An Experimental Design for the Analysis of 5-Caffeoylquinic Acid (5-CQA) in Ethanolic Extracts of Hibiscus (Hibiscus sabdariffa L.) Flower

Authors

  • U. R. Chandimala Department of Food Science, Albert Kázmér Faculty of Agricultural and Food Sciences, Széchenyi István University, Mosonmagyaróvár, Hungary; Department of Food Technology, Institute for Agro-Technology and Rural Sciences, University of Colombo, Hambantota, Sri Lanka https://orcid.org/0000-0002-0647-0615
  • Beatrix Sik Department of Food Science, Albert Kázmér Faculty of Agricultural and Food Sciences, Széchenyi István University, Mosonmagyaróvár, Hungary https://orcid.org/0000-0002-1786-2710
  • Zsolt Ajtony Department of Food Science, Albert Kázmér Faculty of Agricultural and Food Sciences, Széchenyi István University, Mosonmagyaróvár, Hungary https://orcid.org/0000-0002-6484-5147

DOI:

https://doi.org/10.70809/6561

Keywords:

5-caffeoylquinic acid, chlorogenic acid, ethanolic extract, hibiscus, HPLC-PDA

Abstract

This study optimized the extraction and analysis of 5-caffeoylquinic acid (5-CQA), the predominant chlorogenic acid in hibiscus flowers, using a sustainable ethanolic extraction method combined with high-performance liquid chromatography (HPLC) analysis.  To identify the optimal solvent extraction conditions for 5-CQA from hibiscus, Response Surface Methodology (RSM) was employed, incorporating three factors: pH levels (2, 4.5, 7), extraction times (20, 40, 60 minutes) and solvent compositions (25v/v%, 50v/v%, 75v/v% ethanol). The temperature was maintained at the boiling point throughout the extraction period. RSM identified 25% ethanol, pH 7, and 25-minute heat treatment as the optimal extraction conditions for maximizing 5-CQA yield. Two HPLC columns; InertSustain Phenyl (A) and Synergi Polar (B), differing in length, diameter, and particle size were evaluated for chromatographic performance monitoring HPLC-UV signals at a wavelength of 325 nm. The mobile phase for both columns consisted of acetonitrile and acidified water (with orthophosphoric acid). Both HPLC columns achieved high correlation coefficients (0.9908 and 0.9965, A and B) between extracted 5-CQA and standard peaks. Under optimized conditions, the yields of 5-CQA were 1.43 mg/g and 1.48 mg/g dry hibiscus flower for columns A and B, respectively. Compared to literature-reported methods, the developed protocol offers high efficiency, reduced solvent usage, and minimal heat treatment, establishing it as a convenient, and sustainable alternative for 5-CQA extraction.

Author Biography

  • U. R. Chandimala, Department of Food Science, Albert Kázmér Faculty of Agricultural and Food Sciences, Széchenyi István University, Mosonmagyaróvár, Hungary; Department of Food Technology, Institute for Agro-Technology and Rural Sciences, University of Colombo, Hambantota, Sri Lanka

    corresponding author
    rushanthi@uciars.cmb.ac.lk

References

Aree, T. 2023. Atomic-level understanding of conformational flexibility of neochlorogenic and chlorogenic acids and their inclusion complexation with β-cyclodextrin. Food Hydrocolloids. 141 108742. https://doi.org/10.1016/j.foodhyd.2023.108742

Bhandarkar, N. S., Brown, L. and Panchal, S. K. 2019. Chlorogenic acid attenuates high-carbohydrate, high-fat diet–induced cardiovascular, liver, and metabolic changes in rats. Nutrition Research. 62 78–88. https://doi.org/10.1016/j.nutres.2018.11.002

Boukerche, H., Malki, F., Ghaliaoui, N., Bensalem, A. and Mokrane, H. 2024. Combination of ultrasound, microwave and conventional extraction techniques for roselle (Hibiscus Sabdarifa. L.) total anthocyanins and phenolics recovery: effect on antioxidant and structural properties. Biomass Conversion and Biorefinery. 14 (15) 1–13. http://dx.doi.org/10.1007/s13399-023-04029-8

Chaowuttikul, C., Palanuvej, C. and Ruangrungsi, N. 2020. Quantification of chlorogenic acid, rosmarinic acid, and caffeic acid contents in selected Thai medicinal plants using RP-HPLC-DAD. Brazilian Journal of Pharmaceutical Sciences. 56. https://doi.org/10.1590/s2175-97902019000317547

Chongwilaikasem, N., Sithisarn, P., Rojsanga, P. and Sithisarn, P. 2024. Green extraction and partial purification of roselle (Hibiscus sabdariffa L.) extracts with high amounts of phytochemicals and in vitro antioxidant and antibacterial effects. Journal of Food Science. 89 (12) 1–17. https://doi.org/10.1111/1750-3841.17418

