Ferulagonodosa Subsp. geniculata (Guss.) Troia & Raimondo from Sicily (Italy): Isolation of Essential Oil and Evaluation of Its Bioactivity

Ferulago nodosa (L.) Boiss. (Apiaceae) is a species occurring in the Balkan-Tyrrhenian area. The object of the present study is Sicilian F. nodosa subsp. geniculata (Guss.) Troia & Raimondo, classified as an endemic F. nodosa subspecies. Aerial parts of this plant species were subjected to hydrodistillation to obtain an essential oil. A total of 93 compounds were identified with 2,3,6-trimethyl benzaldehyde (19.0%), spathulenol (9.0%), (E)-caryophyllene (5.4%), and caryophyllene oxide (5.4%) as the main components. The biological activities of F. nodosa essential oil were also investigated. This oil showed an interesting antioxidant potential in a 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) test (IC50 of 14.05 μg/mL). Additionally, hypoglycemic and antilipidemic effects were evaluated. Lipase enzyme was inhibited with an IC50 value of 41.99 μg/mL. Obtained data demonstrated that F. nodosa could be considered a promising source of bioactive compounds useful for the treatment and management of obesity.


Introduction
The genus Ferulago W.D.J. Koch (Apiaceae) includes about 50 species distributed in the Mediterranean area, from Portugal to Iran and from Russia to Northwestern Africa [1,2].
The center of origin and diversification of the genus was identified to be in Turkey, which houses 34 species, many of which are exclusive [1,2]. Tomkovich and Pimenov [2] divide the genus Ferulago into two subgenera (Ferulago and Galbanifera) and nine sections.
In Sicily, the genus Ferulago is represented by Ferulago nodosa (L.) Boiss. and Ferulago campestris (Besser) Grecescu, both belonging to the subgenus Galbanifera [2]. In particular, F. nodosa, the object of this contribution, highlights a Balkan-Tyrrhenian distribution, being present in Greece, Albania, and probably also in the Yugoslav Republic of North Macedonia, with a western population exists in Sicily.
Based on morphological characteristics and in consideration of the current geographical and genetic isolation of the Sicilian population, Peruzzi et al. [3] proposed to assign the Sicilian population of Ferulago nodosa to the rank of subspecies, with the combination Ferulago nodosa subsp. geniculata (Guss.) Troia & Raimondo.
The genus Ferulago is recognized in traditional medicine as a remedy for gastrointestinal tract panics, ulcer. Moreover it was known as sedative, a treatment for hemorrhoids, skin infection and spleen disease, as well as a natural preservative in different foods, such as meat and cheese [4].
Oxidative stress is defined as an imbalance between the production of Radical Oxygen Species (ROS) and the antioxidant defensive mechanisms in our bodies, that results in the development of pathological conditions such as pre-diabetes [5,6]. It was reported that 5-10% of people per year with pre-diabetes would progress to diabetes and its associated complications, such as obesity [6]. Therapeutic implications of the pre-diabetes condition include the inhibition of α-amylase and α-glucosidase enzyme suppressing postprandial hyperglycemia and reduced dietary carbohydrate digestion and absorption [7]. In addition, inhibition of pancreatic lipase, with a reduction of fat absorption, is a therapeutic strategy to reduce the risk of obesity [8]. Thus, natural inhibitors of these enzymes and antioxidant agents from dietary plants may be proposed as an effective therapy for management of post-prandial hyperglycemia and obesity diseases.
Essential oils have been used since ancient times due to their biological properties. Many essential oils, in fact, are characterized by antioxidant properties [9]. For this reason, it will be used as a substitute for butylated hydroxyanisole and butylated hydroxytoluene (BHT), which are harmful to human health [9]. For this reason, essential oils from edible plants, alone or inserted in active packaging and edible coatings, may represent a valid instrument to prolong shelf life. Several research papers evidenced that Ferulago extracts and essential oils are characterized by antioxidant activity and are used as medicinal plants or food preservatives [10][11][12].
Moreover, several essential oils have been proven to possess hypoglycemic and hypolipidemic properties [13][14][15][16]. However, no previous study investigated the inhibition of carbohydrate-hydrolyzing enzymes and the hypolipidemic activity of Ferulago subspecies.
For this purpose, this study aimed to investigate the phytochemical composition of Sicilian F. nodosa subsp. geniculata essential oil obtained by the hydrodistillation of aerial parts, as well as its biological properties, including antioxidant, hypoglycemic, and hypolipidemic potentials.
The essential oil obtained from the aerial parts of F. nodosa growing in Sicily has previously been studied by Ruberto et al. [18], who found a different chemical profile, with 2,3,4-trimethylbenzaldehyde (42.2%) and α-pinene (22.4%) as the main constituents. These authors did not detect the 2,3,6-trimethylbenzaldehyde which instead was the major component of our sample. Demetzos et al. [24] studied the essential oil composition of flowering aerial parts from F. nodosa growing in Greece and identified α-pinene (31.1%) as the major component. Notably, they did not detect any aromatic aldehydes. Similar results were published by Evergetis et al. [25], who studied the essential oil profile of F. nodosa growing in Greece, showing α-pinene (30.8%) and β-phellandrene (10.2%) as the main constituents. Thus, the notable chemical polymorphism observed may be the result of significant variability in genetics (e.g., occurrence of different subspecies) together with the influence of geographic factors and the type of processing and extraction of the plant material (e.g., hydrodistillation vs. steam distillation).

