The Antioxidant and Enzyme Inhibitory Potential of n-Hexane-Extracted Oils Obtained from Three Egyptian Cultivars of the Golden Dewdrop Duranta erecta Linn. Supported by Their GC-MS Metabolome Analysis and Docking Studies

Duranta erecta Linn. has a longstanding history for use in folk remedy for several disorders. Its hydroalcoholic extract has been investigated intensely in the treatment of many ailments, but to date very few data are presented to explain the pharmacological use of its oil. In this study, the chemical profiles of the leaf oils extracted from three Egyptian Duranta erecta cultivars, namely ‘Green’, ‘Golden edge’, and ‘Variegata’ are traced using GC-MS analysis. D. erecta ‘Green’ showed predominance of vitamin E (22.7%) and thunbergol (15%) whereas D. erecta ‘Golden edge’ and ‘Variegata’ contained tetratetracontane as a major component in their oils. The highest phenolic and flavonoid contents, displayed as gallic acid and rutin equivalents per gram oil, respectively, were observed in the ‘Golden edge’ and ‘Variegata’ cultivars, which was reflected by their strong DPPH and ABTS scavenging activities as well as the highest reducing power in both CUPRAC and FRAP assays. D. erecta ‘Green’ displayed better metal chelating potential, which may be attributed to its content of vitamin E. All cultivars showed similar enzyme inhibitory profiles. The best inhibition of α-glucosidase and α-amylase was observed by D. erecta ‘Green’. In silico studies of the major constituents docked on the active sites of the target enzymes NADPH oxidase, amylase, glucosidase, butyrylcholinesterase, and tyrosinase revealed high binding scores, which justified the biological activities of the tested oils.


Introduction
Duranta erecta (D. repens) Linn. (Verbenaceae) has long been traditionally used as a folk medicine in countries such as Brazil, Nigeria, India, Philippines, and Bangladesh [1]. The richness of the plant in phytoconstituents (ca. 64 isolated metabolite till now), in particular the iridoids, flavonoids, phenylethanoids, tannins, coumarinolignans, terpenoids, alkaloids, and sterols, make it of particular interest to natural products chemists. D. erecta

Extraction of the Essential Oils from D. erecta Cultivars by n-Hexane
The fresh leaves of three different cultivars of Duranta erecta, viz. 'Green', 'Golden edge', and 'Variegata' (ca. 100 g each) were collected in February 2022 from a botanical garden in Nasr City, Cairo. Authentication of the plant samples were kindly performed by Taxonomy Specialist Terase Labib, Consultant of Plant Taxonomy at the Ministry of Agriculture, and El-Orman Botanical Garden, Giza, Egypt. The leaves were dried, powdered, and macerated overnight in 100 mL n-hexane. The extract was filtered, and the filtrate was concentrated under vacuum. This process was repeated thrice over three consecutive days (3 × 100 mL) to yield 1.31 mg, 1.12 mg, and 1.09 mg of dark yellow (D. erecta 'Green'), light yellow (D. erecta 'Golden edge'), and light yellow (D. erecta 'Variegata') oily residues, respectively, which were subjected to GC-MS analysis and biological investi-gations. Voucher specimens (codes: PHG-P-DE-406, PHG-P-DE-407, and PHG-P-  were deposited at the Department of Pharmacognosy, Faculty of Pharmacy, Ain-Shams University, Cairo, Egypt.

GC-MS Analysis of the Extracted Oils
GC-MS analysis was performed on a Shimadzu QP2010 (Shimadzu Corporation, Kyoto, Japan) coupled to a quadrupole mass spectrometer. Separation was done on the low polarity diphenyl dimethyl polysiloxane Rtx-5MS (30 m × 0.25 mm i.d. × 0.25 µm thickness) capillary column (Restek, Bellefonte, PA, USA) in a split injection mode with a split ratio 1:15. Helium was the carrier gas flowing at a rate of 1.37 mL/min, sample injection volume 1 µL (diluted to 1% v/v in n-hexane), and oven and injector temperatures adjusted to 50 • C and 280 • C, respectively. Initial column temperature was set at 50 • C then gradually increased at a rate of 5 • C/min until reaching 300 • C, allowing metabolites of different boiling points and molecular weights to get separated at 70 eV EI ionization mode. Identification of oil components was based on comparison of their mass spectra and Kovat's indices to those of a series of n-alkanes (C 8 -C 30 ) injected under the same GC condition and to those reported in NIST online mass library, as well as matching with the literature [27][28][29][30].

Total Phenolic and Flavonoid Content
Folin-Ciocalteu and AlCl 3 assays were used to determine the total phenolic and flavonoid contents, respectively [31]. For respective assays, results were expressed as gallic acid equivalents (mg GAEs/g dry extract) and rutin equivalents (mg REs/g dry extract).

Enzyme Inhibitory Assays
The enzyme inhibitory assays were carried out according to previously reported methodologies [32,33]. The acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition was expressed as mg galanthamine equivalents (GALAE)/g extract; tyrosinase inhibition was expressed as mg kojic acid equivalents KAE/g extract; amylase and glucosidase inhibition were expressed as mmol acarbose equivalents (ACAE)/g extract.

