INTRODUCTION:

Propolis is an extremely sticky and resinous substance collected by honeybees. Propolis colour can be different depending on the type of floral source and age¹. The chemical composition of propolis is also depended on the bee species, plant source, environmental conditions, and the region of honeybees²^,³. In North America, the main compounds of propolis from Populus nigra were flavonoid (chrysin, galangin, pinobanksin, and pinocembrin), phenolic acids, and esters⁴. In Mediterranean Sea, propolis from Ferula communis consisted of terpenes as the major component⁵, while propolis from Baccharis dracunculifolia in Brazil had a rich composition of prenylated phenylpropanoids⁶.

The constitutive compounds in propolis could be determined chromatographically through electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS)⁷, liquid chromatography coupled with mass spectroscopy (LC-MS)⁸^,⁴, liquid chromatography/diode array detector/electrospray ionization-mass spectrometry (LC/DAD/ESI-MSn)⁴^,⁹, and liquid chromatography tandem mass spectrometry (LC-MS/MS)¹⁰. Among these techniques, LC-MS/MS is a more favourable tool due to its superior selectivity and sensitivity¹¹^,¹². It offers both qualitative and quantitative analytical technique for multiple clinical laboratories with small samples and simple preparation, thus time-consuming and cost can be reduced. In addition, it also offers to evaluate the low molecular weight compounds without the necessary immunological reagents¹³.

Propolis, containing ample bioactive compounds, is widely used as a health food supplement because it improves immunity and prevents diseases such as heart disease, diabetes, inflammation, stroke, and cancer¹⁴^,². In addition, propolis has also been utilized as antibacterial, antifungal, antiviral, antiparasitic, antiproliferative, and antioxidant¹⁵^,¹⁶. Propolis demonstrated significant inhibition against a variety of bacteria, including S. aureus, Bacillus sp., Streptococcus pyogenes, Aeromonas sp., Micrococcus flavus, Pseudomonas aeruginosa, Vibrio species, E. coli, Salmonella enterica, Enterobacter cloacae, Mycobacterium tuberculosis, and Salmonella typhimurium¹⁷^,¹⁸. It is crucial to highlight that propolis from different sources showed identical effectiveness against bacterial growth, which was then linked to the bioactive composition. Propolis has been reported to inhibit the growth of bacteria by a number of mechanisms, including interfering the synthesis of genetic material¹⁹^,²⁰, reducing the rate of energy metabolism²¹, damaging the function of cell membranes, and inhibiting the formation of intermediate proteins²²^,²³. The mechanism of inhibition is mostly reported from flavonoid activity²², while the other bioactive activities are still poorly understood²⁴. Nowadays, we can investigate this mechanism more specific using molecular docking approaches.

According to Yunta et al²⁵ and Abishad et al²⁶, in silico analysis or molecular docking study is a technique to predict binding efficacy as well as the structure-based drug design. It gives the structure-activity relationship, mode of activity, and additional knowledge from protein-ligand interaction. Because in silico is quicker and less usage reagent and animals than in vitro and in vivo analysis, it is capable of reducing research cost. However, in silico analysis is also has several drawbacks, including the inability to make precise predictions due to factors such as protein flexibility, molecular configuration, and promiscuity²⁷^,²⁸. Hence, the greatest option for the effective development of chemotherapeutic medicines in the medical field is the combination of in vitro and in silico analysis.

In the present study, we determined the bioactive compounds in propolis from Wallacetrigona incisa using LC-MS/MS and analyzed their antibacterial activity by in vitro and molecular docking approaches. W. incisa bee propolis, endemic to South Sulawesi, Indonesia, has not been investigated for its antibacterial potential, particularly the mechanism of action. Only the bioactive content determined using GC-MS with pyrolysis, has been reported in the previous study²⁹. We found that the major bioactive compounds detected in propolis from W. incisa in this study were unique compared to that obtained in propolis from other sources reported in the literature. In this study, six propolis concentrations and five bacteria (P. acnes, S. aureus, S. epidermidis, B. subtilis, and E. coli) were set in the in vitro antibacterial test and molecular docking. In vitro study followed by molecular docking analysis of bioactive compounds in propolis from W. incisa will be able to determine the effectiveness of propolis as antibacterial agent.

MATERIALS AND METHODS:

Materials:

Liquid propolis of W. incisa was obtained from CV. Nutrima Sehatalami. Nutrient agar (Difco™), nutrient broth (Difco™), Clindamycin 300mg, sterile distilled water, bacteria culture of P. acnes, S. aureus, S. epidermidis, B. subtilis, and E. coli were obtained from microbiology laboratory in the Research Center for Biomass and Bioproducts, National Research and Innovation Agency (BRIN).

Determination of bioactive compounds in propolis:

Bioactive compounds in propolis W. incisa were determined using Liquid Chromatography Tandem Mass Spectrophotometry (LC-MS/MS) XEVO G2-XS QTOF, in the laboratory of the Research Center for Chemistry, BRIN.

