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在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
在生命活动的代谢过程中不断产生各种自由基，而自由基的积累会使机体产生氧化损伤而引起衰老、肿瘤、中风、心肌炎和糖尿病等慢性疾病，在众多自由基中2，2-二苯基-1-苦基苯肼(DPPH)是一种较稳定的自由基，当有自由基清除剂存在时颜色由紫色向黄色转变，吸光度随之变小。
阿拉丁DPPH自由基清除率检测试剂盒（比色法）又称氮自由基清除能力检测试剂盒或氮自由基清除率检测试剂盒，其检测原理是DPPH自由基是一种较稳定的含氮自由基，含一个单电子，溶于乙醇后溶液呈紫色，在517nm下有强吸收，如果有其他物质提供一个电子与此单电子配对，溶液会褪色，褪色程度与接受电子的量呈正比，通过吸光度下降的程度来反应样品的氮自由基清除能力，主要用于检测血清、植物组织、抗氧化类食品、保健品及药品等样本。该试剂盒仅用于科研领域，不适用于临床诊断或其他用途。
自备仪器和试剂
操作步骤（仅供参考）：
1、准备样品：
① 植物样品：取新鲜植物样本，清洗干净，研钵研碎(粉碎)，干燥箱烘干后过筛，称取0.05g样品，加入0.8ml氮自由基提取液，40℃水浴浸提30~60min，10000rpm离心10min，上清液待用，4℃保存备用(亦可参考相关资料提取方法提取)。
② 血浆、血清和尿液样品：血浆(用肝素或枸橼酸钠抗凝，不宜使用EDTA抗凝) 4℃ 5000rpm离心10min，取上清液待用；血清或尿液样品可直接测定。
③ 细胞样品：按每5×10⁶ 个细胞加入0.8ml氮自由基提取液，超声破碎(功率200W，超声3s，间隔10s，重复30次)，10000rpm离心10min，取上清液待用。
④ 果汁、葡萄酒等样品：按样品：氮自由基提取液=1：9的比例震荡混匀，10000rpm离心10min，上清液待用，4℃保存备用。
⑤ 高活性样品：如果样品中有效浓度较高，可用提取液进行恰当的稀释。
2、配制维生素C溶液：将一支10mg维生素C充分溶解于1ml氮自由基提取液中，即成10mg/ml维生素C溶液；可分成0.1ml的小份，-20℃保存。
3、配制Vc标准工作液：如需测线性关系，建议用氮自由基提取液将10mg/ml维生素C溶液稀释至50、40、30、20、10、5μg/ml的Vc标准工作液；如需清除率大于90%的阳性对照，建议选用大于50μg/ml的Vc标准工作液。
4、分光光度计开机预热30min，调节波长517nm(500~530nm亦可)，无水乙醇调零。
5、DPPH加样：按照下表设置空白管、样本测定管、样本对照管、阳性对照管，溶液应按照顺序依次加入，混匀，室温避光静置30min。
| 加入物 (ml) | 空白管 | 样本测定管 | 样本对照管 | 阳性对照管 (标准管) |
| 氮自由基提取液 | 0.2 | / | / | / |
| 上清液 | / | 0.2 | 0.2 | / |
| Vc标准工作液 | / | / | / | 0.2 |
| 无水乙醇 | 0.9 | 0.9 | 1.8 | 0.9 |
| DPPH溶液 | 0.9 | 0.9 | / | 0.9 |
6、OD值测定：将各管溶液室温12000rpm离心5min，取上清依次加入到比色皿中，用分光光度计检测各管吸光度值，依次记为A0、A1、A2、A3。
计算：
阳性对照 DPPH清除率(%)= (A0- A3)/A0×100%
待测样本 DPPH清除率(%)=[A0-( A1- A2)]/A0×100%
备注：
A0=空白管的吸光度值
A1=样本测定管的吸光度值
A2=样本对照管的吸光度值
A3=阳性对照管的吸光度值
注意事项：
1、正式测定之前建议选择2~3个预期差异较大的样本做预实验。
2、实验材料应尽量新鲜，当天提取当天测定。
3、不同样本清除DPPH自由基的能力差异很大。如样本DPPH清除率大于90%，应用提取液稀释；如样本DPPH清除率小于5%，应提高样本用量以提高有效成分浓度。
4、如样品活性较低时，可通过提高上清液加入量或减少DPPH溶液加入量来增加检测的灵敏度。
5、不同批次生产的DPPH溶液初始吸光度值可能有差异，根据实际情况，DPPH溶液可用无水乙醇稀释1~2倍后使用。(参见附录2)
6、样品中不宜添加Triton、DTT等可能影响氧化还原反应的成分。
7、本方法受反应时间、DPPH初浓度、样品性质的影响，还受到光照、氧气、pH值、有机离子、无机离子等外界因素的影响。
8、如果样品提取液 pH值偏碱性，将可能导致样本测定管吸光度大于空白管吸光度，使清除率为负值，可用氢氧化钠等调整 pH至中性。
9、为了您的安全和健康，请穿实验服并戴一次性手套操作。
10、试剂开封后请尽快使用，以防影响后续实验效果。
附录1：
标准曲线制作：在室温条件下按说明书操作，对系列Vc标准工作液 (60、50、40、30、20、10μg/ml)进行吸光度的测定，其数值及标准曲线如下 (仅供参考)：
| 初始吸光度值 | 调零吸光度值 | DPPH清除率 | |
| 无水乙醇 | 0.039 | / | / |
| 空白管 | 0.641 | 0.602 | 0 |
