Antioxidant, Cytotoxicity, Antimicrobial Activity, and In Silico Analysis of the Methanolic Leaf and Flower Extracts of Clitoria ternatea

Infectious diseases pose a significant threat to human health worldwide. To address this challenge, we conducted a comprehensive study on the leaf and flower extracts of Clitoria ternatea plants. Our research encompassed in vitro assessments of their antibacterial, antibiofilm, antioxidant, and cytotoxic properties. Additionally, we employed in silico screening to identify promising compounds with potential applications in developing novel anti-Escherichia coli medications. Notably, our investigation revealed a remarkable inhibition zone of 13.00 ± 1 mm when applying the leaf extract (200 μg/ml) against E. coli, showcasing its potent antibacterial properties. Furthermore, both the leaf and flower extracts exhibited substantial biofilm inhibition efficacy against S. aureus, with inhibition percentages of 54% and 58%, respectively. In the realm of antioxidant activity, the leaf and flower extracts of C. ternatea displayed noteworthy DPPH free radical scavenging capabilities. Specifically, the leaf extract exhibited a substantial activity of 62.39% at a concentration of 150 μg/ml, while the flower extract achieved 44.08% at the same concentration. Our study also evaluated the impact on brine shrimp, where the floral extract displayed a significantly higher mortality rate of 93.33% at a dosage of 200 μg/ml compared to the leaf extract. To elucidate potential therapeutic targets, we utilized molecular docking techniques, focusing on the acbR protein (5ENR) associated with antibiotic resistance in E. coli. In this analysis, compounds isolated from the C. ternatea leaf extract, namely D1 (CID-14478556), D2 (CID-6423376), and D3 (CID-20393), exhibited binding energies of −8.2 kcal/mol, −6.5 kcal/mol, and −6.3 kcal/mol, respectively. Additionally, compounds from the flower extract, E1 (CID-5282761), E2 (CID-538757), and E3 (CID-536762), displayed binding energies of −5.4 kcal/mol, −5.3 kcal/mol, and −5.1 kcal/mol, respectively. In conclusion, the leaf and flower extracts derived from C. ternatea represent a promising natural resource with potential therapeutic applications in combating antibiotic-resistant pathogens.


Introduction
It is well-established that people and plants have a strong relationship [1].Humans rely on plants for a variety of purposes, including food, medicine, and domestic use [1].Plants have always been an essential source of drug invention [2].In developing countries, approximately 70-80% of the population still relies on the herbal drug for their primary healthcare [3].Secondary metabolites present in the plant are responsible for benefcial medicinal efects [3].
Conventional herbal medicine is employed globally to treat a wide range of illnesses, such as cancer, diabetes, and heart disease [4].Plants produce a wide range of chemical compounds known as auxiliary metabolites.Tree primary classes of these compounds are terpenes, nitrogencontaining compounds, and phenolic compounds, each possessing unique organic properties that make them valuable in addressing various medical conditions, including cancer, neurological disorders, diabetes, wounds, atherosclerosis, cardiovascular diseases, and injuries [5].Plant extracts fnd extensive applications in various industries, including food, cosmetics, and pharmaceuticals, underscoring the importance of conducting systematic research on medicinal plants to explore their therapeutic potential, biological properties, safety profles, and diverse active compounds [6,7].
Clitoria ternatea belongs to the Fabaceae family and is commonly known as butterfy pea or blue pea fower, with the Bengali name "Aparajita" [8].Tis plant is a long-lived climber and is cultivated as an ornamental plant in many countries [8].Tere are several species of C. ternatea with varying fower colors, including light blue, dark blue, white, and mauve [8].Blue pea plants are found in several countries worldwide, including Tailand, Malaysia, Kenya, Australia, the USA, Sri Lanka, Brazil, Cuba, Sudan, and others.[9].In several Southeast Asian countries, blue pea fowers are consumed as a vegetable [10], and extracts from these fowers are used in desserts and beverages [11].C. ternatea serves various agricultural and medicinal purposes, including use as animal feed, nitrogen-fxing crops, an environmentally friendly insecticide, and food coloring and in traditional medicine for conditions such as anasarca .Recent studies have suggested that diferent parts of C. ternatea exhibit sedative properties and antimicrobial, antiinfammatory, analgesic, antipyretic, and immunomodulatory activities [12].
Te misuse and overuse of antibiotics in human and animal healthcare, along with inadequate infection control measures in medical facilities, have given rise to a signifcant global health concern known as antibiotic resistance [13].Within the feld of biomedical science, there is a strong emphasis on recognizing the value of therapeutic plants [14].
To efectively and economically identify potential drug candidates during the drug discovery process, considerable attention has been given to computer-aided drug design (CADD) methodologies [15].In particular, in silico methods have proven to be valuable in predicting new drugs and identifying potential targets by utilizing compound structures prior to their actual synthesis [16].Consequently, the objective of this research was to investigate the antimicrobial properties of methanolic extracts derived from the leaves and fowers of Clitoria ternatea in controlled laboratory conditions (in vitro).Te selected bacteria for evaluation include Escherichia coli, Salmonella typhi, Staphylococcus aureus, and Pseudomonas sp.Additionally, this study aimed to assess the extracts' ability to inhibit the formation of bioflms, evaluate their antioxidant activity using DPPH, determine their cytotoxic efects on Artemia salina, and identify potent compounds present in the leaf and fower extracts that specifcally target the acrB protein of E. coli.

