Aroma Profile and Biological Effects of Ochradenus arabicus Essential Oils: A Comparative Study of Stem, Flowers, and Leaves

The present analysis explores the chemical constituents and determines the in vitro antimicrobial, antidiabetic, and antioxidant significance of the essential oils (EOs) of the stem, leaves, and flowers of Ochradenus arabicus for the first time. The EOs of the flowers presented seventy-four constituents contributing to 81.46% of the total EOs, with the major compounds being 24-norursa-3,12-diene (13.06%), 24-norursa-3,12-dien-11-one (6.61%), and 24-noroleana-3,12-diene (6.25%). The stem EOs with sixty-one compounds contributed 95.95% of the total oil, whose main bioactive compounds were (+)-camphene (21.50%), eremophilene (5.87%), and δ-selinene (5.03%), while a minimum of fifty-one compounds in the leaves’ EOs (98.75%) were found, with the main constituents being n-hexadecanoic acid (12.32%), octacosane (8.62%), tetradecanoic acid (8.54%), and prehydro fersenyl acetone (7.27%). The antimicrobial activity of the EOs of O. arabicus stem, leaves, and flowers was assessed against two bacterial strains (Escherichia coli and Streptococcus aureus) and two fungal strains (Penicillium simplicissimum and Rhizoctonia solani) via the disc diffusion assay. However, the EOs extracted from the stem were found effective against one bacterial strain, E. coli, and one fungal strain, R. Solani, among the examined microbes in comparison to the standard and negative control. The tested EOs samples of the O. arabicus stem displayed a maximum potential to cure diabetes with an IC50 = 0.40 ± 0.10 µg/mL, followed by leaves and flowers with an IC50 = 0.71 ± 0.11 µg/mL and IC50 = 10.57 ± 0.18 µg/mL, respectively, as compared to the standard acarbose (IC50 = 377.26 ± 1.20 µg/mL). In addition, the EOs of O. arabicus flowers had the highest antioxidant activity (IC50 = 106.40 ± 0.19 µg/mL) as compared to the standard ascorbic acid (IC50 = 73.20 ± 0.17 µg/mL) using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. In the ABTS assay, the EOs of the same sample (flower) depicted the utmost potential to scavenge the free radicals with an IC50 = 178.0 ± 0.14 µg/mL as compared with the ascorbic acid, having an IC50 of 87.34 ± 0.10 µg/mL the using 2,2-Azino-Bis-3-Ethylbenzothiazoline-6-Sulfonic acid (ABTS) assay. The EOs of all parts of O. arabicus have useful bioactive components due to which they present antidiabetic and antioxidant significance. Furthermore, additional investigations are considered necessary to expose the responsible components of the examined biological capabilities, which would be effective in the production of innovative drugs.


Introduction
The genus Ochradenus belongs to the family Resedaceae, is represented by eight species, and is mostly distributed in the desert and arid regions of the Arabian Peninsula,

Essential Oils
The essential oils (EOs) from the stem, leaves, and flowers were extracted via hydrodistillation (three times) employing an 8-quart stove still home distillation unit and Clevengertype device. The Clevenger machine was operated until no further oil could be extracted. The EOs were collected from the top of the hydrosol and the oils collected in the burette were calculated. Moreover, the obtained oils were passed through an anhydrous sodium sulphate to remove any moisture and placed in the refrigerator to avoid the loss of the essential constituents in the essential oils.

GC-MS Chemical Analysis
The EO composition from the various parts (stem, leaves, and flowers) of the plant was profiled via GC-MS analysis. The GC-MS device contains the Perkin Elmer Clarus (PEC) 600 GC system, coupled by Rtx-5MS with the capillary column (30 m × 0.25 mm I.D × 0.25 µm film thickness; a maximum temperature of 260 • C), attached to a PEC 600 MS. Ultra-high purity helium (99.99%) was consumed as a carrier gas with a flow rate of 1.0 mL/min. The temperatures of the injection, transfer line, and ion source were 260, 270, and 280 • C, correspondingly. The ionization energy was observed at 70 eV and the electron multiplier (EM) voltage was attained from auto-tune. All the data of the EOs were achieved by screening the full mass spectra within the 45-550 a.m.u. scan range. The volume of the injected sample was 1 µL, having a split ratio of 10:1. The oven temperature was operated at 60 • C (for 1 min) at a rate of 4 • C/min-260 • C held for 4 min. The sample completed its run in 50 min.

