Integrated chemical and biological characterization of Hypericum perforatum extract using LC-MS/MS and in vitro functional assays

integrated-chemical-and-biological-characterization-of-hypericum-perforatum-extract-using-lc-ms/ms-and-in-vitro-functional-assays
Integrated chemical and biological characterization of Hypericum perforatum extract using LC-MS/MS and in vitro functional assays

References

  1. Budantsev, A. L., Prikhodko, V. A., Varganova, I. V. & Okovityi, S. V. Biological activity of Hypericum perforatum L. (Hypericaceae): a review. Pharm. Pharmacol. 9, 17–31 (2021).

    Google Scholar 

  2. Saddiqe, Z., Naeem, I. & Maimoona, A. A review of the antibacterial activity of Hypericum perforatum L. J. Ethnopharmacol. 131, 511–521. https://doi.org/10.1016/j.jep.2010.07.034 (2010).

    Google Scholar 

  3. Nürk, N. M. & Blattner, F. R. Cladistic analysis of morphological characters in hypericum (Hypericaceae). Taxon 59, 1495–1507. https://doi.org/10.1002/tax.595014 (2010).

    Google Scholar 

  4. Dincel, D. et al. Investigation of antioxidant and anticholinesterase activity of Hypericum perforatum L. extracts. Turk. J. Anal. Chem. 7, 1–8 (2025).

    Google Scholar 

  5. Kisa, O., Oksuz, L., Servi, H. & Aysal, A. I. Antibacterial activity of Hypericum perforatum L. (St. John’s wort) extracts against Gram-positive bacteria and characterisation of its secondary metabolites. Nat. Prod. Res. 39, 1019–1026. https://doi.org/10.1080/14786419.2023.2291702 (2025).

    Google Scholar 

  6. Galeotti, N. Hypericum perforatum (St john’s wort) beyond depression: A therapeutic perspective for pain conditions. J. Ethnopharmacol. 200, 136–146 (2017).

    Google Scholar 

  7. Zlatković, B. K., Bogosavljević, S. S., Radivojević, A. R. & Pavlović, M. A. Traditional use of the native medicinal plant resource of Mt. Rtanj (Eastern Serbia): ethnobotanical evaluation and comparison. J. Ethnopharmacol. 151, 704–713 (2014).

    Google Scholar 

  8. Savikin, K., Dobrić, S., Tadić, V. & Zdunić, G. Antiinflammatory activity of ethanol extracts of hypericum perforatum L., H. barbatum Jacq., H. hirsutum L., H. richeri Vill. And H. Androsaemum L. in rats. Phytother Res. 21, 176–180 (2007).

    Google Scholar 

  9. Mennini, T. & Gobbi, M. The antidepressant mechanism of Hypericum perforatum. Life Sci. 75, 1021–1027 (2004).

    Google Scholar 

  10. Baytop, T. Türkiye’de bitkiler ile tedavi: geçmişte ve bugün (Nobel Tıp Kitabevleri, 1999).

  11. Greeson, J. M., Sanford, B. & Monti, D. A. St. John’s wort (Hypericum perforatum): a review of the current pharmacological, toxicological, and clinical literature. Psychopharmacology 153, 402–414 (2001).

    Google Scholar 

  12. Brondz, I. & Brondz, A. Recent enhancement of the immunity in AIDS and other immunocompromised patients by Hyperforin an antibiotic from Hypericum perforatum L. (in vitro model) part I. J. Biophys. Chem. 3, 304–310 (2012).

    Google Scholar 

  13. Haake, L. R. et al. Viability and radiosensitivity of human tumor cells from breast and colon are influenced by Hypericum perforatum extract HP01. Int. J. Mol. Sci. 26, 622 (2025).

    Google Scholar 

  14. Mete, M. et al. Cytotoxic effects of Hypericum perforatum on glioblastoma cells by inducing oxidative stress, autophagy and apoptosis. Turk. Neurosurg. 35 https://doi.org/10.5137/1019-5149.JTN.45958-23.3 (2025).

  15. You, M. K., Kim, H. J., Kook, J. H. & Kim, H. A. St. John’s wort regulates proliferation and apoptosis in MCF-7 human breast cancer cells by inhibiting AMPK/mTOR and activating the mitochondrial pathway. Int. J. Mol. Sci. 19, 966 (2018).

