DPPH Free Radical Scavenging Assay Kit (Micro Method)

Cat. No.: D1522019

GRADE & PURITY BioReagent, Standard Curve Method

Panoramica

Various free radicals are continuously generated during metabolic processes of life activities. Accumulation of free radicals causes oxidative damage to the organism, leading to chronic diseases such as aging, tumors, stroke, myocarditis and diabetes. Among numerous free radicals, 2,2-diphenyl-1-picrylhydrazyl (DPPH) is a relatively stable free radical. In the presence of free radical scavengers, its color changes from purple to yellow accompanied by a decrease in absorbance.

The DPPH Free Radical Scavenging Capacity Assay Kit (Microplate Method), also known as Nitrogen Radical Scavenging Capacity Assay Kit or Nitrogen Radical Scavenging Rate Assay Kit, is based on the following principle: DPPH free radical is a stable nitrogen-containing free radical with an unpaired single electron. Its ethanol solution displays purple color and exhibits strong absorbance at 517 nm. When other substances donate an electron to pair with this single electron, the solution fades. The degree of discoloration is proportional to the amount of electrons accepted. The nitrogen radical scavenging capacity of samples can be reflected by the reduction of absorbance. This kit is mainly applied to samples including serum, plant tissues, antioxidant foods, health supplements and pharmaceuticals. This kit is for research use only and is not intended for clinical diagnosis or any other applications.

Contents & Storage:

Component No.	Component Name	Appearance	100T	Storage
D1522019A	Nitrogen radical extract	Liquid	100 mL	2-8℃.
D1522019B	DPPH Solution	Liquid	25 mL	2-8℃. Store in the dark.
D1522019C	Vitamin C	Powder	10 mg	2-8℃. Store in the dark.

Materials to be prepared:

1. Experimental materials: plant tissues (celery, corn leaves, etc.), blood, distilled water, anhydrous ethanol, sodium hydroxide, hydrochloric acid, etc.

2. Mortar or homogenizer, grinder or ultrasonicator, centrifuge, centrifuge tubes, thermostatic water bath, constant-temperature drying oven, electronic balance, 30–50 mesh sieve, microplate reader, 96-well plate

Operating Procedures (For Reference Only):

1. Sample Preparation

① Plant samples: Take fresh plant samples, clean thoroughly, grind in a mortar. Dry the ground material in a drying oven and sieve. Weigh 0.05 g sample, add 0.8 mL nitrogen radical extract, extract in a 40 °C water bath for 30–60 min, then centrifuge at 10,000 rpm for 10 min. Retain the supernatant and store at 4 °C for later use (alternative extraction protocols from published references are also acceptable).

② Plasma, serum and urine samples: Plasma samples should be anticoagulated with heparin or sodium citrate (EDTA anticoagulant is not recommended). Centrifuge plasma at 5,000 rpm for 10 min at 4 °C and collect supernatant. Serum or urine samples can be tested directly.

③ Cell samples: Add 0.8 mL nitrogen radical extract per 5×10⁶ cells. Lyse cells by sonication (power: 200 W, sonicate for 3 s, interval 10 s, repeat 30 times). Centrifuge at 10,000 rpm for 10 min, collect supernatant for subsequent assay.

④ Fruit juice, wine and similar samples: Mix sample and nitrogen radical extract at a volume ratio of 1:9 with shaking. Centrifuge at 10,000 rpm for 10 min. Collect supernatant and store at 4 °C.

⑤ Highly active samples: If the effective concentration of the sample is high, appropriately dilute the sample with nitrogen radical extract.

2. Preparation of Vitamin C Stock Solution

Fully dissolve one vial of 10 mg Vitamin C in 1 mL nitrogen radical extract to prepare 10 mg/mL Vitamin C solution. Aliquot into 0.1 mL portions and store at −20 °C.

3. Preparation of  VC Standard Working Solution

If linear relationship measurement is required, dilute the 10 mg/mL Vitamin C stock solution with nitrogen radical extract to prepare  VC standard working solutions at concentrations of 50, 40, 30, 20, 10 and 5 μg/mL. For positive control with DPPH scavenging rate over 90%, standard working solution above 50 μg/mL is recommended.

4. Turn on the microplate reader and preheat for no less than 30 min. Set detection wavelength to 517 nm (500–530 nm is also acceptable). Perform zero calibration using anhydrous ethanol.

5. DPPH Reagent Addition

Set blank tube, sample detection tube, sample control tube and positive control tube (standard tube) according to the table below. Add reagents sequentially, mix thoroughly, incubate at room temperature in the dark for 30 min.

Additive (mL)	Blank Tube	Sample Detection Tube	Sample Control Tube	Positive Control Tube (Standard Tube)
Nitrogen Radical Extract	0.05	/	/	/
Supernatant	/	0.05	0.05	/
VC Standard Working Solution	/	/	/	0.05
Anhydrous Ethanol	0.225	0.225	0.45	0.225
DPPH Solution	0.225	0.225	/	0.225

6. OD Measurement

Centrifuge all tube solutions at 12,000 rpm for 5 min at room temperature. Transfer 300 μL supernatant into a 96-well plate. Measure absorbance values with a microplate reader, recorded sequentially as A₀, A₁, A₂, A₃.

