计算溶液所需的质量、体积或浓度。
还原型维生素C (VC)含量检测试剂盒 (DCPIP, 比色法)
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文献证明
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。
概述
维生素C（Vitamin C）是一种具有抗氧化性质的有机化合物，又称抗坏血酸，是高等灵长类动物与其他少数生物的必需营养素，在生物体内，维生素C是一种抗氧化剂，同时也是一种辅酶，为酸性己糖衍生物，是烯醇式己糖酸内酯，可保护身体免于自由基的威胁，其广泛的食物来源为新鲜的蔬菜和水果，Vc有L-抗坏血酸和D-抗坏血酸，而且两种都具有强还原性，但只有L-抗坏血酸对人体具有生物活性。L-抗坏血酸有还原型抗坏血酸和氧化型抗坏血酸两种，前者含量较高，在一般蔬菜和水果中占90%以上，L-还原型抗坏血酸极易被氧化为L-脱氢抗坏血酸；L-脱氢抗坏血酸亦可被还原为L-还原型抗坏血酸，通常也称作氧化型抗坏血酸。
还原型维生素C (VC)含量检测试剂盒 (DCPIP, 比色法)检测原理是向一定提取液中加入过量的2,6-二氯酚靛酚染料溶液，与还原型抗坏血酸作用后，2,6-二氯酚靛酚被还原，多余的2,6-二氯酚靛酚可用二甲苯萃取比色，待测液中抗坏血酸的含量与二甲苯萃取的颜色呈线性负相关，即待测液中抗坏血酸含量越高，未被还原的染料就越少，二甲苯萃取的颜色就越浅，由于水溶性的花青素不溶于二甲苯，因此不影响测定结果，常用于蔬菜、水果中还原型维生素C含量的测定，特别适用于花青素含量高的蔬菜类样品。
该试剂盒仅用于科研领域，不适用于临床诊断或其他用途。
组分与储存：
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自备材料：
1、新鲜蔬菜或水果等样品、蒸馏水、二甲苯
2、组织捣碎器、匀浆器、滤纸、漏斗、容量瓶（10ml、50ml）、15ml离心管、分光光度计、1ml微量比色皿
操作步骤(仅供参考)：
1、配制 1× 组织匀浆液：按 组织匀浆液 (12×) ：蒸馏水 = 1 ：11 的比例稀释，即得。
2、配制 2× 组织匀浆液：按 组织匀浆液 (12×) ：蒸馏水 = 1 ：5 的比例稀释，即得。
3、制备样品提取液：清洗干净新鲜蔬菜或水果，用布或吸水纸吸干表面水分或自然晾干。称取 1g，加入 1.5ml 的 2× 组织匀浆液，置于组织捣碎器中打成浆状，滤液倒入 50ml 容量瓶内，滤渣可用 1× 组织匀浆液重复匀浆 1～2 次，合并滤液倒入容量瓶内，加入 0.5ml 硫酸锌溶液，摇动容量瓶，再加入 0.5ml 亚铁盐溶液，晃动以除去脂溶性色素，再用 1× 组织匀浆液定容至刻度，摇匀后过滤至干净小烧杯中备用。
4、配制 Vitamin C 标准液：取少量 1× 组织匀浆液加入 10mg Vitamin C 标准中，充分混匀，然后完全转移至 10ml 容量瓶，用 1× 组织匀浆液稀释至刻度，即为 Vitamin C 标准储液 (1mg/ml)：再取 0.5ml 标准储液加入 10ml 容量瓶中，用 1× 组织匀浆液稀释至刻度，即为 Vitamin C 标准液 (0.05mg/ml)；注意：Vc 标准液最好临用前配制。
5、配制二氯酚靛酚溶液：称取 10mg 二氯酚靛酚加入 15ml 离心管中，一次性加入 10ml 二氯酚靛酚稀释液，充分摇匀至完全溶解，即得二氯酚靛酚储液 (1mg/ml) (如有必要，可加热助溶)。临用前，按 二氯酚靛酚储液 (1mg/ml) ：蒸馏水 = 1 ：4 的比例稀释，即得二氯酚靛酚溶液，此溶液每毫升约相当于含有 0.1mg 的维生素C。
6、配置 Vc 梯度标准：按下表分别吸取 Vitamin C 标准液和 1× 组织匀浆液，（单位：ml）
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7、维生素C加样：按照下表设置标准管、测定管，溶液应按照顺序依次加入；如果样品中的Vc浓度过高，可减少样品用量，可补加 1× 组织匀浆液后再进行测定，样品的检测最好能设置平行管。
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8、维生素C测定：迅速摇动约 30s，静置 1min 后二甲苯与水层分离，吸取各管上层二甲苯层萃取溶液倒入 1ml 比色皿中，以二甲苯做空白调零，分光光度计测定各管 500nm 吸光度（即为 A标准、A测定）。
计算：
以系列维生素C浓度（20、25、30、35μg/ml）为横坐标，对应吸光度为纵坐标，绘制标准曲线；A测定代入方程求得待测样品中维生素C浓度。100g 样品中维生素C的质量 (mg/100g) = C × VT × N × 100 / (1000 × m)
式中：
C = （经稀释后）提取液中维生素C浓度（ug/ml）
VT = 样品提取液总体积（ml）
m = 样品鲜质量（g）
N = 样本稀释倍数
1000 = ug 与 mg 的比例换算
注意事项：
1、该试剂盒提供的组织匀浆液可用于大部分的蔬菜和水果等样品，可抑制抗坏血酸氧化酶活性。
2、组织匀浆液 (12×) 久置或低温保存，容易产生乳白色浑浊；如果白色浑浊不明显，可以直接使用，不影响效果；如果白色浑浊较多，应弃用。
