Chemical Profiling of Hedyosmum cumbalense and Hedyosmum spectabile (Chloranthaceae) Essential Oils, and Their Antimicrobial, Antioxidant, and Anticholinesterase Properties

In Ecuador, Hedyosmum cumbalense and Hedyosmum spectabile are valued for their well-known aromatic characteristics and therapeutic benefits. In this study, fresh and dried leaves of these species were steam-distilled to obtain their essential oils (EOs) for chemical characterization and assessment of their antimicrobial, antioxidant, and anticholinesterase properties. Gas chromatography coupled to mass spectrometry (GC-MS) and a flame-ionized detector (GC-FID) with a nonpolar column was employed to determine the chemical composition, along with the enantioselective analysis. The antimicrobial activity was evaluated against three Gram-positive, two Gram-negative, and two sporulated fungi. The radical scavenging properties were evaluated by DPPH and ABTS assays. A total of 66 and 57 compounds were identified for H. cumbalense and H. spectabile, respectively. Two pairs of enantiomers for each species were also detected, with (1R,5R)-(+)-sabinene and (1S,5S)-(–)-sabinene found in both specimens. A poor effect against Gram-positive cocci was observed on H. cumbalense (MIC of 4000 µg/mL). Both oils displayed weak antifungal activities, exhibiting a MIC of 1000 µg/mL. H. cumbalense had a good scavenging effect assessed by the ABTS radical (SC50 = 96.02 ± 0.33 µg/mL). Both EOs showed a strong anticholinesterase potential with an IC50 value of 61.94 ± 1.04 µg/mL for H. cumbalense and 21.15 ± 1.03 µg/mL for H. spectabile.


Introduction
Chloranthaceae is a small family with around 70 species. It is among the oldest lineages of angiosperms. Plants belonging to the Chloranthaceae family can be easily identified by their jagged leaves and primitive odor. The presence of secretory cells in stems and leaves is also a typical feature [1]. The family is composed of species of four genera, Ascarina, Chloranthus, Hedyosmum, and Sarcandra, and they are found in tropical and subtropical regions of South America, East Asia, and the Pacific [2].
In the genus Hedyosmum (Tafalla, Tafallaea, or Tavalla genus, heterotypic synonyms), there are 48 species of tiny trees and shrubs and 45 of them have been taxonomically identified and considered as distinct species [3]. The word "Hedyosmum" is derived from the Greek words "Hedy-," which means sweet, pleasant, fragrant, and "osme," meaning smell [4]. The Hedyosmum genus is primarily found in tropical America, in low and high mountain forests like those of South America's Andes. It is the Chloranthaceae genus with the widest distribution in America, primarily in Ecuador, Peru, Brazil, and central Bolivia [5].
The plants of the genus Hedyosmum are defined as medium-sized shrubs or trees with long-legged roots, segmented, knotty, and fleshy-looking twigs, and unisexual flowers [4]. When crushed, its leaves release an odor that is either aromatic or astringent. Their leaves

Results
The essential oils were obtained by steam-distillation from leaves of Hedyosmum cumbalense and Hedyosmum spectabile. Both oils were obtained as a translucent viscous liquid, with H. cumbalense samples being slightly yellow. The mean percentage yield of EOs was 0.15 ± 0.05% and 0.25 ± 0.05% for H. cumbalense and H. spectabile, respectively.

Antimicrobial Activity of H. cumbalense and H. spectabile
The antibacterial activity of essential oil of H. cumbalense and H. spectabile leaves were assessed through the microdilution broth method. Ampicillin was used as a positive control for Gram-positive, Ciprofloxacin for Gram-negative, and Amphotericin B for yeasts and sporulated fungi. The results of the activity of the essential oils against human pathogenic microorganisms are displayed in Table 4, including the minimum inhibitory concentration (MIC) values and the microorganisms used (three Gram-negative bacteria, three Grampositive bacteria, and two yeasts and sporulated fungi). H. cumbalense demonstrated weak or null activity against Enterococcus faecium and Staphylococcus aureus at a dose of 4000 µg/mL. For Gram-negative bacilli, neither of the essential oils showed antimicrobial activity. A higher effect was displayed on yeasts and sporulated fungus. For Aspergillus niger, both EO exhibited a MIC of 1000 µg/mL. Meanwhile, with Candida albicans, only H. cumbalense afforded a MIC of 1000 µg/mL.

Antioxidant Capacity
The essential oils of both species were tested for antioxidant activity. This was done by using ABTS and DPPH radicals. Trolox was used as a positive reference substance. The data obtained are depicted in Table 5.

Anticholinesterase Activity
The inhibitory effect of both essential oils is depicted in Figure 1. Calculated IC 50 values for H. spectabile and H. cumbalense essential oils were 21.15 ± 1.03 and 60.91 ± 1.04 µg/mL, respectively. The anticholinesterase activity of the positive reference control, donepezil, exhibited an IC 50 value of 12.40 ± 1.35 nM.

