Biological Activities of the Fruit Essential Oil, Fruit, and Root Extracts of Ferula drudeana Korovin, the Putative Anatolian Ecotype of the Silphion Plant

In the present study, preliminary phytochemical investigations were performed on the fruit essential oil and antioxidant-rich methanolic extracts of the fruits and roots of Ferula drudeana, the putative Anatolian ecotype of the Silphion plant, to corroborate its medicinal plant potential and identify its unique characteristics amongst other Ferula species. The essential oil from the fruits of the endemic species Ferula drudeana collected from Aksaray was analyzed by GC and GC/MS. The main components of the oil were determined as shyobunone (44.2%) and 6-epishyobunone (12.6%). The essential oil of the fruits and various solvent extracts of the fruits and roots of F. drudeana were evaluated for their antibacterial and anticandidal activity using microbroth dilution methods. The essential oil of the fruits, methanol, and methylene chloride extracts of the fruits and roots showed weak to moderate inhibitory activity against all tested microorganisms with MIC values of 78–2000 µg/mL. However, the petroleum ether extract of the roots showed remarkable inhibitory activity against Candida krusei and Candida utilis with MIC values of 19.5 and 9.75 µg/mL, respectively. Furthermore, all the samples were tested for their antioxidant activities using DPPH• TLC spot testing, online HPLC–ABTS screening, and DPPH/ABTS radical scavenging activity assessment assays. Methanolic extracts of the fruits and roots showed strong antioxidant activity in both systems.

Ferula drudeana Korovin ( Figure 1) is a rare endemic species growing in the Central Anatolia region of Turkey. This species is the only member of the subgenus Narthex (Falc.) Ferula drudeana Korovin ( Figure 1) is a rare endemic species growing in the Central Anatolia region of Turkey. This species is the only member of the subgenus Narthex (Falc.) Drude of Ferula genus in Turkey [7]. With its unique morphological features and extremely limited local distributions near former Greek villages in Central Anatolia, F. drudeana has been proposed as an Anatolian ecotype of the silphion plant [8]. The silphion plant was used for many medicinal and culinary purposes during ancient times in Mediterranean countries. Pliny the Elder declared that "it would be an endless task to enumerate all the uses to which laser (i.e., the resin of silphion plant) is put" [9]. Theophrastus of Eresus calls the fruits of the silphion plant phyllon (i.e., leaf-like) [10]. Unfortunately, the use of the phyllon name inadvertently created an ambiguous terminology in the literature. The fruits of Ferula drudeana (Figure 2), like the fruits of other Ferula species, is a schizocharpic fruit that splits into two mericarps during their maturity. The fruits of Apiaceae plants are widely accepted sources of medicinal drugs due to their high essential oil/resin content. In contrast, their leaves are rarely used as a source of medicinal drugs [11]. Probably due to the use of ambiguous phyllon names for the fruits of the silphion plant by Theophrastus, instead of the biologically active metabolite-rich fruits, some authors referred to the leaves of the silphion plant as a medicinally used part [12]. In contrast, the medicinal use of the fruits of the silphion plant was not mentioned in the literature. As part of our study investigating the biological activities of the secondary metabolites of Ferula drudeana, we herein report on the analyses of the terpenoid content of the essential oil of the fruits as well as the major phenolic compounds of the methanolic extracts of the fruits and roots.

Volatile Composition of the Fruit Essential Oil of Ferula drudeana
The fruits of F. drudeana were collected in July and air-dried, and the coarsely crushed fruits were subjected to hydrodistillation to obtain its essential oil. The essential oil yield of the fruits of F. drudeana was 3.8%. The essential oil of the fruits was analyzed by GC-GC/MS systems, and 28 compounds representing 89.1% of the essential oil were characterized. The results of the analysis are shown in Table 1 and Figure 3.

