Optimization of the extraction of antioxidants from stinging nettle leaf using response surface methodology


  • Una-Jovana Vajic University of Belgrade, Institute for Medical Research, National Institute of Republic of Serbia
  • Jelena Zivkovic Institute for Medicinal Plant Research “Dr. Josif Pančić”
  • Milan Ivanov University of Belgrade, Institute for Medical Research, National Institute of Republic of Serbia
  • Djurdjica Jovovic University of Belgrade, Institute for Medical Research, National Institute of Republic of Serbia
  • Katarina Savikin Institute for Medicinal Plant Research “Dr. Josif Pančić”
  • Branko Bugarski University of Belgrade, Faculty of Technology and Metallurgy
  • Nevena Mihailovic-Stanojevic University of Belgrade, Institute for Medical Research, National Institute of Republic of Serbia




Urtica dioica L., flavonoids, antioxidant capacity, phenolic compounds, response surface methodology


The aim of this study was to optimize the parameters of the extraction of total flavonoids from stinging nettle leaf. Comparison of the effects of different solvents on total flavonoid content showed that, regardless of extraction time, aqueous-methanolic extracts had higher total flavonoid content than aqueous-ethanolic extracts. So, full factorial design and response surface methodology (RSM) were employed to estimate the effects of methanol content (50, 75, and 100 %) and extraction time (30, 60, and 90 minutes) on total flavonoid content and antioxidant capacities of the extracts. RSM analysis showed that methanol content in the solvent influenced significantly total flavonoid content and FRAP (Ferric Reducing Antioxidant Power Assay) antioxidant capacity, while extraction time had no significant effect on either of the responses. Extraction parameters for maximal total flavonoid content were estimated to be 69 % aqueous-methanol and 67 min, and 65 % aqueous-methanol and 83 min for maximal FRAP antioxidant capacity. DPPH (2,2-diphenyl-1-picrylhydrazyl) antioxidant capacity was not significantly affected by extraction time or methanol percentage in the solvent.


P.-G. Pietta, Flavonoids as antioxidants, J. Nat. Prod. 63, 1035–1042 (2000), DOI: http://dx.doi.org/10.1021/np9904509.

C. Rice-Evans, N. Miller, G. Paganga, Structure-antioxidant activity relationships of flavonoids and phenolic acids, Free Radic. Biol. Med. 20, 933–956 (1996), DOI: http://dx.doi.org/10.1016/0891-5849(95)02227-9.

D. Del Rio, A. Rodriguez-Mateos, J.P.E. Spencer, M. Tognolini, G. Borges, A. Crozier, Dietary (poly)phenolics in human health: Structures, bioavailability, and evidence of protective effects against chronic diseases., Antioxid. Redox Signal. 18, 1818–1892 (2013), DOI: http://dx.doi.org/10.1089/ars.2012.4581.

B. Halliwell, J. Rafter, A. Jenner, Health promotion by flavonoids, tocopherols, tocotrienols, and other phenols: direct or indirect effects? Antioxidant or not?, Am. J. Clin. Nutr. 81, 268–276 (2005), DOI: http://dx.doi.org/10.1093/ajcn/81.1.268S.

N. Di Virgilio, E.G. Papazoglou, Z. Jankauskiene, S. Di Lonardo, M. Praczyk, K. Wielgusz, The potential of stinging nettle (Urtica dioica L.) as a crop with multiple uses, Ind. Crops Prod. 68, 42–49 (2015), DOI: http://dx.doi.org/10.1016/j.indcrop.2014.08.012.

R. Upton, Stinging nettles leaf (Urtica dioica L.): Extraordinary vegetable medicine, J. Herb. Med. 3, 9–38 (2013), DOI: http://dx.doi.org/10.1016/j.hermed.2012.11.001.

U.J. Vajić, J. Grujić-Milanović, J. Živković, K. Šavikin, D. Godevac, Z. Miloradović, et al., Optimization of extraction of stinging nettle leaf phenolic compounds using response surface methodology, Ind. Crops Prod. 74, 912–917 (2015), DOI: http://dx.doi.org/10.1016/j.indcrop.2015.06.032.

J.A. Vinson, Y.A. Dabbagh, M.M. Serry, J. Jang, Plant flavonoids, especially tea flavonols, are powerful antioxidants using an in vitro oxidation model for heart disease, J. Agric. Food Chem. 43, 2800–2802 (1995), DOI: http://dx.doi.org/10.1021/jf00059a005

E.M. Silva, H. Rogez, Y. Larondelle, Optimization of extraction of phenolics from Inga edulis leaves using response surface methodology, Sep. Purif. Technol. 55, 381–387 (2007), DOI: http://dx.doi.org/10.1016/j.seppur.2007.01.008.

D.-O. Kim, C.Y. Lee, Comprehensive study on vitamin C equivalent antioxidant capacity (VCEAC) of various polyphenolics in scavenging a free radical and its structural relationship, Crit. Rev. Food Sci. Nutr. 44, 253–73 (2004), DOI: http://dx.doi.org/10.1080/10408690490464960.

R.H. Myers, D.C. Montgomery, C.M. Anderson-Cook, Response surface methodology: Process and product optimization using designed experiments, 3rd ed., John Wiley & Sons, Inc., New Jersey, 2009.

C. Liyana-Pathirana, F. Shahidi, Optimization of extraction of phenolic compounds from wheat using response surface methodology, Food Chem. 93, 47–56 (2005), DOI: http://dx.doi.org/10.1016/j.foodchem.2004.08.050.

D. Baş, İ.H. Boyacı, Modeling and optimization I: Usability of response surface methodology, J. Food Eng. 78, 836–845 (2007), DOI: http://dx.doi.org/10.1016/j.jfoodeng.2005.11.024.

