Comprehensive Phytochemical Profiling and Antioxidant Assessment of Rhinanthus serotinus subsp. aestivalis: GC-MS and HPLC-DAD Analysis of Volatile and Phenolic Compounds in Leaf and Flower Tissues

Öz

This study represents the first comprehensive phytochemical characterization of Rhinanthus serotinus subsp. aestivalis, an understudied hemiparasitic species from the Orobanchaceae family. Using GC-MS and HPLC-DAD analyses, volatile and phenolic compound profiles were determined in leaf and flower extracts, along with antioxidant capacity evaluation via FRAP and CUPRAC assays. GC-MS analysis identified 16 volatile compounds, with matsutake alcohol (1-octen-3-ol) as the predominant constituent at exceptionally high concentrations (68.42% in flowers, 38.34% in leaves), significantly exceeding levels reported in related species. HPLC analysis revealed distinct tissue-specific phenolic profiles, with leaves demonstrating superior total phenolic content (7.26±0.28 mg GAE/g) and antioxidant capacity compared to flowers. Notable compounds included rutin (828.69 ppm), rosmarinic acid (594.16 ppm), and ascorbic acid (289.89 ppm) in leaves, while flowers were enriched in trans-caffeic acid (459.07 ppm) and pyrogallol (1062.84 ppm). These findings establish R. serotinus subsp. aestivalis as a promising source of bioactive compounds with potential applications in natural product development, functional foods, and pharmaceutical research, while providing fundamental data for future pharmacognostic studies.

Anahtar Kelimeler

• Rhinanthus serotinus
• phytochemical analysis
• matsutake alcohol
• phenolic compounds
• antioxidant activity
• Orobanchaceae

Rhinanthus serotinus subsp. aestivalis'in Kapsamlı Fitokimyasal Profillemesi ve Antioksidan Değerlendirmesi: Yaprak ve Çiçek Dokularında Uçucu ve Fenolik Bileşiklerin GC-MS ve HPLC-DAD Analizi

Öz

Bu çalışma, Orobanchaceae familyasından az çalışılmış bir hemiparazitik tür olan Rhinanthus serotinus subsp. aestivalis'in ilk kapsamlı fitokimyasal karakterizasyonunu temsil etmektedir. GC-MS ve HPLC-DAD analizleri kullanılarak, yaprak ve çiçek ekstraktlarında uçucu ve fenolik bileşik profilleri belirlenmiş, FRAP ve CUPRAC analizleri ile antioksidan kapasite değerlendirmesi yapılmıştır. GC-MS analizi 16 uçucu bileşik tanımlamış olup, matsutake alkolü (1-okten-3-ol) olağanüstü yüksek konsantrasyonlarda (çiçeklerde %68.42, yapraklarda %38.34) baskın bileşen olarak tespit edilmiş ve bu değerler ilgili türlerde rapor edilen seviyelerden önemli ölçüde yüksek bulunmuştur. HPLC analizi dokuma özgü farklı fenolik profiller ortaya koymuş, yapraklar çiçeklere kıyasla üstün toplam fenolik içerik (7.26±0.28 mg GAE/g) ve antioksidan kapasiteye sahip olduğunu göstermiştir. Yapraklarda rutin (828.69 ppm), rosmarinik asit (594.16 ppm) ve askorbik asit (289.89 ppm) gibi önemli bileşikler tespit edilirken, çiçeklerde trans-kafeik asit (459.07 ppm) ve pirogallol (1062.84 ppm) açısından zenginlik gözlenmiştir. Bu bulgular, R. serotinus subsp. aestivalis'i doğal ürün geliştirme, fonksiyonel gıdalar ve farmasötik araştırmalarda potansiyel uygulamaları olan biyoaktif bileşiklerin umut verici bir kaynağı olarak ortaya koyarken, gelecekteki farmakognozi çalışmaları için temel veri sağlamaktadır.

