DPPH
(Synonyms: 2,2-联苯基-1-苦基肼基,2,2-Diphenyl-1-Picrylhydrazyl DPPH radical) 目录号 : GC19475
A colorimetric probe for free radical scavengers
Cas No.:1898-66-4
Sample solution is provided at 25 µL, 10mM.
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DPPH assay [1]
The DPPH free radical scavenging activities of the extracts were analyzed. DPPH solution (40 μg mL-1) was prepared in methanol. To each well on a microtiter plate was added 20 μL of sample (in triplicate) of appropriate concentration (1000, 500, 100, 50, and 10 μg mL-1) and 180 μL of DPPH solution. As a negative control, 20 μL of methanol was used instead of a sample. BHA, BHT, and ascorbic acid (AA) were used as positive controls. The reaction plates were kept in the dark at 37℃ for 30 minutes, after which the absorbance was measured at 517 nm using Multiskan Sky Thermo Scientific Microtiter plate reader. The inhibition of DPPH radicals in the test sample was calculated using the following formula and expressed as a percentage (%):
Where Ab is the absorbance of negative control (without sample) and As is the absorbance of the sample at different concentration and the positive controls, as well. The results are presented as the mean of percentage of DPPH radicals inhibition ± standard error.
This protocol only provides a guideline, and should be modified according to your specific needs
References:
[1]. Mandić M R, Oalđe M M, Lunić T M, et al. Chemical characterization and in vitro immunomodulatory effects of different extracts of moss Hedwigia ciliata (Hedw.) P. Beauv. from the Vršačke Planine Mts., Serbia[J]. PloS one, 2021, 16(2): e0246810.
DPPH (1,1-diphenyl2-picrylhydrazyl) is a stable free radical because of its spare electron delocalization over the whole molecule. The DPPH assay, which is one of the best-known, frequently employed, and accurate methods. The delocalization causes a deep violet color with 竹max around 520 nm. When a solution of DPPH is mixed with a substrate acting as a hydrogen atom donor, a stable nonradical form of DPPH is obtained with simultaneous change of the violet color to pale yellow[1].
DPPH assay has been successfully utilized for investigating antioxidant properties of wheat grain and bran, vegetables, conjugated linoleic acids, herbs, edible seed oils, and flours in several different solvent systems including ethanol, aqueous acetone, methanol, aqueous alcohol and benzene [2,3]. DPPH assay is a convenient method for the antioxidant assay of cysteine, glutathione, ascorbic acid, tocopherol and polyhydroxy aromatic compounds [4], for olive oil, fruits, juices and wines [5].
DPPH（1,1-二苯基-2-丙酰肼基）是一种稳定的自由基，因为它在整个分子上具有多余的电子离域作用。DPPH测定法是最著名、常用且准确的方法之一。离域导致深紫色竹最大约520纳米。当DPPH的溶液与充当氢原子供体的底物混合时，获得稳定的非自由基形式的DPPH，同时紫色变为浅黄色[1]。
DPPH测定法已成功用于研究小麦颗粒和麸皮、蔬菜、共轭亚油酸、草药、食用籽油和面粉在几种不同溶剂体系中的抗氧化性能，包括乙醇、丙酮水溶液、甲醇、乙醇水溶液和苯[2，3]。DPPH测定法是对橄榄油、水果、果汁和葡萄酒中半胱氨酸、谷胱甘肽、抗坏血酸、生育酚和多羟基芳香族化合物[4]进行抗氧化测定的一种方便方法[5]。
References:
[1]. Szabo M, Idi?oiu C, Chambre D, et al. Improved DPPH determination for antioxidant activity spectrophotometric assay[J]. Chemical Papers, 2007, 61(3): 214-216.
[2]. Yu L. Free radical scavenging properties of conjugated linoleic acids[J]. Journal of Agricultural and Food Chemistry, 2001, 49(7): 3452-3456.
[3]. Parry J, Su L, Luther M, et al. Fatty acid composition and antioxidant properties of cold-pressed marionberry, boysenberry, red raspberry, and blueberry seed oils[J]. Journal of agricultural and food chemistry, 2005, 53(3): 566-573.
