Secondary Metabolite Profiling, Antioxidant, Antidiabetic and Neuroprotective Activity of Cestrum nocturnum (Night Scented-Jasmine): Use of In Vitro and In Silico Approach in Determining the Potential Bioactive Compound

This study aims to describe the therapeutic potential of C. nocturnum leaf extracts against diabetes and neurological disorders via the targeting of α-amylase and acetylcholinesterase (AChE) activities, followed by computational molecular docking studies to establish a strong rationale behind the α-amylase and AChE inhibitory potential of C. nocturnum leaves-derived secondary metabolites. In our study, the antioxidant activity of the sequentially extracted C. nocturnum leaves extract was also investigated, in which the methanolic fraction exhibited the strongest antioxidant potential against DPPH (IC50 39.12 ± 0.53 µg/mL) and ABTS (IC50 20.94 ± 0.82 µg/mL) radicals. This extract strongly inhibited the α-amylase (IC50188.77 ± 1.67 µg/mL) and AChE (IC50 239.44 ± 0.93 µg/mL) in a non-competitive and competitive manner, respectively. Furthermore, in silico analysis of compounds identified in the methanolic extract of the leaves of C. nocturnum using GC-MS revealed high-affinity binding of these compounds with the catalytic sites of α-amylase and AChE, with binding energy ranging from −3.10 to −6.23 kcal/mol and from −3.32 to −8.76 kcal/mol, respectively. Conclusively, the antioxidant, antidiabetic, and anti-Alzheimer activity of this extract might be driven by the synergistic effect of these bioactive phytoconstituents.


Introduction
Oxidative stress induced by reactive oxygen species (ROS) is deleterious to proteins, lipids, cell membranes, and DNA, and contributes to the development of several chronic and degenerative disorders [1]. An imbalance between oxidative stress and the antioxidant defense system causes cellular dysfunction, resulting in the development of many chronic diseases, including diabetes mellitus (DM) and neurological disorders [2,3]. DM is a metabolic disorder characterized by impaired carbohydrate metabolism resulting in elevated fasting and postprandial blood sugar levels. During persistent hyperglycemia, glucose can react with proteins nonenzymatically through the process of glycation [4,5].
shown in Table 1. Phytochemical screening revealed significant amounts of bioactive compounds, including flavonoids and polyphenols (Table 2) with free radical quenching ability, in the MeOH extract. The reductones serve as antioxidants by donating a hydrogen to the free radical, often corresponding with the reducing capacity of compounds, which may be a significant signal of its antioxidant potential [29][30][31].

α,α-Diphenyl-β-picrylhydrazyl (DPPH) Assay
DPPH is a relatively stable radical that is widely used to evaluate the quenching ability of antioxidants from natural sources such as fruit and plant extracts. The DPPH scavenging ability of different extracts of the leaves of C. nocturnum, at various concentrations, is presented as % inhibition in Figure 1. The MeOH extract, with an IC 50 value of 39.12 ± 0.53 µg/mL, was found to be the most potent in neutralizing the DPPH radical. The IC 50 of the reference standard, ascorbic acid, was 15.12 ± 0.65 µg/mL (Table 3). nocturnum measured using the DPPH and ABTS assays. Bar graph represents the % inhibition of radicals. The values are represented as mean ± SD of data from triplicate assays.  Free radical scavenging and antioxidant potential of different extracts of the leaves of C. nocturnum measured using the DPPH and ABTS assays. Bar graph represents the % inhibition of radicals. The values are represented as mean ± SD of data from triplicate assays.

ABTS Radical Scavenging Assay
The ABTS radical cation scavenging assay is widely used to evaluate the antioxidant potential of plant and fruit extracts and purified compounds. All the extract of the leaves of C. nocturnum neutralized the ABTS radical in a dose-dependent manner via electron donation to the radical (Figure 1). The inhibition of the ABTS radical was highest for the MeOH fraction (IC 50 20.94 ± 0.82 µg/mL) and that for the standard, ascorbic acid, was 94.33% (IC 50 22.76 ± 0.43 µg/mL) ( Table 3). The percent inhibition by each fraction has been shown in the Figure 1.

