背景介绍：
原花青素 (Oligomeric Proanthocyanidins, OPC) 是一类黄烷醇单体及其聚合体的多酚化合物，广泛存在于植物的各种器官中，具有极强的抗氧化性和清除自由基的作用，广泛应用于医药、食品、化妆品、保健品行业。
植物原花青素 (OPC) 含量检测试剂盒 (微量法) 采用盐酸-香草醛检测法。其原理: 在酸性条件下，植物原花青素A环上的间苯二酚和间苯三酚与香草醛发生缩合反应，产生有色化合物，在500 nm处有特征吸收峰，通过检测 500 nm 处吸光度，可计算植物中原花青素的含量。
该试剂盒用于检测植物样本中原花青素含量。
注意事项：
1. 原花青素标准品未开封 4℃避光保存；甲醇溶解配制后的原花青素标准品溶液尽快使用，4℃避光保存不超过 1 个月。
2. ΔA超出标准曲线线性范围：ΔA高于上限，待测上清使用提取液稀释后重测；ΔA低于下限，可提高样品浓度重新提取测定，结果计算同步调整稀释倍数。
3. 检测设备可选用酶标仪或普通可见分光光度计，仪器需支持 500 nm 波长检测；采用分光光度法时使用微量玻璃比色皿。
4. 甲醇、盐酸具有挥发性与腐蚀性，全部操作尽量在通风橱内开展；实验全程穿戴实验服、一次性手套，注意实验安全。
5. 显色反应受温度、孵育时间影响显著，标准品与待测样品必须同步孵育、同步检测。
6. 花青素含量较高的有色植物样本存在基质干扰；原花青素本身无色，必须设置样品对照孔扣除基质背景。
7. 香草醛-盐酸法易受单宁、部分黄酮类物质干扰；本方法无法区分黄烷醇单体、寡聚原花青素与高聚原花青素。
8. 孵育完成后立即测定吸光度，避免显色衰退造成误差。
9. 本试剂盒仅用于科研领域，不适用于临床诊断或其他用途。
组分货号 | 组分名称 | 物理外观 | 48T | 96T | 储存 | 单次测试使用量 |
- | 提取液：60% 乙醇 (自备) | 液体 | 70 mL | 70 mL×2 | 2-8℃ | 按需 |
- | 试剂 一：8% 盐酸 (自备) | 液体 | 12 mL | 24 mL | 2-8℃ | 按需 |
P1521899A | 香草醛 | 固体 | 62.5 mg | 125 mg | 2-8°C, 避光 | 按需 |
P1521899B | 原花青素标准品 (5 mg) | 固体 | 5 mg | 5 mg | 2-8°C, 避光 | 40 µL |
使用方法
1. 材料准备
额外所需材料：
(1) 实验材料：植物组织。
(2) 所需仪器：酶标仪或可见分光光度计 (能测500nm处的吸光度)。
(3) 其它：96孔板或微量玻璃比色皿、可调节式移液枪及枪头、匀浆器、超声破碎仪、去离子水、甲醇、40目筛。
2. 试剂准配：
(1) 提取液 (自备)
取 30 mL 无水乙醇，加入 20 mL 蒸馏水，再添加 100 μL 浓盐酸，充分混匀；4℃密封保存。
(2) 试剂一：8% 盐酸甲醇溶液 (自备)
1.6 mL 浓盐酸缓慢加入 18.4 mL 无水甲醇中混匀；严禁将甲醇倒入浓盐酸，防止暴沸喷溅；4℃避光保存。
(3) 香草醛 ：
粉剂规格：使用前，48T 加 6.25 mL甲醇溶解，96T 加12.5 mL甲醇溶解。
注：配好的香草醛稳定性差，临用新鲜配制，不宜长期存放。
(4) 测定工作液 & 空白工作液（临用现配，不可久放）
测定工作液: 香草醛和试剂一按 1:1 (体积比) 混合。
空白工作液：试剂一和甲醇按1:1 (体积比) 混合。
(5) 原花青素标准品：
称取 5 mg标准品, 加入 1 mL 提取液溶解，配制成 5 mg/mL 母液；使用前平衡至室温；配制完成后 4℃避光可保存 2 周。
注：本方法采用原花青素B2作为单一标品，若采用其他来源原花青素标准品，响应系数存在差异。
(6) 原花青素标准品设置：按下表所示，用提取液将5 mg/mL标准品母液梯度稀释，得到浓度为2.5、1.25、0.625、0.3125、0.1563、0.0781和 0 mg/mL系列标准溶液。