    Google Scholar 

  16. Orčić, D. Z., Mimica-Dukić, N. M., Francišković, M. M., Petrović, S. S. & Jovin, E. Đ. Antioxidant activity relationship of phenolic compounds in Hypericum perforatum L. Chem. Cent. J. 5, 34 (2011).

    Google Scholar 

  17. Tahirović, I. et al. Total phenolic content and antioxidant capacity in infusions of various herbal teas. Bull. Chem. Technol. Bosnia Herzegovina. 42, 51–55 (2014).

    Google Scholar 

  18. Yousefi, L. Impact of ultrasound pretreatment with different solvents on the antioxidant activity, phenolic and flavonoid compounds of the St. John’s wort (Hypericum perforatum L.) extract. J. Food Sci. Technol. Iran. 20, 126–139 (2023).

    Google Scholar 

  19. Kilibarda, S. et al. Ž. Phytochemical profile and biological activities of Rtanj’s Hypericum perforatum infusion tea and methanolic extracts: insights from LC-MS/MS and HPTLC–Bioautography. Plants 14, 1377 (2025).

  20. Błońska-Sikora, E., Zielińska, A., Dobros, N., Paradowska, K. & Michalak, M. Polyphenol and flavonoid content and antioxidant activity of Hypericum perforatum L. (St. John’s Wort) extracts for potential pharmaceutical and cosmetic applications. Appl. Sci. 15, 2590 (2025).

    Google Scholar 

  21. Re, R. et al. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic. Biol. Med. 26, 1231–1237 (1999).

    Google Scholar 

  22. Škrovánková, S., Mišurcová, L. & Machů, L. Antioxidant activity and protecting health effects of common medicinal plants. Adv. Food Nutr. Res. 67, 75–139 (2012).

    Google Scholar 

  23. Özay, C. & Pehlivan, E. Factors affecting the biosynthesis and accumulation of plant secondary metabolites. J. Fac. Pharm. Ankara. 48 (3), 1248–1263. https://doi.org/10.33483/jfpau.1488042 (2024).

    Google Scholar 

  24. Pandey, P. et al. A. A comprehensive review Uncovering the anticancerous potential of Genkwanin (plant-derived compound) in several human carcinomas. Open. Chem. 22, 20240003 (2024).

    Google Scholar 

  25. Narain, N. K. Spectroscopic studies of a less abundant flavone, Genkwanin. Spectrosc. Lett. 9, 865–875 (1976).

    Google Scholar 

  26. Kim, M. K. et al. Design of experiments-based optimization of flavonoids extraction from Daphne Genkwa flower buds and flavonoids contents at different blooming stages. Plants 11, 925 (2022).

    Google Scholar 

  27. Grayer, R. J. et al. External flavones in sweet basil, Ocimum basilicum, and related taxa. Phytochemistry 43, 1041–1047 (1996).

    Google Scholar 

  28. Santos-Gomes, P. C., Seabra, R. M., Andrade, P. B. & Fernandes-Ferreira, M. Phenolic antioxidant compounds produced by in vitro shoots of Sage (Salvia officinalis L). Plant. Sci. 162, 981–987 (2002).

    Google Scholar 

  29. Del Bano, M. J. et al. Phenolic diterpenes, flavones, and Rosmarinic acid distribution during the development of leaves, flowers, stems, and roots of Rosmarinus officinalis. Antioxidant activity. J. Agric. Food Chem. 51, 4247–4253 (2003).

    Google Scholar 

  30. Kim, A. R. et al. Active components from Artemisia Iwayomogi displaying ONOO⁻ scavenging activity. Phytother Res. 18, 1–7 (2004).

    Google Scholar 

  31. Gao, Y. et al. Genkwanin inhibits Proinflammatory mediators mainly through the regulation of miR-101/MKP-1/MAPK pathway in LPS-activated macrophages. PLoS One. 9, e96741 (2014).

    Google Scholar 

  32. Cottigli, F. et al. Antimicrobial evaluation of coumarins and flavonoids from the stems of Daphne gnidium L. Phytomedicine 8, 302–305 (2001).

    Google Scholar 

  33. Suh, N. A. N. J. O. O., Luyengi, L., Fong, H. H., Kinghorn, A. D. & Pezzuto, J. M. Discovery of natural product chemopreventive agents utilizing HL-60 cell differentiation as a model. Anticancer Res. 15, 233–239 (1995).