Ferric Reducing Antioxidant Power (FRAP)
The FRAP assay was used to evaluate the ferric-reducing potential of distinct extracts of the leaves of C. nocturnum. The outcomes demonstrated that MeOH has considerably higher FRAP values, 478.50 ± 4.56 µmol Fe (II)/g, compared to other extracts ( Figure 2).

ABTS Radical Scavenging Assay
The ABTS radical cation scavenging assay is widely used to evaluate the antioxidant potential of plant and fruit extracts and purified compounds. All the extract of the leaves of C. nocturnum neutralized the ABTS radical in a dose-dependent manner via electron donation to the radical (Figure 1). The inhibition of the ABTS radical was highest for the MeOH fraction (IC50 20.94 ± 0.82 µg/mL) and that for the standard, ascorbic acid, was 94.33% (IC50 22.76 ± 0.43 µg/mL) ( Table 3). The percent inhibition by each fraction has been shown in the Figure 1.

Ferric Reducing Antioxidant Power (FRAP)
The FRAP assay was used to evaluate the ferric-reducing potential of distinct extracts of the leaves of C. nocturnum. The outcomes demonstrated that MeOH has considerably higher FRAP values, 478.50 ± 4.56 µmol Fe (II)/g, compared to other extracts ( Figure 2).

Evaluation of α-Amylase Inhibition and Kinetics Studies to Explore the Mode of Action of the Extract
To investigate the antidiabetic activity, the α-amylase inhibitory potential of different extracts was evaluated. The MeOH extract effectively inhibited α-amylase in a dose-dependent manner and had the lowest IC50 value of 188.77 ± 1.67 µg/mL compared with those of the other extracts ( Figure 3, Table 3). The standard drug, acarbose, showed 75.58% inhibition of -amylase (IC50 41.54 ± 0.54 µg/mL) ( Figure 3, Table 3). Furthermore, kinetics studies revealed noncompetitive inhibition of -amylase by the MeOH extract unlike the competitive inhibition by acarbose ( Figure 4).

Evaluation of α-Amylase Inhibition and Kinetics Studies to Explore the Mode of Action of the Extract
To investigate the antidiabetic activity, the α-amylase inhibitory potential of different extracts was evaluated. The MeOH extract effectively inhibited α-amylase in a dosedependent manner and had the lowest IC 50 value of 188.77 ± 1.67 µg/mL compared with those of the other extracts ( Figure 3, Table 3). The standard drug, acarbose, showed 75.58% inhibition of α-amylase (IC 50 41.54 ± 0.54 µg/mL) ( Figure 3, Table 3). Furthermore, kinetics studies revealed noncompetitive inhibition of α-amylase by the MeOH extract unlike the competitive inhibition by acarbose ( Figure 4).

Evaluation of Acetylcholinesterase Inhibition and Kinetics Studies to Explore the Mode of Action of the Extract
The AChE enzyme activity was evaluated using a colorimetric method in which a yellow-colored 5-thionitrobenzoate anion, with an absorption maximum at 412 nm, is produced when thiocholine reacts with 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB). Amongst the five C. nocturnum leaf extracts, the MeOH extract exhibited the highest AChE inhibitory activity in a dose-dependent manner, with an IC 50 of 239.44 ± 0.93 µg/mL ( Figure 3, Table 3). The standard drug, tacrine, showed the lowest IC 50 of 4.03 ± 0.47 µg/mL ( Figure 3, Table 3). A kinetics study was performed to determine the mode of inhibition by tacrine and the MeOH extract. As is evident from the Lineweaver-Burk double reciprocal plot of 1/V vs. 1/[S] (Figure 4), the MeOH fraction showed a competitive inhibition, whereas Tac exhibited a noncompetitive inhibition, indicating that it binds to the allosteric site of the enzyme (Figure 4).  In vitro inhibition of α-amylase and acetylcholine esterase (AChE) activities by C. nocturnum leaf extracts. The MeOH extract was the most potent in inhibiting α-amylase. Bar graph represents the % inhibition of α-amylase and AChE. The values are represented as mean ± SD of data from triplicate assay.