序号 | 标准品体积（µL） | 提取液体积（µL） | 标准品浓度（mg/mL） |
标品.1 | 100 µL 5 mg/mL (母液) | 0 | 5 |
标品.2 | 取 100 µL 标品.1 | 100 | 2.5 |
标品.3 | 取 100 µL 标品.2 | 100 | 1.25 |
标品.4 | 取 100 µL 标品.3 | 100 | 0.625 |
标品.5 | 取 100 µL 标品.4 | 100 | 0.3125 |
标品.6 | 取 100 µL 标品.5 | 100 | 0.1563 |
标品.7 | 取 100 µL 标品.6 | 100 | 0.0781 |
标品.8 | 0 | 100 | 0 (空白对照) |
注：每次实验都要做一次标准品检测，制作标曲；稀释后的标准品溶液不稳定，必须在4小时内使用。
3. 样本制备：
(1) 纤维较多的植物组织：称取约 0.1 g 样本，加入1 mL 提取液捣碎，冰浴超声波破碎30分钟 (功率 30% 或 300 W，超声 5 s，间隔 8 s)，12,000×g，25℃ 离心 10 分钟，取上清，用提取液定容至1 mL，置冰上待测。
(2) 纤维较少的幼嫩植物组织：称取约 0.1 g 样本，加入1 mL 提取液捣碎，冰浴超声波破碎30分钟 (功率 30% 或 300 W，超声 5 s，间隔 8 s)，12,000×g，25℃ 离心10 分钟，取上清，用提取液定容至1mL，置冰上待测。
4. 实验步骤：
(1) 酶标仪或可见分光光度计预热30分钟以上，调节波长到 500 nm。
(2) 加样体系 (96 孔板或微量比色皿体系，单位：μL)：
试剂 | 标准孔（μL） | 测定孔（μL） | 对照孔（μL） |
样本 | 0 | 40 | 40 |
不同浓度标准品 | 40 | 0 | 0 |
空白工作液 | 0 | 0 | 160 |
工作液 | 160 | 160 | 0 |
(3) 充分混匀，30℃避光恒温孵育 30 分钟，在 500 nm 处测定吸光值。空白孔记为A空、标准孔记为ΔA标、测定孔记为ΔA测、对照孔记为 A对。计算ΔA测 = A测−A对，ΔA标 = A标−A空。
注：空白孔和标准孔只需测定1次。实验之前建议选择2-3个预期差异大的样本做预实验。
5. 结果计算：
标准曲线：y=ax-b
OPC 含量 (mg/g 鲜重) =(ΔA+b)÷a×V反总÷(V样÷V样总×W)×10⁻³
参数定义：
V样总总为加入提取液体积, 1 mL；
V反总为反应总体积, 200 µL；
V样为反应中样品体积, 40 µL；
W为样品质量, g;
6. 结果展示：
注： 此为典型标准曲线，受仪器及操作影响，实际曲线参数会略有偏差。
Background:
Oligomeric Proanthocyanidins (OPC) are a group of polyphenolic compounds consisting of flavanol monomers and their polymers. They are widely distributed in various plant organs and possess potent antioxidant and free radical scavenging activities, with broad applications in the pharmaceutical, food, cosmetic and health product industries.
Plant Oligomeric Proanthocyanidin (OPC) Content Assay Kit (Microassay) adopts the vanillin-hydrochloric acid colorimetric method.
