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Deciphering the multi-functional role of Indian propolis for the management of Alzheimer’s disease by integrating LC–MS/MS, network pharmacology, molecular docking, and in-vitro studies

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The conventional one-drug-one-disease theory has lost its sheen in multigenic diseases such as Alzheimer’s disease (AD). Propolis, a honeybee-derived product has ethnopharmacological evidence of antioxidant, anti-inflammatory, antimicrobial and neuroprotective properties. However, the chemical composition is complex and highly variable geographically. So, to leverage the potential of propolis as an effective treatment modality, it is essential to understand the role of each phytochemical in the AD pathophysiology. Therefore, the present study was aimed at investigating the anti-Alzheimer effect of bioactive in Indian propolis (IP) by combining LC–MS/MS fingerprinting, with network-based analysis and experimental validation. First, phytoconstituents in IP extract were identified using an in-house LC–MS/MS method. The drug likeness and toxicity were assessed, followed by identification of AD targets. The constituent–target–gene network was then constructed along with protein–protein interactions, gene pathway, ontology, and enrichment analysis. LC–MS/MS analysis identified 16 known metabolites with druggable properties except for luteolin-5-methyl ether. The network pharmacology-based analysis revealed that the hit propolis constituents were majorly flavonoids, whereas the main AD-associated targets were MAOB, ESR1, BACE1, AChE, CDK5, GSK3β, and PTGS2. A total of 18 gene pathways were identified to be associated, with the pathways related to AD among the topmost enriched. Molecular docking analysis against top AD targets resulted in suitable binding interactions at the active site of target proteins. Further, the protective role of IP in AD was confirmed with cell-line studies on PC-12, in situ AChE inhibition, and antioxidant assays.

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Alzheimer’s disease


Liquid chromatography hyphenated to mass spectrophotometer


Amyloid-related imaging abnormalities


Indian propolis


Haryana propolis


Protein–protein interaction


Kyoto encyclopedia of genes and genomes


Database for annotation visualization and integrated discovery


Amyloid beta

PC-12 cell line:

Rat pheochromocytoma cell line




Blood–brain barrier


Bioavailability score


False discovery rate


Protein data bank

IC50 :

half maximal inhibitory concentration


Root mean square deviation


Total ion chromatogram


Gene ontology


Biological process


Cellular component


Molecular function


Amyloid precursor protein


Beta-secretase 1


Glycogen synthase kinase-3 beta


Cyclin-dependent kinase 5


Prostaglandin endoperoxide synthase 2


Ascorbic acid


Milli gram equivalent of ascorbic acid/gram


  1. Kumar A, Tiwari A, Sharma A (2018) Changing paradigm from one target one ligand towards multi-target directed ligand design for key drug targets of Alzheimer disease: an important role of in silico methods in multi-target directed ligands design. Curr Neuropharmacol 16:726–739.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Dienstmann R, Vermeulen L, Guinney J et al (2017) Consensus molecular subtypes and the evolution of precision medicine in colorectal cancer. Nat Rev Cancer 17:79–92.

    Article  CAS  PubMed  Google Scholar 

  3. Crews L, Masliah E (2010) Molecular mechanisms of neurodegeneration in Alzheimer’s disease. Hum Mol Genet 19:R12–R20.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Van Dyck CH, Swanson CJ, Aisen P et al (2023) Lecanemab in early Alzheimer’s disease. N Engl J Med 388:142–143.

    Article  CAS  Google Scholar 

  5. Cummings J, Rabinovici GD, Atri A et al (2022) Aducanumab: appropriate use recommendations update. J Prev Alzheimer’s Dis 9:221–230.

    Article  CAS  Google Scholar 

  6. Atri A (2019) Current and future treatments in Alzheimer’s disease. Semin Neurol 39:227–240.

    Article  PubMed  Google Scholar 

  7. Eckert GP (2010) Traditional used plants against cognitive decline and Alzheimer disease. Front Pharmacol 1:1–10.

    Article  Google Scholar 

  8. Diddi S, Lohidasan S, Arulmozhi S, Mahadik KR (2023) Standardization and ameliorative effect of Kalyanaka ghrita in β-amyloid induced memory impairment in wistar rats. J Ethnopharmacol 300:1–18.