ADME Profiling of Compounds Identified via GC-MS Analysis
In this technical era, various computational strategies for the assessment of absorption, distribution, metabolism, excretion, and toxicology (ADMET) have been developed to reduce the time, money, and manpower in the field of drug discovery. In this context, we have performed the ADME analysis via an online web server, SwissADME, to unravel the physiochemical properties and pharmacokinetic profile of compounds identified via GC-MS analysis. In the BOILED-Egg analysis, 3 compounds were in the white region, predicted to have a higher intestinal absorption, whereas 11 compounds were in the yolk region, which were predicted to have a higher potential for penetration across the blood-brain barrier. Four compounds were outside the acceptable range and five compounds did not come under the definition of a "BOILED-Egg." In the analysis of drug-like properties, Lipinski's rule of five, bioactivity profile, and ADMET properties of the selected compounds were determined using the AI-based software. The five criteria in the Lipinski's rule, viz. molecular weight <500 Da, H-bond donors (HBD) <5, H-bond acceptors (HBA) < 10, and Log P (octanol-water partition coefficient) <5, were evaluated for each of the compounds. In the drug-likeness analysis, compounds 1,2,8,11,16,18,21, and 23 violated the one rule (MlogP < 4.15), whereas compounds 15, 17, 20, and 22 violated two rules (MlogP < 4.15 and MW < 500) of Lipinski (Table 5).      1 Log P, octanol-water partition coefficient; 2 TPSA, topological polar surface area; 3 BBB, blood-brain barrier; 4 HIA, human intestinal absorption; 5 HBA, hydrogen-bond acceptor; 6 HBD, hydrogen-bind acceptor.

Toxicity Assessment of the Selected Compounds
The compounds that resided in the BIOLED-Egg region were subjected to the toxicity analysis via ProTox-II, an online web server tool that predicts the toxicity class, LD 50 , and distinct toxicity parameters, such as hepatotoxicity, carcinogenicity, immunogenicity, mutagenicity, and cytotoxicity. Four compounds (dibutyl phthalate, phthalic acid diisobutyl ester, p-chloromethoxybenzene, and precocene I) were predicted to be carcinogenic. Precocene I was also predicted to be immunogenic (Table 6). These compounds were, therefore, eliminated from further docking analysis.