Assay Principle: Under acidic conditions, resorcinol and phloroglucinol on the A-ring of plant proanthocyanidins undergo condensation reaction with vanillin to form colored complexes, which exhibit a characteristic absorption peak at 500 nm. The absorbance at 500 nm is measured to calculate the proanthocyanidin content in plant samples.
This kit is intended for determination of proanthocyanidin content in plant samples.
Precautions
1. Unopened OPC standard powder should be stored at 4°C protected from light. Once dissolved in methanol, prepare OPC standard solution freshly for use; the prepared solution can be stored at 4°C away from light for no more than 1 month.
2. If ΔA exceeds the linear range of the standard curve:
If ΔA is higher than the upper limit, dilute the test supernatant with extraction solution and re-measure; If ΔA is lower than the lower limit, increase sample concentration for re-extraction and determination. Adjust the dilution factor accordingly in result calculation.
3. An enzyme-labeled instrument or general visible spectrophotometer supporting detection at 500 nm can be used. Micro glass cuvettes shall be adopted for spectrophotometric measurement.
4. Methanol and hydrochloric acid are volatile and corrosive. Perform all operations in a fume hood as far as possible. Wear lab coat and disposable gloves throughout the experiment for safety.
5. The color reaction is significantly affected by temperature and incubation time. Standard solutions and test samples must be incubated and measured synchronously.
6. Pigmented plant samples with high anthocyanin content may cause matrix interference. Proanthocyanidins are colorless inherently; sample control wells must be set to subtract matrix background absorption.
7. The Vanillin-HCl method is susceptible to interference from tannins and certain flavonoids. This method cannot distinguish flavanol monomers, oligomeric proanthocyanidins and polymeric proanthocyanidins.
8. Determine the absorbance promptly upon completion of incubation to prevent measurement errors arising from color decay.
9. This kit is for research use only, not for clinical diagnosis or other applications.
Component No. | Component Name | Appearance | 48T | 96T | Storage | Quantity Per Test |
- | Extraction solvent: 60% ethanol (user-supplied) | Liquid | 70 mL | 70 mL×2 | 2-8℃ | As needed |
- | Reagent I: 8% HCl (user-supplied) | Liquid | 12 mL | 24 mL | 2-8℃ | As needed |
P1521899A | Vanillin | Solid | 62.5 mg | 125 mg | 2-8°C, Store in the dark. | As needed |
P1521899B | OPC Standard (5 mg) | Solid | 5 mg | 5 mg | 2-8°C, Store in the dark. | 40 µL |
Procedure
1. Materials Preparation
Additional materials required:
(1) Test sample: Plant tissue
(2) Instruments: Microplate reader or visible spectrophotometer (capable of measuring absorbance at 500 nm).
(3) Others: 96-well microplate or micro glass cuvettes, adjustable pipettes and tips, tissue homogenizer, sonicator, deionized water, methanol, 40-mesh sieve.
2. Reagent Formulation
(1) Extraction solvent (to be prepared by user)
Mix 30 mL absolute ethanol with 20 mL distilled water, then add 100 μL concentrated hydrochloric acid and mix thoroughly. Seal and store at 4°C.
(2) Vanillin: 8% Hydrochloric acid-methanol solution (to be prepared by user)
Slowly add 1.6 mL concentrated hydrochloric acid into 18.4 mL absolute methanol and mix well.
Note: Do not add methanol into concentrated hydrochloric acid to avoid bumping and splashing. Store at 4 °C protected from light.
(3) Vanillin
Powder specification:
Before use, dissolve the powder with methanol: add 6.25 mL methanol for the 48T kit, and 12.5 mL methanol for the 96T kit.
Note 2: Prepared Reagent Vanillin exhibits poor stability. Prepare freshly before use; long-term storage is not recommended.
