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Food Chemistry 113 (2009) 12021205

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

DPPH antioxidant assay revisited


Om P. Sharma *, Tej K. Bhat
Biochemistry Laboratory, Indian Veterinary Research Institute, Regional Station, Palampur, Himachal Pradesh 176 061, India

a r t i c l e i n f o a b s t r a c t

Article history: Scavenging of DPPH free radical is the basis of a common antioxidant assay. A number of protocols have
Received 25 February 2008 been followed for this assay resulting in variation in the results of different laboratories. We present a
Received in revised form 22 June 2008 perspective of the protocols followed by different workers with incongruity in their results and recom-
Accepted 2 August 2008
mend a standard procedure within the sensitivity range of spectrophotometry. Three common standard
antioxidants viz. ascorbic acid, BHT and propyl gallate have been used in this study. The IC50 values for
ascorbic acid and propyl gallate were 11.8 lM and 4.4 lM in methanol and 11.5 lM and 4.7 lM in buf-
Keywords:
fered methanol as reaction medium, respectively. The free radical scavenging by BHT was markedly inu-
DPPH
1,1-Diphenyl-2-picryl-hydrazyl
enced by the reaction medium. The IC50 values were 60.0 lM and 9.7 lM when the reaction was done in
Antioxidant assay methanol and buffered methanol, respectively.
Free radical scavenger 2008 Elsevier Ltd. All rights reserved.

1. Introduction out for developing a standard protocol within the sensitivity range
of spectrophotometric assays (Ayres, 1949; Sloane & William,
Antioxidants are considered important nutraceuticals on 1977). In addition, the free radical scavenging kinetics for three
account of many health benets (Droge, 2002; Lee, Koo, & Min, standard antioxidants viz. ascorbic acid, BHT and propyl gallate
2004; Valko et al., 2007). The requirement of a standard assay is was investigated.
very important in order to compare the results of different
laboratories and validation of the conclusions. 1,1-Diphenyl-2-pic- 2. Materials and methods
ryl-hydrazyl (DPPH) is a stable free radical which has an unpaired
valence electron at one atom of nitrogen bridge (Eklund et al., 1,1-Diphenyl-2-picryl-hydrazyl (DPPH) was purchased from
2005). Scavenging of DPPH radical is the basis of the popular DPPH SigmaAldrich (USA). Ascorbic acid and butylated hydroxytoluene
antioxidant assay (Alma, Mavi, Yildirim, Digrak, & Hirata, 2003; (BHT) were purchased from E. Merck (India). Propyl gallate was
Karioti, Hadjipavlou-Litina, Mensah, Fleischer, & Skaltsa, 2004; purchased from Sisco Research Laboratories, Mumbai, India. Stock
Kordali, Cakir, Mavi, Kilic, & Yildirim, 2005). A perusal of the pub- solutions of DPPH were prepared in methanol, and methanol buf-
lications in the recent past (Table 1) shows that various research fered with acetic acid buffer (0.1 M, pH 5.5), respectively. Buffered
groups have used widely different protocols which differed in the methanol was prepared by mixing 40 ml of 0.1 M acetate buffer
concentration of DPPH (22.5250 lM), incubation time (5 min (pH 5.5) with 60 ml methanol. The solvents and other chemicals
1 h), reaction solvent and pH of the reaction mixture. High concen- were of analytical grade. The reaction tubes, in triplicates, were
trations of DPPH in the reaction mixture give absorbance beyond wrapped in aluminum foil and kept at 30 C for 30 min in dark.
the accuracy of spectrophotometric measurements (Ayres, 1949; All measurements were done under dim light. Spectrophotometric
Sloane & William, 1977). As a result of these differences in reaction measurements were done at 517 nm using Spectronic Genesys 5
conditions, the IC50 values for even the standard antioxidants like spectrophotometer. The data are mean SD.
ascorbic acid and butylated hydroxytoluene (BHT) vary a lot (Table
1). Thus, it is not possible to compare the results of different labo-
3. Results and discussion
ratories (Kano, Takayanagi, Harada, Makino, & Ishikawa, 2005;
Ricci et al., 2005). Light, oxygen and pH of the reaction mixture also
The absorbance proles of DPPH in methanol, ethanol and buf-
affect the absorbance of DPPH (Ozcelik, Lee, & Min, 2003). The
fered methanol are shown in Fig. 1. The order of absorbance was
present investigation on the DPPH antioxidant assay was carried
highest in buffered methanolic solution, followed by methanolic
and ethanolic solutions. Higher absorbance in methanolic solutions
implies better sensitivity vis--vis ethanolic solution of DPPH. The
* Corresponding author. Tel.: +91 1894 230526; fax: +91 1894 233063.
range of accuracy for spectrophotometric measurements falls
E-mail address: omsharma53@yahoo.com (O.P. Sharma). within an absorbance of 0.2210.698 which equals a transmittance