| Vc 标准 10μg/ml | 0.557 | 0.518 | 13.95348837 |
| Vc 标准 20μg/ml | 0.451 | 0.376 | 37.54152824 |
| Vc 标准 30μg/ml | 0.328 | 0.289 | 51.99335548 |
| Vc 标准 40μg/ml | 0.210 | 0.171 | 71.59468439 |
| Vc 标准 50μg/ml | 0.092 | 0.053 | 91.19601329 |
| Vc 标准 60μg/ml | 0.075 | 0.036 | 94.01993355 |
由上图可知：Vc标准工作液在0~50μg/mI时线性关系良好，DPPH清除率与Vc浓度呈正相关，当Vc浓度大于50μg/mI时，DPPH清除率接近最大值，数据有偏差。
附录2：
用无水乙醇将 DPPH溶液分别稀释1倍和2倍后，Vc标准分别在0~28μg/ml和0~20μg/mI时线性关系良好，DPPH清除率与Vc浓度呈正相关，数据参考如下：
| 无水乙醇+DPPH溶液(1+1) | 无水乙醇+DPPH溶液(2+1) | |||
| 调零吸光度值 | DPPH 清除率 | 调零吸光度值 | DPPH 清除率 | |
| 空白管 | 0.304 | 0 | 0.209 | 0 |
| Vc 标准 1μg/ml | 0.274 | 9.868421053 | 0.204 | 2.392344498 |
| Vc 标准 4μg/ml | 0.256 | 15.78947368 | 0.171 | 18.18181818 |
| Vc 标准 6μg/ml | 0.237 | 22.03947368 | 0.143 | 31.57894737 |
| Vc 标准 8μg/ml | 0.219 | 27.96052632 | 0.127 | 39.23444976 |
| Vc 标准 10μg/ml | 0.190 | 37.5 | 0.097 | 53.58851675 |
| Vc 标准 15μg/ml | 0.157 | 48.35526316 | 0.053 | 74.64114833 |
| Vc 标准 20μg/ml | 0.079 | 74.01315789 | 0.012 | 94.25837321 |
| Vc 标准 30μg/ml | 0.018 | 94.07894737 | 0.010 | 95.215311 |
Various free radicals are continuously generated during the metabolic processes of life activities. The accumulation of free radicals can cause oxidative damage to the organism, further 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.
Aladdin DPPH Free Radical Scavenging Assay Kit (Colorimetric Method), also known as the nitrogen radical scavenging capacity detection kit or nitrogen radical scavenging rate detection kit, is based on the following detection principle. DPPH is a stable nitrogen-containing free radical with one unpaired electron. It presents a purple color when dissolved in ethanol and exhibits a strong absorbance at 517 nm. When other substances donate electrons to pair with its unpaired electron, the solution fades, and the degree of discoloration is positively correlated with the amount of accepted electrons. The nitrogen radical scavenging capacity of samples is evaluated according to the reduction in absorbance. This kit is mainly applicable to the detection of samples including serum, plant tissues, antioxidant foods, health products, and pharmaceuticals. This product is only for scientific research use and is not intended for clinical diagnosis or other applications.