Volatile Composition of the Fruit Essential Oil of Ferula drudeana
The fruits of F. drudeana were collected in July and air-dried, and the coarsely crushed fruits were subjected to hydrodistillation to obtain its essential oil. The essential oil yield of the fruits of F. drudeana was 3.8%. The essential oil of the fruits was analyzed by GC-GC/MS systems, and 28 compounds representing 89.1% of the essential oil were characterized. The results of the analysis are shown in Table 1 and Figure 3. Compounds listed in order of their elution in HP Innowax FSC GC column. RRI a : relative retention indices experimentally calculated against n-alkanes; RRI b : RRI from literature (c [13], d [14], e [15], f [16], g [17], h [18], and k [19]) for polar column values, with % calculated from FID data; tr: trace (<0.1%); IM: identification method; tR: identification based on comparison with coinjected standards on an HP Innowax column; MS: identification based on computer matching of the mass spectra libraries. The main components of the essential oil were identified as shyobunone (9) (44.2%), 6-epi-shyobunone (8) (12.6%), epi-isoshyobunone (11) (9.8%), and β-pinene (1) (5.8%). Oxygenated sesquiterpenes (74.5%), sesquiterpene hydrocarbons (8.6%), and monoterpene hydrocarbons (6.0%) were the main groups present in the oil. Oxygenated sesquiterpenes  Compounds listed in order of their elution in HP Innowax FSC GC column. RRI a : relative retention indices experimentally calculated against n-alkanes; RRI b : RRI from literature ( c [13], d [14], e [15], f [16], g [17], h [18], and k [19]) for polar column values, with % calculated from FID data; tr: trace (<0.1%); IM: identification method; t R : identification based on comparison with coinjected standards on an HP Innowax column; MS: identification based on computer matching of the mass spectra libraries.
The essential oil composition of Ferula drudeana is unique amongst the Ferula species. Unlike other investigated Ferula species growing in the Mediterranean, Middle East, and North African countries, it has a very high percentage of oxygenated sesquiterpene compounds [8]. The proportion of rare elemane sesquiterpene ketone compounds in the essential oil is 66.6%.

Antimicrobial Testing of the Fruit Essential Oil of Ferula drudeana
Hydrodistilled fruit essential oil of Ferula drudeana demonstrated weak antimicrobial effects against all tested pathogenic Gram (+) and Gram (−) bacterial strains and the Candida panel with MIC values of 500-2000 µg/mL using CLSI M7-A7 and M27-A2 reference microdilution broth methods. Methanol and methylene chloride extracts of the fruits and roots displayed weak to moderate inhibitory effects against all tested microorganisms at concentrations between 78 and 2500 µg/mL. However, the petroleum ether extract of the roots (R1) showed remarkable inhibitory effects on Candida krusei and Candida utilis with MIC values of 19.5 and 9.75 µg/mL, respectively (Tables 2 and 3). Our results are similar to previous works [25][26][27][28][29][30][31][32][33][34] on different Ferula essential oils and extracts.
In contrast to the essential oil fraction, the presence of strong antifungal activity in the fruit and root petroleum ether extracts suggests that the antifungal activity must be due to nonvolatile component(s) of these extracts. Further studies are in progress to identify the most potent antifungal compound(s) of these extracts. C. albicans, C. tropicalis, and C. glabrata represent the most clinically isolated Candida species. In contrast, other species, such as C. krusei, C. parapsilosis, C. guilliermondii, and C. kefyr, have also been isolated and are thought to be less virulent. However, recent data indicate that >30% of nosocomial Candida infections are due to species other than C. albicans, and in recent years, there has been a significant increase in C. krusei, a human pathogen causing systemic and ocular infections [35]. Identifying potent anticandidal substances in the petroleum ether extracts of F. drudeana may provide a promising antifungal agent for the treatment of such infections.  The methanolic extracts of the fruits and roots of Ferula drudeana Korovin showed strong antioxidant activity by DPPH • reagent treatment on a TLC silica gel plate ( Figure S1, Supplementary Material) [36,37]. Following the application of the fruit essential oil, fruit, and root extract solutions to a silica gel plate, the plate was sprayed with DPPH (2,2-diphenyl-1-picryl-hydrazylhydrate) solution. No antioxidant activity was observed in the petroleum ether and methylene chloride extracts of the fruits and roots. Only a slight discoloration was observed on the periphery of the spot where the essential oil was applied to the silica gel plate. Peripheral discoloration of essential oil is probably related to the microlevel distribution of the essential oil by blank dilution solvent on the silica gel plate at the application point. The strong discoloration of the methanolic extracts of the fruits and roots of F. drudeana indicates the presence of antioxidant compounds in these extracts.

