Free radicals (scavenges DPPH• radicals via hydrogen donation)[1]
As a chemical reagent, DPPH does not have a biological "target" in the context of drug discovery. It acts as a stable, synthetic free radical (radical nitrogen center) that serves as a substrate or probe for measuring the hydrogen-donating or electron-donating capacity of antioxidant compounds .

- Antioxidant activity: DPPH (0.1 mM) in ethanol solution was used to evaluate the free radical scavenging capacity of plant extracts and pure compounds. The reaction was monitored by measuring absorbance at 517 nm, with a decrease in absorbance indicating radical scavenging activity. The method was validated for reproducibility and sensitivity across different sample matrices[1]
Because it has an odd number of electrons, DPPH exhibits a strong absorption band at 517 nm. The solution takes on a dark purple color, and the absorption vanishes as the electron pairs vanish. The decolorization that follows is stoichiometric with respect to the quantity of electrons absorbed. Over the relevant absorption range, alcohol solutions at 0.5 mM exhibit a strong color and follow the Lambert-Beer law [1]. The DPPH assay is a quick, easy, affordable, and popular way to gauge a substance's capacity to donate hydrogen or scavenge free radicals, as well as to evaluate the antioxidant activity of food. Additionally, in complicated biological systems, it can be utilized to quantify antioxidants in liquid or solid sample types. Fruit and vegetable juices can have their total antioxidant capacity and free radical scavenging activity determined using this straightforward and user-friendly approach. The oxidation of wheat grains and bran, vegetables, conjugated linoleic acid, herbs, edible seed oils, and flour in a variety of solvent systems, such as ethanol, aqueous acetone, methanol, hydrous alcohol, and benzene, has all been effectively studied with this assay. characteristics of an antioxidant. Antioxidants include cysteine, glutathione, ascorbic acid, tocopherols, and polyhydroxyaromatic compounds in olive oil, fruits, juices, and wines can be easily measured using this straightforward method [1].

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The DPPH assay is an in vitro chemical method used to assess the radical scavenging ability of antioxidants. In this assay, a DPPH solution is prepared in a solvent (typically methanol or ethanol), and its absorbance is measured at a specific wavelength (515-517 nm). When a sample with antioxidant activity is added, it reduces the DPPH radical, leading to a decrease in absorbance proportional to the sample's concentration. The antioxidant capacity is usually expressed as the half-maximal inhibitory concentration (IC₅₀) or as Trolox equivalent antioxidant capacity (TEAC) .

DPPH is not a therapeutic agent and is not used for in vivo treatment. It is exclusively a laboratory reagent. Its use is strictly limited to in vitro and ex vivo chemical analyses to measure the antioxidant potential of other substances. In vivo studies on living organisms use DPPH only indirectly, to characterize the antioxidant properties of an extract before it is tested in animal models .

DPPH radical scavenging assay: A solution of DPPH (0.1 mM in ethanol) was mixed with sample extracts or compounds. After 30 minutes of incubation in the dark, absorbance was measured at 517 nm. Antioxidant activity was calculated as the percentage decrease in absorbance relative to a control without sample[1]

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The DPPH radical scavenging assay is a standard cell-free, colorimetric protocol .
General Protocol: A working solution of DPPH (e.g., 0.1 mM) is prepared in a suitable organic solvent like methanol .
Reaction: A sample of the test compound or extract is mixed with the DPPH solution. The reaction mixture is incubated in the dark at room temperature for a set period (typically 30-60 minutes) .
Measurement: After incubation, the decrease in absorbance is measured at a wavelength of 515-517 nm using a spectrophotometer or microplate reader. A lower absorbance indicates higher radical scavenging activity .
Controls: Ascorbic acid, Butylated hydroxytoluene (BHT), or Trolox are typically used as positive controls . The negative control is the DPPH solution mixed with the solvent used to dissolve the test sample.
Calculation: The radical scavenging activity percentage is calculated using the formula: [(A_control – A_sample) / A_control] × 100. The IC₅₀ value is then determined from a graph plotting concentration against the percentage of inhibition .