    Google Scholar 

  34. Li, Y. et al. Genkwanin nanosuspensions: a novel and potential antitumor drug in breast carcinoma therapy. Drug Deliv. 24, 1491–1500 (2017).

    Google Scholar 

  35. Wang, X. et al. Antitumor and Immunomodulatory activity of Genkwanin on colorectal cancer in the APC^Min/+ mice. Int. Immunopharmacol. 29, 701–707 (2015).

    Google Scholar 

  36. Nurul-Islam, M., Ishita, I. J., Jung, H. A. & Choi, J. S. Vicenin 2 isolated from Artemisia capillaris exhibited potent anti-glycation properties. Food Chem. Toxicol. 69, 55–62. https://doi.org/10.1016/j.fct.2014.03.042 (2014).

    Google Scholar 

  37. Munirah, M. et al. Plant vegetative stages and drying methods affect flavonoid content of Clinacanthus nutans extracts. Afr. J. Tradit Complement. Altern. Med. 15, 54–63 (2018).

    Google Scholar 

  38. Romero-Benavides, J. C., Atiencie-Valarezo, N. C. & Duarte-Casar, R. Flavonoid composition and antioxidant activity of Tragia volubilis L. methanolic extract. Plants 12, 3139; (2023). https://doi.org/10.3390/plants12173139

  39. Çırak, C., Radušiene, J., Janulis, V., Ivanauskas, L. & Arslan, B. Chemical constituents of some hypericum species growing in Turkey. J. Plant. Biol. 50, 632–635 (2007).

    Google Scholar 

  40. Önlü, Ş. Efficacy of exogenous abscisic acid on cholinesterase enzyme activity and phenolic compound variability in Hypericum perforatum calli. Kahramanmaraş Sütçü İmam Univ. J. Agric. Nat. 28, 1083–1094. https://doi.org/10.18016/ksutarimdoga.vi.1614168 (2025).

    Google Scholar 

  41. Aigbe, F. R. et al. Alterations of haemodynamic parameters in spontaneously hypertensive rats by Aristolochia Ringens Vahl. (Aristolochiaceae). J. Tradit Complement. Med. 8, 72–80 (2018).

    Google Scholar 

  42. Lee, W. J. & Lee, S. H. Protocatechuic acid protects hepatocytes against hydrogen peroxide-induced oxidative stress. Curr. Res. Food Sci. 5, 222–227 (2022).

    Google Scholar 

  43. Kou, Y. et al. 4-Hydroxybenzoic acid restrains Nlrp3 inflammasome priming and activation via disrupting PU.1 DNA binding activity and direct antioxidation. Chem. Biol. Interact. 404, 111262 (2024).

    Google Scholar 

  44. Ullah, R. et al. Vanillic acid, a bioactive phenolic compound, counteracts LPS-induced neurotoxicity by regulating c-Jun N-terminal kinase in mouse brain. Int. J. Mol. Sci. 22, 361 (2020).

    Google Scholar 

  45. Nam, Y. J. & Lee, C. S. Protocatechuic acid inhibits Toll-like receptor-4-dependent activation of NF-κB by suppressing activation of the Akt, mTOR, JNK and p38-MAPK. Int. Immunopharmacol. 55, 272–281 (2018).

    Google Scholar 

  46. Mirzaei, S. et al. Caffeic acid and its derivatives as potential modulators of oncogenic molecular pathways: new hope in the fight against cancer. Pharmacol. Res. 171, 105759 (2021).

    Google Scholar 

  47. Ansari, M. A. et al. Sinapic acid mitigates gentamicin-induced nephrotoxicity and associated oxidative/nitrosative stress, apoptosis, and inflammation in rats. Life Sci. 165, 1–8 (2016).

    Google Scholar 

  48. Basque, A., Touaibia, M. & Martin, L. J. Sinapic and ferulic acid phenethyl esters increase the expression of steroidogenic genes in MA-10 tumor Leydig cells. Toxicol. Vitro. 86, 105505 (2023).

    Google Scholar 

  49. Roghani, M. et al. Alleviation of liver dysfunction, oxidative stress and inflammation underlies the protective effect of ferulic acid in methotrexate-induced hepatotoxicity. Drug Des. Devel. Ther. 14, 1933–1941. (2020).

  50. Rahbardar, M. G., Ferns, G. A. & Mobarhan, M. G. Vanillic acid as a promising intervention for metabolic syndrome: preclinical studies. Iran. J. Basic. Med. Sci. 28, 141 (2025).

