Analysis of Volatile Secondary Metabolites in Ocimum basilicum Cell Suspensions: Inhibition, In Silico Molecular Docking, and an ADMET Analysis against Proteolytic Enzymes of Rhynchophorus ferrugineus

Our study’s overarching goal was to determine which O. basilicum cell suspensions approach yielded the most insecticidal and R. ferrugineus-inhibitory volatile secondary metabolites. After inoculation with Verticillium dahliae as an activator, the growth kinetics were measured, and the extract was identified using GC-MS. Validation was achieved for the insecticidal efficacy of a volatile extract, the pure phenolic content against larva and adult R. ferrugineus, and the inhibitory effect on proteases (in vivo and in vitro). The volatile extract achieved an LC50 of 1229 µg/mL and an LD50 of 13.8 µg/larva. The LC50 values for β-bergamotene, α-eudesmol, β-farnesene, linalool, 1,8-cineole, eugenol, α-guaiene, and β-caryophyllene were 1294, 1312, 1356, 1398, 1426, 1459, 1491, and 1523 g/mL, respectively. The LD50 activities of α-eudesmol, linalool, 1,8-cineole, eugenol, and nerol were 12.4, 13.7, 13.9, 14.2, and 15.6 g/larva, respectively. Active volatile extract of O. basilicum inhibited trypsin proteinase, elastase, cysteine, overall protease, and metalloprotease activity with IC50 values of 89.4, 101.7, 394.7, 112.4, and 535.2 µg/mL and 178.5, 192.4, 547.3, 208.3, and 924.8 µg/mL, in vitro and in vivo, respectively. There was evidence of action against total proteases (in vitro) with IC50 values of 78.9, 81.2, 88.6, 90.7, 91.5, 97.6, 107.4, and 176.3 µg/mL for β-bergamotene, α-eudesmol, β-farnesene, linalool, 1,8-cineole, eugenol, α-guaiene, and β-caryophyllene, respectively. Total proteases (in vivo) are inhibited by the α-eudesmol, linalool, 1,8-cineole, eugenol, nerol, and (E)-β-ocimene, with IC50 values of 162.3, 192.7, 193.1, 201.4, 248.6, and 273.2 µg/mL, respectively. ADMET and molecular docking modeling were the only two methods used to conduct in-depth computational analyses of compounds. The study recommended using an efficient cell suspension method to produce a volatile extract rich in useful secondary metabolites that may be utilized as a bio-insecticide.


Introduction
The various basil cultivars have the genetic ability to develop and preserve volatile compounds in different groups, culminating in a large diversity of chemical constituents in the same basil species [1,2]. Flavonoids, terpenoids, phenols, and alkaloids are some of the different types of secondary metabolites that many plants produce. Each has broad applications and biological impacts, involving anti-aging, antibacterial, and anti-inflammatory The protein content in cell suspension and callus is presented in Table S1. The results from Table S1 indicated a significant elevation in protein content throughout different initiation stages of the callus or cell suspension (5-40 days) with an increase in the presence of stimulation by V. dahliae.
High values of protein content in O. basilicum presence and an absence of infection via V. dahliae were found at the end of the experiment (40 days). Protein content in O. basilicum gradually increased from 68.41 to 305.24 µg/gm in the callus without infection by V. dahliae and from 203.88 to 514.25 µg/gm in the callus in the case of the infection (Table S1). The data showed that the µg protein in the callus of O. basilicum per mL extract from the 1 g callus ratio increased with infection and the age of the callus. The µg protein/g callus ratio increased from 68.41 after five days to 305.24 at 40 days old without infection. In the infection state of O. basilicum, the protein content increased gradually from 203.88 to 514.25, with infection at 5 and 40 days old, respectively.
In the cell suspension, the protein content in O. basilicum gradually increased from 12.73 to 165.42 µg/gm cell suspension without infection by V. dahliae. In contrast, it was increased from 20.70 to 275.12 µg/gm cell suspension in the case of its infection. Generally, the results indicated that V. dahliae significantly increased the protein content in both cell suspension and callus.

