Chemical Profiling and Discrimination of Essential Oils from Six Ferula Species Using GC Analyses Coupled with Chemometrics and Evaluation of Their Antioxidant and Enzyme Inhibitory Potential

The differences in the composition of essential oils obtained from the aerial parts of six Ferula species viz., F. caratavica (Fc), F. kuchistanica (Fk), F. pseudoreoselinum (Fp), F. samarcandica (Fs), F. tenuisecta (Ft) and F. varia (Fv) were detected both qualitatively and semi-quantitatively using GC-MS and GC-FID analyses. One hundred and six metabolites were identified that account for 92.1, 96.43, 87.43, 95.95, 92.90 and 89.48% of Fc, Fk, Fp, Fs, Ft and Fv whole essential oils, respectively. The data from the GC-MS analyses were subjected to unsupervised pattern recognition chemometric analysis utilizing principal component analysis (PCA) to improve the visualization of such differences among the six species. Fk and Ft are very closely related to each other and were gathered together in one cluster. The antioxidant potential was assessed in vitro using different assays including 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), cupric reducing antioxidant capacity (CUPRAC), ferric reducing power (FRAP) and phosphomolybdenum (PM) assays. Ft and Fp exhibited the most notable antioxidant properties as evidenced by their pronounced activities in most of the antioxidant assays performed, followed by Fc. Fk showed the most effective tyrosinase inhibitory potential, which was estimated as 119.67 mgKAE/g oil, while Fp exhibited the most potent α-amylase inhibitory potential, which was equivalent to 2.61 mmol ACAE/g oil. Thus, it was concluded that Ferula species could serve as a promising natural antioxidant drug that could be included in different products and spices to alleviate hyperglycemia and used as a natural ingredient in pharmaceutical cosmetics to counteract hyperpigmentation.


Introduction
Essential oils comprise a mixture of secondary metabolites, which are biosynthesized by aromatic plants as natural protectants [1]. The role of essential oils is not restricted to protection as they also

Qualitative and Semi-quantitative Determinations by GC-MS and GC-FID
The differences in the composition of the essential oils obtained from the aerial parts of Fc, Fk, Fp, Fs, Ft and Fv were detected both qualitatively and semi-quantitatively using GC-MS and GC-FID analyses, respectively. All of the essential oils are yellow in color and possess a characteristic odor. Characterization of the essential oils using GC analyses revealed the presence of 106 metabolites ( Table 1, Figures 1 and 2) that account for 92. 10, 96.43, 87.43, 95.95, 92.90 and 89.48% of Fc, Fk, Fp, Fs, Ft and Fv whole essential oils, respectively. Twenty-nine compounds were detected in Fc with α-pinene (21.17%), 10,13 docosadienoic acid methyl ester (15.20%), β-caryophyllene oxide (13.23%) and caryophyllene (10.88%) representing the predominant compounds. Meanwhile, thirty-nine compounds were identified in Fk essential oil with α-pinene (36.79%) and verbenol (8.49%) being the major compounds. In Fp, forty-five compounds were characterized with 4-terpineol (16.28%), α-pinene (10.99%), β-myrcene (6.04%), β-caryophyllene oxide (5.69%), p-cymen-8-ol (5.36%) and spathulenol (5.34%) as the main metabolites in the oil. Furthermore, 15 compounds were determined in Fs oil with the main compounds, palmitic acid, β-myrecene, heptacosane, octacosane, hexacosane and pentcosane accounting for 39.09, 10.75, 10.27, 9.60, 8.99 and 6.29%, respectively. For Ft, 62 compounds were detected of which α-pinene (42.0%), camphene (8.34%) and α-cadinol (8.14%) exist in high percentages in the oil. Finally, 25 compounds were identified in the Fv oil with 10,13 docosadienoic acid methyl ester (69.61%) constituting the major component ( Figure 3). From the data shown in Table 1, it was concluded that monoterpenes are the predominate class of essential oil metabolites in Fc, Fk and Ft, where they represents 24.90, 42.91 and 61.95%, respectively, while oxygenated monoterpenes are the dominant class of metabolites in Fp (35.60%), and they also exist in a high percentage in Fk (34.82%). On the contrary, fatty acids are highly predominate in Fs and Fv and account for 82.55 and 79.84%, respectively. Compounds were identified based on a comparison of the compounds' mass spectral data and retention indices with those of the NIST Mass Spectral Library (December 2011), the Wiley Registry of Mass Spectral Data, 8th edition and by comparison with the authentic standard (AU). The content (%) was calculated using the normalization method based on the GC-FID data generated from the average of three independent chromatographic runs. Compounds were identified based on a comparison of the compounds' mass spectral data and retention indices with those of the NIST Mass Spectral Library (December 2011), the Wiley Registry of Mass Spectral Data, 8th edition and by comparison with the authentic standard (AU). The content (%) was calculated using the normalization method based on the GC-FID data generated from the average of three independent chromatographic runs.