    Google Scholar 

  51. Díaz-Casado, M. E. et al. Oral β-RA induces metabolic rewiring leading to the rescue of diet-induced obesity. Biochim. Biophys. Acta Mol. Basis Dis. 1870, 167283 (2024).

    Google Scholar 

  52. Ono, K., Tsuji, M., Yamasaki, T. R. & Pasinetti, G. M. Anti-aggregation effects of phenolic compounds on α-synuclein. Molecules 25, 2444 (2020).

    Google Scholar 

  53. López-Herrador, S. et al. Natural hydroxybenzoic and hydroxycinnamic acids derivatives: mechanisms of action and therapeutic applications. Antioxidants 14, 711. https://doi.org/10.3390/antiox14060711 (2025).

    Google Scholar 

  54. Maisch, N. A., Bereswill, S. & Heimesaat, M. M. Antibacterial effects of vanilla ingredients provide novel treatment options for infections with multidrug-resistant bacteria–A recent literature review. Eur. J. Microbiol. Immunol. 12, 53–62. https://doi.org/10.1556/1886.2022.00015 (2022).

    Google Scholar 

  55. Skalkos, D. et al. The lipophilic extract of Hypericum perforatum exerts significant cytotoxic activity against T24 and NBT-II urinary bladder tumor cells. Planta Med. 71, 1030–1035 (2005).

    Google Scholar 

  56. Hostanska, K., Reichling, J., Bommer, S., Weber, M. & Saller, R. Aqueous ethanolic extract of St. John’s wort (Hypericum perforatum L.) induces growth Inhibition and apoptosis in human malignant cells in vitro. Pharmazie 57, 323–331 (2002).

    Google Scholar 

  57. Jain, S. S., Murch, S. J., Bird, R. P. & Saxena, P. K. Optimized St. John’s wort (Hypericum perforatum L.) germplasm lines exert cytotoxicity in HT-29 colon cancer cells via downregulation of NF-κB. J Complement. Integr. Med 7, 1-18 (2010).

  58. Gibbs, C., Duggans, J. S., Malone, E. & Myles, E. L. Cytotoxic effects of hypericum extracts on breast and colorectal tumors. Cancer Res. 74, 5117–5117 (2014).

    Google Scholar 

  59. Martarelli, D. et al. Hypericum perforatum methanolic extract inhibits growth of human prostatic carcinoma cell line orthotopically implanted in nude mice. Cancer Lett. 210, 27–33 (2004).

    Google Scholar 

  60. Matić, I. Z. et al. Cytotoxic activities of Hypericum perforatum L. extracts against 2D and 3D cancer cell models. Cytotechnology 73, 373–389 (2021).

    Google Scholar 

  61. Roscetti, G., Franzese, O., Comandini, A. & Bonmassar, E. Cytotoxic activity of Hypericum perforatum L. on K562 erythroleukemic cells: differential effects between methanolic extract and hypericin. Phytother Res. 18, 66–72 (2004).

    Google Scholar 

  62. Valletta, E., Rinaldi, A., Marini, M., Franzese, O. & Roscetti, G. Distinct Hypericum perforatum L. total extracts exert different antitumour activity on erythroleukemic K562 cells. Phytother Res. 32, 1803–1811 (2018).

    Google Scholar 

  63. Mirmalek, S. A. et al. Cytotoxic and apoptogenic effect of hypericin, the bioactive component of Hypericum perforatum on the MCF-7 human breast cancer cell line. Cancer Cell. Int. 16, 3 (2015).

    Google Scholar 

  64. Celik, E. Apoptotic and anti-inflammatory effects of Hypericum perforatum extract in human basal cell carcinoma TE 354. T cell line. Dicle Med. J. 48, 92–98 (2021).

    Google Scholar 

  65. Pazarcı, P. & Kaplan, H. M. In vitro apoptotic and antiproliferative activity of Hypericum perforatum extract on human osteosarcoma cell line. J. Med. Food. 28, 38–43 (2025).

    Google Scholar 

  66. Shiverick, K., Nseyo, O., Medrano, T., Mejia, M. & Nseyo, U. Antiproliferative effects of Hypericum perforatum L. extract involve induction of p21^CIP1 and Inhibition of Rb expression in human bladder cancer cells. Cancer Res. 67, 3392–3392 (2007).