Anahtar Kelimeler

• Rhinanthus serotinus
• fitokimyasal analiz
• matsutake alkolü
• fenolik bileşikler
• antioksidan aktivite
• Orobanchaceae

Kaynakça

1. N. Kumar, V. Pruthi, Potential applications of ferulic acid from natural sources, Biotechnol Rep, 4, 2014, 86-93. K.H. Kwon, A. Barve, S. Yu, M.T. Huang, A.N. Kong, Cancer chemoprevention by phytochemicals: potential molecular targets, biomarkers and animal models, Acta Pharmacol Sin, 28, 2007, 1409-1421.
2. J. Moore, M. Yousef, E. Tsiani, Anticancer effects of rosemary (Rosmarinus officinalis L.) extract and rosemary extract polyphenols, Nutrients, 8, 2016, 731.
3. A. Trivellini, M. Lucchesini, R. Maggini, H. Mosadegh, T.S.S. Villamarin, P. Vernieri, A. Mensuali-Sodi, A. Pardossi, Lamiaceae phenols as multifaceted compounds: bioactivity, industrial prospects and role of positive-stress, Ind Crops Prod, 83, 2016, 241-254.
4. Y. Sakihama, M.F. Cohen, S.C. Grace, H. Yamasaki, Plant phenolic antioxidant and prooxidant activities: phenolics-induced oxidative damage mediated by metals in plants, Toxicology, 177, 2002, 67-80.
5. W. Watjen, G. Michels, B. Steffan, P. Niering, Y. Chovolou, A. Kampkotter, Q.H. Tran-Thi, P. Proksch, R. Kahl, Low concentrations of flavonoids are protective in rat H4IIE cells whereas high concentrations cause DNA damage and apoptosis, J Nutr, 135, 2005, 525-531. C.M. Ajila, S.K. Brar, M. Verma, R.D. Tyagi, S. Godbout, J.R. Valéro, Extraction and analysis of polyphenols: recent trends, Crit Rev Biotechnol, 31, 2011, 227-249. L. Bravo, Polyphenols: chemistry, dietary sources, metabolism, and nutritional significance, Nutr Rev, 56, 1998, 317-333.
6. L.M. Carvalho, M. Martini, A.P.L. Moreira, A.P. de Lima, D. Correia, T. Falcão, S.C. Garcia, A.V. de Bairros, P.C. do Nascimento, D. Bohrer, Presence of synthetic pharmaceuticals as adulterants in slimming phytotherapeutic formulations and their analytical determination, Forensic Sci Int, 204, 2010, 6-11.
7. L.M. Carvalho, A.P. Moreira, M. Martini, T. Falcão, The illegal use of synthetic pharmaceuticals in herbal formulations: an overview on the adulteration practice and analytical investigations, Forensic Sci Rev, 23, 2011, 73-90.
8. D. Krishnaiah, R. Sarbatly, R. Nithyanandam, A review of the antioxidant potential of medicinal plant species, Food Bioprod Process, 89, 2011, 217-233.

9. M.A.M. Maciel, A.C. Pinto, V.F. Veiga Jr, N.F. Grynberg, A. Echevarria, Plantas medicinais: a necessidade de estudos multidisciplinares, Quím Nova, 25, 2002, 429-438.
10. N. Balasundra, K. Sundram, S. Samman, Phenolic compounds in plants and agro-industrial by-products: antioxidant activity, occurrence, and potential uses, Food Chem, 99, 2006, 191-203.
11. C.D. Stalikas, Extraction, separation, and detection methods for phenolic acids and flavonoids, J Sep Sci, 30, 2007, 3268-3295.
12. I.G. Casella, C. Colonna, M. Contursi, Electroanalytical determination of some phenolic acids by high-performance liquid chromatography at gold electrodes, Electroanalysis, 19, 2007, 1503-1508.