Antioxidant Activity of Essential Oil
The antioxidant potential of F. nodosa subsp. geniculata essential oil was screened using different assays (Table 2). ABTS + radical cation resulted more sensitive to the action of F. nodosa essential oil with an IC 50 value of 14.0 µg/mL, while an IC 50 value of 26.3 µg/mL was observed in a 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. The results can be justified because ABTS + and DPPH . radicals have different stereochemistry, and after a reaction with essential oil these two radicals give diverse responses. Percentages of 39.4% and 32.7% after 30 and 60 min of incubation, respectively, were recorded in a β-carotene bleaching test when the essential oil was tested at maximum concentration. In a FRAP assay, an IC 50 value of 16.9 µg/mL was observed. Several research articles that have Ferulago ssp. as the object of investigation confirmed the antioxidant potential of these plants. Previously, Cecchini et al. [26] compared the DPPH radical scavenging activity of F. campestris fruit and root essential oils, and found IC 50 mean values of 0.23 and 0.24 µg/mL of oil, respectively. In disagreement with our data, both oils were able to protect lipids from peroxidation in a similar manner to the positive control BHT (IC 50 mean values of 0.010 and 0.011 µg/mL for fruit and root essential oils, respectively). A great variability was observed in the DPPH radical scavenging activity of F. angulate essential oils derived from different organs. Among them, unripe seed oil showed the highest radical scavenging potential with an IC 50 value of 162 µg/mL, followed by leaf (IC 50 value of 210 µg/mL). These organs are characterized by a higher phenol and flavonoid content, compared with other parts [10]. Lower DPPH radical scavenging was found for Iranian F. angulata aerial part essential oil (IC 50 value of 488 µg/mL) [27]. A similar situation was observed for F. trifida essential oils with IC 50 values in the range 95-120 µg/mL [28].
Our data are in line, also, with those reported by Karakaya et al. [29], who investigated the antioxidant potential of F. pauciradiata extracts and essential oils derived from roots and aerial parts. The highest DPPH activity was observed in root essential oil and ethyl acetate fraction, with IC 50 values of 4.59 and 6.56 µg/mL, respectively. Shahbazi and Shavisi [30] compared the DPPH radical scavenging activity of sub-fractions of methanol extract and the essential oil obtained from F. bernardii aerial parts, and found IC 50 values of 5.66, 6.88 and 14.81 mg/mL, respectively.
The aqueous and methanol extracts obtained from flowers, stems, and leaves of F. angulata were investigated by Faride et al. [31], who found IC 50 values ranged from 214 to 1606 µg/mL against the DPPH radical. Values in the range from 264 to 393 µmol of Trolox equivalent per gram of dry weight were found for F. angulata ethanol flower extract and leaf methanol extract, respectively [32].
The antioxidant potential of this species was confirmed in vivo. In fact, treatment with F. angulata extract at doses from 200 to 800 mg/kg/day resulted in an increase in catalase, glutathione peroxidase, and super oxide dismutase activities in diabetic Wistar rats [33].
Mileski et al. [34] reported the antioxidant activity of methanol, ethanol, and aqueous extracts obtained from aerial parts and inflorescences of F. macedonica. The DPPH results demonstrated that extract derived from the inflorescences (in the range of 490-1170 µg/mL) were more active than the extract obtained from the aerial parts (630-1810 µg/mL). This evidence was confirmed, also, in an ABTS test. The antioxidant potential of the crude extract and four fractions of F. carduchorum aerial parts, at two vegetative stages, were analyzed by Golfakhrabadi et al. [35]. The highest DPPH radical scavenging activity was found for flower crude extract, with an IC 50 of 0.49 mg/mL, followed by fruit crude extract (IC 50 of 0.62 mg/mL) and flower methanol fraction (IC 50 of 0.68 mg/mL).
Among the identified compounds, spathulenol, the main abundant volatile component of the F. nodosa essential oil, was able to inhibit both DPPH and ABTS radicals, with IC 50 values of 85.60 and 639.25 µg/mL, respectively [36]. Additionally, this tricyclic sesquiterpenoid exhibited protection against lipoperoxidation in rat brains, with an IC 50 value of 26.13 µg/mL and, consequently, decreased the generation of malondialdehyde [36]. Coté et al. [37] demonstrated the DPPH radical scavenging potential of α-pinene and caryophyllene oxide, with IC 50 values of 3.4 and 183 µg/mL, respectively, while IC 50 > 200 µg/mL was found for both β-caryophyllene and bornyl acetate.