O. basilicum Volatile Extract's Formation and Chemical Composition
At 5-6 weeks, the callus was proliferated and then transported to the medium for cell suspension. The liquid LS medium consistently produced more somatic embryos than the solid LS medium used for the same purpose. It took around five or six weeks of The protein content in cell suspension and callus is presented in Table S1. The results from Table S1 indicated a significant elevation in protein content throughout different initiation stages of the callus or cell suspension (5-40 days) with an increase in the presence of stimulation by V. dahliae.
High values of protein content in O. basilicum presence and an absence of infection via V. dahliae were found at the end of the experiment (40 days). Protein content in O. basilicum gradually increased from 68.41 to 305.24 µg/gm in the callus without infection by V. dahliae and from 203.88 to 514.25 µg/gm in the callus in the case of the infection (Table S1). The data showed that the µg protein in the callus of O. basilicum per mL extract from the 1 g callus ratio increased with infection and the age of the callus. The µg protein/g callus ratio increased from 68.41 after five days to 305.24 at 40 days old without infection. In the infection state of O. basilicum, the protein content increased gradually from 203.88 to 514.25, with infection at 5 and 40 days old, respectively.
In the cell suspension, the protein content in O. basilicum gradually increased from 12.73 to 165.42 µg/gm cell suspension without infection by V. dahliae. In contrast, it was increased from 20.70 to 275.12 µg/gm cell suspension in the case of its infection. Generally, the results indicated that V. dahliae significantly increased the protein content in both cell suspension and callus.
In addition, the data in Figure 3 demonstrated that O. basilicum components elevated gradually over the first 25 days in the cell suspension. They kept elevating rapidly during the final 15 days of the test (40 days) ( Figure 3).    (3 compounds, 4.2%), and oxygenated sesquiterpenes (3 compounds, 2%). In HCA, when metabolites are placed close to each other, this demonstrates a high association (Figure 3).     γ-terpinene, and β-terpineol presented low activity against adults with LC 50 of 3014, 3587, 3841, and 3924 µg/mL, respectively. The data showed that O. basilicum volatile extract's serial doses impacted IC 50 values compared to midgut proteases from untreated larvae. Figure 4 revealed that the IC 50 of the midgut rose with increasing levels, while the extract's IC 50 was 112.4 g/mL. All three elastase-like enzymes, proteinases, trypsin, and chymotrypsin, all present in midgut homogenate from 4th instar insects, are shown in Figure 4 to have varying degrees of activity and suppression. The serine proteinases' particular activity in whole homogenate preparations is comparable and is reported as the OD/mg protein/min. The IC 50 values were an O. basilicum volatile extracts' significant impact. Trypsin proteinases and elastase have much higher IC 50 values for suppression by O. basilicum volatile extract in midgut homogenate preparations than chymotrypsin-like serine proteinase. The IC 50 value for the volatile extract is displayed in Figure 4 (in vitro). This demonstrated that suppression differed considerably among midgut homogenates. Both trypsin and elastase proteinase activities were rather high in the fourth instar midgut sample (4.14 and 1.28, respectively). Likewise, the O. basilicum's active volatile extract had an IC 50 of 89.4 and 101.7 µg/mL, respectively. These extracts can potentially inhibit trypsin proteinases originating from the fourth midgut. Metalloprotease and cysteine both have a significant inhibitory reactivity to volatile extract; Figure 4 displays the IC 50 values, which showed that the volatile extract had inhibitory activity at IC 50 concentrations of 535.2 and 394.7 µg/mL, respectively. Chymotrypsin proteinases behaved differently than serine proteinases; the IC 50 values ( Figure 4) indicated that the volatile extract did not suppress chymotrypsin when the IC 50 value was greater than 5000 µg/mL. In Figure 4, we see the effect of the individual com- ponents on the total protease activity measured in the R. ferrugineus larval midgut during its fourth instar. Different dosages significantly affected the IC 50 rates for all examined substances compared to untreated larvae. Figure 4 depicts the influence of compounds on the R. ferrugineus fourth instar larval midgut's overall protease activity (in vitro). Significant influence was exerted by the varying IC 50 values of all substances examined.

