Phytochemical Profile, In Vitro Bioactivity Evaluation, In Silico Molecular Docking and ADMET Study of Essential Oils of Three Vitex Species Grown in Tarai Region of Uttarakhand

A comparative study of volatiles, antioxidant activity, phytotoxic activity, as well as in silico molecular docking and ADMET study, was conducted for essential oils from three Vitex species, viz., V. agnus-castus, V. negundo, and V. trifolia. Essential oils (OEs) extracted by hydrodistillation were subjected to compositional analysis using GC-MS. A total number of 37, 45, and 43 components were identified in V. agnus-castus, V. negundo, and V. trifolia, respectively. The antioxidant activity of EOs, assessed using different radical-scavenging (DPPH, H2O2 and NO), reducing power, and metal chelating assays, were found to be significant as compared with those of the standards. The phytotoxic potential of the EOs was performed in the receptor species Raphanus raphanistrum (wild radish) and the EOs showed different levels of intensity of seed germination inhibition and root and shoot length inhibition. The molecular docking study was conducted to screen the antioxidant and phytotoxic activity of the major and potent compounds against human protein target, peroxiredoxin 5, and 4-hydroxyphenylpyruvate dioxygenase protein (HPPD). Results showed good binding affinities and attributed the strongest inhibitory activity to 13-epi-manoyl oxide for both the target proteins.


Introduction
The plant genus Vitex (family: Verbenaceae) consists of 250 accepted species and has a wide distribution all over the world, ranging from shrubs to trees in the tropical, subtropical regions, and temperate zones [1]. The members of this genus have been widely used in folk medicine and are greatly valued as medicinal plants in several Asian countries, including India, Pakistan, Nepal, China, Sri Lanka, and Bangladesh [2]. The leaves, seeds, flowers, and the whole aerial part of different species of Vitex genus have several external and internal uses. The most popular uses of these plants are in the curing of asthma, ophthalmodynia, headaches, coughs, premenopausal syndrome, etc., but various other uses have also been reported [2]. For instance, V. agnus-castus fruits are being used in the treatment of menstrual disorders (amenorrhea, dysmenorrhea), and in other female conditions like premenstrual dysphoric disorder, infertility, disrupted lactation, acne, breast pain, menopause, and inflammatory conditions [3,4]. V. negundo species is used as tonic, vermifuge, lactagogue, and is also used to treat catarrhal fever, eye diseases, inflammation, skin ulcers, rheumatoid arthritis, and bronchitis [5]. V. trifolia is used as a sedative for headaches, as an anti-inflammatory agent, and for the cure of the common cold. The plant is The fresh leaves of different plant species of the Vitex were collected from Pantnagar (28 • 58 12 N, 79 • 24 36 E), Tarai region of Uttarakhand, India. The plant specimens were identified by one of the authors (D.S. Rawat), a taxonomist. The voucher specimen for different identified Vitex species viz., Vitex agnus-castus, Vitex negundo L., and Vitex trifolia L., with voucher numbers GBPUH-1439, GBPUH-1438, and GBPUH-1440, were deposited at the herbarium of Department of Biological Sciences, for future references.

Extraction of Essential Oil
Fresh leaves of different Vitex species were subjected to hydrodistillation for 4 h to isolate essential oils using a Clevenger-type apparatus, and the isolated essential oils were designated as VAO, VNO, and VTO for Vitex agnus-castus, Vitex negundo, and Vitex trifolia, respectively. The obtained essential oils were dried over anhydrous sodium sulphate (Na 2 SO 4 ) in order to remove any trace of water and then stored in amber color glass vials at a low temperature (4 • C in refrigerator) for further uses. The oil yield (v/w) was recorded as 0.9% (0.45 mL/100 gm dry matter), 0.8% (0.4 mL/100 gm dry matter), and 0.6% (0.3 mL/100 gm dry matter) for VAO, VNO, and VTO, respectively.

Chemical Composition Analysis
To check the chemical diversity in tested Vitex species, the essential oils were analyzed by GC-MS (Shimadzu QP 2010 plus) with GCMS-QP 2010 Ultra DB-5 and GCMS-QP 2010 Ultra Rtx-5MS column (30 m × 0.25 mm i.d., 0.25 µm). The following experimental conditions used helium as the carrier gas (flow rate = 1.21 mL/min, split ratio = 10.0). Oven temperature was programmed at 50-280 • C with a temperature gradient of 3 • C/min up to 210 • C (isotherm for 2 min), then 6 • C/min up to 280 • C. Identification of essential oil components was done by comparing their relative retention index (RI) values with mass spectra NIST (NIST version 2.1) and WILEY (7th edition) libraries, and by matching the fragmentation pattern of the mass spectral data with those reported in the literature [25,26].

Antioxidant Activity
Different in vitro tests were performed to evaluate the antioxidant activity of the essential oils, and the results were presented as mean ± SD of triplicate.

