moisture) where the plant was collected, as reported by Fernández-Sestelo and Carrillo [30], García-Caparrós et al. [31] andŞanli and Karadogan [32].
When the compositions of the volatile fractions obtained by the techniques used (SPME and SDE) were compared, they differed; that is, monoterpenes (~5-65%)/sesquiterpenes (~34-90%) were the predominant constituents in the inflorescences/leaves according to HS-SPME, whilst simple phenols (~14-60%)/monoterpenes (~30-74%)/sesquiterpenes (~9-19%) were present in the fruits/leaves/inflorescences via SDE. These differences could be related to the pre-established parameters in each method per se; e.g., the extraction temperatures and times for SPME were 50 • C and 30 min, whereas for SDE, 100 • C (in the reservoir of the plant) and 2 h were used; furthermore, due to the chemical nature of the extractions, CH 2 Cl 2 was used in SDE and a PDMS fiber (non-polar) in SPME. Thus, applied SDE/SPME techniques provided complementary information on the chemical profiles of volatile fractions of the different parts from P. eriopodon, which would be in agreement with Kung et al. [42].
On the other hand, the 1 H-NMR signals of the leaf EO were contrasted with those of gibbilimbol B and β-caryophyllene, according to the literature reports [43][44][45][46]; consequently, both the signals and their multiplicities together with the coupling constants (J) of all the H-atoms coincided (same multiplicities, signals and J), thus confirming the presence of these two compounds in EO.
The differences found in the LC 50 and CH 50 values for lymphocytes and erythrocytes could be related to the particular structure of these cells, as well as their physiological functions; i.e., erythrocyte has primarily a lipid bilayer (plasma membrane) and few organelles; if this cell is (or is not) sensitive to a xenobiotic, its membrane would suffer greater (or less) damage (morphological abnormalities), causing (or not) cell disruption (hemoglobin release-hemolysis) [47]. While the lymphocyte (a more specialized and complete cell type) is more sensitive to changes at the intracellular level; when the cell is exposed to a xenobiotic, and it affects (or not) some vital function or damages (or not) some organelle, cell viability will decrease (or not) [48]. In accordance with the foregoing, possibly the EO would be causing significant damage inside the lymphocyte, and for this reason, the highest cell mortality (low LC 50 ) occurred compared to cell disruption in erythrocytes.
Furthermore, the obtained antibacterial results were compared with those reported by Ustáriz-Fajardo et al. [14], Guzman et al. [15] and Orjala et al. [43]. Hence, in the case of Venezuelan EO [14], it was not active against E. coli and Klebsiella pneumoniae, gibbilimbol B isolated from the EtOH extract of plant leaves. The same authors stated that the phenol significantly inhibited the growth of S. aureus and was inactive against E. coli and Pseudomonas putida; in addition, Orjala et al. listed MIC values for gibbilimbol B on S. epidermidis and Bacillus cereus of 2 µg/mL and 4 µg/mL, in that order. In spite of this, what was previously described differed from the antibacterial results found in this study, due to (i) the chemical composition of EO (from Venezuela) being different and therefore, its antibacterial effect; (ii) as reported by Guzman et al. and Orjala et al., these authors evaluated the isolated phenol (and not the EO) against the bacterial strains. However, gibbilimbol B (the main constituent of EO from northern Colombia) would be possibly responsible for the notable antibacterial power revealed by the P. eriopodon EO.