0308-8146/$ - see front matter 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2008.08.008
O.P. Sharma, T.K. Bhat / Food Chemistry 113 (2009) 12021205 1203

Table 1
Summary of some representative publications DPPH using antioxidant assay

References
DPPH (lM)
22.5 Mimica-Dukic et al. (2004)
50.0 Karioti et al. (2004)
80.0 Eklund et al. (2005)
100.0 Chen et al. (2005, 2007), Govindarajan et al. (2003), Kano et al. (2005), Saito et al. (2004), and Tepe et al. (2005)
250.0 Alma et al. (2003) and Kim et al. (2004)
Reaction medium
Ethanol Kano et al. (2005), Karioti et al. (2004), and Mimura et al. (2005)
Methanol Alma et al. (2003), Eklund et al. (2005), Govindarajan et al. (2003), and Mimica-Dukic et al. (2004)
Methanol buffered (pH 5.5) Chen et al. (2005)
Ethanol buffered (pH 3.0) Takebayashi, Tai, Gohda, and Yamamoto (2006)
Incubation time (min)
5 Govindarajan et al. (2003)
20 Karioti et al. (2004), Mimura, Yazaki, Sawaki, Ozawa, and Kawaguchi (2005)
30 Alma et al. (2003) and Kim et al. (2004)
60 Karioti et al. (2004) and Mimica-Dukic et al. (2004)
IC50 of ascorbic acid
9.84 (56 lM) Kano et al. (2005)
110.77 (629 lM) Ricci et al. (2005)
IC50 of BHT
5.4 (25 lM) Mimica-Dukic et al. (2004)
19.8 (90 lM) Sokmen et al. (2004)
86.6 (393 lM) Ricci et al. (2005)

2.5 DPPH solution in methanol 100


in methanol
DPPH solution in ethanol
2.0 DPPH solution in in buffered methanol
75
buffered methanol
Inhibition (%)

1.5
A517

50
1.0

25
0.5

0.0 0
0 100 200 300 0 10 20 30
DPPH (M) Ascorbic acid (M)