Chemometric Analysis
It is extremely difficult to identify the qualitative and quantitative differences between the Ferula species under evaluation with the naked eye. So, the data obtained from GC analyses were subjected to unsupervised pattern recognition chemometric analysis utilizing PCA to improve the visualization of these differences. The results of the PCA, as represented by the obtained score plot shown in Figure 4A effectively discriminated the six Ferula species into five clusters along the first component (PC1) and the second component (PC2) that account for 57% and 30%, respectively, or 87% of the total variance. From the obtained results, it is obvious that both Fk and Ft are very closely related to each other as they are gathered together in one cluster in the lower left quadrant. However, PC1 successfully discriminated between Fk and Ft with negative values of PC1 as they are located in the lower left quadrant and  Figure 4B.
The Pearson correlation coefficient (r) between the essential oil contents of different studied samples indicated that Fc had a highly significant positive correlation with Ft (r = 0.71), Fk (r = 0.58), Fv (r = 0.47) and Fp (r = 0.35), while a non-significant negative correlation was observed between Fc and Fs (the highest correlations were observed between Ft and Fk (r = 0.89, p < 0.001), between Fc and Ft (r = 0.71, p < 0.001), and between Fc and Fk (r = 0.58, p < 0.001) as seen in Table 2. These data indicate that three samples, Ft, Fk, and Fc have highly similar essential oil content.
The Pearson correlation coefficient (r) between the essential oil contents of different studied samples indicated that Fc had a highly significant positive correlation with Ft (r = 0.71), Fk (r = 0.58), Fv (r = 0.47) and Fp (r = 0.35), while a non-significant negative correlation was observed between Fc and Fs (the highest correlations were observed between Ft and Fk (r = 0.89, p < 0.001), between Fc and Ft (r = 0.71, p < 0.001), and between Fc and Fk (r = 0.58, p < 0.001) as seen in Table 2. These data indicate that three samples, Ft, Fk, and Fc have highly similar essential oil content.   Table 1 where the major discriminatory signals are α-pinene (4), palmitic acid (95) and 10,13-docosadienoic acid methyl ester (102).  The data is represented as the r value of the correlation coefficient and *** is the level of significance, p < 0.001.

Antioxidant Potential of Different Ferula Species
The antioxidant potential of the different essential oil samples was performed in vitro using the 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), the cupric ion reducing antioxidant capacity (CUPRAC), The ferric reducing antioxidant power (FRAP) and the phosphomolybdenum method (PM) assays. The results displayed in Table 3 reveal that most of the samples showed considerable antioxidant potential in the performed assays. Fc (41.36 mgTE/g oil) exhibited the most antioxidant activity in ABTS assays, followed by Fk (29.12 mgTE/g oil) and Ft (28.03 mgTE/g oil). However, in CUPRAC assay, Fp (289.45 mgTE/g oil) showed the most superior antioxidant potential followed by Ft (278.87 mgTE/g oil) and Fk (120.43 mgTE/g oil). Furthermore, Ft exhibited the most significant antioxidant power in both FRAP and PM assays with antioxidant activity equivalent to 136.81 mgTE/g oil and 78.66 mmolTE/g oil, respectively, followed by Fp, which showed antioxidant potential of 121.64 mgTE/g oil and 50.86 mmolTE/g oil in FRAP and PM assays, respectively. Thus, it can be concluded that the essential oil from both Ft and Fp exhibited the most notable antioxidant properties as evidenced by their pronounced activities in most of the performed antioxidant assays, followed by Fc. α-Pinene, the predominant compound in Ft and Fp has previously been shown to possess notable antioxidant activity [15]. Additionally, the significant antioxidant activity found in this study, which can be interpreted as a result of the synergistic action between the different components that exist in the oils, was in accordance with that previously reported for many other Ferula species such as F. microcolea, F. orantalis and F. communis. Various mechanisms can be used to interpret antioxidant potential including the prohibition of chain initiation, peroxide decomposition, obstruction of continual hydrogen removal as well as the scavenging of free radical and uniting transition metal ion catalysts [3,16,17]. Additionally, α-pinene, the main constituent in both Ft and Fp, has previously been shown to be a potent antioxidant in both DPPH and FRAP assays, displaying EC 50 values equal to 310 and 238 µg/mL, respectively [18]. Table 3.

