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维生素 C（Vitamin C）又称L‑抗坏血酸，是高等灵长类及少数生物的必需营养素，属于酸性己糖衍生物、稀醇式己糖酸内酯。它在生物体内既是抗氧化剂，可清除自由基、保护机体免受氧化损伤，同时也作为辅酶参与多种代谢反应，广泛存在于各类新鲜蔬果中。维生素 C 存在 L‑型和 D‑型两种异构体，仅 L‑型具有生理功能，还原型与氧化型均具有生理活性。
检测原理为：在酸性条件下，维生素 C 将三价铁离子还原为亚铁离子，亚铁离子与菲咯啉形成稳定的红色螯合物；在534 nm处测定吸光度，当样品浓度在0.5～35 μg/ml范围内时，吸光度与维生素 C 含量呈良好线性关系，据此可定量计算维生素 C 含量。
本试剂盒主要用于植物组织中维生素 C（抗坏血酸）的检测，具有反应稳定不易褪色、操作简便、特异性好（还原糖及常见还原物质无干扰）、灵敏度高等优点。
注：本试剂盒仅限科研使用，不可用于临床诊断及其他用途。
自备仪器和试剂
研钵或匀浆器、离心机、 恒温箱或水浴锅
离心管或试管、比色杯
蒸馏水、无水乙醇
操作步骤（仅供参考）
1. 稀释组织匀浆液
按组织匀浆液 (5×)：蒸馏水=1：4的比例稀释，获得1× 组织匀浆液。
2. 准备样品
取待测材料如青菜、水果、松针等，清洗擦干，准确称量2~3 g，加入研磨器内，再加入少量1× 组织匀浆液，研磨碎，留取上清，再次用1× 组织匀浆液研磨，最后一并倒入50 mL离心管，补充1× 组织匀浆液至22 mL，充分混匀，4000g离心5 min，上清液即为待测液。
3. 配制系列Vitamin C标准
将25 mg Vitamin C标准用1 mL 1×组织匀浆液溶解，即得Vitamin C标准 (25 mg/mL)，再用1×组织匀浆液稀释成Vitamin C标准 (50 μg/mL)，取干净离心管或试管，按下表进行操作，依次稀释。配置好的Vitamin C标准 (25 mg/mL) 放置-20℃，短期保存。
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4. Vitamin C加样
按照下表设置空白管、标准管、测定管，溶液应按照顺序依次加入，并注意避免产生气泡；如果样品中的Vitamin C含量过高，可以减少样品用量或适当稀释后再进行测定，样品的检测最好能设置2个平行管，求平均值。
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5. Vitamin C测定
混匀， 30℃温箱中反应60 min，以空白管调零，比色杯光径1 cm，分光光度计测定534 nm系列标准管、测定管的吸光度。
6. 结果计算
以系列标准Vitamin C (0、2、5、8、10、20、30 μg) 为横坐标，以对应的吸光度为纵坐标，绘制标准曲线，求得回归方程。以测定管吸光度代入回归方程求得维生素C含量。Vitamin C, also known as L-ascorbic acid, is an essential nutrient for higher primates and a few other organisms. It is an acidic hexose derivative and a lactone of an enediol hexanoic acid. In organisms, it acts as an antioxidant, scavenging free radicals and protecting against oxidative damage, and also serves as a coenzyme in various metabolic reactions. It is widely found in fresh fruits and vegetables. Vitamin C exists as L- and D-isomers; only the L-form has physiological functions, but both the reduced and oxidized forms are physiologically active.
Assay Principle
Under acidic conditions, Vitamin C reduces ferric ions (Fe³⁺) to ferrous ions (Fe²⁺). The ferrous ions then form a stable red chelate with Ferrozine. The absorbance is measured at 534 nm. When the sample concentration is within the range of 0.5～35 μg/ml, the absorbance shows a good linear relationship with the Vitamin C content, allowing for quantitative determination.