DPPH Radical Scavenging Assay
Previously proposed methods have been followed to perform the assay [27,28]. In brief, different concentrations of VAO, VNO, and VTO (10 µL/mL-50 µL/mL) were added to 5 mL of freshly prepared methanolic solution of DPPH (0.004%), solution was kept for incubation under dark for half an hour, and further, the absorbance was taken in triplicates at 517 nm in a UV spectrophotometer (Thermo Fisher Scientific, Evolution-201, Waltham, MA, USA) against a blank. The standard antioxidant used was BHT, in the same concentrations as the tested essential oils (10 µL/mL-50 µL/mL). The % inhibition of DPPH free radical of the oils and standard was calculated by using the following formula: where A o and A t are the absorbance values of control and test essential oils, respectively. Percent inhibition was plotted against concentrations, and the equation for the line was used to obtain the IC 50 (half-maximal inhibitory concentration) values.

Hydrogen Peroxide (H 2 O 2 ) Radical Scavenging Activity
H 2 O 2 radical scavenging activity of tested samples was performed as per the prescribed protocol reported earlier [29,30]. Here, 0.6 mL of H 2 O 2 solution (40 mM) prepared in phosphate buffer (0.1 M; pH 7.4) was added to 0.4 mL methanolic solution of different concentrations of essential oils and the standard (10-50 µL/mL). The above solution was incubated at room temperature for 10 min. Further, the absorbance was taken at 230 nm against the blank, i.e., methanol. Here, L-ascorbic acid (10-50 µL/mL) was taken as a positive control. The percentage scavenging of H 2 O 2 was calculated by using the following formula: where A o and A t are the absorbance values of control and test essential oils, respectively. Percent inhibition was plotted against concentrations and the equation for the line was used to obtain the IC 50 values.

Nitric Oxide Radical Scavenging Activity
The nitric oxide (NO) radical scavenging activity of the tested essential oils was determined by the method as described earlier [31]. Briefly, 2 mL of sodium nitroprusside (10 mM) prepared in phosphate buffer saline (0.5 mM, pH 7.4) was added to different concentrations of essential oils and the standard (10-50 µL/mL) separately, and incubated at 25 • C for 150 min. Further, 0.5 mL of griess reagent containing 1.0 mL sulphanilic acid Antioxidants 2022, 11, 1911 4 of 27 reagent was added to 0.5 mL of each incubated solution. The mixture was again incubated for 30 min at room temperature and the absorbance was taken at 540 nm. L-ascorbic acid was taken as the standard antioxidant. The percentage scavenging of NO was calculated by using the following formula: where A o and A t are the absorbance values of control and test essential oils, respectively. Percent inhibition was plotted against concentrations and the equation for the line was used to obtain the IC 50 values.

Reducing Power Assay
The reducing power assay of different essential oils was determined by the method developed earlier [32]. Different concentrations of tested samples (essential oils and the standard (10-50 µL/mL)) were added to 2.5 mL of phosphate buffer (200 mM, pH = 6.6). Further, 2.5 mL of 1% potassium ferricyanide, K 3 [FeCN 6 ], was added to the above solution.
The solution was incubated for 20 min at 50 • C and then 2.5 mL of trichloroacetic acid was added to the incubated solution, followed by centrifugation at 650 rpm for 10 min. 5 mL of distilled water and 1 mL of 0.1% ferric chloride were added to the upper layer (1 mL). The absorbance of the final solution was taken at 700 nm, and gallic acid (10-50 µL/mL) was taken as a positive control. The percentage reducing power was calculated by using the following formula: where A o and A t are the absorbance values of control and test essential oils, respectively. RP 50 values were calculated using regression equations for the percent inhibition plotted against concentrations.

Fe 2+ Metal Chelating Activity
The Fe 2+ metal-chelation activity of VAO, VNO, and VTO was measured as per the prescribed and developed protocol [33]. Different concentration of oils (10-50 µL/mL), as well as the standard, were mixed with 0.1 mL of FeCl 2 ·4H 2 O (2 mM) and 0.2 mL of (5 Mm) ferrozine separately. Further, methanol (4.7 mL) was added to the solution, making the final volume 5 mL. The solution was shaken and was incubated for 30 min at 25 • C, and the absorbance was taken at 562 nm using spectrophotometer (Thermo Fisher Scientific, Evolution-201, USA). Na 2 -EDTA (10-50 µL/mL) was used as a standard antioxidant. The ability of the samples to chelate ferrous ion was calculated using the following formula: where A o and A t are the absorbance values of control and test essential oils, respectively. IC 50 values were obtained using regression equations for the plots of percent inhibition against concentrations.

