HYPERICUM PERFORATUM L.'DEN FENOLİK VE FLAVONOİDLER AÇISINDAN ZENGİN BİYOAKTİF MOLEKÜLLERİN OTOMATİK ÇÖZÜCÜ EKSTRAKSİYONU: OPTİMİZASYON VE ÇOK DEĞİŞKENLİ ANALİZ

Öz

Bu çalışmada, Hypericum perforatum L. (sarı kantaron) bitkisinden fenolik ve flavonoid bileşikler açısından zengin özütler elde etmek için otomatik çözücü ekstraksiyonu (ASE) kullanılmıştır. Box-Behnken Tasarımına dayalı tepki yüzey metodolojisi (RSM), üç faktörü (katı kütle, daldırma süresi ve etanol konsantrasyonu) optimize etmek için kullanılmıştır. Toplam fenolik içerik (TPC), toplam flavonoid içerik (TFC) ve antioksidan aktivite (DPPH ve ABTS analizleri) tepki değişkenleri olarak değerlendirilmiştir. Etanol konsantrasyonu TPC ve TFC üzerinde en önemli etkiye sahipken, antioksidan aktivite H. perforatum miktarından önemli ölçüde etkilenmiştir. Optimum koşullar (0.5 g katı kütle, 30 dakikalık daldırma süresi ve ~%30-33 etanol) en yüksek TPC'yi (95.109 mg-GAE/g-DM), TFC'yi (66.998 mg-CE/g-DM), DPPH'nin (38.408 mg-TEAC/g-DM) antioksidan aktivite değerini ve ABTS'nin (42.333 mg-TEAC/g-DM) antioksidan aktivite değerini, 1'e yakın bir arzu edilirlik indeksiyle vermiştir. Temel bileşen analizi (PCA), yanıtlar arasındaki korelasyonları desteklemiş ve ASE'nin sürdürülebilir ve etkili bir ekstraksiyon yöntemi olduğunu göstermiştir.

Anahtar Kelimeler

• Hypericum perforatum L
• antioksidan aktivite
• yeşil ekstraksiyon
• RSM
• PCA

AUTOMATIC SOLVENT EXTRACTION OF BIOACTIVE MOLECULES RICH IN PHENOLICS AND FLAVONOIDS FROM HYPERICUM PERFORATUM L.: OPTIMIZATION AND MULTIVARIATE ANALYSIS

Öz

This study employed automatic solvent extraction (ASE) to obtain extracts rich in phenolic and flavonoid compounds from Hypericum perforatum L. (St. John’s Wort). Response surface methodology (RSM) based on Box-Behnken Design was used to optimize three factors (solid mass, immersion time and ethanol concentration). Total phenolic content (TPC), total flavonoid content (TFC), and antioxidant activity (DPPH and ABTS assays) were evaluated as response variables. Ethanol concentration had the most significant effect on TPC and TFC, while antioxidant activity was significantly influenced by the H. perforatum amount. The optimal conditions (0.5 g solid mass, 30 min immersion time and ~30-33% ethanol) yielded the highest TPC (95.1093 mg-GAE/g-DM), TFC (66.9977 mg-CE/g-DM), antioxidant activity value of DPPH (38.4081 mg-TEAC/g-DM) and antioxidant activity value of ABTS (42.3328 mg-TEAC/g-DM) with a desirability index close to 1. Principal component analysis (PCA) supported correlations among the responses, showing that ASE is a sustainable and effective extraction method.