Collection and Preparation of Plant Extracts.
Fresh leaves and fowers of C. ternatea were harvested from Rajshahi University campus, Bangladesh, and a taxonomist from the Department of Botany, University of Rajshahi, identifed the plant.Te voucher number of the specimen is denoted by an accession no.46 and deposited to the herbarium, Department of Botany, University of Rajshahi.Tese plant parts underwent a process wherein they were fnely chopped and then subjected to shade drying.Te resultant plant materials were then transformed into a powdered form using a grinder.Tese powdered plant components were meticulously placed within two separate conical fasks, with an appropriate quantity of solvent added, adhering minimum ratio of 1 : 3 (1 gram of powder to 3 milliliters of solvent).Subsequently, the conical fasks were placed onto an orbital shaker, employing 90 mm Whatman flter paper for extract fltration.Tis was repeated in triplicate.Following this, the conical fasks were left uncovered for a period spanning from 24 to 48 hours to allow for natural evaporation.Te resulting extracts were then concentrated utilizing a rotary evaporator, maintaining a temperature of 40 °C.Only the residues were collected and placed into labeled glass vials, which were then stored in a refrigerator at 4 °C to ensure preservation.Additionally, the extract was dissolved in dimethyl sulfoxide (DMSO) for the purpose of conducting these experiments.

Antimicrobial Test.
Antimicrobial susceptibility testing was performed using the disc difusion method [17].Four bacteria (S. aureus, E. coli, S. typhi, and Pseudomonas sp.) were streaked on agar plate.Te sterile flter paper discs were impregnated with known amounts of the test substances and dried and placed on plates.Gentamicin (10 μg/ disc) was used as a standard disc.Tese plates were then kept at a low temperature (4 °C) for 24 h to allow maximum difusion.After that, the plates were kept in an incubator (37 °C) for 12−18 h to allow the growth of the organisms.Antimicrobial agents were identifed by the formation of zones of inhibition that kill or inhibit microbial growth [18].

Bioflm Formation Assay.
Te identifed strains of bacteria were inoculated into wells of a 96-well microtiter plate (Tarsons, India) containing 100 μl of Luria-Bertani (LB) liquid medium (Himedia, India) and incubated at 37 °C for 24 hours.Te glass slides were removed from the incubation chamber after the elapsed time, washed twice with double-distilled water, and then oven-dried for an hour at 37 °C.Te recovered wells were stained with crystal violet (0.1%) to assess bioflm formation.Te glass slides were washed twice with double-distilled water and once more after 60 minutes.Te OD 595 was determined using a microplate reader after the slides had been air-dried.Each strain's degree of bioflm development was assessed using OD values [19,20].Te experiment was conducted as planned to see if bioflm formation was inhibited.In this case, 100 μl of plant extract was introduced along with the bacteria.Te following equation was used to get the inhibition percentage: (1) 2.4.Antioxidant Activity Test.Te DPPH (2,2-diphenyl-1picrylhydrazyl) free radical scavenging assay, with BHT (butylated hydroxytoluene) as a control, was used to assess the antioxidant potential of C. ternatea leaf and fower extracts [21].Four autoclaved test tubes were employed for the DPPH scavenging activity test: one for BHT and three for the extract of C. ternatea, each at concentrations of 50, 100, and 150 μg/ml.Ten, methanol was added to each test tube to make 1 ml volume.Finally, each test tube received 1 ml of the DPPH solution, and the total volume was 2 ml.To complete the interaction, the test tubes were kept in the dark for thirty minutes at room temperature.Using a spectrophotometer and a reference blank solution, the absorbance of the solutions was measured at 517 nm.To ensure the experiment's accuracy, each absorbance value was repeated three times before the mean absorbance of the solution was determined.Te percentage (%) inhibition activity was calculated from the following equation [22]: where %I is the percentage of inhibition, A 0 is the absorbance of the control, and A 1 is the absorbance of the sample.