Antibacterial activity of propolis:

Antibacterial activity of propolis was determined by disk diffusion method³⁰ with three replications. The bacteria of P. acnes, S. aureus, and S. epidermidis, B. subtilis, E. coli were used in this analysis with clindamycin 300mg as the positive control and sterile distilled water as the negative control. The concentration variation of propolis was 5%, 7.5%, 10%, 20%, 30%, and 100% w/v while the concentration of clindamycin 300mg was 25% w/v. The bacterial inoculum was first prepared, which was one loop of bacterial culture, and then placed into nutrient broth (NB) media, and incubated at 32-37 ^oC for 18-24 hours. Afterwards, the Nutrient Agar (NA) media was prepared in a petri dish, then the bacteria suspension was spread on the NA media surface using cotton swab sterile. The paper disks that have been soaked in the sample or control solution were placed on the NA media and incubated at 37^oC for 24 hours. Determination of the inhibition zone was performed after finishing the incubation step.

Molecular docking:

Bioactive compounds of propolis determined in this study were used as ligands. The 3D structures of ligands were prepared from PubChem database (https://pubchem.ncbi.nlm.nih.gov/) in a ‘structure data file (SDF)’ format and minimized using PyRx software version 0.8, a virtual screening software for computational drug discovery, then saved in a ‘protein database (PDB)’ format. Furthermore, literature study was conducted in this study to determine the target proteins that play a role in the growth development of B. subtilis, E. coli, P. acnes, S. aureus, and S. epidermidis (Table 1). The FASTA data of target proteins were obtained from NCBI database (https://www.ncbi.nlm.nih.gov/protein) and used for 3D structural modeling at SWISS-MODEL site (https://swissmodel.expasy.org/), a fully automated protein structure homology-modelling server³¹. The 3D structure of ligand and target protein files were input into PyRx software version 0.8 for molecular docking analysis using AutoDock Vina²⁸^,³². The docking results were saved in PDB format and then adjusted to PDB files of target protein using PyMOL (32 bit) software version 1.3.0.0 to evaluate the ligand-protein interactions. The PDB files of ligand-protein were visualized in 3D structure at ProteinsPlus site (https://proteins.plus/) by DoGSiteScorer³³ for predicting potential binding site, while 2D interaction diagrams were drawn using PoseView³⁴.

Table 1: Target proteins

Bacteria Name	Protein Name	NCBI Accession Number	Function	References
Propionibacterium acnes	Christie–Atkins–Munch–Petersen (CAMP) factor 1	APH07537.1	Inflames acne	35
Staphylococcus aureus	Dihydrofolate reductase (DHFR)	BBA23999.1	An essential component of folate metabolism, and necessary for continued nucleic acid synthesis	36
Staphylococcus epidermidis	Accumulation-associated protein (Aap)	AAQ83698.1	Involved in formation of bacterial aggregates (biofilms)	37
Bacillus subtilis	FtsZ	WP_063259738.1	An essential for cell division	38
Escherichia coli	Dispersin	CBG27807.1	Dissemination of bacteria to the intestinal mucosa	26

a)
b)
c)
d)
e)
f)
g)

Figure: 1 Mass spectrum of a) Ganoderic acid R, b) Mulberranol, c) Schizandrin A (Deoxyschizandrin), d) Neoquassin, e) Octahydrocurcumin, f) Isorhamnetin, and g) 2-Methoxyanofinic acid.

Table 2: Bioactive compounds in propolis W. incisa determined by LC-MS/MS

S. No.	Compound	% area	RT (min)	Formula	Fragmentation (m/z)	Classification
1	Ganoderic acid R	6.54	9.82	C₃₄H₅₀O₆	555,3689	Triterpene
2	Mulberranol	6.82	9.38	C₂₅H₂₆O₇	439,1748	Flavone
3	Schizadrin A (Deoxyschizandrin)	6.46	9.55	C₂₄H₃₂O₆	417,2271	Benzene derivatives
4	Neoquassin	10.88	9.47	C₂₂H₃₀O₆	391,2113	Triterpene
5	Octahydrocurcumin	8.52	9.40	C₂₁H₂₈O₆	377,1959	Polyphenol
6	Isorhamnetin	0.22	6.68	C₁₆H₁₂O₇	317,0653	Flavonol
7	2-Methoxyanofinic acid	2.39	8.84	C₁₃H₁₄O₄	235,0962	Phenolic (Lignans)