13. R. Gotti, Capillary electrophoresis of phytochemical substances in herbal drugs and medicinal plants, J Pharm Biomed Anal, 55, 2011, 775-801.
14. D. Argyropoulos, J. Müller, Effect of convective-, vacuum- and freeze drying on sorption behaviour and bioactive compounds of lemon balm (Melissa officinalis L.), J Appl Res Med Aromat Plants, 1, 2014, 59-69. V. Kumar, R.S. Chauhan, H. Sood, C. Tandon, Cost effective quantification of picrosides in Picrorhiza kurroa by employing response surface methodology using HPLC-UV, J Plant Biochem Biotechnol, 24, 2015, 376-384.
15. V. Kumar, H. Sood, R.S. Chauhan, Optimization of a preparative RP-HPLC method for isolation and purification of picrosides in Picrorhiza kurroa, J Plant Biochem Biotechnol, 25, 2016, 208-214. A. Ray, S. Dutta Gupta, S. Ghosh, Isolation and characterization of potent bioactive fraction with antioxidant and UV absorbing activity from Aloe barbadensis Miller gel, J Plant Biochem Biotechnol, 22, 2013, 483-487.
16. M.N. Irakli, V.F. Samanidou, C.G. Biliaderis, I.N. Papadoyannis, Simultaneous determination of phenolic acids and flavonoids in rice using solid-phase extraction and RP-HPLC with photodiode array detection, J Sep Sci, 35, 2012, 1603-1611.
17. B. Shan, Y.Z. Cai, M. Sun, H. Corke, Antioxidant capacity of 26 spice extracts and characterization of their phenolic constituents, J Agric Food Chem, 53, 2005, 7749-7759.
18. S.T. Saito, A. Welzel, E.S. Suyenaga, F. Bueno, A method for fast determination of epigallocatechin gallate (EGCG), epicatechin (EC), catechin (C) and caffeine (CAF) in green tea using HPLC, Food Sci Technol (Campinas), 26, 2006, 394-400.
19. A. Cantalapiedra, M.J. Gismera, M.T. Sevilla, J.R. Procopio, Sensitive and selective determination of phenolic compounds from aromatic plants using an electrochemical detection coupled with HPLC method, Phytochem Anal, 25, 2014, 247-254.
20. E. Barrajon-Catalan, S. Fernandez-Arroyo, C. Roldan, E. Guillen, D. Saura, A. Segura-Carretero, V. Micol, A systematic study of the polyphenolic composition of aqueous extracts deriving from several Cistus genus species: evolutionary relationship, Phytochem Anal, 22, 2011, 303-312.
21. C.S. Harris, A.J. Burt, A. Saleem, P.M. Le, L.C. Martineau, P.S. Haddad, S.A. Bennett, J.T. Arnason, A single HPLC-PAD-APCI/MS method for the quantitative comparison of phenolic compounds found in leaf, stem, root and fruit extracts of Vaccinium angustifolium, Phytochem Anal, 18, 2007, 161-169.
22. K.M. Kalili, A. de Villiers, Recent developments in the HPLC separation of phenolic compounds, J Sep Sci, 34, 2011, 854-876.
23. A. Ribas-Agusti, M. Gratacos-Cubarsi, C. Sarraga, J.A. Garcia-Regueiro, M. Castellari, Analysis of eleven phenolic compounds including novel p-coumaroyl derivatives in lettuce (Lactuca sativa L.) by ultra-high-performance liquid chromatography with photodiode array and mass spectrometry detection, Phytochem Anal, 22, 2011, 555-563.
24. R. Rodriguez-Solana, J.M. Salgado, J.M. Dominguez, S. Cortes-Dieguez, Comparison of Soxhlet, accelerated solvent and supercritical fluid extraction techniques for volatile (GC-MS and GC/FID) and phenolic compounds (HPLC-ESI/MS/MS) from Lamiaceae species, Phytochem Anal, 26, 2015, 61-71. C.D. Stalikas, Extraction, separation, and detection methods for phenolic acids and flavonoids, J Sep Sci, 30, 2007, 3268-3295.
