Anthocyanin Content Assay Kit (Organic Solvent Extraction, Colorimetric Method)

Cat. No.: A1509392

GRADE & PURITY BioReagent

Overview

Anthocyanin, also known as anthocyanin pigment, belongs to the flavonoid class of compounds. It is a water-soluble pigment widely present in plant cell vacuoles, imparting red, purple, blue, and other colors to flowers, fruits, and leaves. Its content in plants is closely related to organ color, developmental stage, and post-harvest senescence, directly affecting the processing properties, storage quality, and nutritional value of agricultural products.

Detection Principle

Anthocyanins are soluble in organic solvents. Crude extraction of anthocyanins is performed using an organic solvent. Anthocyanins appear red in an acidic solution, and the color intensity is proportional to the anthocyanin content. They exhibit a maximum absorption peak at 530 nm. The absorbance can be measured using a spectrophotometer, and the anthocyanin content can be calculated accordingly. This method is simple and easy to perform. Due to the diversity of anthocyanin types in plants (e.g., Cyanidin, Pelargonidin, etc.) and their different molar extinction coefficients, this kit does not provide a single standard. Therefore, results are expressed as relative absorbance or relative content. To determine the absolute content, users are requested to prepare their own pure anthocyanin standard corresponding to the sample. This kit is primarily used for the extraction and relative quantification of anthocyanins in plant tissues or fruits. It is for research use only and is not suitable for clinical diagnosis or other purposes.

Applicable Samples: Plant tissues or fruits

Reagents, consumables and Equipments not provided

• Spectrophotometer (capable of measuring absorbance at 530 nm)
• Mortar or homogenizer, centrifuge tubes, centrifuge, filter paper or gauze, cuvette(s)
• Experimental materials: Peaches, plums, apples, apricots, or other fruits/plant tissues.

Operating Steps (For Reference Only)

1. Anthocyanin Extraction

Method One:

(1) Pre-cool a mortar or homogenizer, as well as the Anthocyanin Assay Buffer, to 4°C.

(2) Take plant tissue, wash and dry it. Weigh 0.25 g of fresh sample, cut into pieces, and place it in the mortar or homogenizer.

(3) Add 2–3 mL of Anthocyanin Assay Buffer. Grind or homogenize thoroughly, then transfer to a 10 mL centrifuge tube. Rinse the mortar/homogenizer with Anthocyanin Assay Buffer and transfer the rinses to the tube. Adjust the final volume to 8 mL with Anthocyanin Assay Buffer.

(4) Incubate at 4°C in the dark for 20 minutes, shaking 2–3 times during incubation. Then filter into a centrifuge tube. Alternatively, centrifuge at 8000 rpm for 3 minutes. The filtrate (supernatant) is the crude anthocyanin extract.

Method Two:

Add the sample to the extraction solution according to the ratio described above. Place in a 32°C incubator for 4–8 hours (shaking several times during incubation) or extract in the dark at room temperature for 24 hours. Centrifuge and collect the supernatant as the crude anthocyanin extract.

2. Sample Addition and Measurement

Using a 1 cm pathlength cuvette, use Anthocyanin Assay Buffer as the blank control. Take the anthocyanin extract (1–2 mL) and read the absorbance at 530 nm using a spectrophotometer. If the anthocyanin concentration is too high, dilute appropriately before measurement. It is recommended to set up duplicate measurements for each sample and take the average.

3. Result Calculation

Definition of Anthocyanin Unit: One unit (U) of anthocyanin is defined as the concentration that yields an OD value of 0.1.

Calculation method:

Anthocyanin content in tissue sample (U/g) = (A_Test - A_Blank) × N / (0.1 × W)

Parameter Explanation:

A_Test: OD value of the anthocyanin extract at 530 nm

A_Blank: OD value of the blank control at 530 nm

W: Fresh weight of the sample (g)

N: Dilution factor

Note: A standard curve can also be prepared using a user-provided anthocyanin standard with gradient dilutions (e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 1.0 mg/mL), using Anthocyanin Assay Buffer as the blank control. The anthocyanin content in the sample can then be calculated from the curve.

Anthocyanin in tissue sample (mg/g) = C × V_T × N / W

Anthocyanin in liquid sample (mg/mL) = C × N

Parameter Explanation:

C: Anthocyanin concentration of the test sample obtained from the standard curve (mg/mL)

V_T: Total volume of the crude anthocyanin extract (mL) = 8

W: Fresh weight of the sample (g)

N: Dilution factor

Precautions

1. To prevent photodegradation of anthocyanins, perform operations in low light as much as possible, and keep grinding/homogenization time short.

2. Sample amount and reagent volumes should be adjusted appropriately based on the anthocyanin content.

3. Anthocyanin Assay Buffer should be stored tightly sealed to prevent evaporation of active components.

4. If a spectrophotometer is unavailable, a microplate reader can also be used for measurement.

5. For your safety and health, please wear a lab coat and disposable gloves during operation.

6. Please use the reagent as soon as possible after opening to avoid affecting subsequent experimental results.

Storage and Shipping

Storage

Room temperature

Shipped In

Normal

Stability And Storage

Store at room temperature long term (12 months).

Contents & Storage

产品号	Component	50T	Storage
A1509392	Anthocyanin Assay Buffer	500 mL	RT.