Essential Oils Composition and Biological Activity of Chamaecyparis obtusa, Chrysopogon nigritanus and Lavandula coronopifolia Grown Wild in Sudan

Generally, there are scant data about the constituents and eventually the biological activity of essential oils (EOs) from aromatic plants that grow naturally in Sudan. The present study aimed to determine the chemical composition, and antioxidant and enzyme inhibitory activities of EO extracted from the fruit of Chamaecyparis obtusa (Siebold and Zucc.) Endl. (family Cupressaceae), root of Chrysopogon nigritanus (Benth.) Veldkampis (family Poaceae) and aerial part of Lavandula coronopifolia Poir (family Lamiaceae). The fruit of C. obtusa contained only monoterpenes, mainly hydrogenated ones, with α-pinene (69.07%) as the major component. Oxygenated sesquiterpenes comprised the highest content of the C. nigritanus root EO with cedr-8-en-15-ol (28.69%) as the major constituent while aerial parts of L. coronopifolia contained both monoterpenes and sesquiterpenes and the oxygenated monoterpene lavandulol (26.56%) as dominant compounds. The EO of the root of C. nigritanus significantly displayed (p < 0.05) the highest anti-DPPH radical, Fe3+- and Cu2+-reducing and metal-chelating activities, while that of C. obtusa fruit significantly exerted (p < 0.05) the best anti-ABTS radical and total antioxidant activity. The two EOs significantly exhibited (p < 0.05) the highest anti-acetylcholinesterase and -butyrylcholinesterase activities, respectively, while EO of L. coronopifolia was the only oil to show a considerable inhibitory effect against the tyrosinase and α-glucosidase enzymes. In conclusion, EOs from these three plants could be natural agents with promising functional properties for food, cosmetics, and pharmaceutical applications.


Introduction
Essential oils (EOs) extracted from various herbs and spices have been a subject of intensive research, partially due to the continuous discoveries of their multifunctional properties beside their classical roles as food additives, preservatives and/or fragrances [1]. More than 3000 plants are known to produce Eos, of which approximately 300 are of commercial importance [2]. They are obtained from all parts of the plant and contain a high variety of volatile, aromatic and low-molecular-weight compounds. They are generally and chelating ability. The enzyme inhibitory property of the essential oils was evaluated on enzymes related with neurodegenerative ailments (acetylcholinesterase (ACh) and butyrylcholinesterase (BCh)), diabetes and obesity (α-glucosidase and α-amylase) and skin hyperpigmentation tyrosinase (Tyr) enzymes.

Chemical Profiles of the EOs
Hydrodistillation of dried fruit of C. obtusa gave a yellow-coloured EO with a sweet odour and a yield of 0.8% (w/w). Results of chemical profiles are depicted in Table 1. Chromatograms are presented in Figures 1-3. The total number of eight compounds belonging to monoterpenes was identified. Hydrogenated and oxygenated monoterpenes represented 87.3% and 11.6% of the total composition of EO. α-Pinene (69.1%) was the dominant compound, followed by δ-3-carene (12.1%) and trans-sabinene hydrate (11.6%), respectively. Sesquiterpenes were not detected. It worth mentioning that this is the first report on EO composition of the fruit. Previous studies on C. obtusa EO were only performed on the leaf of trees grown in South Korea, where it was found to also be rich in monoterpenes [9,16,31].

