GB/T 39100-2020.Determination of antioxidant activity for polypeptides-DPPH and ABTS methods.
1 Scope
GB/T 39100 specifies the methods for determining the antioxidant activity of polypeptides by DPPH and ABTS methods.
GB/T 39100 is applicable to the determination of the antioxidant activity of polypeptides in vitro.
2 Normative references
The following documents are indispensable for the application of this document. For any dated referenced document, only the dated version applies to this document. For any undated referenced document, its latest version (including all amendments) applies to this document.
GB/T 6682 Specifications and test methods for water for analytical laboratories
3 Terms and definitions
The following terms and definitions apply to this document.
3.1
Polypeptides
A class of compounds between amino acids and proteins, formed by two or more amino acids connected by peptide bonds.
3.2
Antioxidant activity
The ability to scavenge free radicals, expressed as the ratio of the half-scavenged amount of the tested polypeptide to the half-scavenged amount of glutathione (AO value).
5 Reagents or materials
Unless otherwise specified, the reagents used in this method are of analytical grade, and the test water is the first-grade water specified in GB/T 6682.
5.1
Glutathione
L-reduced form, purity ≥98%.
5.2 Potassium persulfate
Analytical grade.
5.3 Dimethyl sulfoxide
Analytical grade.
5.4 DPPH solution
Weigh 5.0 mg of DPPH, dissolve it in an appropriate amount of anhydrous ethanol, and dissolve it completely by ultrasound in the dark. Then dilute it to 100.0 mL with anhydrous ethanol to prepare a 50.0μg/mL DPPH solution. This solution should be prepared and used immediately.
5.5 ABTS solution
Weigh 200.0 mg of ABTS and 34.4 mg of potassium persulfate, dissolve them in 50.0 mL of distilled water, shake well, and place them at room temperature in the dark for 24 h to prepare the ABTS mother solution. Take an appropriate amount of ABTS mother solution and dilute it with 95% ethanol until the absorbance value is within 0.70±0.02 (OD734). This is the ABTS determination solution. This solution should be prepared and used immediately.
Note: The ABTS determination solution of appropriate concentration can be prepared according to the sensitivity of the spectrophotometer.
This standard specifies the method for determining the antioxidant properties of polypeptides by DPPH and ABTS methods. This standard is applicable to the determination of the antioxidant properties of polypeptides in vitro.

ICS07.080
National Standard of the People's Republic of China
GB/T39100—2020
Determination of antioxidant activity for polypeptides-DPPH and ABTS methods
Issued on 2020-09-29
State Administration for Market Regulation
National Standardization Administration
Issued
Implementation on 2021-04-01
Foreword
This standard was drafted in accordance with the rules given in GB/T1.1-2009. This standard was proposed and managed by the National Technical Committee for Standardization of Biochemical Testing (SAC/TC387). GB/T39100—2020
The drafting units of this standard are: Hebei University of Science and Technology, Hebei Agricultural University, Beijing Technology and Business University, China Testing and Certification Group Co., Ltd., Beijing Sambo Technology Co., Ltd., Wuhan Mingliao Biotechnology Co., Ltd., Zhejiang Dongjie Niubi Technology Co., Ltd., Food Evaluation Center of the State Administration for Market Regulation, and China Institute of Inspection and Quarantine. The main drafters of this standard are: Gan Zhixin, Tian Yiling, Jia Yingmin, Ma Aijin, Hao Shuai, Peng Hai, Liu Wenqiu, Yang Zhijian, Zheng Gang, Jiang Yu, Zou Mingqiang, Sun Yu, and Qi Xiaohua.
1 Scope
Determination of the antioxidant activity of polypeptides
DPPH and ABTS method
This standard specifies the method for determining the antioxidant activity of polypeptides by DPPH and ABTS method. This standard applies to the determination of the antioxidant activity of dry polypeptides in vitro. Normative references
GB/T39100—2020
The following documents are indispensable for the application of this document. For any dated reference, only the dated version applies to this document. For any undated reference, the latest version (including all amendments) applies to this document. Specifications and test methods for water for analytical laboratories GB/T6682
Terms and definitions
The following terms and definitions apply to this document. 3.1
Polypeptides
A class of compounds between amino acids and proteins, formed by two or more amino acids connected by peptide bonds. 3.2
Antioxidant capacity
antioxidant activity
The ability to scavenge free radicals, expressed as the ratio of the half-scavenged amount of the tested polypeptide to the half-scavenged amount of glutathione (AO value). 4
Abbreviations
The following abbreviations apply to this document.