    Article  CAS  Google Scholar 

  9. Brusotti G, Cesari I, Dentamaro A et al (2014) Isolation and characterization of bioactive compounds from plant resources: the role of analysis in the ethnopharmacological approach. J Pharm Biomed Anal 87:218–228.

    Article  CAS  PubMed  Google Scholar 

  10. Serby MJ, Burns SJ, Roane DM (2011) Treatment of memory loss with herbal remedies. Curr Treat Opt Neurol 13:520–528.

    Article  Google Scholar 

  11. Solanki I, Parihar P, Mansuri ML, Parihar MS (2015) Flavonoid-based therapies in the early management of neurodegenerative diseases. Adv Nutr 6:64–72.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Sforcin JM (2016) Biological properties and therapeutic applications of propolis. Phytother Res 30:894–905.

    Article  PubMed  Google Scholar 

  13. Wagh VD (2013) Propolis: a wonder bees product and its pharmacological potentials. Adv Pharmacol Sci 2013:1–11.

    Article  ADS  CAS  Google Scholar 

  14. Necip A, Demirtas I, Tayhan SE et al (2023) Isolation of phenolic compounds from eco-friendly white bee propolis: antioxidant, wound-healing, and anti-Alzheimer effects. Food Sci Nutr 00:1–12.

    Article  CAS  Google Scholar 

  15. Nakajima Y, Shimazawa M, Mishima S, Hara H (2007) Water extract of propolis and its main constituents, caffeoylquinic acid derivatives, exert neuroprotective effects via antioxidant actions. Life Sci 80:370–377.

    Article  CAS  PubMed  Google Scholar 

  16. Nakajima Y, Shimazawa M, Mishima S, Hideaki H (2009) Neuroprotective effects of Brazilian green propolis and its main constituents against oxygen-glucose deprivation stress, with a gene-expression analysis. Phytother Res 23:1431–1438.

    Article  CAS  PubMed  Google Scholar 

  17. Nanaware S, Shelar M, Sinnathambi A et al (2017) Neuroprotective effect of Indian propolis in β-amyloid induced memory deficit: Impact on behavioral and biochemical parameters in rats. Biomed Pharmacother 93:543–553.

    Article  CAS  PubMed  Google Scholar 

  18. Bankova V, Bertelli D, Borba R et al (2019) Standard methods for Apis mellifera propolis research. J Apic Res 58:1–49.

    Article  Google Scholar 

  19. Katekhaye S, Fearnley H, Fearnley J, Paradkar A (2019) Gaps in propolis research: challenges posed to commercialization and the need for an holistic approach. J Apic Res 58:604–616.

    Article  Google Scholar 

  20. Kasote DM (2017) Propolis: a neglected product of value in the indian beekeeping sector. Bee World 94:80–83.

    Article  Google Scholar 

  21. Shao L, Ding Q, Wang X (2021) “Network target” theory and network pharmacology. In: Li S (ed) Network pharmacology. Springer, Singapore, pp 1–34

    Google Scholar 

  22. Hopkins AL (2008) Network pharmacology: the next paradigm in drug discovery. Nat Chem Biol 4:682–690.

    Article  CAS  PubMed  Google Scholar 

  23. Borse S, Joshi M, Saggam A et al (2021) Ayurveda botanicals in COVID-19 management: an in silico multi-target approach. PLoS ONE 16:1–33.

    Article  CAS  Google Scholar 

  24. Gao X, Li S, Cong C et al (2021) A network pharmacology approach to estimate potential targets of the active ingredients of epimedium for alleviating mild cognitive impairment and treating Alzheimer’s disease. Evid Based Complem Altern Med 2021:1–15.

    Article  Google Scholar 

  25. Ibrahim RS, El-Banna AA (2021) Network pharmacology-based analysis for unraveling potential cancer-related molecular targets of Egyptian propolis phytoconstituents accompanied with molecular docking and in vitro studies. RSC Adv 11:11610–11626.