Hypoglycemic and Hypolipidemic Potential of Essential Oil
F. nodosa essential oil α-amylase, α-glucosidase, and lipase inhibitory activities were concentration-dependent, and results are summarized in Table 3. The essential oil exhibited promising lipase enzyme inhibitory activity, with an IC 50 value of 42.0 µg/mL. This value was 0.8 times higher than that found for the positive control, orlistat. A lower enzyme inhibitory activity was found against α-amylase and α-glucosidase, with IC 50 values of 196.4 and 365.9 µg/mL, respectively.
The α-glucosidase and α-amylase inhibitory activities of F. bracteata root extracts were investigated by Karakaya et al. [38]. Dichloromethane and ethyl acetate extracts exhibited the lowest IC 50 values of 0.95 µg/mL, followed by methanol extract (IC 50 of 4.19 µg/mL), whereas the aqueous extract was inactive. The effects of F. angulata hydroalcoholic extract were evaluated by Musavi-Ezmareh et al. [39] in diabetic male rats. After supplementation of 200 mg/kg body weight of extract for four weeks, a reduction of blood sugar and an improvement of blood lipid profiles were observed. A similar situation was observed with supplementation of Ferulago angulata hydroalcoholic extract in Wistar rats, where a reduction of total cholesterol, low-density lipoproteins, and triglycerides, and an inhibition of lipid peroxidation were observed [39,40]. More recently, Parsamehr et al. [41] confirmed that intraperitoneal injection of F. angulata hydroalcoholic extract for three weeks in diabetic rats could be effective in the treatment of diabetes and at the same time alleviate liver damage. Several terpenes identified in F. nodosa essential oil are able to exert hypoglycemic and hypolipidemic activity. The intraperitoneal administration of α-pinene in diabetic mice at different doses (0.05, 0.10, 0.25 and 0.50 mL/kg) resulted in a reduction of fasting blood glucose levels [42]. Previously, Bae et al. [43] demonstrated that intraperitoneal administration of α-pinene (5, 25, or 50 mg/kg) reduced body weight and the serum levels of α-amylase and lipase. Additionally, the combination of β-caryophyllene (500 µmol) and L-arginine (500 µmol) stabilized glucose tolerance and reduced pancreatic cell damage in diabetic rats [44].
Zhou et al. [45] suggested, also, that β-caryophyllene could be used as a therapeutic target for the treatment of diabetic patients, since this terpene acts at the level of arginine-specific mono-ADP-ribosyltransferase 1. Moreover, β-caryophyllene oral administration at a dose of 200 mg/kg induced a reduction of glucose, increase of plasma insulin levels, and improvement of altered activities of carbohydrate metabolic enzymes [46].

Plant Materials
Aerial parts of F. nodosa subsp. geniculata were harvested in April 2019, immediately before flowering, from different plants growing on a flat-lying litho-soil near Noto Antica, Syracuse, (Sicily, Italy) (36 • 57 27.37 N; 15 • 02 18.76 E) of 378 m above sea level. This plant has a distribution limited to the southeastern sector of Sicily, in particular the complex of the Iblei Mountains. It is a circular-limestone plateau of circular shape, dating back to the Miocene, in which the presence of numerous streams dug deep incisions (canyons), locally known as "cave iblee". The bioclimate of the area is thermo-Mediterranean dry, which favors both aspects of thermo-xerophilous pine forest (Pinus halepensis and Coridothymus capitatus) and all aspects of mesophilic shrub (Myrtus communis, Arbutus unedo, and Pistacia lentiscus). Samples were identified by Prof. Vincenzo Ilardi, University of Palermo, Italy. A voucher specimen (PAL 109707) has been deposited in the Herbarium Mediterraneum Panormitanum of the "Orto Botanico", University of Palermo, Italy.

Isolation of the Essential Oil
Fresh leaves and stems (200 g) of F. nodosa subsp. geniculata were reduced to small pieces and subjected to hydrodistillation according to the standard procedure previously described [47]. The obtained oil (0.22% w/w) was stored in dark vials at −20 • C before analyses.