Statistical Analysis
ANOVA (Tukey's test) was used to determine if there were any differences in the tested extracts. The Pearson correlation test was used to examine the relationship between total bioactive components and biological activities. The statistical procedures were performed by using GraphPad 9.0.

In Silico Data
The X-ray 3D structures of NADPH oxidase, butyrylcholinesterase, tyrosinase, αamylase, and α-glucosidase were downloaded from the protein data bank using the following IDs: 2cdu, 6esj, 5m8q, 4gqq and 3wy2, respectively. All the docking studies were conducted using MOE 2019 ® software [34], which was likewise used to generate the 2D interaction diagrams between the docked ligands and their potential targets. The three identified major compounds were prepared using the default parameters and saved in a single mdb file. The active site of each target was determined from the binding of the corresponding co-crystalized ligand. The mdb file containing the three major compounds was finally docked into the active site of the five enzymes.

Assessment of the Total Phenolic and Flavonoid Content in Duranta-Derived Oils
Phenolic compounds play a key role in the management of many diseases [37][38][39][40]. The total phenolic and flavonoid content of the oils obtained from the three cultivars of D. erecta were determined by spectrophotometric methods. The results are presented in Table  2. The highest total phenolic and flavonoid content was found in D. erecta 'Golden edge', with 12.07 mg GAE/g and 8.51 mg RE/g, respectively. D. erecta 'Green' contained the lowest level of these bioactive compounds, which were ca. 8.51 mg GAE/g and 1.49 mg RE/g, respectively. The content of the total phenolic compounds was clearly dependent on the tested Duranta cultivars. Consistent with our results, the concentration of flavonoids and phenolics were different in the cultivars of a plant species. To the best of our knowledge, there is no scientific data on Duranta oils, although the content of the total bioactive compounds in some of its members have been reported in several publications [5,8,41]. Recently, some concerns were raised about the spectrophotometric assays, where some compounds do not react with the reagents used [42,43]. Therefore, the results obtained from spectrophotometric assays need to be further confirmed by chromatographic and spectroscopic techniques such as HPLC and NMR, respectively.

Assessment of the Total Phenolic and Flavonoid Content in Duranta-Derived Oils
Phenolic compounds play a key role in the management of many diseases [37][38][39][40]. The total phenolic and flavonoid content of the oils obtained from the three cultivars of D. erecta were determined by spectrophotometric methods. The results are presented in Table 2. The highest total phenolic and flavonoid content was found in D. erecta 'Golden edge', with 12.07 mg GAE/g and 8.51 mg RE/g, respectively. D. erecta 'Green' contained the lowest level of these bioactive compounds, which were ca. 8.51 mg GAE/g and 1.49 mg RE/g, respectively. The content of the total phenolic compounds was clearly dependent on the tested Duranta cultivars. Consistent with our results, the concentration of flavonoids and phenolics were different in the cultivars of a plant species. To the best of our knowledge, there is no scientific data on Duranta oils, although the content of the total bioactive compounds in some of its members have been reported in several publications [5,8,41]. Recently, some concerns were raised about the spectrophotometric assays, where some compounds do not react with the reagents used [42,43]. Therefore, the results obtained from spectrophotometric assays need to be further confirmed by chromatographic and spectroscopic techniques such as HPLC and NMR, respectively.

In Vitro Antioxidant Assays
Currently, natural antioxidants are replaced with synthetic alternatives, which appeared to have adverse effects on humans [43]. In this regard, we demonstrated the antioxidant potential of the oils of the three D. erecta cultivars and the results are presented in Tables 2 and 3. Different antioxidant assays are required to get a complete picture of the antioxidant potential of the tested samples. Therefore, six complementary assays (DPPH, ABTS, CUPRAC, FRAP, MCA, and PBD) were performed to assess the antioxidant properties of Duranta oils. The free radical scavenging potential was assessed using DPPH and ABTS. D. erecta 'Golden edge' showed the strongest DPPH scavenging ability (12.93 mg TE/g), whereas D. erecta 'Variegata' displayed the best ABTS scavenging capacity (8.98 mg TE/g). The results of DPPH and ABTS revealed that D. erecta 'Green' had no ability to scavenge free radicals. The reducing power of the oils, which is their ability to donate an electron to stabilize free radicals, were studied using CUPRAC (ability to reduce Cu 2+ to Cu + ) and FRAP (ability to reduce Fe 3+ to Fe 2+ ) assays. The best reducing power was observed for D. erecta 'Variegata' (CUPRAC: 36.40 mg TE/g; FRAP: 21.07 mg TE/g), followed by 'Golden edge' and 'Green'. Table 3. Reducing abilities, metal chelating, and total antioxidant ability (by phosphomolybdenum assay) of the tested oils. Apparently, the free radical scavenging and reducing abilities of the tested oils are consistent with their levels of total bioactive compounds. This fact was also confirmed by Pearson's correlation analysis, and strong correlation values (R > 0.7) were determined ( Figure 3). Consistent with our results, good correlation values between these parameters have been reported by several researchers [44][45][46]. The phoshomolybdenum assay involves the conversion of Mo (VI) to Mo (V) by antioxidants in the acidic condition. Thus, the assay is known as one of reducing power assays. In addition, since all antioxidant compounds could be active, the assay is known as the total antioxidant assay [43]. As can be seen in Table 3, the tested samples exhibited very similar effects in the assay (p > 0.05).