Anahtar Kelimeler

• Hypericum perforatum L.
• antioxidant activity
• green extraction
• RSM
• PCA

Kaynakça

1. Ahmad, A., Rehman, M. U., Wali, A. F., El-Serehy, H. A., Al-Misned, F. A., Maodaa, S. N., Aljawdah, H. M., Mir, T. M., Ahmad, P. (2020). Box–Behnken Response Surface Design of Polysaccharide Extraction from Rhododendron arboreum and the Evaluation of Its Antioxidant Potential. Molecules, 25(17), 3835. https://doi.org/10.3390/MOLECULES25173835
2. Alahmad, A., Alghoraibi, I., Zein, R., Kraft, S., Dräger, G., Walter, J. G., Scheper, T. (2022). Identification of Major Constituents of Hypericum perforatum L. Extracts in Syria by Development of a Rapid, Simple, and Reproducible HPLC-ESI-Q-TOF MS Analysis and Their Antioxidant Activities. ACS Omega, 7(16), 13475–13493. https://doi.org/10.1021/ ACSOMEGA.1C06335/ASSET/IMAGES/LARGE/AO1C06335_0016.JPEG
3. Bhadange, Y. A., Carpenter, J., Saharan, V. K. (2024). A Comprehensive Review on Advanced Extraction Techniques for Retrieving Bioactive Components from Natural Sources. ACS Omega, 9(29), 31274–31297. https://doi.org/10.1021/ ACSOMEGA.4C02718/ASSET/IMAGES/LARGE/AO4C02718_0007.JPEG
4. Chemat, F., Abert-Vian, M., Fabiano-Tixier, A. S., Strube, J., Uhlenbrock, L., Gunjevic, V., Cravotto, G. (2019). Green extraction of natural products. Origins, current status, and future challenges. TrAC Trends in Analytical Chemistry, 118, 248–263. https://doi.org/10.1016/J.TRAC.2019.05.037
5. Chen, H.-Y.and Chen, C. (2025). A Study of the Response Surface Methodology Model with Regression Analysis in Three Fields of Engineering. Applied System Innovation 2025, Vol. 8, Page 99, 8(4), 99. https://doi.org/ 10.3390/ASI8040099
6. Cossuta, D., Vatai, T., Báthori, M., Hohmann, J., Keve, T., Simándi, B. (2012). Extraction of hyperforin and hypericin from St. John’s wort (Hypericum perforatum L.) with different solvents. Journal of Food Process Engineering, 35(2), 222–235. https://doi.org/10.1111/J.1745-4530.2010.00583.X;PAGE:STRING:ARTICLE/CHAPTER
7. Harkat-Madouri, L., Touati, H., Boulekbache-Makhlouf, L., Madani, K., Haddadi-Guemghar, H. (2025). Optimization of the Microwave-Assisted Extraction of Total Phenolic Compounds (TPCs) From Almond Skins Through Artificial Neural Networks (ANNs) and Assessment of the Antioxidant and Antihyperglycemic Activity of the Extracts. Journal of Food Processing and Preservation, 2025(1), 3294168. https://doi.org/10.1155/ JFPP/3294168
8. Imenšek, N., Kristl, J., Šumenjak, T. K., Ivančič, A. (2021). Antioxidant Activity of Elderberry Fruits during Maturation. Agriculture 2021, Vol. 11, Page 555, 11(6), 555. https://doi.org/ 10.3390/AGRICULTURE11060555

