This standard specifies the determination method of superoxide dismutase (SOD) activity in food. This standard is applicable to the determination of superoxide dismutase (SOD) activity in various foods. The detection limit of the first method is 1.17U/mL; the detection limit of the second method is 0.033U/mL. GB/T 5009.171-2003 Determination of superoxide dismutase (SOD) activity in health foods GB/T5009.171-2003 Standard download decompression password: www.bzxz.net

ICS67.040
National Standard of the People's Republic of China
GB/T5009.171—2003
Determination of the action of superoxide dismutase (SOD) in health foods
Determination of the action of superoxide dismutase in health foods Issued on August 11, 2003
Ministry of Health of the People's Republic of China
Standardization Administration of the People's Republic of China
Implementation on January 1, 2004
This standard is proposed and managed by the Ministry of Health of the People's Republic of China. The responsible drafting units of this standard are: Jilin City Health and Epidemic Prevention Station, Jilin Medical College. The main drafters of the first method of this standard are: Yuan Yanhua, Luo Su, Yu Jingwei, Sun Lianjun, Zhao Fengming. The main drafters of the second method of this standard are: Luo Su, Yuan Yanhua, Zhang Jilin, Lu Zhongkui. GB/T5009.171—2003
GB/T5009.171—2003
Superoxide free radical O can cause cell damage in the body. Superoxide dismutase (SOD) can remove O in the body. There is no standard test method for the determination of SOD activity in food at home and abroad. Therefore, the Ministry of Health's Ninth Five-Year Standardization Plan has determined the research topic of SOD activity determination in food.
This standard determines SOD activity in food by modified Marklund method and chemiluminescence method. The minimum detection concentration of SOD activity determination in the former is 1.17U/mL, equivalent to 1.1×10-mol/L. The method is sensitive, the instrument is cheap, the operation is simple, and it is easy to promote. The minimum detection concentration of SOD activity determination in the latter is 0.033U/mL, equivalent to 3.0×101°mol/L, and the sensitivity is about two orders of magnitude higher than the former. It has the advantages of being more sensitive, rapid and less interfering, but the reagents are more expensive. It can be used for arbitration or scientific research. There was no significant difference in the results of the two determination methods for the same SOD enzyme (P>0.05). 412
1 Scope
Determination of superoxide dismutase (SOD) activity in health foods
This standard specifies the determination method of superoxide dismutase (SOD) activity in foods. This standard is applicable to the determination of superoxide dismutase (SOD) activity in various foods. The detection limit of the first method of this method is 1.17U/mL, and the detection limit of the second method is 0.033U/mL. The first method is a modified Marklund method
2 Definition
The amount of SOD required to inhibit the self-oxidation rate of pyrogallol by 50% at 25℃ is one activity unit. 3 Principle
GB/T5009.171—2003
Under alkaline conditions, pyrogallol will undergo self-oxidation, and the SOD activity can be determined based on the ability of SOD to inhibit the self-oxidation of pyrogallol. 4 Reagents
4.1 Liquid A: pH 8.200.1mol/L Tris (Tris)-hydrochloric acid buffer solution (containing 1mmol/EDTA·2Na). Weigh 1.2114g Tris and 37.2mg EDTA·2Na and dissolve in 62.4mL 0.1mol/L hydrochloric acid solution, and dilute to 100mL with distilled water.
4.2 Liquid B: 4.5mmol/L pyrogallol hydrochloric acid solution. Weigh 56.7mg pyrogallol (AR) and dissolve in a small amount of 10mmol/L hydrochloric acid solution, and dilute to 100mL.
4.310mmol/L hydrochloric acid solution.
4.40.200mg/mL superoxide dismutase (SOD). 4.5 Distilled water: double quartz distilled water.
5 Instruments
5.1 UV-visible spectrophotometer.
5.2 Precision acidometer, accuracy 0.01 pH. 5.3 Centrifuge.
5.4 10 mL colorimetric tube.
5.5 10 mL centrifuge tube.
Glass mortar.
6 Preparation of samples
6.1 Solid sample (tea, pollen, etc.) Weigh 1.00 g of sample and place it in a glass mortar. Add 9.0 mL of distilled water and grind for 5 min. Transfer to a 10 mL centrifuge tube. Rinse the mortar with a small amount of distilled water, wash and add it to the centrifuge tube, add distilled water to the scale, centrifuge at 4000 r/min for 15 min, and take the supernatant for determination.