ABTS: 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonicacid) diammonium salt AO: antioxidant activity DPPH: 1,1-diphenyl-2-picrylhydrazyl DMSO: dimethylsulfoxide ECso: half maximal effective concentration 5
Reagents or materials
Unless otherwise specified, the reagents used in this method are of analytical grade, and the test water is the first-grade water specified in GB/T6682. 5.1
Glutathione
L-reduced, purity ≥98%.
GB/T39100—2020
2 Potassium persulfate
Analytically pure.
3 Dimethyl sulfoxide
Analytically pure.
DPPH solution
Weigh 5.0 mg of DPPH, dissolve it in an appropriate amount of anhydrous ethanol, and dissolve it completely by ultrasonic treatment in the dark. Then use anhydrous ethanol to make the volume to 100.0 mL to prepare a 50.0 μg/mL DPPH solution. This solution should be prepared and used immediately. 5 ABTS solution
Weigh 200.0 mg of ABTS and 34.4 mg of potassium persulfate, dissolve them in 50.0 mL of distilled water, shake well, and place them at room temperature in the dark for 24 hours to prepare the ABTS mother solution. Take an appropriate amount of ABTS mother solution and dilute it with 95% ethanol until the absorbance value is within 0.70 ± 0.02 (OD734). This is used as the ABTS determination solution. This solution should be prepared and used immediately.
Note: The ABTS determination solution of appropriate concentration can be prepared according to the sensitivity of the spectrophotometer. 6. Instruments and equipment
Spectrophotometer: wavelength range is 400nm800nm, absorbance value is accurate to 0.001. 2. Electronic balance: sensitivity is 0.0001g.
DPPH·free radical scavenging method
7.1 Principle
DPPH is a dark purple prismatic crystal, which is dark purple in anhydrous ethanol solution. The DPPH·free radical has a strong absorption peak at 517nm in the visible light region. This method combines a stable DPPH·free radical provided by DPPH with an ion provided by the antioxidant polypeptide, so that the characteristic purple color of the DPPH·free radical disappears and becomes colorless or light yellow. The absorbance after the reaction is measured by a UV-visible spectrophotometer. By comparing with the scavenging ability of glutathione, the relative half-scavenging amount is used to determine the antioxidant capacity AO value of the polypeptide.
Test steps
7.2.1 Sample preparation
7.2.1.1 Sample solution to be tested
Weigh 10.0 mg of polypeptide sample. Dissolve and dilute the sample with good water solubility with distilled water. Dissolve the sample with poor water solubility with DMSO and dilute to 1.0 mL with distilled water. Mix thoroughly to prepare a 10.0 mg/mL mother solution. Dilute the mother solution with distilled water to different multiples to obtain sample solutions of different concentrations. The concentration of the sample solution to be tested should be selected so that its clearance rate is between 35% and 65%, and the R2 of the linear equation is ≥0.9500.
7.2.1.2 Glutathione solution
Weigh 10.0 mg of L-reduced glutathione, dilute to 1.0 mL with distilled water, mix thoroughly to prepare a 10.0 mg/mL mother solution. Dilute the mother solution with distilled water to different multiples to obtain glutathione solutions of different concentrations. The concentration of glutathione solution should be selected so that its clearance rate is between 35% and 65%, and the R2 of the linear equation is ≥0.9500.7.2.2 Determination
7.2.2.1
Control group
Take 3 test tubes, numbered 1, 2, and 3 respectively, and add reagents to each test tube according to the combination in Table 1. Table 1 Amount of reagent added
Solution name
DPPH solution
Glutathione solution
Sample solvent solution
Absolute ethanol solution
No. 1 (A)
3.0mL
1.0mL
No. 2 (A)
1.0mL
3.0mL
GB/T39100—2020
No. 3 (A)
3.0mL
1.0mL
Add 3.0mL DPPH solution and 1.0mL glutathione solution to test tube No. 1 as the test group (A); add 1.0mL glutathione solution and 3.0mL anhydrous ethanol solution to test tube No. 2 as the control group (A); add 3.0mL DPPH solution and 1.0mL sample solvent solution to test tube No. 3 as the blank group (A,). After fully mixing, react at room temperature in the dark for 30 minutes, and measure the absorbance value with a UV spectrophotometer at a wavelength of 517nm (sample solvent zero calibration). 7.2.2.2 Sample group
Replace the glutathione solution with the sample solution to be tested, and the other operations are the same as 7.2.2.1. 7.2.3 Experimental data processing
Calculate according to formula (1):
Wherein:
——clearance rate;
1_A, -A