3、样品提取液要避免日光直射，否则会加速抗坏血酸的氧化。
4、配好的 Vitamin C 标准液 4℃ 避光保存，4周有效（最好临用前配制），也可 -20℃ 保存。
5、配制好的二氯酚靛酚储液 4℃ 避光保存，二氯酚靛酚溶液 4℃ 避光保存，1周有效；如有条件可称取二氯酚靛酚 10mg，加入 10ml 二氯酚靛酚稀释液充分溶解，可避免浪费试剂。
6、二氯酚靛酚应充分溶解才能使用，如溶解不完全，可导致测定数据不稳定。
7、反应体积中加入二甲苯后，应充分摇动各管至少 30s，并静置 1min 以上，使二甲苯充分抽提出未反应的二氯酚靛酚。
8、测定溶液中含有二甲苯等有机溶剂，不能用塑料比色皿或普通塑料酶标板测定。
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。
Vitamin C is an organic compound with antioxidant properties, also known as ascorbic acid. It is an essential nutrient for higher primates and a small number of other organisms. In vivo, vitamin C acts as an antioxidant as well as a coenzyme. It is an acidic hexose derivative and an enolic hexonoic acid lactone, protecting the body from free radical damage. Fresh vegetables and fruits serve as abundant dietary sources of vitamin C. Vitamin C exists in two forms: L‑ascorbic acid and D‑ascorbic acid, both of which possess strong reducing properties, yet only L‑ascorbic acid exhibits biological activity in humans. L‑ascorbic acid has two states: reduced ascorbic acid and oxidized ascorbic acid. The reduced form accounts for over 90% of total vitamin C in common vegetables and fruits. L‑reduced ascorbic acid is readily oxidized into L‑dehydroascorbic acid; reversely, L‑dehydroascorbic acid (generally referred to as oxidized ascorbic acid) can also be reduced back to L‑reduced ascorbic acid.
For the Reduced Vitamin C (VC) Content Assay Kit (DCPIP, Colorimetric Method), an excess amount of 2,6‑dichlorophenolindophenol dye solution is added to the sample extract. The 2,6‑dichlorophenolindophenol is reduced upon reaction with reduced ascorbic acid. The residual unreacted 2,6‑dichlorophenolindophenol is extracted with xylene for colorimetric detection. The concentration of ascorbic acid in the test sample shows a negative linear correlation with the color intensity of the xylene extract: higher ascorbic acid concentration in the sample leads to less residual unreduced dye and lighter color of the xylene layer. Since water‑soluble anthocyanins are insoluble in xylene, they will not interfere with detection results. This kit is commonly applied to quantify reduced vitamin C in vegetables and fruits, especially suitable for vegetable samples with high anthocyanin content.