Herbicidal (Phytotoxic) Activity
The herbicidal activity on the receptor plant, Raphanus raphanistrum, was carried out with essential oils of Vitex species. Different parameters were used, such as inhibition of seed germination, inhibition of shoot, and root length growth, using the method reported earlier [34,35]. For the experiment, radish seeds were obtained from the VRC (Vegetable Research Centre), G.B.P.U.A. & T. Pantnagar, Uttarakhand, India. For evaluating the seed germination inhibition, different concentrations of essential oils (50-200 µL/mL) were prepared in Tween-20 (1%) solution of distilled water. In order to break dormancy, radish seeds were surface sterilized in 5% hypochlorite solution for 15 min. Ten sterilized seeds of radish were placed in each petri plates, which were lined with sheets of qualitative filter papers. Further, 2 mL of various concentrations of the tested sample (50-200 µL/mL) were applied onto the plates and the seeds were allowed to germinate at controlled condition of 25 ± 1 • C and a photoperiod of 12 h in an incubator. Seeds with a root length of 2 mm were considered germinated. Distilled water was taken as the control while pendimethalin (50-200 µL/mL) was used as a standard herbicide, and the bioassay was performed in triplicate. After 120 h, the numbers of germinated seeds in each petri dish were counted, followed by the calculation of percent seed germination inhibition values using the following formula: Inhibition of seed germination (% Inhibition) = 100 × (1 − Gt/Gc) where Gt = no. of seeds germinates in treatment, Gc = No. of seeds germinate in control.

Inhibition of Shoot and Root Elongation
Assessment of shoot and root elongation were performed at controlled condition of 25 • C for a photoperiod of 24 h. Each Petri dish received 2.0 mL of the test solution, and two pre-germinated seeds were placed in each petri plate. The EOs were tested at the same concentrations as the germination bioassay. At the end of the 120 h of incubation, the length of the shoot and root were measured. Distilled water was taken as the controlled treatment while pendimethalin (50-200 µL/mL) was used as a standard herbicide, and the bioassays were performed in triplicate. The formulae used for determining the inhibition of shoot and root growth were as follows: Inhibition of hypocotyl (shoot length) growth (% Inhibition) = 100 × (1 − Ct/Cc) where, Ct = shoot length growth in treatment, Cc = shoot length growth in control.

Molecular Docking Studies
Virtual ligand screening is an in silico method used to dock small molecules (ligand) to macromolecule (protein) to discover potent compounds that have the necessary biological effect [36]. The molecular docking study of the selected volatiles from VAO, VNO, and VTO was carried out on 4-hydroxyphenylpyruvate dioxygenase (HPPD) receptors, as this protein has been reported as a molecular target for compounds with post-emergence herbicidal activity [37,38], and the second protein taken was human peroxiredoxin 5, which has a broader activity against reactive oxygen species [39]. The three-dimensional (3D) structures of the HPPD and human peroxiredoxin 5 proteins were obtained from the RCSB ProteinData Bank with PDB ID: 6J63 and 1HD2, respectively. The 3D structures of the selected proteins converted into PDB formats by deleting the water molecules, HETATOMS, and adding polar hydrogens using Biovia Discovery Studio-2021 Client. The compounds from the essential oils for docking studies were selected based on their higher percentage contents and their concerned structures were obtained from the PUBCHEM database (https: //pubchem.ncbi.nlm.nih.gov/, accessed on 12 August 2022) in the SDF (structure data file) format. The selected compounds were 1 Energy minimization (optimization) was performed by adding charges and optimizing the universal force field. Further, the ligands were converted into AutoDock Ligand format (PDBQT). To find out the binding affinity and to know the various ligandreceptor interactions responsible for the antioxidant and phytotoxic activity, the molecular docking of the selected major constituents was performed using PyRx with Vina Wizard tool. The protein and multiple ligands to be docked were selected in the PyRx software using the Vina Wizard Control. The "Run Vina" control was selected to initiate the docking process. The results were observed by selecting the "Analyze Vina" tool and exported as CSV files [36]. Biovia Discovery Studio-2021 Client was used for the visualization of 2D and 3D interactions of docking poses.

In Silico ADMET Study
The structures of the selected compounds from the essential oils were drawn using ChemDraw Ultra 8.0 for the pharmacokinetics (absorption, distribution, metabolism, and excretion (ADME)) studies. The legends were converted into the SMILES format and then the drug-like and pharmacokinetic properties of the selected compounds were predicted using ADME tool by a SwissADME online server (http://www.swissadme.ch/, accessed on 12 August 2022), as per the developed protocol [40]. ProTox-II webserver (http://tox.charite.de/protox_II, accessed on 12 August 2022) was used to study the toxicity profile. It calculates the prediction based on different parameters such as organ toxicity (hepatotoxicity), oral toxicity, and toxicological endpoints (cytotoxicity, mutagenicity, carcinotoxicity, and immunotoxicity).

Statistical Analysis
Two and one factor Analysis of variance (ANOVA), followed by the Tukey test, was performed using RStudio (Version 2021.09.2) developed by RStudio team, PBC, Boston, MA and OriginPro, Version 2022b developed by OriginLab, Northampton, MA, USA Student trial version software, respectively, to analyze the significant difference among the treatment means. The p value < 0.05 was considered to show the significant difference. All the data in the experiment were reported as mean ± SD (standard deviation). To define the variability in different essential oils based on chemical composition, Chemometric Analysis was performed based on the heatmap clustering using heatmapper, free web server available (http://www.heatmapper.ca, accessed on 12 August 2022) developed at University of Alberta, Canada [41]. We performed Principal Component Analysis (PCA) on chemical composition for the three Vitex species under investigation to identify the most significant features in the dataset and Pearson's correlation test to analyze the correlation among the chemical compounds of essential oils, and their biological activities were performed using OriginPro, Version 2022b.
In addition, the compounds identified in the tested essential oils have potent biological applications. 1,8-cineole is used in cosmetic products and as a flavoring agent because of its pleasant aroma and taste. The compound has several other properties: insecticidal, antioxidant, and anti-inflammatory [61]. Viridiflorol has prominent use as an anti-inflammatory, antioxidant, and anti-tuberculosis agent [62]. Sabinene has antimicrobial, anti-inflammatory, and antioxidant properties described in literature [63]. Further, the diterpene, 13-epi-manoyl oxide, has cytotoxic antibacterial and antifungal activities [64].