This kit is primarily intended for the detection of Vitamin C (ascorbic acid) in plant tissues. It features a stable reaction (color does not fade easily), simple operation, good specificity (no interference from reducing sugars and common reducing substances), and high sensitivity.
Note: For research use only. Not for use in diagnostic procedures or other purposes.
Reagents, consumables and Equipments not provided
Spectrophotometer (capable of measuring absorbance at 534 nm)
Mortar and pestle or homogenizer, Centrifuge, Incubator or water bath
Centrifuge tubes or test tubes, Cuvettes (1 cm path length)
Distilled water, Anhydrous ethanol
Assay Procedure (For Reference Only)
1. Dilution of Tissue Homogenate
Dilute the Tissue Homogenate (5×) by mixing it with distilled water at a ratio of 1:4 (e.g., 1 part homogenate to 4 parts water) to obtain 1× Tissue Homogenate.
2. Sample Preparation
Take the plant material to be tested (e.g., green vegetables, fruits, pine needles). Wash and dry them. Accurately weigh 2-3 g of the sample.
Place the sample in a mortar or grinder. Add a small amount of 1× Tissue Homogenate and grind thoroughly. Collect the supernatant.
Grind the residue again with more 1× Tissue Homogenate.
Combine all extracts and transfer into a 50 mL centrifuge tube. Add 1× Tissue Homogenate to bring the total volume to 22 mL. Mix well.
Centrifuge at 4000g for 5 minutes. The resulting supernatant is the Test Solution.
3. Preparation of Vitamin C Standards
Dissolve the 25 mg Vitamin C Standard in 1 mL of 1× Tissue Homogenate to obtain a Vitamin C Standard (25 mg/mL). This stock can be stored at -20°C for short-term preservation.
Dilute this stock with 1× Tissue Homogenate to prepare a Vitamin C Standard (50 μg/mL).
Use clean centrifuge tubes or test tubes to prepare a series of working standards according to the table below:
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4. Assay Setup
Set up Blank, Standard, and Test tubes according to the table below. Add solutions in the order listed, avoiding bubbles. If the Vitamin C content in the sample is expected to be very high, reduce the volume of the Test Solution used or dilute the Test Solution appropriately. It is recommended to run samples in duplicate and calculate the average.
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5. Measurement
Mix well. Incubate at 30°C for 60 minutes.
Zero the spectrophotometer with the Blank Tube at 534 nm using a 1 cm path length cuvette.
Measure the absorbance of all Standard Tubes and Test Tubes.
6. Calculation of Results
Plot a standard curve with the Vitamin C mass (μg) of the series standards (0, 2, 5, 8, 10, 20, 30 μg) on the x-axis and their corresponding absorbance values on the y-axis. Obtain the regression equation.
Substitute the absorbance value of the Test Tube into the regression equation to obtain the mass of Vitamin C in the tested sample aliquot (m₀, in μg).
Calculate the Vitamin C content in the original sample using the following formula:
Vitamin C Content (mg/100g) = (m₀ × VT × 100) / (m₁ × VS × 1000)
Parameter Explanation:
m₀: Mass of Vitamin C in the tested sample aliquot obtained from the standard curve (μg).
VT: Total volume of the Test Solution (mL). (According to the procedure, this is 22 mL).
m₁: Mass of the sample used for extraction (g). (e.g., 2-3 g).
VS: Volume of the Test Solution used in the assay (mL). (According to the procedure, this is 1.0 mL).
100: Conversion factor for content per 100g of sample.
1000: Conversion factor for micrograms (μg) to milligrams (mg).
Notes
| V1515894 | Component | 100T | Storage |
| V1515894A | Vitamin C标准 | 25 mg | RT. Store in the dark. |
| V1515894B | 组织匀浆液 (5×) | 500 mL | RT. Store in the dark. |
| V1515894C | 酸性缓冲液 | 25 mL | RT. |
| V1515894D | Vitamin C Assay Buffer | 25 mL | RT. |
| V1515894E | 菲咯啉显色液 | 50 mL | RT. Store in the dark. |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0434305 |
分析证书 | V1515894 |
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