Table 3: Bioactive compounds in different propolis

Bioactive compound	Kind of propolis	Type of analysis	Reference
Isoflavone-C-glycoside	Guinea-Bissau red propolis from the genus Dalbergia	LC/DAD/ESI-MS	⁹
Hydroxy-gebistein-C-glycoside
Genistein-C-glycoside
Vesticarpan
Vestitone
Sativanone
Mucronulatol
Trilhydroxy-methoxyisoflavone
Cinnamyl ester	Cameroonian propolis from Nyambaka (Adamawa) and Mveg (Nord-West)	LC-MS	³⁹
dimethylkuradine
Pinocembrin
Caffeic acid	Romanian propolis	LC-MS/MS	⁴⁰
Gallic acid
t-ferulic acid
Kaempferol
Quercetin
Chrysin
Pinocembrin
Caffeic acid phenethyl ester (CAPE)
Gallangin	Romanian propolis, Cameroonian propolis from Nyambaka (Adamawa) and Mveg (Nord-West)	LC-MS, LC-MS/MS	³⁹^,⁴⁰
p-coumaric acid

RESULT AND DISCUSSION:

Analysis of bioactive compounds in propolis:

Bioactive compounds of liquid propolis from W. incisa were determined using LC-MS/MS and the seven major components were listed in Table 2. To the best of our knowledge, these compounds have not been found to be the major components of propolis from other sources in the previously reported works. The chemical structures and the fragmentation patterns of the seven bioactive compounds were summarized in Figure 1.

LC-MS is a technique generally used to determine the bioactive compounds contained in organic material, such as plants, animals, and fungi. Some previous researches utilized LC-MS to determine the bioactive compounds in propolis (Table 3).

Antibacterial activity of propolis:

The propolis of W. incisa given the better efficacy was observed on Gram-positive bacterial species S. aureus rather than S. epidermidis and P. acnes (Table 4). Inhibition of S. aureus showed strongly at concentrations of 30% and 100%. Ability of the propolis to inhibit the bacterial growth was presumably due to the presence of triterpenes, flavonoids (flavonol and flavone), and phenolics. The triterpenes found in W. incisa propolis consisted of ganoderic R and neoquassin. Ganoderic R was known to originate from the medicinal mushroom Ganoderma lucidum⁴¹^,⁴², but in our study the specific Ganoderma species attached to the W. incisa bees was unknown. Furthermore, neoquassin has been studied to be beneficial as antimalarial, antiamoebic, antibacterial, antiviral, and antiulcer⁴³. The presence of this neoquassin compound should be able to reduce the growth of P. acnes bacteria as in the study by Diehl, et al⁴³ stated that P. acnes could be inhibited for 58%. The flavonol isorhamnetin was estimated to inhibit the RNA polymerase process in bacteria, especially S. aureus²² through inhibiting alpha-hemolysin HLA transcription and reducing RNA III expression⁴⁴. This compound is known as the inhibitor of Bacillus sp. growth, but the mechanism has not been investigated⁴⁵. Isorhamnetin was recorded to be present in the plants Ginkgo biloba L., Hippopahe rhamniodes L, some of fruit as apple, cherry, and pear, fennel leaves, dill weed, chives, turnip greens, and red onion⁴⁶^,⁴⁷. Isorhamnetin is a flavonol, and scientifically has the potential to inhibit the activity of E. coli, S. aures, Salmonella spp., Bacillus spp., Pseudomonas flourescens, Clostridium botulinum, and Listeria monocytogenes⁴⁸^,⁴⁹. However, its antibacterial ability is more affected on Gram-positive bacteria than Gram-negative. The other compound in W. incisa propolis, mulberranol, is the main flavone compound from the stem bark of the Morus alba L tree. This compound showed good antibacterial activity on S. aureus, S. epidermidis, and Salmonella typhimurium⁵⁰. The inhibitory ability of our propolis on bacteria might also be due to the presence of deoxyschizandrin, a phenolic compound commonly found in the Schisandra chinensis plant. Intan and Zahro⁵¹ mentioned that this compound inhibited the growth of Gram-positive bacteria better than Gram-negative ones, Bargah et al⁵² also revealed that the phenolic from Cassia auriculata strongly inhibited the S. aureus colonies. Deoxyschizandrin changed the structure of cell wall and outer membrane of bacteria distracting the activity of AKPase in intracellular bacteria and increasing the activity of the ATPase pump⁵³. Phenolic compound octahydrocurcumin exhibited quite broad activities as antibacterial, anticancer, antitumor, antioxidant, and anti-inflammatory⁵⁴. The other phenolic compound, 2-methoxyanofinic acid, has only been discovered to be in W. incisa propolis and not in other variants. Hence, there has not been much research related to its potency and mechanism as an antibacterial.

Molecular docking approach:

Molecular docking, a computational method, is commonly used for screening the therapeutic potential of a compound by identifying the interaction events with its target protein. This method uses a scoring function to estimate the binding affinity between two molecules (ligand-protein) based on their preferential orientation⁵⁵^,⁵⁶^,⁵⁷. The value of binding affinity determines the drug efficacy of a compound and is influenced by changes in free energy, interactions of different functional groups with amino acid residues, and binding properties²⁵^,⁵⁸. In our docking study, all the seven major bioactive compounds of propolis were able to interact with the entire target proteins (Table 5). This interaction caused inhibition of target protein activity, thereby explaining the antibacterial activity of the propolis.

