Phytochemical characterization of Styrax benzoin resin extract, molecular docking, ADME, and antibacterial activity study

Abstract Styrax benzoin fumes have a spiritual aspect from ancient times, magical essence like a pleasant perfume, and are employed in religious ceremonies in India. This study aims to identify the volatile compounds in S. benzoin extract, their binding affinity to the bacterial target proteins, and study the antibacterial activity of the potential extract. The compounds obtained from GC-MS analysis of S. benzoin extract were subjected to molecular docking studies against DHFR of Staphylococcus aureus, tRNA synthetase of Escherichia coli, DHPS of Mycobacterium tuberculosis. Molecular docking studies revealed that seventeen compounds out of 20 compounds exhibited higher binding affinity than co-ligand (-7.00 kcal/mol) against the Staphylococcus aureus enzyme DHFR. Consequently, the crude extracts were evaluated for antibacterial activity against S. aureus, and the acetone extract showed promising findings. S. benzoin fumes might replace synthetic room fresheners, and promising compounds could be exploited in the cosmetics industry. Graphical Abstract


Introduction
Gum Benzoin is a fragrant dried balsamic resin that is excreted from the bark of various Styrax species.Styrax benzoin (Styracaceae) is a perennial tree indigenous to subtropical or tropical parts of South-East or East Asia and North America.There are around 150 Styrax species.The genus Styrax contains a large variety of bioactive molecules (Pazar and Akg€ ul 2015;Venditti et al. 2018;Xu et al. 2019;Liu et al. 2020;Son et al. 2021) with cytotoxic activities (Teles et al. 2005;Tra et al. 2022), cyclooxygenase inhibitory activities (Bertanha et al. 2012).S. benzoin is endemic to Sumatra in Indonesia and is mainly exploited for aromatic resin Loban manufacturing.S. benzoin is also known as gum benjamin tree, loban tree, Sumatra benzoin tree, and kemenyan (Indonesia and Malaysia).It has been cultivated for thousands of years in various regions of the world for incense and therapeutic purposes.Loban crystals are burned in places of worship throughout the Indian subcontinent to produce a holy, sanctified environment.S. benzoin generally contains cinnamic acid, benzoic acid, methyl cinnamate, coniferyl benzoate, cinnamaylcinnamate, phenylethylene, phenylpropylic alcohol, and vanillin (Popravko et al. 1984;Hovaneissian et al. 2008).S. benzoin resin is used in traditional herbal medicine to treat a number of skin ailments, skin ulcers, wounds, and bedsores.It is also used to treat psoriasis and eczema.Benzoin oil is used as a flavoring agent as well as a preservative for beverages and food.Benzoin oil is used to treat throat infections and various respiratory ailments (Sharif et al. 2016).It is also used in tincture as a mouthwash to treat cold sores.After the tooth extraction, dentists use benzoin resins tincture as an anti-inflammatory agent.Considering the bioactivities, benzoin compounds are widely used in various pharmacological preparations (Abdallah and Khalid 2012).In Ayurveda, Loban smoke is used to prevent airborne diseases (Gond and Bhutada 2020).In eastern India and Bangladesh, rural people use Loban smoke to reduce the spread of the Cowpox virus.Several researchers have reported the various activities of resin.To the best of our knowledge, this is the first report on the identification of volatile chemicals in S. benzoin resin and molecular docking-guided activity analysis.
The enzyme dihydrofolate reductase (DHFR) is a crucial enzyme related to biosynthetic pathways of purines, thymidylate, methionine, serine, glycine, and N-formylmethionyl tRNA.Inhibition of DHFR creates a disparity in the biosynthesis of these molecules; as a result, disruption of DNA replication in the microbial cell (Rashid et al. 2016).Therefore, DHFR is an attractive target for anticancer, antibacterial, and antimicrobial agents (Heaslet et al. 2009).Considering this information and traditional uses, methanol, and acetone extracts of Styrax resign are analyzed with GC-MS.Molecular docking studies were performed with the compounds derived in GC-MS with DHFR of Staphylococcus aureus, tRNA synthetase of Escherichia coli, Dihydropteroate synthase of Mycobacterium tuberculosis, and the predicted outcome was validated in the laboratory using the in-vitro disc diffusion technique.

