AN OVERVIEW OF ADVANCED 2,2-Diphenyl-1-picrylhydrazyl (DPPH) ANALYSIS TECHNIQUES
DOI:
https://doi.org/10.21580/wjc.v8i2.26028Keywords:
antioxidant analysis, DPPH Assay, analytical techniquesAbstract
Antioxidants play a crucial role in preventing oxidative damage, necessitating reliable analytical
techniques for their evaluation. Oxidative stress, resulting from an imbalance between free radicals
and antioxidants in the body, has been associated with various chronic diseases, including cancer,
cardiovascular disorders, and neurodegenerative conditions. Therefore, accurately assessing
antioxidant activity is essential for developing health-related products and validating their efficacy.
This study reviewed and compared various analytical methods used to determine antioxidant
activity, emphasizing their advantages, limitations, and applicability across different sample types.
Traditional spectrophotometric assays, such as the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method,
remain widely used because of their simplicity and cost-effectiveness, although they often suffer
from matrix interferences. Advanced techniques, including gas chromatography–mass
spectrometry (GC–MS), high-performance liquid chromatography coupled with DPPH (HPLC
DPPH), electrochemical methods, microfluidic systems, and miniaturized paper-based assays,
provide improved sensitivity, accuracy, and efficiency. These methods enable a more comprehensive
assessment of antioxidant activity by offering deeper insights into reaction mechanisms and
compound interactions. The integration of multiple analytical approaches can further enhance
antioxidant characterization, supporting applications in the food, pharmaceutical, and biomedical
industries. This review highlights the importance of selecting appropriate analytical techniques
based on research objectives and sample characteristics, while also outlining future directions for
advancing antioxidant detection methodologies.
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References
Aeinehvand, M., Yusof, R., Madou, M., 2018. A microfluidic lab-on-a-disc (LOD) for antioxidant activities of plant extracts. Micromachines (Basel) 9, 140. https://doi.org/10.3390/mi9040140
Abdulwahab, M.K., Ariffin, A., Yehye, W.A., Abdul‐Aziz, A., Kareem, H.S., Nordin, N., 2015. Synthesis of the hydrazones of 2‐((3,5‐Di‐tert‐butyl‐4‐hydroxybenzyl)thio) acetohydrazide and the study of their radical scavenging activity by the DPPH assay and the computational method. Bull Korean Chem Soc 36, 2716–2724. https://doi.org/10.1002/bkcs.10553
Abo El-Nasr, A., Mokhtar, F., 2023. Antioxidant, antimicrobial, and anticancer cells line of Aspergillus flavus ON764430 extracts isolated from Al Mudawara Mountain, El Fayum governorate. Advances in Basic and Applied Sciences 1, 33–45. https://doi.org/10.21608/abas.2023.194936.1005
Adiyanto, S.A., Chriscensia, E., Wibowo, E.C., Nathanael, J., Crystalia, A.A., Putra, A.B.N., Priadi, G., Fahrurrozi, F., Hartrianti, P., 2023. In vitro cytoprotective activity study of cocoa (Theobroma cacao L.) pod husk ethanolic extract against blue light irradiation. Pharmacognosy Res 15, 537–543. https://doi.org/10.5530/pres.15.3.056
Ahmad, J., Bagheri, R., Bashir, H., Baig, M.A., Al-Huqail, A., Ibrahim, M.M., Qureshi, M.I., 2018. Organ-specific phytochemical profiling and antioxidant analysis of Parthenium hysterophorus L. Biomed Res Int 2018, 1–10. https://doi.org/10.1155/2018/9535232