Upon inhibition of B. subtilis, mulberranol possessed the highest binding affinity value (−9.0kcal/mol) when interacting with FtsZ, an essential protein in cell division³⁸, at the amino acid residues of Ala77A, Glu145A, Gly23A, Gly110A, and Thr115A (Figure 2). This interaction prohibited the growth of B. subtilis by deactivating FtsZ, stopping the cell division failed. In E. coli, the inhibition occurred by affecting dispersin activity, the 10.2 kDa immunogenic protein found in biofilm and mediated the dissemination of E. coli to the intestinal mucosa²⁶. In this study, ganoderic acid R had the highest binding affinity value (−6.8kcal/mol) when interacting with dispersin at the amino acid residues of Gln95A and Thr100A (Figure 3). The inhibition of dispersin activity would prevent the biofilm formation in E. coli.

Table 4: Antibacterial activity of propolis.

Concentration of propolis (%)	Inhibition Zone (mm)
Concentration of propolis (%)	*P. acnes*	*S. aureus*	*S. epidermidis*	*B. subtilis*	*E. coli*
5	0.67 ± 0.05	0.67 ± 0.05	0.60 ± 0.00	0.68 ± 0.08	0.62 ± 0.04
7.5	0.73 ± 0.08	0.72 ± 0.10	0.70 ± 0.09	0.72 ± 0.12	0.62 ± 0.04
10	0.77 ± 0.05	0.70 ± 0.00	0.68 ± 0.04	0.72 ± 0.08	0.65 ± 0.08
20	0.72 ± 0.12	0.78 ± 0.15	0.75 ± 0.10	0.80 ± 0.09	0.65 ± 0.08
30	0.75 ± 0.14	1.00 ± 0.23*	0.75 ± 0.08	0.85 ± 0.14*	0.65 ± 0.05
100	0.97 ± 0.10	1.05 ± 0.21*	1.17 ± 0.36*	1.12 ± 0.19*	0.68 ± 0.08
Positive control	4.27 ± 0.86*	4.17 ± 0.76*	3.95 ± 0.07*	3.80 ± 0.23*	2.90 ± 0.39*
Negative control	0.60 ± 0.00	0.60 ± 0.00	0.60 ± 0.00	0.60 ± 0.00	0.60 ± 0.00

(Note: *) significant inhibition zone based on Dunnet Test

Table 5: The results of molecular docking analysis

Propolis compounds	*Propionibacterium acnes*	*Staphylococcus aureus*	*Staphylococcus epidermidis*	*Bacillus subtilis*	*Escherichia coli*
	Binding Affinity (kcal/mol)
	CAMP Factor 1	DHFR	Aap	FtsZ	Dispersin
Ganoderic acid R	−7.2	−8.5	−6.5	−8.0	−6.8
Mulberranol	−6.8	−9.6	−7.3	−9.0	−6.4
Schisandrin A (Deoxyschizandrin)	−5.2	−6.0	−5.5	−6.6	−5.2
Neoquassin	−6.4	−9.1	−6.4	−7.9	−6.0
Octahydrocurcumin	−5.8	−7.9	−5.9	−7.6	−6.0
Isorhamnetin	−6.3	−8.6	−7.0	−8.0	−6.1
2-Methoxyanofinic acid	−6.0	−7.5	−6.1	−7.1	−6.0

Upon inhibition of P. acnes, the bioactive compounds inhibited the activity of CAMP factor 1, an essential protein contributing to acne inflammation³⁵. Our docking analysis showed that ganoderic acid R formed the binding interactions at Gln206A of CAMP factor 1 (Figure 4) with the binding affinity value of −7.2 kcal/mol. These compounds inhibited CAMP factor 1 and reduced P. acnes virulence, so that inflammation could be avoided. In S. aureus, inhibition occurred by interacting with DHFR, an essential protein of folate metabolism and important to continue nucleic acid synthesis³⁶. Mulberranol had the highest binding affinity value (−9.6 kcal/mol) when interacting with DHFR at the amino acid residue of Thr47A (Figure 5). In the case of S. epidermidis, inhibition of Aap activity would prevent the formation of bacterial aggregates (biofilms)³⁷. Mulberranol also showed the best performance in interaction with Aap at the amino acid residues of Asn584A, Gly583A, Leu446A, Tyr470A, Tyr580A (Figure 6) and had the highest binding affinity value (−7.3 kcal/mol).

Figure. 2 The 3D and 2D binding interactions of mulberranol in the active site of FtsZ.

Figure 3: The 3D and 2D binding interactions of ganoderic acid R in the active site of disperin.