25. J.L. Rambla, A. Trapero-Mozos, G. Diretto, A. Rubio-Moraga, A. Granell, L. Gómez-Gómez, O. Ahrazem, Gene-metabolite networks of volatile metabolism in Airen and Tempranillo grape cultivars revealed a distinct mechanism of aroma bouquet production, Front Plant Sci, 7, 2016, 1619.
26. J. Zhang, J. Zhao, Y. Xu, J. Liang, P. Chang, F. Yan, M. Li, Y. Liang, Z. Zou, Genome-wide association mapping for tomato volatiles positively contributing to tomato flavor, Front Plant Sci, 6, 2015, 1042.
27. A. Slegers, P. Angers, É. Ouellet, T. Truchon, K. Pedneault, Volatile compounds from grape skin, juice and wine from five interspecific hybrid grape cultivars grown in Québec (Canada) for wine production, Molecules, 20, 2015, 10980-11016.
28. X.W. Ma, M.Q. Su, H.X. Wu, Y.G. Zhou, S.B. Wang, Analysis of the volatile profile of core Chinese mango germplasm by headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry, Molecules, 23, 2018, 1480.
29. R. Marsili, Flavor, Fragrance, and Odor Analysis (2. edition), 2012, Boca Raton, CRC Press. S. Van Nocker, S.E. Gardiner, Breeding better cultivars, faster: applications of new technologies for the rapid deployment of superior horticultural tree crops, Hortic Res, 1, 2014, 14022.
30. S. Yang, J. Fresnedo-Ramírez, M. Wang, L. Cote, P. Schweitzer, P. Barba, E.M. Takacs, M. Clark, J. Luby, D.C. Manns, A next-generation marker genotyping platform (AmpSeq) in heterozygous crops: a case study for marker-assisted selection in grapevine, Hortic Res, 3, 2016, 16002.
31. L.A. Chaparro-Torres, M.C. Bueso, J.P. Fernández-Trujillo, Aroma volatiles obtained at harvest by HS-SPME/GC-MS and INDEX/MS-E-nose fingerprint discriminate climacteric behaviour in melon fruit, J Sci Food Agric, 96, 2016, 2352-2365.
32. J.M. Obando-Ulloa, J. Ruiz, A.J. Monforte, J.P. Fernández-Trujillo, Aroma profile of a collection of near-isogenic lines of melon (Cucumis melo L.), Food Chem, 118, 2010, 815-822.
33. F. Dunemann, D. Ulrich, A. Boudichevskaia, C. Grafe, W.E. Weber, QTL mapping of aroma compounds analysed by headspace solid-phase microextraction gas chromatography in the apple progeny "Discovery" × "Prima", Mol Breed, 23, 2009, 501-521.
34. J. Vogt, D. Schiller, D. Ulrich, W. Schwab, F. Dunemann, Identification of lipoxygenase (LOX) genes putatively involved in fruit flavour formation in apple (Malus × domestica), Tree Genet Genomes, 9, 2013, 1493-1511.
35. J. Battilana, L. Costantini, F. Emanuelli, F. Sevini, C. Segala, S. Moser, R. Velasco, G. Versini, M.S. Grando, The 1-deoxy-d-xylulose 5-phosphate synthase gene co-localizes with a major QTL affecting monoterpene content in grapevine, Theor Appl Genet, 118, 2009, 653-669.
36. A. Doligez, E. Audiot, R. Baumes, P. This, QTLs for muscat flavor and monoterpenic odorant content in grapevine (Vitis vinifera L.), Mol Breed, 18, 2006, 109-125.
37. S. Guillaumie, A. Ilg, S. Réty, M. Brette, C. Trossat-Magnin, S. Decroocq, C. Léon, C. Keime, T. Ye, R. Baltenweck-Guyot, Genetic analysis of the biosynthesis of 2-methoxy-3-isobutylpyrazine, a major grape-derived aroma compound impacting wine quality, Plant Physiol, 162, 2013, 604-615.