Angeli, L., Morozova, K., Scampicchio, M., 2023. A kinetic-based stopped-flow DPPH• method. Sci Rep 13, 7621. https://doi.org/10.1038/s41598-023-34382-7
Angulo‐López, J.E., Losoya‐Sifuentes, C., Palmieri, S., Torres‐León, C., Flores‐Gallegos, A.C., Fanti, F., Sergi, M., Ascacio‐Valdes, J.A., Contreras Esquivel, J.C., Rúelas‐Chácon, X., Aguilar, C.N., Compagnone, D., 2023. Dietary fibre concentrates from avocado and mango by‐products; antioxidant capacity and polyphenols evaluation by HPLC‐IDA‐EPI‐MS. Int J Food Sci Technol 58, 6609–6620. https://doi.org/10.1111/ijfs.16775
Arce-Amezquita, P.M., Beltrán-Morales, F.A., Manríquez-Rivera, G.A., Cota-Almanza, M.E., Quian-Torres, A., Peralta-Olachea, R.G., 2019. Nutritional value of conventional, wild and organically produced fruits and vegetables available in Baja California Sur markets. Revista Terra Latinoamericana 37, 401. https://doi.org/10.28940/terra.v37i4.524
Arvapalli, D.M., Sheardy, A.T., Bang, J.J., Wei, J., 2021. Antiproliferative and ROS regulation activity of photoluminescent curcumin-derived nanodots. ACS Appl Bio Mater 4, 8477–8486. https://doi.org/10.1021/acsabm.1c00991
Ayuni, D., Neri, L., Pittia, P., Sirikantaramas, S., Devahastin, S., Borompichaichartkul, C., 2024. Impact of extraction methods on bioactive compounds and antioxidant activities of Thai jasmine rice leaf extracts. Int J Food Sci Technol 59, 7865–7872. https://doi.org/10.1111/ijfs.17062
Baldan, V., Sut, S., Faggian, M., Dalla Gassa, E., Ferrari, S., De Nadai, G., Francescato, S., Baratto, G., Dall’Acqua, S., 2017. Larix decidua bark as a source of phytoconstituents: An LC-MS study. Molecules 22, 1974. https://doi.org/10.3390/molecules22111974
Becker, M., Nunes, G., Ribeiro, D., Silva, F., Catanante, G., Marty, J., 2019. Determination of the antioxidant capacity of red fruits by miniaturized spectrophotometry assays. J Braz Chem Soc. https://doi.org/10.21577/0103-5053.20190003
Braham, F., Carvalho, D.O., Almeida, C.M.R., Zaidi, F., Magalhães, J.M.C.S., Guido, L.F., Gonçalves, M.P., 2020. Online HPLC-DPPH screening method for evaluation of radical scavenging phenols extracted from Moringa oleifera leaves. South African Journal of Botany 129, 146–154. https://doi.org/10.1016/j.sajb.2019.04.001
Bottoni, M., Baron, G., Gado, F., Milani, F., Santagostini, L., Colombo, L., Colombo, P.S., Caporali, E., Spada, A., Biagi, M., Giuliani, C., Bruschi, P., Aldini, G., Fico, G., 2022. Achillea moschata wulfen: from ethnobotany to phytochemistry, morphology, and biological activity. Molecules 27, 8318. https://doi.org/10.3390/molecules27238318
Ceylan, F.D., Adrar, N., Günal-Köroğlu, D., Gültekin Subaşı, B., Capanoglu, E., 2023. Combined neutrase–alcalase protein hydrolysates from hazelnut meal, a potential functional food ingredient. ACS Omega 8, 1618–1631. https://doi.org/10.1021/acsomega.2c07157
Darachai, P., Limpawattana, M., Hawangjoo, M., Klaypradit, W., 2019. Effects of shrimp waste types and their cooking on properties of extracted astaxanthin and its characteristics in liposomes. Journal of Food and Nutrition Research 7, 530–536. https://doi.org/10.12691/jfnr-7-7-7
Deng, M., Deng, Y., Dong, L., Ma, Y., Liu, L., Huang, F., Wei, Z., Zhang, Y., Zhang, M., Zhang, R., 2018. Effect of storage conditions on phenolic profiles and antioxidant activity of litchi pericarp. Molecules 23, 2276.