Discussion
The essential oils of H. cumbalense and H. spectabile exhibited a poor yield of 0.15 ± 0.05% and 0.25 ± 0.05%, respectively. H. spectabile is one of the most recent species registered in the genus, which explains the lack of information about its essential oil extraction. In the same way, although H. cumbalense is more recognizable among native people, there is still not enough knowledge about its EO. In general, the majority of Hedyosmum species presented a low yield of essential oil by hydrodistillation; however, this value is very variable and depends on several factors such as the part of the plant used, the preparation of the material (dried or fresh leaves), or the extraction time and method, including hydrodistillation, steam-distillation, etc. [12].
Despite having different main constituents, some minor components are shared in different proportions. A total of 14 common compounds were identified, which are α-

Discussion
The essential oils of H. cumbalense and H. spectabile exhibited a poor yield of 0.15 ± 0.05% and 0.25 ± 0.05%, respectively. H. spectabile is one of the most recent species registered in the genus, which explains the lack of information about its essential oil extraction. In the same way, although H. cumbalense is more recognizable among native people, there is still not enough knowledge about its EO. In general, the majority of Hedyosmum species presented a low yield of essential oil by hydrodistillation; however, this value is very variable and depends on several factors such as the part of the plant used, the preparation of the material (dried or fresh leaves), or the extraction time and method, including hydrodistillation, steam-distillation, etc. [12].
Despite having different main constituents, some minor components are shared in different proportions. A total of 14 common compounds were identified, which are α- According to Radice et al., the above-mentioned compounds are recurrent constituents in many essential oils of the Hedyosmum species [7]. It is also indicated that terpenes are the primary chemical group present in this genus, which includes sesquiterpenoids and monoterpenes. These types of compounds represent another difference between the EOs. Based on the data obtained, the H. spectabile dominant group of compounds was sesquiterpenes (71.30%), which includes mainly hidrocarbonated sesquiterpenes. On the contrary, more than 50% of H. cumbalense essential oil was constituted by monoterpenes (61.59%), mostly by oxygenated monoterpenes.
The All chiral compounds except sabinene found in H. spectabile, which has an e.e. of 4.25%, have significant levels of enantiomeric excess. The chiral chemicals found in H. spectabile make up 1.4% of the essential oil's overall composition but are not among its major constituents. On the other hand, H. cumbalense chiral compounds account for 16.8% of its total oil content. This percentage is significant enough to raise the possibility that chiral chemicals may influence the biological activity observed. This is the first time either plant has undergone an enantioselective examination.
Regarding Gram-negative bacilli, none of the essential oils showed activity against the two strains of bacteria examined: Escherichia coli (O157:H7) and Pseudomonas aeruginosa. On the other hand, a weak effect against Gram-positive cocci was reported for the EO of H. cumbalense, which showed a MIC of 4000 µg/mL against Enterococcus faecium and Staphylococcus aureus. H. spectabile was inactive. According to the classification of the biological activity of essential oils proposed by Van Vuuren and Holl, D., the MIC values equal to or over 1000 µg/mL should be considered noteworthy of publication [14]. Contrary to the results of antibacterial activity, the effects obtained for yeasts and sporulated fungi were presented in both essential oils. For Aspergillus niger, both plants presented a MIC of 1000 µg/mL. However, for Candida albicans, the MIC value for H. spectabile was higher (2000 µg/mL).
Although H. cumbalense and H. spectabile have not previously been studied, the findings are comparable to those of other Hedyosmum species since it can also be observed that the antimicrobial activity is equally low in different plants of the genus. Kirchner et al., for instance, assessed the essential oil extracted from fresh leaves of Hedyomum brasiliense Miq. for its antibacterial properties. The study demonstrated no activity against the Gramnegative bacteria, Escherichia coli and Pseudomonas aeruginosa, but the EO was described as an antibacterial agent against Gram-positive microorganisms, Staphylococcus aureus, Staphylococcus saprophyticus and Bacillus subtilis, with a MIC value of 0.312% (v/v). This research, along with more recent studies provided by Murakami et al., also confirmed the antifungal properties of H. brasiliense against the dermatophytes M. canis, M. gypseum, T. mentagrophytes, and T. rubrum, and the yeasts Candida albicans and C. parapsilosis [12,15]. Another investigation into the genus is a comparison of male and female specimens of H. racemosum Ruiz & Pav. by Valarezo et al. The essential oil extracted from their leaves also exhibited low activity against pathogenic bacteria and fungi. As opposed to the other species described, female plants proved to be effective against Klebsiella pneumoniae, which is a Gram-negative bacterium [16].