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  26. Sankaran S, Dubey R, Lohidasan S (2023) Optimization of extraction conditions using response surface methodology and HPTLC fingerprinting analysis of Indian propolis. J Biol Act Prod Nat 13:76–93.

    Article  CAS  Google Scholar 

  27. Ruch RJ, Cheng S, Klaunig JE (1989) Prevention of cytotoxicity and inhibition of intercellular communication by antioxidant catechins isolated from chinese green tea. Carcinogenesis 10:1003–1008.

    Article  CAS  PubMed  Google Scholar 

  28. Oyaizu M (1986) Studies on products of browning reaction. Antioxidative activities of products of browning reaction prepared from glucosamine. Jpn J Nutr Diet 44:307–315.

    Article  CAS  Google Scholar 

  29. Tolosa L, Donato MT, Gómez-Lechón MJ (2015) General cytotoxicity assessment by means of the MTT assay. In: Vinken M, Rogiers V (eds) Methods in molecular biology. Humana Press Inc, New York, pp 333–348

    Google Scholar 

  30. Dash UC, Sahoo AK (2017) In vitro antioxidant assessment and a rapid HPTLC bioautographic method for the detection of anticholinesterase inhibitory activity of Geophila repens. J Integr Med 15:231–241.

    Article  PubMed  Google Scholar 

  31. Alzheimer’s Association (2023) 2023 Alzheimer’s disease facts and figures. Alzheimers Dement 19:1598–1695.

    Article  Google Scholar 

  32. Avula B, Sagi S, Masoodi MH et al (2020) Quantification and characterization of phenolic compounds from northern Indian propolis extracts and dietary supplements. J AOAC Int 103:1378–1393.

    Article  PubMed  Google Scholar 

  33. Jerković I, Marijanović Z, Kuś PM, Tuberoso CIG (2016) Comprehensive study of Mediterranean (Croatian) propolis peculiarity: headspace, volatiles, anti-varroa-treatment residue, phenolics, and antioxidant properties. Chem Biodivers 13:210–218.

    Article  CAS  PubMed  Google Scholar 

  34. Saftić L, Peršurić Ž, Fornal E et al (2019) Targeted and untargeted LC–MS polyphenolic profiling and chemometric analysis of propolis from different regions of Croatia. J Pharm Biomed Anal 165:162–172.

    Article  CAS  PubMed  Google Scholar 

  35. Monteiro AFM, De Viana JO, Nayarisseri A et al (2018) Computational studies applied to flavonoids against Alzheimer’s and Parkinson’s diseases. Oxid Med Cell Longev 2018:1–21.

    Article  CAS  Google Scholar 

  36. Mohd Sairazi NS, Sirajudeen KNS (2020) Natural products and their bioactive compounds: neuroprotective potentials against neurodegenerative diseases. Evid Based Complement Altern Med 2020:1–30.

    Article  Google Scholar 

  37. Shahinozzaman M, Taira N, Ishii T et al (2018) Anti-inflammatory, anti-diabetic, and anti-Alzheimer’s effects of prenylated flavonoids from Okinawa propolis: an investigation by experimental and computational studies. Molecules 23:1–18.

    Article  CAS  Google Scholar 

  38. Rafieian-Kopaei M, Hamedi A, Dehkordi ES et al (2020) Phytochemical investigation on volatile compositions and methoxylated flavonoids of Agrostis gigantea Roth. Iran J Pharm Res 19:360–370.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Zahoor M, Khan I, Zeb A et al (2021) Pharmacological evaluation and in-silico modeling study of compounds isolated from Ziziphus oxyphylla. Heliyon 7:1–7.

    Article  CAS  Google Scholar 

  40. Youn K, Jun M (2012) Inhibitory effects of key compounds isolated from Corni fructus on bace1 activity. Phytother Res 26:1714–1718.

    Article  CAS  PubMed  Google Scholar 

  41. Yu XD, Zhang D, Xiao CL et al (2022) P-coumaric acid reverses depression-like behavior and memory deficit via inhibiting AGE-RAGE-mediated neuroinflammation. Cells 11:1–15.

    Article  CAS  Google Scholar 

  42. Liu R, Li J, Song J et al (2014) Pinocembrin improves cognition and protects the neurovascular unit in Alzheimer related deficits. Neurobiol Aging 35:1275–1285.