Djitieu Deutchoua, A.D., Ngueumaleu, Y., Kenne Dedzo, G., Kenfack Tonle, I., Ngameni, E., 2019. Electrochemical study of DPPH incorporated in carbon paste electrode as potential tool for antioxidant properties determination. Electroanalysis 31, 335–342. https://doi.org/10.1002/elan.201800606
Fontoura, B.H., Perin, E.C., Teixeira, S.D., de Lima, V.A., Carpes, S.T., 2023. Multivariate and machine learning approaches for prediction of antioxidant potential in Bertholletia excelsa barks. Journal of King Saud University-Science 35, 102792. https://doi.org/10.1016/j.jksus.2023.102792
Fu, L., Zheng, Y., Zhang, P., Lai, G., 2021. Quantification of silicon in rice based on an electrochemical sensor via an amplified electrocatalytic strategy. Micromachines (Basel) 12, 1048. https://doi.org/10.3390/mi12091048
Gandhi, Y., Rawat, H., Singh Dhanjal, D., Kumar, V., Charde, V., Soni, H., Mishra, S.K., Singh, G., Singh, S., Sharma, P., Shakya, S.K., Narsimhaji, C. V., Meena, A.K., Singh, A., Singh, R., Srikanth, N., Acharya, R., 2023. A Comparative analysis of phytochemicals, metal ions, volatile metabolites in heart wood, stem bark and leaves of Salix alba L. along with in vitro antioxidant, antacid, antimicrobial activities for sake of environment conservation by substitution of stem. Chem Biodivers 20. https://doi.org/10.1002/cbdv.202301234
Gómez, J., Simirgiotis, M.J., Lima, B., Paredes, J.D., Villegas Gabutti, C.M., Gamarra-Luques, C., Bórquez, J., Luna, L., Wendel, G.H., Maria, A.O., Feresin, G.E., Tapia, A., 2019. Antioxidant, gastroprotective, cytotoxic activities and UHPLC PDA-Q orbitrap mass spectrometry identification of metabolites in Baccharis grisebachii decoction. Molecules 24, 1085. https://doi.org/10.3390/molecules24061085
Grover, M., Behl, T., Virmani, T., 2021. Phytochemical screening, antioxidant assay and cytotoxic profile for different extracts of Chrysopogon zizanioides roots. Chem Biodivers 18. https://doi.org/10.1002/cbdv.202100012
Guillen Quispe, Y., Hwang, S., Wang, Z., Zuo, G., Lim, S., 2017. Screening in vitro targets related to diabetes in herbal extracts from Peru: Identification of active compounds in Hypericum laricifolium juss. by Offline High-Performance Liquid Chromatography. Int J Mol Sci 18, 2512. https://doi.org/10.3390/ijms18122512
Gulcin, İ., Alwasel, S.H., 2023. DPPH radical scavenging assay. Processes 11, 2248. https://doi.org/10.3390/pr11082248
Hosseini, S., Azari, P., Aeinehvand, M., Rothan, H., Djordjevic, I., Martinez-Chapa, S., Madou, M., 2016. Intrant ELISA: A novel approach to fabrication of electrospun fiber mat-assisted biosensor platforms and their integration within standard analytical well plates. Applied Sciences 6, 336. https://doi.org/10.3390/app6110336
Hu, W., Peng, Y., Wei, Y., Yang, Y., 2023. Application of electrochemical impedance spectroscopy to degradation and aging research of lithium-ion batteries. The Journal of Physical Chemistry C 127, 4465–4495. https://doi.org/10.1021/acs.jpcc.3c00033
Hu, W., Zhou, J., Shen, T., Wang, X., 2019. Target-guided isolation of three main antioxidants from Mahonia bealei (Fort.) Carr. leaves using HSCCC. Molecules 24, 1907. https://doi.org/10.3390/molecules24101907
Ionita, P., Dinoiu, V., Munteanu, C., Turcu, I.M., Tecuceanu, V., Zaharescu, T., Oprea, E., Ilie, C., Anghel, D., Ionita, G., 2015. Antioxidant activity of rosemary extracts in solution and embedded in polymeric systems. Chemical Papers 69. https://doi.org/10.1515/chempap-2015-0024
Jalili Safaryan, M., Ganjloo, A., Bimakr, M., Zarringhalami, S., 2016. Optimization of ultrasound-assisted extraction, preliminary characterization and in vitro antioxidant activity of polysaccharides from green pea pods. Foods 5, 78. https://doi.org/10.3390/foods5040078
Kim, H.H., Jeong, S.H., Park, M.Y., Bhosale, P.B., Abusaliya, A., Kim, H.W., Seong, J.K., Ahn, M., Park, K. Il, Kim, G.S., 2023. Antioxidant effects of polyphenolic compounds in through the distillation of Lonicera japonica & Chenpi Extract and anti-inflammation on skin keratinocyte. https://doi.org/10.21203/rs.3.rs-3198087/v1
Koc, T.B., Savan, E.K., Karabulut, I., 2022. Determination of antioxidant capacity and total phenolic and ascorbic acid contents of some fruits and vegetables with an electrochemical approach. https://doi.org/10.21203/rs.3.rs-1654076/v2
Lee, K.J., Oh, Y.C., Cho, W.K., Ma, J.Y., 2015. Antioxidant and anti-inflammatory activity determination of one hundred kinds of pure chemical compounds using offline and online screening HPLC assay. Evidence-Based Complementary and Alternative Medicine 2015, 1–13. https://doi.org/10.1155/2015/165457
Li, X., Fan, X., Li, Z., Shi, L., Liu, J., Luo, H., Wang, L., Du, X., Chen, W., Guo, J., Li, C., Liu, S., 2022. Application of microfluidics in drug development from traditional medicine. Biosensors (Basel) 12, 870. https://doi.org/10.3390/bios12100870
Liu, C., Lei, Y., Liu, Y., Guo, J., Chen, X., Tang, Y., Dang, J., Wu, M., 2023. An integrated strategy for investigating antioxidants from Ribes himalense Royle ex Decne and their potential target proteins. Antioxidants 12, 835. https://doi.org/10.3390/antiox12040835
Liu, S. C., Yoo, P. B., Garg, N., Lee, A. P., Rasheed, S., 2021. A microfluidic device for blood plasma separation and fluorescence detection of biomarkers using acoustic microstreaming. Sensors and Actuators A: Physical 317, 112482.