In Vivo Effect of O. basilicum Volatile Extract and Pure Components on the Serine, Metalloprotease, and Cysteine Protease Activities from Fourth R. ferrugineus Instar Midgut Preparations
The R. ferrugineus' IC50 value's fourth instar larvae midgut was elevated progressively with O. basilicum volatile extract's concentrations ( Figure 5). The O. basilicum volatile extract demonstrated a significant impact as per the IC50 value. The preparations made from the midgut of a fourth-instar larva showed an activity of 1.98 OD/mg protein/min.
In the fourth midgut preparation, the volatile extract inhibited the trypsin proteinase, elastase proteinases, metalloprotease, and cysteine proteases to a larger extent than in vitro values. Activity versus trypsin proteinase, total proteases, and elastase was observed at IC50 values of 178.5, 208.3, and 192.4 µg/mL, respectively, for the volatile extract of O. basilicum. The inhibition impact of cysteine and metalloprotease demonstrated that the volatile extract has the greatest inhibitory impact with values of 547.3 and 924.8 µg/mL, respectively ( Figure 5). Figure 5 indicates the compound's impact on overall in vivo protease activities.
had the poorest cysteine protease binding ability (    Table 3, Figures 8 and S5 illustrate the chemicals' docking ratings to metalloprotease (PDB:1KAP). It was discovered, based on the findings of the docking investigation, that the compounds under research had a low docking energy, ranging from −4.2706 (1,8cineole) to −5.6725 (β-farnesene) kcal/mol, and a strong affinity for the binding sites (target) of metalloprotease (Table 3). β-Farnesene, nerol, germacrene D, α-guaiene, βbergamotene, and α-eudesmol had a higher affinity for binding than molecules with lower docking energies, with values of −5.    Table 3 and Figures 9 and S6 illustrate the chemicals' docking ratings to trypsin proteinase (PDB:1FN8). It was discovered, based on the findings of the docking investigation, that the compounds under research had a low docking energy, ranging from −4.2897 (1,8-cineole) to −5.2044 (β-Bergamotene) kcal/mol, and a strong affinity for the binding sites (target) of trypsin proteinase (Table 3). β-Bergamotene, α-eudesmol, βfarnesene, linalool, α-guaiene, and β-caryophyllene has a higher affinity for binding than molecules with lower docking energies, with values of   Table 3, Figures 9 and S6 illustrate the chemicals' docking ratings to trypsin proteinase (PDB:1FN8). It was discovered, based on the findings of the docking investigation, that the compounds under research had a low docking energy, ranging from −4.2897 (1,8cineole) to −5.2044 (β-Bergamotene) kcal/mol, and a strong affinity for the binding sites (target) of trypsin proteinase (Table 3). β-Bergamotene, α-eudesmol, β-farnesene, linalool, α-guaiene, and β-caryophyllene has a higher affinity for binding than molecules with lower docking energies, with values of −5.