    Google Scholar 

  67. Oezmen, A. et al. In vitro anti-neoplastic activity of the ethno-pharmaceutical plant Hypericum adenotrichum Spach endemic to Western Turkey. Oncol. Rep. 22, 845–852 (2009).

    Google Scholar 

  68. Li, Y. et al. Quality markers of Polygala fallax Hemsl Decoction based on qualitative and quantitative analysis combined with network Pharmacology and chemometric analysis. Phytochem Anal. 35, 1496–1508 (2024).

    Google Scholar 

  69. Zhao, X. et al. In vitro antioxidant, anti-mutagenic, anti-cancer and anti-angiogenic effects of Chinese bowl tea. J. Funct. Foods. 7, 590–598 (2014).

    Google Scholar 

  70. Orhan, I. E. et al. Assessment of antimicrobial and antiprotozoal activity of the Olive oil macerate samples of Hypericum perforatum and their LC–DAD–MS analyses. Food Chem. 138, 870–875 (2013).

    Google Scholar 

  71. Süntar, I., Oyardı, O., Akkol, E. K. & Ozçelik, B. Antimicrobial effect of the extracts from Hypericum perforatum against oral bacteria and biofilm formation. Pharm. Biol. 54, 1065–1070 (2016).

    Google Scholar 

  72. Nezhad, S. K., Zenouz, A. T., Aghazadeh, M. & Kafil, H. S. Strong antimicrobial activity of Hypericum perforatum L. against oral isolates of Lactobacillus spp. Cell. Mol. Biol. 63, 58–62 (2017).

    Google Scholar 

  73. Uğur, Y. Extraction and quantification of melatonin in Cornelian Cherry (Cornus Mas L.) by ultra-fast liquid chromatography coupled to fluorescence detector (UFLC-FD). Acta Chromatogr. 35, 219–226. https://doi.org/10.1556/1326.2022.01052 (2023).

    Google Scholar 

  74. Uğur, Y. et al. Changes in the phenolic, melatonin, sugar contents and antioxidant capacity, depending on ripening stage in different Cornelian Cherry (Cornus Mas L.) fruits. Chem. Select. 9, e202304682. https://doi.org/10.1002/slct.202304682 (2024).

    Google Scholar 

  75. Uğur, Y. & Güzel, A. Determination of phytochemical content by LC-MS/MS, investigation of antioxidant capacity, and enzyme Inhibition effects of nettle (Urtica dioica). Eur. Rev. Med. Pharmacol. Sci. 27, 1793–1800. https://doi.org/10.26355/eurrev_202303_31540 (2023).

    Google Scholar 

  76. Zengin, R. et al. Sun-drying and melatonin treatment effects on apricot color, phytochemical, and antioxidant properties. Appl. Sci. 15, 508. https://doi.org/10.3390/app15020508 (2025).

    Google Scholar 

  77. Dursun, İ. et al. Antioxidant and antimicrobial activities of different extracts of Tragopogon dubius and Tragopogon porrifolium L. subsp. Longirostris: determination of their phytochemical contents by UHPLC-Orbitrap®-HRMS analysis. Food Biosci. 63, 105604 (2025).

    Google Scholar 

  78. Sharma, N., Arya, G., Kumari, R. M., Gupta, N. & Nimesh, S. Evaluation of anticancer activity of silver nanoparticles on the A549 human lung carcinoma cell lines through Alamar blue assay. Bio-protocol 9, e3131 (2019).

    Google Scholar 

  79. Wlodkowic, D., Skommer, J. & Darzynkiewicz, Z. Flow cytometry-based apoptosis detection. In Apoptosis: Methods and Protocols, Second Edition, 19–32 (Humana Press, 2009); https://doi.org/10.1007/978-1-60327-017-5_2

  80. Pozarowski, P. & Darzynkiewicz, Z. Analysis of cell cycle by flow cytometry. In Checkpoint Controls and Cancer: Volume 2: Activation and Regulation Protocols, 301–311 (Humana Press, 2004); https://doi.org/10.1385/1-59259-811-0:301

  81. Uğur, Y. et al. Comparative chemical and biological evaluation of urtica dioica extracts obtained by methanol and hexane: antioxidant, cytotoxic, apoptotic, and antimicrobial potentials. BMC Complement. Med. Ther. 26, 13. https://doi.org/10.1186/s12906-025-05211-3 (2026).

    Google Scholar 

Download references