Several EOs from known aromatic species like Lavender, Thyme or Peppermint have demonstrated the antimicrobial potential of volatile components occurring in plants. Particularly, thymol and γ-terpinene have shown good antifungal profiles, being p-thymol the most prominent compound, interfering at the cell wall level, modifying the permeability of the lipidic bilayer of fungal strains [17]. Despite being the antiviral effect the most cited effect for EOs, the antimicrobial effect displayed by several aromatic species, including Hedyosmum, encouraged us to value the antimicrobial potential of these two related species.
The H. spectabile EO proved a null or deficient scavenging effect for DPPH, with an SC 50 of 2366.6 ± 2.99 µg/mL and moderate activity for ABTS, exerting an SC 50 of 214.41 ± 4.03 µM; meanwhile, the reported antioxidant activity for H. cumbalense EO was greater, with an SC 50 of 209.99 ± 1.33 µg/mL on the DPPH assay, and a good scavenging effect on the ABTS radical (SC 50 of 96.02 ± 0.33 µM). It was discovered that the Hedyosmum species generally exhibit strong antioxidant activity by comparing it to previous studies on the genus. For instance, Guerrini et al. examined the antioxidant activity of Hedyosmum sprucei Solms-Laub. essential oil isolated from aerial portions of the plant and found that it had an IC 50 value of 230 ± 10 µg/mL for DPPH scavenging [18].
Compared to the other biological activities assessed in this study, the anticholinesterase activity of both essential oils was the most notable. H. cumbalense demonstrated strong inhibitory activity against AChE with an IC 50 value of 61.94 ± 1.04 µg/mL, while H. spectabile displayed an even greater effect with a value of 21.15 ± 1.03 µg/mL. It is suggested that its primary constituents may be responsible for the potent anticholinesterase activity. In particular, the single compound acyphypllene, found in H. spectabile (5.37 ± 0.27%), was identified by Lobato et al. to be a promising metabolite that showed signs of inhibiting acetylcholinesterase receptors [19]. According to Bonesi et al., the compound sabinene, which is found in both essential oils and is a prominent component of H. cumbalense (14.37 ± 1.64%), also exhibited high activity against AChE with an IC 50 value of 176.5 µg/mL [20].
It is of vital importance when assessing anticholinesterase effects, which are used to find anti-Alzheimer's compounds, to asses also the antioxidant potential of substances present in aromatic species. Our research group, besides investigating the antimicrobial potential of EOs, also determined their potential as possible chemopreventive Alzheimer's agents. In order to determine this, antiradical assays with inhibitory in vitro studies of acetylcholinesterase were conducted.
It is well known the relationship between inflammation and Alzheimer's progress in patients and also that the inflammation mostly occurs by a disbalance between oxidants and antioxidants. In Alzheimer's disease, the activity of antioxidant enzymes is reduced; for that reason, the presence of endogenous or exogenous antioxidants is vital because they prevent or slow the damage of cells caused by free radicals. According to Sinyor et al., while in vitro or in vivo studies have demonstrated that antioxidants rich nutrients can protect the brain from oxidative damage, there are limited data about epidemiological and clinical trial studies; however, the evidence suggests that antioxidants could reduce the risk of inflammatory events that lead to AD progress [21].
Our research group has performed extensive research about the biological properties of essential oils of aromatic species from southern Ecuador, but at the moment, we have investigated only H. racemosum male and female specimens and H. strigosum EO and found that the antimicrobial activity is relevant only in the case of H. strigosum were it was demonstrated a strong antimicrobial effect over Campylobacter jejuni and two dermatophytes fungi and again, thymol, was found to be the most abundant compound [16,22] which according to literature present antifungal properties.