Discussion
Oxidative stress induced by ROS damages proteins, lipids, and DNA and is one of the major causes of several chronic diseases, such as DM [29,32]. The prevalence of DM is rising globally and this trend is predicted to continue in the coming decades [8]. Oxidative stress and DM are independent risk factors for several complications, including cardiovascular diseases, diabetic encephalopathy, and AD [2,[32][33][34][35]. Antioxidants from natural sources, such as plants, and their secondary metabolites are efficient quenchers of free radicals and interrupt their production. Their consumption helps in the management of oxidative stress and in preventing the onset of several diseases such as DM and AD [2,[36][37][38][39]. Numerous in vitro and in vivo studies have shown that C. nocturnum leaf extracts have antifungal, antibacterial, antidiabetic, and wound healing properties [22,24,27,28,40].
In this study, sequential extraction of C. nocturnum leaves was performed using n-Hexane, DCM, EtOAc, MeOH, and water. Phytochemical screening showed that the MeOH extract contains significant amounts of bioactive compounds, including flavonoids and polyphenols (Table 2), which are known for their free radical quenching ability, and that it has the highest TPC ( Figure 2). The reductones serve as antioxidants by donating a hydrogen atom to free radicals, and their content corresponds with the reducing capacity of the extracts and their antioxidant potential [29][30][31]. The MeOH extract exhibited significant total antioxidant activity ( Figure 1). These results are in concordance with those of previously studies [22,23,28,40]. A recently published study also showed that leaves of C. nocturnum are a rich source of phytochemical constituents [40]. Because phenolic compounds are believed to be responsible for the majority of antioxidant properties of plant extracts, the antioxidant potential of the MeOH extract might be attributable to polyphenolic compounds [32,41]. Compounds with the ability to reduce oxidative stress via quenching of free radicals can delay or stop the progression of several chronic diseases [1,8,42,43]. The MeOH extract of C. nocturnum leaves exhibited strong DPPH and ABTS radical quenching ability ( Figure 1, Table 3), indicating its potent antioxidant activity. These results are in agreement with previously published reports [23,44,45].
Several strategies have been developed to manage DM, among which, the strategies based on the inhibition of key enzymes are the most common. The inhibition of the most important carbohydrate metabolizing enzymes (α-amylase and α-glucosidase) is the first line drug therapy for the management of blood glucose levels in DM patients [29,46,47]. Oxidative stress and DM contribute to the development of several complications, including cognitive disorders, such as AD. AD is the most common cause of dementia. Epidemiological studies suggest that DM patients are more prone to develop AD [9,48]. The most prominent therapeutic strategy for AD is the inhibition of cholinesterase, as this enzyme catalyzes the conversion of ACh into choline and acetate. Several studies have established a strong relationship between DM, particularly type 2 DM, and AD, as they share common pathophysiological features, such as oxidative stress, abnormal signaling events related to insulin, advanced glycation end products, and mitochondrial anomalies [49,50]. Although the initial management of hyperglycemia is performed through diet control and exercise, this is not sufficient, and oral drug therapy is recommended [51]. Several synthetic drugs are commercially available for the management of hyperglycemia (glinides, carbohydrate metabolizing enzyme inhibitors, sulfonylureas, and thiazolidinediones) [52] and AD (tacrine, donepezil, rivastigmine, and galantamine) [16]. Several studies have shown that these antihyperglycemic drugs reduce the risk of dementia [53,54]. Despite their excellent profile, the long-term use of these anti-diabetes and anti-Alzheimer's medications causes several prominent side effects, including hepatotoxicity, nephrotoxicity, and hypoglycemia [55,56]. To date, there is no FDA approved drug that can manage both hyperglycemia and AD via targeting of α-amylase and AChE. In this context, our findings that the sequentially extracted C. nocturnum extracts exhibit antidiabetic and anti-Alzheimer's activity via targeting α-amylase and AChE are significant. Among all the extracts, the MeOH extract exhibited the most potent α-amylase inhibitory action. This extract inhibited the α-amylase activity in a dose-dependent manner (Figure 3, Table 3), consistent with previous reports that α-amylase inhibitory potential was higher in more polar plant extracts [32,57,58]. Thus, the enzyme inhibitory potential of the MeOH extract could be due to the presence of polyphenols, flavonoids, and glycosides. Interestingly, the DCM extract also showed marked inhibition at a concentration of 50 µg/mL. Besides this further increasing, the concentration did not show significant inhibitory potential. It might be due to lower number of phytoconstituents in the DCM extract. Preliminary screening also revealed that the MeOH extract of leaves of C. nocturnum inhibited the AChE activity in a dose-dependent manner (Figure 3, Table 3).
