Plant Soluble Sugar Content Assay Kit (Anthrone, Colorimetric Method)

Cat. No.: P1507813

GRADE & PURITY BioReagent

Descripción general

Soluble sugars in plants mainly refer to monosaccharides and oligosaccharides that are soluble in water and ethanol. The carbon nutritional status in plants and the quality and characteristics of agricultural products are often evaluated based on sugar content. To adapt to adverse conditions such as drought and low temperature, plants actively accumulate soluble sugars to lower osmotic potential and freezing point, thereby adjusting to environmental changes. Methods for determining soluble sugars in plants include chemical approaches such as the anthrone colorimetric method, 3,5‑dinitrosalicylic acid method, phenol‑sulfuric acid method, and Fehling's reagent colorimetric method.

Detection Principle:

Under the action of concentrated sulfuric acid, reducing sugars undergo dehydration to form furfural or hydroxymethylfurfural. These products react with anthrone to produce a blue‑green furfural derivative. Within a certain range, the intensity of the color is proportional to the content of reducing sugars, with a maximum absorption peak at 630 nm. This method can detect almost all carbohydrates in a sample. It is applicable not only to pentoses (xylose, ribose, arabinose), hexoses (glucose, fructose, sorbose, galactose), sucrose, glycogen, and polyglucose, but also to all oligosaccharides and polysaccharides, including starch and cellulose. In practice, this product can measure the total carbohydrate content in a sample in a single assay, eliminating the need for separate measurements of individual components and offering unique application value.

This kit is intended for research use only and is not suitable for clinical diagnosis or other purposes.

P1507813	Component	50T	Storage
P1507813A	Sucrose Standard Solution (10 mg/mL)	1 mL	2-8℃
P1507813B	Anthrone Reagent	30 mL	2-8℃. Store in the dark.

Reagents, consumables and Equipments not provided

1. Distilled water, concentrated sulfuric acid

2. Electronic balance, water bath or hot plate, spectrophotometer, cuvette, scissors, mortar or homogenizer

3. 50 mL beaker or conical flask, volumetric flask, 20 mL graduated test tubes or 10 mL screw-cap centrifuge tubes

Procedure

1. Extraction of Soluble Sugars

1.1 Weigh 0.5–1 g of fresh plant sample (dry sample is also acceptable), cut into small pieces, add about 3 mL of distilled water for homogenization, and transfer to a graduated tube. Rinse the homogenizer 2–3 times with 12 mL of distilled water and transfer the rinsate to the same container.

1.2 Seal the opening with plastic film and extract in a boiling water bath for 30 minutes. After cooling, filter the extract and transfer the filtrate to a 50 mL volumetric flask.

1.3 Collect the residue, repeat homogenization and extraction with water, combine all filtrates, and adjust to volume.

2. Dilution of Sucrose Standard

Take 1 mL of Sucrose Standard Solution (10 mg/mL) and add to a 100 mL volumetric flask. Adjust to volume with distilled water to obtain the Sucrose Standard (100 µg/mL). Using clean centrifuge tubes or test tubes, prepare a series of sucrose standards with different masses according to the table below.

Tube No.	1	2	3	4	5
Sucrose Standard (100 µg/mL) (mL)	0.2	0.4	0.6	0.8	1
Distilled water (mL)	1.8	1.6	1.4	1.2	1
Equivalent sucrose mass (µg)	20	40	60	80	100
Sucrose standard concentration (µg/mL)	10	20	30	40	50

3. Sample Loading

Take 10 mL screw-cap centrifuge tubes and set up blank tubes, standard tubes, and test tubes according to the table below. Reagents should be added in the specified order, avoiding bubble formation, and mixed carefully. If the sugar concentration in the sample is too high, reduce the sample volume or dilute appropriately before measurement. It is recommended to set up 2–3 replicate tubes for each sample and calculate the average (the volumes of all reagents can be scaled down proportionally, but the minimum required amount should be maintained).

Additive (mL)	Blank Tube	Standard Tube	Test Tube
Distilled water	2	－	－
Series sucrose standards (No. 1–5)	－	2	－
Extraction solution	－	－	2
Anthrone Reagent	0.5	0.5	0.5
Concentrated sulfuric acid	5	5	5

Mix thoroughly, boil in a water bath for 1 minute, remove, and allow to cool naturally to room temperature.

4. Determination of Soluble Sugars

Mix well, set the blank tube to zero, and measure the absorbance at 630 nm for the standard tubes and test tubes using a spectrophotometer with a 1 cm light path cuvette.

5. Calculation of Results

Plot a standard curve with the mass of the series sucrose standards (No. 1–5) in µg as the x‑axis and the corresponding absorbance as the y‑axis, and determine the linear regression equation. Calculate the mass of soluble sugar in the test tube based on its absorbance, and then compute the content. Alternatively, plot the standard curve using sucrose standard concentration (µg/mL) versus absorbance and determine the soluble sugar concentration of the sample. The soluble sugar content, expressed as a mass percentage (%), is calculated as follows:

Soluble sugar content (%) = (m₁ × V_T× N) / (m₀ × V_S× 1,000,000) × 100%

= (c × V_T× N) / (m₀ × 1,000,000) × 100%

Parameter Description:

m₁: Mass of soluble sugar obtained from the standard curve (µg)

V_T: Total volume of extraction solution (mL)

N: Dilution factor of the sample extraction solution

m₀: Mass of the plant sample (g)

V_S: Volume of sample extraction solution taken for measurement (mL)

c: Soluble sugar concentration of the sample (µg/mL)

1,000,000: Conversion factor between µg and g

Notes

1. The test solution must be clear and transparent; no protein precipitation should occur after heating. If the sample color is too dark, decolorize with activated carbon before measurement.

2. If the soluble sugar concentration of the sample is too high, dilute with distilled water. The optimal sugar concentration range is 10–100 µg/mL.

3. Concentrated sulfuric acid (specific gravity 1.84) is highly oxidizing and corrosive, posing significant hazards. Handle with extreme care. Add concentrated sulfuric acid slowly to avoid violent boiling due to heat generation, which may cause skin or clothing burns. In case of contact, rinse immediately with running water and seek medical attention if necessary.

4. The results of this method are influenced by sulfuric acid concentration and heating time; perform operations accurately and carefully.

5. Different sugars produce different color intensities with the anthrone reagent: fructose gives the deepest color, followed by glucose, while galactose and mannose produce lighter colors, and pentoses even lighter. Therefore, errors may arise when measuring sugar mixtures due to varying proportions of different sugars. This error does not occur when measuring a single sugar type.

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

Appendix

Standard Curve Preparation:

Following the instructions, measure the absorbance of the series standards using a spectrophotometer. Example data and standard curve are provided below (for reference only):

Sucrose standard (µg/mL)	0	10	20	30	40	50	100	250
Absorbance	0.07	0.191	0.284	0.394	0.519	0.620	1.192	3.467

The results indicate a good linear gradient for sucrose standards within the range of 10–100 µg/mL.

Almacenamiento y envío

Condiciones de almacenamiento de almacenamiento

Store at 2-8°C,Protected from light

Enviado en

Wet ice

Estabilidad y almacenamiento

Store at 2-8℃ long term (12 months). Store in the dark.

Contents & Storage

P1507813	Component	50T	Storage
P1507813A	Sucrose Standard Solution (10 mg/mL)	1 mL	2-8℃
P1507813B	Anthrone Reagent	30 mL	2-8℃. Store in the dark.