Based on the investigation above, ganoderic acid R and mulberranol were the most potential bioactive compounds of propolis from W. incisa because these two compounds displayed good performance in interacting with target proteins of several bacteria. Ganoderic acid R is a triterpene compound first isolated from Ganoderma lucidum and possessed an anti-hepatotoxic activity⁵⁹. Meanwhile mulberranol is a flavonoid compound first reported to be contained in the bark of mulberry (Morus alba L.)⁶⁰. In 2015, mulberranol was detected in the root extract of mulberry by LC/MS analysis⁶¹. Mulberranol had been studied to have anti-inflammatory activity because it showed an inhibitory effect on macrophage RAW 264.7 cells with an IC₅₀value of 7.9±1.1μg/mL⁶². However, other bioactive constituents in the propolis from W. incisa were also able to interact with target proteins contributing to the antibacterial activity.

Figure 4: The 3D and 2D binding interactions of ganoderic acid R in the active site of CAMP factor 1.

Figure 5: The 3D and 2D binding interactions of mulberranol in the active site of DHRF.

Figure 6: The 3D and 2D binding interactions of mulberranol in the active site of Aap.

CONCLUSION:

This study revealed seven bioactive compounds contained in the propolis W. incisa, an unexplored propolis, as ganoderic acid R, mulberranol, schizandrin A (deoxyschizandrin), neoquassin, octahydrocurcumin, isorhamnetin, and 2-methoxyanofinic acid. This propolis displayed antibacterial activity, particularly in inhibiting the growth of Gram-positive bacteria, at the optimum inhibition concentration of 30% and 100%. This bioactivity might be arise from the binding interaction of the seven chemical constituents with the protein targets of the bacteria, causing interference in the bacterial cell division, biofilm formation, and virulence. Future research may involve using propolis W. incisa as an antibacterial feature in pharmaceutically-developed products, including antiseptic and acne treatments.

CONFLICT OF INTEREST:

The authors declare that no conflict of interest exists.

ACKNOWLEDGMENTS:

The authors thank the CV. Nutrima Sehatalami, Bogor, for the propolis W. incisa and the Integrated Laboratory of Bioproduct (iLab), National Research and Innovation Agency (BRIN) for the research facility.

REFERENCES:

1. Hossain R, Quispe C, Khan RA, Saikat ASM, Ray P, Ongalbek D, Yeskaliyeva B, Jain D, Smeriglio A, Trombetta D, et al. Propolis : An update on its chemistry and pharmacological applications. Chin. Med. 2022; 17(100): 1–60. doi.org/10.1186/s13020-022-00651-2

2. Zullkiflee N, Taha H, Usman A. Propolis : Its Role and Efficacy in Human Health and Diseases. Molecules. 2022; 27(6120):1–21. doi.org/https://doi.org/10.3390/molecules27186120

3. Kapare HS, Sathiyanarayanan L. Nutritional and Therapeutic potential of Propolis: A Review. Res. J. Pharm. Technol. 2020; 13(7): 3545–3549. doi.org/10.5958/0974-360X.2020.00627.7

4. Falcão SI, Vale N, Gomes P, Domingues MRM, Freire C, Cardoso SM, Vilas-boas M. Phenolic Profiling of Portuguese Propolis by LC–MS Spectrometry: Uncommon Propolis Rich in Flavonoid Glycosides. Phytochem. Anal. 2013; 24(4): 309–318. doi.org/10.1002/pca.2412

5. Popova M, Trusheva B, Antonova D, Cutajar S, Mifsud D, Farrugia C, Tsvetkova I, Najdenski H, Bankova V. The specific chemical profile of Mediterranean propolis from Malta. Food Chem. 2011; 126: 1431–1435. doi.org/10.1016/j.foodchem.2010.11.130

6. Bankova V, Popova M, Trusheva B. Plant Sources of Propolis : an Update from a Chemist’s Point of View. Nat. Prod. Commun. 2006; 1(11): 1023–1028.

7. Medana C, Carbone F, Aigotti R, Appendino G, Baiocchi C. Selective Analysis of Phenolic Compounds in Propolis by HPLC-MS/MS. Phytochem. Anal. 2008; 19: 32–39. doi.org/10.1002/pca.1010

8. Pellati F, Orlandini G, Pinetti D, Benvenuti S. HPLC-DAD and HPLC-ESI-MS / MS methods for metabolite profiling of propolis extracts. J. Pharm. Biomed. Anal. 2011; 55: 934–948. doi.org/10.1016/j.jpba.2011.03.024

9. Falcão SI, Lopes M, Vilas-boas M. A First Approach to the Chemical Composition and Antioxidant Potential of Guinea-Bissau Propolis. Nat. Prod. Commun. 2019: 1–5. doi.org/10.1177/1934578X19844138

10. Nichitoi MM, Costache T, Josceanu AM, Isopescu R, Isopencu G, Lavric V. Development and Application of an LC-MS/MS Method for Identification of Polyphenols in Propolis Extract. Proceedings. 2020; 55(10): 1–5. doi.org/10.3390/proceedings2020055010

11. K P, A AD, K P, A MS. An Overview : LC-MS as Tool of sample Extraction and Quantification in Bioanalytical Laboratories. Asian J. Pharm. Anal. 2020; 10(3): 165–166. doi.org/10.5958/2231-5675.2020.00030.7