This kit is for research use only, not for clinical diagnosis or other purposes.
Components & Storage:
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Materials to be Prepared by User:
1. Fresh vegetable or fruit samples, distilled water, xylene
2. Tissue homogenizer, mortar, filter paper, funnel, volumetric flasks (10 mL, 50 mL), 15 mL centrifuge tubes, spectrophotometer, 1 mL microcuvettes
Operating Procedure (For Reference Only):
1. Preparation of 1× Tissue Homogenate Solution: Dilute Tissue Homogenate Solution (12×) with distilled water at a ratio of 1 : 11.
2. Preparation of 2× Tissue Homogenate Solution: Dilute Tissue Homogenate Solution (12×) with distilled water at a ratio of 1 : 5.
3. Preparation of sample extract: Rinse fresh vegetable or fruit samples thoroughly, blot surface moisture with absorbent paper or air‑dry naturally. Weigh 1 g sample and add 1.5 mL of 2× Tissue Homogenate Solution, then homogenize in tissue homogenizer. Filter the homogenate into a 50 mL volumetric flask. Re‑homogenize the filter residue 1–2 times using 1× Tissue Homogenate Solution, combine all filtrates into the volumetric flask. Add 0.5 mL Zinc Sulfate Solution and shake well, then add 0.5 mL Ferrous Salt Solution and mix to remove fat‑soluble pigments. Make up to the mark with 1× Tissue Homogenate Solution, mix thoroughly and filter into a clean beaker for later use.
4. Preparation of Vitamin C Standard Solution: Add a small volume of 1× Tissue Homogenate Solution to 10 mg Vitamin C Standard and mix completely. Transfer the whole mixture into a 10 mL volumetric flask and dilute to volume with 1× Tissue Homogenate Solution to prepare Vitamin C standard stock solution (1 mg/mL). Pipette 0.5 mL of standard stock solution into a 10 mL volumetric flask, dilute to volume with 1× Tissue Homogenate Solution to obtain Vitamin C working standard solution (0.05 mg/mL). Note: Prepare VC standard solution freshly before use if possible.
5. Preparation of 2,6‑Dichlorophenolindophenol Solution: Weigh 10 mg 2,6‑dichlorophenolindophenol into a 15 mL centrifuge tube. Add 10 mL 2,6‑Dichlorophenolindophenol Diluent at once, mix thoroughly until fully dissolved to obtain 2,6‑dichlorophenolindophenol stock solution (1 mg/mL) (heating can assist dissolution if necessary). Before use, dilute the stock solution (1 mg/mL) with distilled water at a ratio of 1 : 4. The prepared dye solution contains approximately 0.1 mg vitamin C per milliliter.
6. Preparation of VC standard gradient: Pipette Vitamin C working standard solution and 1× Tissue Homogenate Solution according to the table below (Unit: mL).
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7. VC loading: Set standard tubes and assay tubes as listed in the table; add reagents sequentially in the specified order. If the VC concentration of the sample is excessively high, reduce sample volume and supplement with 1× Tissue Homogenate Solution before detection. Parallel tubes are recommended for sample testing.
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8. VC measurement: Vortex vigorously for ~30 sec, stand for 1 min to allow phase separation between xylene and aqueous layer. Transfer the upper xylene extract from each tube into 1 mL microcuvette. Zero the spectrophotometer with pure xylene blank, then record absorbance at 500 nm (Astandard, Asample).
Calculation:
Plot the standard curve with serial vitamin C concentrations (20, 25, 30, 35 μg/mL) on the x‑axis and corresponding absorbance values on the y‑axis.