Samples CUPRAC (mg TE/g) FRAP (mg TE/g) MCA (mg EDTAE/g) PBD (mmol TE/g)
The chelation of transition metals is an important mechanism and prevents the formation of the most dangerous hydroxyl radical. The best ability for metal chelation was observed in D. erecta 'Green' with 16.37 mg EDTAE/g, followed by 'Golden edge' (10.71 mg EDTAE/g) and 'Variegata' (2.77 mg EDTAE/g). The observed metal chelating ability could be explained by the presence of tocopherol in the tested oil. These findings are in accordance with those in the literature where tocopherol has been reported as a good metal chelator [47][48][49]. Although few previous works [36] have explained the significant antioxidant properties of the methanol extracts of some members of Duranta, the antioxidant activities of the oils of the three cultivars of D. erecta are moderate to some extent.

Enzyme Inhibitory Assay
Some diseases, such as Alzheimer's disease, diabetes mellitus, or obesity, affect millions of people, and their prevalence is increasing every day. With this in mind, new and effective strategies are becoming increasingly popular as a subject of study. Enzymes are critical in managing the diseases mentioned above. The symptoms of the diseases could be controlled by inhibiting important enzymes. Some enzymes, such as acetylcholinesterase, amylase, and lipase, have been identified as targets for certain diseases, such as Alzheimer's, diabetes, and obesity, respectively. In this regard, several synthetic enzyme inhibitors have been approved in the treatment of diseases, but the majority of which have unpleasant side effects [50][51][52][53]. Therefore, natural sources could be considered as treasures that need detailed exploration. We tested the enzyme inhibitory properties of the oils obtained from the three Duranta erecta cultivars against cholinesterases (AChE and BChE), tyrosinase, amylase, and glucosidase. The results are presented in Table 4. In general, the tested oils exhibited similar inhibitory activities against the enzymes. Although BChE was inhibited by all the tested oils (6.41-6.65 mg GALAE/g), AChE was only inhibited by D. erecta 'Variegata'. Tyrosinase is the main catalyst in the synthesis of melanin and its inhibition is important to treat hyperpigmentation problems. As presented in Table 4, the tested oils exhibited, to large extent, similar anti-tyrosinase effects (56.92-61.96 mg KAE/g, p > 0.05). Amylase and glucosidase were selected as targets for antidiabetic effects and the best amylase inhibition was recorded by D. erecta 'Green' and 'Golden edge' with 0.32 mmol ACAE/g. However, the strongest glucosidase inhibition was observed in D. erecta 'Green' (1.31 mmol ACAE) and 'Variegata' (1.24 mmol ACAE). It is clear from Figure 3 that the total content of phenols and flavonoids weakly correlate or even did not correlate with the abilities to inhibit enzymes. Consistent with our findings, several researchers also reported a weak correlation between total bioactive compounds and enzyme inhibition effects [54][55][56]. In the literature, available data on the enzyme inhibitory properties of the members of the genus Duranta is limited [22,23,56] and no previous study has focused on its oils. Thus, the presented results could be a scientific basis for further applications with D. erecta oils.

Conclusions
Duranta erecta is an important herb with several medicinal and industrial applications. Phytochemical and pharmacological studies have previously reported on its hydroalcoholic extract; however, few documented its oil and properties. GC-MS investigations on three different cultivars of D. erecta growing in Egypt showed significant variations in their constituent profiles. Mono-and sesquiterpene hydrocarbons were almost absent in Duranta oil with predominance in fatty esters, acids, alkanes, alkenes, sterols, alcohols, tocopherols, and some phenolics. In vitro antioxidant assays showed that the oils displayed good radical scavenging activities and promising reducing power, which was likewise supported by the in silico data. Therefore, we strongly recommend further investigations on Duranta oils, such as toxicity studies, animal testing, and bioavailability.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

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

Conclusions
Duranta erecta is an important herb with several medicinal and industrial applications. Phytochemical and pharmacological studies have previously reported on its hydroalcoholic extract; however, few documented its oil and properties. GC-MS investigations on three different cultivars of D. erecta growing in Egypt showed significant variations in their constituent profiles. Mono-and sesquiterpene hydrocarbons were almost absent in Duranta oil with predominance in fatty esters, acids, alkanes, alkenes, sterols, alcohols, tocopherols, and some phenolics. In vitro antioxidant assays showed that the oils displayed good radical scavenging activities and promising reducing power, which was likewise supported by the in silico data. Therefore, we strongly recommend further investigations on Duranta oils, such as toxicity studies, animal testing, and bioavailability.