Principal Component Analysis
Principal component analysis (PCA) is one of the greatest multivariate statistical techniques used to identify a dataset's most important features. To assess the chemical profiling changes caused by interspecies as well as altitudinal influences, distinct essential oils can be used in PCA pattern recognition. The PCA approach determined that the cumulative contribution rate of variance of the first two principal components (PC1 and PC2) could account for 81.2% of the variance information for changes in chemical composition. In order to define the compositional variations in the essential oils, PC1 and PC2 were used. PC1 was favorably linked with terpinen-4-ol, β-iraldeine, β-caryophyllene, viridiflorol, and sabinene, and contributed 48.7% of the total variance. However, PC2 makes up 32.5% of the variation and has a strong positive correlation with α-pinene and 1,8cineole ( Figure 2).

Principal Component Analysis
Principal component analysis (PCA) is one of the greatest multivariate statistical techniques used to identify a dataset's most important features. To assess the chemical profiling changes caused by interspecies as well as altitudinal influences, distinct essential oils can be used in PCA pattern recognition. The PCA approach determined that the cumulative contribution rate of variance of the first two principal components (PC1 and PC2) could account for 81.2% of the variance information for changes in chemical composition. In order to define the compositional variations in the essential oils, PC1 and PC2 were used. PC1 was favorably linked with terpinen-4-ol, β-iraldeine, β-caryophyllene, viridiflorol, and sabinene, and contributed 48.7% of the total variance. However, PC2 makes up 32.5% of the variation and has a strong positive correlation with α-pinene and 1,8-cineole (Figure 2).

Antioxidant Activity
The antioxidant activity was determined by using different chemical-based methodologies. Figure 3A-E depict the antioxidant activity of tested essential oil in terms of percent inhibition. Results revealed that all the antioxidant activities were in a concentrationdependent manner. The percent inhibition of free radicals (DPPH, H2O2, NO), reducing power, and metal chelation increased, with increasing concentration from 10 µL/mL to 50 µL/mL. Further, the percent inhibition by the tested essential oils and the standards for different antioxidant assays were plotted against concentrations, and the equation for the line was used to obtain the IC50 (half-maximal inhibitory concentration) values.

