DPPH Antioksidan Analizinin Yeniden Değerlendirilmesi

Year 2019, Volume: 9 Issue: 2, 125 - 135, 25.12.2019

Serap Ayaz Seyhan

Abstract

Antioksidanların uygulamaları, oksidatif stresin zararlı etkilerini en aza indirmedeki çeşitli rolleri nedeniyle artmaktadır. DPPH· (1,1-Difenil-2-pikrilhidrazil radikali; C18H12N5O6) antioksidan analizi, farklı bileşiklerin antioksidan aktivitesini değerlendirmek için yaygın olarak kullanılan hızlı, basit ve ucuz bir yöntemdir. Ayrıntılı literatür taraması, çeşitli araştırma gruplarının farklı DPPH· derişimlerinde (22,5–400µM), inkübasyon süresinde (5 dk-120 dk), reaksiyon çözücüsünde (etanol, metanol) ve reaksiyon karışım pH'sında (pH= 3 ve 5.5 tampon ortamı) olan oldukça farklı protokoller kullandıklarını göstermektedir. Farklı laboratuvarların sonuçlarının karşılaştırılması ve sonuçların doğrulanması için standart bir analizin gerekliliği çok önemlidir. Bu çalışmada DPPH· antioksidan analizini etkileyen DPPH· derişimi, standart madde derişimi, reaksiyon/inkübasyon süresi gibi önemli faktörler spektrofotometrik yöntem ile incelenerek standart bir DPPH· antioksidan analiz yöntemi oluşturulmuştur. Bu yöntem kapsamında ölçümler, reaksiyon ortamındaki DPPH· derişimi 25-75 µM aralığında, çalkalayıcılı inkübatörde 30oC’de, 30dk’da ve karanlık ortamda gerçekleştirilmelidir.

Keywords

DPPH, Antioksidan, Antioksidan aktivite, Yöntem

Supporting Institution

Marmara Üniversitesi Bilimsel Araştırma Projeleri Birimi

Project Number

SAG-A-131113-0423

Thanks

Bu çalışma, M. Ü. Bilimsel Araştırma Projeleri Birimi tarafından SAG-A-131113-0423 nolu proje ile desteklenmiştir.

Re-Evaluating of The DPPH• Antioxidant Assay

Abstract

Applications of antioxidants are increasing due to
their various roles in minimising harmful effects of oxidative stress. DPPH^·
(1,1-Diphenyl-2-picrylhydrazyl radical) antioxidant assay is a fast, simple,
inexpensive method commonly used to evaluate the antioxidant activity of
different compounds. A detailed literature survey shows that various research
groups have used widely different protocols differed in the concentration of
DPPH^·
(22,5–400µM), incubation time (5 min–120 min), reaction solvent (ethanol,
methanol) and pH of the reaction mixture (pH=3 and 5.5 buffer) . The
requirement of a standard assay is very important in order to compare the
results of different laboratories and validation of the conclusions. In this
study, a standard DPPH• antioxidant analysis procedure was performed by
analyzing the important factors such as DPPH• concentration, standard substance
concentration, reaction / incubation time by spectrophotometric method. Under
this procedure, the measurements should be carried out at DPPH concentration in
the reaction medium in the range of 25-75 µM, 30 ° C in a shaker incubator, 30
minutes and dark medium.

