Antimicrobial potentials of Pandanus amaryllifolius Roxb.: Phytochemical profiling, antioxidant, and molecular docking studies

The emergence of antimicrobial resistance has led to an urgent need for novel antimicrobial drugs. This study aimed to determine the antioxidant and antimicrobial potentials in silico and in vitro of Pandanus amaryllifolius Roxb. ethanolic extract. The extracts were subjected to gas chromatography-mass spectrometry (GC-MS) analysis to identify the compounds. In silico antimicrobial studies were performed to gain insights into the possible mechanism of action of the active compounds as antimicrobials. The antimicrobial activities of the ethanolic extracts were assessed using the agar well diffusion method against the Surabaya strain of Escherichia coli and Staphylococcus aureus. Antioxidant properties of the extract were done using DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) and ABTS [2,2’-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid)] inhibition assays. The phytochemical screening revealed that the extract has high flavonoids and polyphenols contents. The GC-MS analysis detected the presence of 52 bioactive substances, with n-hexadecanoic acid, 9, 12, 15-octadecatrienoic acid, benzofuran 2,3-dihydro-. quinic acid, neophytadiene as major compound. Molecular docking studies showed that these compounds have a high binding affinity towards the target proteins, thereby inhibiting their activities. The ethanolic extract of P. amaryllifolius Roxb. exhibited antioxidant and antimicrobial activities. The IC50 were 11.96 ± 4.01 μg/ml and 26.18 ± 7.44 μg/ml for DPPH and ABTS. The diameters of inhibition zones (DIZ) and percentage of inhibition (PI) were calculated and varied for every single pathogen 16.44 ± 1.21mm/66.76 ± 4.92% (50%) and 21.22 ± 0.11mm/82.49 ± 3.91% (50%) for E. coli and S. aureus (DIZ/PI) respectively. Overall, this study provides information on the mechanism responsible for P. amaryllifolius Roxb. extract as a natural antimicrobe and lays the foundation for further studies to isolate and characterize the active compounds as antimicrobial candidates.


Introduction
Antimicrobial resistance (AMR) severely impacts the foundation of contemporary medicine and the viability of an efficient, worldwide healthcare response to the persistent threat posed by infectious diseases [1][2][3].The quest for novel and potent antimicrobial drugs is urgently required since antimicrobial resistance has turned into a worldwide health problem.
With the frustratingly slow development of new medications and pharmaceutical company investment, the abuse of antimicrobials like antibiotics has become a significant problem for both medicine and agriculture [4].By 2050, according to O'Neill (2016), AMR will lead to death of 10 million population per year surpassing cancer as the leading cause of death.This unsettling prediction and current trend in AMR has motivated researchers to isolate and discover new bioactive compounds from plants that targeted against microbial resistance [5] also notably given that around 50% of existing pharmaceuticals and nutraceuticals are naturally derived products [6,7].
The historic usage of plants to cure a variety of illnesses, including infectious diseases, and their potential as a source of novel antimicrobial agents [8].Moreover, chemically complex substances have excellent therapeutic potential since they have less adverse effects than manufactured medications and also have a low likelihood of acquiring resistance [9].Around 1,340 floras have been identified for specific antibacterial properties, and >30,000 antimicrobial chemicals have been extracted from various plant species [6].In the battle against AMR, herbal remedies have proven to be an effective tool that may be used alone or in conjunction with existing antibiotic strategies [4].Pandanus amaryllifolius Roxb.ex Lindl. is indigeneous and widely available in Indonesia.Using indigeneous plants for extraction of essentials for betterment of lives is the sustainable approach to achieve sustainable development goals (SDGs) [10].
A plant from the Pandanaceae family that is mostly found in Southeast Asian countries, Pandan has been utilized for traditional medicine and ethnobotanical products [16].Because of their distinctive and pleasant scent, pandan leaves are frequently used in Southeast Asia to flavor a variety of foods, including baked goods, desserts, and even home cooking.The only P. amaryllifolius Roxb.species with fragrant leaves is remaining to the chemical 2-acetyl-1-pyrroline (2AP), which is responsible for the perfume.Several studies have shown that P. amaryllifolius Roxb. is a great source of phenolic and flavonoid chemicals.Several studies have found that the leaves and roots of P. amaryllifolius Roxb.contain bioactive substances such phenolic compounds and flavonoids, which function as antioxidants and may scavenge free superoxide radicals [16,17].