Compound Identification
The chemical constituents in the EOs of all parts of the selected plant O. arabicus were determined through GC-MS and the unknown constituents were identified using mass spectral library software (NIST 2011 v.2.3, Gaithersburg, MD, USA) and with the already reported literature. Moreover, the use of RI (obsd.) compared with RI (Lit.) and co-injection with an available authentic sample of detected compounds to GC or GC/MS were also used for the identification of chemical constituents. The quantification was performed by using an external standard technique via calibration curves produced by operating the GC profiling of the representative compounds.

Antimicrobial Activity
The significance of the EOs in the O. arabicus stem, leaves, and flowers were comparatively examined via disc diffusion using a systematic approach [23]. The cultures for the three fungal and two bacterial strains were refreshed through potato dextrose agar (PDA) and nutrient agar (NA), respectively. Petri plates were covered with a parafilm to avoid contamination, and the fungal (28 • C) and bacterial (37 • C) cultures were checked after 72 and 24 h to screen the antimicrobial bioassay for the tested samples. The inoculum was prepared by the addition of a small colony obtained from the fungal and bacterial strains with normal saline water (0.9% NaCl) in a 1 mL Eppendorf tube and vortexed to make it homogenous. Moreover, the turbidity of the Eppendorf tube was compared with McFarland standard solution. Then, a sterile cotton swab was dipped into the normal saline suspension and well streaked over the entire surface of the plate containing PDA and NA medium to ensure the uniform distribution of the inoculum. Afterwards, the bacterial and fungus inoculation was performed on the plates.

Microbial Strains
The bacterial strains (ATCC, E. coli, and S. aureus) and clinically isolated fungal strains (P. simplicissimum, R. solani, and F. fujikuroi) were used in the current study. Streptomycin and Topsin-M 70 WP were applied as the positive control and dimethylsulfoxide (DMSO) was used as the negative control in antimicrobial activity.

Lawn Preparation
The fungal and bacterial lawn was achieved using PDA and NA media, respectively, and sterilized cotton swabs were used. The EOs from each sample (stem, leaves, and flower) at concentrations of 25, 50, and 100 µL per well were obtained using the disc diffusion method [24]. Furthermore, the bacterial Petri plates were placed at 37 • C and the fungal strains plates were kept at 25 • C in incubators for 24-48 h and inhibition zones (mm) were measured. Streptomycin was used as the positive control in antibacterial activity and Topsin-M 70 WP was used in antifungal activity. DMSO was used as the negative control using the same concentrations.

α-Glucosidase Activity
The α-Glucosidase activity was performed using 0.1 M phosphate buffer having pH 6.8 at 37 • C [25,26]. The α-glucosidase enzyme (0.2 µg/mL) was incubated in phosphatebuffered saline with various concentrations of EOs at 37 • C for 15 min followed by the addition of 0.7 mM substrate (p-nitrophenyl-α-D-glucopyranoside) and the alteration in absorbance was observed for 30 min at 400 nm using a spectrophotometer (Spectra Max M2, Molecular Devices, San Jose, CA, USA). The tested oils were replaced with deuterated

Antioxidant Evaluation
The antioxidant significance of the EOs of the studied samples was proceeded using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and (ABTS) 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid assays [1][2][3]. To proceed with the DPPH assay, around 3 mg of DPPH was standardized in 100 mL distilled methanol to form the free radicals and the mixture was placed in the dark for 30 min. The tested samples at dosages of 1000, 500, 250, 125, and 62.5 µg/mL were well-prepared. Next, about 2 mL from the tested samples were added up to 2 mL of the already prepared DPPH stock solution and then again incubated for approximately 20 min in the dark. The absorbance of the tested samples was examined at 517 nm applying UV/Vis spectrophotometer and the % potential was determined via Equation (2). % Antioxidant activity = 1 − 2/1 × 100 (2) where 1 signifies the control absorbance and 2 denotes the standard absorbance. The ABTS assay was carried out by taking around 383 mg of ABTS and about 66.2 mg of the K 2 S 2 O 8 separately in 100 mL of MeOH and properly homogenized and, after that, both were combined. Later, around 2 mL of the mixture were arranged to incubate with around 2 mL of examined samples at dosages of 1000, 500, 250, 125, and 62.5 µg/mL for 25 min. Lastly, the absorbance of the analyzed samples was calculated at 746 nm using a UV spectrophotometer, and the significance was estimated using the same Equation (2).

Statistical Analysis
The following programs were utilized to analyze the attained results for biological activity. SoftMax Pro package and Excel were utilized. EZ-FIT (Perrella Scientific, Inc., Amherst, NH, USA) was used for the IC 50 calculations of all the tested samples. To overcome the expected errors, all experiments were performed in triplicate, and variations in the results are reported as standard error of mean values (SEM), as reported by Akhter et al. [27].