Cytotoxicity Test.
Te cytotoxicity test was conducted on brine shrimp (Artemia Salina) nauplii [23,24].Brine shrimp nauplii were hatched at ambient temperature in simulated seawater in a beaker.Five test tubes were then prepared with 25, 50, 100, 150, and 200 μg/ml concentrations of the extracts.Subsequently, 10 freshly hatched nauplii (Artemia salina) were placed in each test tube, and the tubes were kept at room temperature for 24 hours.Finally, the LC 50 values of methanolic leaf and fower extracts were determined and recorded.

Molecular Docking Study
2.6.1.Ligand Preparation.For the docking experiment, the chemical compounds obtained through GC-MS analysis of C. ternatea [25] were selected as ligands (Tables S1 and S2).
Tese chemical compounds of C. ternatea were retrieved from the PubChem database of chemical molecules, and their activities against biological assays were identifed through an extensive literature review [26,27].Te compounds underwent three-dimensional extraction and energy minimization using the Merck molecular force feld (MMFF94) to optimize the objective function.

Protein Preparation.
Using a protein data bank (PDB ID: 5ENR), the crystalline structure of E. coli proteins was obtained.Te shape of the protein was refned, and heteroatoms were removed using Discovery Studio software (version 4.5.0) and PyMol software (version 2.4.0).In Swiss PDB Viewer software (version 4.1), the washed proteins were reduced in energy and simplifed by employing the Groningen molecular simulation (GROMOS) 431B force feld [28].Te quality and geometry of the protein structure were evaluated with the help of the Ramachandran plot analysis.

Molecular Docking.
Te AutoDock Vina software (version 1.1.2) was used to conduct a molecular docking study to accurately understand the binding kinetics of the target protein and the retrieved compounds of C. ternatea [29,30].Te software was utilized to generate molecular models from the protein's structure.Te PDBQT layout was then applied to the ligand molecules.Te software was utilized to generate molecular models from the protein's structure.To group and rank the molecules, the most advantageous binding free energy and docking directions within a 2.0 RMSD range were chosen.Molecular docking was successfully performed with an exhaustiveness of 8 and a range of energy modes set at 10 and 20.Ultimately, no bonded interactions between the AutoDock structures were examined using Discovery Studio and PyMol [31].

Statistical Analysis.
GraphPad Prism (version 8.4) was used for the analysis and preparation of all fgures, in which all values were reported as the mean ± standard error of the mean.eight hydrophobic bonds at Ile729, Leu750, Phe727, Trp754, and Pro783 positions (Table 5 and Figure 5).

Antibacterial Activity
In the case of fower extracts, lower binding scores were obtained for three phytochemicals from C. ternatea, which were marked as E1, E2, and E3, referring to CIDs 5282761, 538757, and 536762, respectively.E1 demonstrated a higher binding afnity (−5.4 kcal/mol) than any other C. ternatea fower substance (Table 6).E1 interacted with E. coli acrB protein and formed eight hydrophobic bonds at Leu750, Ile786, Trp754, Phe727, and Pro783 positions.E2 interacted with E. coli acrB protein and formed two hydrogen bonds at the Pro50 and Tyr758 positions and two hydrophobic bonds at the Pro50 and Tyr49 positions.E3 interacted with E. coli acrB protein and formed two hydrogen bonds at Asn274 and Arg620 positions and a hydrophobic bond at Pro50 positions (Table 6 and Figure 6).