6.2 Clear liquid samples can be directly measured in the original solution. Turbid liquid samples can be centrifuged at 4000 r/min for 15 min, and then the supernatant can be measured. 413
GB/T 5009.171—2003
7 Analysis steps
7.1 Determination of the self-oxidation rate of pyrogallol At about 25°C, add 2.35mL of solution A, 2.00ml of steamed stuffing water, and 0.15mL of solution B to a 10mL colorimetric tube. Add solution B and mix immediately and pour into colorimetric IIIL. Determine the absorbance at the initial time and after 1min at a wavelength of 325nm, respectively. The difference between the two is the self-oxidation rate of pyrogallol △Azzs (min-\). This test determines that △A32s (min-1) is 0.060. 7.2 Determination of the self-oxidation rate of pyrogallol inhibited by sample solution and SOD enzyme solution According to step 7.1, add a certain amount of sample solution or enzyme solution to inhibit the self-oxidation rate of pyrogallol to about 1/2AAs25 (min-1), that is, △A325 (min\1) is 0.030. The sample addition procedure for SOD activity determination is shown in Table 1.
Table 1 SOD activity determination sample addition table
Liquid A/mL
Distilled water/ml
Sample solution or SOD solution/μL
Liquid B/mL
8 Results
8.1 Result calculation
8.1.1 Liquid sample was calculated according to formula (1):
AA325-AA's25×100%
SOD activity (U/mL)=
Wherein:
SOD enzyme activity unit;
pyrogallol self-oxidation rate;
-sample solution or SOD enzyme solution inhibiting pyrogallol self-oxidation rate; V—-volume of enzyme solution or sample solution added, in milliliters (mL); D——dilution multiple of enzyme solution or sample solution;
4.5—total volume of reaction solution, in milliliters (mL). The calculation result shall be retained to three significant figures.
8.1.2 The solid sample shall be calculated according to formula (2):
SOD activity (U/g)
Wherein:
AA'325
AAs =AA'an ×100%
-volume of enzyme solution or sample solution added, in mL; pyrogallol self-oxidation rate;
the inhibition rate of pyrogallol self-oxidation by the same solution or SOD enzyme solution; - dilution multiple of enzyme solution or sample solution;
V, total volume of the same solution, in mL; m
-mass of sample, in g;
4.5-total volume of reaction solution, in mL. The calculation result shall be retained to three significant figures.
8.2 Precision
SOD solution
The absolute difference between two independent determination results obtained under repeatability conditions shall not exceed 10% of the arithmetic mean. 414
9 Definition
Method 2 Chemiluminescence
The amount of SOD required to inhibit 50% of the luminescence intensity under analytical conditions is one activity unit. 10 Principle
SOD can catalyze the following reaction:
O:-+O:+2H+SOPH,O.+0
GB/T 5009.171—2003
Under aerobic conditions, xanthine oxidase can catalyze the oxidation of xanthine (or hypoxanthine) into uric acid, and O is produced simultaneously in the reaction process. O2 can further react with luminol (3-aminophthalhydrazide) to excite the luminescent agent luminol, and when it returns to the ground state, it emits light outward. Since SOD can eliminate O;-, it can inhibit the luminescence of mino. Through this reaction process, the luminescence intensity value of the blank control is 100%, and the SOD activity is determined by the degree of inhibition of luminescence after adding SOD. 11 Reagents
11.10.05mol/L carbonate buffer (pH10.2)11.1.10.1mol/L sodium carbonate (NazCO,) solution Weigh 10.599g of sodium carbonate (AR) and dissolve it in distilled water and make up to 1000mL. 11.1.20.1mol/L sodium bicarbonate (NaHCO,) solution Weigh 8.401g of sodium bicarbonate (AR) and dissolve it in distilled water and make up to 1000mL.
11.1.30.1mol/L sodium carbonate-sodium bicarbonate buffer (pH10.2) Dissolve 0.1mol/L sodium carbonate (NazCO,) solution and 0.1mol/L sodium bicarbonate (NaHCO.) solution in a 6+4 ratio. 11.1.4 0.05mol/L sodium carbonate-sodium bicarbonate buffer (pH10.2) 0.1mol/L pH10.2 sodium carbonate-sodium bicarbonate buffer (11.1.3) and distilled water in a 1+1 ratio. 11.2 0.05mol/L sodium carbonate-sodium bicarbonate (containing 0.1mmol/EDTA2Na) buffer (pH10.2) Weigh 37.2mg EDTA·2Na and dissolve it in 0.05mol/L sodium carbonate-sodium bicarbonate pH10.2 buffer (11.1.4) and dilute to 1000mL. 11.3 0.1mmol/L Luminol solution Weigh 3.54mg luminol and dissolve it in distilled water and dilute to 200mL. 11.4 0.1mmol/L hypoxanthine solution (HX) Weigh 2.76mgHX and dissolve it in distilled water and dilute to 200mL. 11.5 0.1mmol/L xanthine oxidase (XO) 0.1mgXO is diluted to 1.0mL with 0.05mol/L carbonate buffer (11.2) containing 0.1mmol/LEDTA·2Na. 11.6 0.001mg/mL superoxide dismutase (SOD) Weigh 0.1mgSOD accurately and dilute to 100mL with 0.05mol/L carbonate buffer (11.2) containing 0.1mmol/LEDTA.2Na. 11.7 HX-L solution 0.1mmol/LHX solution and 0.1mmol/L luminol solution 1+1 (V/V) (mix before use) 12 Instrument
Biochemiluminescence instrument.