[4]. Nishizawa M, Kohno M, Nishimura M, et al. Non-reductive scavenging of 1, 1-diphenyl-2-picrylhydrazyl (DPPH) by peroxyradical: a useful method for quantitative analysis of peroxyradical[J]. Chemical and Pharmaceutical bulletin, 2005, 53(6): 714-716.
[5]. S芍nchez-Moreno C. Methods used to evaluate the free radical scavenging activity in foods and biological systems[J]. Food science and technology international, 2002, 8(3): 121-137.
| Cas No. | 1898-66-4 | SDF | |
| 别名 | 2,2-联苯基-1-苦基肼基,2,2-Diphenyl-1-Picrylhydrazyl DPPH radical | ||
| 化学名 | 2,2-diphenyl-1-(2,4,6-trinitrophenyl)-hydrazinyl | ||
| Canonical SMILES | O=[N+](C1=C(C([N+]([O-])=O)=CC([N+]([O-])=O)=C1)[N]N(C2=CC=CC=C2)C3=CC=CC=C3)[O-] | ||
| 分子式 | C18H12N5O6 | 分子量 | 394.3 |
| 溶解度 | 10mg/mL in DMF, 10mg/mL in Ethanol | 储存条件 | Store at 2-8°C, protect from light |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
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1 mg | 5 mg | 10 mg |
| 1 mM | 2.5361 mL | 12.6807 mL | 25.3614 mL |
| 5 mM | 0.5072 mL | 2.5361 mL | 5.0723 mL |
| 10 mM | 0.2536 mL | 1.2681 mL | 2.5361 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
Paper-based DPPH Assay for Antioxidant Activity Analysis
Anal Sci2018;34(7):795-800.PMID: 29998961DOI: 10.2116/analsci.18P014
We report on a paper-based 2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl (DPPH) assay for a simple, inexpensive, low reagent and sample consumption and high throughput analysis of antioxidant activity. The paper-based device was fabricated using a lamination method to create a 5-mm in diameter circular test zone that was embedded with a DPPH reagent. The analysis was carried out in one-step by dropping an antioxidant/sample onto the test zone. After reduction by the antioxidant, the DPPH radicals become stable DPPH molecules, resulting in a change in color from deep violet to pale yellow. The violet color intensity of DPPH was inversely proportional to the antioxidant activity of the samples, and was measured using imaging software. A high precision and a low limit of detection were found in the analysis of six standard antioxidants including gallic acid, trolox, ascorbic acid, caffeic acid, vanilliic acid and quercetin. The device was then validated against the traditional spectrophotometric DPPH assay by analyzing the antioxidant activity of 7 tea samples. The results showed no significant difference for gallic acid equivalent for all 7 samples obtained from the two methods at the 95% confidence level, indicating that the developed method was reliable for antioxidant activity analysis of real samples. Finally, the paper-based DPPH device was found to be stable over 10 days when stored in a refrigerator (2 - 4～C), making it an easy-to-use device for end-users.
The Chemistry of DPPHﹿFree Radical and Congeners
Int J Mol Sci2021 Feb 3;22(4):1545.PMID: 33546504DOI: 10.3390/ijms22041545
Since the discovery in 1922 of 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl stable free radical (DPPH·), the chemistry of such open-shell compounds has developed continuously, allowing for both theoretical and practical advances in the free radical chemistry area. This review presents the important, general and modern aspects of the chemistry of hydrazyl free radicals and the science behind it.