Results and Discussion
Medicinal plants and their products have promising therapeutic capabilities and serve as a local remedy as well as providing valuable innovative constituents for the mass production of medicines [26,28]. Plant constituents are different since the diverse range of ecological amplitudes alters the composition of the chemical components, which are the promising sources to treat various human health complications [29][30][31].

Essential Oil Composition
The EOs obtained from the various parts (flowers, stems, and leaves) of O. arabicus had a mild scent and had the appearance of a yellow fluid lighter than water. The GC-MS analysis revealed that the stem, leaves, and flowers of the selected plant are a prominent source of bioactive chemical components. However, a maximum of 74 bioactive compounds were noticed in flowers, contributing 81.46% of the total and whose dominant constituents were 24-norursa-3,12-diene (13.06%), 24-norursa-3,12-dien-11-one (6.61%), and 24-noroleana-3,12diene (6.25%), followed by the stems with 62 compounds producing 89.52%, with the main constituents being (+)-camphene (21.50%), eremophilene (5.87%), and δ-selinene (5.03%). The least number of compounds were detected in leaves (46), which yields 98.75% of the total oil, with the key compounds being n-hexadecanoic acid (12.32%), followed by octacosane (8.62%) and tetradecanoic acid (8.54%). Chromatograms are shown in Figure 1. The chemical components 24-norursa-3,12-diene, 24-norursa-3,12-dien-11-one, 24-noroleana-3,12diene, methyl pimar-8-en-18-oate, and 24-norursa-3,9(11),12-triene) are reported, up to our best knowledge, for the first time from the flowers essential oil of O. arabicus, while having been earlier documented in the literature for the genus Boswellia [18][19][20].   The aforementioned bioactive components have significant potential to inhibit the microbial resistance to overcome microbial infections, neutralize the free radicals to prevent cellular damage, and also have promising capacities to act as an anti-cancerous agent, as shown by Hussain et al. [32] and Sonigra et al. [33]. The O. arabicus stem contains compounds such as (+)-camphene, eremophilene, δ-selinene, β-elemene, and β-eudesmol, already reported by Chandra et al. [34] in Callicarpa macrophylla and Utegenova et al. [35] in some species of the genus Ferula, who also highlight their potential against human pathogenic microbes. The EOs of the O. arabicus leaves are characterized by n-hexadecanoic acid, which was reported earlier by Ravi et al. [36], and is well known for its therapeutic significance. The same parts of O. arabicus also contained octaconsane, tetradecanoic acid, perhydro farnesyl acetone, and hexacosane, which have been previously reported by Swamy et al. [37] in Plectranthus amboinicus leaves, and have an antimicrobial effect. Moreover, in the present study, it was revealed that all parts (flowers, stems, and leaves) have eleven common compounds, which accounted for 41.85%, 25.82%, and 12.96%, respectively, indicating that these are the prominent compounds present in the plant ( Table 1). Some of bioactive compounds showed similarity between the flowers', stems', and leaves' oil constituents ( Figure 2). Among these constituents, (+)-camphene with 21.50% is the most dominant compound in the stem of O. arabicus and has significant potential against various health complications, which was reported by Vallianou et al. [38], as well as, especially, regulating metabolic disorder, as shown by Noma et al. [39].

Antimicrobial Capabilities
To discover a novel antimicrobial agent, we used EOs of O. arabicus stems, leaves, and flowers against the human pathogenic bacterial strains Escherichia coli and Streptococcus aureus, and the fungal strains Penicillium simplicissimum, Rhizoctonia solani, and Fusarium fujikuroi. As different parts of the same plant have different bioactive compounds, some EOs were active as compared to others ( Table 2). Among the tested samples, the EOs of the O. arabicus stem showed a significant capacity against the Gram-negative bacteria E. coli. This activity was due to the presence of bioactive compounds such as α-campholenal, L-pinocarveol, and myrtenol, which have been earlier reported to have antibacterial potential (Zhang et al. [40], Bansal et al. [41], and Cordeiro et al. [42]). The essential oil of O. arabicus leaves also showed better antifungal activity against P. simplicissimum. Our results are supported by Sousa et al. [43] as the dominant compound spathulenol in the EOs of Eugenia calycina leaf exhibited antibacterial capabilities against the anaerobic Gram-negative bacteria prevotella nigrescens in a concentration of 100 µg/mL. Moreover, the antifungal activity of the EOs of the leaves of O. arabicus against P. simplicissimum and R. solani is in complete agreement with the documented results of Yu et al. [44] and Golus et al. [45]. The plant contains caryophyllene, β-caryophyllene, and dodecanoic acid, which have antifungal significance, as reported by Ghaffari et al. [46], Dahham et al. [47], and Wu et al. [48].