Online HPLC-ABTS •+ Identification of Major Antioxidant Compounds of the Methanolic Extracts of Ferula drudeana
The methanolic extracts of the fruits and roots of F. drudeana were subjected to online high-performance liquid chromatography (HPLC)-2,2 -azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS •+ )-based screening assay for the identification of phenolic antioxidants of the fruits and roots of F. drudeana [38]. HPLC elute of the methanolic extracts of the fruits and roots of F. drudeana was split into two lines. The elute of one line mixed with a stabilized solution of ABTS •+ reagent, so the formation of negative peaks, indicating the antioxidant activity of the corresponding compound peaks, were monitored by measuring the decrease in absorbance at 734 nm ( Figure 4). The elute of the second line was subjected to electrospray ionization mass spectrometry (EIMS) to identify the phenolic compounds responsible for the antioxidant activity of the methanolic extracts of fruits and roots of F. drudeana.   The methanolic extract of the fruits of F. drudeana was subjected to high-performance liquid chromatography-MS/MS analysis to identify the phenolic compounds responsible for the antioxidant activity ( Figure 5).  Figure S3, Supplementary Material) suggested that these compounds could be 1,3-, 1,5-, or 3,5-dicaffeoyl-quinic acid derivatives [40]. Furthermore, the presence of a strong m/z 179 ion (i.e., [caffeoyloxy-H] − ) fragment vs. the lack of a weak m/z 335 ion fragment suggested that the structure of 15 should be 3,5-dicaffeoylquinic acid [40]. In contrast, the presence of m/z 179 ion and weak m/z 335 ion fragments in the mass spectrum of 16 ( Figure S3, Supplementary Material) confirmed its structure as 1,5dicaffeoylquinic acid (i.e., cynarine) [41].
The mass spectrum of compound 17 showed the main fragment ion at m/z 173, indicating the presence of a 4-OH substituted quinic acid. Consequently, this compound could be 3,4-dicaffeoylquinic acid or 4,5-dicaffeoylquinic acid. As the mass spectrum of 3,4dicaffeoylquinic acid has an additional fragment at m/z 335 [41] and this fragment was absent in the mass spectrum of 17, its structure should be 4,5-dicaffeoylquinic acid.
The mass spectrum of compound 17 showed the main fragment ion at m/z 173, indicating the presence of a 4-OH substituted quinic acid. Consequently, this compound could be 3,4-dicaffeoylquinic acid or 4,5-dicaffeoylquinic acid. As the mass spectrum of 3,4-dicaffeoylquinic acid has an additional fragment at m/z 335 [41] and this fragment was absent in the mass spectrum of 17, its structure should be 4,5-dicaffeoylquinic acid.
The mass spectrum of compound 19 showed m/z 431 [M-H] − pseudo molecular ion and m/z 268 (apigenin aglycone) base peak ion, which suggested the structure of 19 was apigenin glucoside. Apigenin-7-glucoside (19) has previously been identified in other Ferula species [43]. The mass spectroscopic data of compound 20 displayed m/z 461 [M-H] − pseudo molecular and m/z 299 (hispidulin aglycone) ions as well as m/z 446, 283, and 255 fragment ions, which are in agreement with previously reported data. Thus, the structure of compound 20 was identified as hispidulin-7-glucoside [44].