Antioxidant Activity
The antioxidant activity was determined by using different chemical-based methodologies. Figure 3A-E depict the antioxidant activity of tested essential oil in terms of percent inhibition. Results revealed that all the antioxidant activities were in a concentrationdependent manner. The percent inhibition of free radicals (DPPH, H 2 O 2 , NO), reducing power, and metal chelation increased, with increasing concentration from 10 µL/mL to 50 µL/mL. Further, the percent inhibition by the tested essential oils and the standards for different antioxidant assays were plotted against concentrations, and the equation for the line was used to obtain the IC 50 (half-maximal inhibitory concentration) values. Figure 4A-E represent the antioxidant activity of tested essential oils in terms of their IC 50 values. In the DPPH assay, the reduction of the stable radical DPPH (violet) to the yellow-colored DPPH-H is employed to measure the potential of an antioxidant molecule to act as a donor of hydrogen atoms or electrons. Figure 4A shows that VNO reduced DPPH with an IC 50 value of 23.16 ± 0.5 µL/mL, which is close to the standard antioxidant taken for the assay, BHT (18.84 ± 0.6 µL/mL). VAO and VTO displayed moderate and weak antioxidant activity, with IC 50 25.39 ± 0.0 µL/mL and 32.49 ± 0.5 µL/mL, respectively. H 2 O 2 can cross the biological membrane, and as a result it can damage the human body by forming reactive OH· radicals following Fenton reaction [65]. In H 2 O 2 radical scavenging assay, VAO (IC 50 = 24.49 ± 0.1 µL/mL) displayed good scavenging activity when compared to the standard, ascorbic acid (28.33 ± 0.5 µL/mL), followed by VNO (32.38 ± 0.5 µL/mL) and VTO (34.30 ± 0.5 µL/mL). The extent of nitrite scavenging by the samples was compared with ascorbic acid and showed IC 50 values as: ascorbic acid (24.49 ± 0.1 µL/mL) > VNO (27.58 ± 0.1 µL/mL) > VTO (32.27 ± 0.1 µL/mL) > VAO (32.95 ± 0.5 µL/mL). The reducing power of a compound is related to its ability to transfer electrons, which indicates its significant antioxidant potential. As shown in Figure 4D, VNO displayed good reducing capability (RP 50 = 19.05 ± 0.6 µL/mL) that is very close and lower than that of the standard gallic acid (20.22  ologies. Figure 3A-E depict the antioxidant activity of tested essential oil in terms of per-cent inhibition. Results revealed that all the antioxidant activities were in a concentrationdependent manner. The percent inhibition of free radicals (DPPH, H2O2, NO), reducing power, and metal chelation increased, with increasing concentration from 10 µL/mL to 50 µL/mL. Further, the percent inhibition by the tested essential oils and the standards for different antioxidant assays were plotted against concentrations, and the equation for the line was used to obtain the IC50 (half-maximal inhibitory concentration) values.  Figure 4A-E represent the antioxidant activity of tested essential oils in terms of their IC50 values. In the DPPH assay, the reduction of the stable radical DPPH (violet) to the yellow-colored DPPH-H is employed to measure the potential of an antioxidant molecule to act as a donor of hydrogen atoms or electrons. Figure 4A shows that VNO reduced DPPH with an IC50 value of 23.16 ± 0.5 µL/mL, which is close to the standard antioxidant taken for the assay, BHT (18.84 ± 0.6 µL/mL). VAO and VTO displayed moderate and weak antioxidant activity, with IC50 25.39 ± 0.0 µL/mL and 32.49 ± 0.5 µL/mL, respectively. H2O2 can cross the biological membrane, and as a result it can damage the human body by forming reactive OH· radicals following Fenton reaction [65]. In H2O2 radical scavenging assay, VAO (IC50 = 24.49 ± 0.1 µL/mL) displayed good scavenging activity when compared to the standard, ascorbic acid (28.33 ± 0.5 µL/mL), followed by VNO (32.38 ± 0.5 µL/mL) and VTO (34.30 ± 0.5 µL/mL). The extent of nitrite scavenging by the samples was compared with ascorbic acid and showed IC50 values as: ascorbic acid (24.49 ± 0.1 µL/mL) > VNO (27.58 ± 0.1 µL/mL) > VTO (32.27 ± 0.1 µL/mL) > VAO (32.95 ± 0.5 µL/mL). The reducing power of a compound is related to its ability to transfer electrons, which indicates its significant antioxidant potential. As shown in Figure 4D, VNO displayed good reducing capability (RP50 = 19.05 ± 0.6 µL/mL) that is very close and lower than that of the standard gallic acid (20. Such high antioxidant activity of VNO for the DPPH and NO radical scavenging is Such high antioxidant activity of VNO for the DPPH and NO radical scavenging is likely due to high amount of sabinene as well as other constituents of VNO such as β-caryophyllene, terpinen-4-ol, 1,8-cineole, which already possess antioxidant potential via different parameters [66][67][68]. Additionally, Kazemi [69] showed that sabinene exhibited potent NO-scavenging effect and inhibited the expression of inducible NO synthase. Similar results were observed in previous studies in antioxidant activity of V. negundo essential oil in which the major component was sabinene [49]. In H 2 O 2 radical scavenging assay, VAO showed good scavenging activity, which may be due to the presence of 1,8-cineole, sabinene, and β-caryophyllene as the major constituents [69,70]. In earlier reports, essential oil and extracts of aerial parts of V. agnus castus have been tested for antioxidant activity as having a high amount of 1,8-cineole and β-caryophyllene in their composition, and the samples showed good antioxidant activity [64,71,72]. Since essential oils are complexed mixtures of number of compounds, their whole biological activity is hard to explain. Therefore, research on the antioxidant activity of essential oils typically indicates that other minor chemical constituents that may interact synergistically or antagonistically to produce an additive and effective system against free radicals may also be responsible for the antioxidant activity [68,73]. activity. Statistically significant differences were examined using one-way ANOVA and Tukey posthoc tests. *** p < 0.001, ** p < 0.005, * p < 0.05 above columns indicate significant differences between treated groups. Values are mean ± SD, n = 3.

Herbicidal (Phytotoxic) Activity
The tested samples demonstrated notable phytotoxic activity against seed germination and seedling growth of the wild radish (R. raphanistrum) in a concentration-dependent manner. At the highest concentration (100 µL/mL), VAO showed inhibition of seed activity. Statistically significant differences were examined using one-way ANOVA and Tukey posthoc tests. *** p < 0.001, ** p < 0.005, * p < 0.05 above columns indicate significant differences between treated groups. Values are mean ± SD, n = 3.

