Chemical Composition, Enantiomeric Distribution and Anticholinesterase and Antioxidant Activity of the Essential Oil of Diplosthephium juniperinum

The aim of this study was to extract and identify the chemical compounds of Diplosthephium juniperinum essential oil (EO) from Ecuador and to assess its anticholinesterase and antioxidant properties. The EO chemical composition was determined by GC–MS. A total of 74 constituents of EO were identified, representing 97.27% in DB-5ms and 96.06% in HP-INNOWax of the total EO. The major constituents (>4.50%) identified were: α-pinene (21.52, 22.04%), geranyl acetate (10.54, 7.78%), silphiper-fol-5-ene (8.67, 7.38%), α-copaene (8.26, 8.18%), 7-epi-silphiperfol-5-ene (4.93, 5.95%), and germacrene D (4.91, 6.00%). Enantioselective analysis of the volatile fraction of D. juniperinum showed: (+)-α-pinene as a pure enantiomer and 5 pairs of enantiomeric compounds. Among them, (−)-β-Pinene and (−)-Germacrene D presented a high enantiomeric excess of 93.23 and 84.62%, respectively, while (−)-α-Thujene, (−)-Sabinene and (S)-4-Terpineol with a lower enantiomeric excess of 56.34, 47.84 and 43.11%, respectively. A moderate inhibitory effect was observed for Acetylcholinesterase (AChE) and Butyrylcholinesterase (BuChE) enzymes with IC50 values of 67.20 ± 7.10 and 89.00 ± 9.90 µg/mL, respectively. A lower antioxidant potential was observed for the EO measured through DPPH and ABTS radical scavenging assays with SC50 values of 127.03 and >1000 µg/mL, respectively. To the best of our knowledge, this is the first report of the chemical composition, enantiomeric distribution and, anticholinesterase and antioxidant potential of the EO of D. juniperinum. As future perspective, further in-vivo studies could be conducted to confirm the anticholinesterase potential of the EO.


Introduction
Asteraceae family is the largest group of vascular plants in the world and is composed mainly of flowering plants (angiosperms). Also called Compositae, the Asteraceae family comprises approximately 32,205 species belonging to 1911 plant genera [1], and grouped into 13 subfamilies [2,3]. Many species of this family are mainly herbaceous plants, however it can include trees, shrubs and sub-shrubs to vines [4]. Asteraceae occurs on all continents except Antarctica. On a global scale, the diversity of Asteraceae reported, is distributed as follows: South America (6316 species), Asia (6016 species), North America (5404 species), Africa (4631 species), Europe (2283 species), Oceania (1444 species), and the Pacific Islands (174 species) [5].
Despite its large number of species, a small number of them have been used for human and animal consumption as weeds (Bidens, Cirsium, Hypochaeris and Sonchus genera) [6,7], for its toxic and insecticidal properties, in gardening, for ornamental use (Aster, Bellis, Cosmos, Chrysanthemum, Gazania and Gerbera genera), in the food industry as oil plants

Anticholinesterase Activity
In this study, we evaluated for the first time the anti-cholinesterase activity of D. juniperinum EO by measuring the rate of reaction. Results showed a moderate inhibition effect with IC 50 values of 67.20 ± 7.10 and 89.00 ± 9.90 µg/mL against AChE and BuChE, respectively. Donepezil hydrochloride was used as a positive control and their value of IC 50 is presented in Table 3.

Antioxidant Activity
The results obtained for DPPH and ABTS radical scavenging of the D. juniperinum EO as presented in Table 4, and expressed as the concentration of the EO that scavenge or decrease the concentration of the radical at 50% (SC 50 ). Trolox was used as a positive control.