Fig. 1. Absorbance at 517 nm of DPPH solutions prepared in methanol, ethanol and Fig. 2. Scavenging of DPPH radical by ascorbic acid. Ascorbic acid solution (1 mM)
buffered methanol. was prepared in methanol or buffered methanol. Different aliquots were taken and
the volume was made to 3.0 ml with methanol or buffered methanol. The reaction
was started by addition of 1.0 ml solution of 200 lM DPPH solution in methanol or
buffered methanol. The reaction mixture was kept at 30 C for 30 min and the
of 2060% (Ayres, 1949). This corresponds to the DPPH concentra- absorbance was measured at 517 nm.
tion of nearly 2570 lM (Fig. 1). A number of workers have used
DPPH concentrations far beyond the spectrophotometric accuracy,
even up to 250 lM (Table 1). We used a DPPH concentration of methanol as solvents. However, the IC50 values were fairly low as
50 lM, in consonance with the requirements of the accuracy of compared to those reported by earlier workers as 56 lM (Kano
spectrophotomteric measurements (Ayres, 1949; Sloane & et al., 2005), and 629 lM (Ricci et al., 2005). The DPPH radical scav-
William, 1977). The absorbance of DPPH without any additions enging prole of BHT is shown in Fig. 3. The IC50 value was 60.0 lM
was stable over 30 min. In addition, the suitable solvent for the and 9.7 lM when the reaction was done in methanol and buffered
DPPH assay was methanol or buffered methanol for the assay of methanol, respectively. The earlier reports of IC50 of BHT are 25 lM
antioxidant activity of non-polar/less polar and polar com- (Mimica-Dukic et al., 2004), 90 lM (Mimica-Dukic et al., 2004;
pounds/extracts, respectively. Further, we determined the DPPH Sokmen et al., 2004) and 393 lM (Ricci et al., 2005). The DPPH
radical scavenging activity of ascorbic acid, BHT and propyl gallate scavenging prole of propyl gallate is shown in Fig. 4. The IC50 val-
(Figs. 24), three common antioxidants used as standards for com- ues were 4.4 lM and 4.7 lM in methanol and buffered methanol as
paring the antioxidant potential (Kano et al., 2005; Mimica-Dukic, reaction medium, respectively. DPPH scavenging activity of phen-
Bozin, Sokovic, & Simin, 2004; Ricci et al., 2005; Soares, Andreazza, olics is positively correlated with the number of hydroxyl groups
& Salvador, 2003; Sokmen et al., 2004). The IC50 for ascorbic acid (Sroka & Cisowski, 2003). This observation explains the relative
was 11.8 lM and 11.5 lM when the reaction was done in metha- IC50 values of BHT and propyl gallate. Propyl gallate with three hy-
nol and buffered methanol, respectively. The radical scavenging droxyl groups has lower IC50 values as compared to BHT with one
prole of ascorbic acid was comparable in methanol and buffered hydroxyl group. Unlike ascorbic acid and propyl gallate, the DPPH
1204 O.P. Sharma, T.K. Bhat / Food Chemistry 113 (2009) 12021205

100 in methanol DPPH (50 M) in buffered methanol without


any addition
in buffered methanol
DPPH (50 M) in buffered methanol in the presence
75 of ascorbic acid
Inhibition (%)

DPPH (50 M) in buffered methanol in the presence


of BHT
50
DPPH (50 M) in buffered methanol in the presence
0.75 of propyl gallate

25

0.50
0

A517
0 25 50 75 100
BHT (M)
0.25
Fig. 3. Scavenging of DPPH radical by BHT. BHT solution (1 mM) was prepared in
methanol or buffered methanol. Different aliquots were taken and the volume was
made to 3.0 ml with methanol or buffered methanol. The reaction was started by
addition of 1.0 ml solution of 200 lM DPPH solution in methanol or buffered
methanol. The reaction mixture was kept at 30 C for 30 min and the absorbance 0.00
was measured at 517 nm. 0 25 50 75 100
Time (min)

100 in methanol Fig. 5. Time course of scavenging of DPPH free radical by ascorbic acid, BHT and
propyl gallate. The reaction mixture (4.0 ml) in buffered methanol contained DPPH
in buffered methanol (50 lM) alone or in the presence of ascorbic acid (11.5 lM), BHT (9.7 lM) or propyl
gallate (4.7 lM). The absorbance was measured at 517 nm.
75
Inhibition (%)