In an effort to discover and explore the bioactivity compounds which can be antimicrobial agent candidate, there has been an increasing interest in examining the possible antibacterial activity of P. amaryllifolius Roxb.and its components [16,18].This present study intended to explore the antioxidant and antimicrobial potential of P. amaryllifolius Roxb.ethanolic extract by evaluating its activity in silico and in vitro methods.The findings could provide valuable outcome for understanding the pharmaceutical potential of P. amaryllifolius Roxb.as a source of new antioxidant and aid in the development of innovative therapies for the treatment of infectious diseases.Moreover, this study is well contributing to about 5 Sustainable Development Goals (SDGs 17): viz.good health and well-being, sustainable cities and communities, quality education, life on land, and responsible consumption and production etc. designed and adopted to serve as a "shared blueprint for peace and prosperity for people and the planet, now and into the future." in 2015.

Collection and authentication of plant material
Pandanus amaryllifolius Roxb.was collected from Taman Husada Graha Famili (Plant Medicinal Garden) Surabaya, East Java, Indonesia (7º18'12.2"S112º41'12.7"7E).The healthy and green leaves sample without indications of insect or microbial damage were collected form the site.The sample plant material was authenticated by the Purwodadi Botanical Garden (Indonesian Institute of Sciences, Jakarta, Indonesia).The voucher specimen was placed in the Plant Systematics Laboratory, Department of Biology, Faculty Science and Technology Universitas Airlangga, Indonesia with reference No. PA.0116022023.

Sample extraction
The leaves of P. amaryllifolius Roxb.were allowed to dry in open air and then ground into a powder with electric grinder and sieved by using 60-mesh size sieves.Each 100 g of powder was separately soaked with ethanol for 24 hrs at room temperature (28±2˚C), same procedure was repeated thrice subsequently, followed by filtration with filter paper (pore diameter 110 mm); Merck KGaA, Darmstadt, Germany, and the filtrate obtained was evaporated in a rotary evaporator at 60˚C to acquire crude extracts.The volume of the extract (w/w) was measured before storage at 4˚C in the refrigerator [2,15].

Phytochemical profiling by gas chromatography-mass spectrometry (GC-MS)
Gas chromatography-mass spectrometry (GC-MS) analysis was done by using "Agilent Technologies".The specification of GC-MS was agilent technologies model 19091N-136HP-INNO-Wax, 5% phenyl methyl silox agilent technologies, Initial temperature was 150˚C held for 2 min, final temperature was 260˚C at the rate of 20˚C/min, 1 μl of 0.2 g/ml fraction was injected.Temperature of heater was 300˚C, pressure was 27, 213psi, column (60mol/ L×250μmol/L×0.25μmol/L) and carrier gas (helium, 99.9999% purity, flow rate = 1,7583mL/ min; average velocity was 34,171cm/sec).The constituents of compounds were compared with the retention times and mass spectrum of the samples obtained using gas chromatography with the mass spectra from the National Institute of Standards and Technology (NIST) Version 14MS database library [3,19].

In silico antimicrobial activity
Ligand retrieval.Ligand from P. amaryllifolius Roxb.ethanol leaves in this study refers to the results of GC-MS.GC-MS data showed fifty-two compounds from P. amaryllifolius Roxb.3D structure of ligand, Collision induced dissociation (CID), formula, molecular weight (g/ mol), and SMILE Canonical obtained from PubChem database (https://pubchem.ncbi.nlm.nih.gov/).Structure optimization and energy minimization of the ligands were performed through Open Babel v2.3.1 to obtain the PDB file [2,20,21].