Antioxidant Activity
Antioxidants have been widely used as food additives to provide protection against oxidative degradation of foods by free radicals, and they also have many beneficial effects for human health. Six complementary assays were performed to evaluate the antioxidant activity of the three essential oils under investigation. Results are presented in Table 4. The antiradical activity was evaluated by testing the capacity of the EOs to scavenge the free DPPH and ABTS radicals and results showed that EO extracted from the root of C. nigritanus displayed significantly (p < 0.05) higher anti-DPPH activity than that extracted from the aerial parts of L. coronopifolia, while EO from C. obtusa was not active. On the other hand, the EO from the latter significantly exerted (p < 0.05) the highest anti-ABTS activity, followed by those obtained from the L. coronopifolia aerial part and C. nigritanus fruit, respectively. Furthermore, EO from C. nigritanus root significantly exhibited (p < 0.05) the highest Cu ++ and Fe +++ ion-reducing capacity, followed by EO from the C. obtusa fruit and L. coronopifolia aerial parts, respectively. EOs from both C. obtusa fruit and C. nigritanus root were not significantly (p ≥ 0.05) different in their metal-chelating capacity, while that of L. coronopifolia aerial parts revealed lower activity. The total antioxidant activity from the phosphomolybdenum assay showed that the EO of C. obtusa fruit was significantly (p < 0.05) 2.6-and 7.1-fold highly active than those recorded from the C. nigritanus root and L. coronopifolia aerial parts, respectively. Overall, the high antioxidant activity of C. nigritanus root in most assays could have been due to the richness of the EO with oxygenated sesquiterpenes (64.3%) [40], among them α-bisabolol which was reported to Extraction of EO from C. nigritanus root yielded 1.1% (w/w) of a yellow-coloured oil with an aromatic-spicy odor. Chemical analysis revealed a total of 19 compounds ( Table 2). The majority of compounds were oxygenated sesquiterpenes (64.3%) with Cedr-8-en-15ol (28.7%) as the major constituent. Sesquiterpene hydrocarbons represented 4.0% of the EO while monoterpenes were not detected. Moreover, the alkane triacontane (20.1%) was found in a relatively abundant amount. These results were contradictory to those obtained by Khalil and Ayoub [32] and Semde et al. [33] but generally in agreement with that performed by Champagnat et al. [20]. The latter found that the EO from the root of a plant grown in Mali contained only sesquiterpene terpenes among them the alcohols preziza-7(15)-en-12-ol (9.5%), cedren-8-en-15-ol (6.2%), preziza-7(15)-en-3α-ol (6.0%), preziza-7(15)-ene (1.7%) and β-cedrene (1.6%). In the present study, Cedr-8-en-15-ol (28.7%) and α-Cedrene (2.0%) were the major oxygenated and hydrocarbon sesquiterpenes, respectively, and preziza-7(15)-ene was not detected. Khalil and Ayoub [32] and Semde et al. [33] reported that the sesquiterpene hydrocarbons longifolene-D (25.2%) and dehydronigritene (25.1%) were the main components of the EO root of C. nigritanus grown in Sudan and Burkina Faso, respectively. Additionally, α-and β-vetivone and khusimol, which are considered the "fingerprint" of vetiver oil, were not detected in the present study, thus suggesting the presence of a different chemotype of C. nigritanus characterized by the presence of high amounts of Cedr-8-en-15-ol (28.7%). Evidently, this aspect needs additional confirmations with further studies. On the other hand, this variation could also be attributed to maturity of the plant and environmental factors. In fact, studies have shown that the terpenoid composition of aromatic plants can be affected by the photoperiod, light intensity, temperature, rainfall and harvesting season or month [34,35]. De Almeida et al. [36] reported that multiple environmental factors change the specialized metabolism on plants, resulting in production of different terpenes. Aerial parts of L. coronopifolia yielded 0.2% (w/w) of a nicely perfumed faint yellowcoloured EO. The chemical profile of the EO is presented in Table 3. Nineteen compounds were identified and sesquiterpenes comprising 58.6% of the EO with the majority belonged to oxygenated sesquiterpenes (41.5%), while monoterpenes forming 32.9% of the EO with only one oxygenated monoterpenes lavandulol (26.6%) was detected and represented the main constituent of the EO. This was followed respectively by caryophyllene oxide (18.4%), farnesyl acetone (8.0%) and β-gurjunene (7.4%). Only one previous study by Alizadeh and Aghaee [37] reported the composition of EO of L. coronopifolia. They found that the EO of the aerial part of a plant grown in the Genow and Rodan regions in Iran was mainly composed of hydrocarbon monoterpenes (73.7-75.1%) and oxygenated monoterpenes (24.6-25.8%) with α-pinene (58.3-63.5%) as the main component. In this study, although lavandulol (26.6%), an oxygenated monoterpenes, was the major compound, the EO was mainly dominated by oxygenated sesquiterpenes compounds and only two hydrogenated monoterpenes; namely, α-pinene (5.0%) and δ-3-carene (1.3%) were detected. The prevalence of sesquiterpenes could be associated to the harsh environmental conditions of the plant, as a previous study showed that sesquiterpenes are effective in the defense against biotic and abiotic stresses, thereby protecting the plants [38]. The characteristic nice fra-grance of the EO can be due to the dominance of lavandulol, which is known to play a significant role in the perfume and cosmetics industry [39].