D. Baş, İ.H. Boyacı, Modeling and optimization II: Comparison of estimation capabilities of response surface methodology with artificial neural networks in a biochemical reaction, J. Food Eng. 78, 846–854 (2007), DOI: http://dx.doi.org/10.1016/j.jfoodeng.2005.11.025.

J. Zhishen, T. Mengcheng, W. Jianming, The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals, Food Chem. 64, 555–559 (1999), DOI: http://dx.doi.org/10.1016/S0308-8146(98)00102-2.

N. Lisov, A. Petrović, U. Čakar, M. Jadranin, V. Tešević, L. Bukarica-Gojković, Extraction kinetics of some phenolic compounds during Cabernet Sauvignon alcoholic fermentation and antioxidant properties of derived wines, Maced. J. Chem. Chem. Eng. 39, 185–196 (2020), DOI: http://dx.doi.org/10.20450/mjcce.2020.2060.

A.A. Jovanović, V. Đorđević, G.M. Zdunić, K.P. Šavikin, D. Pljevljakušić, B.M. Bugarski, Ultrasound-assisted extraction of polyphenols from Thymus serpyllum and its antioxidant activity, Hem. Ind. 70, 391–398 (2016) DOI: http://dx.doi.org/10.2298/HEMIND150629044J.

A.A. Jovanović, V.B. Đorđević, G.M. Zdunić, D.S. Pljevljakušić, K.P. Šavikin, D.M. Gođevac, et al., Optimization of the extraction process of polyphenols from Thymus serpyllum L. herb using maceration, heat- and ultrasound-assisted techniques, Sep. Purif. Technol. 179, 369–380 (2017), DOI: http://dx.doi.org/10.1016/j.seppur.2017.01.055.

A. Khoddami, M.A. Wilkes, T.H. Roberts, Techniques for analysis of plant phenolic compounds, Molecules, 18, 2328–2375 (2013), DOI: http://dx.doi.org/10.3390/molecules18022328.

Y. Liu, H. Wang, X. Cai, Optimization of the extraction of total flavonoids from Scutellaria baicalensis Georgi using the response surface methodology, J. Food Sci. Technol. 52, 2336–2343 (2015), DOI: http://dx.doi.org/10.1007/s13197-014-1275-0.

W. Liu, Y. Yu, R. Yang, C. Wan, B. Xu, S. Cao, Optimization of total flavonoid compound extraction from Gynura medica leaf using response surface methodology and chemical composition analysis, Int. J. Mol. Sci. 11, 4750–4763 (2010), DOI: http://dx.doi.org/10.3390/ijms11114750.

A. Jouyban, M.A.A. Fakhree, A. Shayanfar, T. Ghafourian, QSPR modeling using Catalan solvent and solute parameters, J. Braz. Chem. Soc. 22, 684–692 (2011), DOI: http://dx.doi.org/10.1590/S0103-50532011000400011.

M. Naczk, F. Shahidi, Extraction and analysis of phenolics in food, J. Chromatogr. A. 1054, 95–111 (2004), DOI: http://dx.doi.org/10.1016/j.chroma.2004.08.059.

C.D. Stalikas, Extraction, separation, and detection methods for phenolic acids and flavonoids, J. Sep. Sci. 30, 3268–3295 (2007), DOI: http://dx.doi.org/10.1002/jssc.200700261.

M.C. Tan, C.P. Tan, C.W. Ho, Effects of extraction solvent system, time and temperature on total phenolic content of henna (Lawsonia inermis) stems, Int. Food Res. J. 20, 3117–3123 (2013).

P. Mladěnka, L. Zatloukalová, T. Filipskỳ, R. Hrdina, Cardiovascular effects of flavonoids are not caused only by direct antioxidant activity, Free Radic. Biol. Med. 49, 963–975 (2010), DOI: http://dx.doi.org/10.1016/j.freeradbiomed.2010.06.010.

A. Alberti, A.A.F. Zielinski, D.M. Zardo, I.M. Demiate, A. Nogueira, L.I. Mafra, Optimisation of the extraction of phenolic compounds from apples using response surface methodology, Food Chem. 149, 151–158 (2014), DOI: http://dx.doi.org/10.1016/j.foodchem.2013.10.086.

M. Biesaga, K. Pyrzyńska, Stability of bioactive polyphenols from honey during different extraction methods, Food Chem. 136, 46–54 (2013), DOI: http://dx.doi.org/10.1016/j.foodchem.2012.07.095.

R. Tsao, Z. Deng, Separation procedures for naturally occurring antioxidant phytochemicals, J. Chromatogr. B. 812, 85–99 (2004), DOI: http://dx.doi.org/10.1016/j.jchromb.2004.09.028.

J. Dai, R.J. Mumper, Plant phenolics: Extraction, analysis and their antioxidant and anticancer properties, Molecules, 15, 7313–7352 (2010), DOI: http://dx.doi.org/10.3390/molecules15107313.

M. de L. Reis Giada, Food phenolic compounds: Main classes, sources and their antioxidant power, in: Oxidative Stress and Chronic Degenerative Diseases-A Role for Antioxidants, J.A. Morales-González (Ed), InTech, Rijeka, 2013, pp. 87–112.

D. Huang, O. Boxin, R.L. Prior, The chemistry behind antioxidant capacity assays, J.



2022-05-30 — Updated on 2022-07-01


How to Cite

Vajic, U.-J., Zivkovic, J., Ivanov, M., Jovovic, D., Savikin, K., Bugarski, B., & Mihailovic-Stanojevic, N. (2022). Optimization of the extraction of antioxidants from stinging nettle leaf using response surface methodology. Macedonian Journal of Chemistry and Chemical Engineering, 41(1), 119–128. https://doi.org/10.20450/mjcce.2022.2238 (Original work published May 30, 2022)