12. Gelb MH, Basheeruddin K, Burlina A, Chen H, Chien Y, Dizikes G, Dorley C, Giugliani R, Hietala A, Hong X, et al. Liquid Chromatography – Tandem Mass Spectrometry in Newborn Screening Laboratories. Int. J. Naonatal Screen. 2022; 8(62): 1–15. doi.org/doi.org/10.3390/ ijns8040062

13. Adaway JE, Keevil BG, Owen LJ. Liquid chromatography tandem mass spectrometry in the clinical laboratory. Ann. Clin. Biochem. 2015; 52(1): 18–38. doi.org/10.1177/0004563214557678

14. Braakhuis A. Evidence on the Health Benefits of Supplemental Propolis. Nutrients. 2019; 11(2705): 1–15. doi.org/10.3390/nu11112705

15. Przybyłek I, Karpinski TM. Antibacterial Properties of Propolis. Molecules. 2019; 24(2047): 1–17. doi.org/10.3390/molecules24112047

16. Sharma N, Sanadhya S, Nagarajappa R, Ramesh G, Naik D. Antifungal activity of Propolis, Fluconazole and Chlorhexidine against Oral Candida albicans – A Comparative in-vitro Study. Res. J. Pharm. Technol. 2022; 15(8): 3589–3594. doi.org/10.52711/0974-360X.2022.00601

17. Guzman JD. Natural Cinnamic Acids, Synthetic Derivatives and Hybrids with Antimicrobial Activity. Molecules. 2014; 19: 19292–19349. doi.org/10.3390/molecules191219292

18. Kalia P, Kumar NR, Harjai K. Preventive effect of Honey bee propolis on Salmonella enterica serovar Typhimurium infected BALB / c mice : A Hematological Study. Res. J. Pharm. Technol. 2020; 13(7): 3389–3391. doi.org/10.5958/0974-360X.2020.00602.2

19. Veloz JJ, Alvear M, Salazar LA. Antimicrobial and Antibiofilm Activity against Streptococcus mutans of Individual and Mixtures of the Main Polyphenolic Compounds Found in Chilean Propolis. Biomed Res. Int. 2019; 7602343: 1–8. doi.org/https://doi.org/10.1155/2019/7602343

20. Kharsany K, Viljoen A, Leonard C, Vuuren S Van. The new buzz : Investigating the antimicrobial interactions between bioactive compounds found in South African propolis. J. Ethnopharmacol. 2019; 238: 111867. doi.org/10.1016/j.jep.2019.111867

21. Yilmaz S, Sova M, Ergun S. Antimicrobial Activity of Trans-Cinnamic Acid and Commonly Used Antibiotics Against Important Fish Pathogens and Non-Pathogenic Isolates. J. Appl. Microbiol. 2018; 125(6): 1714–1727. doi.org/10.1111/jam.14097

22. Almuhayawi MS. Saudi Journal of Biological Sciences Propolis as a novel antibacterial agent. Saudi J. Biol. Sci. 2020; 27(11): 3079–3086. doi.org/10.1016/j.sjbs.2020.09.016

23. Olegario LS, Andrade JKS, Andrade GRS, Denadai M, Cavalcanti RL, Silva MAAP da, Narain N. Chemical characterization of four Brazilian brown propolis : An insight in tracking of its geographical location of production and quality control. Food Res. Int. 2019; 123: 481–502. doi.org/10.1016/j.foodres.2019.04.004

24. Alanazi S, Alenzi N, Alenazi F, Tabassum H, Watson D. Chemical characterization of Saudi propolis and its antiparasitic and anticancer properties. Sci. Rep. 2021; 11(5390): 1–9. doi.org/10.1038/s41598-021-84717-5

25. Yunta MJR. Docking and Ligand Binding Affinity : Uses and Pitfalls. Am. J. Model. Optim. 2016; 4(3): 74–114. doi.org/10.12691/ajmo-4-3-2

26. Abishad P, Niveditha P, Unni V, Vergis J, Kurkure NV, Chaudhari S, Rawool DB, Barbuddhe SB. In silico molecular docking and in vitro antimicrobial efficacy of phytochemicals against multi‑drug‑resistant enteroaggregative Escherichia coli and non‑typhoidal Salmonella. Gut Pathog. 2021; 13(46): 1–11. doi.org/10.1186/s13099-021-00443-3

27. Ekins S, Mestres J, Testa B. In silico pharmacology for drug discovery applications to targets and beyond. Br. J. Pharmacol. 2007; 152: 21–37.

28. Trott O, Olson A. Autodock Vina: improving the speed and accuracy of docking with a scoring function, efficient optimazion and multithrearing. J. Comput. Chem. 2010; 31(2): 455–461. doi.org/10.1002/jcc.21334.AutoDock

29. Mahani, Faturachman F, Michelle, Lembong E, Sulaeman A, Hardinsyah, Nurjanah N, Sunarno, Sariadji K. Antiemetic Activities of Indonesian Stingless Bee Propolis on Emetic Induced By Anti-Tuberculosis Drugs. Int. J. Pharm. Pharm. Sci. 2021; 13(4): 39–44.