Substitute Asample into the regression equation to calculate vitamin C concentration in sample extract.Vitamin C mass in 100 g fresh sample (mg/100g) = C × VT × N × 100 / (1000 × m)
Where:
C = vitamin C concentration in diluted extract (μg/mL)
VT = total volume of sample extract (mL)
m = fresh weight of sample (g)
N = sample dilution factor
1000 = unit conversion factor from μg to mg
Precautions:
1. The supplied Tissue Homogenate Solution is applicable for most vegetable and fruit samples, and inhibits the activity of ascorbate oxidase.
2. Tissue Homogenate Solution (12×) may turn milky white upon long‑term storage or low‑temperature preservation. Slight turbidity does not interfere with experiments; discard the reagent if heavy precipitation appears.
3. Avoid direct sunlight exposure of sample extract, as light accelerates oxidation of ascorbic acid.
4. Prepared Vitamin C Standard Solution can be stored at 4°C protected from light for up to 4 weeks (fresh preparation before use is preferred); long‑term storage at -20°C is also available.
5. Prepared 2,6‑dichlorophenolindophenol stock solution should be stored at 4°C protected from light. The diluted working dye solution is stable for 1 week at 4°C protected from light. To save reagents, weigh 10 mg 2,6‑dichlorophenolindophenol and dissolve in 10 mL 2,6‑Dichlorophenolindophenol Diluent as needed.
6. 2,6‑dichlorophenolindophenol must be fully dissolved before use; incomplete dissolution will lead to unstable assay data.
7. After adding xylene into reaction mixture, vortex each tube for at least 30 sec and stand for ≥1 min to ensure complete extraction of unreacted 2,6‑dichlorophenolindophenol into xylene phase.
8. The assay solution contains organic solvent xylene. Plastic cuvettes and ordinary plastic microplates are NOT compatible for absorbance reading.
9. For personal safety, wear lab coat and disposable gloves during operation.
10. Use opened reagents promptly to avoid compromised experimental performance.
规格
规格或纯度
BioReagent, 适用于分析
英文名称
Reduced Vitamin C (VC) Content Assay Kit (DCPIP, Colorimetric Method)
样品类型
植物
检测方法
比色法
定量&半定量
定量
检测仪器
可见光分光光度计
检测波长
500nm
敏感性
Light-sensitive
储存与运输
储存条件
2-8°C储存,避光,室温
运输条件
常规运输,低温运输
稳定性与储存
各组分在相应的储存条件下保质期12个月。
组分与储存
| 储运单元货号 | 储运单元名称 | 组分货号 | 组分名称 | 物理外观 | 50T | 储存温度 |
| R1515898NT | 还原型维生素C (VC)含量检测试剂盒 (DCPIP, 比色法)(常温组分) | R1515898A | Vitamin C 标准 | 粉末 | 10mg | 室温，避光 |
| R1515898B | 组织匀浆液 (12×) | 液体 | 250ml | 室温 | ||
| R1515898C | 二氯酚靛酚稀释液 | 液体 | 20ml | 室温 | ||
| R1515898D | 硫酸锌溶液 | 液体 | 25ml | 室温 | ||
| R1515898E | 亚铁盐溶液 | 液体 | 25ml | 室温 | ||
| R1515898WT | 还原型维生素C (VC)含量检测试剂盒 (DCPIP, 比色法)(2-8℃组分) | R1515898F | 二氯酚靛酚 | 粉末 | 20mg | 2-8℃，避光 |
注：发货时各组分依据储存温度（室温/2-8℃）独立包装，差异化温控运输以保障产品性能。同一订单含多温区物料可能分批次送达，请以实际收货为准。收货后按组分表标注温度分类存放，有效期自生产之日起 12 个月。
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常见问题
什么是「BioReagent」？
「BioReagent」表示该产品经过加工和检测，适用于生物和生化应用。规格可能包括内毒素限值、微生物控制、无菌性、低 DNase/RNase/蛋白酶活性，以及在相关情况下的生物活性验证。
本产品应如何储存？
请于2–8 °C、避光条件下保存。本品对光敏感，请保存在原始的避光或棕色容器中。
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本产品采用湿冰冷藏运输。收货后请立即拆包，并按上述储存条件保存。
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