Calculations:

Positive control ·DPPH Scavenging Rate (%)=(A₀-A₃)/A₀×100%

Test sample ·DPPH Scavenging Rate (%) =[A₀-(A₁-A₂)]/A₀×100%

Notes:

A₀ = Absorbance value of blank tube

A₁ = Absorbance value of sample detection tube

A₂ = Absorbance value of sample control tube

A₃ = Absorbance value of positive control tube

Precautions:

1. It is recommended to conduct a preliminary experiment with 2–3 samples with expected significant differences before formal detection.

2. All experimental materials should be kept fresh. Perform extraction and determination on the same day.

3. The DPPH radical scavenging capacity varies greatly among different samples. If the DPPH scavenging rate exceeds 90%, dilute the sample with extraction solution; if the scavenging rate is lower than 5%, increase the sample dosage to raise the concentration of active ingredients.

4. For samples with low activity, the detection sensitivity can be improved by increasing the addition amount of supernatant or reducing the dosage of DPPH solution.

5. The initial absorbance of DPPH solution may vary between different batches. If necessary, DPPH solution can be diluted 1–2 times with absolute ethanol before use (see Appendix 2).

6. Avoid adding substances that interfere with redox reactions, such as Triton and DTT, to samples.

7. This method is affected by reaction time, initial DPPH concentration and sample properties, as well as external factors including light, oxygen, pH value, organic ions and inorganic ions.

8. Alkaline sample extract may result in a higher absorbance of the sample assay tube than the blank tube, leading to a negative scavenging rate. Adjust the pH to neutral with sodium hydroxide or other neutralizers.

9. Wear laboratory coats and disposable gloves during operation for personal safety and health protection.

10. Use reagents as soon as possible after opening to avoid affecting subsequent experimental results.

Appendix 1:

Standard Curve Preparation: Operate in accordance with the instructions at room temperature. Determine the absorbance of a series of  VC standard working solutions (60, 50, 40, 30, 20, 10 μg/mL). The measured data and standard curve are shown below (for reference only).

	Initial absorbance value	Zero-set absorbance value	DPPH scavenging rate
Absolute ethanol	0.039	/	/
Blank Tube	0.641	0.602	0
VC  Standard 10μg/ml	0.557	0.518	13.95348837
VC  Standard 20μg/ml	0.451	0.376	37.54152824
VC  Standard 30μg/ml	0.328	0.289	51.99335548
VC  Standard 40μg/ml	0.210	0.171	71.59468439
VC  Standard 50μg/ml	0.092	0.053	91.19601329
VC  Standard 60μg/ml	0.075	0.036	94.01993355

As shown in the figure above, the  VC standard working solution presents a good linear relationship within the concentration range of 0–50 μg/mL, and the DPPH scavenging rate is positively correlated with  VC concentration. When the  VC concentration exceeds 50 μg/mL, the DPPH scavenging rate approaches the maximum value, resulting in data deviation.

Appendix 2:

After the DPPH solution is diluted 1-fold and 2-fold with absolute ethanol respectively, the linearity remains favorable for st  VC andards at 0–28 μg/mL and 0–20 μg/mL correspondingly. The DPPH scavenging rate is positively correlated with  VC concentration. Relevant reference data are listed below.

	Absolute Ethanol + DPPH Solution (1+1)		Absolute Ethanol + DPPH Solution (2+1)
	Zero-set absorbance value	DPPH scavenging rate	Zero-set absorbance value	DPPH scavenging rate
Blank Tube	0.304	0	0.209	0
VC  Standard 1μg/ml	0.274	9.868421053	0.204	2.392344498
VC  Standard 4μg/ml	0.256	15.78947368	0.171	18.18181818
VC  Standard 6μg/ml	0.237	22.03947368	0.143	31.57894737
VC  Standard 8μg/ml	0.219	27.96052632	0.127	39.23444976
VC  Standard 10μg/ml	0.190	37.5	0.097	53.58851675
VC  Standard 15μg/ml	0.157	48.35526316	0.053	74.64114833
VC  Standard 20μg/ml	0.079	74.01315789	0.012	94.25837321
VC  Standard 30μg/ml	0.018	94.07894737	0.010	95.215311

Specifications

Sinonimi

DPPH Free Radical Scavenging Capacity Assay Kit | DPPH Free Radical Scavenging Capacity Assay Kit, Colorimetric Method

Specifiche e purezza

BioReagent, Standard Curve Method

Grado

BioReagent, Standard Curve Method

Applicazione

Cell Metabolism

Tipo di campione

Siero, Tessuti vegetali

Metodo di rilevamento

Colorimetric Method

Quantitative

Quantitative

Detection instrument

Microplate reader

Detection wavelength

517nm

Sensibilità

Light-sensitive

Stoccaggio e spedizione

Condizioni di conservazione di stoccaggio

Store at 2-8°C,Protected from light

Spedito in

Wet ice

Stabilità e conservazione

Store at 2-8℃ long term (6 months). Store in the dark.

Contents & Storage

Component No.	Component Name	Appearance	100T	Storage
D1522019A	Nitrogen radical extract	Liquid	100 mL	2-8℃.
D1522019B	DPPH Solution	Liquid	25 mL	2-8℃. Store in the dark.
D1522019C	Vitamin C	Powder	10 mg	2-8℃. Store in the dark.