Discussion
The present study investigated the volatile secondary metabolites from O. basilicum's cell suspension extract. β-Bergamotene, α-eudesmol, β-farnesene, linalool, 1,8-cineole, eugenol, α-guaiene, and β-caryophyllene had a significant insecticidal action versus R. ferrugineus' adults. α-Eudesmol, linalool, 1,8-cineole, eugenol, and nerol revealed the greatest objective application activities versus larvae. The research reported that volatile extracts were effective versus overall proteases (in vitro and in vivo) and particular activities versus trypsin proteases and elastases. In cell suspensions obtained from O. basilicum, especially after V. dahliae activator inoculation, the synthesis of secondary metabolites such as oxygenated sesquiterpenes, oxygenated monoterpenes, oxygenated sesquiterpenes, monoterpene hydrocarbons, and phenylpropanoids compounds increased rapidly. It was established that the volatile extract from O. basilicum effectively and efficiently improved deterrence and decreased R. ferrugineus larvae feeding. However, the antifeedant impact is strongest in adults.
Moreover, according to our data, the overall rate of antifeedant activities was observed and discovered in optimal circumstances. The volatile extract demonstrated substantial insecticidal action, which may be due to the bioactive metabolites' range present in the extract. O. basilicum oxygenated monoterpenes, sesquiterpene hydrocarbons, oxygenated sesquiterpenes, and monoterpene hydrocarbons from cell suspension raised progressively during the first 25 days of the test, then quickly accelerated and kept growing quickly for the final fifteen days. For the substances tested using the IC 50 rate, various dosages had a significant impact. Comparing the compounds' influence against midgut protease activities (in vitro) generated by fourth-stage R. ferrugineus, larvae's midgut revealed the effective action of O. basilicum's volatile extract.
In addition, as these volatile extracts are content antifeedant compounds, site targets and mechanisms of action in these insects have been unknown until now. Based on the research [31], insecticidal action pathways may entail changing feeding physiology, longterm toxicity, or repellency.
Importantly, these secondary molecules are acknowledged as important components once they develop in cells and play a critical role in pathogen defense [32][33][34][35]. PGR is needed to control how plants develop and differentiate, which could control how anabolism destroys phenolic contents [36,37]. Therefore, cutting-edge methods like bioactive secondary metabolite green biosynthesis are highly sought after now [38][39][40]. Furthermore, due to its short response time, ability to divide cells, and ease of use, cell suspension culture is a quicker and more efficient method than callus culture for boosting the production of bioactive chemicals [9,26,40,41].
The suppression of trypsin proteinase, cysteine protease, metalloprotease, and elastase proteinases via the O. basilicum's volatile extract was definitively proven fourth midgut levels were lower than in vitro values. Numerous in vivo values exhibit the equivalent pattern and are less than their in vitro counterparts. The in vivo test of β-farnesene, β-bergamotene, α-guaiene, and β-caryophyllene activity versus overall protease revealed that compounds suppress overall protease the least in vitro.
Through the study of a molecule's orientation or position on a possible target, molecular docking may be used to make predictions about the binding affinities and dynamics between the molecules. Putting the chemicals in a docking onto serine proteinases, metalloprotease, cysteine, and elastase protease exhibited several interactions, notably van der Waals interactions, H-bonds, and H-pi hydrophobic bonds. These bond interactions aided in comprehending the biological roles of numerous compounds in various fields, including pharmaceuticals and insecticides [44,45].
As per LC 50 results, the O. basilicum's volatile extract was active versus R. ferrugineus. The volatile extract exhibited clear insecticidal, antifeedant properties, and suppressed proteinases extracted from midgut preparation from the fourth instar. These findings shed light on substances that could be used to construct biochemical markers that reflect the insect resistance of specific plant varieties.

Plant Materials
The O. basilicum seed was procured from a commercial nursery in the eastern region of Saudi Arabia. Seeds were decontaminated and seeded in an MS medium involving agar and sucrose at pH = 5.8, with 0.6 and 3% (w/w), respectively. The medium was cultured for 7-8 weeks, with seedling lengths of 19-20 cm in a climatic chamber at the optimum conditions of 26 ± 3 • C, 16 h light at King Faisal University, Saudi Arabia.

