GC/MS Profiling of the Essential Oil and Lipophilic Extract of Moricandia sinaica Boiss. and Evaluation of Their Cytotoxic and Antioxidant Activities

The genus Moricandia (Brassicaceae) comprises about eight species that were used in traditional medicine. Moricandia sinaica is used to alleviate certain disorders such as syphilis and exhibits analgesic, anti-inflammatory, antipyretic, antioxidant, and antigenotoxic properties. Throughout this study, we aimed to figure out the chemical composition of lipophilic extract and essential oil obtained from M. sinaica aerial parts using GC/MS analysis, as well as their cytotoxic and antioxidant activities correlated with the major detected compounds’ molecular docking. The results revealed that both the lipophilic extract and the oil were found to be rich in aliphatic hydrocarbons, accounting for 72.00% and 79.85%, respectively. Furthermore, the lipophilic extract’s major constituents are octacosanol, γ-sitosterol, α-amyrin, β-amyrin acetate, and α-tocopherol. Contrarily, monoterpenes and sesquiterpenes accounted for the majority of the essential oil. The essential oil and the lipophilic extract of M. sinaica showed cytotoxic properties towards human liver cancer cells (HepG2) with IC50 values of 126.65 and 220.21 µg/mL, respectively. The lipophilic extract revealed antioxidant activity in the DPPH assay with an IC50 value of 2679 ± 128.13 µg/mL and in the FRAP assay, moderate antioxidant potential was expressed as 44.30 ± 3.73 µM Trolox equivalent/mg sample. The molecular docking studies revealed that ꞵ-amyrin acetate, α -tocopherol, γ-sitosterol, and n-pentacosaneachieved the best docking scores for NADPH oxidase, phosphoinositide-3 kinase, and protein kinase B. Consequently, M. sinaica essential oil and lipophilic extract can be employed as a viable management strategy for oxidative stress conditions and the formulation of improved cytotoxic treatment regimens.


Introduction
For millennia, the spotlight has been directed toward medicinal plants as a plentiful supply of bioactive compounds, and many of the therapeutic medications currently in use are natural products or compounds derived from plants [1][2][3][4][5]. According to the World Health Organization (WHO), about 80% of the world's population lives in developing and underdeveloped countries and relies on medicines of natural origin as a remedy for medical ailments [6]. Plants are considered a potential source for the finding of new candidate compounds. Natural plants and their isolated chemicals provide us with a great source of

GC/MS Analysis of the Essential Oil and Lipophilic Extract
The GC/MS analysis investigation of the lipophilic extract and essential oil of M. sinaica is shown in Table 1 (Supplementary Figures S1 and S2). The evaluation of the lipophilic extract and essential oil contents revealed the identification of 15 and 22 chemicals, representing 97.80% and 99.90%, respectively. Both the lipophilic extract and the essential oil were found to be rich in aliphatic hydrocarbons, accounting for 72.00% and 79.85%, respectively, where n-pentacosane (78.01%) is the major hydrocarbon in the essential oil and n-nonacosane (65.66%) is the major hydrocarbon in the lipophilic extract. In the lipophilic extract, octacosanol (12.67%), γ-sitosterol (6.60%), α-amyrin (3.51%), β-amyrin acetate (0.81%), and α-tocopherol (0.54%) are the major constituents. In contrast, monoterpenes in the essential oil (8.10%) are represented as tricyclene, camphene, β-citronellene, octanal, linalool, and α-terpineol, in addition to sesquiterpenes (2.82%) such as α-cadinene, caryophylla-4(12),8(13)-dien-5α-ol, and β-eudesmol. The chemical structures of the major compounds as well as the percentage distribution of volatile components of the lipophilic extract and essential oil of M. sinaica are displayed in Figures 1 and 2. Compounds are listed in the order of elution on the DB-5 GC column. The compound's mass spectral data (MS) and retention indices (RI) were compared to those in the NIST Mass Spectral Library 2011), Wiley Registry of Mass Spectral Data 8th edition, and the literature for identification [22][23][24][25][26]. a Retention index calculated experimentally on DB-5 GC column in comparison to n-alkane series (C8-C28). b Published retention indices.
Prior studies on the chemical characteristics of M. arvensis aerial parts essential oil obtained from two areas in Algeria revealed the richness of the oil of the Southern Setif population with palmitic acid (13.2-12.9%) and phytol (7.9-10.5%). In comparison, the oil of the Northern population is rich with 3-butenylisothiocyanate and octadecanoic acid, 2-hydroxy-1,3-p [27]. Another report by Marrelli et al. revealed the n-hexane extract's chemical composition of M. arvensis using GC/MS analysis. The findings revealed the existence of different fatty acids such as palmitic, stearic, and myristic acids, together with phytosterols such as β-Sitosterol, 22,24-dimethylcholesterol, and stigmasta-3,5-dien-7-one [28]. Comparing the current findings to the previously published findings, both the essential oil and the lipophilic extract revealed variations in the components and their relative quantities that might be utilized as a chemical fingerprint to assess the validity and to discriminate between the given oils or extracts.
ical composition of M. arvensis using GC/MS analysis. The findings revealed the existence of different fatty acids such as palmitic, stearic, and myristic acids, together with phytosterols such as ꞵ-Sitosterol, 22,24-dimethylcholesterol, and stigmasta-3,5-dien-7-one [28]. Comparing the current findings to the previously published findings, both the essential oil and the lipophilic extract revealed variations in the components and their relative quantities that might be utilized as a chemical fingerprint to assess the validity and to discriminate between the given oils or extracts.