13 Preparation of sample
Follow the method specified in Chapter 6 of Method 1. Except that 0.05 mol/L sodium carbonate-sodium bicarbonate (containing 0.1 mmol/LEDTA·2Na) buffer (11.2) is used instead of distilled water for solid sample. 14 Analysis steps
14.1 Plotting the inhibition luminescence curve
The operation procedure is shown in Table 2 and Figure 1.
GB/T5009.171—2003
[SOD]/(ng/mL)
Figure 1 SOD inhibition chemiluminescence curve
Table 2 SOD inhibition chemiluminescence curve preparation steps0 (control)
0.05mol/L sodium carbonate-sodium bicarbonate buffer solution (pH10.2)/μL0.1mg/mLXO/μL
HX-L solution/μL
Different concentrations of SOD (or test solution)/μuL
SOD concentration/(ng/mL) or (sample solution volume/μL)Relative light intensity
Uninhibited/%
Inhibited/%
14.2 Determination of SOD activity
Determine the relative luminescence intensity of the sample, calculate the inhibition luminescence rate, and check the SOD inhibition luminescence curve to obtain the SOD amount (ng). 15 Results
15.1 Calculation of results
15.1.1 Liquid samples were calculated according to formula (1):
SOD activity (U/mL) = m×10×3.5×3300Vxcso
Wherein:
SOD amount in the inhibition curve, in nanograms (ng); - the volume of the sample solution, in milliliters (mL); XD
SOD concentration when the standard SOD inhibits 50% of the luminescence, in nanograms per milliliter (ng/ml.); SOD standard specific activity U/mg protein;
-SOD concentration when the SOD enzyme solution inhibits 50% of the chemiluminescence rate, in nanograms per milliliter (ng/mL); dilution factor of the sample solution.
The calculation result is rounded to three significant figures.
15.1.2 For solid samples, calculate SOD activity (U/g) according to formula (2): SOD activity (U/g) × 10 × V × 3.5 × 3300mXV.Xc5o
Wherein:
SOD amount in the inhibition curve, in nanograms (ng); sample mass, in grams (g);
Total volume of sample solution, in milliliters (mL): XD
(2)
V,-volume of sample solution, in milliliters (mL): GB/T5009.171—2003
3.5—SOD concentration when standard SOD inhibits 50% luminescence, in nanograms per milliliter (ng/mL); 3300--SOD standard specific activity U/mg protein; Cso--SOD concentration when SOD enzyme solution inhibits 50% chemiluminescence, in nanograms per milliliter (ng/mL); D-dilution multiple of sample solution.
The calculation result shall retain three significant figures.
15.2 Precision
The absolute difference between two independent determination results obtained under repeatability conditions shall not exceed 10% of the arithmetic mean. 417Xc5o
Where:
SOD amount in the inhibition curve, in nanograms (ng); sample mass, in grams (g);
Total volume of sample solution, in milliliters (mL):XD
(2)
V,-volume of sample solution, in milliliters (mL):GB/T5009.171—2003
3.5—SOD concentration when standard SOD inhibits 50% luminescence, in nanograms per milliliter (ng/mL);3300--SOD standard specific activity U/mg protein;Cso--SOD concentration when SOD enzyme solution inhibits 50% chemiluminescence, in nanograms per milliliter (ng/mL);D-dilution multiple of sample solution.
The calculation result shall retain three significant figures.
15.2 Precision
The absolute difference between two independent determination results obtained under repeatability conditions shall not exceed 10% of the arithmetic mean. 417Xc5obzxz.net
Where:
SOD amount in the inhibition curve, in nanograms (ng); sample mass, in grams (g);
Total volume of sample solution, in milliliters (mL):XD
(2)
V,-volume of sample solution, in milliliters (mL):GB/T5009.171—2003
3.5—SOD concentration when standard SOD inhibits 50% luminescence, in nanograms per milliliter (ng/mL);3300--SOD standard specific activity U/mg protein;Cso--SOD concentration when SOD enzyme solution inhibits 50% chemiluminescence, in nanograms per milliliter (ng/mL);D-dilution multiple of sample solution.
The calculation result shall retain three significant figures.
15.2 Precision
The absolute difference between two independent determination results obtained under repeatability conditions shall not exceed 10% of the arithmetic mean. 417
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