Herbicidal (Phytotoxic) Activity
The tested samples demonstrated notable phytotoxic activity against seed germination and seedling growth of the wild radish (R. raphanistrum) in a concentration-dependent manner. At the highest concentration (100 µL/mL), VAO showed inhibition of seed germination, root growth, and shoot growth of R. raphanistrum by 66  100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 VAO = V. agnus-castus; VNO = V. negundo; VTO = V. trifolia; SD = standard deviation; According to Tukey's test (p < 0.05), mean values that are followed by the same letter inside a column are not statistically different from one another.  The phytotoxic potential of EOs from various Vitex species such as V. agnus castus, V. negundo V. simplicifolia has also been reported previously in other plants and weeds [15]. However, there is no study reported on phytotoxic potential of V. trifolia. Based on the present study, it was evident that VTO was more effective against R. raphanistrum than VNO and VAO. The suppressing effect of VTO on R. raphanistrum could be due to high amounts of β-caryophyllene (16.2%) and the synergetic effect of β-caryophyllene with other major and minor compounds present in the oil. In previous reports, β-caryophyllene was found to be responsible for the inhibition of germination and seedling growth of several plant species such as Brassica campestris, Raphanus sativus, Vigna radiata, and Solanum lycopersicum [22]. VNO also showed good inhibition values for seed germination and shoot growth, while VAO showed better inhibition value for root growth. The inhibition effect of samples could be due to the presence of phytotoxic compounds such as β-caryophyllene, 1,8-cineole, and sabinene, which are the main components in essential oil possessing phytotoxic activity [22,74]. In addition, 1,8-cineole was reported to interfere with the normal growth Nicotiana tabacum by blocking the DNA synthesis in their cell nuclei and organelles in root apical meristem cells [75]. Studies have also demonstrated that the terpenoids in EOs have a phytotoxic effects on plants, resulting in morphological and physiological alterations in the cells that impair plant growth [76].

Molecular Docking
From in vitro studies, it was found that essential oils have potent antioxidant and phytotoxic activity. We also examined whether the major phytoconstituents from VAO, VNO, and VTO physically bind with antioxidant protein (human peroxiredoxin 5, PDB: 1HD2) and 4-hydroxyphenylpyruvate dioxygenase (HPPD, PDB: 6J63) receptors. The tested essential oils displayed good inhibition of the free radicals, for which the enzyme human peroxiredoxin 5 was selected, as it has broader activity against the reactive oxygen species (ROS) and is mostly involved in the stress protection mechanism [80,81]. The reason for selecting HPPD is that it is known to be the target protein for compounds with post-emergence herbicidal activity. In our results, the tested essential oils were found to have good post-emergence herbicidal activity against the receptor species, for which HPPD was selected as a target enzyme [18,38]. Among all selected phytocompounds, 13epi-manoyl oxide demonstrated the best binding affinity with human peroxiredoxin 5 (−6.2 kcal mol) and HPPD (−8.7 kcal/mol). By introspecting the multiple dock poses, the best docked pose was selected as having the lowest binding energy. The best docked pose of 13-epi-manoyl oxide exhibited 2 pi-alkyl interaction, 1 pi-sigma interaction, and other Van der Waal interactions with 6J63 containing amino acid residues such as Phe A:424, Phe A:419, and Phe A:381, as represented in Figure 6B. Similarly, the best docked pose of Figure 5. Correlation among chemical components of essential oils and biological activities of Vitex species (here, DPPH = percent inhibition of DPPH radical scavenging activity at 50 µL/mL; H 2 O 2 = percent inhibition of H 2 O 2 radical scavenging activity at 50 µL/mL; NO = percent inhibition of NO radical scavenging activity at 50 µL/mL; RPA = percent inhibition of reducing power activity at 50 µL/mL; FeMCA = percent inhibition of Fe 2+ metal chelating activity at 50 µL/mL; SGI = percent inhibition of seed germination at 100 µL/mL; RLI = percent inhibition of root length at 100 µL/mL; SLI = percent inhibition of shoot length at 100 µL/mL.