To find the mechanism of inhibition of α-amylase and AChE by the MeOH extract, we performed enzyme kinetics studies. The MeOH extract was found to be a noncompetitive inhibitor of α-amylase and a competitive inhibitor of AChE (Figure 4). On the contrary, the standard drugs, acarbose and tacrine, showed competitive and noncompetitive inhibition of α-amylase and AChE, respectively, which is in agreement with previous reports [32,39]. It is evident that plant extracts exhibit competitive and noncompetitive inhibition due to the presence of a variety of bioactive compounds [59]. A decrease in V max and no change in K m are characteristics that differentiate noncompetitive inhibition from competitive (no change in V max and an increase in K m ) and uncompetitive (decrease in both V max and K m ) inhibition [60].
Using GC-MS analysis, 23 compounds were identified as the bioactive substances probably responsible for the aforementioned effects of the MeOH extract (Table 4). Several studies have reported the antioxidant, antidiabetic, antifungal, and antibacterial activities of these compounds present in C. nocturnum [23,40]. However, our GC-MS analysis did not record the flavonol glycoside and steroidal saponins described in an NMR analysis of methanolic extract of leaves by Mimaki et al., (2001) [61]. However, these chemicals were also not documented in a previously published publication either, although our data are consistent with the same class of substances reported by Chaskar et. al. (2017), such as hexadecenoic acid, 1-Hexadecanol, and carboxylic acid [62].
Based on our results, we surmise that the bioactive compounds in the MeOH extract of C. nocturnum leaves, either individually or in combination, substantially ameliorate the oxidative damage and inhibit the activities of α-amylase and AChE. However, the most persuasive step in the development of drugs is the prediction of the pharmacological properties of a chemical entity using several AI-based software. Among the various AI-based strategies, ADMET is currently being used to avoid wastage of time, resources, and manpower [61,62]. For this reason, we performed the ADMET analysis to investigate the drug-likeness properties of the bioactive components of the MeOH extract predicted using GC-MS. The SWISS ADME generates results in the form of a BOILED-Egg graph. The white region denotes high gastrointestinal tract absorption of the compounds and the yellow region (yolk) indicates high BBB penetration. The ADMET analysis revealed that all the compounds had acceptable drug-likeness properties and conformed to Lipinski's rule of five [29]. However, some compounds violated either one or two of these rules, but these violations do not warrant exclusion of these compounds as potential candidates. Only 13 compounds were localized in the BOILED-Egg graph, and these were subjected to toxicity analysis ( Table 5). All the compounds were in the range of classified LD 50 values. Four compounds (dibutyl phthalate, phthalic acid di-isobutyl ester, p-chloromethoxybenzene, and precocene I) were active against the carcinogenicity parameter. Precocene I was also active against the immunogenicity parameter. These compounds were eliminated at this level from further docking analysis.
Molecular docking analysis was performed to determine the interactions of the selected constituents of the MeOH extract that interact with the active site of α-amylase and AChE, and consequently inhibit their activity. Such docking analyses to search for molecular targets of constituents in plant extracts have been reported previously [32]. Molecular docking is a crucial tool for examining the interaction of ligands with a target protein and helps in comprehending the mechanisms underlying their binding and inhibitory activities. Redocking co-crystallized acarbose and tacrine into their respective binding sites in α-amylase and AChE allowed us to validate the docking approach (Figures 5 and 6). We found that all the redocked structures interacted with the same amino acids as in the respective crystal structure. The molecular docking study was carried out using Pyrex and further validated using Autodock 4.2. Furthermore, our results illustrated that the selected ten compounds were strongly occupied the active pocket of the α-amylase crystal structure with binding energy (∆G) values ranging from −3.10 to −6.23 kcal/mol, which is quite a bit better than the standard (∆G -2.71 kcal/mol) as well as their substrate (∆G −2.79 kcal/mol). Among these compounds, 7,9-di-tert-butyl-1-oxaspiro [4,5] deca-6,9-diene-2,8-dione was most potent inhibitor of α-amylase, as evidenced by its lowest binding energy. Its binding to the active pocket was stabilized by interaction with nine amino acid residues (Leu165, Gln63, Thr163, Trp58, Trp59, Asp300, His299, Arg195, Tyr62) ( Figure 5). Interestingly, the same compound also showed the lowest binding affinity for AChE, and its binding was stabilized through interactions with 17 amino acid residues (Ser122, Asp72, Asn85, Trp84, Ser81, Gly80, Phe330, Tyr334, Tyr442, Trp432, Ile439, Ser200, His440, Glu199, Ile444, Gly441, Tyr121) ( Figure 6). The other selected compounds also interacted efficiently with the active pocket of AChE, showing varied ∆G values (Table 8). Although all the selected compounds interacted with the catalytic site of the both the target enzymes, resulting in inhibition of their activity, we cannot comment if all or few of these compounds are responsible for the actual inhibitory activity of the extract. Nonetheless, the results of our in vitro and in silico studies convincingly highlight the antidiabetic and anti-Alzheimer potential of the MeOH extract of C. nocturnum leaves.