O. basilicum Callus and Cell Suspension Initiation with V. dahliae as a Biotic Elicitor
For this experiment, we sterilized, delinted, and cultivated O. basilicum seeds in Petri dishes with sterile blotted paper at a temperature of 28 ± 1 • C with a light intensity of 30 Einsteins/(m 2 .s) [26]. The O. basilicum's explants (hypocotyls, cotyledonary, and epicotyls, length 4~6 mm) were seeded in an MS medium containing 0.1 mg/L 2,4-D, 0.5 mg/L kinetin, 1 mg/L IBA, and 0.5 mg/L NAA, as well as sucrose (3% w/v) as per Darrag et al.,with changes [26]. Plant growth regulators were absent from the control treatment. Sub-cultures were started every three weeks while all cultures were maintained in a climate chamber (16 h, light, and 26 ± 3 • C) for seven weeks. Callus growth enhancement was evaluated using V. dahliae as an initiator. Callus was removed from individual cultures utilizing vacuum filtration 72 h after infection, and for 40 days, Callus was given a visual assessment every five days. Sub-cultivation of the V. dahliae occurred every 35 days in PDA (at 21 • C). For ten days, conidia were cultivated on potato dextrose (PD) medium at 22 degrees Celsius using a 240 rpm rotary shaker. After centrifugation, three washes were performed with a 0.1 M K 2 HPO 4 -KH 2 PO 4 solution at a pH of 6.5 to remove any remaining debris from the conidia. Under a microscope, conidia concentration was measured with a hemocytometer. We injected either 25 µL of conidial suspension ((3-5) × 10 7 conidia/mL) or control (sterile water) into 8 mL of freshly prepared MS solid medium.

Total Protein Assay in O. basilicum's Callus and Cell Suspension
As per Darrag et al., with changes [26], using LS media, callus was initiated and detected for 56 weeks. The medium was passed via filters with various mesh sizes. A conidial suspension ((2-5) × 10 7 conidia/mL) was added to 25 mL of LS culture medium (200 mL total volume), transmitted to 30 flasks (Erlenmeyer, 500 mL), and calibrated to a 250 mL final volume utilizing the liquid medium. After 72 h, the protein content of the cultures was evaluated. (250 mL) conical flasks with agar-free LS medium (100 mL) were used to produce suspension media, which was then maintained in a climate chamber for six weeks at 30 ± 2 • C, 16 h light circumstances, and shaking at 110 rpm, with subculture every fourteen days.
Protein content increases in cell suspension and callus were determined every five days until the callus was 40 days old. At intervals of 5-40 days of cell suspension, cultures were collected 72 h after inoculation to identify protein content. Protein estimation was carried out according to the method of Bradford [37].
In brief, aliquots of protein extract were placed in clean test tubes (protein concentration was evaluated at intervals ranging from 5 to 40 days after callus and cell suspension formation). Mix one hundred microliters of protein extract and 1 mL of working solution, add distilled water (3 mL), give the mixture a good shake, and let it sit for 2 min at 25 • C. The optical density was evaluated spectrophotometrically at 595 nm; the protein concentration was recorded using a standard curve of BSA. The standard curve was established by using different concentrations of BSA (5-100 µg), similar to that described above.

Characterization Using GC-MS Analysis
After 40 days, the cell suspension hydro distillate extract was diluted with n-hexane, employing an auto-sampler injector and administrated (1 µL) with an automatic injector (GC grade, 2 µL: 1 mL) (Model automatic sampler: Varian, Combi Pal). The GC-MS was used to detect that connection (GC, CP-3800, Walnut Creek, Varian, CA, USA, and MS, Varian, Saturn 2200). Its parameters were 0.25 µm thickness, 5% phenyl-dimethylpolysilox-ane, 30 m length, and 0.25 mm inside diameter, with VF-5ms, a column of fused silica capillaries [9]. The ionization energy of the electron impact (EI) used in the ionization detector was 70 eV. The carrying gas was helium, with a constant flow rate of 1 mL/min maintained throughout the experiment. The transfer line and injector temperatures were 300 and 240 • C, respectively. For the first minute, the oven was maintained at 50 degrees Celsius, then for the next 50 min, it was heated to 230 degrees Celsius at a rate of 30 degrees Celsius per minute, then for the next 5 min, it was heated to 290 degrees Celsius at a rate of 10 degrees Celsius per minute, and finally, it was kept at isothermal conditions for 6 min. The total injection duration was 54.3 min, and the specimen injection split ratio was 1/500. The Wiley and NIST electronic libraries were used to develop a compound standard for n-alkanes (C6-C26) and determine the identities of the individual components.