Protein preparation.Target proteins from microorganisms for identification of antimicrobial activity through a computational approach consist of Bacillus subtilis-FtsZ, Candida albicans-acetohydroxyacid synthase (AHAS), Escherichia coli-Rhomboid Protease (Rpro), and Staphylococcus aureus-Sortase A (SA).The RCSB database (http://www.rcsb.org/pdb/home/home.do)was used for retrieval of the four target proteins with the program database (pdb) file.Water molecules and native ligands are removed from targets through PyMol v2.5 software [3,22,23].
Drug likeness prediction.The similarity of drug properties from query compounds in this study was identified through drug likeness analysis.Lipinski Rule of Five from SCFBio server (http://www.scfbio-iitd.res.in/software/drugdesign/lipinski.jsp) was used for the drug like-molecule assessment in this study.Parameters including molecular mass, high lipophilicity (LOGP), hydrogen bond acceptors-donors, and molar refractivity are considered as determinants of the drug like-molecule properties of the query compound [24,25].
Virtual screening.The method used to identify activity from query compound by referring to interactions on targets through a computational approach is virtual screening.The type of virtual screening method used in this study is molecular docking.The Docking study mainly aims to identify the antimicrobial potential of P. amaryllifolius Roxb.Compounds from P. amaryllifolius Roxb.extract act as ligands and targets are proteins from microbes such as Bacillus subtilis-FtsZ, Candida albicans-AHAS, Escherichia coli-Rhomboid Protease (Rpro), and Staphylococcus aureus-Sortase A (SA).PyRx v0.9.9 software was performed to simulate ligandprotein docking [25,26].The docking grid in this study consist FtsZ-RCSB ID: 2VAM-Bacillus substilis Center (Å) X: 28 Ligand-protein interaction.In the present study, the molecular interactions were identified with the help of Discovery Studio 2016 software.Weak bond interactions can be identified in ligand-protein complexes such as hydrogen, van der Waals, hydrophobic, and pi/alkyl.These interactions serve to trigger activity such as an inhibitory response on the target [27,28].
3D molecular visualization.Molecular complexes exhibited through PyMol v2.5 software with coloring and structural selection methods.Transparent surfaces and sticks are selected for visualization.Color selection refers to the type of atom and 3D representation [22,29].

In vitro antioxidant activity
The DDPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) inhibition assay.The DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) inhibition assay was carried out in concurrence of Prieto (2012) and Wahyuni et al. (2023) with required modifications.During analysis 100μl samples at different concentrations from 1.075 to 200 μg/ml in ethanol were added to 100 μl DPPH reagent (0.2 mM) and incubated for 30 min at room temperature (28±2˚C), theses mixtures are further used as samples [2,30].Whereas, Ascorbic acid (reference standard) was positive control in the experiment.The resulting absorbance was measured at wavelength 517 nm by using microplate reader (Thermo Scientific, USA).Furthermore, the percentage of sample inhibition was calculated by using following Formula (1): Where 'A sample' is the absorbance of sample (the mixture of DPPH reagent and sample), whereas, 'A control' is the absorbance of only reference standard.The percentage of inhibition at all concentrations were plotted and linear regression analysis was carried out for determination of the half-maximal inhibitory concentration (IC 50 ) value.
The ABTS [2,2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid)] inhibition assay.The ABTS [2,2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid)] inhibition assay was performed as per the method illustrated by Fu et al. (2022) and Wahyuni et al. (2023).However, the ABTS reagent was prepared by adding 7 mM ABTS solution with 2.4 mM potassium persulphate solution and mixed well, then stored at room temperature (28±2˚C) for 12-16 hours in the dark place.After the waiting period, the absorbance of the solution at wavelength 734 nm was measured (stock solution diluted to obtain absorbance between 0.7-0.72).100 μl sample at different concentrations from 1.075 to 200 μg/ml in ethanol were mixed with 100 μl of ABTS reagent.Whereas, trolox and ascorbic acid were used as positive control (reference standard).After incubating for 5 mins in the dark place at room temperature (28±2˚C), the absorbance was assessed at wavelength 734 nm by using microplate reader (Thermo Scientific, USA).The percent inhibition and IC 50 value were calculated by using same Formula 1 as given above [2,31].