on scavenging of DPPH radical at a DPPH concentration of 50 lM


50
in methanol or buffered methanol, depending upon the solubility
of the compound under investigation, is recommended. All opera-
tions must be done in dark or dim light (Ozcelik et al., 2003). The
25
extent of inhibition is inuenced by the solvent. This is evident
from the data for DPPH radical scavenging by BHT in methanolic
or buffered methanolic solution (Fig. 3). Besides, our data on the
0 comparative reaction of ascorbic acid, BHT and propyl gallate
0.0 2.5 5.0 7.5 10.0 (Fig. 5) indicates that the time course of inhibition also has to be
Propyl gallate (M) determined. The protocol described here, thus, takes care of the
Fig. 4. Scavenging of DPPH radical by propyl gallate. Propyl gallate solution spectrophotometric sensitivity range, besides sensitivity of DPPH
(100 lM) was prepared in methanol or buffered methanol. Different aliquots were to light, pH and solubility of the compound.
taken and the volume was made to 3.0 ml with methanol or buffered methanol. The
reaction was started by addition of 1.0 ml solution of 200 lM DPPH solution in Acknowledgement
methanol or buffered methanol. The reaction mixture was kept at 30 C for 30 min
and the absorbance was measured at 517 nm.
We thank Ms Jyoti Dhar, Technical Assistant, for excellent tech-
nical support in these studies.
radical scavenging activity of BHT was markedly high in buffered
methanol as compared to that in methanol alone (Fig. 3). DPPH References
radical scavenging activity is inuenced by the polarity of the reac-
Alma, M. H., Mavi, A., Yildirim, A., Digrak, M., & Hirata, T. (2003). Screening chemical
tion medium, chemical structure of the radical scavenger, and the composition and in vitro antioxidant and antimicrobial activities of the essential
pH of the reaction mixture ( Saito, Okamoto, & Kawabata, 2004; oils from Origanum syriacum L. growing in Turkey. Biological and Pharmaceutical
Shizuka & Jun Kawabata, 2005; Ozcelik et al., 2003). The difference Bulletin, 26, 17251729.
Ayres, G. H. (1949). Evaluation of accuracy in photometric analysis. Analytical
in the IC50 values of BHT in methanol and buffered methanol could
Chemistry, 21, 652657.
be due to one or more of these factors. Chen, Y. C., Sugiyama, Y., Abe, N., Kuruto-Nima, R., Nozawa, R., & Hirota, A. (2005).
The radical scavenging reaction of ascorbic acid with DPPH was, DPPH radical scavenging compounds from Dou-Chi, a soybean fermented food.
essentially, instantaneous (Fig. 5). On the other hand, the radical Bioscience Biotechnology and Biochemistry, 69, 9991006.
Chien, P. J., Sheu, F., Huang, W. T., & Su, M. S. (2007). Effect of molecular weight of
scavenging reaction of BHT with DPPH was rather slow and the chitosans on their antioxidative activities in apple juice. Food Chemistry, 102,
absorbance continued to decrease till a period of 90 min of obser- 11921198.
vation (Fig. 5). The reaction of DPPH with propyl gallate was also Droge, W. (2002). Free radicals in the physiological control of cell function.
Physiological Reviews, 82, 4795.
quite fast but slower as compared to that with ascorbic acid Eklund, P. C., Langvik, O. K., Warna, J. P., Salmi, T. O., Willfor, S. M., & Sjoholm, R. E.
(Fig. 5). For the sake of uniformity, a time interval of 30 min was (2005). Chemical studies on antioxidant mechanisms and free radical
taken for ascorbic acid, BHT and propyl gallate radical scavenging scavenging properties of lignans. Organic and Bimolecular Chemistry, 21,
33363347.
capacity measurements. It is important to do a time course of rad- Govindarajan, R., Rastogi, S., Vijayakumar, M., Shirwaikar, A., Rawat, A. K. S.,
ical scavenging activity while using DPPH radical for the assay of Mehrotra, S., et al. (2003). Studies on the antioxidant activities of Desmodium
antioxidant activity. In conclusion, the antioxidant assay based gangeticum. Biological and Pharmaceutical Bulletin, 26, 14241427.
O.P. Sharma, T.K. Bhat / Food Chemistry 113 (2009) 12021205 1205