GC-FID Analysis of the Essential Oil
An Agilent 4890D gas chromatograph (GC) coupled with an ionization flame detector (FID) (Santa Clara, CA, USA) was used to analyze the essential oil. The separation stationary phase was represented by an HP-5 capillary column (5% phenylmethylpolysiloxane, 25 m, 0.32 mm i.d.; 0.17 µm f.t.) (Agilent, Folsom, CA, USA). The mobile phase was helium (99.999%) flowing at 1.0 mL/min. The oven temperature programmer was as follows: 60 • C isothermal for 5 min, then ramp (4 • C/min) to 220 • C, and ramp (11 • C/min) to 280 • C. The essential oil was diluted 1:100 in hexane and the volume injected was 1 µL in split mode (1:34). The injector and detector temperatures were set to 280 • C. A commercial mix of n-alkanes (C 8 -C 30 ) purchased from Supelco (Bellefonte, CA, USA) was used to determine the peak linear retention index (RI). Quantitative values, expressed as percentages, were obtained following the procedure of Cecchini et al. [26].

GC-MS Analysis of the Essential Oil
An Agilent 6890N GC equipped with a 5973N single quadrupole mass spectrometer (MS) (Santa Clara, CA, USA) was employed. The stationary phase was an HP-5MS capillary column (30 m, 0.25 mm i.d., 0.1 µm f.t.) (Agilent, Folsom, CA, USA). The operative conditions and the mobile phase were the same as those reported above. The injector and transfer line temperatures were 280 and 250 • C, respectively. The same dilution as that reported above was injected into the GC-MS system in split mode (1:50). Mass spectra were acquired in electron impact (EI) mode in the range of 29-400 m/z. The identification was carried out by a combination of MS matching and RI overlapping against the Adams 2007 [17], NIST 17 (NIST 17, 2017) [48], and FFNSC2 (FFNSC2) [49] libraries. The comparison with available authentic standards (Sigma-Aldrich, Milan, Italy) was also used.

Antioxidant Activity
The in vitro antioxidant potential of F. nodosa subsp. geniculata essential oil samples was screened by ABTS, DPPH, β-carotene bleaching test, and FRAP assay. Both DPPH and ABTS tests were applied to examine the radical scavenging activity of the essential oil, using the procedure previously described by Loizzo et al. [50]. In both cases, ascorbic acid was used as a positive control.
In the β-carotene bleaching test a mixture of linoleic acid, β-carotene, and Tween 20 was prepared and the resulting emulsion was mixed with the essential oil at different concentrations (2.5-100 µg/mL) [50,51]. The absorbance was read after 30 min of incubation at 470 nm. Propyl gallate was used as a positive control. The essential oil at a concentration of 2.5 mg/mL was tested, also, to evaluate the ability of samples to protect iron from a redox reaction [52]. BHT was used as a control.

Hypoglycemic and Hypolipidemic Potential
In the α-amylase inhibitory test, a mixture of α-amylase, starch, and essential oil was prepared as previously reported [52]. In the α-glucosidase inhibitory test, a mixture of maltose, α-glucosidase, o-dianisidine, peroxidase/glucose oxidase, and samples was prepared. In both cases, essential oil was tested at different concentrations, ranging from 25 to 1000 µg/mL, and absorbance was measured at 540 and 500 nm, respectively [50].
The pancreatic lipase inhibitory test was performed as previously reported [53]. A mixture of pnitrophenyl caprylate and porcine pancreatic lipase enzyme was prepared and added to 96-well-plate-contained essential oil at different concentrations (2.5-40 µg/mL). After that, the absorbance was measured (405 nm).

Conclusions
The goal of this study was to chemically characterize and biologically investigate the essential oil obtained from F. nodosa subsp. geniculata aerial parts collected in Sicily (Italy).
The major components of essential oil were 2,3,6-trimethyl benzaldehyde, spathulenol, (E)-caryophyllene, and caryophyllene oxide. Among monoterpenoids, bornyl acetate and α-pinene were the most representative compounds. F. nodosa subsp. geniculata essential oil showed, also, an interesting antioxidant potential, with particular reference to ABTS testing. Additionally, hypoglycemic and antilipidemic effects were evaluated with interesting activity against lipase. Obtained data demonstrated that F. nodosa subsp. geniculata essential oil could be considered a promising phytocomplex useful for formulation of nutraceutical products to prevent diseases associated with oxidative stress, such as type 2 diabetes and obesity.

Conflicts of Interest:
The authors declare that they have no conflict of interest.