Antimicrobial Capabilities
To discover a novel antimicrobial agent, we used EOs of O. arabicus stems, leaves, and flowers against the human pathogenic bacterial strains Escherichia coli and Streptococcus aureus, and the fungal strains Penicillium simplicissimum, Rhizoctonia solani, and Fusarium fujikuroi. As different parts of the same plant have different bioactive compounds, some EOs were active as compared to others ( Table 2). Among the tested samples, the EOs of the O. arabicus stem showed a significant capacity against the Gram-negative bacteria E. coli. This activity was due to the presence of bioactive compounds such as α-campholenal, L-pinocarveol, and myrtenol, which have been earlier reported to have antibacterial potential (Zhang et al. [40], Bansal et al. [41], and Cordeiro et al. [42]). The essential oil of O. arabicus leaves also showed better antifungal activity against P. simplicissimum. Our results are supported by Sousa et al. [43] as the dominant compound spathulenol in the EOs of Eugenia calycina leaf exhibited antibacterial capabilities against

In Vitro Antidiabetic Potential
Medicinal plants are rich sources of chemical components that have promising potential to serve as a remedy for various human health complications, including diabetes mellitus. Hence, due to a lack of scientific literature, the EOs of O. arabicus stems, leaves, and flowers were investigated for their antidiabetic ability against α-glucosidase ( Figure 3). As is known, different parts of plants have variations in the constituents, which is why the EOs extracted from the stem displayed the most potent inhibitory activity (IC 50 = 0.40 ± 0.10 µg/mL) in comparison with the leaves' EOs with an IC 50 value of 0.71 ± 0.11 µg/mL and the EOs of the flowers of O. arabicus, with a value of IC 50 = 10.57 ± 0.18 µg/mL, compared to standard (IC 50 = 377.26 ± 1.20 µg/mL). Therefore, our findings are the same as those described by Tahir et al. [49] and Akolade et al. [50]. The outcomes of our project also are in accordance with the results of Numonov et al. [51]. The antidiabetic potential is due to the presence of common compounds present in the plant, such as cembrene A, caryophyllene oxide, linalool, nonanal, and dibutyl phthalate, which has been reported before by Marshall et al. [52], Kaur et al. [53] More et al. [54], Chhikara et al. [55], and Keerthana et al. [56], respectively.

Antioxidant Significance
The essential oils of the studied O. arabicus samples (flowers, leaves, and stems) had the free-radical scavenging capabilities, which were assessed using DPPH and ABTS bioassay. The EOs had considerable potential in both assays ( Figure 4A,B). The flowers of the plant under study presented the highest significance with IC50 = 106.40 ± 0.19 µg/mL, followed by the leaves and stems with an IC50 = 143.80 ± 0.22 µg/mL and 159.60 ± 0.32 µg/mL, respectively, as compared with the standard, with an IC50 = 73.20 ± 0.17 µg/mL. Moreover, in the ABTS assay, the flowers of the selected plant had, followed by the leaves and stems, an IC50 = 178.0 ± 0.14, 205.50 ± 0.15, and 226.60 ± 0.20 µg/mL, respectively. Furthermore, the utmost effect was detected in the DPPH assay ( Figure 4A) with IC50 = 73.20 ± 0.17 µg/mL in comparison with the ABTS assay IC50 = 87.34 ± 0.10 µg/mL ( Figure 4B). The capacity to scavenge free radicals is mainly attributed due to the presence of α-pinene, (+)camphene, and thymol, which have promising capacities to neutralize free radicals as stated by Wang et al. [4], Yang et al. [5], and Yildiz et al. [6], respectively. The plant extract has already been reported for its antioxidant impact, as stated by Alshamsi et al. [7], but the essential oils composition through GC-MS analysis of the selected plant parts were applied here for the first time. Our findings are in agreement with the data of Ben Nouri et al. [8] for Cupressus sempervirens and of Nigella sativa as reported before by [9]; other essential oils extracted from other plants were described by Anthony et al. [10]. However, our outcomes have little variation from the literature, especially that presented by Thusoo et al. [11] for Valeriana jatamansi and Diniz do Nascimento et al. [12] for some spice plants, which were studied for their free-radical scavenging potential. The chemical components in a plant are responsible for addressing various ailments, including antioxidant potential [13,14], whose contents are influenced by various factors such as edaphic, climatic, and topographic factors, as shown by Sampaio et al. [15]. The quantity of the active ingredients may be affected due to the quality of water, as shown in the work of Ghani et al. [16].