Postharvest Treatments
Both plants were processed upon their arrival at the laboratory: the leaves were separated from foreign material. The leaves of H. spectabile were dried in an incubator at 25 • C for 24 h. The vegetal material obtained from H. cumbalense was analyzed 1 or 2 h after it arrived at the laboratory.

Essential Oil Extraction
The method used for the extraction of the essential oil was steam distillation, performed in a Clevenger-type apparatus for 4 h. After the procedure, the 2 remaining layers, corresponding to water on the bottom and the oil on the top, were separated using a pipette. The oil extracted was stored in an amber vial at 4 • C to prevent the loss of its content and the alteration of the compounds. This method was used for both plants.

Qualitative Analysis
Both samples were diluted (1/100, v/v, EO/DCM) and analyzed using a Thermo Scientific Gas Chromatograph (TRACE 1300 Series) coupled to a Single Quadrupole Mass Spectrometer (ISQ 7000 Series). A non-polar DB-5 ms column of 0.25 mm × 30 m, a thickness of 0.25 µm (5%-phenyl-methylpolyxilosane) was used. For each run, 1 µL of the EO dilutions were injected with a split ratio of 1:40. The equipment operated with electronic ionization (70 eV). Methane was used as a carrier gas at 1 mL/min in constant flow mode. The initial oven temperature was 60 • C, following a gradient until 230 • C was reached. Each run lasted 58 min. The Retention Index was calculated using alkanes from C9 to C10, which were previously injected under the same conditions. The compounds were identified through a comparison between the CRI and reference literature [23].

Quantitative Analysis
An Agilent Gas Chromatograph (model 6890N series) equipped with a flame ionization (FID) detector was used. The GC-FID analyses were performed using the same method and instrumental configuration as GC-MS, which was previously described. The run time was 58 min.

Enantioselective Analysis
For the enantioselective analysis, a Thermo Scientific Gas Chromatograph (TRACE 1300 Series) using a chiral 2,3-diethyl-6-tert-butyldimethylsilyl-β-cyclodextrin-based column was used. The running conditions were similar to those applied for the qualitative and quantitative analyses. The run time was 88 min.  Table 4). The essential oil was dissolved in DMSO, and the bacterial strains were cultured in Müeller-Hinton (MH) broth. Ampicillin (1 g/mL), Ciprofloxacin (1 mg/mL), and Amphotericin B (250 g/mL) were employed as positive controls for Gram-positive cocci, Gram-negative bacilli, and yeasts and sporulated fungi, respectively. Minimum inhibitory concentration (MIC) findings were obtained using DMSO as a negative control (the lowest concentration of the sample capable of inhibiting all visible signs of growth of the microorganism) [24]. The methodology presented by Salinas et al. [25], which summarizes the DPPH radical scavenging assay put out by Thaipong et al. [26] with just minor changes, was used to evaluate DPPH. The 2,2-diphenyl-1-picrylhydryl free radical (DPPH-) was used. For the creation of the solution used, 24 mg of DPPH was dissolved in 100 mL of methanol. This solution was stabilized at 515 nm in an EPOCH 2 microplate reader until an absorbance of 1.1 ± 0.01 was attained. For the antiradical reaction, different concentrations of the essential oil were used (1, 0.5, and 0.25 mg/mL). Then, 30 mL of the EO sample and 270 mL of the DPPH-adjusted working solution were each added to a 96-microwell plate. The conditions in which the reaction was observed were 515 nm for 1 hour at room temperature. The values of SC 50 were computed according to the curve generated from previous data, in which methanol was used as blank. Trolox was employed as a positive control. The essential oils of H. cumbalense and H. spectabile were both analyzed using the same process. 4.6.2. The 2,2-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) Radical Scavenging Assay

Antimicrobial Activity
The procedure used was based on the method employed to estimate the antioxidant power evaluated against the ABTS•+ cation (2,2 -azinobis-3-ethylbenzothiazoline-6-sulfonic acid) described by Salinas et al. [25]. The technique was created using the information provided by Arnao et al. [27] and Thaipong et al. [26], with a few minor alterations. As a first step, a stock solution of the radical was made by stirring equal volumes of potassium persulfate (2.6 M) and ABTS (7.4 M) for 12 h. For the creation of the standard solution, it was dissolved in methanol until an absorbance of 1.1 ± 0.02 at 734 nm was reached using an EPOCH 2 microplate reader. The antiradical response was examined over the course of 60 min at room temperature. It was done by plating 270 mL of the ABTS working adjusted solution and 30 mL of the essential oils at various doses (1, 0.5, and 0.25 mg/mL). The blank, positive control, and calculations were the same as the DPPH assay.

Anticholinesterase Assay
The anticholinesterase assay was based on the procedure presented by Andrade et al. [28], in which they followed the methodology proposed by Ellman et al. [29], with minor modifications as recommended by Rhee et al. [30]. For the reaction mixture, 40 µL of Buffer Tris were incorporated into 20 µL of the tested sample solution, along with 20 µL of acetylthiocholine (ATCh, 15 mM, PBS pH 7.4), and 100 L of DTNB (3 Mm, Buffer Tris). Then, the pre-incubation was executed for 3 min at room temperature, under constant shaking. The reaction was then begun by adding 20 µL of 0.5 U/mL AChE. The quantity of product produced was measured using an EPOCH 2 microplate reader at 405 nm for an hour at room temperature. Ten milligrams of essential oil were dissolved in MeOH to create EO sample solutions. To reach final concentrations of 1000, 100, and 10 g/mL, three more 10-factor dilutions were added. The corresponding curve fitting of the data received from the computed rate of reactions was used to get the IC50 value. The calculated IC 50 value for donepezil-hydrochloride, which served as a positive control, was 12.40 ± 1.35 nM. The H. cumbalense and H. spectabile essential oils were assessed under the same methodology.