Discussion
Within the feld of biological sciences, plants have been utilized as potent sources of substances to control and treat human diseases.Antimicrobial medications are currently used to treat a large number of bacterial and fungal infections.According to Kone et al. [32], bacteria and fungi are known to cause opportunistic diseases.Antibiotic resistance has emerged in many virulently pathogenic microbial species as a result of widespread and indiscriminate usage of antibacterial drugs [32].Many antimicrobials now in use have negative side efects such as toxicity, hypersensitivity, immunosuppression, and tissue residues, constituting a public health risk.Tese restrictions reduce the therapeutic efcacy of currently available antibiotics, encouraging the search for alternative therapies for the treatment of bacterial and fungal infections.As the ecosystem shifts toward nontoxic and environmentally friendly products, there is a growing need to prioritize the development of contemporary pharmaceuticals derived from traditional medicinal plants.Tese can be used for the treatment of a variety of human and animal diseases.C. ternatea is a plant known for its various therapeutic characteristics.Te indigenous medicine uses several parts of C. ternatea plant, including the leaves, roots, stems, and fowers, to cure a wide variety of human afictions.Te purpose of the current study was to use C. ternatea leaf and fower extracts to control four distinct bacterial strains (S. aureus, E. coli, S. typhi, and Pseudomonas sp).We found that C. ternatea leaf extracts formed a signifcant zone of inhibition against E. coli, with a diameter of 13 mm.Te inhibition zones for the standard Gentamicin (10 μg/disc) ranged from 15 mm to 20 mm against four selected bacteria for the above-mentioned extracts.Upon examination, it was observed that the leaf and fower extracts of C. ternatea displayed intermediate resistance against all four selected bacteria, primarily at concentrations of 150 μg/ml and 200 μg/ml.In contrast, they exhibited higher resistance at concentrations of 50 μg/ml and 100 μg/ml against the same bacteria.Based on the data presented, the study concluded that the leaf and fower extracts of C. ternatea have antibacterial properties against the tested bacteria.As a result, the expected dose (150 μg/ml or 200 μg/ml) was sufcient to slow down bacterial development, while C. ternatea leaf extract was the most efective against the bacteria.Tese fndings corroborate earlier research fndings wherein the leaf extracts exhibited comparable antibacterial efcacy against S. aureus (11 mm), E. coli (13.3 mm), Pseudomonas sp.(13.3 mm), and S. typhi (12.7 mm), while the fower extracts demonstrated efectiveness against S. aureus (11 mm), E. coli (13.33 mm), Pseudomonas sp.(11.3 mm), and S. typhi (10.3 mm).[33].
Tis study suggests that both leaf and fower extracts of C. ternatea have strong abilities to inhibit bioflm formation, which aligns with previous research fndings [34].We found that the extract of C. ternatea leaves and fowers exhibited signifcant antioxidant activity, peaking at 62.39% and 44.08%, respectively, at a concentration of 150 μg/ml.Te majority of diseases and ailments are caused by oxidative stress, which is generated by free radicals [35].Antioxidants have been shown to protect cells from oxidative damage caused by free radicals, which may help to avoid diseases such as cancer and aging.By interacting with free radicals, chelating metals, and serving as oxygen scavengers, they can disrupt the oxidation process [36].Some of these are alcohol, tobacco, prescription drugs, smoked and barbecued food, pesticides, insecticides, harmful agrochemicals, additives in the foods we eat, and pollutants in the air we breathe [37].Several studies have shown that synthetic antioxidants such as butylated hydroxytoluene and butylated hydroxyanisole are suspected of being carcinogenic.As a result, antioxidants of natural origin have become a hot topic among modern researchers [38].In this study, the highest DPPH scavenging activity (62.39%) was observed in C. ternatea leaf extracts at a concentration of 150 μg/ml, while the lowest (19.43%) was found in C. ternatea fower extracts at 50 μg/ml.Te leaf and fower extracts exhibited lower antioxidant activity compared to the standard BHT.Specifcally, the leaf extracts showed relatively higher antioxidant activity than the fower extracts.In a study by Fu et al. [39], the antioxidant properties of C. ternatea's fower extract were investigated in vitro.Te DPPH results for their methanol extract (58%) were found to be lower compared to the methanolic extract used in the current study.Similarly, Jadhav et al. [40] also evaluated the antioxidant properties of C. ternatea's fower extract through in vitro testing.Teir methanol extract yielded DPPH results (52%) that were slightly higher than those obtained from the methanolic extract utilized in the   Biochemistry Research International present study.Te Brine shrimp assay is a vital tool in biology, efciently assessing the toxicity of various substances.It is a swift and cost-efective method for examining chemicals, natural compounds, and environmental samples for potential harm to Its applications span drug discovery, environmental monitoring, phytochemical research, and initial safety assessments [41][42][43].Te leaf extract concentration and brine shrimp mortality were positively correlated, as indicated by the LC 50 values of C. ternatea's leaf and fower extracts.Our fndings confrmed that the mortality percentages for the leaf and fower extracts were 83.33% and 93.33%, respectively.Rahman et al. [44] performed an in-vitro evaluation to assess the cytotoxic efects of C. ternatea's leaf extracts.Te cytotoxicity test results from their methanol extract (95%) were similar compared to the methanolic extract utilized in the current study.Utilizing molecular docking, the binding confguration of two interacting molecules with known structures is identifed.Tis process predicts how the receptor and ligand should ideally align to form a stable complex [20].Te evaluation of docking studies is an efective approach to drug development [45].Utilizing molecular docking, we assessed the efects of phytochemicals from C. ternatea plants on target proteins.From a range of datasets, molecular docking and molecular dynamics investigations can aid in the identifcation of efcient inhibitors.Our understanding of ligand-protein interactions and target binding afnity is improved by this research [46].Furthermore, extensive research has been conducted to discover potent inhibitors of target proteins, which is essential for advancing computer modeling and investigating the precise dynamics of ligandprotein interactions [47].Chian et al. suggest that successful ligand candidates can be distinguished through docking simulations [48].By intercepting at a protein's active region, the targeted protein can be blocked.10 chemicals (Table S1) were retrieved from the GC-MS analysis of methanolic leaf extracts, and 15 chemicals (Table S2) were retrieved from the GC-MS analysis of methanolic fower extracts of C. ternatea [25,49].Our research fndings were paired with an in silico technique to identify a possible component in leaf and fower   5).Similarly, the most potent compounds against the E. coli acrB protein (5ENR) were identifed in C. ternatea's fower extracts, which included 2,4-dihydroxy-2,5-dimethyl-3(2H)-furan-3-one, 1,2-dioxolan-3-one, 5-ethyl-5-methyl-4-methylene, and acetic acid, 1-(2-methyltetrazol-5-yl)ethenyl ester (Table 6).5ENR is a transport protein of E. coli that binds to the structure of bacterial efux pumps, and these pumps are crucial antibacterial drug development targets because efux pumps play a signifcant role in multidrug resistance (MDR) [50].as a valuable source of novel natural products possessing antimicrobial properties.However, further investigations are necessary to identify the active compounds within the extracts and assess their efectiveness in vivo.