Moon, J.-K., Shibamoto, T., 2009. Antioxidant assays for plant and food components. J Agric Food Chem 57, 1655–1666. https://doi.org/10.1021/jf803537k
Munishamappa, V., Sankar, S., Kumar, V., 2017. Evaluation of antioxidant activity of ormeloxifene: in vitro study. Int J Basic Clin Pharmacol 6, 2507–2509. https://doi.org/10.18203/2319-2003.ijbcp20174385
Munteanu, I.G., Apetrei, C., 2021. Analytical methods used in determining antioxidant activity: a review. Int J Mol Sci 22, 3380. https://doi.org/10.3390/ijms22073380
Ngueumaleu, Y., Deutchoua, A.D.D., Hanga, S.S.P., Liendji, R.W., Dedzo, G.K., Ngameni, E., 2023. Probing the reactivity of 2,2-diphenyl-1-picrylhydrazyl (DPPH) with metal cations and acids in acetonitrile by electrochemistry and UV-Vis spectroscopy. Physical Chemistry Chemical Physics 25, 5282–5290. https://doi.org/10.1039/D2CP05296B
Nguyen, M.P., Meredith, N.A., Kelly, S.P., Henry, C.S., 2018. Design considerations for reducing sample loss in microfluidic paper-based analytical devices. Anal Chim Acta 1017, 20–25. https://doi.org/10.1016/j.aca.2018.01.036
Oliveira, G.K.F., Tormin, T.F., Sousa, R.M.F., de Oliveira, A., de Morais, S.A.L., Richter, E.M., Munoz, R.A.A., 2016. Batch-injection analysis with amperometric detection of the DPPH radical for evaluation of antioxidant capacity. Food Chem 192, 691–697. https://doi.org/10.1016/j.foodchem.2015.07.064
Pedan, V., Fischer, N., Rohn, S., 2016. An online NP-HPLC-DPPH method for the determination of the antioxidant activity of condensed polyphenols in cocoa. Food Research International 89, 890–900. https://doi.org/10.1016/j.foodres.2015.10.030
Phonchai, A., Kim, Y., Chantiwas, R., Cho, Y.-K., 2016. Lab-on-a-disc for simultaneous determination of total phenolic content and antioxidant activity of beverage samples. Lab Chip 16, 3268–3275. https://doi.org/10.1039/C6LC00646A
Quanson, J.L., Stander, M.A., Pretorius, E., Jenkinson, C., Taylor, A.E., Storbeck, K.-H., 2016. High-throughput analysis of 19 endogenous androgenic steroids by ultra-performance convergence chromatography tandem mass spectrometry. Journal of Chromatography B 1031, 131–138. https://doi.org/10.1016/j.jchromb.2016.07.024
Rajesh, R., 2021. Phytochemical screening, HPTLC finger print and in vitro antioxidant activity of bark extracts of Lannea coromandelica (Houtt.) Merr. Indian Journal of Pharmaceutical Education and Research 55, 498–506. https://doi.org/10.5530/ijper.55.2.88
Ruiz-Rivera, A., Chin, K.-W., Soh, S., Raad, R., 2015. On the performance of online and offline green path establishment techniques. EURASIP J Wirel Commun Netw 2015, 193. https://doi.org/10.1186/s13638-015-0417-z
Sayad, A.A., Ibrahim, F., Uddin, S.M., Pei, K.X., Mohktar, M.S., Madou, M., Thong, K.L., 2016. A microfluidic lab-on-a-disc integrated loop mediated isothermal amplification for foodborne pathogen detection. Sens Actuators B Chem 227, 600–609. https://doi.org/10.1016/j.snb.2015.10.116
Siddartha, A.G., Gupta, V.K., 2020. Green synthesis of MgO nanoparticles prepared by Ficus religiosa and monitoring of their antimicrobial activity against Pseudomonas aeruginosa. Solid State Technol 63.