Cytotoxic Activity Using SRB Assay
Previous studies on essential oils and lipophilic extracts of M. sinaica reveale richness with potent secondary metabolites that have an effect on numerous cance [23,29]. The cytotoxicity results of the essential oil and the lipophilic extract of M. aerial parts revealed their inhibitory activities toward human liver cancer cells (H with IC50 values of 126.65 and 220.21 µg/mL, respectively ( Figure 3). In agreemen our results, GC/MS evaluation demonstrated the abundance of n-pentacosane in

Cytotoxic Activity Using SRB Assay
Previous studies on essential oils and lipophilic extracts of M. sinaica revealed their richness with potent secondary metabolites that have an effect on numerous cancer cells [23,29]. The cytotoxicity results of the essential oil and the lipophilic extract of M. sinaica aerial parts revealed their inhibitory activities toward human liver cancer cells (HepG2) with IC 50 values of 126.65 and 220.21 µg/mL, respectively ( Figure 3). In agreement with our results, GC/MS evaluation demonstrated the abundance of n-pentacosane in the essential oil of M. sinaica aerial parts (78.01%); it is a 25-carbon unbranched chain that was discovered in several essential oils with antimicrobial properties [30]. Additionally, it was reported to contain anticancer, antifungal, anti-inflammatory, antioxidant, and antiviral agents [31,32]. α-amyrin (3.51%) and β-amyrin acetate (0.81%) are also found to have antioxidant, antimicrobial, anti-inflammatory, and anticancer properties [23]. Octacosanol and 1-Octacosanol were also found to account for 12.67% and 0.50%, respectively. Studies have shown that octacosanol is an antiangiogenic substance that inhibits angiogenesis. Octacosanol prevents neovascularization and the proliferation of endothelial cells [33].

Antioxidant Activity
Natural polyphenols from the Brassicaceae family are abundant, provide a wide range of health implications, and are known for their antioxidant effects [34]. That gives us an interest in exploring the essential oils' and the lipophilic extract's potential as antioxidants using DPPH and FRAP assays. The lipophilic extract in the DPPH assay showed an IC50 value of 2679 ± 128.13 µg/mL as compared to the Trolox reference drug (IC50 = 4.94 ± 0.263 µg/mL). On the other hand, the lipophilic extract showed considerable antioxidant activity in the FRAP assay, expressed as 44.30 ± 3.73µM Trolox equivalent/mg sample. The essential oil of M. sinaica aerial parts did not show any significant results in both assays. Previous reports regarding the biological activity of different Moricandia species revealed that M. nitens green synthesized GNPs showed substantial anti-Helicobacter pylori and anticancer activity against HepG2 and HCT-116 and were reported to be an effective α-glucosidase inhibitor [35]. Moreover, the methanolic extract of M. arvensis exhibited potent lipase-in-

Antioxidant Activity
Natural polyphenols from the Brassicaceae family are abundant, provide a wide range of health implications, and are known for their antioxidant effects [34]. That gives us an interest in exploring the essential oils' and the lipophilic extract's potential as antioxidants using DPPH and FRAP assays. The lipophilic extract in the DPPH assay showed an IC 50 value of 2679 ± 128.13 µg/mL as compared to the Trolox reference drug (IC 50 = 4.94 ± 0.263 µg/mL). On the other hand, the lipophilic extract showed considerable antioxidant activity in the FRAP assay, expressed as 44.30 ± 3.73 µM Trolox equivalent/mg sample. The essential oil of M. sinaica aerial parts did not show any significant results in both assays. Previous reports regarding the biological activity of different Moricandia species revealed that M. nitens green synthesized GNPs showed substantial anti-Helicobacter pylori and anticancer activity against HepG2 and HCT-116 and were reported to be an effective α-glucosidase inhibitor [35]. Moreover, the methanolic extract of M. arvensis exhibited potent lipase-inhibitory activity and antioxidant activity [28].