A.——absorbance of the mixture of the test solution and DPPH solution; A.——absorbance of the mixture of the test solution and anhydrous ethanol solution; X100%
A.——absorbance of the mixture of DPPH solution and sample solvent solution. (1)
Use the natural logarithm of the concentration of the test solution as the horizontal axis and the clearance rate as the vertical axis to establish a linear equation between the natural logarithm of the concentration of the test solution and the clearance rate (R≥0.9500), calculate the half clearance ECs0, and calculate the antioxidant capacity AO value of the polypeptide sample according to formula (2). AO
Wherein:
ECso(S)
ECsa(R)
Antioxidant capacity
ECso(S)
ECso(R)
The half clearance of the polypeptide sample, in milligrams per liter (mg/L); the half clearance of glutathione, in milligrams per liter (mg/L). The calculation results are expressed as the arithmetic mean of the parallel determination values, retaining three significant figures. 8
ABTS+ free radical scavenging method
8.1 Principle
. (2)
ABTS generates relatively stable blue-green ABTS+ free radicals after oxidation, with a maximum absorption peak at 734nm in the visible light region. Antioxidant peptides react with ABTS+ free radicals to make them fade, and the absorbance at 734nm decreases. The absorbance after the reaction is measured by a UV-visible spectrophotometer. By comparing with the scavenging ability of glutathione, the relative half-scavenging amount is used to determine the antioxidant capacity AO value of the peptide.
8.2 Test steps
Sample preparation
8.2.1
8.2.1.1 Sample solution to be tested
Weigh 10.0 mg of polypeptide sample. Dissolve and dilute the sample with good water solubility with distilled water. Dissolve the sample with poor water solubility in DMSO and dilute to 1.0 mL with distilled water. Mix thoroughly to prepare a 10.0 mg/mL mother solution. Dilute the mother solution to different multiples with 95% ethanol to obtain solutions of different concentrations to be tested. The concentration of the sample solution to be tested should be selected so that its clearance rate is between 35% and 65%, and the R of the linear equation is ≥0.9500.
8.2.1.2 Glutathione solution
Weigh 10.0 mg of L-reduced glutathione, dilute to 1.0 mL with distilled water, mix thoroughly to prepare a 10.0 mg/mL mother solution. Dilute the mother solution with 95% ethanol to different multiples to obtain different concentrations of test solutions. The concentration of glutathione solution should be selected so that its clearance rate is between 35% and 65%, and the R2 of the linear equation is ≥ 0.9500. 8.2.2 Determination
8.2.2.1 Control group
Take 2 test tubes, numbered 1 and 2 respectively, and add reagents to each test tube according to the combination in Table 2. Table 2 Reagent addition amount
Solution name
ABTS solution
Glutathione solution
Sample solvent solution
No. 1 (A,)
3.6mL
0.4mL
No. 2 (Ah)
3.6mL
0.4mL
Add 3.6mLABTS solution and 0.4mL glutathione solution to test tube No. 1 as the experimental group (A); add 3.6mLABTS solution and 0.4mL sample solvent solution to test tube No. 2 as the blank group (A); after fully mixing, react at room temperature in the dark for 5 minutes, and measure the absorbance value with an ultraviolet spectrophotometer at a wavelength of 734nm (sample solvent zero calibration). 8.2.2.2 Sample group
Replace the glutathione solution with the sample solution to be tested, and the other operations are the same as 8.2.2.1. 8.2.3 Experimental data processing
Calculate according to formula (3):
Where:
——clearance rate;
Ab-A.
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A——absorbance of the mixture of ABTS solution and sample solvent solution; a
. (3)
A, -—absorbance of the mixture of the test solution and ABTS solution. GB/T39100—2020
Use the natural logarithm of the concentration of the test solution as the abscissa and the clearance rate as the ordinate to establish a linear equation between the natural logarithm of the concentration of the test solution and the clearance rate (R2≥0.9500), calculate the half clearance EC50, and calculate the antioxidant capacity AO value of the polypeptide sample according to formula (2) in 7.2.3. The calculation results are expressed as the arithmetic mean of the parallel determination values, retaining three significant figures. Repeatability
The absolute difference of no less than 3 independent determination results obtained under repeatability conditions does not exceed 20% of the arithmetic mean. 5
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