Materials and Methods
Plant material. Samples of fresh leaves/inflorescences (or fruits) from Piper eriopodon were collected from the sidewalk "Mundo Nuevo", Bonda village in the city of Santa Marta Volatile fractions. The fractions from different parts (inflorescences/fruits and leaves) of the plants were obtained by two methods: simultaneous distillation-solvent extraction (SDE) according to the methodology described by Godefroot et al. [54], using CH 2 Cl 2 (2 mL) as solvent; and headspace solid phase micro-extraction (HS-SPME) based on the procedure reported by Muñoz-Acevedo et al. [55], using PDMS (100 µm)-coated fiber. All extracts (SDE) and trapped volatiles (in fiber) were analyzed with GC-MS.
Isolation of essential oil. Essential oil was obtained from fresh leaves using microwave radiation-assisted hydrodistillation with a Clevenger-type apparatus, a Dean-Stark reservoir and a modified microwave oven [for home, at 700 W, during 1 h (one cycle/15 min)]. Once the EO was decanted and dehydrated, it was prepared for the spectroscopic analysis (GC-MS and 1 H-NMR) [56].
Simple maceration process (SMP). Total extracts of ethyl acetate (ACS reagent grade) from P. eriopodon inflorescences/leaves were obtained via simple maceration. The plant part (0.5-1 g) was sunk in the solvent (5 mL) for seven days at 25 • C (under stirring). The extracts were concentrated (up to 1 mL) and analyzed using GC-MS.
Linear retention indices were calculated using C 7 -C 35 aliphatic hydrocarbons and analyzed in the same conditions. The chemical constituents were recognized/identified by comparing their mass spectra together with the linear retention indices with those of the available databases (NIST11, NIST Retention Index and Wiley9) and the consulted/existing literature [57][58][59].
Analysis by NMR. The NMR spectrum of hydrogen ( 1 H) was acquired to 400 MHz, in an Avance-400 Bruker spectrometer. Chemical shifts were reported in ppm using TMS as the internal reference (δ scale), and CDCl 3 was used as the solvent and internal standard ( 1 H: δ 7.26 ppm). The coupling constants (J) were expressed in Hz.
In vitro biological properties. All assays were carried out 3-5 times, including the positive/negative controls, as well as the suitable statistical treatment of the data.
Cytotoxic, acetylcholinesterase enzyme inhibition and repellent capabilities. The cytotoxicity (on human erythrocytes and lymphocytes, and Hep-2 cell line) along with acetylcholinesterase enzyme inhibition assays were carried out based on the methodology described by Muñoz-Acevedo et al. [60]. The repellent test was carried out based on the preferred area procedure reported by Tapondjou et al. [61].
ABTS +• radical-cation scavenging capacity. The assessment of the antioxidant capacity equivalent to Trolox ® , expressed as total antioxidant capacity [TAA, mmol Trolox ® /kg SE (substance evaluated: EO or "control" antioxidant)] was carried out following the procedure described by Muñoz-Acevedo et al. [63].
Statistical treatment. The data obtained from the biological tests of the EO were treated with the corresponding figures of merit (average, standard deviation and relative standard deviation). In addition, the analysis tools used to determine the significance between the data were F-test two-sample for variance (p < 0.05, F < 0.05), the t-test (paired two samples for means (p < 0.05, F < 0.05)) and two-factor analysis of variance (Anova) (p < 0.05, F > F crit ) in Statistica software (version 10, StatSoft, Inc., Tulsa, OK, USA).

Conclusions
From this research, the following could be concluded: (i) This is the first report of volatile fractions of the different parts of P. eriopodon from Colombia that could be related to some ecological role (defense or chemical messenger) in agreement to the established chemical compositions; (ii) based on the antibacterial efficacy of EO, it could be used as a food protectant/preservative, an attribute that would be reinforced by its anti-radical (antioxidant) capacity equivalent to BHA (synthetic antioxidant used in the industry [64]), which could be replaced by this EO as a natural antioxidant; as well as an antiseptic considering that it could be moderately irritating (as per its CH 50 value). In addition, the selective cytotoxicity (SI:~2) of the EO on the Hep-2 line regarding lymphocytes would suggest its probable use as a chemotherapeutic agent (anticancer/anti-proliferative) against cervical adenocarcinoma. Lastly, despite the moderate degree of repellency and significant inhibition of AChE by EO, it could be applied as a bio-pesticide. (iii) As the main component found in the leaf EO was gibbilimbol B (~72% and probably responsible for the bioproperties), the final use/application could be carried out with the mixture (EO matrix) more easily than with the isolated alkenyl-phenol because the technique used (MWHD) would be much faster (1 h) and less tedious than the process with solvents (extraction/concentration/purification).  Institutional Review Board Statement: Not applicable, but an informed consent statement was evaluated by the ethics committee of the Universidad del Norte (in 2014) for the isolation of human blood.

Informed Consent Statement:
The informed consent statement (version 1, 16 May 2014) was approved by the Ethics Committee of the Universidad del Norte to isolate erythrocytes/lymphocytes from human peripheral blood.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.