Molecular Docking
The essential oil and lipophilic extract of M. sinaica, which has potential cytotoxic and antioxidant properties, led us to perform a docking investigation of the major components against the enzymes NADPH oxidase, phosphoinositide-3 kinase (PI3K), and Akt, also known as protein kinase B (PKB). This research aimed to determine the probable binding mechanisms by which the major investigated metabolites may function. Therefore, the major components were docked into the NADPH oxidase, phosphoinositide-3 kinase (PI3K), and Akt 3D coordinates that were downloaded from the protein data bank using the following PDB IDs: 2cdu, 1E90, and 3cwq, respectively. Re-docking each co-crystallized ligand into its associated active site enabled us to confirm the docking parameters applied. The estimated RMSD values between the co-crystallized pose and the docked pose were 1.02, 0.97, and 1.03 Å NADPH oxidase, phosphoinositide-3 kinase (PI3K), and Akt, respectively, helping to ensure the docking technique is valid. The co-crystallized ligand's re-docking resulted in docking scores of −11.3, −12.1, and −13.4 Kcal/mole for NADPH oxidase, phosphoinositide-3 kinase (PI3K), and Akt, respectively. The docking of the main metabolites to the three enzymes showed acceptable results corresponding to those of the reference compounds. Interestingly, in the docking with NADPH oxidase, β-amyrin acetate, α-tocopherol and γ-sitosterol were the best compounds, achieving docking scores of −14.19, −13.12, and −12.65 Kcal/Mol, respectively ( Figure 4). Similarly, in the docking with PI3K, n-pentacosane, α-tocopherol and β-amyrin acetate were the best compounds, achieving docking scores of −13. 91, −14.15, and −14.05 Kcal/Mol, respectively ( Figure 5). Worth noting, in the docking with AKT, n-pentacosane, α-tocopherol, and γ-sitosterol were the best compounds, achieving docking scores of −11. 9, −13.19, and −12.05 Kcal/Mol, respectively ( Figure 6). The major compounds' docking scores against the three proposed target enzymes are represented in Table 2

The Essential Oil Isolation
The leaves were finely chopped and hydro-distilled for 5 h with a Clevenger apparatus. The produced oil is yellowish orange in color with a fragrant odor; the yield was 0.21% (21 mg/100 g). It was collected and maintained at −4 • C in a tight, dark glass vial for GC/MS analysis.

Preparation of the Lipophilic Extract
Dry leaves (100 g) of Moricandia sinaica Boiss. were extracted three times with n-hexane. To obtain the dried residue of the lipophilic extract, the filtrate was fully evaporated in vacuo at 40 • C until dryness, and (2.41 g) of the lipophilic extract was obtained. The lipophilic extract was kept in a tightly sealed container for subsequent examination.

Gas Chromatography-Mass Spectrometry (GC/MS)
A Shimadzu GCMS-QP 2010 chromatograph (Kyoto, Japan) with a DB-5 capillary column (30 m × 0.25 mm i.d. × 0.25 µm film thickness; Restek, Bellefonte, PA, USA) was used for gas chromatography/mass spectrometry (GC/MS) analysis. The oven temperature was set at 50 • C for 3 min (isothermal), programmed to 30 • C at a rate of 5 • C/min, and kept constant at 300 • C for 10 min (isothermal); the temperature of the injector was 280 • C. Helium was employed as the carrier gas, with a flow rate of 1.40 mL/min. Diluted samples (1% v/v) were injected at a split ratio of 15:1 in a volume of 1 µL. The following were the MS running specifications: 280 • C for the interface, 220 • C for the ion source, 70 eV for the EI mode, and 35-500 amu for the scan range.

Characterization of the Essential Oil and Lipophilic Extract Components
The volatile constituents were characterized based on their retention indices and fragmentation patterns matching with NIST Mass Spectral Library, Wiley library database and published in the literature [23,26,29,[36][37][38][39]. Retention indices (RI) were estimated in comparison to homologous series of n-alkanes (C8-C30) injected under the same conditions.