9. Jakubczyk, A., Kiersnowska, K., Ömeroğlu, B., Gawlik-Dziki, U., Tutaj, K., Rybczyńska-Tkaczyk, K., Szydłowska-Tutaj, M., Złotek, U., Baraniak, B. (2021). The influence of hypericum perforatum l. Addition to wheat cookies on their antioxidant, anti-metabolic syndrome, and antimicrobial properties. Foods, 10(6), 1379. https://doi.org/ 10.3390/FOODS10061379/S1
10. Kakouri, E., Trigas, P., Daferera, D., Skotti, E., Tarantilis, P. A., Kanakis, C. (2023). Chemical Characterization and Antioxidant Activity of Nine Hypericum Species from Greece. Antioxidants, 12(4), 899. https://doi.org/ 10.3390/ANTIOX12040899/S1
11. Kaplan, M., Köprü, S., Say, R., Karaman, K., Yılmaz, M. M. (2021). Characterization of in vitro bioactive performance of Hypericum perforatum using response surface methodology. Sigma Journal of Engineering and Natural Sciences, 39(4), 392–403. https://doi.org/10.14744/sigma.2021.00027
12. Karabegoić, I., Stojanović, S. S., Mančić, S., Đorđević, N., Malićanin, M., Danilović, B. (2023). Optimization of food-grade bioactive compound extraction from plant sources by response surface methodology approach: A comprehensive review. International Journal of Food Science and Technology, https://doi.org/10.2478/mjfst-2023-0004
13. Knez, E., Kadac-Czapska, K., Grembecka, M. (2025). Evaluation of Spectrophotometric Methods for Assessing Antioxidant Potential in Plant Food Samples-A Critical Approach. Applied Sciences 2025, Vol. 15, Page 5925, 15(11), 5925. https://doi.org/10.3390/APP15115925
14. Lim, J. S., Seong, Y. S., Oh, G., Im, J. H., Fu, X., Kim, M. H., Roh, J. H., Lee, O. H. (2025). Analysis of Anti-Stress and Sleep-Inducing Effects of a Zizyphus jujuba Mill.-Hypericum perforatum L. Mixture and Its Bioactive Compounds. Journal of the Korean Society of Food Science and Nutrition, 54(6), 489–497. https://doi.org/10.3746/JKFN.2025.54.6.489
15. Lohvina, H., Sándor, M., Wink, M. (2021). Effect of Ethanol Solvents on Total Phenolic Content and Antioxidant Properties of Seed Extracts of Fenugreek (Trigonella foenum-graecum L.) Varieties and Determination of Phenolic Composition by HPLC-ESI-MS. Diversity 2022, Vol. 14, Page 7, 14(1), 7. https://doi.org/ 10.3390/D14010007
16. Milutinović, M., Miladinović, M., Gašić, U., Dimitrijević-Branković, S., Rajilić-Stojanović, M. (2024). Recovery of bioactive molecules from Hypericum perforatum L. dust using microwave-assisted extraction. Biomass Conversion and Biorefinery, 14(5), 7111–7123. https://doi.org/ 10.1007/S13399-022-02717-5/TABLES/5
17. Mišina, I., Perkons, I., Siger, A., Soliven, A., Górnaś, P. (2025). Residues of St. John’s Wort (Hypericum perforatum) Tea Infusions/Water Extracts as a Valuable Source of Tocotrienols: An Extraction Study. Applied Sciences (Switzerland), 15(4), 2047. https://doi.org/10.3390/ APP15042047/S1
18. Özbek, H. N., Halahlih, F., Göğüş, F., Koçak Yanık, D., Azaizeh, H. (2020). Pistachio (Pistacia vera L.) Hull as a Potential Source of Phenolic Compounds: Evaluation of Ethanol–Water Binary Solvent Extraction on Antioxidant Activity and Phenolic Content of Pistachio Hull Extracts. Waste and Biomass Valorization, 11(5), 2101–2110. https://doi.org/10.1007/S12649-018-0512-6
19. Ozdemir, M., Gungor, V., Melikoglu, M., Aydiner, C. (2024). Solvent selection and effect of extraction conditions on ultrasound-assisted extraction of phenolic compounds from galangal (Alpinia officinarum). Journal of Applied Research on Medicinal and Aromatic Plants, 38, 100525. https://doi.org/10.1016/J.JARMAP.2023.100525
20. Pan, Y., He, C., Wang, H., Ji, X., Wang, K., Liu, P. (2010). Antioxidant activity of microwave-assisted extract of Buddleia officinalis and its major active component. Food Chemistry, 121(2), 497–502. https://doi.org/10.1016/ J.FOODCHEM.2009.12.072
21. Pinelo, M., Sineiro, J., Núñez, M. J. (2006). Mass transfer during continuous solid–liquid extraction of antioxidants from grape byproducts. Journal of Food Engineering, 77(1), 57–63. https://doi.org/10.1016/J.JFOODENG.2005.06.021