Result and discussion
According to the results of the GC-MS analysis (Table S1), the acetone and methanol extracts included 20 compounds.Compounds 1-9 were present in both acetone and methanol extracts.Compounds 10-15 were solely obtained in acetone extract, and 16-20 were only found in methanol extract (Table S1, Figure S1).It is observed that nine components, namely a-copaene or copaene, b-elemene, trans-caryophyllene, a-humulene, germacrene D, cadina-1(10),4-diene, trans-longipinocarveol, isospathulenol, 5,5,8a-trimethyl-4-methyleneoctahydro-2H-1,4a-methanonaphthalen-3-ol, were present in both the acetone as well as methanol extracts which should be considered as chemotaxonomy marker components of S. benzoin resin.Prior to the wet laboratory experiment, a molecular docking study was conducted with target proteins from S. aureus (Figure S2), E. coli, and M. tuberculosis.In the current study, the docking technique was validated using RMSD.The RMSD values were 0.8490, 0.7462, and 1.1873 for co-ligands of 2W9G, 1EYE, and 6P26, respectively, when the docked posture of coligands was superimposed on their respective native crystallographic bound conformation (Figure S3).These low RMSD results indicated a trust-worthy docking protocol (Huang and Zou 2006).The binding affinity values of S. benzoin resin compounds predicted by Autodock Vina towards DHFR varied from À6.5 to À9.4 kcal/mol.The docking score of co-ligand trimethoprim was À7.0, predicted by Autodock Vina against DHFR.The majority of the molecules had a higher docking score than co-ligand and were found to be deeply inserted into the active region of the protein.The binding affinity predicted by Autodock Vina is shown in Table S1, and binding poses are shown in Figure S1.The binding affinity values for compounds against tRNA synthetase of E. coli predicted by Autodock Vina ranged from À5.3 to À9.8 kcal/mol.Although the highest docking score of two hits, 10 and 15 bounded outside the active site.Other hits have a lower binding affinity than its co-ligand N-benzyl-2-(cyclohex-1-en-1-yl)ethan-1-amine (-8.8 kcal/mol).S. benzoin compounds bind to tRNA synthetase with a lesser affinity than DHFR.The binding affinity of two compounds 10 and 15 for M. tuberculosis DHPS is higher than that of the co-ligand pterin-6-ylmethyl-monophosphate of 1EYE (-8.1 kcal/mol).The binding affinity of compounds for tRNA synthetase and DHFR are shown in Table S1.The docking analysis revealed that the S. benzoin compounds had a higher binding affinity against the S. aureus target protein.
The Lipinski rule of five suggests that poor absorption is more likely in drug discovery when there are more than five hydrogen bond donors and ten hydrogen bond acceptors, the molecular weight is greater than 500 dalton, and the expected Log P (CLogP) is greater than 5 (or MlogP > 4.15) (Lipinski et al. 2001).All of the compounds in this study, with the exception of compound 16, have molecular weights 500, hydrogen bond donor 5, and hydrogen bond acceptor 10.Compounds 1,2,3,4,5,6,7,8,20,and 21 had MLogP values greater than 4.15, while other compounds had MLogP values less than 4.15.The log Po/w (n-octanol/water partition coefficient), abbreviated as iLOGp here, is a crucial physicochemical parameter in drug development (Daina 2014).The log n-octanol/water partition coefficient for a drug-like molecule should be less than 5, and the polar surface area should be less than 140 Å 2 (Ojima 2009).All the hits had iLOGp values < 5 and TPSA < 140.There were no violations of the Ghose and Veber criterion for any of the molecules.According to the radar map, with the exception of compounds 16 and 21, all other molecules are found in the colored zone (Figure S4).For the majority of compounds, physiochemical space is adequate for oral bioavailability.Based on molecular docking analysis and ADME properties, in vitro activity of S. benzoin extracts was performed against S. aureus (Table S2).
Using the disc diffusion method, the antimicrobial activity of acetone extract of S. benzoin against three pathogenic strains (S. aureus, E. coli, and M. tuberculosis) revealed that the extract exhibited growth suppression capacity on S. aureus at different dosages (Figure 1).Despite the fact that the methanol extract had low growth suppression activity.This finding suggested that the compound found solely in acetone extract was responsible for the inhibitory activity.The inhibitory effect of the extract was started from the concentration of 50 mg/ml (MIC) with an inhibition zone of 12 mm (Table S3).Interestingly, the activity of the extract reduced with storage time at room temperature.This finding suggests that the activity of the crude extract may be attributable to the presence of a volatile compound in the extract at room temperature.

Conclusion
The present investigation revealed 20 terpenoid compounds in the acetone and methanol extract of S. benzoin resin.The methanol extract showed no significant antimicrobial activity, but the acetone extract showed activity.The component of acetone extract, 10-14, binds significantly to the dihydrofolate reductase of S. aureus.The ADME properties of the compounds were also impressive.The in-vitro antibacterial activity of the extract complements authenticates the docking result.The outcome of various theoretical and experimental studies concludes that the smoke of S. benzoin resin might replace synthetic room fresheners, and promising compounds could be exploited in the pharmaceutical industry.Although resin has been used in religious ceremonies in India since ancient times, this is the first report of volatile component identification and docking-guided activity analysis against Staphylococcus aureus.

Figure 1 .
Figure 1.A: Antimicrobial activity of acetone extract of S. benzoin against S. aureus B: Ampicillin (100 ml/ml), C: Plot of different dilutions of acetone extract against S. aureus.