Antimicrobial activity.The antimicrobial activity was analyzed by using agar well diffusion method against selected gram-positive bacteria and gram-negative bacteria viz.Staphylococcus aureus and Escherichia coli respectively [2,3].Both bacteria were isolated from Surabaya City, East Java, Indonesia.Nutrient agar media was used to culture test microorganism.Whereas, potato dextrose agar was used for cultivating the microbes.The 30 mL sterilized agar medium was used to pour into presterile petri plates with 10cm diameter and all plates are allowed to solidify.Then 100 μL of inoculate with optical density (OD) 0.1 from each selected strain was spread on the solidified agar plates carefully with the help of glass spreader.On the other side a stock solution of plant extract was prepared and diluted serially (25% and 50%).The well of 5 mm diameter, was made in solidified medium in petri plates with the help of a cork borer.Whereas 20% of dimethyl sulfoxide (DMSO) and Chloramphenicol were used as negative control, and positive control respectively.All wells were filled with 30 μL of prepared extract and control accordingly in respective agar petry plates.All plates are incubated for 24 hrs at 37˚C, and antimicrobial activity was assessed and diameter of the inhibition zone (DIZ) was measured around the wells in the nutrient agar medium.The percentage of inhibition i.e.PI was calculated as by using Formula (2):

Phytochemical profiling
The yield of extract.The yield of ethanolic extract of Pandanus amaryllifolius Roxb.was 16.24 g.According to preliminary phytochemical analysis, ethanol extract P. amaryllifolius Roxb.leaves contained high amounts of flavonoid and polyphenols, and moderate level of alkaloid and terpenoids.However, saponin was not detected in the ethanol extract as shown in S1 Table .Flavonoids and polyphenols are well known for their antioxidant and anti-inflammatory properties [32].Alkaloids and terpenoids have also been shown to have several biological activities including anti-inflammatory, anticancer, and antimicrobial properties [33].The absence of saponins in the ethanol extract is an indication that it may not have any cholesterollowering properties [34].The GC-MS analysis was carried out for the identification of the specific compounds that responsible for antimicrobial properties of the extract.
Gas chromatography-mass spectrometry (GC-MS) analysis.The gas chromatogram of the constituent compounds from the ethanol extract is shown in Fig 1 .The GC-MS analysis strongly supported to find 52 bioactive compounds.The active principles with their respective retention time (RT) and concentration (peak area %) are given in S2 Table .The chromatogram showed that compound contained n-hexadecanoic acid (19.31%); 9,12,15-octadecatrienoic acid (17.82%); and benzofuran, 2,3-dihydro-(6.84%)as major compound.Therefore, the compounds were shown to have antimicrobial activity based on the references [2,3].

In silico antimicrobial activity
Ligand retrieval and protein preparation.The compounds from P. amaryllifolius Roxb.were attained from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/), which provides information consisting of compound name, Collision-induced dissociation (CID), formula, structure data format (sdf) file, and SMILE Canonical in Table 1.SMILE Canonical and sdf file were used as input data for identifying drug-like molecules from P. amaryllifolius Roxb.through Lipinski's Rule of Five.The protein samples used in this study consisted of FtsZ -Bacillus subtilis (RCSB ID: 2VAM), acetohydroxyacid synthase (AHAS)-Candida albicans (RCSB ID: 6DEK), rhomboid protease (Rpro)-Escherichia coli (RCSB ID: 3ZMI), and Sortase A (SA)-Staphylococcus aureus (RCSB ID: 2MLM), which were obtained from RCSB (http:// www.rcsb.org/pdb/home/home.do).Bacillus subtilis uses the protein FTsZ to regulate the cell division process, while the AHAS enzyme in Candida albicans plays a role in the virulence, invasion, and pathogenicity of the fungus [35,36].The activity of Rpro in Escherichia coli and SA in Staphylococcus aureus as targets for antibiotic binding is essential in the mechanism of bacterial infection in humans [37].The 3D structure visualization of the targets, consisting of FtsZ, AHAS, Rpro, and SA, was done through PyMol v2.5 using coloring by secondary structure (Fig 2).