Tyrosinase and α-Amylase Inhibitory Potential
Tyrosinase enzyme is an oxidase enzyme containing copper that assists in the completion of the first two steps of mammalian melanogenesis, which leads to undesirable hyperpigmentation. Thus, the search for effective tyrosinase inhibitors has recently become vital so that they can be incorporated in cosmetics for effective skin whitening and to counteract hyperpigmentation [19]. Fk showed the most effective tyrosinase inhibitory potential, which was estimated as 119.67 mgKAE/g oil followed by Fv, which showed an inhibitory potential equivalent to 118.42 mgKAE/g oil, where KAE is a Kojic acid equivalent, a potent tyrosinase inhibitory drug. Fv oil is rich in 10,13 docosadienoic acid methyl ester, a polyunsaturated fatty acid, which greatly accounts for its promise as a tyrosinase inhibitor [20]. The underlying tyrosinase inhibitory mechanism mainly relies on the essential oils being rich in components that possess a hydrophobic portion that competitively inhibits the active sites of tyrosinase enzyme with subsequent interference of melanin synthesis. This inhibition may be achieved via interaction with Cu +2 that exists in the active sites of tyrosinase in addition to the prohibition of tautomerization to dopachrome triggered by the oil, which behaves as a reducing agent and blocks of the oxidation reaction during the formation of melanin intermediates during the conversion of tyrosinase/DOPA into melanin, thus reducing skin pigmentation [21]. The α-amylase enzyme is critical in assisting in the catalysis of the first steps in the conversion of starch into maltose, and subsequently to glucose [22,23]. Nowadays, α-amylase inhibitors are used in therapeutic approaches to counteract hyperglycemia. Fp and Fv exhibited the most potent α-amylase inhibitory potential as evidenced by their pronounced inhibitory activity, which was equivalent to 2.61 and 1.40 mmol ACAE/g oil, respectively, in which ACAE is the acarbose equivalent, a potent α-amylase inhibitor ( Figure 5). 4-Terpineol as well as α-pinene, which predominate the essential oil of Fp, were previously reported to possess considerable α-amylase inhibitory activity [24]. Similarly, the potent α-amylase inhibitory potential is mainly due to the synergistic action between the different components, which is in accordance to different previously reported studies that confirmed the α-amylase inhibitory effect of different terpenes and different Ferula species such as F. gummosa essential oil [24,25]. [20]. The underlying tyrosinase inhibitory mechanism mainly relies on the essential oils being rich in components that possess a hydrophobic portion that competitively inhibits the active sites of tyrosinase enzyme with subsequent interference of melanin synthesis. This inhibition may be achieved via interaction with Cu +2 that exists in the active sites of tyrosinase in addition to the prohibition of tautomerization to dopachrome triggered by the oil, which behaves as a reducing agent and blocks of the oxidation reaction during the formation of melanin intermediates during the conversion of tyrosinase/DOPA into melanin, thus reducing skin pigmentation [21].
The α-amylase enzyme is critical in assisting in the catalysis of the first steps in the conversion of starch into maltose, and subsequently to glucose [22,23]. Nowadays, α-amylase inhibitors are used in therapeutic approaches to counteract hyperglycemia. Fp and Fv exhibited the most potent αamylase inhibitory potential as evidenced by their pronounced inhibitory activity, which was equivalent to 2.61 and 1.40 mmol ACAE/g oil, respectively, in which ACAE is the acarbose equivalent, a potent α-amylase inhibitor ( Figure 5). 4-Terpineol as well as α-pinene, which predominate the essential oil of Fp, were previously reported to possess considerable α-amylase inhibitory activity [24]. Similarly, the potent α-amylase inhibitory potential is mainly due to the synergistic action between the different components, which is in accordance to different previously reported studies that confirmed the α-amylase inhibitory effect of different terpenes and different Ferula species such as F. gummosa essential oil [24,25].