Evaluation of the Extracted Secondary Metabolites' Contact-Insecticide and Antifeedant Efficacy against R. ferrugineus
Final methanolic volatile extract concentrations were prepared by serial dilution (1, 10, 50, 100, 500, 1000, 5000, and 7000 g/mL). Moreover, compounds (estragole, (E)-β-ocimene, 1,8-cineole, β-terpineol, α-guaiene, eugenol, β-bergamotene, β-farnesene, germacrene D, α-eudesmol, nerol, linalool, and γ-terpinene) were dissolved in acetone and then added to 0.1% TritonX-100 [26]. Cells were suspended in the methanolic extract at serially increasing concentrations for 40 days. To dilute the compounds, TritonX-100 (0.1%) was added to acetone after the chemicals were dissolved. Adults or larvae of R. ferrugineus were purchased. They were then cultured using long lengths of sugar cane stem. The extracts' anti-larvae action was assessed by applying topically to larvae and keeping them at 4-5 • C for 5 min. Utilizing a manually controlled micro-applicator, each larva had extract injected into its dorsum in three separate 10 µL doses using a 50-ll micro-syringe from Hertfordshire, UK's Burkard Manufacturing Co., Ltd. (MS-N50; Shizuoka, Japan's Ito Corp.). Five larvae in a box with three replications were fed on portions of long sugarcane stem measuring 10 cm. Larval mortality was assessed 24, 48, 72, and 96 h after topical medication to calculate the LD 50 . The sugarcane stem's ten-centimeter-long portions cut into equal longitudinal halves were used to measure the antifeedant activity against adults. Long sections of sugarcane stem with a surface area of approximately 32.2 cm 2 were dried in air at room temperature, assuming a dipping time of 10 s and an initial serial concentration of (10 mL). Each treated object remained in a plastic container. Each box included an additional pair (male and female). Each therapy was administered ten times. In total, 24-, 48-, 72-, and 96-h feeding observations were assessed.

Assessment of O. basilicum Cell Suspension Extract and Pure Components on R. ferrugineus Larvae's Overall Proteolytic Enzyme Activity (In Vitro)
Protein concentrations were measured utilizing the Lowry approach [46]. The homogenate of R. ferrugineus 4th midgut instar larvae (lab strain) was assayed for total proteolytic enzyme activity using azocasein, as Darrag et al. [26], with modifications. Ten 4th midgut larval homogenates were carefully removed, incised, and then rinsed in a solution containing 0.9% NaCl before even being homogenized in a buffer (500 µL) containing 5 mM dithiothreitol (DTT), 0.1% (v/v) Triton X-100, and 50 mM N-2-hydroxyethyl piperazine-N -2-ethane sulphonic acid (HEPS), and 8.0 pH. It was spun in a centrifuge (Sigma 3k30), and the remaining homogenates were from a preliminary phase for 30 min at 5000 rpm. Using the supernatants, the protein concentration and proteolytic enzyme activity were determined. A total, including assay buffer (60 µL), was used to incubate the supernatant (10 µL) at 37.5 • C for 20 min before azocasien (200 µL, 2% (w/w)) was added (pH = 8). Before each reaction experiment, 40-day-old methanolic extract of cell suspension, pure chemicals, and 10 µL of enzyme specimens were incubated for 10 min. The addition of the substrate initiated the reaction, which was allowed for 20 min in the case of leupeptin before being terminated 180 min later by adding 300 µL of cold tri-chloroacetic acid (TCA) (10%, v/v). Twenty minutes were spent centrifuging the mixture at 5000 rpm (Sigma 3k30). Overall, 10 µL of NaOH (10 N) was added to the supernatant, and the absorbance was measured using an ELISA plate reader at 450 nm to quantify the enzymes' specific activity as OD450/mg protein/h.