Discussion
Average yields of EO were calculated based on the fresh plant material of the aerial parts of D. juniperinum was similar with D. antioquense EO with 0.16% and much higher that the reported in D. rosmarinifolius with a very low yield 0.0045% [33].
Germacrene D, is one of the main components identified in the D. juniperinum EO, and the enantiomer (−)-germacrene D was found with an e.e. of 92.31 %. Biologically, this sesquiterpene exerted promising results, potentially influence in the attraction and oviposition of females of the species Heliothis virescens [42]. Chiral compounds have great importance for the identification of adulterations due to EOs have different proportions of each enantiomer [43] and this enantiomeric characterization is also important in the olfactory profile [44].
Natural acetylcholinesterase inhibitors, such as galantamine, are usually used in the pharmacological industry as a drug to treat Alzheimer's disease [45], the search for future AChE and BuChE inhibitors guarantee the alleviation of symptoms related to the aforementioned disease and the reduction of mortality rates [46]. Several studies on the anticholinesterase activity of EOs and almost none on their main components showed that EOs are complex mixtures and their final activities are due to the combined effects of the all components [47], therefore, the inhibitory activity of the EO is probably the result of a complex interaction of its chemical components, producing synergistic or antagonistic inhibitory responses [48].
Anti-cholinesterase effect of EO from Diplostephium genus, has not been reported, the monoterpenes are the kind of compounds predominant in them. As mentioned by Aazza and collaborators [49] the α-pinene, limonene and sabinene, are responsible for the anticholinesterase effect. Additionally, (+)-α-Pinene as reported by Miyazawa and Yamafuji [50], presented an IC 50 of 0.40 mM against acetylcholinesterase and, this compound was identified in the EO of D. juniperinum at a concentration of 21% and enantiomerically pure, which could explain the moderate effect observed for this EO against AChE and BuChE enzymes. Therefore, it is important to know the main constituents of the EOs, their proportion and chiral composition because they are the ones that give their biological potential.
A literature review on the Diplostephium genus indicates that few studies have been conducted on its species, one of them is on the ethanolic extract of D. phylicoides, which shows a high antioxidant activity (IC 50 = 13.80 µg/mL) attributed to the presence of flavonoids in its composition [51].
In other study, α-pinene reported a lower antioxidant effect, with an IC 50 of 12.57 ± 0.18 mg /mL [52]. Similar results for the EO of D. juniperinum for ABTS and DPPH assays with an SC 50 of ca. 120 µg/mL and >1000 µg/mL were observed. The importance of knowing the antioxidant properties of EO is due to their implication in counteracting the harmful effects on biological entities by free radicals or reactive oxygen species [53].
Several studies have demonstrated that extracts of Astareaceae species have a positive impact on human health, thanks to their anti-inflammatory, antimicrobial and antioxidant, and antimicrobial [54]. Recently, species of the Asteraeceae family have been considered as a sustainable planning tool in cities for their phytoremediation properties as air pollutant removal, soil protection, shaping landscapes, etc. [55]. Further studies can be conducted to validate the anticholinesterase effect in in vivo studies, however, the low yield obtained for this species could difficult such approximation. In order to obtain a better amount needed for in vivo assays, oil extraction optimization studies could be carried out, including the study of intrinsic and extrinsic parameters related to the species, such as plant age, phenological stage, soil type, amount of shade and season of the year when the species is harvested [56]. This further research could complement the current one.

Distillation of the Essential Oil
The EO from fresh aerial parts of D. juniperinum was extracted by steam hydrodistillation in a Clevenger-type apparatus for approximately 3 h. Three distillations were carried out with 1300, 1320 and 1410 g of fresh plant material, respectively. After obtaining the EO it was separated from the aqueous phase and dried with anhydrous sodium sulfate, filtered and stored in an amber sealed vial at −4 • C, until its analytical and biological assays. The procedure was performed three times [57].

Physical Properties of Essential Oil
The relative density, refractive index and optical rotation of the EO of D. juniperinum were determined in triplicate at 20 • C. The relative density was determined according to the AFNOR NF T 75-11 method (equivalent to ISO 279: 1998, using a pycnometer of 1 mL capacity and an analytical balance (Mettler AC 100), the refractive index according to AFNOR method NF 75-112 (ISO 280:1998) in a refractometer model ABBE (BOECO, Hamburg, Germany). The specific optical rotation was determined with the ISO 592-1998 standard method in an automatic polarimeter (Hanon P-810) [23].

Sample Preparation of EO
Quantitative and qualitative characterization of EO from D. juniperinum required sample preparation of the volatile fractions. Ten µL of EO was diluted in 990 µL in dichloromethane (CH 2 Cl 2 ) obtaining a 1:100 v/v solution. The samples were used in the chemical analyses described below [25].

Qualitative and Quantitative Analysis
Qualitative identification was performed using the analytical technique of Gas Chromatography coupled to Mass Spectrometry (GC/MS). One µL of each sample was injected in duplicate in split mode (40:1) at 20 • C into an Agilent Technologies model 6890N gas chromatograph (GC) with an autoinjector model 7683 and a mass spectrometer model 5973 INERT (Santa Clara, CA, USA). The GC equipment operates in electron-ionization mode at 70 eV, with helium as carrier gas (1.00 mL/min in constant flow), the GC oven operated with temperature ramp from 60 • C to 250 • C with a gradient of 3 • C/min and the ion source at 250 • C. Additionally, the capillary columns DB-5ms (5%-phenyl-methyl polysiloxane, 30 m × 0.25 mm i.d., 0.25 µm film thickness;) and HP-INNOWax, (polyethylene glycol, 30 m × 0.25 mm i.d., 0.25 µm film thickness both purchased from J & W Scientific, Folsom, CA, USA, were used. The procedure was performed for triplicate. The identification of the aromatic compounds was performed by comparison of the mass spectra and the linear retention index (LRI) with those reported in literature. The LRI was determined experimentally according to Van Den Dool and Krats [58], for which it was necessary to inject a homologous series of C 9 to C 24 alkanes in the same conditions of the EO.
Quantitative analysis of the EO of D. juniperinum was performed using a gas chromatography coupled to a flame ionization detector (GC/FID). The previously prepared samples were injected under the same analytical conditions as the qualitative GC/MS method, and the chromatography columns were the same. The percentage of aromatic compounds was determined by comparing the GC peaks with the total area of the identified peaks [59]. A calibration curve was built for each column as previously described by Gilardoni et al. [60], using isopropyl caproate (0. 6, 1.8, 4.3, 8.3, 16.8, and 34.3 mg of isopropyl caproate in 10 mL of cyclohexane) and n-nonane (7 mg) as calibration standard and internal standard respectively. The LOD (0.4 µg/mL) and LOQ (1.2 µg/mL) were stablished. Both calibration curves generated a correlation coefficient of 0.995.