ORAC and DPPH assay comparison to assess antioxidant capacity of tea infusions: relationship between total polyphenol and individual catechin content
Int J Food Sci Nutr2010 Mar;61(2):109-24.PMID: 20109129DOI: 10.3109/09637480903292601
Commercially available tea infusions are the major source of catechins for preparing bottled tea beverages and tea supplements available in the market today. In the present study, we analyzed five tea infusions to measure the total antioxidant capacity (TAC) by oxygen radical absorbance capacity (ORAC) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity (DRSC) assays, total polyphenol content by the colorimetric method and individual catechin content by high-performance liquid chromatography. Four major tea catechins were also analyzed for their TAC to reveal differential antioxidant behavior of the tea infusions, resulting in the ORAC and DRSC methods. The correlation coefficients between DRSC and the total polyphenol or total catechin content of the tea infusions were 1.0 and 0.99. However, the values fall to 0.73 and 0.69, respectively, while the ORAC activity was correlated with total polyphenol and total catechin content. Determining the TAC of individual tea catechins showed that ORAC of epicatechin was seven-fold higher than that of epigallocatechin gallate; on the contrary, epigallocatechin gallate showed significantly (P < 0.05) stronger DRSC activity than epicatechin. By evaluating the structure-activity relationship, this study further revealed that OH substitution at the 3' position in pyrogallol moieties contributes to the lower ORAC value of epigallocatechin and epigallocatechin gallate comparing with their non-3'-OH counterparts, such as epicatechin and epicatechin gallate, respectively. Also, numbers of OH substitutions were poorly correlated with the observed ORAC value unlike the DRSC. Overall, results of this study enabled us to hypothesize that substances having a lower TAC value in the ORAC assay compared with that in DPPH assays may pertain to a pro-oxidant effect by generating reactive oxygen species in an aqueous buffer, at a physiological pH. We also propose that substances exhibiting lower TAC value in the ORAC assay compared with that in the DPPH assay are powerful pro-oxidants compared with the substances showing a higher TAC value in the ORAC assay than that in the DPPH assay.
Use and Abuse of the DPPH(•) Radical
J Agric Food Chem2015 Oct 14;63(40):8765-76.PMID: 26390267DOI: 10.1021/acs.jafc.5b03839
The 2,2-diphenyl-1-picrylhydrazyl (DPPH(•)) radical is approaching 100 years from its discovery in 1922 by Goldschmidt and Renn. This radical is colored and remarkably stable, two properties that have made it one of the most popular radicals in a wide range of studies. First, there is the evaluation of the antioxidant abilities of phenols and other natural compounds (A-H) through a "test" that-at a closer look-is utterly inappropriate. In fact, the test-derived EC50, that is, the concentration of A-H able to scavenge 50% of the initial DPPH(•), is not a kinetic parameter and hence its purported correlation with the antioxidant properties of chemicals is not justified. Kinetic measurements, such as the second-order rate constants for H-atom abstraction from A-H by DPPH(•), in apolar media, are the only useful parameters to predict the antioxidant ability of A-H. Other applications of DPPH(•) include kinetic and mechanistic studies, kinetic solvent effects, EPR spectroscopy, polymer chemistry, and many more. In this review these applications are evaluated in detail by showing the usefulness of some and the uselessness of others. The chemistry of DPPH(•) is also briefly reviewed.
Methods to Determine Chain-Breaking Antioxidant Activity of Nanomaterials beyond DPPH•. A Review
Antioxidants (Basel)2021 Sep 29;10(10):1551.PMID: 34679687DOI: 10.3390/antiox10101551
This review highlights the progress made in recent years in understanding the mechanism of action of nanomaterials with antioxidant activity and in the chemical methods used to evaluate their activity. Nanomaterials represent one of the most recent frontiers in the research for improved antioxidants, but further development is hampered by a poor characterization of the ''antioxidant activity'' property and by using oversimplified chemical methods. Inhibited autoxidation experiments provide valuable information about the interaction with the most important radicals involved in the lipid oxidation, namely alkylperoxyl and hydroperoxyl radicals, and demonstrate unambiguously the ability to stop the oxidation of organic materials. It is proposed that autoxidation methods should always complement (and possibly replace) the use of assays based on the quenching of stable radicals (such as DPPH• and ABTS•+). The mechanisms leading to the inhibition of the autoxidation (sacrificial and catalytic radical trapping antioxidant activity) are described in the context of nanoantioxidants. Guidelines for the selection of the appropriate testing conditions and of meaningful kinetic analysis are also given.