Conclusion
Te emergence of new disease-causing pathogens and the development of antibiotic resistance pose signifcant concerns for the healthcare industry.In this study, methanolic extracts of C. ternatea leaves and fowers exhibited signifcant antibacterial activity.Te leaf had the highest antibacterial activity against the four bacterial strains.Although the antioxidant activity of both leaves and fowers was lower than that of the BHT standard, the leaf exhibited higher antioxidant activity compared to the fower.Terefore, the methanolic extract of C. ternatea leaves and fowers holds potential as a valuable source of natural antioxidant and antibacterial compounds, ofering opportunities for the development of novel pharmaceuticals to combat various human diseases.Te fower extracts exhibited higher toxicity to brine shrimp than the leaf extracts, as indicated by the cytotoxicity results.Two compounds, i.e., CID 14478556 from the leaf and CID 5282761 from the fower, displayed higher binding afnities in the active region of the acrB protein (5ENR) during molecular docking tests.Tus, these two could be used for future drug development against E. coli infections.

Figure 3 :Figure 4 :
Figure 3: Bioflm forming inhibition assay of C. ternatea plants' leaves and fowers extracts.(a) Inhibition assay of leaves extract against four selected bacterial strains and (b) inhibition assay of fowers extract against four selected bacterial strains.

Figure 5 :
Figure 5: Molecular docking of the acrB protein (5ENR) of E. coli and C. ternatea leaf chemicals.(a-c) Te cartoon view, 2D view, and surface view of the CID-14478556 and acrB protein complex; (d-f ) the cartoon view, 2D view, and surface view of the CID-6423376 and acrB protein complex; and (g-i) the cartoon view, 2D view, and surface view of the CID-20393 and acrB protein complex, respectively.

Figure 6 :
Figure 6: Molecular docking of the acrB protein (5ENR) of E. coli and C. ternatea fower chemicals.(a-c) Te cartoon view, 2D view, and surface view of the CID-5282761 and acrB protein complex;(d-f) the cartoon view, 2D view, and surface view of the CID-538757 and acrB protein complex; and (g-i) the cartoon view, 2D view, and surface view of the CID-536762 and acrB protein complex, respectively.

Table 1 :
Antibacterial activity of C. ternatea leaf extracts against four selected bacteria.

Table 2 :
Antibacterial activity of C. ternatea fower extracts against four selected bacteria.

Table 4 :
Cytotoxicity activity of C. ternatea leaves and fowers extract and LC 50 values.

Table 5 :
Noncovalent connections of C. ternatea leaf compounds with specifc proteins as well as their binding connections by hydrogen and hydrophobic bonds.
Te relationship distance was calculated in Å.

Table 6 :
Noncovalent connections of C. ternatea fower compounds with specifc proteins as well as their binding connections by hydrogen and hydrophobic bonds.