Preparation of Essential Oil Samples
All the plant materials were air-dried in the shade for 7 days at room temperature and powdered using a mortar and pestle to get particles of a uniform, reduced size. Preparation of the essential oil samples was achieved by hydrodistillation of the air-dried aerial parts of the different Ferula species, F. caratavica (Fc), F. kuchistanica (Fk), F. pseudoreoselinum (Fp), F. samarcandica (Fs), F. tenuisecta (Ft) and F. varia (Fv) for 2 h by Clevenger-type apparatus. Anhydrous Na 2 SO 4 was used to dehydrate the prepared essential oils, yielding 0.4, 0.7, 0.3, 0.3, 0.8 and 0.5 % v/w of dry weight for Fc, Fk, Fp, Fs, Ft and Fv, respectively. Then the various oil samples were maintained at −30 • C in dark-colored stoppered glasses until their analyses [26,27].

GC-FID and GC-MS Analyses
A Shimadzu GC-17A gas chromatograph (Shimadzu Corporation, Kyoto, Japan) with an FID detector and DB-5 fused-bonded cap column (Phenomenex; 29 m × 0.25 mm i.d., film thickness 0.25 µm; Torrance, California, USA) was utilized for the semi-quantitative determination of the different components of the essential oils using the normalization method to get the relative percentage of each component and applying GC-FID data that is highly sensitive using GC solution ® software ver. 2.4 (Shimadzu Corporation, Kyoto, Japan). The areas under the peaks (AUP) were determined using three independent runs where the total area is considered as 100%. Meanwhile, the Shimadzu GC-2010 plus gas chromatograph (Shimadzu Corporation, Kyoto, Japan) supplied with Rtx-5MS (Restek, Bellefonte, PA, USA) in addition to a quadrupole mass spectrometer was used for the identification of the essential oil different metabolites. Instrument settings were adjusted according to what was previously reported [28,29]. The Wiley Registry of Mass Spectral Data 8th edition, NIST MassSpectral Library (December 2011), and previously reported data were employed to confirm the identity of the compounds and the retention indexes were calculated to corroborate the identification of the volatile compounds [30,31].

Chemometric and ANOVA Analysis
To examine the differences between the essential oils' components prepared from different Ferula species, the data collected from the different GC-MS spectra were subjected to chemometric analysis of unsupervised pattern recognition represented by PCA, which was processed by employing Unscrambler 9.7 (CAMO SA, Oslo, Norway) [28,32]. Meanwhile, other statistical analyses used for biological assessment were performed using ANOVA assay (with Tukey's test, significant value: p < 0.05) and Xlstat 2017 software.

Determination of the Antioxidant Potential
The antioxidant activity of the different essential oil samples from different Ferula species was evaluated using ABTS, CUPRAC, FRAP and PM assays. These assays were performed following the methods described by Mamadalieva et al. [33]. The antioxidant activities were reported as Trolox equivalents and the samples were analyzed in triplicate.

Determination of Enzyme Inhibitory Effects
The possible inhibitory potential of the essential oil samples was investigated against tyrosinase and α-amylase enzymes using standard in vitro bioassays as previously reported by Mamadalieva et al. [33] in which all the samples were analyzed in triplicate. Results are expressed in mgKAE/g oil for tyrosinase inhibitory activity and in mmol ACAE/g oil for α-amylase inhibition.

Conclusions
The essential oils obtained from different Ferula species, F. caratavica, F. kuchistanica, F. pseudoreoselinum, F. samarcandica, F. tenuisecta and F. varia showed significant variation as revealed by GC analyses. Furthermore, this variation became more clearly observable when coupled with a chemometric approach as represented by PCA used as an unsupervised pattern recognition technique. Additionally, the obtained essential oils showed notable antioxidant as well as tyrosinase and α-amylase inhibitory activities with variable degrees, which is mainly related to the differences in the secondary metabolites that predominate in the oils. Thus, it was concluded that the different Ferula species could serve as a promising natural antioxidant drug that could be included in different products and used as spices to alleviate hyperglycemia and as a natural ingredient in pharmaceutical cosmetics to counteract hyperpigmentation. Chemometric study based on gathering the different biological activities of many additional Ferula species will be considered. It is recommended that further in vivo studies such as animal and bioavailability studies be carried out to confirm the obtained results.