Enantioselective Analysis of Essential Oil
Enantiomeric compounds present in the EO of D. juniperinum were determined by GC/MS on a capillary column with 2,3-diethyl-6-tert-butyldimethylsilyl-β-cyclodextrin stationary phase. The injection conditions used were the same in GC/MS. In addition, enantiomerically pure standards were injected under the same conditions to determine the elution order of the EO enantiomers [61].

AChE and BuChE Inhibition Spectrophotometric Analysis
Cholinesterase (ChEs) inhibition of EO was determined for the enzymes (i) acetylcholinesterase (AChE) and (ii) butyrylcholinesterase (BuChE). The procedure was followed as described by Ellman et al. [62] and Calva et al. [57]. Phosphate buffered saline (pH = 7.4), DTNB (5,5 -dithiobis-(2-nitrobenzoic acid) ion (1.5 mM) a reagent that reacts with thiocholine to give the yellow coloration and the EO sample in DMSO (1% v/v) were prepared. The reaction of DTNB is monitored by measuring its absorption at 412 nm. AChE, from Electrophorus electricus (Sigma-Aldrich, C3389, St. Louis, MO, USA) and BuChE, from horse serum, (Sigma-Aldrich, SRE020, St. Louis, MO, USA) are dissolved in PBS (pH = 7.4) at 24 mU/mL. Preincubation was carried out for 10 min and acetylcholine iodide (1.5 mM) is added to initiate the reaction. The reaction is monitored for 30 min at 30 • C in a PherastarFS detection system (BMG Labtech). Inhibitory concentration (IC 50  The DPPH radical scavenging assay was developed according to the metodologhy proposed by Thaipong et al. [63] with slight modifications, using 2,2-diphenyl-1-picrylhydryl free radical (DPPH-). A working solution was prepared dissolving 24 mg of DPPH in 100 mL methanol and was stabilized in an EPOCH 2 microplate reader (BIOTEK, Winooski, VT, USA) at 515 nm until an absorbance of 1.1 ± 0.01 was reached. The antiradical reaction between EO and free radical was performed at different concentrations of EO (1, 0.5 and 0.25 mg/mL). In a 96-microwell plate, 270 µL of DPPH adjusted working solution and 30 µL of EO sample was placed. The reaction was monitored at 515 nm for 60 min at room temperature. Trolox and methanol were used as positive control and blank control, respectively. The results were expressed as SC 50 (scavenging concentration of the radical at 50%) and calculated according to the corresponding curve fitting of data with GraphPadPrism v.8.0.1. Measurements were performed in triplicate.

ABTS Assay
The antioxidant power measured against ABTS •+ cation (2,2 -azinobis-3-ethylbenzothiazoline-6-sulfonic acid) was determined as reported by Arnao et al. [64] and Thaipong et al. [63] with slight modifications as described. Briefly, the assay started with the preparation of a stock solution of the radical by reacting equal volumes of ABTS (7.4 µM) and potassium persulfate (2.6 µM) for 12 h under stirring. The standard solution was prepared by dissolution in methanol to an absorbance of 1.1 ± 0.02 measured at 734 nm in an EPOCH 2 microplate reader (BIOTEK, Winooski, VT, USA). The antiradical reaction was evaluated over a time of 1 h in the dark at room temperature by plating 270 µL of ABTS working adjusted solution and 30 µL of EO from D. juniperinum at different concentrations (1, 0.5 and 0.25 mg/mL). Trolox and methanol were used as positive control and blank control, respectively. The results were expressed as SC 50 (scavenging concentration of the radical at 50%) and calculated according to the corresponding curve fitting of data with GraphPadPrism v.8.0.1. Measurements were performed in triplicate

Conclusions
The fresh aerial parts of D. juniperinum afforded, an essential oil in quite a low yield (0.12% by weight). The EO obtained was composed exclusively of sesquiterpenes and monoterpenes hydrocarbons, whose major constituents were α-pinene (about 22%) and geranyl acetate (about 10%). The enantioselective analysis showed (+)-α-pinene as a pure enantiomer and 5 pairs of enantiomeric compounds. The EO also manifested a moderate inhibition activity against AChE and BuChE and a lower antioxidant potential was observed for the EO measured through DPPH and ABTS radical scavenging assays. As future perspective, further in-vivo studies could be conducted to confirm the anticholinesterase potential of the EO. In addition, this genus that reported bioactive compounds, could be of interest for the development of new applications such as in the food industry, as enrichment of the food matrix to enhance their beneficial properties and also the substitution of synthetic antioxidants.