Assessment of Cytotoxic Activity Using SRB Assay
Nawah Scientific Inc., (Mokatam, Cairo, Egypt) provided the Hepatocellular carcinoma (HepG2). Cells were cultured in DMEM media treated with 100 mg/mL of streptomycin, 100 units/mL of penicillin, and 10% heat-inactivated fetal bovine serum in a humidified, 5% (v/v) CO 2 atmosphere at 37 • C. The SRB assay was employed to assess cell viability. In 96-well plates, aliquots of 100 µL cell suspension (5 × 10 3 cells) were incubated in complete media for 24 h. Another aliquot of 100 µL of media containing different doses of drugs was used to treat the cells. Cells were fixed by replacing media with 150 µL of 10% TCA and incubated at 4 • C for 1 h, after 72 h of drug exposure. After removing the TCA solution, the cells were washed 5 times with distilled water. Aliquots of 70 µL SRB solution (0.4% w/v) were added and incubated in a dark place at room temperature for 10 min. Plates were washed with 1% acetic acid for 3 times before being let to air dry overnight. Then, 150 µL of TRIS (10 mM) was added to dissolve protein-bound SRB stain; with the use of a BMGLABTECH®-FLUO star Omega microplate reader (Ortenberg, Germany), the absorbance was determined at 540 nm [40].

DPPH Assay
Samples were prepared using DMSO at a concentration of 50 mg/mL. Then, from this stock, the oil was prepared at a concentration of 2 mg/mL in methanol, and the lipophilic extract was prepared at a concentration of 10 mg/mL in methanol. A stock solution of 20 µg/mL of Trolox was prepared in methanol, from which 5 concentrations were prepared, including 12.5, 7.5, 6.25, 2.5, and 1.25 µg/mL. DPPH (2,2-diphenyl-1-picrylhydrazyl-hydrate) free radical assay was carried out according to the method of [41]. Briefly, at room temperature, 100 µL of freshly prepared DPPH reagent (0.1% in methanol) was added to 100 µL of the sample in 96-well plates (n = 6), which was incubated for 30 min in the dark. The resulting reduction in DPPH color intensity was measured at 540 nm after the incubation time. The data are displayed as means ± SD according to the following equation: Percentage inhibition = Average absorbance of blank − average absorbance of the test Average absorbance of blank × 100 The microplate reader FluoStar Omega was used for recording the results. The data were processed using Microsoft Excel ® and the IC 50 value calculation was performed using Graph pad Prism 6®by converting the concentrations to their logarithmic value and selecting a non-linear inhibitor regression equation (log (inhibitor) vs. normalized response-variable slope equation).

FRAP Assay
Trolox Standard for FRAP assay Trolox stock solution of 3 mM in methanol was prepared, and the following dilutions were prepared at the concentrations of 1200, 1000, 800, 500, 400, 300, 200, and 100 µM. Samples were prepared at a concentration of 50 mg/mL in DMSO. Then, the oil was prepared at a concentration of 2 mg/mL in methanol, and the lipophilic extract was prepared at a concentration of 10 mg/mL in methanol. The assay was carried out in accordance with the method of Benzie et al. [42] with some modifications to be carried out in microplates. A freshly prepared TPTZ reagent (300 mM Acetate Buffer (PH = 3.6), 10 mM TPTZ in 40 mM HCl, and 20 mM FeCl 3 , in a ratio of 10:1:1 v/v/v, respectively). About 190 uL of the freshly prepared TPTZ reagent were mixed with 10 µL of the sample in 96-well plates (n = 3), the reaction was incubated at room temperature for 30 min in the dark. The resulting blue color was measured at 593 nm at the end of the incubation period. The data are represented as means ± SD. The microplate reader FluoStar Omega was used to record the results. The ferric-reducing ability of the samples is presented as µM TE/ mg sample using the linear regression equation obtained from the following calibration curve (linear dose-response curve of Trolox) (Figure 7). the use of a BMGLABTECH®-FLUO star Omega microplate reader (Ortenberg, Germany), the absorbance was determined at 540 nm [40].