Antioxidant Activity
Antioxidants have been widely used as food additives to provide protection against oxidative degradation of foods by free radicals, and they also have many beneficial effects for human health. Six complementary assays were performed to evaluate the antioxidant activity of the three essential oils under investigation. Results are presented in Table 4. The antiradical activity was evaluated by testing the capacity of the EOs to scavenge the free DPPH and ABTS radicals and results showed that EO extracted from the root of C. nigritanus displayed significantly (p < 0.05) higher anti-DPPH activity than that extracted from the aerial parts of L. coronopifolia, while EO from C. obtusa was not active. On the other hand, the EO from the latter significantly exerted (p < 0.05) the highest anti-ABTS activity, followed by those obtained from the L. coronopifolia aerial part and C. nigritanus fruit, respectively. Furthermore, EO from C. nigritanus root significantly exhibited (p < 0.05) the highest Cu ++ and Fe +++ ion-reducing capacity, followed by EO from the C. obtusa fruit and L. coronopifolia aerial parts, respectively. EOs from both C. obtusa fruit and C. nigritanus root were not significantly (p ≥ 0.05) different in their metal-chelating capacity, while that of L. coronopifolia aerial parts revealed lower activity. The total antioxidant activity from the phosphomolybdenum assay showed that the EO of C. obtusa fruit was significantly (p < 0.05) 2.6-and 7.1-fold highly active than those recorded from the C. nigritanus root and L. coronopifolia aerial parts, respectively. Overall, the high antioxidant activity of C. nigritanus root in most assays could have been due to the richness of the EO with oxygenated sesquiterpenes (64.3%) [40], among them α-bisabolol which was reported to ameliorate the antioxidant network and re-establish the redox balance by antagonising oxidative stress [41]. Results of L. coronopifolia aerial parts supported those obtained by Messaoud et al. [24] who studied the anti-DPPH (IC 50 162.2 µgmL −1 ), Ferricreducing (60.7 µmol g −1 ) and metal-chelating (IC 50 6.1 µgmL −1 ) activities of a species grown in Tunisia. They related this antioxidant activity to the richness of EO in hydrocarbon monoterpenes and/or to the synergistic effect of more than one individual oil compound as well as compounds like carvacrol, thymol methyl ether, and carvacrol methyl ether [42]. Furthermore, α-pinene might partly contribute to the observed antioxidant activity of EO of C. obtusa fruit [43].

Enzyme-Inhibitory Activity
The capacity of the three EOs under investigation to inhibit the AChE, BChE, Tyr, α-glucosidase and α-amylase enzymes was also examined and results are presented in Table 5. The EO of C. obtusa fruit significantly exerted (p < 0.05) the highest AChE inhibition activity followed by that of L. coronopifolia aerial parts. This high activity could be attributed to α-pinene and δ-3-carene, the major components of C. obtusa fruit EO, which was known for their potential as anti-AChE [44,45]. Although the EO of the C. nigritanus root did not show any anti-AChE activity, it significantly exerted (p < 0.05) the highest BChE inhibition activity followed by EO of C. obtusa fruit, while that from L. coronopifolia aerial parts was not active. Only, EO of the latter species displayed considerable anti-Tyr activity. However, water extracts of dried lavender leaves from L. angustifolia, L. stoechas, L. dentata and L. latifolia grown in northern Iran have been found to possess anti-Tyr activity suggesting that Lavandula spp. could be an interesting source of tyrosine inhibitor agents [46]. Results of the inhibition property against the two enzymes associated with diabetes and obesity showed that EOs of both C. obtusa fruit and L. coronopifolia aerial part had the same capacity to inhibit the α-amylase with a value significantly (p < 0.05) higher than that exerted by the EO of C. nigritanus root. However, only EO from L. coronopifolia aerial parts exerted inhibition activity against α-glucosidase enzyme in concurrence with the findings of Elsbaey et al. [26]. Moreover, Boutahiri et al. [47] reported that the aqueous crude extract of flower tops of L. pedunculate inhibited the pancreatic α-amylase and intestinal α-glucosidase enzyme activities, suggesting that this genus may be a good source of antidiabetic agents. Values are reported as mean ±SD; * GALAEs, galanthamine equivalents; ** KAEs, kojic acid equivalents; *** ACEs, acarbose equivalents; Different superscript letters in the same column indicate significant difference (p < 0.05); na, not active.