Deineka, V. I., Oleinits, E. Y., Blinova, I. P. and Deineka, L. A. 2019. Selectivity of the Separation of Isomeric Chlorogenic Acids under the Conditions of Reversed-Phase HPLC. Journal of Analytical Chemistry, 74 778–783. http://dx.doi.org/10.1134/S1061934819080057

Grujic, N., Rakic, B., Sefer, E., Rakic, D., Nedeljkovic, I., Kladar, N. V. and Bozin, B. 2015. Determination of 5-caffeoylquinic acid (5-CQA) as one of the major classes of chlorogenic acid in commercial tea and coffee samples. Vojnosanitetski Pregled. 72 (11) 1018–1023. https://doi.org/10.2298/VSP130915096G

Jeon, J. S., Kim, H. T., Jeong, I. H., Hong, S. R., Oh, M. S., Park, K. H., Shim, J. H. and Abd El-Aty, A. M. 2017. Determination of chlorogenic acids and caffeine in homemade brewed coffee prepared under various conditions. Journal of Chromatography. B, 1064 115–123. https://doi.org/10.1016/j.jchromb.2017.08.041

Mok, H. W., Ko, M. J., Choi, H. J. and Chung, M. S. 2022. Extraction of chlorogenic acids from hibiscus (Hibiscus syriacus L.) by subcritical water. Journal of Industrial and Engineering Chemistry. 11 255–262. https://doi.org/10.1016/j.jiec.2022.04.005

Mukherjee, P. K., Harwansh, R. K. and Bhattacharyya, S. 2015. Bioavailability of Herbal Products: Approach Toward Improved Pharmacokinetics. pp. 217–245. In Mukherjee, P. K. (eds). Evidence-Based Validation of Herbal Medicine. Elsevier, https://doi.org/10.1016/B978-0-12-800874-4.00010-6

Paraı´so, C. M., Santos, S. S. D., Correa, V. G., Magon, T., Peralta, R. M., Visentainer, J. V. and Madrona, G. S. 2019. Ultrasound-assisted extraction of hibiscus (Hibiscus sabdariffa L.) bioactive compounds for application as potential functional ingredient. Journal of Food Science and Technology. 56 (10) 4667–4677. https://doi.org/10.1007/s13197-019-03919-y

Peng, Y., Liu, F. and Ye, J. 2005. Determination of Phenolic Acids and Flavones in Lonicera japonica Thumb by Capillary Electrophoresis with Electrochemical Detection. Electroanalysis. 17 (4) 356–362. https://doi.org/10.1002/elan.200403102

Pimentel-Moral, S., Borrás-Linares, I., Lozano-Sánchez, J., Arráez-Romána, D., Martínez-Férezc, A. and Segura-Carretero, A. 2018. Microwave-assisted extraction for Hibiscus sabdariffa bioactive compounds. Journal of Pharmaceutical and Biomedical Analysis. 156. 313–322. https://doi.org/10.1016/j.jpba.2018.04.050

Plazas, M., Andujar, I., Vilanova, S., Hurtado, M., Gramazio, P. J., Herraiz, F. J. and Prohens, J. 2013. Breeding for chlorogenic acid content in eggplant. Notulae Botanicae Horti Agrobotanici. 41 (1) 26–35. https://doi.org/10.15835/nbha4119036

Sakai, E., Farhana, F., Yamaguchi, Y. and Tsukuba, T. 2022. Potentials of natural antioxidants from plants as antiosteoporotic agents. In: Atta-ur-Rahman, ed. Studies in Natural Products Chemistry. 72 1–28. https://doi.org/10.1016/B978-0-12-823944-5.00002-8

Yilmaz, P. K. and Kolak, U. 2017. SPE-HPLC Determination of Chlorogenic and Phenolic Acids in Coffee. Journal of Chromatographic Science, 55 (7) 712–718. https://doi.org/10.1093/chromsci/bmx025

Downloads

Published

2025-01-30

Issue

Vol. 29 No. Suppl 1 (2025): 34th Plant Protection Forum held at Keszthely

Section

Articles

License

Copyright (c) 2025 UR Chandimala, Sik Beatrix, Ajtony Zsolt

Creative Commons License

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Cikkre a Creative Commons 4.0 standard licenc alábbi típusa vonatkozik: CC-BY-NC-ND-4.0. Ennek értelmében a mű szabadon másolható, terjeszthető, bemutatható és előadható, azonban nem használható fel kereskedelmi célokra (NC), továbbá nem módosítható és nem készíthető belőle átdolgozás, származékos mű (ND). A licenc alapján a szerző vagy a jogosult által meghatározott módon fel kell tüntetni a szerző nevét és a szerzői mű címét (BY).