DPPH Assay
Samples were prepared using DMSO at a concentration of 50 mg/ml. Then, from this stock, the oil was prepared at a concentration of 2 mg/ml in methanol, and the lipophilic extract was prepared at a concentration of 10 mg/ml in methanol. A stock solution of 20 μg/mL of Trolox was prepared in methanol, from which 5 concentrations were prepared, including 12.5, 7.5, 6.25, 2.5, and 1.25 μg/mL. DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) free radical assay was carried out according to the method of [41]. Briefly, at room temperature, 100 μL of freshly prepared DPPH reagent (0.1% in methanol) was added to 100 μL of the sample in 96-well plates (n=6), which was incubated for 30 min in the dark. The resulting reduction in DPPH color intensity was measured at 540 nm after the incubation time. The data are displayed as means ± SD according to the following equation:

Percentage inhibition
Average absorbance of blank average absorbance of the test Average absorbance of blank 100 The microplate reader FluoStar Omega was used for recording the results. The data were processed using Microsoft Excel® and the IC50 value calculation was performed using Graph pad Prism 6® by converting the concentrations to their logarithmic value and selecting a non-linear inhibitor regression equation (log (inhibitor) vs. normalized response-variable slope equation).

FRAP Assay
Trolox Standard for FRAP assay Trolox stock solution of 3 mM in methanol was prepared, and the following dilutions were prepared at the concentrations of 1200, 1000, 800, 500, 400 ,300, 200, and 100 μM. Samples were prepared at a concentration of 50 mg/ml in DMSO. Then, the oil was prepared at a concentration of 2 mg/ml in methanol, and the lipophilic extract was prepared at a concentration of 10 mg/ml in methanol. The assay was carried out in accordance with the method of Benzie et al. [42] with some modifications to be carried out in microplates. A freshly prepared TPTZ reagent (300 mM Acetate Buffer (PH=3.6), 10 mM TPTZ in 40 mM HCl, and 20 mM FeCl3, in a ratio of 10:1:1 v/v/v, respectively). About 190 uL of the freshly prepared TPTZ reagent were mixed with 10 uL of the sample in 96-well plates (n=3), the reaction was incubated at room temperature for 30 min in the dark. The resulting blue color was measured at 593 nm at the end of the incubation period. The data are represented as means ± SD. The microplate reader FluoStar Omega was used to record the results. The ferric-reducing ability of the samples is presented as μM TE/ mg sample using the linear regression equation obtained from the following calibration curve (linear dose-response curve of Trolox) (Figure 7).

Molecular Docking
Applying the software Molecular Operating Environment (MOE 2019.02), docking investigations were carried out [43,44]. The X-ray crystal structures of NADPH oxidase, phosphoinositide-3 kinase (PI3K), and Akt, known as protein kinase B (PKB) were obtained from the protein data bank www.pdb.org (accessed on 10 December 2022) using the following PDB IDs: 2CDU, 1E90, and 3CWQ, respectively [45][46][47]. Hydrogens and charges of the receptors were adjusted through AMBER10: EHT implanted in MOE software. The relevant co-crystallized ligand is bound at the established binding sites of three enzymes. Five major compounds identified in the essential oil (n-pentacosane, camphene, β-citronellene, linalool, and α-terpineol) and six compounds from the lipophilic extract (n-nonacosane (65.66%), octacosanol, γ-sitosterol, α-amyrin and β-amyrin acetate, and α-tocopherol) were produced using the 2D builder of MOE2019 and transformed into 3D structures with the same software. Utilizing the triangular matcher and London dg, the compounds were docked onto the three enzyme binding sites as placement and scoring methods, respectively. Finally, MOE produced 2D interaction diagrams to analyze the docking observations.

Conclusions
In the present research, the chemical investigation of Moricandia sinaica lipophilic extract and essential oil was analyzed using the GC/MS technique. The results revealed that both are rich in aliphatic hydrocarbons, where n-pentacosane (78.01%), and n-nonacosane (65.66%) are the major hydrocarbons in the essential oil and the lipophilic extract. It is worth noting that γ-sitosterol, α-amyrin, β-amyrin acetate, and α-tocopherol are the main important constituents in the lipophilic extract that correlate with its antioxidant activity using DPPH and FRAP assays. In addition, monoterpenes and sesquiterpenes are present in the essential oil, which correlates with its cytotoxic activity against HEPG-2 cells. The biological investigations were supported by the molecular docking study and interactions of the major constituents with NADPH oxidase, phosphoinositide-3 kinase (PI3K), and Akt, known as protein kinase B (PKB) enzymes. Accordingly, the lipophilic extract and essential oil of M. sinaica would be a promising alternative for the production of cytotoxic and antioxidant agents, supported by further in vivo investigations as well as pharmacodynamic and pharmacokinetic studies.

Conflicts of Interest:
The authors declare no conflict of interest.