Singh, A.K., Kumar, V., Singh, K., Fatima, S., 2023. RSM and fuzzy modeling approach for optimization of extraction of antioxidant compounds from Asparagus racemosus roots. Food Bioengineering 2, 75–86. https://doi.org/10.1002/fbe2.12040
Sirivibulkovit, K., Nouanthavong, S., Sameenoi, Y., 2018. Paper-based DPPH assay for antioxidant activity analysis. Analytical Sciences 34, 795–800. https://doi.org/10.2116/analsci.18P014
Soum, V., Park, S., Brilian, A.I., Kwon, O.-S., Shin, K., 2019. Programmable paper-based microfluidic devices for biomarker detections. Micromachines (Basel) 10, 516. https://doi.org/10.3390/mi10080516
Tay, U.J., Goh, M., Hui, J.C.W., Arumugam, P., 2022. Floating sphere assay: a rapid qualitative method for microvolume analysis of gelation. https://doi.org/10.1101/2022.03.21.485096
Valle, D.L., Puzon, J.J.M., Cabrera, E.C., Rivera, W.L., 2016. Thin Layer Chromatography‐bioautography and gas chromatography‐mass spectrometry of antimicrobial leaf extracts from Philippine Piper betle L. against multidrug‐resistant bacteria. Evidence-Based Complementary and Alternative Medicine 2016. https://doi.org/10.1155/2016/4976791
Viet, T.D., Xuan, T.D., Anh, L.H., 2021. α-amyrin and β-amyrin isolated from Celastrus hindsii leaves and their antioxidant, anti-xanthine oxidase, and anti-tyrosinase potentials. Molecules 26, 7248. https://doi.org/10.3390/molecules26237248
Wang, D., Du, N., Wen, L., Zhu, H., Liu, F., Wang, X., Du, J., Li, S., 2017. An efficient method for the preparative isolation and purification of flavonoid glycosides and caffeoylquinic acid derivatives from leaves of Lonicera japonica Thunb. Using High Speed Counter-Current Chromatography (HSCCC) and Prep-HPLC Guided by DPPH-HPL. Molecules 22, 229. https://doi.org/10.3390/molecules22020229
Ward, K., Fan, Z.H., 2015. Mixing in microfluidic devices and enhancement methods. Journal of Micromechanics and Microengineering 25, 094001. https://doi.org/10.1088/0960-1317/25/9/094001
Xie, J., Schaich, K.M., 2014. Re-evaluation of the 2,2-diphenyl-1-picrylhydrazyl free radical (DPPH) assay for antioxidant activity. J Agric Food Chem 62, 4251–4260. https://doi.org/10.1021/jf500180u
Xu, B., Guo, Jiuchuan, Fu, Y., Chen, X., Guo, Jinhong, 2020. A review on microfluidics in the detection of food pesticide residues. Electrophoresis 41, 821–832. https://doi.org/10.1002/elps.201900209
Yang, S., Berdine, G., 2023. Interpretable artificial intelligence (AI) – saliency maps. The Southwest Respiratory and Critical Care Chronicles 11, 31–37. https://doi.org/10.12746/swrccc.v11i48.1209
Yoon, Y., Kim, S., Lee, J., Choi, J., Kim, R.-K., Lee, S.-J., Sul, O., Lee, S.-B., 2016. Clogging-free microfluidics for continuous size-based separation of microparticles. Sci Rep 6, 26531. https://doi.org/10.1038/srep26531
Zhang, D., Xiao, D., Yin, T., Zhao, S., Zhur, O., Xiao, X., He, H., Chen, L., 2024. The extraction of effective components and an antioxidant activity study of Tulipa edulis. Food Science and Human Wellness 13, 276–286. https://doi.org/10.26599/FSHW.2022.9250023
Ziyatdinova, G., Kalmykova, A., 2023. Electrochemical characterization of the antioxidant properties of medicinal plants and products: A review. Molecules 28, 2308. https://doi.org/10.3390/molecules28052308
Zuo, G., Kim, H.-Y., Guillen Quispe, Y.N., Wang, Z., Kim, K.-H., Gonzales Arce, P.H., Lim, S.-S., 2021. Valeriana rigida Ruiz & Pav. root extract: A new source of caffeoylquinic acids with antioxidant and aldose reductase inhibitory activities. Foods 10, 1079. https://doi.org/10.3390/foods10051079
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