38. Y. Bezman, F. Mayer, G.R. Takeoka, R.G. Buttery, G. Ben-oliel, H.D. Rabinowitch, M. Naim, Differential effects of tomato (Lycopersicon esculentum Mill) matrix on the volatility of important aroma compounds, J Agric Food Chem, 51, 2003, 722-726.
39. T. Vandendriessche, B.M. Nicolai, M.L.A.T.M. Hertog, Optimization of HS SPME fast GC-MS for high-throughput analysis of strawberry aroma, Food Anal Methods, 6, 2013, 512-520.
40. L. García-Vico, A. Belaj, A. Sánchez-Ortiz, J.M. Martínez-Rivas, A.G. Pérez, C. Sanz, Volatile compound profiling by HS-SPME/GC-MS-FID of a core olive cultivar collection as a tool for aroma improvement of virgin olive oil, Molecules, 22, 2017, 141.
41. R. Soó, D. Webb, Rhinanthus L., Flora Europaea, Editörler: T. Tutin, V. Heywood, N. Burges, D. Valentine, D. Moore, 1972, Cambridge, Cambridge University Press.
42. H.S. Heide-Jorgensen, Introduction: The parasitic syndrome in higher plants, Parasitic Orobanchaceae: Parasitic Mechanisms and Control Strategies, Editörler: D.M. Joel, J. Gressel, L.J. Musselman, 2013, Berlin/Heidelberg, Springer.
43. C.R. Clarke, M.P. Timko, J.I. Yoder, M.J. Axtell, J.H. Westwood, Molecular dialog between parasitic plants and their hosts, Annu Rev Plant Biol, 57, 2019, 279-299.
44. D.L. Nickrent, Parasitic angiosperms: How often and how many?, Taxon, 69, 2020, 5-27. H. Bouwmeester, N. Sinha, J. Scholes, Parasitic plants: Physiology, development, signaling, and ecosystem interactions, Plant Physiol, 185, 2021, 1267-1269.
45. Y. Krasylenko, J. Tšitel, G. Ceccantini, M. Oliveira-da-Silva, V. Dvoˇrák, D. Steele, Y. Sosnovsky, R. Piwowarczyk, D.M. Watson, L. Teixeira-Costa, Parasites on parasites: Hyper-, epi-, and autoparasitism, Amer J Bot, 108, 2021, 8-21.
46. J.M. Mutuku, S. Cui, S. Yoshida, K. Shirasu, Orobanchaceae parasite--host interactions, New Phytol, 230, 2021, 46-59. J. Tešitel, A.R. Li, K. Knotková, R. McLellan, P.C.G. Bandaranayake, D.M. Watson, The bright side of parasitic plants: What are they good for?, Plant Physiol, 185, 2021, 1309-1324.
47. J. Tešitel, P. ˇRíha, Š. Svobodova, T. Malinová, M. Štech, Phylogeny, life history evolution and biogeography of the Rhinanthoid Orobanchaceae, Folia Geobot, 45, 2010, 347-367.
48. A. Uysal, G. Zengin, Y. Durak, A. Aktumsek, Screening for antioxidant and antimutagenic properties of extracts from Centaurea pterocaula as well as theirs enzyme inhibitory potentials, Marmara Pharm J, 20, 2016, 232-242.
49. İ. Akbulut, E. Gürbüz, A. Rayman Ergün, T. Baysal, Drying of apricots treated with Ginkgo biloba plant extract and determination of the quality properties, J Adv Res Nat Appl Sci, 7, 2021, 145-159.
50. J. Tsuji, K. Tsuruoka, K. Yamamoto, Preparation of matsutake alcohol (1-octen-3-ol) from a butadiene telomer, Bull Chem Soc Jpn, 49, 1976, 1701-1702.