Assessment of the Cell Suspension Extract and Components on Serine Proteinase (In Vitro) Specific Activity of R. ferrugineus Larvae
We used a serine protease assay buffer and a modified version of the fast microplate method to analyze the serine proteinase's specific activity in mixtures (150 µL) [26]. After 30 min of centrifugation (8000 rpm), the test buffer (100 mM Tris-HCl) was removed from the midgut homogenate of fourth instar larvae (pH 8.1) (Rotors, Model No. 12158, Sigma 3K30) [26]. The sample (10 µL) was diluted using an assay buffer to 50 µL to determine the presence of trypsin proteinase, chymotrypsin proteinase, and elastase. Assay buffer was used to dilute stock substrates of BAρNA (100 mg/mL DMSO), both SAAPFρNA and SAAPLρNA (100 mg/mL DMF), to 1.0 mg/mL. To halt the reaction after 15 min of incubation at 37 • C, 50 µL of acetic acid (30%, v/v) was added. The intensity of the activity was measured using a 405 nm ELISA plate reader. A denaturation enzyme was used in place of an active enzyme in the experiment mixture. For three substrates (SAAPLρNA, BAρNA, and SAAPFρNA), activity levels of certain proteinases were assessed as OD mg −1 protein min −1 .

Assessment of the Cell Suspension Extract and Pure Components on the Specific Activity of Metalloproteinase (In Vitro) of R. ferrugineus Larvae
The 4th midgut instar larval homogenate was used to test metalloproteinases activity using the substrate CEGR (DABCYL-Cys-Glu-Gly-Arg-Ser-Ala-EDANS-NH2) [26], with modifications. The ten midgut larvae from the fourth stage were neatly eliminated, dissected, and repeatedly washed with 0.9% NaCl solution. A 500 µL protease test solution, including 50 mM HEPS, 0.1% Triton X-100, and 5 mM DTT (pH 8.0), was utilized to homogenize the midgut instar. The homogenates were then centrifuged employing a Sigma 3k30 (cooling centrifuge) at 5000 rpm for 30 min to evaluate the supernatant's enzyme activity and protein content. Before introducing CEGR (125 µL, 2% (w/v)), each experiment's supernatant (10 µL) was incubated in an assay buffer (60 µL) at 37 • C for 20 min (pH 8). During a 10 min incubation period without a substrate, 10 µL enzyme samples were tested (20 min. for EDTA, EGTA, 1.10, Phenanthroline). After 180 min at 37 • C, 300 µL of cold TCA, 10% (v/v), was used to terminate the reaction. For the next 20 min, the mixture was spun (5000 rpm, Sigma 3K30). The absorbance was measured at 450 nm after mixing the supernatant with hydroxide solution (10 µL, 10 N) using an ELISA reader. An enzyme-free assay combination was utilized as a blank, and particular activity was evaluated utilizing the OD493 mg −1 protein min −1 and an enzyme-free blank specimen.