Keywords

DPPH• radical, Antioxidant, Antioxidant activity, Mthod

Project Number

SAG-A-131113-0423

References

• 1 Sellappan, S., Akoh, C.C., Krewer, G. (2002). Phenolic compounds and antioxidant capacity of Georgia-grown blueberries and blackberries. Journal of Agricultural and Food Chemistry, 50 (8): 2432-2438.
• 2 Huang, D., Ou, B., Prior, R.L. (2005). The chemistry behind antioxidant capacity assays. Journal of Agricultural and Food Chemistry, 53 (6): 1841-1856.
• 3 Niki, E. (2010). Assessment of antioxidant capacity in vitro and in vivo. Free Radical Biology & Medicine, 49 (4): 503–515.
• 4 Deng, J., Cheng, W., Yang, G. (2011). A novel antioxidant activity index (AAU) for natural products using the DPPH assay. Food Chemistry, 125(4): 1430–1435.
• 5 Prior, R.L., Wu, X., Scaich, K. (2005). Standardized methods for the determination antioxidant capacity and phenolics in foods and dietary supplements. Journal of Agricultural and Food Chemistry, 53(8): 4290-4302.
• 6 Scherer, R., Godoy, H.T. (2009). Antioxidant activity index (AAI) by the 2,2-diphenyl-1-picrylhydrazyl method. Food Chemistry, 112(3): 654–658.
• 7 Sharma, OP., Bhat, T.K. (2009). DPPH antioxidant assay revisited. Food Chemistry, 113(4): 1202–1205.
• 8 Kedare, S.B., Singh, R.P. (2011). Genesis and development of DPPH method of antioxidant assay. Journal of Food Science and Technology, 48 (4): 412-422.
• 9 Benzie, I.F.F., Strain, J.J. (1999). Ferric reducing/antioxidant power assay: direct measure of total antioxidant activity of biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration. Methods in Enzymology, 299: 15-27.
• 10 Prior, R.L., Cao, G. (1999). In vivo total antioxidant capacity: comparison of different analytical methods. Free Radic Biol Med. 27(11-12):1173-81.
• 11 Çıkla Yılmaz, D., Ayaz Seyhan, S. (2017). Antioxidant potential of Cydonia oblonga Miller leaves. Istanbul J Pharm, 47 (1): 9-12.
• 12 Mishra, K., Ojha, H., Chaudhury, N.K. (2012). Estimation of antiradical properties of antioxidants using DPPH assay: A critical review and results. Food Chemistry, 130 (4): 1036–1043.
• 13 Mimica-Dukic, N., Bozin, B., Sokovic, M., Simin, N. (2004). Antimicrobial and antioxidant activities of Melissa officinalis L. (Lamiaceae) essential oil. Journal of Agricultural and Food Chemistry, 52: 2485–2489.
• 14 Karioti, A., Hadjipavlou-Litina, D., Mensah, M. L., Fleischer, T. C., & Skaltsa, H. (2004). Composition and antioxidant activity of the essential oils of Xylopia aethiopica (Dun) A. Rich. (Annonaceae) leaves, stem bark, root bark, and fresh and dried fruits, growing in Ghana. Journal of Agricultural and Food Chemistry, 52: 8094–8098.
• 15 Budaraju, S., Mallikarjunan, K., Annor, C., Schoenfuss, T., Raun, R. (2018). Effect of pre-treatments on the antioxidant potential of phenolic extracts from barley malt rootlets. Food Chemistry, 266: 31-37.
• 16 Chen, G. L., Chen, S. G., Xiao, Y., Fu, N. L. (2018). Antioxidant capacities and total phenolic contents of 30 flowers. Industrial Crops and Products, 111: 430–445.
• 17 Köse, Ş., Ocak, E. (2018). Antimicrobial and antioxidant properties of sırmo (allium vineale l.), mendi (chaerophyllum macropodum boiss.) and sıyabo (ferula rigidula dc.). The Journal of Food, 43 (2): 294-302.
• 18 Avalos-Llano, K. R., Martín-Belloso, O., Soliva-Fortuny, R. (2018). Effect of pulsed light treatments on quality and antioxidant properties of fresh-cut strawberries. Food Chemistry, 264: 393–400.