30. Rosalina R, Ningrum RS, Lukis PA. Aktifitas Antibakteri Ekstrak Jamur Endofit Mangga Podang (Mangifera indica L.) Asal Kabupaten Kediri Jawa Timur. Maj. Ilm. Biol. Biosf. A Sci. J. 2018; 35(3): 139–144. doi.org/10.20884/1.mib.2018.35.3.757

31. Waterhouse A, Bertoni M, Bienert S, Studer G, Tauriello G, Gumienny R, Heer FT, Beer TAP De, Rempfer C, Bordoli L, et al. SWISS-MODEL : homology modelling of protein structures and complexes. Nucleic Acids Res. 2018; 46(1): 296–303. doi.org/10.1093/nar/gky427

32. Pawar RP, Rohane SH. Role of Autodock vina in PyRx Molecular Docking. Asian J. Res. Chem. 2021; 14(2): 132–134. doi.org/10.5958/0974-4150.2021.00024.9

33. Volkamer A, Griewel A, Grombacher T, Rarey M. Analyzing the Topology of Active Sites: On the Prediction of Pockets and Subpockets. J Chem Inf Model. 2010; 50: 2041–2052. doi.org/https://doi.org/10.1021/ci100241y

34. Fricker PC, Gastreich M, Rarey M. Automated Drawing of Structural Molecular Formulas under Constraints. J Chem Inf Comput Sci. 2004; 44(3): 1065–1078. doi.org/DOI: https://doi.org/10.1021/ci049958u

35. Mayslich C, Grange PA, Castela M, Marcelin AG, Calvez V, Dupin N. Characterization of a Cutibacterium acnes Camp Factor 1-Related Peptide as a New TLR-2 Modulator in In Vitro and Ex Vivo Models of Inflammation. Int. J. Mol. Sci. 2022; 23(9): 5065. doi.org/https://doi.org/10.3390/ijms23095065

36. Ragi K, Kakkassery JT, Raphael VP, Johnson R, K VT. In vitro antibacterial and in silico docking studies of two Schiff bases on Staphylococcus aureus and its target proteins. Futur. J. Pharm. Sci. 2021; 7(78): 1–9. doi.org/https://doi.org/10.1186/s43094-021-00225-3

37. Alabdullatif M, Ramirez-arcos S. Biofilm-associated accumulation-associated protein (Aap): A contributing factor to the predominant growth of Staphylococcus epidermidis in platelet concentrates. VoxSanguinis. 2019; 114(1): 28–37.

38. Sharavanan VJ, Sivaramakrishnan M, Kothandan R, Muthusamy S, Kandaswamy K. Molecular Docking Studies of Phytochemicals from Leucas aspera Targeting Escherichia coli and Bacillus subtilis Subcellular Proteins. Pharmacogn J. 2019; 11(2): 278–285. doi.org/10.5530/pj.2019.11.43 Article

39. Balingui CF, Noel NJ, Emmanuel T, Benoit NM, Gervais HM, Boubakary A. LC-MS Analysis, Total Phenolics Content, Phytochemical Study and DPPH Antiradical Scavenging Activity of Two Cameroonian Propolis Samples. Med. Chem. (Los. Angeles). 2019;9(8):100–106.

40. Nichitoi MM, Josceanu AM, Isopescu RD, Isopencu G, Lavric V. Romanian Propolis Extracts : Characterization and Statistical Analysis And Modelling. U.P.B. Sci. Bull., Ser. B. 2019;81(4):149–162.

41. Duru ME, Çayan GT. Biologically Active Terpenoids from Mushroom origin : A Review. ACG Publ. 2015; 9(4): 456–483.

42. Rahangdale P, Wankhade AM, Vyas J V, Paithnakar V. A Review on Management of Varius Disease by Traditional Chinese Medicine G anoderma lucidum. Res. J. Pharmacogn. Phytochem. 2023; 15(2): 167–172. doi.org/10.52711/0975-4385.2023.00026

43. Diehl C, Reznichenko N, Casero R, Faenza L, Cuffini C, Palacios S. Novel antibacterial, antifungal and antiparasitic activities of Quassia amara wood extract. Int. J. Pharmacol. Phytochem. Ethnomedicine. 2016; 2: 62–71.

44. Jiang L, Li H, Wang L, Song Z, Shi L, Li W, Deng X, Wang J. Isorhamnetin Attenuates Staphylococcus aureus -Induced Lung Cell Injury by Inhibiting Alpha-Hemolysin Expression. J.Microbiol.Biotechnol. 2016; 26(3): 596–602.