51. A. Mosandl, G. Heusinger, M. Gessner, Analytical and sensory differentiation of l-octen-3-ol enantiomers, J Agric Food Chem, 34, 1986, 119-122.
52. R. Zawirska-Wojtasiak, Optical purity of (R)-()-1-octen-3-ol in the aroma of various species of edible mushrooms, Food Chem, 86, 2004, 113-118.
53. Y. Kim, G. Rhee, S. Heo, K. Nam, Q. Li, C.K. Yoo, Human health risk, environmental and economic assessment based on multimedia fugacity model for determination of best available technology (BAT) for VOC reduction in industrial complex, Korean Chem Eng Res, 58, 2020, 325-345.
54. C.C. Pant, A.B. Melkani, L. Mohan, V. Dev, Composition and antibacterial activity of essential oil from Scutellaria grossa Wall ex Benth, Nat Prod Res, 26, 2012, 190-192.
55. A. Rahman, Z.S. Shanta, M.A. Rashid, T. Parvin, S. Afrin, M.K. Khatun, M.A. Sattar, In vitro antibacterial properties of essential oil and organic extracts of Premna integrifolia Linn, Arab J Chem, 9, 2016, S475-S479. A. Shafaghat, Antibacterial activity and GC/MS analysis of the essential oils from flower, leaf and stem of Origanum vulgare ssp. viride growing wild in north-west Iran, Nat Prod Commun, 6, 2011, 1351-1352.
56. C. Xiong, Q. Li, S. Li, C. Chen, Z. Chen, W. Huang, In vitro antimicrobial activities and mechanism of 1-octen-3-ol against food-related bacteria and pathogenic fungi, J Oleo Sci, 66, 2017, 1041-1049.
57. L. Khodaei, A. Delazar, H. Nazemiyeh, S. Asnaashari, L. Nahar, S.D. Sarker, Composition of the volatile oils of the aerial parts of Pedicularis sibthorpii and P. wilhelmsiana growing in Iran, J Essent Oil Bear Plants, 15, 2012, 352-356. P. Novy, H. Davidova, C.S. Serrano-Rojero, J. Rondevaldova, J. Pulkrabek, L. Kokoska, Composition and antimicrobial activity of Euphrasia rostkoviana Hayne essential oil, Evid Based Complement Altern Med, 2015, 734101.
58. W. Takken, D.L. Kline, Carbon dioxide and 1-octen-3-ol as mosquito attractants, J Am Mosq Control Assoc, 5, 1989, 311-316.
59. A. Watentena, I.C. Okoye, Species specificity of carbon dioxide, 1-octen-3-ol, l-lactic acid and 2-butanone as mosquito chemo-attractants in mosquito surveillance: A review, Int J Mosq Res, 6, 2019, 138-142.
60. S. Khoja, K.M. Eltayef, I. Baxter, A. Myrta, J.C. Bull, T. Butt, Volatiles of the entomopathogenic fungus, Metarhizium brunneum, attract and kill plant parasiti nematodes, Biol Control, 152, 2021, 104472.
61. S. Olson, An analysis of the biopesticide market now and where it is going, Outlooks Pest Manag, 26, 2015, 203-206.
62. E.C. Aytar, Glaucium flavum-derived phytochemical compounds and their molecular interactions with SIRT1, ChemistrySelect, 9, 2024, e202403811.
63. B. Halliwell, J.M.C. Gutteridge, Free radicals in Biology and Medicine, 1999, Oxford, Oxford University Press. R. Raviadaran, M.H. Ng, D. Chandran, K.K. Ooi, S. Manickam, Stable W/O/W multiple nanoemulsion encapsulating natural tocotrienols and caffeic acid with cisplatin synergistically treated cancer cell lines (A549 and HEP G2), and reduced toxicity on normal cell line (HEK 293), Mater Sci Eng C, 121, 2021, 111808.