• 19 Wang, Q., Rehman, M., Peng, D., Liu, L.(2018). Antioxidant capacity and α-glucosidase inhibitory activity of leaf extracts from ten ramie cultivars. Industrial Crops and Products, 122: 430-437.
• 20 Eklund, P. C., Langvik, O. K., Warna, J. P., Salmi, T. O., Willfor, S. M., & Sjoholm, R. E. (2005). Chemical studies on antioxidant mechanisms and free radical scavenging properties of lignans. Organic and Bimolecular Chemistry, 21: 3336–3347.
• 21 Kano, M., Takayanagi, T., Harada, K., Makino, K., & Ishikawa, F. (2005). Antioxidative activity of anthocyanins from purple sweet potato Ipomoera batatas cultivar Ayamurasaki. Bioscience Biotechnology and Biochemistry, 69: 979–988.
• 22 Tepe, B., Sokmen, M., Akpulat, H. A., & Sokmen, A. (2005). In vitro antioxidant activities of the methanol extracts of four Helichrysum species from Turkey. Food Chemistry, 90: 685–689.
• 23 Kanmaz, E. Ö., Saral, Ö. (2017). The effect of extractıon parameters on antıoxıdant actıvıty of subcrıtıcal water extracts obtaıned from mandarın peel. The Journal of Food, 42 (4): 405-412.
• 24 Türker, İ., İşleroğlu H. (2017). Mahlep püresinin kızılötesi ışınım ile kurutulması işleminde antosiyanin, fenolik madde ve antioksidan kapasite değişim kinetiği. The Journal of Food, 42 (4): 422-430.
• 25 Alma, M. H., Mavi, A., Yildirim, A., Digrak, M., & Hirata, T. (2003). Screening chemical composition and in vitro antioxidant and antimicrobial activities of the essential oils from Origanum syriacum L. growing in Turkey. Biological and Pharmaceutical Bulletin, 26: 1725–1729.
• 26 Kim, H. J., Chen, F., Wu, C., Wang, X., Chung, H. Y., & Jin, Z. (2004). Evaluation of antioxidant activity of Australian tea tree (Melaleuca alternifolia) oil and its components. Journal of Agricultural and Food Chemistry, 52: 2849–2854.
• 27 Sarker, U., Oba, S. (2018). Response of nutrients, minerals, antioxidant leaf pigments, vitamins, polyphenol, flavonoid and antioxidant activity in selected vegetable amaranth under four soil water content. Food Chemistry, 30: 72-83.
• 28 Ko, C. H., Lee, J. Y., Jang, M. G., Song, H., Kim, S. (2018). Seasonal variations in the phenolic compounds and antioxidant activity of Sasa quelpaertensis. Industrial Crops and Products,122: 506-512.
• 29 Mimura, T., Yazaki, K., Sawaki, K., Ozawa, T., Kawaguchi, M. (2005). Hydroxyl radical scavenging effects of guaiacol used in traditional dental pulp sedation: Reaction kinetic study. Biomedical Research, 26: 139–145.
• 30 Da Silva Andrade, L. B., da Silva Julião, M. S., Vera Cruz, R. C., Soares Rodrigues, T. H., dos Santos Fontenelle, R. O., Coelhoda Silva, A. L. (2018). Antioxidant and antifungal activity of carnauba wax powder extracts. Industrial Crops and Products, 125: 220-227.
• 31 Takebayashi, J., Tai, A., Gohda, E., & Yamamoto, I. (2006). Characterization of the radical-scavenging reaction of 2-O-substituted ascorbic acid derivatives, AA-2G, AA-2P, and AA-2S: A kinetic and stoichiometric study. Biological and Pharmaceutical Bulletin, 29: 766–771.
• 32 Chen, Y. C., Sugiyama, Y., Abe, N., Kuruto-Nima, R., Nozawa, R., Hirota, A. (2005). DPPH radical scavenging compounds from Dou-Chi, a soybean fermented food. Bioscience Biotechnology and Biochemistry, 69, 999–1006.
• 33 Govindarajan, R., Rastogi, S., Vijayakumar, M., Shirwaikar, A., Rawat, A. K. S., Mehrotra, S. (2003). Studies on the antioxidant activities of Desmodium gangeticum. Biological and Pharmaceutical Bulletin, 26: 1424–1427.