Molecular Docking
All docked compounds bound to all five enzymes, with a potential preference for AChE and BChE (Figure 4). The detailed protein-ligand interactions provided insights into the binding patterns of some selected compounds. All the study compounds were quite small molecules, hence freely bound to the active sites of the target proteins. α-Bisabolol was accommodated in the catalytic channel of AChE via an H-bond with Phe295 and multiple hydrophobic interactions, while van der Waals interactions reinforced the binding ( Figure 5A). Similarly, cedr-8-en-13-ol occupied the catalytic pocket of BChE, mainly via an H-bond with His438, and multiple hydrophobic interactions, with a couple of van der Waals interactions contributing to the overall binding affinity ( Figure 5B). Interestingly, trans-sabinene hydrate fit in the relatively narrow catalytic channel of tyrosinase, forming an H-bond via a hydroxyl group with Ser375, a hydrophobic interaction, and a couple of van der Waals interactions; however, the active-site copper metal ions were not engaged in any interactions ( Figure 6A). In the case of α-amylase, cedr-8-en-15-ol formed two H-bonds with Trp59 and Gln63 via a hydroxyl group, a couple of hydrophobic interactions, and van der Waals interactions with the active site residues ( Figure 6B). Additionally, caryophyllene oxide is completely buried in the catalytic cavity of α-glucosidase, forming an H-bond with Arg267, multiple hydrophobic and van der Waals interactions all over the channel ( Figure 6C). Taken together, H-bonds and hydrophobic contacts are the key interactions with which the selected phytochemicals from C. obtusa, C. nigritanus, and L. coronopifolia extracts bind to, and potentially inhibit these enzymes.

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via an H-bond with His438, and multiple hydrophobic interactions, with a couple of van der Waals interactions contributing to the overall binding affinity ( Figure 5B). Interestingly, trans-sabinene hydrate fit in the relatively narrow catalytic channel of tyrosinase, forming an H-bond via a hydroxyl group with Ser375, a hydrophobic interaction, and a couple of van der Waals interactions; however, the active-site copper metal ions were not engaged in any interactions ( Figure 6A). In the case of α-amylase, cedr-8-en-15-ol formed two H-bonds with Trp59 and Gln63 via a hydroxyl group, a couple of hydrophobic interactions, and van der Waals interactions with the active site residues ( Figure 6B). Additionally, caryophyllene oxide is completely buried in the catalytic cavity of α-glucosidase, forming an H-bond with Arg267, multiple hydrophobic and van der Waals interactions all over the channel ( Figure 6C). Taken together, H-bonds and hydrophobic contacts are the key interactions with which the selected phytochemicals from C. obtusa, C. nigritanus, and L. coronopifolia extracts bind to, and potentially inhibit these enzymes.  x FOR PEER REVIEW 10 of 16

Extraction of Essential Oils
EO from each plant sample (400 g) was extracted by hydrodistillation using a Clevenger-type apparatus for 4 h. The extracted oils were dried over anhydrous sodium sulphate and stored at 4 • C, in amber-coloured bottles, before use.

Chemical Analysis of the EOs
The essential oil composition was characterized by gas chromatography-flame ionization detector (GC-FID) and gas chromatography-mass spectrophotometry (GC-MS) techniques. GC-MS analysis was performed by using an Agilent 5975 GC-MS system coupled to an Agilent 7890 A GC. To separate chemical components, the HP-Innowax column (60 m × 0.25 mm, 0.25 µm film thickness) was used. Other analytical parameters were reported in our earlier paper [48].
The retention index (RI) calculated by co-injection with reference to a homologous series of n-alkanes (C8-C30) under the identical experimental circumstances was used to identify the components. In order to make more accurate identifications, RI values and mass spectra of the compounds were compared with those of the literature and NIST 05 and Wiley eighth edition, respectively.