Assessment of Cell Suspension Extract and Components on Cysteine Proteinase Activity of R. ferrugineus Larvae (In Vitro)
Utilizing the substrate N-benzoyl-Phe-Val-Arg-p-nitroanilide hydrochloride and certain modifications, the cysteine proteinases of R. ferrugineus homogenate from the fourth midgut instar was tested for activity. Ten larvae of the fourth midgut were carefully taken out, dissected, and periodically washed with 0.9% (w/v) NaCl solution, assay buffer containing 50 mM HEPS, 5 mM DTT, and 0.1% Triton X-100 (v/v) (pH 8.0, 500 µL) was used to homogenize the samples. The homogenates were spun using a cooling Sigma 3k30 centrifuge for 30 min at 5000 rpm. The supernatant was tested and found to have a verifiable amount of protein and an active form of all the proteolytic enzymes. Before introducing 100 µL of the substrate, 10 µL of the supernatant was incubated in assay buffer (60 µL, pH = 8) at 37 • C for 30 min. Enzyme samples in 10 µL were incubated for 10 min with iodoacetic acid before the addition of the substrate. We added mersalyl (1.5 mL, 5 mM), garnet rapid reactive solution (0.02 mg/mL), and Tween 20 (2%, v/v) to terminate the reaction at 37.5 degrees Celsius after 60 min. The absorbance at 520 nm was measured after spinning the reaction for 6 min at 5000 rpm. The activity in the midgut was assayed at OD520.60 min −1 mg −1 of protein with an ELISA rapid reader.
Various inhibitors were applied to understand the proteases found in the larval fourth of instars' homogenate midgut preparations of R. ferrugineus. In all protease activity assay studies, 10 µL of enzyme samples specimens were treated with a pre-incubation period of extract or pure substances for 10 min (estragole, β-bergamotene, (E)-β-ocimene, β-terpineol, α-guaiene, 1,8-cineole, eugenol, germacrene D, β-farnesene, α-eudesmol, nerol, linalool, and γ-terpinene) or inhibitors at concentrations of 1, 10, 50, 100, 500, 1000, 5000, and 7000 mg/L. The impact of previous concentrations of each enzyme's activity in the presence of an extract or substances in treated larvae was assessed in vivo after a 24-h treatment period. An experiment that was set up free of potential inhibitors served as a control. An ELISA plate fast reader measured the enzymes' absorption at various wavelengths. For each inhibitor, the control enzyme's percentage activity was assessed for each enzyme. Four instars midgut larvae were treated with previous concentrations of extracts or compounds, assessed as previously mentioned, to measure the protease activity in vivo. Larvae were subjected to extract-and compound-treated leaf discs for 24 h.

Docking of Experimented Compounds into Enzymes
Using the protein data bank (PDB), we obtained serine-(PDB:3F7O) [47], metallo-(PDB:1KAP) [48], cysteine-(PDB:3IOQ) [44], and trypsin proteinase (PDB:1FN8] [49], which was thereafter transferred to the Molecular Operating Environment (MOE). To compensate for the lack of hydrogen, heteroatoms and crystallographic molecules of water were eliminated from the protein's chemistry [50]. The compounds (estragole, β-terpineol, (E)β-ocimene, α-guaiene, β-bergamotene, germacrene D, eugenol, 1,8-cineole, β-farnesene, α-eudesmol, nerol, linalool, and γ-terpinene) were drawn using Chem Draw professional 15 builder module. After reducing the number of ligands, creating three-dimensional structures, removing duplications, and inserting bonds, the molecule was ready for docking. After inputting the default configuration and producing structures with the minimum energy, the ligands were made elastic and manually positioned within the catalytic site cavity presented in the enzyme model. The MOE 2015.10 (Molecular Operating Environment (MOE), Montreal, QC, Canada) was utilized for the protein-ligand docking, and a caused fit approach was used, which indicates that the receptor is stiff and the ligand is elastic [46]. The full-force field was used to determine the binding energy and scoring techniques that estimated free-binding interaction energies using terms from the molecular force field to calculate the affinity between the ligand and the protein. After obtaining the docking data, we recorded calculations and RMSD values and confirmed that the optimal ligand interaction had been achieved.

Statistical Design
Statistics and probit analysis were performed using SPSS 25.0, as mentioned by Finney [51]. To sum up, all numerical predictions of toxicity variables and findings were presented as the mean ± SE. We regressed mortality and translated the resulting dosage to an LC 50 value (µg/mL). Using the relative growth rate's least-squares regression analysis (control %) with the extract level's logarithm, the range of LC 50 was determined with a confidence interval of 95 percent. Enzyme activity data were analyzed using an ANOVA. Means were separated using the SNK (Student-Newman-Keuls) test, and deviations at the p < 0.05 level were considered noteworthy.