22. Rodríguez De Luna, S. L., Ramírez-Garza, R. E., Serna Saldívar, S. O. (2020). Environmentally Friendly Methods for Flavonoid Extraction from Plant Material: Impact of Their Operating Conditions on Yield and Antioxidant Properties. The Scientific World Journal, 2020(1), 6792069. https://doi.org/10.1155/2020/6792069
23. Şahin, S. (2015). A novel technology for extraction of phenolic antioxidants from mandarin (Citrus deliciosa Tenore) leaves: Solvent-free microwave extraction. Korean Journal of Chemical Engineering, 32(5), 950–957. https://doi.org/10.1007/S11814-014-0293-Y/METRICS
24. Sakanaka, S., Tachibana, Y., Okada, Y. (2005). Preparation and antioxidant properties of extracts of Japanese persimmon leaf tea (kakinoha-cha). Food Chemistry, 89(4), 569–575. https://doi.org/10.1016/J.FOODCHEM.2004.03.013
25. Sánchez-Muniz, F. J., Olivero-David, R., Triki, M., Salcedo, L., González-Muñoz, M. J., Cofrades, S., Ruiz-Capillas, C., Jiménez-Colmenero, F., Benedi, J. (2012). Antioxidant activity of Hypericum perforatum L. extract in enriched n-3 PUFA pork meat systems during chilled storage. Food Research International, 48(2), 909–915. https://doi.org/10.1016/ J.FOODRES.2012.07.002
26. Seyrekoğlu, F and Temiz, H. (2020). Effect of Extraction Conditions on the Phenolic Content and DPPH Radical Scavenging Activity of Hypericum perforatum L. Turkish Journal of Agriculture - Food Science and Technology, 8(1), 226–229. https://doi.org/10.24925/ TURJAF.V8I1.226-229.3013
27. Seyrekoglu, F., Temiz, H., Eser, F., Yildirim, C. (2022). Comparison of the antioxidant activities and major constituents of three Hypericum species (H. perforatum, H. scabrum and H. origanifolium) from Turkey. South African Journal of Botany, 146, 723–727. https://doi.org/10.1016/J.SAJB.2021.12.012
28. Song, Y., Lee, J., Kwon, H. K., Kim, M., Shin, S., Kim, S., Son, H., Park, C., Yoo, H. Y. (2025). Efficient Ultrasound-Assisted Extraction of Bioactive Molecules from Brown Macroalga Sargassum horneri: Optimal Extraction, Antioxidant and Cytotoxicity Evaluation. International Journal of Molecular Sciences 2025, Vol. 26, Page 2749, 26(6), 2749. https://doi.org/ 10.3390/IJMS26062749
29. Szpisják-Gulyás, N., Al-Tayawi, A. N., Horváth, Z. H., László, Z., Kertész, S., Hodúr, C. (2023). Methods for experimental design, central composite design and the Box–Behnken design, to optimise operational parameters: A review. Acta Alimentaria, 52(4), 521–537. https://doi.org/10.1556/066.2023.00235
30. Kaloteraki, C., Almpounioti, K., Potsaki, P., Bousdouni, P., Kandyliari, A., Koutelidakis, A. E. (2021). Total Antioxidant Capacity and Phenolic Content of 17 Mediterranean Functional Herbs and Wild Green Extracts from North Aegean, Greece. Biology and Life Sciences Forum 2021, Vol. 6, Page 43, 6(1), 43. https://doi.org/10.3390/ FOODS2021-11003
31. Tumbarski, Y., Ivanov, I., Todorova, M., Gerasimova, A., Dincheva, I., Makedonski, L., Nikolova, K. (2024). Chemical Composition and Biological Activities of St John’s Wort (Hypericum perforatum L.) Essential Oil from Bulgaria. Applied Sciences 2024, Vol. 14, Page 11754, 14(24), 11754. https://doi.org/10.3390/ APP142411754
32. Vo, T. P., Pham, T. V., Weina, K., Tran, T. N. H., Vo, L. T. V., Nguyen, P. T., Bui, T. L. H., Phan, T. H., Nguyen, D. Q. (2023). Green extraction of phenolics and flavonoids from black mulberry fruit using natural deep eutectic solvents: optimization and surface morphology. BMC Chemistry, 17(1), 1–15. https://doi.org/10.1186/ S13065-023-01041-X/FIGURES/7
33. Wong, B. Y., Tan, C. P., Ho, C. W. (2013). Effect of solid-to-solvent ratio on phenolic content and antioxidant capacities of “Dukung Anak” (Phyllanthus niruri). International Food Research Journal, 20(1), 325–330. https://openurl.ebsco.com/contentitem/obo:91515356?sid=ebsco:plink:crawler&id=ebsco:obo:91515356&crl=c
34. Wu, H., Zhao, W., Zhou, J., Xie, X., Zhong, X., Liu, Y., Shi, L. (2024). Extraction, analysis of antioxidant activities and structural characteristics of flavonoids in fruits of Diospyros lotus L. LWT, 201, 116248. https://doi.org/10.1016/ J.LWT.2024.116248