Antioxidant and Enzyme Inhibitory Assays
The antioxidant and enzyme inhibitory activity of comfrey root extracts was determined according to previously described methods [49,50]. DPPH and ABTS radical scavenging activity, cupric ion-reducing antioxidant capacity (CUPRAC), and ferric ion-reducing antioxidant power (FRAP) were expressed as mg trolox equivalents (TE)/g oil. The metal chelating ability (MCA) was reported as mg EDTA equivalents (EDTAE)/g oil, whereas the total antioxidant activity (phosphomolybdenum assay, PBD) was expressed as mmol TE/g extract. AChE and BChE inhibitory activities were given as mg galanthamine equivalents (GALAE)/g oil; tyrosinase inhibitory activity was expressed as mg kojic acid equivalents (KAE)/g extract; amylase and glucosidase inhibitory activities were presented as mmol acarbose equivalents (ACAE)/g oil.

Molecular Modeling
The crystal structures of target enzymes: AChE (PDB ID: 6O52) [51], BChE (PDB ID: 6EQP) [52], and α-amylase (PDB ID: 1B2Y) [53] were retrieved from the protein data bank (PDB) (https://www.rcsb.org/) (accessed on 6 October 2022). However, since the crystal structures of human α-tyrosinase and α-glucosidase are not available, those of Priestia megaterium tyrosinase (6QXD) [54] and Mus musculus α-glucosidase (7KBJ) [55] were used as templates to build their human models using their respective UniProt sequences P14679, and P0DUB6. The details of the homology modeling are available in the Ref. [56]. All protein structures were prepared according to the protocol described in the Ref. [57]. The 3D structures of selected ligands were downloaded from the PubChem database (https: //pubchem.ncbi.nlm.nih.gov/) (accessed on 23 December 2022) and geometry optimization was done using Biovia Discovery Studio Visualizer v4.5 (Dassault Systèmes Biovia Software Inc., San Diego, CA, USA, 2012). The cocrystal ligand in each complex was used to define the docking grid box dimension and coordinates using AutoDockTools 1.5.6, and each ligand was docked into the binding pocket of each protein using AutoDock 4.2.6 (https://autodock.scripts.edu accessed on 15 December 2022) [58]. The details of the docking protocol have been described in the Ref. [59]. The docking score of each ligand against each protein was estimated in terms of binding energy, and protein-ligand interactions were visualized using Biovia Discovery Studio Visualizer v4.5 (Dassault Systèmes Biovia Software Inc., San Diego, CA, USA, 2012).

Statistical Analysis
Data are presented as mean ± standard deviation of the number (n = 3) of replicates. One-way analysis of variance with Tukey's post hoc test was conducted; p < 0.05 was con-sidered statistically significant. The statistical evaluation was performed using Graphpad version 9.0.

Conclusions
Chemical analysis of the three EOs indicated that the fruit of C. obtusa contained only monoterpenes, mainly hydrogenated ones, with α-pinene as the major component. The C. nigritanus root composed only of sesquiterpenes with a majority of compounds are oxygenated ones and Cedr-8-en-15-ol as a dominant component, while aerial parts of L. coronopifolia contained both monoterpenes and sesquiterpenes, but with higher content in the latter and lavandulol as the major compound. It was clear that the three investigated EOs were varied in their antioxidant and enzyme inhibition properties, and this is possible due to the variation in their chemical composition. Generally, the EO of the C. nigritanus root displayed the best antioxidant activity (4/6 assays). The EO of the L. coronopifolia aerial part was the only EO to reveal anti-Tyr and-α-glucosidase activities. Additionally, it displayed, like that of the C. obtusa fruit, the best α-amylase inhibition capacity. EOs of the C. obtusa fruit and C. nigritanus root revealed the best anti-AChE and -BChE activities, respectively. Molecular docking was performed to get insights into the binding mode and interaction of the bioactive compounds with the studied enzyme targets. The results showed that the docked compounds bound to all five enzymes, with a potential preference for AChE and BChE by α-Bisabolol and cedr-8-en-13-ol, respectively. Together, these data show that EOs could have significant potential in different food, cosmetic and pharmaceutical applications. Further, studies are recommended to evaluate their biosafety and determine whether these biological activities are attributed to their main components of the EOs or the synergistic effect of more than one compounds. Additionally, understanding the genetic factors and environmental conditions that alter the composition and quantity of volatile compounds is essential for pharmacological studies.