Collection, Identification, and Preparation of Cestrum nocturnum Extract
The C. nocturnum leaves were collected (voucher no. IU/PHAR/HRB/22/21) and washed to remove filth and dust particles and shed dried for seven days. After drying, leaves were ground to powder form. The dried powder (25 g) was sequentially extracted with the appropriate amount of n-Hexane, dichloromethane (DCM), ethyl acetate (EtOAc), methanol (MeOH), and water using the Soxhlet apparatus. The filtered crude extract was scratched out and kept at −20 • C for further analytical use. The following formula was used to determine the percentage yield of various extracts. % yield = Weight of crude extract Weight of raw material × 100

Qualitative Screening of Phytochemicals
Each extract of leaves of C. nocturnum was qualitatively screened for the presence of phytoconstituents, such as phenols, glycosides, and steroids, following the methods described previously [63].

DPPH Radical Scavenging Activity
The method described by Brand-Williams et al. [64] was used to assess the DPPH radical quenching ability of the extracts. The reference standard ascorbic acid was used for the comparative study. The percent inhibition of the DPPH was calculated using the equation below: %DPPH = ∆Absorbance of control − ∆Absorbance of sample ∆Absorbance of control × 100

ABTS Radical Scavenging Activity
The ABTS stock solution (7 mM) was prepared by mixing it with 2.45 mM potassium persulfate. Before the experiment, the solution was suitably diluted to yield an absorbance of 0.70 at 734 nm. Different concentrations of the extracts (in a 100 µL volume) were added to 900 µL of ABTS solution and the mixtures were incubated for 30 min at 37 • C. The absorbance was taken at 734 nm using an Eppendorf Bio-spectrophotometer. The reference standard used was ascorbic acid [65]. The equation used for calculating the % inhibition was the same as that used for DPPH.

Ferric Reducing Antioxidant Potential
The ferric reducing potential was determined according to the standard protocol [66] with a slight modification [32]. The absorbance was taken at 593 nm. The results were calculated using the standard curve of FeSO 4 and indicated as µmol Fe (II)/g dry weight of the C. nocturnum leaves powder.

Total Phenolic Content
The total phenolic content was determined by using the Follin-Ciocalteu standard protocol [32]. The results were calculated using standard gallic acid curve. The results are manifested as µg GA equivalent/mg extract.

α-Amylase Inhibition Assay
The α-amylase inhibitory potential of the different C. nocturnum leaf extracts was determined according to the standard protocol [29,32]. The enzyme (5 unit/mL) was freshly prepared in 20 mM of ice-cold PBS (pH 6.7) containing 6.7 mM NaCl. The enzyme (250 µL) was mixed with different concentrations of the inhibitors (acarbose or extract), except in the blank, and incubated for 20 min at 37 • C. Thereafter, starch solution (0.5% w/v) was added and the mixture was incubated for 15 min at 37 • C. Following the addition of the DNS reagent, the mixture was vortexed and incubated at 100 • C for 10 min in a water bath. At the end of incubation, the absorbance at 540 nm was measured using an Eppendorf Bio-spectrophotometer. The % inhibition rate was evaluated using the following equation: % inhibition = 100 − % reaction where % reaction = (mean product in sample/mean product in control) × 100

Determination of Anti-Acetylcholinesterase Activity
The acetylcholinesterase test was prepared according to Ellman et al. (1961) with a slight modification [67]. For use as a blank control, 33 µL of 10 mM DTNB, 100 µL of 1 mM AChI, 767 µL of 50 mM Tris HCl buffer (pH 8.0), and 100 µL of extract (different concentrations) were mixed in a 2 mL cuvette. For the test reaction, 300 µL of the buffer was replaced with an equal volume of AChE solution (0.28 U/mL). Tacrine was used as a reference standard. The reaction was monitored for 20 min by measuring the OD at 405 nm every minute. The values are presented as the mean of three replicates. The % inhibition of enzyme activity was calculated using the following equation: % inhibition = ∆Absorbance of control − ∆Absorbance of sample ∆Absorbance of control × 100

Kinetics Studies to Assess the Mode of Inhibition of α-Amylase Activity by the MeOH Extract of C. nocturnum Leaves
Michaelis-Menten kinetics (the Lineweaver-Burk plot) [30,32] were determined to decipher the mode of inhibition of α-amylase activity by the MeOH extract of C. nocturnum leaves. α-Amylase was preincubated with the inhibitor (extract/acarbose) for 20 min. One hundred microliters of starch (0.625-5 mg/mL) was added to each tube, including the blank, and incubated at 37 • C for 15 min. After the addition of DNS solution, the absorbance was recorded at 540 nm. The Lineweaver-Burk plot was made to determine the effect of the extract or acarbose on V max and K m .