Drug likeness prediction.Drug-like molecule analysis refers to an evaluation, characterization of a potential drug candidate molecule, and determination of the physicochemical properties of the molecule such as lipophilicity, bioavailability, and stability [38,39].This analysis is important for the drug development process because it helps determine the potential of a molecule as an effective drug and minimizes the risk of side effects.Drug likeness analysis in this study aims to determine the drug-like molecule of the query compound.Lipinski Rule's of Five (http://www.scfbio-iitd.res.in/software/drugdesign/lipinski.jsp) is used for drug likeness analysis, this method determines drug like-molecule through parameters consisting of molecular mass (D), high lipophilicity (LogP), hydrogen bond donors-acceptors, and molar refractivity.Molecular mass affects the mobility of a drug molecule with a value of �500 D, LogP value must be �5 and the number of hydrogen bonds (�5 donors and �10 acceptors) affects the physicochemical activity and absorption of drug molecules.The activity of drug molecules is also affected by molar refractivity which refers to the ability to induce charge mobility in the target domain with a value of 40-130.All compounds from P. amaryllifolius Roxb.leaves extract can act as a drug-like molecule (Table 2).All compounds may trigger specific activities such as inhibition and selective permeable passage to reach the target.
Virtual screening.Virtual screening is the application of computational methods such as molecular docking to determine the activity of natural compounds.Molecular docking plays a role in predicting ligand-target interactions [40].Ligand activity is determined from energy calculations or binding affinity values to understand the mechanism of this molecular interaction.Binding affinity is a negative bond energy formed due to molecular interactions, the binding affinity value must be negative because it increases the ligand-target interaction strength [41,42].Binding affinity is influential in the drug development process because it helps determine the potential of a ligand to become an effective drug.Inhibition of drug molecule activity is determined by the binding affinity value [41].Docking in this study targets to identify antimicrobial activity of P. amaryllifolius Roxb.compounds.P. amaryllifolius Roxb.compounds act as ligands and targets are protein from microbes consisting of Bacillus subtilis -FtsZ, Candida albicans-AHAS, Escherichia coli-Rhomboid Protease (Rpro), and Staphylococcus aureus-Sortase A (SA).  3) has a more negative binding affinity than other compounds.These three compounds have the potential as inhibitors of protein activity in microbes.Dual inhibitor refers to the activity of a ligand with more negative binding affinity for the two targets [43,44].-Pyrrolidinepropanol, alpha-cyclohexyl-alpha-  Ligand-protein interaction.Analysis of molecular interactions in protein-ligand complexes aims to determine the position and type of chemical bonds formed.Weak chemical bonds such as van der Waals, electrostatic, hydrophobic, hydrogen and pi/alkyl bonds are produced by ligands.Weak bonds can trigger ligand activity on targets such as inhibitors.The number of unfavorable interactions on the protein-ligand complex must be <3 to be stable.The results of this study indicate that all ligands can form weak bond interactions (Table 4) such as van der Waals, hydrogen, and Pi/alkyl in the target domain.An unfavorable interaction was formed between 4-amino-1-.beta.[45], with IC 50 value 11.96 ± 4.01μg/ml and 26.18 ± 7.44 μg/ml for DPPH and ABTS assays, respectively.The IC 50 value of antioxidant activity is a little bit more than ascorbic acid as a positive control.Suwannakul et al. [46] reported DPPH value of IC 50 of 110.57± 36.42 μg/ml for ethanol extract of Pandanus sp.Leaves, while Quyen et al. [47] reported IC 50 129.327 and 104.31 μg/ml for DPPH and ABTS respectively compared to IC 50 of 11.96 ± 4.01 μg/ml and 26.18 ± 7.44 μg/ml for DPPH and ABTS obtained from this current study.The observed variations in antioxidants properties are likely due to location of the plant used for these studies.Moreover, compared to the other studies, the leaf extract from P. amaryllifolius Roxb.was lower than Pterocarpus macrocarpus Kurz.bark extract [3], Trifolium pratense L. [48], Callisia fragrance leaf juice [45], and Centella asiatica L. leaf [49] that have been previously reported as high antioxidant compound.In addition, the antioxidant activity of this study was higher than Sonchus arvensis L. [2,15].The potent antioxidant activity of the P. amaryllifolius Roxb.extract was probably due to the presence of active ingredients with antioxidant activities.