(4) Assay Working Solution&Blank Working Solution (prepare freshly before use; long-term storage is not allowed)
Assay Working Solution: Vanillin and Reagent I at a volume ratio of 1:1.
Blank Working Solution: Reagent I and methanol at a volume ratio of 1:1.
(5) OPC Standard
Weigh 5 mg standard and dissolve in 1 mL extraction solution to prepare 5 mg/mL stock solution. Equilibrate to room temperature before use. The prepared stock solution can be stored at 4°C away from light for 2 weeks.
Note: This method employs procyanidin B2 as the sole standard. Proanthocyanidin standards from alternative sources may exhibit different response coefficients.
(6) Preparation of OPC standard series
Serially dilute the 5 mg/mL standard stock solution with extraction solution according to the table below to obtain standard solutions at concentrations of 2.5, 1.25, 0.625, 0.3125, 0.1563, 0.0781 and 0 mg/mL.
No. | Volume of Standard (μL) | Volume of Extraction Solution (μL) | Standard Concentration (mg/mL) |
Std.1 | 100 µL 5 mg/mL (Stock Solution) | 0 | 5 |
Std.2 | 100 µL of Std.1 | 100 | 2.5 |
Std.3 | 100 µL of Std.2 | 100 | 1.25 |
Std.4 | 100 µL of Std.3 | 100 | 0.625 |
Std.5 | 100 µL of Std.4 | 100 | 0.3125 |
Std.6 | 100 µL of Std.5 | 100 | 0.1563 |
Std.7 | 100 µL of Std.6 | 100 | 0.0781 |
Std.8 | 0 | 100 | 0 (Blank Control) |
Note: A standard curve must be generated with standard samples in each independent assay. Diluted standard solutions are unstable and shall be used within 4 hours.
3. Sample Preparation
(1) Fibrous plant tissues
Weigh approximately 0.1 g sample, add 1 mL extraction solution and homogenize. Perform ice-bath sonication for 30 min (power: 30% or 300 W; pulse: 5s on, 8s off). Centrifuge at 12,000×g, 25 °C for 10 min. Collect the supernatant, make up to 1 mL with extraction solution, and keep on ice until measurement.
(2) Young plant tissues with low fiber content
Weigh approximately 0.1 g sample, add 1 mL extraction solution and homogenize. Perform ice-bath sonication for 30 min (power: 30% or 300 W; pulse: 5s on, 8s off). Centrifuge at 12,000×g , 25 °C for 10 min. Collect the supernatant, make up to 1 mL with extraction solution, and keep on ice until measurement.
4. Assay Procedure
(1) Pre-warm the microplate reader or visible spectrophotometer for no less than 30 min, and set the detection wavelength to 500 nm.
(2) Reaction loading system (for 96-well plate or micro cuvette; unit: μL):
Reagent | Standard Wells (μL) | Assay Wells（μL） | Control Wells（μL） |
Sample | 0 | 40 | 40 |
Standard solutions of various concentrations | 40 | 0 | 0 |
Blank Working Solution | 0 | 0 | 160 |
Assay Working Solution | 160 | 160 | 0 |
(3) Mix thoroughly, incubate at a constant temperature of 30°C protected from light for 30 min, then measure absorbance at 500 nm.
Record absorbance values:
Ablank=blank well, Astd=standard well, Atest=assay well, Actrl=control well.
Calculate:
ΔAtest = Atest−Actrl.
ΔAstd = Astd−Ablank.
5. Result Calculation
Standard curve: y=ax-b
OPC content (mg/g fresh weight )=(ΔA+b)÷a×Vtotal rxn÷(Vsample÷V sample‑total×W)×10⁻³
Parameter definition:
Vsample‑total: total volume of extraction solution added, 1 mL
Vtotal‑rxn: total reaction volume, 200 µL
Vsample: volume of sample in the reaction, 40 µL
W: mass of plant sample (g)
6. Representative Figures
Note: This is a typical standard curve, actual curve parameters may vary slightly due to instrument and operation differences.
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