Determination of the Antioxidant Potential of the Methanolic Extracts of Ferula drudeana by DPPH and ABTS Free Radical Scavenging Activity Assessment
The antioxidant potential of the methanolic extracts of the fruits and roots of F. drudeana as well as two of their major antioxidant compounds, namely, chlorogenic acid and luteolin 7-glucoside, were determined by DPPH and ABTS radical scavenging activity tests. The results are shown in Table 5. The DPPH radical scavenging test results indicated that the methanolic fruit extract of F. drudeana was about 2.2 times more active than the root extract. Chlorogenic acid and luteolin-7-glucoside, two of the major antioxidant compounds of methanolic extracts, were more potent antioxidants than butylated hydroxytoluene (BHT) but were not as effective as gallic or ascorbic acids.
The results of the TEAC experiment (i.e., ABTS radical scavenging activity) suggested that the methanolic extract of the fruits of F. drudeana was approximately 1.6 times more effective than the root extract at 1 mg/mL concentration. However, at 0.1 mg/mL concentration, none of the methanolic extracts was active. The antioxidant activity of luteolin-7-glucoside was higher than both BHT and ascorbic acid at 0.1 mg/mL concentration but lower than gallic acid. In this assay, chlorogenic acid was found to be a more potent antioxidant than any of the standards used.

Biological Activities of the Ferula drudeana Metabolites
The biological activities of the main essential oil terpenoids and phenolic compounds of the methanolic extracts of Ferula drudeana are summarized in Table 6 below. Table 6. Biological activities of the compounds identified in the fruit essential oil, fruit, and root methanol extracts of Ferula drudeana.

General Experimental Procedures
The GC-FID analyses were carried out with capillary GC using an Agilent 6890N GC system (Agilent, Santa Clara, CA, USA), and the GC/MS analyses were performed on an Agilent 5975 GC-MSD system (Agilent, Santa Clara, CA, USA). An HP-Innowax FSC column (60 m × 0.25 mm, 0.25 µm film thickness, Agilent, Wilmington, DE, USA) was used for the analyses. The HPLC chromatographic separations were carried out using Shimadzu LC 20 System (Shimadzu, Tokyo, Japan). The mass spectra were recorded with AB Sciex 3200 Q TRAP mass spectrometer (AB Sciex, Toronto, Canada). GL Science Inertsil ODS 250 × 4.6 mm, 5 µm i.d. particle size, analytical column (GL Sciences, Tokyo, Japan) was used for the HPLC analyses. The turbidity of the standardized microbial sample solutions was measured using McFarland densitometer (Biosan McFarland Densitometer, Model Den-1B, Riga, Latvia). Antioxidant activity absorbances were recorded with a Biotek microplate reader (BioTek, Winooski, Vermont, USA). Chlorogenic acid, luteolin 7-glucoside, gallic acid, butylated hydroxytoluene (BHT), and L-ascorbic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Material
The plant material was collected (07 July 2012) near Mount Hasan in Aksaray, Turkey. A voucher specimen identified by Prof. Dr. H. Duman (Gazi University, Ankara) was deposited in the Herbarium of Gazi University (GAZI Nr. 9898000001568).

Extraction
Essential oil of the fruits of Ferula drudeana Korovin was obtained by hydrodistillation as described in Section 2.1 and subjected to GC-GC/MS analyses and antimicrobial testing. Air-dried and coarsely powdered fruits and roots (each 20 g) of F. drudeana were extracted in a Soxhlet extractor successively with petroleum ether (600 mL, 8 h), methylene chloride (600 mL, 8 h), and methanol (600 mL, 8 h). Each extract was concentrated in vacuo to remove the extraction solvent using a rotary evaporator and subjected to antimicrobial and antioxidant activities testing. The extract yields were as follows: fruit petroleum ether extract (F1) 1.916 g, fruit methylene chloride extract (F2) 0.265 g, fruit methanol extract (F3) 1.252 g, root petroleum ether extract (R1) 2.268 g, root methylene chloride extract (R2) 0.299 g, and root methanol extract (R3) 1.224 g.