DPPH is not used in live-cell assays. However, the DPPH assay is often compared to or used alongside cellular antioxidant activity (CAA) assays. A CAA assay typically uses human cell lines (e.g., HepG2 liver cancer cells) and a fluorescent probe (e.g., DCFH-DA). Cells are loaded with the probe and then treated with the test sample and a radical initiator (e.g., ABAP). The fluorescence, which is quenched by cellular antioxidants, is measured over time. The results from chemical DPPH assays do not always correlate with those from CAA assays, as the latter account for factors like bioavailability and membrane permeability .

DPPH is not administered to animals. It is solely an analytical tool. Animal studies that mention DPPH use it to pre-screen the antioxidant activity of a substance in vitro before evaluating the substance's efficacy in an animal model of disease (e.g., oxidative stress) . There is no standard protocol for the "administration of DPPH" to animals.

2735032 The intravenous LD50 in mice is 1800 μg/kg. (U.S. Army Armaments Research and Development Command Chemical Systems Laboratory, NIOSH Exchange Chemicals, NX#06207)

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DPPH is a chemical reagent intended for research use only and is not for human consumption. According to safety data, DPPH can cause skin and respiratory sensitization . Standard laboratory safety practices, including the use of gloves, goggles, and a fume hood, should be followed when handling this compound. It has no defined toxicological profile for therapeutic use as it is not a pharmaceutical agent.

[1]. Genesis and development of DPPH method of antioxidant assay. J Food Sci Technol. 2011 Aug;48(4):412-22.

Methodological developments: The DPPH assay has been optimized to improve accuracy and precision, and recommendations for standardizing reaction conditions (e.g., solvent selection, incubation time, and wavelength selection) have been proposed. This method has been shown to be effective in quantifying antioxidant activity in food, plant, and biological samples [1]
- Mechanism of action: DPPH is a stable free radical that accepts an electron or hydrogen atom from an antioxidant to form a non-free radical DPPH-H. The degree of color fading (from purple to yellow) is related to antioxidant capacity [1]
- Applications: Widely used in food science, pharmacology, and cosmetics for screening natural antioxidants, evaluating dietary supplements, and assessing oxidative stress in biological systems [1]
The DPPH assay is one of the most popular, rapid, and low-cost methods for the initial screening of antioxidant activity in natural products and synthetic compounds . However, its results are chemical in nature and do not necessarily reflect the biological activity of a compound inside a living cell or organism. The reaction mechanism can involve either a hydrogen atom transfer (HAT) or a single electron transfer (SET), depending on the antioxidant's structure and the reaction conditions .

C18H12N5O6

394.32

394.078

1898-66-4

2735032

Brown to black solid powder

136-138ºC

5.97

0

7

4

29

549

0

C1=CC=C(C=C1)N(C2=CC=CC=C2)[N]C3=C(C=C(C=C3[N+](=O)[O-])[N+](=O)[O-])[N+](=O)[O-]

HHEAADYXPMHMCT-UHFFFAOYSA-N

InChI=1S/C18H12N5O6/c24-21(25)15-11-16(22(26)27)18(17(12-15)23(28)29)19-20(13-7-3-1-4-8-13)14-9-5-2-6-10-14/h1-12H

1,1-Diphenyl-2-picrylhydrazyl (free radical)

2,2-Diphenyl-1-picrylhydrazyl; UNII-DFD3H4VGDH; DFD3H4VGDH; 1,1-Diphenyl-2-picrylhydrazyl (free radical); EINECS 217-591-8; Hydrazyl, 2,2-diphenyl-1-picryl-; NSC 12562; ...; 1898-66-4;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 25 mg/mL (63.40 mM)
Ethanol: 1 mg/mL (2.54 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.27 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)