Antimicrobial activity.Staphylococcus aureus and Escherichia coli collected in Surabaya were mainly used in this study as representatives of pathogenic bacteria and yeast as representatives of fungi for the analysis of antimicrobial activity.Antimicrobial tests were carried out with all the extracts against bacteria (Table 6, Fig 5), the ethanol extract has antimicrobial activities against all the pathogens.The diameter of inhibition zone (DIZ) and percentage of inhibition were varied for every single pathogen, 13.88 ± 0.  2014) observed antimicrobial activity of ethanolic extracts of Baeckea frutescens leaves against E. coli and S. aureus bacteria.They observed inhibition zone (in mm) are of 7 for 20% and 7.5 for 50% against E. coli and that of 7.5 for 20% and 11.5 for 50% for S. aureus respectively [51].Based on these values we can say that our results are in line with previous literature.Hence, P. amaryllifolius Roxb.possesses potential of antimicrobial activity.
Microbial infection will increase free radicals (reactive oxygen intermediates/ROI, reactive oxygen species/ROS, and nitric oxide synthetize/NO).Free radicals are molecules with one unpaired electron in their outer orbit which makes the molecule unstable [52].Free radicals can cause oxidative stress.It has implications for various pathological conditions [53].The involvement of oxidative stress can cause the amount of antioxidant status to decrease [52].Oxidative stress condition is defined as an imbalance condition between antioxidants and free   radicals, where the state of free radicals is higher than antioxidants [52].The number of antioxidants decreases because the body used to balance the high free radicals due to the presence of parasites.The more severe the infection from microbe, the use of antioxidants in the body will increase, causing the number of antioxidants in the body to decrease [52].It is very valuable for the further investigation of efficacious the P. amaryllifolius Roxb.leaf as antimicrobial agent candidate.The in silico molecular docking studies supported the experimental findings and provided insight into the mechanism of action of the bioactive compounds.The results showed that the compounds in P. amaryllifolius Roxb.extract can effectively bind to the target proteins of the selected pathogens, inhibiting their growth and replication.The in silico studies revealed that the extract can serves as an antimicrobial against Bacillus subtilis, Candida albicans, Escherichia coli, and Staphylococcus aureus by inhibiting the activity of FtsZ, AHAS, Rpro, and SA via 4-amino-1-.beta.-D-ribofuranosyl-2(1H)-pyrimidinone,-Pyrrolidinepropanol,.alpha.-cyclohexyl-.alpha.-phenyl,and (1S,15S)-Bicyclo[13.1.0]hexadecan-2-onewith more negative binding affinity and form stable interactions.The results of this study also revealed that -Pyrrolidinepropanol, alpha.-cyclohexyl-.alpha.-phenylhas dual inhibitory activity on AHAS and SA.Overall, this study provides compelling evidence that P. amaryllifolius Roxb.leaves is a promising candidate for the expansion of new antimicrobial agents.

Conclusion
This study presents a wide-raging exploration of the antimicrobial potential of Pandanus amaryllifolius Roxb.phytochemical screening uncovered the existence of several bioactive compounds in the plant extract, which may contribute to its antimicrobial activity.Additionally, antioxidant assays demonstrated the plant's potential to scavenge free radicals, which may further enhance its therapeutic properties.These findings are a significant step forward in the search for novel and effective alternatives to conventional antimicrobial agents.
The inhibition zone of the sample ðcmÞ=The zone of positive control ðcmÞ � 100% ð2Þ Data analysis.Data are given as the mean ± standard deviation.The IC 50 values for in vitro antioxidant and linear regression studies was carried out by using Microsoft Excel version 20.0 (The Microsoft Corporation, Redmond, Washington, USA).