Molecular Docking
From in vitro studies, it was found that essential oils have potent antioxidant and phytotoxic activity. We also examined whether the major phytoconstituents from VAO, VNO, and VTO physically bind with antioxidant protein (human peroxiredoxin 5, PDB: 1HD2) and 4-hydroxyphenylpyruvate dioxygenase (HPPD, PDB: 6J63) receptors. The tested essential oils displayed good inhibition of the free radicals, for which the enzyme human peroxiredoxin 5 was selected, as it has broader activity against the reactive oxygen species (ROS) and is mostly involved in the stress protection mechanism [80,81]. The reason for selecting HPPD is that it is known to be the target protein for compounds with post-emergence herbicidal activity. In our results, the tested essential oils were found to have good post-emergence herbicidal activity against the receptor species, for which HPPD was selected as a target enzyme [18,38]. Among all selected phytocompounds, 13-epi-manoyl oxide demonstrated the best binding affinity with human peroxiredoxin 5 (−6.2 kcal mol) and HPPD (−8.7 kcal/mol). By introspecting the multiple dock poses, the best docked pose was selected as having the lowest binding energy. The best docked pose of 13-epi-manoyl oxide exhibited 2 pi-alkyl interaction, 1 pi-sigma interaction, and other Van der Waal interactions with 6J63 containing amino acid residues such as Phe A:424, Phe A:419, and Phe A:381, as represented in Figure 6B. Similarly, the best docked pose of 13-epi-manoyl oxide exhibited alkyl interaction with 1HD2 containing amino acid Ala A:90, Arg A:86, and exhibited Van der Waal interaction. For comparison purposes, a docking study of Nitisinone (CID:115355) was also performed with HPPD. Nitisinone (2-[2-nitro-4-(trifluoromethyl)benzoyl]cyclohexane-1,3-dione, (NTBC)) is a known inhibitor of HPPD. The docking study of ascorbic acid (CID:54670067), a known antioxidant, was performed with 1HD2. The binding energy for NTBC complexed with 6J63 was −8.9 kcal/mol, which is very close to that of 13-epi-manoyl oxide (−8.7 kcal/mol). On the other hand, binding energy of ascorbic acid complexed with 1HD2 came out to be −5.7 kcal/mol, which was higher than most of the compounds such as 13-epi-manoyl oxide (−6.2 kcal/mol), caryophyllene oxide (−6.1 kcal/mol), 5-(1-isopropenyl-4,5-dimethylbicyclo [4.3.0]nonan-5-yl)-3-methyl-2-pentenol acetate (−6.1 kcal/mol), β-caryophyllene (−6.0 kcal/mol), and viridiflorol (−5.9 kcal/mol), as shown in Figure 7. The lower values of binding free energy demonstrate more significant interaction between the receptor and the ligand. Our results were consistent with previous in silico studies reported by Alminderej et al. [73], where a phenylpropanoid-rich Piper cubeba EO gave similar results in terms of a proposed in vitro antioxidant activity by targeting human periredoxin 5. In this study, the compounds viridiflorol and caryophyllene oxide showed significant interaction with 1HD2 receptor as in the present study. In a recent study, focusing on the phytotoxic potential of Calycolpus goetheanus EO, it was found that the major components of the specimen, 1,8-cineole and β-caryophyllene interacted favorably with the HPPD protein [18]. These results are in general agreement with those obtained in the present study. 13-epi-manoyl oxide exhibited alkyl interaction with 1HD2 containing amino acid Ala A:90, Arg A:86, and exhibited Van der Waal interaction. For comparison purposes, a docking study of Nitisinone (CID:115355) was also performed with HPPD. Nitisinone (2-[2-nitro-4-(trifluoromethyl)benzoyl]cyclohexane-1,3-dione, (NTBC)) is a known inhibitor of HPPD. The docking study of ascorbic acid (CID:54670067), a known antioxidant, was performed with 1HD2. The binding energy for NTBC complexed with 6J63 was −8.9 kcal/mol, which is very close to that of 13-epi-manoyl oxide (−8.7 kcal/mol). On the other hand, binding energy of ascorbic acid complexed with 1HD2 came out to be −5.7 kcal/mol, which was higher than most of the compounds such as 13-epi-manoyl oxide (−6.2 kcal/mol), caryophyllene oxide (−6.1 kcal/mol), 5-(1-isopropenyl-4,5-dimethylbicyclo [4.3.0]nonan-5-yl)-3-methyl-2-pentenol acetate (−6.1 kcal/mol), β-caryophyllene (−6.0 kcal/mol), and viridiflorol (−5.9 kcal/mol), as shown in Figure 7. The lower values of binding free energy demonstrate more significant interaction between the receptor and the ligand. Our results were consistent with previous in silico studies reported by Alminderej et al. [73], where a phenylpropanoid-rich Piper cubeba EO gave similar results in terms of a proposed in vitro antioxidant activity by targeting human periredoxin 5. In this study, the compounds viridiflorol and caryophyllene oxide showed significant interaction with 1HD2 receptor as in the present study. In a recent study, focusing on the phytotoxic potential of Calycolpus goetheanus EO, it was found that the major components of the specimen, 1,8-cineole and β-caryophyllene interacted favorably with the HPPD protein [18]. These results are in general agreement with those obtained in the present study.
The listed binding energies of the volatiles docked with human peroxiredoxin 5 and HPPD ( Figure 7) were found to be in the range −6.2 to −4.3 kcal/mol and −8.7 to −5.4 kcal/mol, respectively. Based on the study, it was observed that the major constituents interacted favorably with the receptors-most of which are the Van der Waal interactions. The analysis of ligand recognition reveals that the compounds can be good antioxidant and phytotoxic agents. Figure 6A-H shows the interaction of selected volatiles with the receptors (6J63 and 1HD2) having the least binding energies (higher docking scores), along with their 2D interaction with amino acid residues.

ADMET Analysis
The forecasting of ADME (absorption, distribution, metabolism, and excretion) properties of the selected compounds, including their pharmacokinetic and drug-like properties, have been estimated using SwissADME online server (http://www.swissadme.ch/, accessed on 12 August 2022). The collective laws of Lipinski's [82], Egan's [83], and Veber's [84], which determine the properties of a drug, were followed. According to the rule that the compound should not violate more than 1 Lipinski rule, molecular weight (MW) < 500, topological surface area (TPSA) < 140, number of H-bond acceptors (nOHA)