Gas Chromatography-Gas Chromatography/Mass Spectrometry Analyses of Ferula drudeana Essential Oil
The oil was analyzed by capillary GC and GC/MS using an Agilent GC-MSD system (Agilent Technologies Inc., Santa Clara, CA, USA).
The GC/MS analysis was carried out with an Agilent 5975 GC-MSD system. Innowax FSC column (60 m × 0.25 mm, 0.25 µm film thickness) was used with helium as carrier gas (0.8 mL/min). GC oven temperature was kept at 60 • C for 10 min and programmed to 220 • C at a rate of 4 • C/min and kept constant at 220 • C for 10 min and then programmed to 240 • C at a rate of 1 • C/min. The split ratio was adjusted to 40:1. The injector temperature was set at 250 • C. MS were taken at 70 eV. The mass range was from m/z 35 to 450.
The GC analysis was carried out with an Agilent 6890N GC system fitted with a FID detector set at a temperature of 300 • C. To obtain the same elution order with GC/MS, simultaneous autoinjection was carried out on a duplicate of the same column applying the same operational conditions. Relative percentages of the separated compounds were calculated from FID chromatograms.
The components of essential oils were identified by comparing their mass spectra with those in the Baser Library of Essential Oil Constituents, Wiley GC/MS Library, Adams Library, and Mass Finder Library and confirmed by comparison of their retention indices. Alkanes were used as reference points to calculate the relative retention indices (RRI). Relative percentages of the separated compounds were calculated from FID chromatograms. The results of the analysis are shown in Table 1.

Antioxidant Activity
The antioxidant activity of Ferula drudeana essential oil and extracts were evaluated by DPPH • TLC spot testing assay, the major antioxidant compounds of the active extracts were identified by online HPLC-ABTS •+ screening assay coupled with HPLC-UV-MS/MS system, and the antioxidant potential of methanolic extracts of F. drudeana was determined with DPPH and ABTS radical scavenging activity testing.

DPPH • TLC Spot Testing
The DPPH • TLC spot testing method was used to find the active antioxidant extracts. The extract and essential oil solutions (10 µL of 1 mg/mL) of Ferula drudeana were spotted on a silica gel TLC plate, then 2.54 mM DPPH-methanol solution was sprayed using a Camag TLC sprayer, and the results were evaluated after 30 min. Spots with the DPPH solution scavenging activity were observed as white-yellow spots on a purple background ( Figure S1, Supplementary Material) [36,37].

HPLC-ABTS •+ Derivatization
Online HPLC-ABTS screening and HPLC-UV-MS/MS method were used, and the extract's active compounds were identified using the method developed by Koleva and He [30,162]. HPLC coupled with ABTS assay was performed using a stock solution containing 3.5 mM potassium persulphate, and 2 mM ABTS was prepared and kept at room temperature in darkness for 16 h to stabilize the radical. The radical reagent was prepared by diluting the stock solution with pure water to an absorbance of 0.70 ± 0.02 at 734 nm. The extracts (at 10 mg/mL concentration, 10 µL) were injected into a Shimadzu HPLC system. HPLC separation was carried out as described in the previous section. HPLC analytes from the column arrived at a T-junction, where the ABTS reagent was added. A Shimadzu reagent pump delivered the ABTS reagent at a 0.7 mL/min flow rate. After the analytes were mixed with ABTS reagent in a reaction coil (15 m-0.25 mm i.d. PEEK tubing), DAD measured the negative peaks at 734 nm. Data were analyzed using LC Solution Software.

LC-MS/MS Analysis
AbSciex 3200 MS/MS detector was used for LC-MS/MS analysis. A negative ionization mode was preferred for ionization. Chromatographic separations were carried out with GL science Inertsil ODS 250 × 4.6 mm, i.d., 5 µm column using Shimadzu 20A HPLC. The column oven temperature was set to 40 • C, and the flow rate was adjusted to 1 mL/minute. Mobile phases were (A) methanol: water: formic acid (10:89:1, v/v/v) and (B) methanol: water: formic acid (89:10:1, v/v/v). The B concentration increased from 15% to 40% in 15 min, then increased to 45% within 3 min; it was at 45% B at 12 min, then increased to 75% within 5 min and 100% within 5 min. For mass scanning (EMS), a mass range of 100-1000 amu was chosen.

