DPPH (2,2-diphenyl-1-picrylhydrazyl radical, 2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl) is a relatively stable lipid radical with a free electron on its N. Its ethanol solution is purple in color, with a maximum absorption peak at 517 nm. After the addition of antioxidants, DPPH capture an electron and free electron pairing, purple faded to colorless material, the absorption at 517 nm disappeared, the degree of fading and its acceptance of the number of electrons into a quantitative relationship. According to this principle with a spectrophotometer to detect the DPPH free radicals and sample liquid reaction after the change in absorbance value, can test the sample to provide hydrogen atoms, scavenging free radicals antioxidant capacity. This method is simple, easy to implement, sensitive and reliable, and it is one of the good methods for screening antioxidants of natural products.

Operation method

DPPH method

Principle

DPPH (2,2-diphenyl-1-picrylhydrazyl radical, 2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl) is a relatively stable lipid radical with a free electron on its N. Its ethanol solution is purple in color, with a maximum absorption peak at 517 nm. After the addition of antioxidants, DPPH capture an electron and free electron pairing, purple faded to colorless material, the absorption at 517 nm disappeared, the degree of fading and its acceptance of the number of electrons into a quantitative relationship. According to this principle with a spectrophotometer to detect the DPPH free radicals and sample liquid reaction after the change in absorbance value, can test the sample to provide hydrogen atoms, scavenging free radicals antioxidant capacity. This method is simple, easy to implement, sensitive and reliable, and it is one of the good methods for screening antioxidants of natural products.

Materials and Instruments

Huperzia japonica, Scutellaria baicalensis, Scutellaria baicalensis, Radix et Rhizoma Pinelliae, Radix et Rhizoma Gastrodiae, Radix et Rhizoma Chrysanthemi, Radix Paeoniae Alba
DPPH Anhydrous ethanol Dimethyl sulfoxide Vitamin C
Round-bottomed flask Rotary evaporator Vacuum dryer Enzyme plate Enzyme labeler

Move

1. Selection of herbs: 4 herbs of the student's choice

Alternative herbs: Panax quinquefolium, Phellodendron amurense, Scutellaria baicalensis, Radix et Rhizoma Dioscoreae, Radix et Rhizoma Gynostemma, Ophiopogonis paniculata, Radix et Rhizoma Gastrodiae, Flos Chrysanthemi, Paeoniae Alba

2. Chinese herbal medicine extraction

Put 20 g of chopped Chinese herbal medicine into 250 ml round-bottomed flask, add 10 times the amount of ethanol, reflux extraction for 1 time, filtered extract was concentrated by rotary evaporator, and dried by vacuum dryer, standby.

3. Preparation of reagents

DPPH solution: weigh 3.2 mg DPPH dissolved in 40 ml of anhydrous ethanol, shaking, spare.
Extract solution: use dimethyl sulfoxide (DMSO) as solvent to formulate a solution with a concentration of 5 mg/ml, and spare. Dilute it to the corresponding concentration when measuring.
Vitamin C solution: DMSO was prepared as a solvent at a concentration of 0.5 mg/ml and set aside.

4. Screening of active herbs

Take 10 μL of the sample solution into the enzyme plate, then add 90 μL of DPPH solution, use vitamin C solution as a positive control, use DPPH solution as a standard, the sample solution as a blank, and leave it at room temperature for 30 min, and then measure it by the enzyme marker at 517 nm, and use the following formula to calculate the clearance (k) of various samples, and each sample was measured 2 times (adding samples as shown below).

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