45. Habtamu A, Melaku Y. Antibacterial and Antioxidant Compounds from the Flower Extracts of Vernonia amygdalina. Adv. Pharmacol. Sci. 2018; 4083736: 1–6. doi.org/10.1155/2018/4083736

46. Gong G, Guan Y, Zhang Z, Rahman K, Wang S, Zhou S, Luan X, Zhang H. Isorhamnetin : A review of pharmacological effects. Biomed. Pharmacother. 2020; 128: 110301. doi.org/10.1016/j.biopha.2020.110301

47. Kandakumar S, Manju D V. Pharmacological Applications of Isorhamnetin: A Short Review. Int. J. Trend Sci. Res. Dev. 2017; 1(4): 672–678.

48. Ren X, Bao Y, Zhu Y, Liu S, Peng Z, Zhang Y, Zhou G. Isorhamnetin, Hispidulin, and Cirsimaritin Identified in Tamarix ramosissima Barks from Southern Xinjiang and Their Antioxidant and Antimicrobial Activities. Molecules. 2019; 24(390): 1–15. doi.org/10.3390/molecules24030390

49. Chauhan AK, Kim J, Lee Y, Balasubramanian PK, Kim Y. Isorhamnetin Has Potential for the Treatment of Escherichia coli -Induced Sepsis. Molecules. 2019; 24(3984): 1–13. doi.org/doi:10.3390/molecules24213984

50. Abubakar M, Mehvish R, Shahid S. Novel Pharmacological Activities and Agents of Morusalba. Int. J. Res. Pharm. Chem. 2020; 10(4): 318–330. doi.org/https://dx.doi.org/ 10.33289/IJRPC.10.4.2020.10(78)

51. Intan AEK, Zahro L. Pharmacological Activities Of Schisandra Chinensis. J. Info Kesehat. 2020; 10(1): 244–252.

52. Bargah RK, Kushwaha A, Tirkey A, Hariwanshi B. In Vitro Antioxidant and Antibacterial Screening of flowers Extract from Cassia auriculata Linn. Res. J. Pharm. Technol. 2020;13(6): 2624–2628. doi.org/10.5958/0974-360X.2020.00466.7

53. Cui SM, Li T, Wang Q, He KK, Zheng YM, Liang HY, Song LY. Antibacterial Effects of Schisandra chinensis Extract on Escherichia coli and its Applications in Cosmetic. Curr. Microbiol. 2020; 77: 865–874.

54. Pandey A, Chaturvedi M, Mishra S, Kumar P, Somvanshi P, Chaturvedi R. Reductive metabolites of curcumin and their therapeutic effects. Heliyon. 2020; 6: e05469. doi.org/10.1016/j.heliyon.2020.e05469

55. Morris GM, Lim-Wilby M. Molecular Modeling of Proteins. Methods Molecular BiologyTM. In: Molecular Docking. In: Kukol, A. (eds). 443rd ed. UK: Humana Press; 2008. 1–389.

56. Patil NS, Rohane SH. Organization of Swiss Dock : In study of Computational and Molecular Docking Study. J. Res. Chem. 2021;14(2):145–148. doi.org/10.5958/0974-4150.2021.00027.4

57. Satpute UM, Rohane SH. Efficiency of AUTODOCK: Insilico study of Pharmaceutical Drug Molecules. Asian J. Res. Chem. 2021;14(1):92–94. doi.org/10.5958/0974-4150.2021.00016.X

58. Bagal A, Borkar T, Ghige T, Kulkarni A, Kumbhar A, Devane G, Rohane S. Molecular Docking – Useful Tool in Drug Discovery. Asian J. Res. Chem. 2022; 15(2): 129–132.

59. Shiao M-S, Lin L-J, Yeh S-F, Chou C-S. Two new triterpenes of the fungus Ganoderma lucidum. J. Nat. Prod. 1987; 50(5): 886–890. doi.org/doi:10.1021/np50053a019

60. Rama Rao AV, Deshpande V, Shastri RK, Tavale SS, Dhaneshwar NN. Structures of albanols A and B, two novel phenols from Morus alba bark. Tetrahedron Lett. 1983; 24(29): 3013–3016. doi.org/doi:10.1016/s0040-4039(00)88083-1

61. Kim K, Chung W, Kim Y, Kim K, Lee I, Park JY, Jeong H, Na Y, Lee C, Jang H. Transcriptomic Analysis Reveals Wound Healing of Morus alba Root Extract by Up-Regulating Keratin Filament and CXCL12/CXCR4 Signaling. Phyther. Res. 2015; 29(8): 1251–1258. doi.org/doi:10.1002/ptr.5375

62. Tran HNK, Nguyen VT, Kim JA, Rho SS, Woo MH, Choi JS, Lee JH, Min BS. Anti-inflammatory activities of compounds from twigs of Morus alba. Fitoterapia. 2017; 120: 17–24. doi.org/10.1016/j.fitote.2017.05.004

Received on 19.06.2023 Modified on 16.10.2023

Research J. Pharm. and Tech 2024; 17(6):2522-2530.

DOI: 10.52711/0974-360X.2024.00395