ADMET Analysis
The forecasting of ADME (absorption, distribution, metabolism, and excretion) properties of the selected compounds, including their pharmacokinetic and drug-like properties, have been estimated using SwissADME online server (http://www.swissadme.ch/, accessed on 12 August 2022). The collective laws of Lipinski's [82], Egan's [83], and Veber's [84], which determine the properties of a drug, were followed. According to the rule that the compound should not violate more than 1 Lipinski rule, molecular weight (MW) < 500, topological surface area (TPSA) < 140, number of H-bond acceptors (nOHA) The listed binding energies of the volatiles docked with human peroxiredoxin 5 and HPPD ( Figure 7) were found to be in the range −6.2 to −4.3 kcal/mol and −8.7 to −5.4 kcal/mol, respectively. Based on the study, it was observed that the major constituents interacted favorably with the receptors-most of which are the Van der Waal interactions. The analysis of ligand recognition reveals that the compounds can be good antioxidant and phytotoxic agents. Figure 6A-H shows the interaction of selected volatiles with the receptors (6J63 and 1HD2) having the least binding energies (higher docking scores), along with their 2D interaction with amino acid residues.
Some of the compounds interacted mainly with two isoenzymes of the cytochrome (CYP) family, namely CYP2C19 and CYP2C9, suggesting their efficiency while having minimal toxicity. Drug-like properties and GI absorption of selected compounds from VAO, VNO, and VTO were also represented by the boiled-egg prediction ( Figure 8) and bioavailability radar graph (Figure 9). The compounds present in the yellow zone in the boiled-egg graph can permeate through the blood-brain barrier (BBB), and the pink area of the bioavailability radar graphs shows the drug-likeness of the compounds. share TPSA values less than 30 Å 2 , indicating good brain penetration and good lipophilicity behavior, with the consensus Log Po/w coming in the range 2.60-5.14 ( Table 5). There was no P-glycoprotein (P-gp) substrate found, suggesting the good intestinal absorption of compounds. Except sabinene, α-pinene, β-farnesene, β-caryophyllene, 13-epi-manoyl acetate, and α-phellandrene, all compounds showed high gastrointestinal absorption. The compounds that were predicted to not cross the blood-brain barrier (BBB) were β-farnesene, β-caryophyllene, 5-(1-isopropenyl-4,5-dimethylbicyclo[4.3.0]nonan-5-yl)-3methyl-2-pentenol acetate, and 13-epi-manoyl oxide. Some of the compounds interacted mainly with two isoenzymes of the cytochrome (CYP) family, namely CYP2C19 and CYP2C9, suggesting their efficiency while having minimal toxicity. Drug-like properties and GI absorption of selected compounds from VAO, VNO, and VTO were also represented by the boiled-egg prediction ( Figure 8) and bioavailability radar graph (Figure 9). The compounds present in the yellow zone in the boiled-egg graph can permeate through the blood-brain barrier (BBB), and the pink area of the bioavailability radar graphs shows the drug-likeness of the compounds.   The toxicity parameters of selected phytocompounds were predicted using web server ProTox II (Table 6). All the selected compounds were predicted not to be hepatotoxic, carcinogenic, cytotoxic, immunotoxic, and mutagenic, except α-terpinyl acetate (hepatotoxic), 5-(1-isopropenyl-4,5-dimethylbic clo[4.3.0]nonan-5-yl)-3-methyl-2-pentenol acetate (carcinogenic), β-iraldiene, and caryophyllene oxide (immunotoxic). The LD50 values were also calculated to ensure the safety of the selected compounds as shown in Table 6. The compounds with LD50 > 2000 mg/kg, suggesting their safety for biological administration and as potential drugs. The toxicity parameters of selected phytocompounds were predicted using web server ProTox II (Table 6). All the selected compounds were predicted not to be hepatotoxic, carcinogenic, cytotoxic, immunotoxic, and mutagenic, except α-terpinyl acetate (hepatotoxic), 5-(1-isopropenyl-4,5-dimethylbic clo[4.3.0]nonan-5-yl)-3-methyl-2-pentenol acetate (carcinogenic), β-iraldiene, and caryophyllene oxide (immunotoxic). The LD 50 values were also calculated to ensure the safety of the selected compounds as shown in Table 6. The compounds with LD 50 > 2000 mg/kg, suggesting their safety for biological administration and as potential drugs.     50 ≤ 5000), Class VI: non-toxic (LD 50 > 5000)).

Conclusions
In this study, the chemical diversity among the EOs obtained from three Vitex species from Tarai region, India, was revealed and analyzed. The chemical profile of EOs was characterized by high content of terpenoids. Moreover, the in vitro antioxidant and phytotoxic activities of the EOs were investigated to check the biological potentials of the plant-derived products of these Vitex species. All the tested EOs showed moderate to good antioxidant and phytotoxic potentials as assessed with different assays. The molecular docking study suggested that the compounds from the EOs can be good antioxidant and phytotoxic agents by the analysis of ligand interaction with the proteins. The ADMET analysis revealed the safety of most of the major compounds in the EOs. Overall, this study unveiled some interesting biological activities of these EOs, especially as natural antioxidants and phytotoxic agents, which justifies the use of the plant species in traditional medicine, as well as in the crop protection field. However, the in vivo study is necessary to investigate and assess the potency and safety of these EOs and their active components.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/antiox11101911/s1, Supplementary Material S1, ion-chromatograms, and mass spectra of the major compounds can be observed.