Kano, M., Takayanagi, T., Harada, K., Makino, K., & Ishikawa, F. (2005). Antioxidative Saito, S., Okamoto, Y., & Kawabata, J. (2004). Effect of alcoholic solvents on
activity of anthocyanins from purple sweet potato Ipomoera batatas cultivar antiradical abilities of protocatechuic acid and its alkyl esters. Bioscience
Ayamurasaki. Bioscience Biotechnology and Biochemistry, 69, 979988. Biotechnology and Biochemistry, 68, 12211227.
Karioti, A., Hadjipavlou-Litina, D., Mensah, M. L., Fleischer, T. C., & Skaltsa, H. (2004). Shizuka, S., & Jun Kawabata, J. (2005). Effects of electron-withdrawing substituents
Composition and antioxidant activity of the essential oils of Xylopia aethiopica on DPPH radical scavenging reactions of protocatechuic acid and its analogues
(Dun) A. Rich. (Annonaceae) leaves, stem bark, root bark, and fresh and dried in alcoholic solvents. Tetrahedron, 61, 81018108.
fruits, growing in Ghana. Journal of Agricultural and Food Chemistry, 52, Sloane, H. J., & William, S. G. (1977). Spectrophotometric accuracy, linearity and
80948098. adherence to Beers law. Applied Spectroscopy, 31, 2530.
Kim, H. J., Chen, F., Wu, C., Wang, X., Chung, H. Y., & Jin, Z. (2004). Evaluation of Soares, D. G., Andreazza, A. C., & Salvador, M. (2003). Sequestering ability of
antioxidant activity of Australian tea tree (Melaleuca alternifolia) oil and its butylated hydroxytoluene, propyl gallate, resveratrol, and vitamins C and E
components. Journal of Agricultural and Food Chemistry, 52, 28492854. against ABTS, DPPH, and hydroxyl free radicals in chemical and biological
Kordali, S., Cakir, A., Mavi, A., Kilic, H., & Yildirim, A. (2005). Screening of chemical systems. Journal of Agricultural and Food Chemistry, 51, 10771080.
composition and antifungal and antioxidant activities of the essential oils from Sokmen, M., Serkedjieva, J., Daferera, D., Gulluce, M., Polissiou, M., Tepe, B., et al.
three Turkish Artemisia species. Journal of Agricultural and Food Chemistry, 53, (2004). In vitro antioxidant, antimicrobial, and antiviral activities of the
14081416. essential oil and various extracts from herbal parts and callus cultures of
Lee, J., Koo, N., & Min, D. B. (2004). Reactive oxygen species, aging, and antioxidative Origanum acutidens. Journal of Agricultural and Food Chemistry, 52, 33093312.
nutraceuticals. Comprehensive Reviews in Food Science and Food Safety, 3, 2133. Sroka, Z., & Cisowski, W. (2003). Hydrogen peroxide scavenging, antioxidant and
Mimica-Dukic, N., Bozin, B., Sokovic, M., & Simin, N. (2004). Antimicrobial and antiradical activity of some phenolic acids. Food and Chemical Toxicology, 41,
antioxidant activities of Melissa ofcinalis L. (Lamiaceae) essential oil. Journal of 753758.
Agricultural and Food Chemistry, 52, 24852489. Takebayashi, J., Tai, A., Gohda, E., & Yamamoto, I. (2006). Characterization of the
Mimura, T., Yazaki, K., Sawaki, K., Ozawa, T., & Kawaguchi, M. (2005). Hydroxyl radical-scavenging reaction of 2-O-substituted ascorbic acid derivatives, AA-2G,
radical scavenging effects of guaiacol used in traditional dental pulp sedation: AA-2P, and AA-2S: A kinetic and stoichiometric study. Biological and
Reaction kinetic study. Biomedical Research (Tokyo), 26, 139145. Pharmaceutical Bulletin, 29, 766771.
Ozcelik, B., Lee, J. H., & Min, D. B. (2003). Effects of light, oxygen, and pH on the Tepe, B., Sokmen, M., Akpulat, H. A., & Sokmen, A. (2005). In vitro antioxidant
absorbance of 2,2-diphenyl-1-picrylhydrazyl. Journal of Food Science, 68, activities of the methanol extracts of four Helichrysum species from Turkey.
487490. Food Chemistry, 90, 685689.
Ricci, D., Fraternale, D., Giamperi, L., Bucchini, A., Epifano, F., Burini, G., et al. (2005). Valko, M., Leibfritz, D., Moncol, J., Cronin, M. T., Mazur, M., & Telser, J. (2007). Free
Chemical composition, antimicrobial and antioxidant activity of the essential oil radicals and antioxidants in normal physiological functions and human disease.
of Teucrium marum (Lamiaceae). Journal of Ethnopharmacology, 98, 195200. International Journal of Biochemistry and Cell Biology, 39, 4484.

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