    Article  CAS  PubMed  Google Scholar 

  43. Huang L, Lin M, Zhong X et al (2019) Galangin decreases p-tau, Aβ 42 and β-secretase levels, and suppresses autophagy in okadaic acid-induced PC12 cells via an Akt/GSK3β/mTOR signaling-dependent mechanism. Mol Med Rep 19:1767–1774.

    Article  CAS  PubMed  Google Scholar 

  44. Giacomeli R, de Gomes MG, Reolon JB et al (2020) Chrysin loaded lipid-core nanocapsules ameliorates neurobehavioral alterations induced by β-amyloid1-42 in aged female mice. Behav Brain Res 390:1–12.

    Article  CAS  Google Scholar 

  45. Ghaderi S, Gholipour P, Komaki A et al (2022) p-Coumaric acid ameliorates cognitive and non-cognitive disturbances in a rat model of Alzheimer’s disease: the role of oxidative stress and inflammation. Int Immunopharmacol.

    Article  PubMed  Google Scholar 

  46. Bu J, Zhang Y, Mahan Y et al (2022) Acacetin improves cognitive function of APP/PS1 Alzheimer’s disease model mice via the NLRP3 inflammasome signaling pathway. Transl Neurosci 13:390–397.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Boisard S, Le Ray AM, Gatto J et al (2014) Chemical composition, antioxidant and anti-AGEs activities of a French poplar type propolis. J Agric Food Chem 62:1344–1351.

    Article  CAS  PubMed  Google Scholar 

  48. Gu L, Lu J, Li Q et al (2020) A network-based analysis of key pharmacological pathways of Andrographis paniculata acting on Alzheimer’s disease and experimental validation. J Ethnopharmacol 251:1–12.

    Article  CAS  Google Scholar 

  49. Zhang YW, Thompson R, Zhang H, Xu H (2011) APP processing in Alzheimer’s disease. Mol Brain 4:1–13.

    Article  CAS  Google Scholar 

  50. Coimbra JRM, Marques DFF, Baptista SJ et al (2018) Highlights in BACE1 inhibitors for Alzheimer’s disease treatment. Front Chem 6:1–10.

    Article  CAS  Google Scholar 

  51. Chen JJ, Liu Q, Yuan C et al (2015) Development of 2-aminooxazoline 3-azaxanthenes as orally efficacious β-secretase inhibitors for the potential treatment of Alzheimer’s disease. Bioorg Med Chem Lett 25:767–774.

    Article  CAS  PubMed  Google Scholar 

  52. Halder D, Das S (2022) Role of multi-targeted bioactive natural molecules and their derivatives in the treatment of Alzheimer’s disease: an insight into structure-activity relationship. J Biomol Struct Dyn 0:1–38.

    Article  CAS  Google Scholar 

  53. Liu SL, Wang C, Jiang T et al (2016) The role of Cdk5 in Alzheimer’s disease. Mol Neurobiol 53:4328–4342.

    Article  CAS  PubMed  Google Scholar 

  54. Sayas CL, Ávila J (2021) GSK-3 and tau: a key duet in Alzheimer’s disease. Cells 10:1–19.

    Article  CAS  Google Scholar 

  55. Trott O, Olson AJ (2009) AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem 31:455–461.

    Article  CAS  Google Scholar 

  56. Syaifie PH, Hemasita AW, Nugroho DW et al (2022) In silico investigation of propolis compounds as potential neuroprotective agent. Biointerface Res Appl Chem 12:8285–8306.

    Article  CAS  Google Scholar 

  57. de Sousa NF, Scotti L, de Moura ÉP et al (2021) Computer aided drug design methodologies with natural products in the drug research against Alzheimer’s disease. Curr Neuropharmacol 20:857–885.

    Article  CAS  Google Scholar 

  58. Shrestha S, Natarajan S, Park JH et al (2013) Potential neuroprotective flavonoid-based inhibitors of CDK5/p25 from Rhus parviflora. Bioorg Med Chem Lett 23:5150–5154.