Kinetics Studies to Assess the Mode of Inhibition of AChE Activity by the MeOH Extract of C. nocturnum Leaves
The kinetic study was carried out using the varied concentration of substrate, AchI (i.e., 0.5, 1.0, and 2.0 mM), and inhibitor C. nocturnum leaves extract (0.0, 50, and 100 µg/mL of reaction). The hydrolysis of AChI by AChE, either in the absence or presence of an inhibitor, was spectrophotometrically monitored for 20 min at 405 nm. The absorbance was taken at 1 min intervals. The mode of inhibition was determined according to the Michaelis-Menten kinetics [39].

GC-MS Analysis of the MeOH Extract
The phytoconstituents in the MeOH extract, which exhibited the maximum inhibitory potential against α-amylase and AChE, were identified using GC-MS. The GC-MS analysis was performed at the Central Instrumentation Laboratory Facility (CIL), Central University of Punjab, Bhatinda, India. The sample was injected into a Restek column (30 m × 0.25 mm; film thickness, 0.25 µm) on a GC-MS system (Shimadzu QP 2010 Ultra GC-MS). The constant column flow of the carrier gas (helium) was 1 mL/min. The mass spectra peaks were compared against the reference National Institute of Standards and Technology (NIST) libraries to identify the compounds.

Retrieval and Preparation of Ligands Structure
Numerous organic compounds' structures and their functional details are available in the PubChem database (http://pubchem.ncbi.nlm.nih.gov) that accessed on 10 November 2022. A unique identification number (CID) has been designated for each compound in the database. The 3D-structures of GC-MS-identified compounds were retrieved in 3D SDF file format. Using BIOVIA Discovery Studio Visualizer, the SDF file of ligands was converted into PDB file format. The CHARMM force field was applied in order to singe step minimization using the steepest descent method for 500 steps and an RMS gradient of 0.01.

Target Protein and Ligands Preparation
The target protein was prepared by deleting heteroatoms and adding polar hydrogen, as well as kollman charges, by using Autodock 4.2 [69]. Further 3D structure of the proteins was converted into PDBQT file format. The ligands were prepared according to the welldefined standard protocol [70].

ADME and Drug-Likeness Studies of Selected Ligands
The selected ligands were subjected to pharmacokinetic profiling by using a web-based tool, as defined in earlier studies [71]. Furthermore, the ligands' drug-likeness properties were also depicted by the Swiss ADME tool (http://www.swissadme.ch) that has been accessed on 20 November 2022.

Predicted Toxicity of the Selected Compounds
Toxicity prediction was performed by the ProTox-II (https://tox-new.charite.de/ protox_II/index.php?site=compound_input) on 25 November 2022, an online web-based server for the prediction of toxicities of small molecules. It provides the numerous details of the compounds about the toxicity such as LD 50 , Carcinogenicity, Immunotoxicity, Mutagenicity Cytotoxicity, as well as, most importantly, Hepatotoxicity [72].

Molecular Interaction Analysis
To determine the antidiabetic and anti-Alzheimer's potential of the selected compounds, we performed in silico molecular docking of these compounds at the catalytic sites of α-amylase and AChE, respectively, using the standard protocol [73]. For validating the results of docking, the structures of acarbose and tacrine were extracted from the structures of their respective complexes with α-amylase and AChE and redocked within the active pocket of the respective targets using Autodock. After the completion of docking, the structures of the complexes were visualized using the BIOVIA Discovery Studio Visualizer and ranked on the basis of binding energies.

Conclusions
For the first time, we demonstrate the potent antidiabetic and anti-Alzheimer's activities of sequentially extracted C. nocturnum methanolic leaf extracts via the inhibition of α-amylase and AChE, respectively. The results of our in vitro analyses show that the methanolic extract of C. nocturnum leaves has potent antioxidant, antidiabetic, and anti-Alzheimer's activities. These results were further corroborated by the antidiabetic and anti-Alzheimer's properties of the bioactive compounds identified using GC-MS. The findings suggest that 7,9-di-tert-butyl-1-oxaspiro [4,5] deca-6,9-diene-2,8-dione alone or in combination with other compounds inhibits the activities of both α-amylase and AChE. Thus, it is a good approach to alleviate oxidative stress and hyperglycemia, as well as Alzheimer's, with the whole of these compounds/extracts. A thorough and comprehensive in vivo study is also required to fully understand the function of these extracts and their bioactive constituents.