64. G.T. Tabakam, T. Kodama, A.R.N. Donfack, Y.M.M. Nguekeu, B. Nomin-Erdene, Z.P. Htoo, K.M. Do, S.A. Ngouela, M. Tene, H. Morita, A new caffeic acid ester and a new ceramide from the roots of Eriosema glomeratum, Phytochem Lett, 45, 2021, 82-87.
65. R. Salsabila, M.S. Perdani, N.S. Kitakawa, H. Hermansyah, Production of methyl caffeate as an intermediate product to produce caffeic acid phenethyl ester by esterification using cation-exchange resin, Energy Rep, 6, 2020, 528-533.
66. S. Budavari, The Merck Index (12. edition), 1996, Whitehall, Merck & Co. Inc. Y.K. Gupta, M. Sharma, G. Chaudhary, Pyrogallol-induced hepatotoxicity in rats: a model to evaluate antioxidant hepatoprotective agents, Methods Find Exp Clin Pharmacol, 24, 2002, 497-500.
67. M. Sharma, K. Rai, S.S. Sharma, Y.K. Gupta, Effect of antioxidants on pyrogallolinduced delay in gastric emptying in rats, Pharmacology, 60, 2000, 90-96.
68. S. Kerkar, C. Speyer, J. Tyburski, C. Steffes, Reactive oxygen metabolites induce a biphasic contractile response in microvascular lung pericytes, J Trauma, 51, 2001, 440-445.
69. G. Upadhyay, A. Kumar, M.P. Singh, Effect of silymarin on pyrogallol- and rifampicin-induced hepatotoxicity in mouse, Eur J Pharmacol, 565, 2007, 190-201.
70. G. Upadhyay, A.K. Singh, A. Kumar, O. Prakash, M.P. Singh, Resveratrol modulates pyrogallol-induced changes in hepatic toxicity markers, xenobiotic metabolizing enzymes and oxidative stress, Eur J Pharmacol, 596, 2008, 146-152.
71. M.A. Bianco, A. Handaji, H. Savolainen, Quantitative analysis of ellagic acid inhardwood samples, Sci Tot Environ, 222, 1998, 123-126.
72. H. Sutanto, B.H. Susanto, M. Nasikin, Solubility and antioxidant potential of a pyrogallol derivative for biodiesel additive, Molecules, 24, 2019, 2439.
73. J.A. Ross, C.M. Kasum, Dietary flavonoids: bioavailability, metabolic effects, and safety, Annu Rev Nutr, 22, 2002, 19-34.
74. R. Mauludin, R.H. Müller, C.M. Keck, Development of an oral rutin nanocrystal formulation, Int J Pharm, 370, 2009, 202-209.
75. W. Panasiak, M. Wleklik, A. Oraczewska, M. Luczak, Influence of flavonoids on combined experimental infections with EMC virus and Staphylococcus aureus in mice, Acta Microbiol Pol, 38, 1989, 185-188.
76. V. Mihailović, S. Kreft, E. Tavčar Benković, N. Ivanović, M.S. Stanković, Chemical profile, antioxidant activity and stability in stimulated gastrointestinal tract model system of three Verbascum species, Ind Crops Prod, 89, 2016, 141-151.
77. D. Benedec, I. Oniga, D. Hanganu, A.M. Vlase, I. Ielciu, G. Crișan, N. Fițt, M. Niculae, T. Bab, E. Pall, Revealing the Phenolic Composition and the Antioxidant, Antimicrobial and Antiproliferative Activities of Two Euphrasia sp. Extracts, Plants, 13, 2024, 1790.
78. E.C. Aytar, E.I. Torunoglu, A. Gümrükçüoğlu, A. Durmaz, S. Al-Farraj, M. Sillanpää, Molecular docking analyses on the chemical profile and antioxidant potential of Cakile maritima using GC–MS and HPLC, Scientific Reports, 15, 2025, 11937.