Determination of the Antioxidant Potential of Methanolic Extracts DPPH Radical Scavenging Activity
Using a modified version of the Brand-Williams method [163,164], the DPPH radical scavenging capacity of the fruit and root methanolic extracts, standards, and positive control standards were assessed.
Solutions of the methanolic extracts of fruits and roots of F. drudeana (1.25 mg/mL), two standard compounds (0.1 mg/mL of each luteolin-7-glucoside and chlorogenic acid), and three positive control standards (0.1 mg/mL of each gallic acid and ascorbic acid and 1 mg/mL BHT) in methanol were prepared. In 96-well flat-bottom plates, 100 µL of the sample solutions (extracts, standards, and positive control standards) were serially diluted with 100 µL of methanol. Then, diluted samples were mixed with 100 µL of DPPH solution (0.08 mg/mL in methanol). As a control, 100 µL of methanol and 100 µL of DPPH solution were combined. The mixtures were kept in the dark for 30 min. Absorbance of each well was recorded at 517 nm. The percentage of inhibition was calculated using the following equation: where A control is the absorbance of the solution that contains all reagents with the exception of extract or standard chemical. The 50% inhibition concentration (IC 50 ) values of DPPH radical of each sample were calculated using SigmaPlot (Version 12.0).

ABTS Radical Scavenging Activity
ABTS radical scavenging test was performed following the method described by Re et al. [165] with slight modification. In order to produce the ABTS •+ free radical cation, Plants 2023, 12, 830 14 of 21 7 mM ABTS and 2.5 mM K 2 S 2 O 8 were dissolved in 10 mL ultrapure water and incubated at room temperature for 16 h in the dark. The ABTS •+ solution was diluted with 100% ethanol prior to the experiment to obtain an absorbance of 0.7-0.8 at 734 nm. Solutions were prepared for extracts (0.1-1 mg/mL), standards (0.1-1 mg/mL), and Trolox (3.0, 2.0, 1.0, 0.5, 0.25, and 0.125 mM). During the experiment, 990 µL of ABTS •+ solution was combined with 10 µL of the sample (extract/standard) solution, and the combined mixtures were allowed to incubate for 30 min at room temperature in the dark. Trolox equivalent antioxidant capacity (TEAC) of the extracts and standards were calculated using the linear equation obtained for Trolox (y = 20.227x + 4.1663 R 2 _ = 0.9968).

Conclusions
This is the first report on the bioactivities of terpenoids present in the essential oil and major phenolic compounds of methanolic extracts of Ferula drudeana. The fruit essential oil of F. drudeana was analyzed by GC-MS, and 28 terpenoid compounds were identified. About 75% of the essential oil consisted of oxygenated sesquiterpene compounds. The presence of unusually high levels of shyobunone derivatives with reported anti-Alzheimer and neuroprotective activities in the essential oil of F. drudeana clearly highlights the medicinal value of this species. Although the antimicrobial activity of the fruit essential oil of F. drudeana was weak to moderate, the presence of strong anticandidal activity in the fruit and root petroleum ether extracts suggested the source of anticandidal activity was probably the nonvolatile component(s) of this extract. The results of DPPH •· TLC spot testing, online HPLC-ABTS •+ screening assay, and DPPH/ABTS radical scavenging tests indicated that F. drudeana was rich in polyphenolic compounds and exhibited good antioxidant activity. Three flavonoid glucosides and five hydroxycoumaric acid esters of quinic acid were determined in the methanol extracts of the fruits and roots of F. drudeana by HPLC-MS/MS analyses, and their antioxidant activities were detected simultaneously by online coupled HPLC-ABTS •+ based assay. Furthermore, based on the literature survey, known biological activities of the major essential oil terpenoids and methanolic extract phenolic compounds of F. drudeana were reviewed to confirm the medicinal values of F. drudeana secondary metabolites.