Reagents, consumables and Equipments not provided
⁕Spectrophotometer (capable of measuring absorbance at 517 nm)
⁕Mortar or homogenizer, grinder, ultrasonic crusher, centrifuge, centrifuge tube, constant temperature water bath, constant temperature drying oven, electronic balance, 30–50 mesh sieve, cuvettes
⁕Experimental materials: plant tissues (celery, corn leaves, etc.), blood, distilled water, absolute ethanol, sodium hydroxide, hydrochloric acid, and other related materials
Protocol (for reference only):
1. Sample Preparation:
① Plant samples: Collect fresh plant specimens and clean thoroughly. Grind the samples with a mortar, dry them in a drying oven, and sieve afterward. Weigh 0.05 g of the processed sample, add 0.8 mL of nitrogen radical extraction solution, and conduct water bath extraction at 40 ℃ for 30–60 min. Centrifuge at 10,000 rpm for 10 min, and collect the supernatant for subsequent use; store the supernatant at 4 ℃ for later application. Alternative extraction methods described in relevant references are also acceptable.
② Plasma, serum and urine samples: For plasma samples (anticoagulated with heparin or sodium citrate; EDTA anticoagulation is not recommended), centrifuge at 5,000 rpm for 10 min at 4 ℃ and collect the supernatant. Serum and urine samples can be directly detected without pre-treatment.
③ Cell samples: Add 0.8 mL of nitrogen radical extraction solution per 10⁶ cells. Perform ultrasonic crushing (power: 200 W; ultrasonic time: 3 s; interval: 10 s; repeat 30 times). Centrifuge at 10,000 rpm for 10 min, and collect the supernatant for later use.
④ Fruit juice, wine and other liquid samples: Mix the sample with nitrogen radical extraction solution at a ratio of 1:9 by shaking thoroughly. Centrifuge at 10,000 rpm for 10 min, collect the supernatant, and store at 4 ℃ for standby.
⑤ High-activity samples: If the effective concentration of the sample is excessively high, appropriately dilute the sample with the extraction solution.
2. Preparation of Vitamin C Solution:
Fully dissolve 10 mg of vitamin C powder in 1 mL of nitrogen radical extraction solution to prepare a 10 mg/mL vitamin C solution. Aliquot the solution into 0.1 mL portions and store at -20 ℃.
3. Preparation of Vc Standard Working Solution:
For linear relationship measurement, dilute the 10 mg/mL vitamin C solution with nitrogen radical extraction solution to prepare a series of Vc standard working solutions at concentrations of 50, 40, 30, 20, 10 and 5 μg/mL. For a positive control with a scavenging rate higher than 90%, use Vc standard working solution with a concentration above 50 μg/mL.
4. Preheat the spectrophotometer for 30 minutes after startup. Adjust the wavelength to 517 nm (500–530 nm is also applicable), and set zero with absolute ethanol.
5. DPPH Loading:
Set blank tubes, sample detection tubes, sample control tubes and positive control tubes in accordance with the following table. Add each solution in sequence and mix well. Let the mixture stand for 30 minutes at room temperature under dark conditions.
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6. OD value determination: Centrifuge the solution in each tube at 12000 rpm for 5 minutes at room temperature. Transfer the supernatant into cuvettes in sequence, then measure the absorbance values of each tube with a spectrophotometer, which are recorded as A0、A1、A2、A3 respectively.
Calculation:
Positive control DPPH scavenging rate (%) = (A0- A3)/A0×100%
Test sample DPPH scavenging rate (%) =[A0-( A1- A2)]/A0×100%
Notes:
A0=Absorbance of the blank tube
A1=Absorbance of the sample assay tube
A2=Absorbance of the sample control tube
A3=Absorbance of the 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 Vc standards 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 |
项目号 | 物理外观 | 组件 | 50T | 100T | Storage |
D1522020A | 液体 | 氮自由基提取液 | 50 mL | 100 mL | 2-8℃. |
D1522020B | 液体 | DPPH溶液 | 50 mL | 100 mL | 2-8℃. Store in the dark. |
D1522020C | 固体 | 维生素C | 10 mg | 10 mg | 2-8℃. Store in the dark. |
通过匹配包装上的批号来查找并下载产品的 COA，每批产品都进行了严格的验证，您可放心使用！
| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0939121 |
分析证书 | D1522020 |
| ZJ26F0535648 |
分析证书 | D1522020 |
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