    Article  CAS  PubMed  Google Scholar 

  59. Ma SL, Tang NLS, Zhang YP et al (2008) Association of prostaglandin-endoperoxide synthase 2 (PTGS2) polymorphisms and Alzheimer’s disease in Chinese. Neurobiol Aging 29:856–860.

    Article  CAS  PubMed  Google Scholar 

  60. Ye J, Li L, Hu Z (2021) Exploring the molecular mechanism of action of yinchen wuling powder for the treatment of hyperlipidemia, using network pharmacology, molecular docking, and molecular dynamics simulation. Biomed Res Int 2021:1–14.

    Article  CAS  Google Scholar 

  61. Laskar RA, Sk I, Roy N, Begum NA (2010) Antioxidant activity of Indian propolis and its chemical constituents. Food Chem 122:233–237.

    Article  CAS  Google Scholar 

  62. Bonamigo T, Campos JF, Alfredo TM et al (2017) Antioxidant, cytotoxic, and toxic activities of propolis from two native bees in Brazil: Scaptotrigona depilis and Melipona quadrifasciata anthidioides. Oxid Med Cell Longev 2017:1–12.

    Article  CAS  Google Scholar 

  63. Ramnath S, Venkataramegowda S (2016) Antioxidant activity of Indian propolis—an in vitro evaluation. Int J Pharmacol Phytochem Ethnomed 5:79–85.

    Article  Google Scholar 

  64. Kasote DM, Pawar MV, Bhatia RS et al (2017) HPLC, NMR based chemical profiling and biological characterisation of Indian propolis. Fitoterapia 122:52–60.

    Article  CAS  PubMed  Google Scholar 

  65. Hou XQ, Yan R, Yang C et al (2014) A novel assay for high-throughput screening of anti-Alzheimer’s disease drugs to determine their efficacy by real-time monitoring of changes in PC12 cell proliferation. Int J Mol Med 33:543–549.

    Article  CAS  PubMed  Google Scholar 

  66. Park SY, Kim HS, Cho EK et al (2008) Curcumin protected PC12 cells against beta-amyloid-induced toxicity through the inhibition of oxidative damage and tau hyperphosphorylation. Food Chem Toxicol 46:2881–2887.

    Article  CAS  PubMed  Google Scholar 

  67. Khan H, Marya, Amin S et al (2018) Flavonoids as acetylcholinesterase inhibitors: current therapeutic standing and future prospects. Biomed Pharmacother 101:860–870.

    Article  CAS  PubMed  Google Scholar 

  68. Guo AJY, Xie HQ, Choi RCY et al (2010) Galangin, a flavonol derived from Rhizoma Alpiniae Officinarum, inhibits acetylcholinesterase activity in vitro. Chem Biol Interact 187:246–248.

    Article  ADS  CAS  PubMed  Google Scholar 

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The authors are grateful to the All-India Council for Technical Education (AICTE) for providing financial assistance to Sandeep Sankaran through the AICTE Doctoral Fellowship Scheme (ADF). The authors would like to acknowledge Dr Sangram Patil, Mr Amol Kadam, and the Centre of Food Testing Laboratories, Pune for their support in LC-MS/MS work. The authors are thankful to Dr Amol Jadhav, and Nirav BioSolutions, Pune for their help in carrying out the cell viability assay.


This research did not receive any specific grant from funding agencies for carrying out this work.

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Authors and Affiliations



S.S: performed LC-MS/MS, network pharmacology, molecular docking, in-vitro experiments, and wrote the manuscript. R.D: helped in LC-MS/MS and in-vitro experiments. A.G: assisted in molecular docking studies, Writing - review. RC: helped in network pharmacology analysis. A.K: helped in in-vitro experiments. S.L: conceptualized and designed the work, provided reagents and facilities, Supervision, Writing - review and editing.

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Correspondence to Sathiyanarayanan Lohidasan.

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Sankaran, S., Dubey, R., Gomatam, A. et al. Deciphering the multi-functional role of Indian propolis for the management of Alzheimer’s disease by integrating LC–MS/MS, network pharmacology, molecular docking, and in-vitro studies. Mol Divers (2024).

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