Antioxidant Significance
The essential oils of the studied O. arabicus samples (flowers, leaves, and stems) had the free-radical scavenging capabilities, which were assessed using DPPH and ABTS bioassay. The EOs had considerable potential in both assays ( Figure 4A,B). The flowers of the plant under study presented the highest significance with IC 50 = 106.40 ± 0.19 µg/mL, followed by the leaves and stems with an IC 50 = 143.80 ± 0.22 µg/mL and 159.60 ± 0.32 µg/mL, respectively, as compared with the standard, with an IC 50 = 73.20 ± 0.17 µg/mL. Moreover, in the ABTS assay, the flowers of the selected plant had, followed by the leaves and stems, an IC 50 = 178.0 ± 0.14, 205.50 ± 0.15, and 226.60 ± 0.20 µg/mL, respectively. Furthermore, the utmost effect was detected in the DPPH assay ( Figure 4A) with IC 50 = 73.20 ± 0.17 µg/mL in comparison with the ABTS assay IC 50 = 87.34 ± 0.10 µg/mL ( Figure 4B). The capacity to scavenge free radicals is mainly attributed due to the presence of α-pinene, (+)-camphene, and thymol, which have promising capacities to neutralize free radicals as stated by Wang et al. [4], Yang et al. [5], and Yildiz et al. [6], respectively. The plant extract has already been reported for its antioxidant impact, as stated by Alshamsi et al. [7], but the essential oils composition through GC-MS analysis of the selected plant parts were applied here for the first time. Our findings are in agreement with the data of Ben Nouri et al. [8] for Cupressus sempervirens and of Nigella sativa as reported before by [9]; other essential oils extracted from other plants were described by Anthony et al. [10]. However, our outcomes have little variation from the literature, especially that presented by Thusoo et al. [11] for Valeriana jatamansi and Diniz do Nascimento et al. [12] for some spice plants, which were studied for their free-radical scavenging potential. The chemical components in a plant are responsible for addressing various ailments, including antioxidant potential [13,14], whose contents are influenced by various factors such as edaphic, climatic, and topographic factors, as shown by Sampaio et al. [15]. The quantity of the active ingredients may be affected due to the quality of water, as shown in the work of Ghani et al. [16].
which were studied for their free-radical scavenging potential. The chemical components in a plant are responsible for addressing various ailments, including antioxidant potential [13,14], whose contents are influenced by various factors such as edaphic, climatic, and topographic factors, as shown by Sampaio et al. [15]. The quantity of the active ingredients may be affected due to the quality of water, as shown in the work of Ghani et al. [16].

Conclusions
EOs contain bioactive constituents that serve as a basis for the pharmaceutical trade nutraceutical supplements, and due to their fragrance, are required for producing cosmet ics and perfumes. They also have a significant role in resisting microbes as well as regu lating metabolic disorders and scavenging free radicals, acting as a natural remedy. The

Conclusions
EOs contain bioactive constituents that serve as a basis for the pharmaceutical trade, nutraceutical supplements, and due to their fragrance, are required for producing cosmetics and perfumes. They also have a significant role in resisting microbes as well as regulating metabolic disorders and scavenging free radicals, acting as a natural remedy. The EOs of O. arabicus presented significant bioactive compounds in all parts (stems, leaves, and flowers). Among the tested samples, the O. arabicus flowers' EOs had seventy-four chemical constituents, ensued by the stem EOs with sixty-one compounds, while a minimum of fifty-one compounds were found in the leaves' EOs. In conclusion, (+)-camphene (21.50%) was the major compound detected in the stems' EOs, hexadecanoic acid (12.32%) in the EOs of leaves, and 24-Norursa-3,12-diene (13.06%) in the flowers' oils. The stems' EOs of O. arabicus were found effective against the human pathogenic bacterial strain E. coli and the fungal strain R. solani. In addition, the other tested samples were observed to be inactive against the examined microbes, while substantial in vitro α-glucosidase activity was noticed in all parts of the plant and, particularly, the EOs of the O. arabicus stems can act out as an antidiabetic agent. The essential oils have a promising potential to scavenge free radicals. However, additional studies are still essential to emphasize and isolate new chemical constituents responsible for the observed activities.